Prof. Dr. Matthias Epple

Inorganic Chemistry
University of Duisburg-Essen

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  • Controlling the Surface Functionalization of Ultrasmall Gold Nanoparticles by Sequence-Defined Macromolecules
    van der Meer, S.B. and Seiler, T. and Buchmann, C. and Partalidou, G. and Boden, S. and Loza, K. and Heggen, M. and Linders, J. and Prymak, O. and Oliveira, C.L.P. and Hartmann, L. and Epple, M.
    Chemistry - A European Journal 27 (2021)
    Ultrasmall gold nanoparticles (diameter about 2 nm) were surface-functionalized with cysteine-carrying precision macromolecules. These consisted of sequence-defined oligo(amidoamine)s (OAAs) with either two or six cysteine molecules for binding to the gold surface and either with or without a PEG chain (3400 Da). They were characterized by 1H NMR spectroscopy, 1H NMR diffusion-ordered spectroscopy (DOSY), small-angle X-ray scattering (SAXS), and high-resolution transmission electron microscopy. The number of precision macromolecules per nanoparticle was determined after fluorescent labeling by UV spectroscopy and also by quantitative 1H NMR spectroscopy. Each nanoparticle carried between 40 and 100 OAA ligands, depending on the number of cysteine units per OAA. The footprint of each ligand was about 0.074 nm2 per cysteine molecule. OAAs are well suited to stabilize ultrasmall gold nanoparticles by selective surface conjugation and can be used to selectively cover their surface. The presence of the PEG chain considerably increased the hydrodynamic diameter of both dissolved macromolecules and macromolecule-conjugated gold nanoparticles. © 2020 The Authors. Published by Wiley-VCH GmbH
    view abstract10.1002/chem.202003804
  • Gene transfection achieved by utilizing antibacterial calcium phosphate nanoparticles for enhanced regenerative therapy
    Xiang, C. and Tenkumo, T. and Ogawa, T. and Kanda, Y. and Nakamura, K. and Shirato, M. and Sokolova, V. and Epple, M. and Kamano, Y. and Egusa, H. and Sasaki, K.
    Acta Biomaterialia 119 (2021)
    Protamine-coated multi-shell calcium phosphate (CaP) was developed as a non-viral vector for tissue regeneration therapy. CaP nanoparticles loaded with different amounts of plasmid DNA encoding bone morphogenetic protein 2 (BMP-2) and insulin-like growth factor 1 (IGF-1) were used to treat MC3T3E1 cells, and the yield of the released BMP-2 or IGF-1 was measured using ELISA 3 days later. Collagen scaffolds containing CaP nanoparticles were implanted into rat cranial bone defects, and BMP-2 and IGF-1 yields, bone formation, and bone mineral density enhancement were evaluated 28 days after gene transfer. The antibacterial effects of CaP nanoparticles against Streptococcus mutans and Aggregatibacter actinomycetemcomitans increased with an increase in the protamine dose, while they were lower for Staphylococcus aureus and Porphyromonas gingivalis. In the combination treatment with BMP-2 and IGF-1, the concentration ratio of BMP-2 and IGF-1 is an important factor affecting bone formation activity. The calcification activity and OCN mRNA of MC3T3E1 cells subjected to a BMP-2:IGF-1 concentration ratio of 1:4 was higher at 14 days. During gene transfection treatment, BMP-2 and IGF-1 were released simultaneously after gene transfer; the loaded dose of the plasmid DNA encoding IGF-1 did not impact the BMP-2 or IGF-1 yield or new bone formation ratio in vitro and in vivo. In conclusion, two growth factor-releasing systems were developed using an antibacterial gene transfer vector, and the relationship between the loaded plasmid DNA dose and resultant growth factor yield was determined in vitro and in vivo. © 2020
    view abstract10.1016/j.actbio.2020.11.003
  • Hybrid chitosan/gelatin/nanohydroxyapatite scaffolds promote odontogenic differentiation of dental pulp stem cells and in vitro biomineralization
    Vagropoulou, G. and Trentsiou, M. and Georgopoulou, A. and Papachristou, E. and Prymak, O. and Kritis, A. and Epple, M. and Chatzinikolaidou, M. and Bakopoulou, A. and Koidis, P.
    Dental Materials 37 (2021)
    Objective: Hybrid chitosan/gelatin/nanohydroxyapatite (CS/Gel/nHA) scaffolds have attracted considerable interest in tissue engineering (TE) of mineralized tissues. The present study aimed to investigate the potential of CS/Gel/nHA scaffolds loaded with dental pulp stem cells (DPSCs) to induce odontogenic differentiation and in vitro biomineralization. Methods: CS/Gel/nHA scaffolds were synthesized by freeze-drying, seeded with DPSCs, and characterized with flow cytometry. Scanning Electron Microscopy (SEM), live/dead staining, and MTT assays were used to evaluate cell morphology and viability; real-time PCR for odontogenesis-related gene expression analysis; SEM-EDS (Energy Dispersive X-ray spectroscopy), and X-ray Diffraction analysis (XRD) for structural and chemical characterization of the mineralized constructs, respectively. Results: CS/Gel/nHA scaffolds supported viability and proliferation of DPSCs over 14 days in culture. Gene expression patterns indicated pronounced odontogenic shift of DPSCs, evidenced by upregulation of DSPP, BMP-2, ALP, and the transcription factors RunX2 and Osterix. SEM-EDS showed the production of a nanocrystalline mineralized matrix inside the cell-based and - to a lesser extent - the cell-free constructs, with a time-dependent production of net-like nanocrystals (appr. 25−30 nm in diameter). XRD analysis gave the crystallite size (D = 50 nm) but could not distinguish between the initially incorporated and the biologically produced nHA. Significance: This is the first study validating the potential of CS/Gel/nHA scaffolds to support viability and proliferation of DPSCs, and to provide a biomimetic microenvironment favoring odontogenic differentiation and in vitro biomineralization without the addition of any inductive factors, including dexamethasone and/or growth/morphogenetic factors. These results reveal a promising strategy towards TE of mineralized dental tissues. © 2020 The Academy of Dental Materials
    view abstract10.1016/j.dental.2020.09.021
  • New tools to probe the protein surface: Ultrasmall gold nanoparticles carry amino acid binders
    van der Meer, S.B. and Hadrovic, I. and Meiners, A. and Loza, K. and Heggen, M. and Knauer, S.K. and Bayer, P. and Schrader, T. and Beuck, C. and Epple, M.
    Journal of Physical Chemistry B (2021)
    A strategy toward epitope-selective functionalized nanoparticles is introduced in the following: ultrasmall gold nanoparticles (diameter of the metallic core about 2 nm) were functionalized with molecular tweezers that selectively attach lysine and arginine residues on protein surfaces. Between 11 and 30 tweezer molecules were covalently attached to the surface of each nanoparticle by copper-catalyzed azide alkyne cycloaddition (CuAAC), giving multiavid agents to target proteins. The nanoparticles were characterized by high-resolution transmission electron microscopy, differential centrifugal sedimentation, and 1H NMR spectroscopy (diffusion-ordered spectroscopy, DOSY, and surface composition). The interaction of these nanoparticles with the model proteins hPin1 (WW domain; hPin1-WW) and Survivin was probed by NMR titration and by isothermal titration calorimetry (ITC). The binding to the WW domain of hPin1 occurred with a KD of 41 ± 2 μM, as shown by ITC. The nanoparticle-conjugated tweezers targeted cationic amino acids on the surface of hPin1-WW in the following order: N-terminus (G) ≈ R17 > R14 ≈ R21 > K13 > R36 > K6, as shown by NMR spectroscopy. Nanoparticle recognition of the larger protein Survivin was even more efficient and occurred with a KD of 8 ± 1 μM, as shown by ITC. We conclude that ultrasmall nanoparticles can act as versatile carriers for artificial protein ligands and strengthen their interaction with the complementary patches on the protein surface. © XXXX American Chemical Society
    view abstract10.1021/acs.jpcb.0c09846
  • A Novel Branched Copolymer-Containing Anticancer Drug for Targeted Therapy: In Vitro Research
    Yurchenko, A. and Nikitina, N. and Sokolova, V. and Prylutska, S. and Kuziv, Y. and Virych, P. and Chumachenko, V. and Kutsevol, N. and Ponomarenko, S. and Prylutskyy, Y. and Epple, M.
    BioNanoScience 10 (2020)
    The viability of cancer cell lines (human transformed cervix epithelial cells, HeLa, and osteoblastic cell line from a C57BL/6 mouse calvaria, MC3T3, as models for cancer cells) was studied using MTT and live/dead assays after incubation with a branched copolymer dextran-graft polyacrylamide in anionic form (D-g-PAAan) as nanocarrier for drugs, doxorubicin (Dox), cisplatin (Cis), as well as their D-g-PAAan+Dox and D-g-PAAan+Cis mixtures, as a function of the concentration. Fourier transform infrared spectroscopy clearly indicates the complex formation of Cis and Dox with the D-g-PAAan branched copolymer. The size distribution of particles in aqueous solution and its stability were determined by dynamic light scattering. The in vitro uptake of studied particles into cancer cells was demonstrated by confocal laser scanning microscopy. It was found that D-g-PAAan+Dox particles in contrast to Dox alone showed higher toxicity towards cancer cells. This indicates the possibility of further preclinical studies of the water-soluble D-g-PAAan+Dox particles on animal tumor models in vivo as a promising anticancer agent. © 2019, Springer Science+Business Media, LLC, part of Springer Nature.
    view abstract10.1007/s12668-019-00700-5
  • A pH-sensitive fluorescent protein sensor to follow the pathway of calcium phosphate nanoparticles into cells
    Kollenda, S. and Kopp, M. and Wens, J. and Koch, J. and Schulze, N. and Papadopoulos, C. and Pöhler, R. and Meyer, H. and Epple, M.
    Acta Biomaterialia 111 (2020)
    Calcium phosphate nanoparticles (100 nm) were fluorescently labelled with poly(ethyleneimine) (PEIATTO490LS; red fluorescence). They were loaded with a Tandem fusion protein consisting of mRFP1-eGFP (red and green fluorescence in the same molecule)that acts as smart biological pH sensor to trace nanoparticles inside cells. Its fluorescence is also coupled to the structural integrity of the protein, i.e. it is also a label for a successful delivery of a functional protein into the cell. At pH 7.4, the fluorescence of both proteins (red and green) is detectable. At a pH of 4.5-5 inside the lysosomes, the green fluorescence is quenched due to the protonation of the eGFP chromophore, but the pH-independent red fluorescence of mRFP1 remains. The nanoparticles were taken up by cells (cell lines: HeLa, Caco-2 and A549) via endocytic pathways and then directed to lysosomes. Time-resolved confocal laser scanning microscopy confirmed mRFP1 and nanoparticles co-localizing with lysosomes. The fluorescence of eGFP was only detectable outside lysosomes, i.e. most likely inside early endosomes or at the cell membrane during the uptake, indicating the neutral pH at these locations. The Tandem fusion protein provides a versatile platform to follow the intracellular pathway of bioactive nanocarriers, e.g. therapeutic proteins. The transfection with a Tandem-encoding plasmid by calcium phosphate nanoparticles led to an even intracellular protein distribution in cytosol and nucleoplasm, i.e. very different from direct protein uptake. Neither dissolved protein nor dissolved plasmid DNA were taken up by the cells, underscoring the necessity for a suitable carrier like a nanoparticle. Statement of Significance: A pH-sensitive protein ("tandem") was used to follow the pathway of calcium phosphate nanoparticles. This protein consists of a pH-sensitive fluorophore (eGFP; green) and a pH-independent fluorophore (mRFP1; red). This permits to follow the pathway of a nanoparticle inside a cell. At a low pH inside an endolysosome, the green fluorescence vanishes but the red fluorescence persists. This is also a very useful model for the delivery of therapeutic proteins into cells. The delivery by nanoparticles was compared with the protein expression after cell transfection with plasmid DNA encoding for the tandem protein. High-resolution image analysis gave quantitative data on the intracellular protein distribution. © 2020 Acta Materialia Inc.
    view abstract10.1016/j.actbio.2020.05.014
  • Ab initio calculations and a scratch test study of RF-magnetron sputter deposited hydroxyapatite and silicon-containing hydroxyapatite coatings
    Surmenev, R.A. and Grubova, I.Y. and Neyts, E. and Teresov, A.D. and Koval, N.N. and Epple, M. and Tyurin, A.I. and Pichugin, V.F. and Chaikina, M.V. and Surmeneva, M.A.
    Surfaces and Interfaces 21 (2020)
    A crucial property for implants is their biocompatibility. To ensure biocompatibility, thin coatings of hydroxyapatite (HA) are deposited on the actual implant. In this study, we investigate the effects of the addition of silicate anions to the structure of hydroxyapatite coatings on their adhesion strength via a scratch test and ab initio calculations. We find that both the grain size and adhesion strength decrease with the increase in the silicon content in the HA coating (SiHA). The increase in the silicon content to 1.2 % in the HA coating leads to a decrease in the average crystallite size from 28 to 21 nm, and in the case of 4.6 %, it leads to the formation of an amorphous or nanocrystalline film. The decreases in the grain and crystallite sizes lead to peeling and destruction of the coating from the titanium substrate at lower loads. Further, our ab initio simulations demonstrate an increased number of molecular bonds at the amorphous SiHA-TiO2 interface. However, the experimental results revealed that the structure and grain size have more pronounced effects on the adhesion strength of the coatings. In conclusion, based on the results of the ab initio simulations and the experimental results, we suggest that the presence of Si in the form of silicate ions in the HA coating has a significant impact on the structure, grain size, and number of molecular bonds at the interface and on the adhesion strength of the SiHA coating to the titanium substrate. © 2020 Elsevier B.V.
    view abstract10.1016/j.surfin.2020.100727
  • An: In situ SAXS investigation of the formation of silver nanoparticles and bimetallic silver-gold nanoparticles in controlled wet-chemical reduction synthesis
    Garcia, P.R.A.F. and Prymak, O. and Grasmik, V. and Pappert, K. and Wlysses, W. and Otubo, L. and Epple, M. and Oliveira, C.L.P.
    Nanoscale Advances 2 (2020)
    We present a study on the formation of silver (Ag) and bimetallic silver-gold (AgAu) nanoparticles monitored by in situ SAXS as well as by ex situ TEM, XRD and UV-vis analysis in a flow reactor at controlled reaction temperature. The formation mechanism of the nanoparticles is derived from the structural parameters obtained from the experimental data. The evolution of the average particle size of pure and alloyed nanoparticles shows that the particle growth occurs initially by a coalescence mechanism. The later growth of pure silver nanoparticles is well described by Ostwald ripening and for the alloyed nanoparticles by a process with a significantly slower growth rate. Additionally, the SAXS data of pure silver nanoparticles revealed two major populations of nanoparticles, the first one with a continuous crystal growth to a saturation plateau, and the second one probably with a continuous emergence of small new crystals. The particle sizes obtained by SAXS agree well with the results from transmission electron microscopy and X-ray diffraction. The present study demonstrates the capability of an in situ investigation of synthesis processes using a laboratory based SAXS instrument. Online monitoring of the synthesis permitted a detailed investigation of the structural evolution of the system. This journal is © The Royal Society of Chemistry.
    view abstract10.1039/c9na00569b
  • Bactericidal activity and recovery effect of hydroxyl radicals generated by ultraviolet irradiation and silver ion application on an infected titanium surface
    Tenkumo, T. and Ishiyama, K. and Prymak, O. and Nakamura, K. and Shirato, M. and Ogawa, T. and Miyashita, M. and Takahashi, M. and Epple, M. and Kanno, T. and Sasaki, K.
    Scientific Reports 10 (2020)
    This study investigated the bactericidal effect, the underlying mechanisms of treatment, and recovery of biocompatibility of the infected titanium surface using a combination treatment of silver ion application and ultraviolet-A (UV-A) light irradiation. Streptococcus mutans and Aggregatibacter actinomycetemcomitans were used in suspension and as a biofilm on a titanium surface to test for the bactericidal effect. The bactericidal effect of the combination treatment was significantly higher than that of silver ion application or UV-A light irradiation alone. The bactericidal effect of the combination treatment was attributable to hydroxyl radicals, which generated from the bacterial cell wall and whose yield increased with the silver concentration. To assess the biocompatibility, proliferation and calcification of MC3T3E1 cells were evaluated on the treated titanium surface. The treated titanium screws were implanted into rat tibias and the removal torques were measured 28 days post-surgery. The titanium surface that underwent the combination treatment exhibited recovery of biocompatibility by allowing cellular proliferation or calcification at levels observed in the non-infected titanium surfaces. The removal torque 28 days after surgery was also comparable to the control values. This approach is a novel treatment option for peri-implantitis. © 2020, The Author(s).
    view abstract10.1038/s41598-020-65411-4
  • Cell-biological effects of zinc oxide spheres and rods from the nano- to the microscale at sub-toxic levels
    Olejnik, M. and Kersting, M. and Rosenkranz, N. and Loza, K. and Breisch, M. and Rostek, A. and Prymak, O. and Schürmeyer, L. and Westphal, G. and Köller, M. and Bünger, J. and Epple, M. and Sengstock, C.
    Cell Biology and Toxicology (2020)
    Zinc oxide particles were synthesized in various sizes and shapes, i.e., spheres of 40-nm, 200-nm, and 500-nm diameter and rods of 40∙100 nm2 and 100∙400 nm2 (all PVP-stabilized and well dispersed in water and cell culture medium). Crystallographically, the particles consisted of the hexagonal wurtzite phase with a primary crystallite size of 20 to 100 nm. The particles showed a slow dissolution in water and cell culture medium (both neutral; about 10% after 5 days) but dissolved within about 1 h in two different simulated lysosomal media (pH 4.5 to 4.8). Cells relevant for respiratory exposure (NR8383 rat alveolar macrophages) were exposed to these particles in vitro. Viability, apoptosis, and cell activation (generation of reactive oxygen species, ROS, release of cytokines) were investigated in an in vitro lung cell model with respect to the migration of inflammatory cells. All particle types were rapidly taken up by the cells, leading to an increased intracellular zinc ion concentration. The nanoparticles were more cytotoxic than the microparticles and comparable with dissolved zinc acetate. All particles induced cell apoptosis, unlike dissolved zinc acetate, indicating a particle-related mechanism. Microparticles induced a stronger formation of reactive oxygen species than smaller particles probably due to higher sedimentation (cell-to-particle contact) of microparticles in contrast to nanoparticles. The effect of particle types on the cytokine release was weak and mainly resulted in a decrease as shown by a protein microarray. In the particle-induced cell migration assay (PICMA), all particles had a lower effect than dissolved zinc acetate. In conclusion, the biological effects of zinc oxide particles in the sub-toxic range are caused by zinc ions after intracellular dissolution, by cell-to-particle contacts, and by the uptake of zinc oxide particles into cells. [Figure not available: see fulltext.]. © 2020, The Author(s).
    view abstract10.1007/s10565-020-09571-z
  • Comparative study of the structure, properties, and corrosion behavior of Sr-containing biocoatings on Mg0.8Ca
    Sedelnikova, M.B. and Sharkeev, Y.P. and Tolkacheva, T.V. and Khimich, M.A. and Bakina, O.V. and Fomenko, A.N. and Kazakbaeva, A.A. and Fadeeva, I.V. and Egorkin, V.S. and Gnedenkov, S.V. and Schmidt, J. and Loza, K. and Prymak, O. and Epple, M.
    Materials 13 (2020)
    A comparative analysis of the structure, properties and the corrosion behavior of the micro-arc coatings based on Sr-substituted hydroxyapatite (Sr-HA) and Sr-substituted tricalcium phosphate (Sr-TCP) deposited on Mg0.8Ca alloy substrates was performed. The current density during the formation of the Sr-HA coatings was higher than that for the Sr-TCP coatings. As a result, the Sr-HA coatings were thicker and had a greater surface roughness Ra than the Sr-TCP coatings. In addition, pore sizes of the Sr-HA were almost two times larger. The ratio (Ca + Sr + Mg)/P were equal 1.64 and 1.47 for Sr-HA and Sr-TCP coatings, respectively. Thus, it can be assumed that the composition of Sr-HA and Sr-TCP coatings was predominantly presented by (Sr, Mg)-substituted hydroxyapatite and (Sr, Mg)-substituted tricalcium phosphate. However, the average content of Sr was approximately the same for both types of the coatings and was equal to 1.8 at.%. The Sr-HA coatings were less soluble and had higher corrosion resistance than the Sr-TCP coatings. Cytotoxic tests in vitro demonstrated a higher cell viability after cultivation with extracts of the Sr-HA coatings. © 2020 by the authors.
    view abstract10.3390/MA13081942
  • Development of a bone substitute material based on additive manufactured Ti6Al4V alloys modified with bioceramic calcium carbonate coating: Characterization and antimicrobial properties
    Surmeneva, M.A. and Chudinova, E.A. and Chernozem, R.V. and Lapanje, A. and Koptyug, A.V. and Rijavec, T. and Loza, K. and Prymak, O. and Epple, M. and Wittmar, A. and Ulbricht, M. and Surmenev, R.A.
    Ceramics International 46 (2020)
    This investigation shows that composite structures based on additive manufactured electron beam melted Ti6Al4V scaffolds coated with calcium carbonate particles can be used as a potential biocomposites for bone substitutes. A continuous bioceramic coating of CaCO3 was deposited on additive manufactured titanium alloy under the influence of ultrasound. XRD analysis revealed the formation of a mixture of calcite and vaterite phases. CaCO3 coating led to decreasing roughness of additively manufactured (AM) scaffolds and improved surface hydrophilicity. In vitro assay demonstrated enhanced inorganic bone phase formation on the surface of CaCO3-coated AM scaffolds compared to as-manufactured ones. The short-term adhesion of S. aureus onto sample surface was evaluated by fluorescent microscopy 0, 3, and 72 h after cell seeding. It revealed that the surface modification resulted in the decreased number of bacteria attached to the surface after CaCO3 deposition. The morphology, roughness, solubility and superhydrophilic character of the CaCO3 coated EBM-manufactured Ti6Al4V alloy surface are suggested as factors contributing to preventing S. aureus adhesion. Thus, the developed biocomposites based on additively manufactured Ti6Al4V alloy scaffolds and CaCO3 coating can be successfully used in bone tissue regeneration providing the effective growth of inorganic bone phase and preventing the bacteria adhesion. © 2020 Elsevier Ltd and Techna Group S.r.l.
    view abstract10.1016/j.ceramint.2020.07.041
  • Effective activation of human antigen-presenting cells and cytotoxic CD8+ T cells by a calcium phosphate-based nanoparticle vaccine delivery system
    Scheffel, F. and Knuschke, T. and Otto, L. and Kollenda, S. and Sokolova, V. and Cosmovici, C. and Buer, J. and Timm, J. and Epple, M. and Westendorf, A.M.
    Vaccines 8 (2020)
    The ability of vaccines to induce T cell responses is crucial for preventing diseases caused by viruses. Nanoparticles (NPs) are considered to be efficient tools for the initiation of potent immune responses. Calcium phosphate (CaP) NPs are a class of biodegradable nanocarriers that are able to deliver immune activating molecules across physiological barriers. Therefore, the aim of this study was to assess whether Toll-like receptor (TLR) ligand and viral antigen functionalized CaP NPs are capable of inducing efficient maturation of human antigen presenting cells (APC). To achieve this, we generated primary human dendritic cells (DCs) and stimulated them with CpG or poly(I:C) functionalized CaP NPs. DCs were profoundly stronger when activated upon NP stimulation compared to treatment with soluble TLR ligands. This is indicated by increased levels of costimulatory molecules and the secretion of proinflammatory cytokines. Consequently, coculture of NP-stimulated APCs with CD8+ T cells resulted in a significant expansion of virus-specific T cells. In summary, our data suggest that functionalized CaP NPs are a suitable tool for activating human virus-specific CD8+ T cells and may represent an excellent vaccine delivery system. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
    view abstract10.3390/vaccines8010110
  • Enhanced dissolution of silver nanoparticles in a physical mixture with platinum nanoparticles based on the sacrificial anode effect
    Breisch, M. and Loza, K. and Pappert, K. and Rostek, A. and Rurainsky, C. and Tschulik, K. and Heggen, M. and Epple, M. and Tiller, J.C. and Schildhauer, T.A. and Köller, M. and Sengstock, C.
    Nanotechnology 31 (2020)
    A strategy to reduce implant-related infections is the inhibition of the initial bacterial implant colonization by biomaterials containing silver (Ag). The antimicrobial efficacy of such biomaterials can be increased by surface enhancement (nanosilver) or by creating a sacrificial anode system for Ag. Such a system will lead to an electrochemically driven enhanced Ag ion release due to the presence of a more noble metal. Here we combined the enlarged surface of nanoparticles (NP) with a possible sacrificial anode effect for Ag induced by the presence of the electrochemically more noble platinum (Pt) in physical mixtures of Ag NP and Pt NP dispersions. These Ag NP/Pt NP mixtures were compared to the same amounts of pure Ag NP in terms of cell biological responses, i.e. the antimicrobial activity against Staphylococcus aureus and Escherichia coli as well as the viability of human mesenchymal stem cells (hMSC). In addition, Ag NP was analyzed by ultraviolet-visible (UV-vis) spectroscopy, cyclic voltammetry, and atomic absorption spectroscopy. It was found that the dissolution rate of Ag NP was enhanced in the presence of Pt NP within the physical mixture compared to a dispersion of pure Ag NP. Dissolution experiments revealed a fourfold increased Ag ion release from physical mixtures due to enhanced electrochemical activity, which resulted in a significantly increased toxicity towards both bacteria and hMSC. Thus, our results provide evidence for an underlying sacrificial anode mechanism induced by the presence of Pt NP within physical mixtures with Ag NP. Such physical mixtures have a high potential for various applications, for example as antimicrobial implant coatings in the biomedicine or as bactericidal systems for water and surface purification in the technical area. © 2019 IOP Publishing Ltd.
    view abstract10.1088/1361-6528/ab4e48
  • Enteric Coating Systems for the Oral Administration of Bioactive Calcium Phosphate Nanoparticles Carrying Nucleic Acids into the Colon
    Hosseini, S. and Wey, K. and Epple, M.
    ChemistrySelect 5 (2020)
    A system to deliver bioactive calcium phosphate nanoparticles (about 60 nm, loaded with nucleic acids) across the stomach into the colon was developed by freeze-drying the nanoparticles in the presence of the cryoprotectant trehalose, incorporation into gelatin capsules, and coating the capsules with an enteric polymer. These capsules were stable at pH 1 (stomach conditions) and dissolved at pH 7.1 (colon conditions). The nanoparticles were released after about 60–120 min at pH 7.1. Cell culture experiments with HeLa and Caco-2 cells showed an uptake of the nanoparticles, a transfection with EGFP-encoding plasmid DNA, and a gene-silencing with anti-EGFP siRNA. From a variety of enteric coating polymers, the systems cellulose acetate phthalate (CAP), hydroxypropylmethylcellulose-Eudragit L100, and hydroxypropylmethylcellulose-polyvinylalcohol-Eudragit L100 were the most suitable to accomplish the desired biological effect. The presented method is applicable for colon therapy, e. g. for an oral administration of bioactive nanoparticles which are sensitive to acidic conditions. © 2020 The Authors. Published by Wiley-VCH GmbH
    view abstract10.1002/slct.202002846
  • Functional adaptations in the tergite cuticle of the desert isopod Hemilepistus reaumuri (Milne-Edwards, 1840)
    Ernst, F. and Fabritius, H.-O. and Griesshaber, E. and Reisecker, C. and Neues, F. and Epple, M. and Schmahl, W.W. and Hild, S. and Ziegler, A.
    Journal of Structural Biology 212 (2020)
    To survive in its extreme habitat, the cuticle of the burrowing desert isopod Hemilepistus reaumuri requires properties distinct from isopods living in moist or mesic habitats. In particular, the anterior tergites are exposed to high mechanical loads and temperatures when individuals guard the entrance of their burrow. We have, therefore, investigated the architecture, composition, calcite texture and local mechanical properties of the tergite cuticle, with particular emphasis on large anterior cuticle tubercles and differences between the anterior and posterior tergite. Unexpectedly, structure and thickness of the epicuticle resemble those in mesic isopod species. The anterior tergite has a thicker endocuticle and a higher local stiffness than the posterior tergite. Calcite distribution in the cuticle is unusual, because in addition to the exocuticle the endocuticle distally also contains calcite. The calcite consists of a distal layer of dense and highly co-oriented crystal-units, followed proximally by irregularly distributed and, with respect to each other, misoriented calcite crystallites. The calcite layer at the tip of the tubercle is thicker relative to the tubercle slopes, and its crystallites are more misoriented to each other. A steep decrease of local stiffness and hardness is observed within a distal region of the cuticle, likely caused by a successive increase in the ACC/calcite ratio rather than changes in the degree of mineralisation. Comparison of the results with other isopods reveals a much lower ACC/calcite ratio in H. reaumuri and a correlation between the degree of terrestriality of isopod species and the magnesium content of the cuticle. © 2020 Elsevier Inc.
    view abstract10.1016/j.jsb.2020.107570
  • Genetic immunization against hepatitis B virus with calcium phosphate nanoparticles in vitro and in vivo
    Rojas-Sánchez, L. and Zhang, E. and Sokolova, V. and Zhong, M. and Yan, H. and Lu, M. and Li, Q. and Yan, H. and Epple, M.
    Acta Biomaterialia 110 (2020)
    Calcium phosphate nanoparticles were loaded with plasmid DNA and toll-like receptor ligands (TLR), i.e. CpG or flagellin, to activate antigen-presenting cells (APCs) like dendritic cells (DCs). The functionalized nanoparticles were studied in vitro on HeLa, C2C12 and BHK-21 cell lines, focusing on the expression of two specific proteins. EGFP-DNA, encoding for enhanced green fluorescent protein (EGFP), was used as a model plasmid to optimize the transfection efficiency in vitro by fluorescence microscopy and flow cytometry. Calcium phosphate nanoparticles loaded with TLR ligands and plasmid DNA encoding for the hepatitis B virus surface antigen (pHBsAg) were evaluated by in vitro and in vivo immunization experiments to identify a possible candidate for a prophylactic hepatitis B virus (HBV) vaccine. The nanoparticles induced a strong expression of HBsAg in the three cell lines. In splenocytes, the expression of the co-stimulatory molecules CD80 and CD86 was enhanced. After intramuscular injection in mice, the nanoparticles induced the expression of HBsAg, the antigen-specific T cell response, and the antigen-specific antibody response (IgG1). Statement of Significance: Hepatitis B is one of the most frequent viral infections worldwide. For preventive immunization, nanoparticles can be used which carry both an adjuvant (a stimulatory molecule) and DNA encoding for a viral antigen. After administration of such nanoparticles to cells, they are taken up by cells where the DNA is transcribed into the viral antigen (a protein). This viral antigen is inducing a virus-specific immune response. This was shown both by in vitro cell culture as well as by an extensive in vivo study in mice. © 2020 Acta Materialia Inc.
    view abstract10.1016/j.actbio.2020.04.021
  • In situ synthesis of a binary Ti–10at% Nb alloy by electron beam melting using a mixture of elemental niobium and titanium powders
    Surmeneva, M.A. and Koptyug, A. and Khrapov, D. and Ivanov, Y.F. and Mishurova, T. and Evsevleev, S. and Prymak, O. and Loza, K. and Epple, M. and Bruno, G. and Surmenev, R.A.
    Journal of Materials Processing Technology 282 (2020)
    This study reports the results of the preliminary assessment to fabricate Ti-10at% Nb alloy by electron beam melting (EBM®) from a blend of elemental Nb and Ti powders. The microstructure of the EBM-manufactured Ti-10at% Nb alloys is sensitive to the following factors: different sintering properties of Nb and Ti powders, powder particle properties, material viscosities at varying melt pool temperatures, β-stabilizer element content and the EBM® process parameters. Three phases were observed in as-manufactured Ti-10at% Nb alloy: μm-size Nb phase, a Nb-rich β-solid solution surrounding Nb phase, lamellar structured α-phase and β-solid solution with different distribution and volume fraction. Thus, the combination of powder particle characteristics, very short time material spends in molten condition and sluggish kinetics of mixing and diffusional process in Ti-Nb alloy results in heterogeneous microstructures depending on the local Nb content in the powder blend and the EBM® process conditions. © 2020 Elsevier B.V.
    view abstract10.1016/j.jmatprotec.2020.116646
  • In vivo biodistribution of calcium phosphate nanoparticles after intravascular, intramuscular, intratumoral, and soft tissue administration in mice investigated by small animal PET/CT
    Kollenda, S.A. and Klose, J. and Knuschke, T. and Sokolova, V. and Schmitz, J. and Staniszewska, M. and Costa, P.F. and Herrmann, K. and Westendorf, A.M. and Fendler, W.P. and Epple, M.
    Acta Biomaterialia 109 (2020)
    Calcium phosphate nanoparticles were covalently surface-functionalized with the ligand DOTA and loaded with the radioisotope 68Ga. The biodistribution of such 68Ga-labelled nanoparticles was followed in vivo in mice by positron emission tomography in combination with computer tomography (PET-CT). The biodistribution of 68Ga-labelled nanoparticles was compared for different application routes: intravenous, intramuscular, intratumoral, and into soft tissue. The particle distribution was measured in vivo by PET-CT after 5 min, 15 min, 30 min, 1 h, 2 h, and 4 h, and ex vivo after 5 h. After intravenous injection (tail vein), the nanoparticles rapidly entered the lungs with later redistribution into liver and spleen. The nanoparticles remained mostly at the injection site following intramuscular, intratumoral, or soft tissue application, with less than 10 percent being mobilized into the blood stream. Statement of Significance: The in vivo biodistribution of DOTA-terminated calcium phosphate nanoparticles was followed by PET/CT. To our knowledge, this is the first study of this kind. Four different application routes of clinical relevance were pursued: Intravascular, intramuscular, intratumoral, and into soft tissue. Given the high importance of calcium phosphate as biomaterial and for nanoparticular drug delivery and immunization, this is most important to assess the biofate of calcium phosphate nanoparticles for therapeutic application and also judge biodistribution of nanoscopic calcium phosphate ceramics, including debris from endoprostheses and related implants. © 2020
    view abstract10.1016/j.actbio.2020.03.031
  • In Vivo Effects of a Hydroxyapatite-Based Oral Care Gel on the Calcium and Phosphorus Levels of Dental Plaque
    Sudradjat, H. and Meyer, F. and Loza, K. and Epple, M. and Enax, J.
    European Journal of Dentistry 14 (2020)
    Objectives Particulate hydroxyapatite (HAP; Ca 5 (PO 4) 3 (OH)) is a biomimetic oral care ingredient. One mode of action in caries-susceptible individuals may be the adhesion/incorporation of HAP into dental plaque. Therefore, the aim of this in vivo study was to analyze the 3-day effects of a newly developed hydroxyapatite-based oral care gel on the calcium and phosphorus levels within the dental plaque of children. Materials and Methods This study was conducted in Kebon Padangan at Bali in Indonesia. Thirty-four children (mean age, 8.9 years; mean DMF-T [decayed, missing, and filled teeth; permanent teeth], 0.6; mean dmft-t [primary teeth] 4.5) were included in the study. The gel was applied thrice for 3 days by an experienced dentist. Dental plaque was collected at baseline and after the study. Levels of calcium and phosphorus of plaque samples were analyzed by energy-dispersive X-ray spectroscopy. Statistical Analysis Medians for both calcium and phosphorus levels were calculated (baseline and 3-day application of HAP-gel). Results The calcium level increased after 3 days of application of the HAP-gel from 0.25 wt% (median) to 0.40 wt% (median), while the phosphorus level increased from 1.17 wt% (median) to 1.41 wt% (median). However, variations in both calcium and phosphorus levels measured in the pooled dental plaque samples were high. Conclusion Within the limitations of the study, the 3-day application of the oral HAP-gel in children increased the median of both calcium and phosphorus levels in plaque. Consequently, a positive influence on the remineralization/demineralization process is very likely. © 2020 Dental Investigation Society.
    view abstract10.1055/s-0040-1708456
  • MFsim- A n open Java all-in-one rich-client simulation environment for mesoscopic simulation
    Van Den Broek, K. and Daniel, M. and Epple, M. and Hein, J.-M. and Kuhn, H. and Neumann, S. and Truszkowski, A. and Zielesny, A.
    Journal of Cheminformatics 12 (2020)
    MFsim is an open Java all-in-one rich-client computing environment for mesoscopic simulation with Jdpd as its default simulation kernel for Molecular Fragment (Dissipative Particle) Dynamics. The new environment comprises the complete preparation-simulation-evaluation triad of a mesoscopic simulation task and especially enables biomolecular simulation tasks with peptides and proteins. Productive highlights are a SPICES molecular structure editor, a PDB-to-SPICES parser for particle-based peptide/protein representations, a support of polymer definitions, a compartment editor for complex simulation box start configurations, interactive and flexible simulation box views including analytics, simulation movie generation or animated diagrams. As an open project, MFsim allows for customized extensions for different fields of research. [Figure not available: See fulltext.] © 2020 The Author(s).
    view abstract10.1186/s13321-020-00432-9
  • Reduction of inflammation in a chronic periodontitis model in rats by TNF-α gene silencing with a topically applied siRNA-loaded calcium phosphate paste
    Tenkumo, T. and Rojas-Sánchez, L. and Vanegas Sáenz, J.R. and Ogawa, T. and Miyashita, M. and Yoda, N. and Prymak, O. and Sokolova, V. and Sasaki, K. and Epple, M.
    Acta Biomaterialia 105 (2020)
    We developed a calcium phosphate-based paste containing siRNA against TNF-α and investigated its anti-inflammatory and bone-healing effects in vitro and in vivo in a rat periodontitis model. The bioactive spherical CaP/PEI/siRNA/SiO2 nanoparticles had a core diameter of 40–90 nm and a positive charge (+23 mV) that facilitated cellular uptake. The TNF- α gene silencing efficiency of the nanoparticles in J774.2 monocytes, gingival-derived cells, and bone marrow-derived cells was 12 ± 2%, 36 ± 8%, and 35 ± 22%, respectively. CaP/PEI/siRNA/SiO2 nanoparticles cancelled the suppression of alkaline phosphatase (ALP) activity in LPS-stimulated bone marrow-derived cells. In vivo, ALP mRNA was up-regulated, TNF-α mRNA was down-regulated, and the amount of released TNF-α was significantly reduced after topical application of the calcium phosphate-based paste containing siRNA-loaded nanoparticles. The number of TNF-α-positive cells in response to CaP/PEI/siRNA/SiO2 nanoparticle application was lower than that observed in the absence of siRNA. Elevated ALP activity and numerous TRAP-positive cells (osteoclasts) were observed in response to the application of all calcium phosphate pastes. These results demonstrate that local application of a paste consisting of siRNA-loaded calcium phosphate nanoparticles successfully induces TNF-α silencing in vitro and in vivo and removes the suppression of ALP activity stimulated by inflammation. Statement of significance: We developed a calcium phosphate-based paste containing nanoparticles loaded with siRNA against TNF-α. The nanoparticles had a core diameter of 40–90 nm and positive charge (+23 mV). The anti-inflammatory and osteoinductive effects of the paste were investigated in vitro and in vivo in a rat periodontitis model. In vitro, the TNF-α gene silencing efficiency of the nanoparticles in J774.2 monocytes, gingival-derived cells, and bone marrow-derived cells was 12 ± 2%, 36 ± 8%, and 35 ± 22%, respectively. The ALP activity of bone marrow-derived cells was recovered. In vivo, TNF-α mRNA was down-regulated and the amount of released TNF-α was significantly reduced, whereas the ALP mRNA was up-regulated. Elevated ALP activity and TRAP-positive cells were observed by immunohistochemistry. © 2020
    view abstract10.1016/j.actbio.2020.01.031
  • Subtoxic cell responses to silica particles with different size and shape
    Kersting, M. and Olejnik, M. and Rosenkranz, N. and Loza, K. and Breisch, M. and Rostek, A. and Westphal, G. and Bünger, J. and Ziegler, N. and Ludwig, Al. and Köller, M. and Sengstock, C. and Epple, M.
    Scientific Reports 10 (2020)
    Health risks from particles are a priority challenge to health protection at work. Despite the ubiquitous exposure to a wide range of particles and the many years of research in this field, there are fundamental unresolved questions regarding the prevention of particle-related respiratory diseases. Here, the highly relevant particulate material silicon dioxide was analyzed with emphasis on defined size and shape. Silica particles were prepared with different size and shape: Spheres (NS nanospheres 60 nm; SMS submicrospheres 230 nm; MS microspheres 430 nm) and rods (SMR submicrorods with d = 125 nm, L = 230 nm; aspect ratio 1:1.8; MR microrods with d = 100 nm, L = 600 nm; aspect ratio 1:6). After an in-depth physicochemical characterization, their effects on NR8383 alveolar macrophages were investigated. The particles were X-ray amorphous, well dispersed, and not agglomerated. Toxic effects were only observed at high concentrations, i.e. ≥ 200 µg mL−1, with the microparticles showing a stronger significant effect on toxicity (MS≈MR > SMR≈SMS≈NS) than the nanoparticles. Special attention was directed to effects in the subtoxic range (less than 50% cell death compared to untreated cells), i.e. below 100 µg mL−1 where chronic health effects may be expected. All particles were readily taken up by NR8383 cells within a few hours and mainly found associated with endolysosomes. At subtoxic levels, neither particle type induced strongly adverse effects, as probed by viability tests, detection of reactive oxygen species (ROS), protein microarrays, and cytokine release (IL-1β, GDF-15, TNF-α, CXCL1). In the particle-induced cell migration assay (PICMA) with leukocytes (dHL-60 cells) and in cytokine release assays, only small effects were seen. In conclusion, at subtoxic concentrations, where chronic health effects may be expected, neither size and nor shape of the synthesized chemically identical silica particles showed harmful cell-biological effects. © 2020, The Author(s).
    view abstract10.1038/s41598-020-78550-5
  • Synthesis and characterization of PLGA/HAP scaffolds with DNA-functionalised calcium phosphate nanoparticles for bone tissue engineering
    Sokolova, V. and Kostka, K. and Shalumon, K.T. and Prymak, O. and Chen, J.-P. and Epple, M.
    Journal of Materials Science: Materials in Medicine 31 (2020)
    Porous scaffolds of poly(lactide-co-glycolide) (PLGA; 85:15) and nano-hydroxyapatite (nHAP) were prepared by an emulsion-precipitation procedure from uniform PLGA–nHAP spheres (150–250 µm diameter). These spheres were then thermally sintered at 83 °C to porous scaffolds that can serve for bone tissue engineering or for bone substitution. The base materials PLGA and nHAP and the PLGA–nHAP scaffolds were extensively characterized by X-ray powder diffraction, infrared spectroscopy, thermogravimetry, differential scanning calorimetry, and scanning electron microscopy. The scaffold porosity was about 50 vol% as determined by relating mass and volume of the scaffolds, together with the computed density of the solid phase (PLGA–nHAP). The cultivation of HeLa cells demonstrated their high cytocompatibility. In combination with DNA-loaded calcium phosphate nanoparticles, they showed a good activity of gene transfection with enhanced green fluorescent protein (EGFP) as model protein. This is expected enhance bone growth around an implanted scaffold or inside a scaffold for tissue engineering. [Figure not available: see fulltext.] © 2020, The Author(s).
    view abstract10.1007/s10856-020-06442-1
  • Synthesis and intracellular tracing surface-functionalized calcium phosphate nanoparticles by super-resolution microscopy (STORM)
    Rojas-Sánchez, L. and Loza, K. and Epple, M.
    Materialia 12 (2020)
    Calcium phosphate nanoparticles with covalently attached fluorescent dye molecules were prepared by copper-catalysed azide-alkyne cycloaddition (CuAAC) and by strain-promoted azide-alkyne cycloaddition (SPAAC), i.e. click chemistry. The reaction kinetics and labeling degrees of the two click reactions were compared and quantified. For oxidation-sensitive molecules, the SPAAC method is preferable due to the milder reaction conditions and the absence of copper(II). The highly fluorescent AF-488-labeled nanoparticles were applied for super-resolution fluorescence microscopy, i.e. stochastic optical reconstruction microscopy (STORM). By this method, individual nanoparticles (diameter about 60 nm) were observed in endolysosomes inside HeLa cells after cellular uptake at a resolution of 20–30 nm. © 2020
    view abstract10.1016/j.mtla.2020.100773
  • Synthesis, Structure, Properties, and Applications of Bimetallic Nanoparticles of Noble Metals
    Loza, K. and Heggen, M. and Epple, M.
    Advanced Functional Materials 30 (2020)
    Bimetallic nanoparticles of noble metals are of high interest in imaging, biomedical devices, including nanomedicine, and heterogeneous catalysis. Synthesis, properties, characterization, biological properties, and practical applicability of nanoparticles on the basis of platinum group metals and the coin metals Ag and Au are discussed, also in comparison with the corresponding monometallic nanoparticles. In addition to the parameters that are required to characterize monometallic nanoparticles (mainly size, size distribution, shape, crystallographic nature, surface functionalization, charge), further information is required for a full characterization of bimetallic nanoparticles. This concerns the overall elemental composition of a bimetallic nanoparticle population (ratio of the two metals) and the internal distribution of the elements in individual nanoparticles (e.g., the presence of homogeneous alloys, core–shell systems, and possible intermediate stages). It is also important to ensure that all particles are identical in terms of elemental composition, that is, that the homogeneity of the particle population is given. Macroscopic properties like light absorption, antibacterial effects, and catalytic activity depend on these properties. The currently available methods for a full characterization of bimetallic nanoparticles are discussed, and future developments in this field are outlined. © 2020 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
    view abstract10.1002/adfm.201909260
  • Temperature-Induced Stress Relaxation in Alloyed Silver-Gold Nanoparticles (7-8 nm) by in Situ X-ray Powder Diffraction
    Prymak, O. and Grasmik, V. and Loza, K. and Heggen, M. and Epple, M.
    Crystal Growth and Design 20 (2020)
    Alloyed silver-gold nanoparticles (spherical, 8 nm) were wet-chemically prepared by reduction with sodium citrate/tannic acid and colloidally stabilized by poly(N-vinylpyrrolidone) (PVP), in steps of 10 atom %, including pure silver nanoparticles (35 nm) and pure gold nanoparticles (7 nm). The nanoparticles were subjected to in situ X-ray powder diffraction up to 850 °C to induce internal stress relaxation and recrystallization. The stress-induced negative deviation from Vegard's rule that was present in the original alloyed nanoparticles vanished between 150 and 250 °C, indicating the internal healing of defects. Simultaneously, a discontinuous increase in the crystallite size and a drop in the microstrain were observed. After heating to 850 °C, the original gradient structure (silver-rich shell, gold-rich core) had changed to a homogeneous elemental distribution as shown by high-angle annular dark-field scanning transmission electron microscopy/energy-dispersive X-ray spectroscopy (STEM/EDX). Thus, there is a considerable mobility of the metal atoms inside the nanoparticles that starts as low as 150 °C. © 2019 American Chemical Society.
    view abstract10.1021/acs.cgd.9b00728
  • The impact of post manufacturing treatment of functionally graded Ti6Al4V scaffolds on their surface morphology and mechanical strength
    Khrapov, D. and Koptyug, A. and Manabaev, K. and Léonard, F. and Mishurova, T. and Bruno, G. and Cheneler, D. and Loza, K. and Epple, M. and Surmenev, R. and Surmeneva, M.
    Journal of Materials Research and Technology 9 (2020)
    An ultrasonic vibration post-treatment procedure was suggested for additively manufactured lattices. The aim of the present research was to investigate mechanical properties and the differences in mechanical behavior and fracture modes of Ti6Al4V scaffolds treated with traditional powder recovery system (PRS) and ultrasound vibration (USV). Scanning electron microscopy (SEM) was used to investigate the strut surface and the fracture surface morphology. X-ray computed tomography (CT) was employed to evaluate the inner structure, strut dimensions, pore size, as well as the surface morphology of additively manufactured porous scaffolds. Uniaxial compression tests were conducted to obtain elastic modulus, compressive ultimate strength and yield stress. Finite element analysis was performed for a body-centered cubic (BCC) element-based model and for CT-based reconstruction data, as well as for a two-zone scaffold model to evaluate stress distribution during elastic deformation. The scaffold with PRS post treatment displayed ductile behavior, while USV treated scaffold displayed fragile behavior. Double barrel formation of PRS treated scaffold was observed during deformation. Finite element analysis for the CT-based reconstruction revealed the strong impact of surface morphology on the stress distribution in comparison with BCC cell model because of partially molten metal particles on the surface of struts, which usually remain unstressed. © 2019 The Authors.
    view abstract10.1016/j.jmrt.2019.12.019
  • Transport of ultrasmall gold nanoparticles (2 nm) across the blood–brain barrier in a six-cell brain spheroid model
    Sokolova, V. and Mekky, G. and van der Meer, S.B. and Seeds, M.C. and Atala, A.J. and Epple, M.
    Scientific Reports 10 (2020)
    The blood–brain barrier (BBB) is an efficient barrier for molecules and drugs. Multicellular 3D spheroids display reproducible BBB features and functions. The spheroids used here were composed of six brain cell types: Astrocytes, pericytes, endothelial cells, microglia cells, oligodendrocytes, and neurons. They form an in vitro BBB that regulates the transport of compounds into the spheroid. The penetration of fluorescent ultrasmall gold nanoparticles (core diameter 2 nm; hydrodynamic diameter 3–4 nm) across the BBB was studied as a function of time by confocal laser scanning microscopy, with the dissolved fluorescent dye (FAM-alkyne) as a control. The nanoparticles readily entered the interior of the spheroid, whereas the dissolved dye alone did not penetrate the BBB. We present a model that is based on a time-dependent opening of the BBB for nanoparticles, followed by a rapid diffusion into the center of the spheroid. After the spheroids underwent hypoxia (0.1% O2; 24 h), the BBB was more permeable, permitting the uptake of more nanoparticles and also of dissolved dye molecules. Together with our previous observations that such nanoparticles can easily enter cells and even the cell nucleus, these data provide evidence that ultrasmall nanoparticle can cross the blood brain barrier. © 2020, The Author(s).
    view abstract10.1038/s41598-020-75125-2
  • Ultrasmall gold and silver/gold nanoparticles (2 nm) as autofluorescent labels for poly(D,L-lactide-co-glycolide) nanoparticles (140 nm)
    Wey, K. and Epple, M.
    Journal of Materials Science: Materials in Medicine 31 (2020)
    Ultrasmall metallic nanoparticles show an efficient autofluorescence after excitation in the UV region, combined with a low degree of fluorescent bleaching. Thus, they can be used as fluorescent labels for polymer nanoparticles which are frequently used for drug delivery. A versatile water-in-oil-in-water emulsion-evaporation method was developed to load poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles with autofluorescent ultrasmall gold and silver/gold nanoparticles (diameter 2 nm). The metallic nanoparticles were prepared by reduction of tetrachloroauric acid with sodium borohydride and colloidally stabilised with 11-mercaptoundecanoic acid. They were characterised by UV–Vis and fluorescence spectroscopy, showing a large Stokes shift of about 370 nm with excitation maxima at 250/270 nm and emission maxima at 620/640 nm for gold and silver/gold nanoparticles, respectively. The labelled PLGA nanoparticles (140 nm) were characterised by dynamic light scattering (DLS), scanning electron microscopy (SEM), and UV–Vis and fluorescence spectroscopy. Their uptake by HeLa cells was followed by confocal laser scanning microscopy. The metallic nanoparticles remained inside the PLGA particle after cellular uptake, demonstrating the efficient encapsulation and the applicability to label the polymer nanoparticle. In terms of fluorescence, the metallic nanoparticles were comparable to fluorescein isothiocyanate (FITC). [Figure not available: see fulltext.]. © 2020, The Author(s).
    view abstract10.1007/s10856-020-06449-8
  • Ultrasmall gold nanoparticles (2 nm) can penetrate and enter cell nuclei in an in vitro 3D brain spheroid model
    Sokolova, V. and Nzou, G. and van der Meer, S.B. and Ruks, T. and Heggen, M. and Loza, K. and Hagemann, N. and Murke, F. and Giebel, B. and Hermann, D.M. and Atala, A.J. and Epple, M.
    Acta Biomaterialia 111 (2020)
    The neurovascular unit (NVU) is a complex functional and anatomical structure composed of endothelial cells and their blood-brain barrier (BBB) forming tight junctions. It represents an efficient barrier for molecules and drugs. However, it also prevents a targeted transport for the treatment of cerebral diseases. The uptake of ultrasmall nanoparticles as potential drug delivery agents was studied in a three-dimensional co-culture cell model (3D spheroid) composed of primary human cells (astrocytes, pericytes, endothelial cells). Multicellular 3D spheroids show reproducible NVU features and functions. The spheroid core is composed mainly of astrocytes, covered with pericytes, while brain endothelial cells form the surface layer, establishing the NVU that regulates the transport of molecules. After 120 h cultivation, the cells self-assemble into a 350 µm spheroid as shown by confocal laser scanning microscopy. The passage of different types of fluorescent ultrasmall gold nanoparticles (core diameter 2 nm) both into the spheroid and into three constituting cell types was studied by confocal laser scanning microscopy. Three kinds of covalently fluorophore-conjugated gold nanoparticles were used: One with fluorescein (FAM), one with Cy3, and one with the peptide CGGpTPAAK-5,6-FAM-NH2. In 2D cell co-culture experiments, it was found that all three kinds of nanoparticles readily entered all three cell types. FAM- and Cy3-labelled nanoparticles were able to enter the cell nucleus as well. The three dissolved dyes alone were not taken up by any cell type. A similar situation evolved with 3D spheroids: The three kinds of nanoparticles entered the spheroid, but the dissolved dyes did not. The presence of a functional blood-brain barrier was demonstrated by adding histamine to the spheroids. In that case, the blood-brain barrier opened, and dissolved dyes like a FITC-labelled antibody and FITC alone entered the spheroid. In summary, our results qualify ultrasmall gold nanoparticles as suitable carriers for imaging or drug delivery into brain cells (sometimes including the nucleus), brain cell spheroids, and probably also into the brain. Statement of significance: 3D brain spheroid model and its permeability by ultrasmall gold nanoparticles. We demonstrate that ultrasmall gold nanoparticles can easily penetrate the constituting cells and sometimes even enter the cell nucleus. They can also enter the interior of the blood-brain barrier model. In contrast, small molecules like fluorescing dyes are not able to do that. Thus, ultrasmall gold nanoparticles can serve as carriers of drugs or for imaging inside the brain. © 2020
    view abstract10.1016/j.actbio.2020.04.023
  • Zn- or Cu-containing CaP-based coatings formed by micro-arc oxidation on titanium and Ti-40Nb Alloy: Part I-Microstructure, composition and properties
    Komarova, E.G. and Sharkeev, Y.P. and Sedelnikova, M.B. and Prosolov, K.A. and Khlusov, I.A. and Prymak, O. and Epple, M.
    Materials 13 (2020)
    Zn- and Cu-containing CaP-based coatings, obtained by micro-arc oxidation process, were deposited on substrates made of pure titanium (Ti) and novel Ti-40Nb alloy. The microstructure, phase, and elemental composition, as well as physicochemical and mechanical properties, were examined for unmodified CaP and Zn- or Cu-containing CaP coatings, in relation to the applied voltage that was varied in the range from 200 to 350 V. The unmodified CaP coatings on both types of substrates had mainly an amorphous microstructure with a minimal content of the CaHPO4 phase for all applied voltages. The CaP coatings modified with Zn or Cu had a range from amorphous to nano- and microcrystalline structure that contained micro-sized CaHPO4 and Ca(H2PO4)2·H2O phases, as well as nano-sized β-Ca2P2O7, CaHPO4, TiO2, and Nb2O5 phases. The crystallinity of the formed coatings increased in the following order: CaP/TiNb < Zn-CaP/TiNb < Cu-CaP/TiNb < CaP/Ti < Zn-CaP/Ti < Cu-CaP/Ti. The increase in the applied voltage led to a linear increase in thickness, roughness, and porosity of all types of coatings, unlike adhesive strength that was inversely proportional to an increase in the applied voltage. The increase in the applied voltage did not affect the Zn or Cu concentration (~0.4 at%), but led to an increase in the Ca/P atomic ratio from 0.3 to 0.7. © 2020 by the authors.
    view abstract10.3390/ma13184116
  • Zn- or Cu-containing CaP-Based Coatings Formed by Micro-Arc Oxidation on Titanium and Ti-40Nb Alloy: Part II—Wettability and Biological Performance
    Komarova, E.G. and Sharkeev, Y.P. and Sedelnikova, M.B. and Prymak, O. and Epple, M. and Litvinova, L.S. and Shupletsova, V.V. and Malashchenko, V.V. and Yurova, K.A. and Dzyuman, A.N. and Kulagina, I.V. and Mushtovatova, L.S. and Bochkareva, O.P. and Karpova, M.R. and Khlusov, I.A.
    Materials 13 (2020)
    This work describes the wettability and biological performance of Zn-and Cu-containing CaP-based coatings prepared by micro-arc oxidation on pure titanium (Ti) and novel Ti-40Nb alloy. Good hydrophilic properties of all the coatings were demonstrated by the low contact angles with liquids, not exceeding 45◦ . An increase in the applied voltage led to an increase of the coating roughness and porosity, thereby reducing the contact angles to 6◦ with water and to 17◦ with glycerol. The free surface energy of 75 ± 3 mJ/m2 for all the coatings were determined. Polar component was calculated as the main component of surface energy, caused by the presence of strong polar PO43− and OH− bonds. In vitro studies showed that low Cu and Zn amounts (~0.4 at.%) in the coatings promoted high motility of human adipose-derived multipotent mesenchymal stromal cells (hAMMSC) on the implant/cell interface and subsequent cell ability to differentiate into osteoblasts. In vivo study demonstrated 100% ectopic bone formation only on the surface of the CaP coating on Ti. The Zn-and Cu-containing CaP coatings on both substrates and the CaP coating on the Ti-40Nb alloy slightly decreased the incidence of ectopic osteogenesis down to 67%. The MAO coatings showed antibacterial efficacy against Staphylococcus aureus and can be arranged as follows: Zn-CaP/Ti > Cu-CaP/TiNb, Zn-CaP/TiNb > Cu-CaP/Ti. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
    view abstract10.3390/ma13194366
  • A bioactive nano-calcium phosphate paste for in-situ transfection of BMP-7 and VEGF-A in a rabbit critical-size bone defect: results of an in vivo study
    Schlickewei, C. and Klatte, T.O. and Wildermuth, Y. and Laaff, G. and Rueger, J.M. and Ruesing, J. and Chernousova, S. and Lehmann, W. and Epple, M.
    Journal of Materials Science: Materials in Medicine 30 (2019)
    The aim of this study was to prepare an injectable DNA-loaded nano-calcium phosphate paste that is suitable as bioactive bone substitution material. For this we used the well-known potential of calcium phosphate in bone contact and supplemented it with DNA for the in-situ transfection of BMP-7 and VEGF-A in a critical-size bone defect. 24 New Zealand white rabbits were randomly divided into two groups: One group with BMP-7- and VEGF-A-encoding DNA on calcium phosphate nanoparticles and a control group with calcium phosphate nanoparticles only. The bone defect was created at the proximal medial tibia and filled with the DNA-loaded calcium phosphate paste. As control, a bone defect was filled with the calcium phosphate paste without DNA. The proximal tibia was investigated 2, 4 and 12 weeks after the operation. A histomorphological analysis of the dynamic bone parameters was carried out with the Osteomeasure system. The animals treated with the DNA-loaded calcium phosphate showed a statistically significantly increased bone volume per total volume after 4 weeks in comparison to the control group. Additionally, a statistically significant increase of the trabecular number and the number of osteoblasts per tissue area were observed. These results were confirmed by radiological analysis. The DNA-loaded bone paste led to a significantly faster healing of the critical-size bone defect in the rabbit model after 4 weeks. After 12 weeks, all defects had equally healed in both groups. No difference in the quality of the new bone was found. The injectable DNA-loaded calcium phosphate paste led to a faster and more sustained bone healing and induced an accelerated bone formation after 4 weeks. The material was well integrated into the bone defect and new bone was formed on its surface. The calcium phosphate paste without DNA led to a regular healing of the critical-size bone defect, but the healing was slower than the DNA-loaded paste. Thus, the in-situ transfection with BMP-7 and VEGF-A significantly improved the potential of calcium phosphate as pasty bone substitution material. [Figure not available: see fulltext.]. © 2019, Springer Science+Business Media, LLC, part of Springer Nature.
    view abstract10.1007/s10856-019-6217-y
  • A critical review of modern concepts for teeth whitening
    Epple, M. and Meyer, F. and Enax, J.
    Dentistry Journal 7 (2019)
    Besides prevention of caries and periodontitis, an increasing number of oral care products focus on teeth whitening. The aim of this review is to summarize and discuss frequently used whitening agents and their efficacy from a chemical viewpoint. Therefore, a comprehensive literature survey on teeth whitening agents and products was conducted. The current whitening methods are analyzed and discussed from a chemist’s viewpoint. Frequently used whitening agents are abrasives (mechanical removal of stains), antiredeposition agents (prevention of deposition of chromophores), colorants (intended to lead to a white color), proteases (degradation of proteins), peroxides (oxidation of organic chromophores), and surfactants (removal of hydrophobic compounds from tooth surface). In-office bleaching using peroxides is effective, but side effects like tooth sensitivity or a damage of the natural organic matrix of enamel and dentin may occur. The applicability of abrasives in teeth whitening is limited due to potential tooth wear, especially when toothpastes with high RDA values are used. The effect of other whitening agents in vivo is often unclear because of a shortage of placebo-controlled clinical trials. © 2019 by the authors.
    view abstract10.3390/dj7030079
  • A Novel Nanoconjugate of Landomycin A with C 60 Fullerene for Cancer Targeted Therapy: In Vitro Studies
    Bilobrov, V. and Sokolova, V. and Prylutska, S. and Panchuk, R. and Litsis, O. and Osetskyi, V. and Evstigneev, M. and Prylutskyy, Y. and Epple, M. and Ritter, U. and Rohr, J.
    Cellular and Molecular Bioengineering 12 (2019)
    Introduction: Landomycins are a subgroup of angucycline antibiotics that are produced by Streptomyces bacteria and possess strong antineoplastic potential. Literature data suggest that enhancement of the therapeutic activity of this drug may be achieved by means of creating specific drug delivery systems. Here we propose to adopt C 60 fullerene as flexible and stable nanocarrier for landomycin delivery into tumor cells. Methods: The methods of molecular modelling, dynamic light scattering and Fourier transform infrared spectroscopy were used to study the assembly of C 60 fullerene and the anticancer drug Landomycin A (LA) in aqueous solution. Cytotoxic activity of this nanocomplex was studied in vitro towards two cancer cell lines in comparison to human mesenchymal stem cells (hMSCs) using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) test and a live/dead assay. The morphology of the cells incubated with fullerene–drug nanoparticles and their uptake into target cells were studied by scanning electron microscopy and fluorescence light microscopy. Results: The viability of primary cells (hMSCs, as a model for healthy cells) and cancer cell lines (human osteosarcoma cells, MG-63, and mouse mammary cells, 4T1, as models for cancer cells) was studied after incubation with water-soluble C 60 fullerenes, LA and the mixture C 60 + LA. The C 60 + LA nanocomplex in contrast to LA alone showed higher toxicity towards cancer cells and lower toxicity towards normal cells, whereas the water-soluble C 60 fullerenes at the same concentration were not toxic for the cells. Conclusions: The obtained physico-chemical data indicate a complexation between the two compounds, leading to the formation of a C 60 + LA nanocomposite. It was concluded that immobilization of LA on C 60 fullerene enhances selectivity of action of this anticancer drug in vitro, indicating on possibility of further preclinical studies of novel C 60 + LA nanocomposites on animal tumor models. © 2018, Biomedical Engineering Society.
    view abstract10.1007/s12195-018-0548-5
  • A Novel Nanoconjugate of Landomycin A with C60 Fullerene for Cancer Targeted Therapy: In Vitro Studies
    Bilobrov, V. and Sokolova, V. and Prylutska, S. and Panchuk, R. and Litsis, O. and Osetskyi, V. and Evstigneev, M. and Prylutskyy, Y. and Epple, M. and Ritter, U. and Rohr, J.
    Cellular and Molecular Bioengineering 12 (2019)
    Introduction: Landomycins are a subgroup of angucycline antibiotics that are produced by Streptomyces bacteria and possess strong antineoplastic potential. Literature data suggest that enhancement of the therapeutic activity of this drug may be achieved by means of creating specific drug delivery systems. Here we propose to adopt C60 fullerene as flexible and stable nanocarrier for landomycin delivery into tumor cells. Methods: The methods of molecular modelling, dynamic light scattering and Fourier transform infrared spectroscopy were used to study the assembly of C60 fullerene and the anticancer drug Landomycin A (LA) in aqueous solution. Cytotoxic activity of this nanocomplex was studied in vitro towards two cancer cell lines in comparison to human mesenchymal stem cells (hMSCs) using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) test and a live/dead assay. The morphology of the cells incubated with fullerene–drug nanoparticles and their uptake into target cells were studied by scanning electron microscopy and fluorescence light microscopy. Results: The viability of primary cells (hMSCs, as a model for healthy cells) and cancer cell lines (human osteosarcoma cells, MG-63, and mouse mammary cells, 4T1, as models for cancer cells) was studied after incubation with water-soluble C60 fullerenes, LA and the mixture C60 + LA. The C60 + LA nanocomplex in contrast to LA alone showed higher toxicity towards cancer cells and lower toxicity towards normal cells, whereas the water-soluble C60 fullerenes at the same concentration were not toxic for the cells. Conclusions: The obtained physico-chemical data indicate a complexation between the two compounds, leading to the formation of a C60 + LA nanocomposite. It was concluded that immobilization of LA on C60 fullerene enhances selectivity of action of this anticancer drug in vitro, indicating on possibility of further preclinical studies of novel C60 + LA nanocomposites on animal tumor models. © 2018, Biomedical Engineering Society.
    view abstract10.1007/s12195-018-0548-5
  • A tumor-peptide–based nanoparticle vaccine elicits efficient tumor growth control in antitumor immunotherapy
    Heße, C. and Kollenda, S. and Rotan, O. and Pastille, E. and Adamczyk, A. and Wenzek, C. and Hansen, W. and Epple, M. and Buer, J. and Westendorf, A.M. and Knuschke, T.
    Molecular Cancer Therapeutics 18 (2019)
    Recognition of immunoactive oligonucleotides by the immune system, such as Toll-like receptor ligand CpG, leads to increased antibody and T-cell responses. Systemic application often results in unwanted generalized nonantigen-specific activation of the immune system. Nanoparticles are ideal carriers for small and large molecules. Recently, we have demonstrated that calcium phosphate (CaP) nanoparticles functionalized with CpG, and viral antigens are able to induce specific T-cell immunity that protects mice against viral infection and efficiently reactivates the exhausted CD8þ T-cell compartment during chronic retroviral infection. Therefore, CaP nanoparticles are promising vaccine vehicles for therapeutic applications. In this study, we investigated the therapeutic potential use of these nanoparticles in a murine xenograft colorectal cancer model. Therapeutic vaccination with CaP nanoparticles functionalized with CpG and tumor model antigens increased the frequencies of cytotoxic CD8þ T cells in the tumor in a type I interferon-dependent manner. This was accompanied with significantly repressed tumor growth in contrast to the systemic administration of soluble CpG and antigens. Combination therapy of CaP nanoparticles and immune checkpoint blocker against PD-L1 further enhanced the cytotoxic CD8þ T-cell response and eradicated the tumors. Strikingly, vaccination with CaP nanoparticles functionalized with CpG and a primary tumor cell lysate was also sufficient to control the tumor growth. In conclusion, our results represent a translational approach for the use of CaP nanoparticles as a potent cancer vaccine vehicle. © 2019 American Association for Cancer Research.
    view abstract10.1158/1535-7163.MCT-18-0764
  • Adhesion, proliferation, and osteogenic differentiation of human mesenchymal stem cells on additively manufactured Ti6Al4V alloy scaffolds modified with calcium phosphate nanoparticles
    Chudinova, E.A. and Surmeneva, M.A. and Timin, A.S. and Karpov, T.E. and Wittmar, A. and Ulbricht, M. and Ivanova, A. and Loza, K. and Prymak, O. and Koptyug, A. and Epple, M. and Surmenev, R.A.
    Colloids and Surfaces B: Biointerfaces 176 (2019)
    In the present study, biocomposites based on 3D porous additively manufactured Ti6Al4V (Ti64) scaffolds modified with biocompatible calcium phosphate nanoparticles (CaPNPs) were investigated. Ti64 scaffolds were manufactured via electron beam melting technology using an Arcam machine. Electrophoretic deposition was used to modify the scaffolds with CaPNPs, which were synthesized by precipitation in the presence of polyethyleneimine (PEI). Dynamic light scattering revealed that the CaP/PEI nanoparticles had an average size of 46 ± 18 nm and a zeta potential of +22 ± 9 mV. Scanning electron microscopy (SEM) revealed that the obtained spherical CaPNPs had an average diameter of approximately 90 nm. The titanium-based scaffolds coated with CaPNPs exhibited improved hydrophilic surface properties, with a water contact angle below 5°. Cultivation of human mesenchymal stem cells (hMSCs) on the CaPNPs-coated Ti64 scaffolds indicated that the improved hydrophilicity was beneficial for the attachment and growth of cells in vitro. The Ti6Al4V/CaPNPs scaffold supported an increase in the alkaline phosphatase (ALP) activity of cells. In addition to the favourable cell proliferation and differentiation, Ti6Al4V/CaPNPs scaffolds displayed increased mineralization compared to non-coated Ti6Al4V scaffolds. Thus, the developed composite 3D scaffolds of Ti6Al4V functionalized with CaPNPs are promising materials for different applications related to bone repair. © 2018 Elsevier B.V.
    view abstract10.1016/j.colsurfb.2018.12.047
  • Assessment of cytotoxicity and antibacterial effects of silver nanoparticle-doped titanium alloy surfaces
    Kirmanidou, Y. and Sidira, M. and Bakopoulou, A. and Tsouknidas, A. and Prymak, O. and Papi, R. and Choli-Papadopoulou, T. and Epple, M. and Michailidis, N. and Koidis, P. and Michalakis, K.
    Dental Materials 35 (2019)
    Objectives: This study aimed to develop silver nanoparticle (AgNP)-doped Ti6Al4V alloy surfaces and investigate their antibacterial properties against representative periopathogens and potential cytotoxicity on osteoblastic cells. Methods: AgNPs of different size distributions (5 and 30 nm) were incorporated onto the Ti6Al4V surfaces by electrochemical deposition, using colloid silver dispersions with increasing AgNP concentrations (100, 200 and 300 ppm). The time-course silver release from the specimen surfaces to cell culture media was assessed by Atomic Absorption Spectroscopy (AAS). Cell attachment, viability and proliferation were investigated by SEM, live/dead staining MTT and BrdU assays. The antibacterial effects were assessed against P. gingivalis and P. intermedia by serial dilution spotting assays. Results: A time- and concentration-dependent silver release from the experimental surfaces was observed. Overall, cell viability and attachment on the AgNP-doped surfaces, suggested adequate cytocompatibility at all concentrations. A transient cytotoxic effect was detected at 24 h for the 5 nm-sized groups that fully recovered at later time-points, while no cytotoxicity was observed for the 30 nm-sized groups. A statistically significant, concentration-dependent decrease in cell proliferation rates was induced at 48 h in all AgNP groups, followed by recovery at 72 h in the groups coated with 5 nm-sized AgNPs. A statistically significant, concentration-dependent antibacterial effect up to 30% was confirmed against both periopathogens. Significance: This study sheds light to the optimal size-related concentrations of AgNP-doped Ti6Al4V surfaces to achieve antibacterial effects, without subsequent cytotoxicity. These results significantly contribute to the development of antibacterial surfaces for application in oral implantology. © 2019 The Academy of Dental Materials
    view abstract10.1016/j.dental.2019.06.003
  • Bimetallic silver-platinum nanoparticles with combined osteo-promotive and antimicrobial activity
    Breisch, M. and Grasmik, V. and Loza, K. and Pappert, K. and Rostek, A. and Ziegler, N. and Ludwig, Al. and Heggen, M. and Epple, M. and Tiller, J.C. and Schildhauer, T.A. and Köller, M. and Sengstock, C.
    Nanotechnology 30 (2019)
    Bimetallic alloyed silver-platinum nanoparticles (AgPt NP) with different metal composition from Ag10Pt90 to Ag90Pt10 in steps of 20 mol% were synthesized. The biological effects of AgPt NP, including cellular uptake, cell viability, osteogenic differentiation and osteoclastogenesis as well as the antimicrobial activity towards Staphylococcus aureus and Escherichia coli were analyzed in comparison to pure Ag NP and pure Pt NP. The uptake of NP into human mesenchymal stem cells was confirmed by cross-sectional focused-ion beam preparation and observation by scanning and transmission electron microscopy in combination with energy-dispersive x-ray analysis. Lower cytotoxicity and antimicrobial activity were observed for AgPt NP compared to pure Ag NP. Thus, an enhanced Ag ion release due to a possible sacrificial anode effect was not achieved. Nevertheless, a Ag content of at least 50 mol% was sufficient to induce bactericidal effects against both Staphylococcus aureus and Escherichia coli. In addition, a Pt-related (≥50 mol% Pt) osteo-promotive activity on human mesenchymal stem cells was observed by enhanced cell calcification and alkaline phosphatase activity. In contrast, the osteoclastogenesis of rat primary precursor osteoclasts was inhibited. In summary, these results demonstrate a combinatory osteo-promotive and antimicrobial activity of bimetallic Ag50Pt50 NP. © 2019 IOP Publishing Ltd.
    view abstract10.1088/1361-6528/ab172b
  • Biomimetic fabrication of mineralized composite films of nanosilver loaded native fibrillar collagen and chitosan
    Socrates, R. and Prymak, O. and Loza, K. and Sakthivel, N. and Rajaram, A. and Epple, M. and Narayana Kalkura, S.
    Materials Science and Engineering C 99 (2019)
    Silver nanoparticles loaded fibrillar collagen-chitosan matrix (CC) was prepared by biomimetic approach by blending silver nanoparticles (tAgNPs), collagen fibril and chitosan hydrogel followed by cross-linking and biomineralization. Electron micrograph showed that the surface of the composites exhibited native fibrillar morphology of collagen and their cross-section revealed layer-like arrangement of native fibrillar collagen. The mineralized composites exhibited surface mineralization of calcium phosphates incorporated with magnesium. FT-IR ATR analysis revealed the uniform blending of collagen and chitosan without any chemical interaction between them. XRD analysis showed incorporation of silver nanoparticles and lamellar structure of collagen and chitosan. The mechanical property of the dry composite film showed increase in tensile strength with the addition of chitosan and raised to 4.6 fold in M-CC4 composite. The incorporation of chitosan in M-CC3 led to 2.2 fold increase in mineralization as confirmed by the TGA analysis. Contact angle analysis revealed the hydrophilic nature of the composite. Hemolysis analysis of the composites verified the hemocompatible nature of composites with hemolysis < 5%. MTT assay for the composites was carried by seeding MG-63 cells and indicated cell viability > 80%. Antibacterial activity analysis showed the percent growth inhibition of about 27% and 37% for S. aureus and E. coli respectively. The prepared composite would possess silver nanoparticles loaded collagen fibril in the native state and the formed biomineral will be similar to the bone mineral. Hence the fabricated composite -could be used as a biomaterial for bone tissue engineering applications. © 2019 Elsevier B.V.
    view abstract10.1016/j.msec.2019.01.101
  • Calcium phosphate bone graft substitutes with high mechanical load capacity and high degree of interconnecting porosity
    Hettich, G. and Schierjott, R.A. and Epple, M. and Gbureck, U. and Heinemann, S. and Mozaffari-Jovein, H. and Grupp, T.M.
    Materials 12 (2019)
    Bone graft substitutes in orthopedic applications have to fulfill various demanding requirements. Most calcium phosphate (CaP) bone graft substitutes are highly porous to achieve bone regeneration, but typically lack mechanical stability. This study presents a novel approach, in which a scaffold structure with appropriate properties for bone regeneration emerges from the space between specifically shaped granules. The granule types were tetrapods (TEPO) and pyramids (PYRA), which were compared to porous CaP granules (CALC) and morselized bone chips (BC). Bulk materials of the granules were mechanically loaded with a peak pressure of 4 MP; i.e., comparable to the load occurring behind an acetabular cup. Mechanical loading reduced the volume of CALC and BC considerably (89% and 85%, respectively), indicating a collapse of the macroporous structure. Volumes of TEPO and PYRA remained almost constant (94% and 98%, respectively). After loading, the porosity was highest for BC (46%), lowest for CALC (25%) and comparable for TEPO and PYRA (37%). The pore spaces of TEPO and PYRA were highly interconnected in a way that a virtual object with a diameter of 150 m could access 34% of the TEPO volume and 36% of the PYRA volume. This study shows that a bulk of dense CaP granules in form of tetrapods and pyramids can create a scaffold structure with load capacities suitable for the regeneration of an acetabular bone defect. © 2019 by the authors. Licensee MDPI, Basel, Switzerland.
    view abstract10.3390/ma12213471
  • Calcium phosphate nanoparticle-based vaccines as a platform for improvement of HIV-1 env antibody responses by intrastructural help
    Damm, D. and Rojas-Sánchez, L. and Theobald, H. and Sokolova, V. and Wyatt, R.T. and Überla, K. and Epple, M. and Temchura, V.
    Nanomaterials 9 (2019)
    Incorporation of immunodominant T-helper epitopes of licensed vaccines into virus-like particles (VLP) allows to harness T-helper cells induced by the licensed vaccines to provide intrastructural help (ISH) for B-cell responses against the surface proteins of the VLPs. To explore whether ISH could also improve antibody responses to calcium phosphate (CaP) nanoparticle vaccines we loaded the nanoparticle core with a universal T-helper epitope of Tetanus toxoid (p30) and functionalized the surface of CaP nanoparticles with stabilized trimers of the HIV-1 envelope (Env) resulting in Env-CaP-p30 nanoparticles. In contrast to soluble Env trimers, Env containing CaP nanoparticles induced activation of naïve Env-specific B-cells in vitro. Mice previously vaccinated against Tetanus raised stronger humoral immune responses against Env after immunization with Env-CaP-p30 than mice not vaccinated against Tetanus. The enhancing effect of ISH on anti-Env antibody levels was not attended with increased Env-specific IFN-γ CD4 T-cell responses that otherwise may potentially influence the susceptibility to HIV-1 infection. Thus, CaP nanoparticles functionalized with stabilized HIV-1 Env trimers and heterologous T-helper epitopes are able to recruit heterologous T-helper cells induced by a licensed vaccine and improve anti-Env antibody responses by intrastructural help. © 2019 by the authors. Licensee MDPI, Basel, Switzerland.
    view abstract10.3390/nano9101389
  • Calcium phosphate nanoparticle-mediated transfection in 2D and 3D mono- and co-culture cell models
    Sokolova, V. and Rojas-Sánchez, L. and Białas, N. and Schulze, N. and Epple, M.
    Acta Biomaterialia 84 (2019)
    The transfer of nucleic acids into living cells, i.e. transfection, is a major technique in current molecular biology and medicine. As nucleic acids alone are not able to penetrate the cell membrane, an efficient carrier is needed. Calcium phosphate nanoparticles can serve as carrier due to their biocompatibility, biodegradability and high affinity to nucleic acids like DNA or RNA. Their application was extended here from two-dimensional (2D) to three-dimensional (3D) cell culture models, including co-cultures. Compared to 2D monolayer cell cultures, a 3D culture system represents a more realistic spatial, biochemical and cellular environment. The uptake of fluorescent calcium phosphate nanoparticles (diameter 40–70 nm; cationic) was studied in 2D and 3D cell culture models by confocal laser scanning microscopy. The transfection of eGFP by calcium phosphate nanoparticles was compared in 2D and 3D cell culture, including co-cultures of green fluorescing HeLa-eGFP cells and MG-63 cells in 2D and in 3D models with the red fluorescent protein mCherry. This permitted a cell-specific assessment of the local transfection efficiency. In general, the penetration of nanoparticles into the spheroids was significantly higher than that of a model oligonucleotide carried by Lipofectamine. The transfection efficiency was comparable in 3D cell cultures with 2D cell cultures, but it occurred preferentially at the surface of the spheroids, following the uptake pathway of the nanoparticles. Statement of significance: Three-dimensional cell culture models can serve as a bridge between the in-vitro cell cultures and the in-vivo situation, especially when mass transfer effects have to be considered. This is the case for nanoparticles where the incubation effect in a two-dimensional cell culture strongly differs from a three-dimensional cell culture or a living tissue. We have compared the uptake of nanoparticles and a subsequent transfection of fluorescent proteins in two-dimensional and three-dimensional cell culture models. An elegant model to investigate the transfection in co-cultures was developed using HeLa-eGFP cells (green fluorescent) together with MG-63 cells (non-fluorescent) that were transfected with the red-fluorescing protein mCherry. Thereby, the transfection of both cell types in the co-culture was easily distinguished. © 2018 Acta Materialia Inc.
    view abstract10.1016/j.actbio.2018.11.051
  • Click Chemistry on the Surface of Ultrasmall Gold Nanoparticles (2 nm) for Covalent Ligand Attachment Followed by NMR Spectroscopy
    Van Der Meer, S.B. and Loza, K. and Wey, K. and Heggen, M. and Beuck, C. and Bayer, P. and Epple, M.
    Langmuir 35 (2019)
    Ultrasmall gold nanoparticles (core diameter 2 nm) were surface-conjugated with azide groups by attaching the azide-functionalized tripeptide lysine(N3)-cysteine-asparagine with ∼117 molecules on each nanoparticle. A covalent surface modification with alkyne-containing molecules was then possible by copper-catalyzed click chemistry. The successful clicking to the nanoparticle surface was demonstrated with 13C-labeled propargyl alcohol. All steps of the nanoparticle surface conjugation were verified by extensive NMR spectroscopy on dispersed nanoparticles. The particle diameter and the dispersion state were assessed by high-resolution transmission electron microscopy (HRTEM), differential centrifugal sedimentation (DCS), and 1H-DOSY NMR spectroscopy. The clicking of fluorescein (FAM-alkyne) gave strongly fluorescing ultrasmall nanoparticles that were traced inside eukaryotic cells. The uptake of these nanoparticles after 24 h by HeLa cells was very efficient and showed that the nanoparticles even penetrated the nuclear membrane to a very high degree (in contrast to dissolved FAM-alkyne alone that did not enter the cell). About 8 fluorescein molecules were clicked to each nanoparticle. © 2019 American Chemical Society.
    view abstract10.1021/acs.langmuir.9b00295
  • Combining Small-Angle X-ray Scattering and X-ray Powder Diffraction to Investigate Size, Shape and Crystallinity of Silver, Gold and Alloyed Silver-Gold Nanoparticles
    Garcia, P.R.A.F. and Loza, K. and Daumann, S. and Grasmik, V. and Pappert, K. and Rostek, A. and Helmlinger, J. and Prymak, O. and Heggen, M. and Epple, M. and Oliveira, C.L.P.
    Brazilian Journal of Physics 49 (2019)
    The combination of simultaneous measurements of small and wide angle X-ray scattering (SAXS/WAXS–SWAXS) to investigate the overall size, shape, and crystallinity of silver nanoparticles and alloyed silver-gold nanoparticles in the size range of 8 to 80 nm is shown. The obtained results for overall size are in agreement with the particle size obtained by differential centrifugal sedimentation (DCS) and transmission electron microscopy (TEM). In addition to the overall size, SWAXS provided precise information about the crystallographic internal structure of the particles, providing a powerful multi-scale tool for structural characterization of the studied systems. © 2019, Sociedade Brasileira de Física.
    view abstract10.1007/s13538-019-00642-z
  • Decreased bacterial colonization of additively manufactured Ti6Al4V metallic scaffolds with immobilized silver and calcium phosphate nanoparticles
    Surmeneva, M. and Lapanje, A. and Chudinova, E. and Ivanova, A. and Koptyug, A. and Loza, K. and Prymak, O. and Epple, M. and Ennen-Roth, F. and Ulbricht, M. and Rijavec, T. and Surmenev, R.
    Applied Surface Science 480 (2019)
    The design of an ideal bone graft substitute has been a long-standing effort, and a number of strategies have been developed to improve bone regeneration. Electron beam melting (EBM) is an additive manufacturing method allowing for the production of porous implants with highly defined external dimensions and internal architectures. The increasing surface area of the implant may also increase the abilities of pathogenic microorganisms to adhere to the surfaces and form a biofilm, which may result in serious complications. The aim of this study was to explore the modifications of Ti6Al4V alloy scaffolds to reduce the abilities of bacteria to attach to the EBM-manufactured implant surface. The layers composed of silver (Ag), calcium phosphate (CaP) nanoparticles (NPs) and combinations of both were formed on the EBM-fabricated metallic scaffolds by electrophoretic deposition in order to provide them with antimicrobial properties. The assay of bacterial colonization on the surface was performed with the exposure of scaffold surfaces to Staphylococcus aureus cells for up to 17 h. Principal component analysis (PCA) was used to assess the relationships between different surface features of the studied samples and bacterial adhesion. The results indicate that by modifying the implant surface with appropriate nanostructures that change the hydrophobicity and the surface roughness at the nano scale, physical cues are provided that disrupt bacterial adhesion. Our results clearly show that AgNPs at a concentration of approximately 0.02 mg/сm 2 that were deposited together with CaPNPs covered by positively charge polyethylenimine (PEI) on the surface of EBM-sintered Ti6Al4V scaffolds hindered bacterial growth, as the total number of attached cells (NAC) of S. aureus remained at the same level during the 17 h of exposure, which indicates bacteriostatic activity. © 2019 Elsevier B.V.
    view abstract10.1016/j.apsusc.2019.03.003
  • Dental pulp stem cells in chitosan/gelatin scaffolds for enhanced orofacial bone regeneration
    Bakopoulou, A. and Georgopoulou, Α. and Grivas, I. and Bekiari, C. and Prymak, O. and Loza, Κ. and Epple, M. and Papadopoulos, G.C. and Koidis, P. and Chatzinikolaidou, Μ.
    Dental Materials 35 (2019)
    Objective: Biomimetic chitosan/gelatin (CS/Gel) scaffolds have attracted great interest in tissue engineering of several tissues. However, limited information exists regarding the potential of combining CS/Gel scaffolds with oral cells, such as dental pulp stem cells (DPSCs), to produce customized constructs targeting alveolar/orofacial bone reconstruction, which has been the aim of the present study. Methods: Two scaffold types, designated as CS/Gel-0.1 and CS/Gel-1, were fabricated using 0.1 and 1% (v/v) respectively of the crosslinker glutaraldehyde (GTA). Scaffolds (n = 240) were seeded with DPSCs with/without pre-exposure to recombinant human BMP-2. In vitro assessment included DPSCs characterization (flow cytometry), evaluation of viability/proliferation (live/dead staining, metabolic-based tests), osteo/odontogenic gene expression analysis (qRT-PCR) and structural/chemical characterization (scanning electron microscopy, SEM; energy dispersive X-ray spectroscopy, EDX; X-ray powder diffraction, XRD; thermogravimetry, TG). In vivo assessment included implantation of DPSC-seeded scaffolds in immunocompromised mice, followed by histology and SEM-EDX. Statistical analysis employed one/two-way ANOVA and Tukey's post-hoc tests (significance for p < 0.05). Results: Both scaffolds supported cell viability/proliferation over 14 days in culture, showing extensive formation of a hydroxyapatite-rich nanocrystalline calcium phosphate phase. Differential expression patterns indicated GTA concentration to significantly affect the expression of osteo/odontogenic genes, with CS/Gel-0.1 scaffolds being more effective in upregulating DSPP, IBSP and Osterix. In vivo analysis demonstrated time-dependent production of a nanocrystalline, mineralized matrix at 6, 8 and 10 weeks, being more prominent in constructs bearing rhBMP-2 pre-treated cells. The latter showed higher amounts of osteoid and fully mineralized bone, as well as empty space reduction. Significance: These results reveal a promising strategy for orofacial bone tissue engineering. © 2018 The Academy of Dental Materials
    view abstract10.1016/j.dental.2018.11.025
  • Determination of the properties and loading efficiency of encapsulated BSA-FITC and dexamethasone for drug delivery systems
    Chudinova, E. and Surmeneva, M. and Koptyug, A. and Sokolova, V. and Prymak, O. and Bouckercha, S. and Epple, M. and Surmenev, R.
    IOP Conference Series: Materials Science and Engineering 597 (2019)
    In this work porous microparticles of calcium carbonate were synthesized with bovine serum albumin - fluorescein isothiocyanate conjugate (BSA-FITC) and dexamethasone, and then used for encapsulation in polymer microcapsules by means of layer-by-layer assembly (LbL). The properties of the obtained microcapsules were characterized by scanning electron microscopy, dynamic light scattering, infrared-, ultraviolet- and visible spectroscopy. According to the performed DLS measurements, an average hydrodynamic diameter ranged from 4 to 8 m and zeta-potential for all types of capsules was determined as -18 and -21 mV. BSA-FITC was encapsulated using this approach yielded a loading efficiency of 49 % protein. This value calculated for dexamethasone was of 38%. The microcapsules filled with an encapsulated drug may find applications in the field of biotechnology, biochemistry, and medicine. © Published under licence by IOP Publishing Ltd.
    view abstract10.1088/1757-899X/597/1/012056
  • Functionalization of titania nanotubes with electrophoretically deposited silver and calcium phosphate nanoparticles: Structure, composition and antibacterial assay
    Chernozem, R.V. and Surmeneva, M.A. and Krause, B. and Baumbach, T. and Ignatov, V.P. and Prymak, O. and Loza, K. and Epple, M. and Ennen-Roth, F. and Wittmar, A. and Ulbricht, M. and Chudinova, E.A. and Rijavec, T. and Lapanje, A. and Surmenev, R.A.
    Materials Science and Engineering C 97 (2019)
    Herein TiO2 nanotubes (NTs) were fabricated via electrochemical anodization and coated with silver and calcium phosphate (CaP) nanoparticles (NPs) by electrophoretic deposition. Scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX) revealed that Ag and CaP NPs were successfully deposited onto the TiO2 NTs. Using X-ray diffraction, only anatase and Ti were observed after deposition of Ag and CaP NPs. However, X-ray photoelectron spectroscopy (XPS) analysis revealed that the binding energy (BE) of the Ag and CaP NP core levels corresponded to metallic Ag, hydroxyapatite and amorphous calcium phosphate, based on the knowledge that CaP NPs synthesized by precipitation have the nanocrystalline structure of hydroxyapatite. The application of Ag NPs allows for decreasing the water contact angle and thus increasing the surface free energy. It was concluded that the CaP NP surfaces are superhydrophilic. A significant antimicrobial effect was observed on the TiO2 NT surface after the application of Ag NPs and/or CaP NPs compared with that of the pure TiO2 NTs. Thus, fabrication of TiO2 NTs, Ag NPs and CaP NPs with PEI is promising for diverse biomedical applications, such as in constructing a biocompatible coating on the surface of Ti that includes an antimicrobial effect. © 2018 Elsevier B.V.
    view abstract10.1016/j.msec.2018.12.045
  • Glancing angle deposition of Zn-doped calcium phosphate coatings by RF magnetron sputtering
    Prosolov, K.A. and Belyavskaya, O.A. and Linders, J. and Loza, K. and Prymak, O. and Mayer, C. and Rau, J.V. and Epple, M. and Sharkeev, Y.P.
    Coatings 9 (2019)
    Zn-substituted hydroxyapatite with antibacterial effect was used in radiofrequency (RF) magnetron deposition of calcium phosphate coating onto Ti- and Si-inclined substrates. The development of surface nanopatterns for direct bacteria killing is a growing area of research. Here, we combined two approaches for possible synergetic antibacterial effect by manufacturing a patterned surface of Zn-doped calcium phosphate using glancing angle deposition (GLAD) technique. A significant change in the coating morphology was revealed with a substrate tilt angle of 80°. It was shown that an increase in the coating crystallinity for samples deposited at a tilt angle of 80° corresponds to the formation of crystallites in the bulk structure of the thin film. The variation in the coating thickness, uniformity, and influence of sputtered species energy on Si substrates was analyzed. Coatings deposited on tilted samples exhibit higher scratch resistance. The coating microand nano-roughness and overall morphology depended on the tilt angle and differently affected the rough Ti and smooth Si surfaces. GLAD of complex calcium phosphate material can lead to the growth of thin films with significantly changed morphological features and can be utilized to create self-organized nanostructures on various types of surfaces. © 2019 by the authors.
    view abstract10.3390/coatings9040220
  • In vitro study of the anticancer activity of various doxorubicin-containing dispersions
    Matvienko, T. and Sokolova, V. and Prylutska, S. and Harahuts, Y. and Kutsevol, N. and Kostjukov, V. and Evstigneev, M. and Prylutskyy, Y. and Epple, M. and Ritter, U.
    BioImpacts 9 (2019)
    Introduction: The aim of this research was to study the impact of various doxorubicin (Dox)-containing nanofluids, e.g. single-walled carbon nanotube (SWCNT)+Dox, graphene oxide (GO)+Dox and Dextran-PNIPAM (copolymer)+Dox mixtures on HeLa cells (human transformed cervix epithelial cells, as a model for cancer cells) depending on their concentration. Methods: Structural analysis of GO+Dox complex was accomplished using Hartree-Fock level of theory in 6-31G** basis set in Gaussian. Dynamic light scattering (DLS), zeta-potential, scanning electron microscopy and confocal laser scanning microscopy were used. The cell viability was analyzed by the MTT assay. Results: The viability of HeLa cells was studied with the MTT assay after the incubation with various Dox-containing dispersions depending on their concentration. The size of the particles was determined by DLS. The morphology of the nanoparticles (NPs) was studied by scanning electron microscopy and their uptake into cells was visualized by confocal laser scanning microscopy. It was found that the Dextran-PNIPAM+Dox nanofluid in contrast to Dox alone showed higher toxicity towards HeLa cells up to 80% after 24 hours of incubation, whereas the SWCNT+Dox and GO+Dox nanofluids at the same concentrations protected cells from Dox. Conclusion: The importance of Dextran-PNIPAM copolymer as a universal platform for drug delivery was established, and the huge potential of Dextran-PNIPAM+Dox NPs as novel anticancer agents was noted. Based on the in vitro study of the SWCNT+Dox and GO+Dox nanofluids, it was concluded that SWCNT and GO NPs can be effective cytoprotectors against the highly toxic drugs. © 2019 The Author(s).
    view abstract10.15171/bi.2019.07
  • Induction of herpes simplex virus type 1 cell-to-cell spread inhibiting antibodies by a calcium phosphate nanoparticle-based vaccine
    Kopp, M. and Aufderhorst, U.W. and Alt, M. and Dittmer, U. and Eis-Hübinger, A.-M. and Giebel, B. and Roggendorf, M. and Epple, M. and Krawczyk, A.
    Nanomedicine: Nanotechnology, Biology, and Medicine 16 (2019)
    Herpes simplex viruses 1 and 2 are among the most ubiquitous human infections and persist lifelong in their host. Upon primary infection or reactivation from ganglia, the viruses spread by direct cell–cell contacts (cell-to-cell spread) and thus escape from the host immune response. We have developed a monoclonal antibody (mAb 2c), which inhibits the HSV cell-to-cell spread, thereby protecting from lethal genital infection and blindness in animal models. In the present study we have designed a nanoparticle-based vaccine to induce protective antibody responses exceeding the cell-to-cell spread inhibiting properties of mAb 2c. We used biodegradable calcium phosphate (CaP) nanoparticles coated with a synthetic peptide that represents the conformational epitope on HSV-1 gB recognized by mAb 2c. The CaP nanoparticles additionally contained a TLR-ligand CpG m and were formulated with adjuvants to facilitate the humoral immune response. This vaccine effectively protected mice from lethal HSV-1 infection by inducing cell-to-cell spread inhibiting antibodies. © 2018 The Authors
    view abstract10.1016/j.nano.2018.12.002
  • Influence of Calcium-Phosphate Coating on Wettability of Hybrid Piezoelectric Scaffolds
    Zviagin, A. and Chernozem, R. and Surmeneva, M. and Loza, K. and Prymak, O. and Ulbricht, M. and Epple, M. and Surmenev, R.
    IOP Conference Series: Materials Science and Engineering 597 (2019)
    Herein, electrospun biodegradable scaffolds based on polycaprolactone (PCL), poly(3-hydroxybutyrate) (PHB) and polyaniline (PANi) polymers were fabricated. A calcium-phosphate (CaP) coating was deposited on the surface of the scaffolds via an improved soaking process. Influence of the deposition cycles and ethanol concentration in the solution on the relative increase of the scaffolds weight and water contact angle (WCA) are determined. The characterization of the molecular and crystal structure confirmed the formation of CaP phase. Importantly, WCA results showed that the pristine scaffolds have the hydrophobic surface, while the deposition of CaP coating onto scaffolds allows to significantly improve the surface wetting behavior, and infiltration of the water droplets into the CaP-coated scaffolds was observed. Thus, the fabricated hybrid biodegradable piezoelectric scaffolds can be utilized for regenerative medicine. © Published under licence by IOP Publishing Ltd.
    view abstract10.1088/1757-899X/597/1/012061
  • Nanoscopic Porous Iridium/Iridium Dioxide Superstructures (15 nm): Synthesis and Thermal Conversion by In Situ Transmission Electron Microscopy
    Pappert, K. and Loza, K. and Shviro, M. and Hagemann, U. and Heggen, M. and Dunin-Borkowski, R.E. and Schierholz, R. and Maeda, T. and Kaneko, K. and Epple, M.
    Chemistry - A European Journal 25 (2019)
    Porous particle superstructures of about 15 nm diameter, consisting of ultrasmall nanoparticles of iridium and iridium dioxide, are prepared through the reduction of sodium hexachloridoiridate(+IV) with sodium citrate/sodium borohydride in water. The water-dispersible porous particles contain about 20 wt % poly(N-vinylpyrrolidone) (PVP), which was added for colloidal stabilization. High-resolution transmission electron microscopy confirms the presence of both iridium and iridium dioxide primary particles (1–2 nm) in each porous superstructure. The internal porosity (≈58 vol%) is demonstrated by electron tomography. In situ transmission electron microscopy up to 1000 °C under oxygen, nitrogen, argon/hydrogen (all at 1 bar), and vacuum shows that the porous particles undergo sintering and subsequent compaction upon heating, a process that starts at around 250 °C and is completed at around 800 °C. Finally, well-crystalline iridium dioxide is obtained under all four environments. The catalytic activity of the as-prepared porous superstructures in electrochemical water splitting (oxygen evolution reaction; OER) is reduced considerably upon heating owing to sintering of the pores and loss of internal surface area. © 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
    view abstract10.1002/chem.201901623
  • Piezoelectric 3-D Fibrous Poly(3-hydroxybutyrate)-Based Scaffolds Ultrasound-Mineralized with Calcium Carbonate for Bone Tissue Engineering: Inorganic Phase Formation, Osteoblast Cell Adhesion, and Proliferation
    Chernozem, R.V. and Surmeneva, M.A. and Shkarina, S.N. and Loza, K. and Epple, M. and Ulbricht, M. and Cecilia, A. and Krause, B. and Baumbach, T. and Abalymov, A.A. and Parakhonskiy, B.V. and Skirtach, A.G. and Surmenev, R.A.
    ACS Applied Materials and Interfaces (2019)
    Elaboration of novel biocomposites providing simultaneously both biodegradability and stimulated bone tissue repair is essential for regenerative medicine. In particular, piezoelectric biocomposites are attractive because of a possibility to electrically stimulate cell response. In the present study, novel CaCO3-mineralized piezoelectric biodegradable scaffolds based on two polymers, poly[(R)3-hydroxybutyrate] (PHB) and poly[3-hydroxybutyrate-co-3-hydroxyvalerate] (PHBV), are presented. Mineralization of the scaffold surface is carried out by the in situ synthesis of CaCO3 in the vaterite and calcite polymorphs using ultrasound (U/S). Comparative characterization of PHB and PHBV scaffolds demonstrated an impact of the porosity and surface charge on the mineralization in a dynamic mechanical system, as no essential distinction was observed in wettability, structure, and surface chemical compositions. A significantly higher (4.3 times) piezoelectric charge and a higher porosity (∼15%) lead to a more homogenous CaCO3 growth in 3-D fibrous structures and result in a two times higher relative mass increase for PHB scaffolds compared to that for PHBV. This also increases the local ion concentration incurred upon mineralization under U/S-generated dynamic mechanical conditions. The modification of the wettability for PHB and PHBV scaffolds from hydrophobic (nonmineralized fibers) to superhydrophilic (mineralized fibers) led to a pronounced apatite-forming behavior of scaffolds in a simulated body fluid. In turn, this results in the formation of a dense monolayer of well-distributed and proliferated osteoblast cells along the fibers. CaCO3-mineralized PHBV surfaces had a higher osteoblast cell adhesion and proliferation assigned to a higher amount of CaCO3 on the surface compared to that on PHB scaffolds, as incurred from micro-computed tomography (μCT). Importantly, a cell viability study confirmed biocompatibility of all the scaffolds. Thus, hybrid biocomposites based on the piezoelectric PHB polymers represent an effective scaffold platform functionalized by an inorganic phase and stimulating the growth of the bone tissue. © 2019 American Chemical Society.
    view abstract10.1021/acsami.9b04936
  • Session 5: Biomaterials - Inorganic
    Sokolova, V. and Shi, Z. and Huang, S. and Du, Y. and Kopp, M. and Frede, A. and Knuschke, T. and Buer, J. and Yang, D. and Westendorf, A.M. and Epple, M.
    Biomedizinische Technik. Biomedical engineering 64 (2019)
    view abstract10.1515/bmt-2019-7005
  • Solution NMR Spectroscopy with Isotope-Labeled Cysteine ( 13 C and 15 N) Reveals the Surface Structure of l -Cysteine-Coated Ultrasmall Gold Nanoparticles (1.8 nm)
    Ruks, T. and Beuck, C. and Schaller, T. and Niemeyer, F. and Zähres, M. and Loza, K. and Heggen, M. and Hagemann, U. and Mayer, C. and Bayer, P. and Epple, M.
    Langmuir 35 (2019)
    Ultrasmall gold nanoparticles with a diameter of 1.8 nm were synthesized by reduction of tetrachloroauric acid with sodium borohydride in the presence of l-cysteine, with natural isotope abundance as well as 13 C-labeled and 15 N-labeled. The particle diameter was determined by high-resolution transmission electron microscopy and differential centrifugal sedimentation. X-ray photoelectron spectroscopy confirmed the presence of metallic gold with only a few percent of oxidized Au(+I) species. The surface structure and the coordination environment of the cysteine ligands on the ultrasmall gold nanoparticles were studied by a variety of homo- and heteronuclear NMR spectroscopic techniques including 1 H- 13 C-heteronuclear single-quantum coherence and 13 C- 13 C-INADEQUATE. Further information on the binding situation (including the absence of residual or detached l-cysteine in the solution) and on the nanoparticle diameter (indicating the well-dispersed state) was obtained by diffusion-ordered spectroscopy ( 1 H-, 13 C-, and 1 H- 13 C-DOSY). Three coordination environments of l-cysteine on the gold surface were identified that were ascribed to different crystallographic sites, supported by geometric considerations of the nanoparticle ultrastructure. The particle size data and the NMR-spectroscopic analysis gave a particle composition of about Au 174 (cysteine) 67 . © 2018 American Chemical Society.
    view abstract10.1021/acs.langmuir.8b03840
  • Solution NMR Spectroscopy with Isotope-Labeled Cysteine (13C and 15N) Reveals the Surface Structure of l -Cysteine-Coated Ultrasmall Gold Nanoparticles (1.8 nm)
    Ruks, T. and Beuck, C. and Schaller, T. and Niemeyer, F. and Zähres, M. and Loza, K. and Heggen, M. and Hagemann, U. and Mayer, C. and Bayer, P. and Epple, M.
    Langmuir 35 (2019)
    Ultrasmall gold nanoparticles with a diameter of 1.8 nm were synthesized by reduction of tetrachloroauric acid with sodium borohydride in the presence of l-cysteine, with natural isotope abundance as well as 13C-labeled and 15N-labeled. The particle diameter was determined by high-resolution transmission electron microscopy and differential centrifugal sedimentation. X-ray photoelectron spectroscopy confirmed the presence of metallic gold with only a few percent of oxidized Au(+I) species. The surface structure and the coordination environment of the cysteine ligands on the ultrasmall gold nanoparticles were studied by a variety of homo- and heteronuclear NMR spectroscopic techniques including 1H-13C-heteronuclear single-quantum coherence and 13C-13C-INADEQUATE. Further information on the binding situation (including the absence of residual or detached l-cysteine in the solution) and on the nanoparticle diameter (indicating the well-dispersed state) was obtained by diffusion-ordered spectroscopy (1H-, 13C-, and 1H-13C-DOSY). Three coordination environments of l-cysteine on the gold surface were identified that were ascribed to different crystallographic sites, supported by geometric considerations of the nanoparticle ultrastructure. The particle size data and the NMR-spectroscopic analysis gave a particle composition of about Au174(cysteine)67. © 2018 American Chemical Society.
    view abstract10.1021/acs.langmuir.8b03840
  • Stability of nanoparticle dispersions and particle agglomeration
    Loza, K. and Epple, M. and Maskos, M.
    NanoScience and Technology (2019)
    The stability of colloids is an important issue of colloid-based processes and formulations. Due to the large specific surface area, particles have a low thermodynamic stability and tend to agglomerate over time. Furthermore, the physicochemical properties of nanomaterials depend on the size, morphology, and surface state of the system, therefore in-depth characterization techniques are essential to predict the degree of variation in properties. In this chapter, the colloidal stability of nanoparticle dispersions as well as the basic stabilization mechanisms will be discussed both at the theoretical and at the experimental level. Relevant characterization methods will be presented and illustrated with suitable examples, including their limitations. © 2019, Springer Nature Switzerland AG.
    view abstract10.1007/978-3-030-12461-8_4
  • Surface functionalization of titanium with silver nanoparticles
    Sharonova, A. and Surmeneva, M. and Loza, K. and Prymak, O. and Epple, M. and Surmenev, R.
    Journal of Physics: Conference Series 1145 (2019)
    This study aims to investigate the most efficient ways for metallic samples functionalization with silver nanoparticles (AgNPs). Three different techniques of surface functionalization have been used for the coating of titanium metal, i.e. the sessile drop method (evaporation), dip-coating and electrophoretic deposition (EPD). AgNPs stabilized with polyvinylpyrrolidone had a spherical shape and the metallic core diameter, charge and polydispersity index were 70 20 nm, -15 mV and 0.192, respectively. SEM analysis revealed that AgNPs were homogeneously distributed over the entire surface and did not form the particle agglomerates only in case of EPD. Thus, EPD method and spherical AgNPs can be used for further investigation concerning the preparation of biocomposites with antibacterial and bioactive properties. © Published under licence by IOP Publishing Ltd.
    view abstract10.1088/1742-6596/1145/1/012032
  • Surface modification of Ti6Al4V alloy scaffolds manufactured by electron beam melting
    Chudinova, E. and Surmeneva, M. and Koptyug, A. and Loza, K. and Prymak, O. and Epple, M. and Surmenev, R.
    Journal of Physics: Conference Series 1145 (2019)
    In this paper, the results of the surface functionalization of the Ti6Al4V alloy scaffolds with different structures for use as a material for medical implants are presented. Radio frequency magnetron sputtering was used to modify the surface of the porous structures by deposition of the biocompatible hydroxyapatite (HA) coating with the thickness of 86050 nm. The surface morphology, elemental and phase composition of the HA-coated scaffolds were studied. According to energy-dispersive X-ray spectroscopy, the stoichiometric ratio of Ca/P for flat, orthorhombic and cubic scaffolds is 1.65, 1.60, 1.53, respectively, which is close to that of stoichiometric ratio for HA (Ca/P = 1.67). It was revealed that this method of deposition makes it possible to obtain the homogeneous crystalline coating both on the dense sample and in the case of scaffolds of complex geometry with different lattice cell structure. © Published under licence by IOP Publishing Ltd.
    view abstract10.1088/1742-6596/1145/1/012030
  • Synthesis of metallic and metal oxide particles
    Loza, K. and Epple, M.
    NanoScience and Technology (2019)
    The diversity of applications in catalysis, energy storage and medical diagnostics utilizes unique and fascinating properties of metal and metal oxide nanostructures. Confined to the nanometer scale, materials may display properties that are different from the equivalent bulk compounds. To meet the requirements for various applications, numerous production techniques were developed to control particle size, morphology, aggregation state, crystal structure, surface charge and composition. This chapter presents an overview of the preparation of metallic and metal oxide nanoparticles by bottom-up and top-down approaches. We describe basic synthetic routes for prominent cases of metals (gold, silver, platinum and copper) and metal oxides (zinc oxide, titania, and silica). © 2019, Springer Nature Switzerland AG.
    view abstract10.1007/978-3-030-12461-8_1
  • X-ray powder diffraction to analyse bimetallic core-shell nanoparticles (gold and palladium; 7-8 nm)
    Rostek, A. and Loza, K. and Heggen, M. and Epple, M.
    RSC Advances 9 (2019)
    A comparative X-ray powder diffraction study on poly(N-vinyl pyrrolidone) (PVP)-stabilized palladium and gold nanoparticles and bimetallic Pd-Au nanoparticles (both types of core-shell nanostructures) was performed. The average diameter of Au and Pd nanoparticles was 5 to 6 nm. The two types of core-shell particles had a core diameter of 5 to 6 nm and an overall diameter of 7 to 8 nm, i.e. a shell thickness of 1 to 2 nm. X-ray powder diffraction on a laboratory instrument was able to distinguish between a physical mixture of gold and palladium nanoparticles and bimetallic core-shell nanoparticles. It was also possible to separate the core from the shell in both kinds of bimetallic core-shell nanoparticles due to the different domain size and because it was known which metal was in the core and which was in the shell. The spherical particles were synthesized by reduction with glucose in aqueous media. After purification by multiple centrifugation steps, the particles were characterized with respect to their structural, colloid-chemical, and spectroscopic properties, i.e. particle size, morphology, and internal elemental distribution. Dynamic light scattering (DLS), differential centrifugal sedimentation (DCS), atomic absorption spectroscopy (AAS), ultraviolet-visible spectroscopy (UV-vis), high-angle annular dark field imaging (HAADF), and energy-dispersed X-ray spectroscopy (EDX) were applied for particle characterization. This journal is © The Royal Society of Chemistry.
    view abstract10.1039/c9ra05117a
  • 3D biodegradable scaffolds of polycaprolactone with silicate-containing hydroxyapatite microparticles for bone tissue engineering: High-resolution tomography and in vitro study
    Shkarina, S. and Shkarin, R. and Weinhardt, V. and Melnik, E. and Vacun, G. and Kluger, P. and Loza, K. and Epple, M. and Ivlev, S.I. and Baumbach, T. and Surmeneva, M.A. and Surmenev, R.A.
    Scientific Reports 8 (2018)
    To date, special interest has been paid to composite scaffolds based on polymers enriched with hydroxyapatite (HA). However, the role of HA containing different trace elements such as silicate in the structure of a polymer scaffold has not yet been fully explored. Here, we report the potential use of silicate-containing hydroxyapatite (SiHA) microparticles and microparticle aggregates in the predominant range from 2.23 to 12.40 μm in combination with polycaprolactone (PCL) as a hybrid scaffold with randomly oriented and well-aligned microfibers for regeneration of bone tissue. Chemical and mechanical properties of the developed 3D scaffolds were investigated with XRD, FTIR, EDX and tensile testing. Furthermore, the internal structure and surface morphology of the scaffolds were analyzed using synchrotron X-ray μCT and SEM. Upon culturing human mesenchymal stem cells (hMSC) on PCL-SiHA scaffolds, we found that both SiHA inclusion and microfiber orientation affected cell adhesion. The best hMSCs viability was revealed at 10 day for the PCL-SiHA scaffolds with well-aligned structure (~82%). It is expected that novel hybrid scaffolds of PCL will improve tissue ingrowth in vivo due to hydrophilic SiHA microparticles in combination with randomly oriented and well-aligned PCL microfibers, which mimic the structure of extracellular matrix of bone tissue. © 2018 The Author(s).
    view abstract10.1038/s41598-018-27097-7
  • A systematic electron microscopic study on the uptake of barium sulphate nano-, submicro-, microparticles by bone marrow-derived phagocytosing cells
    Sokolova, V. and Loza, K. and Knuschke, T. and Heinen-Weiler, J. and Jastrow, H. and Hasenberg, M. and Buer, J. and Westendorf, A.M. and Gunzer, M. and Epple, M.
    Acta Biomaterialia 80 (2018)
    Nanoparticles can act as transporters for synthetic molecules and biomolecules into cells, also in immunology. Antigen-presenting cells like dendritic cells are important targets for immunotherapy in nanomedicine. Therefore, we have used primary murine bone marrow-derived phagocytosing cells (bmPCs), i.e. dendritic cells and macrophages, to study their interaction with spherical barium sulphate particles of different size (40 nm, 420 nm, and 1 µm) and to follow their uptake pathway. Barium sulphate is chemically and biologically inert (no dissolution, no catalytic effects), i.e. we can separate the particle uptake effect from potential biological reactions. The colloidal stabilization of the nanoparticles was achieved by a layer of carboxymethylcellulose (CMC) which is biologically inert and gives the particles a negative zeta potential (i.e. charge). The particles were made fluorescent by conjugating 6-aminofluoresceine to CMC. Their uptake was visualized by flow cytometry, confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), transmission electron microscopy (TEM), and correlative light and electron microscopy (CLEM). Barium sulphate particles of all sizes were readily taken up by dendritic cells and even more by macrophages, with the uptake increasing with time and particle concentration. They were mainly localized inside phagosomes, heterophagosomes, and in the case of nanoparticles also in the nearby cytosol. No particles were found in the nucleus. In nanomedicine, inorganic nanoparticles from the nanometer to the micrometer size are therefore well suited as transporters of biomolecules, including antigens, into dendritic cells and macrophages. The presented model system may also serve to describe the aseptic loosening of endoprostheses caused by abrasive wear of inert particles and the subsequent cell reaction, a question which relates to the field of nanotoxicology. Statement of Significance: The interaction of particles and cells is at the heart of nanomedicine and nanotoxicology, including abrasive wear from endoprostheses. It also comprises the immunological reaction to different kinds of nanomaterials, triggered by an immune response, e.g. by antigen-presenting cells. However, it is often difficult to separate the particle effect from a chemical or biochemical reaction to particles or their cargo. We show how chemically inert barium sulphate particles with three different sizes (nano, sub-micro, and micro) interact with relevant immune cells (primary dendritic cells and macrophages). Particles of all three sizes are readily taken up into both cell types by phagocytosis, but the uptake by macrophages is significantly more prominent than that by dendritic cells. The cells take up particles until they are virtually stuffed, but without direct adverse effect. The uptake increases with time and particle concentration. Thus, we have an ideal model system to follow particles into and inside cells without the side effect of a chemical particle effect, e.g. by degradation or ion release. © 2018 Acta Materialia Inc.
    view abstract10.1016/j.actbio.2018.09.026
  • Calcium phosphate particles stimulate interleukin-1β release from human vascular smooth muscle cells: A role for spleen tyrosine kinase and exosome release
    Dautova, Y. and Kapustin, A.N. and Pappert, K. and Epple, M. and Okkenhaug, H. and Cook, S.J. and Shanahan, C.M. and Bootman, M.D. and Proudfoot, D.
    Journal of Molecular and Cellular Cardiology 115 (2018)
    Aims Calcium phosphate (CaP) particle deposits are found in several inflammatory diseases including atherosclerosis and osteoarthritis. CaP, and other forms of crystals and particles, can promote inflammasome formation in macrophages leading to caspase-1 activation and secretion of mature interleukin-1β (IL-1β). Given the close association of small CaP particles with vascular smooth muscle cells (VSMCs) in atherosclerotic fibrous caps, we aimed to determine if CaP particles affected pro-inflammatory signalling in human VSMCs. Methods and results Using ELISA to measure IL-1β release from VSMCs, we demonstrated that CaP particles stimulated IL-1β release from proliferating and senescent human VSMCs, but with substantially greater IL-1β release from senescent cells; this required caspase-1 activity but not LPS-priming of cells. Potential inflammasome agonists including ATP, nigericin and monosodium urate crystals did not stimulate IL-1β release from VSMCs. Western blot analysis demonstrated that CaP particles induced rapid activation of spleen tyrosine kinase (SYK) (increased phospho-Y525/526). The SYK inhibitor R406 reduced IL-1β release and caspase-1 activation in CaP particle-treated VSMCs, indicating that SYK activation occurs upstream of and is required for caspase-1 activation. In addition, IL-1β and caspase-1 colocalised in intracellular endosome-like vesicles and we detected IL-1β in exosomes isolated from VSMC media. Furthermore, CaP particle treatment stimulated exosome secretion by VSMCs in a SYK-dependent manner, while the exosome-release inhibitor spiroepoxide reduced IL-1β release. Conclusions CaP particles stimulate SYK and caspase-1 activation in VSMCs, leading to the release of IL-1β at least in part via exosomes. These novel findings in human VSMCs highlight the pro-inflammatory and pro-calcific potential of microcalcification. © 2017 The Authors
    view abstract10.1016/j.yjmcc.2017.12.007
  • Comparative biological effects of spherical noble metal nanoparticles (Rh, Pd, Ag, Pt, Au) with 4-8 nm diameter
    Rostek, A. and Breisch, M. and Pappert, K. and Loza, K. and Heggen, M. and Köller, M. and Sengstock, C. and Epple, M.
    Beilstein Journal of Nanotechnology 9 (2018)
    For a comparative cytotoxicity study, nanoparticles of the noble metals Rh, Pd, Ag, Pt, and Au (spherical, average diameter 4 to 8 nm) were prepared by reduction in water and colloidally stabilized with poly(N-vinyl pyrrolidone) (PVP). Thus, their shape, size, and surface functionalization were all the same. Size and morphology of the nanoparticles were determined by dynamic light scattering (DLS), analytical disc centrifugation (differential centrifugal sedimentation, DCS), and high-resolution transmission electron microscopy (HRTEM). Cell-biological experiments were performed to determine the effect of particle exposure on the viability of human mesenchymal stem cells (hMSCs). Except for silver, no adverse effect of any of the metal nanoparticles was observed for concentrations up to 50 ppm (50 mg L-1) incubated for 24 h, indicating that noble metal nanoparticles (rhodium, palladium, platinum, gold) that do not release ions are not cytotoxic under these conditions. © 2018 Rostek et al.
    view abstract10.3762/bjnano.9.258
  • Covalent Surface Functionalization of Calcium Phosphate Nanoparticles with Fluorescent Dyes by Copper-Catalysed and by Strain-Promoted Azide-Alkyne Click Chemistry
    Rojas-Sánchez, L. and Sokolova, V. and Riebe, S. and Voskuhl, J. and Epple, M.
    ChemNanoMat (2018)
    Spherical calcium phosphate nanoparticles with a solid core diameter around 90 nm (from scanning electron microscopy, SEM) were coated with a silica shell and then covalently functionalized by azide groups. To these azide groups, all kinds of alkyne-carrying molecules can be covalently attached by copper-catalysed azide-alkyne cycloaddition (CuAAC) and by strain-promoted azide-alkyne cycloaddition (SPAAC) at a very high density. This was demonstrated for a number of dyes (FAM, TAMRA, Cy5, Alexa Fluor™ 488, and an aromatic thioether with aggregation-induced emission (AIE) properties). It was also possible to attach more than one molecule to the surface of one particle by two-step click reaction, permitting the synthesis of multimodal nanoparticles that are stable under biological conditions. The nanoparticles have a hydrodynamic diameter of around 200 nm (from dynamic light scattering, DLS), which makes them suitable for uptake by cells. The strongly fluorescing nanoparticles were easily taken up by cells as demonstrated by fluorescence microscopy, confocal laser scanning microscopy (CLSM), and structured illuminated microscopy (SIM). © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
    view abstract10.1002/cnma.201800509
  • Crystallographic characterization of laser-generated, polymer-stabilized 4 nm silver-gold alloyed nanoparticles
    Prymak, O. and Jakobi, J. and Rehbock, C. and Epple, M. and Barcikowski, S.
    Materials Chemistry and Physics 207 (2018)
    Monometallic silver and gold nanoparticles and bimetallic silver-gold (AgAu) nanoparticles were prepared by laser ablation in liquids in the atomic composition range of Ag:Au from 0:100 to 100:0 with steps of 10 at% and colloidally stabilized with poly(N-vinylpyrrolidone) (PVP). As metallic bulk targets for laser ablation, pure silver, pure gold, and alloyed AgAu foils with the desired composition were used. Size separation by centrifugation and freeze-drying gave monodisperse spherical nanoparticles with a diameter of 4 nm as determined by differential centrifugal sedimentation (DCS) and transmission electron microscopy (TEM). A crystallographic characterization of the nanoparticles was carried out by X-ray powder diffraction (XRD) and Rietveld refinement, leading to highly precise cubic lattice parameters (fcc crystal system) and crystallite sizes. For comparison, the same analysis including the determination of the microstrain was carried out for the bulk target materials (AgAu alloys in the full concentration range). Both nanoparticles and bulk target materials obeyed Vegard's rule, with only slight deviations. The fact that the crystallite size as determined by XRD was identical to the hydrodynamic diameter by DCS and the Feret diameter by TEM indicates that the particles consist of only one domain, i.e. they are single crystals. The combination of UV-vis spectroscopy with energy-dispersive X-ray spectroscopy (EDX) as line scan along the nanoparticle showed a homogenous distribution of the gold and silver inside the nanoparticles, indicating solid solution alloys, in contrast to what was observed earlier for chemically prepared AgAu nanoparticles by reduction of metal ions in water. © 2018 Elsevier B.V.
    view abstract10.1016/j.matchemphys.2017.12.080
  • Cytotoxicity of doxorubicin-conjugated poly[N-(2-hydroxypropyl)methacrylamide]-modified ?-Fe2O3 nanoparticles towards human tumor cells
    Plichta, Z. and Kozak, Y. and Panchuk, R. and Sokolova, V. and Epple, M. and Kobylinska, L. and Jendelová, P. and Horák, D.
    Beilstein Journal of Nanotechnology 9 (2018)
    Doxorubicin-conjugated magnetic nanoparticles containing hydrolyzable hydrazone bonds were developed using a non-toxic poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) coating, which ensured good colloidal stability in aqueous media and limited internalization by the cells, however, enabled adhesion to the cell surface. While the neat PHPMA-coated particles proved to be non-toxic, doxorubicin-conjugated particles exhibited enhanced cytotoxicity in both drug-sensitive and drug-resistant tumor cells compared to free doxorubicin. The newly developed doxorubicin-conjugated PHPMA-coated magnetic particles seem to be a promising magnetically targeted vehicle for anticancer drug delivery. © 2018 Plichta et al.
    view abstract10.3762/bjnano.9.236
  • Deciphering the Surface Composition and the Internal Structure of Alloyed Silver–Gold Nanoparticles
    Grasmik, V. and Rurainsky, C. and Loza, K. and Evers, M.V. and Prymak, O. and Heggen, M. and Tschulik, K. and Epple, M.
    Chemistry - A European Journal 24 (2018)
    Spherical bimetallic AgAu nanoparticles in the molar ratios 30:70, 50:50, and 70:30 with diameters of 30 to 40 nm were analyzed together with pure silver and gold nanoparticles of the same size. Dynamic light scattering (DLS) and differential centrifugal sedimentation (DCS) were used for size determination. Cyclic voltammetry (CV) was used to determine the nanoalloy composition, together with atomic absorption spectroscopy (AAS), energy-dispersive X-ray spectroscopy (EDX) and ultraviolet-visible (UV/Vis) spectroscopy. Underpotential deposition (UPD) of lead (Pb) on the particle surface gave information about its spatial elemental distribution and surface area. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM) were applied to study the shape and the size of the nanoparticles. X-ray powder diffraction gave the crystallite size and the microstrain. The particles form a solid solution (alloy) with an enrichment of silver on the nanoparticle surface, including some silver-rich patches. UPD indicated that the surface only consists of silver atoms. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
    view abstract10.1002/chem.201800579
  • Deposition of polycrystalline zinc substituted hydroxyapatite coatings with a columnar structure by RF magnetron sputtering: Role of in-situ substrate heating
    Prosolov, K.A. and Belyavskaya, O.A. and Rau, J.V. and Prymak, O. and Epple, M. and Sharkeev, Y.P.
    Journal of Physics: Conference Series 1115 (2018)
    Zn incorporation into hydroxyapatite structure leads to enhanced osteointegration and antibacterial activity of deposited coatings. Radiofrequency magnetron sputtering is a physical vapor deposition technique which can be used to create thin coatings with a controlled level of crystallinity. The material state is a crucial parameter for biocoatings as it governs cell response. Bioactive Zn substituted hydroxyapatite coatings were deposited onto Ti by radiofrequency magnetron sputtering at increased substrate temperatures (100, 200, 300 and 400°C). XRD showed crystallization of the coatings at elevated substrate temperatures starting from 300°C. Cross-section transmission electron microscopy showed a polycrystalline columnar grain structure of Zn substituted coatings deposited at 400°C substrate temperature. An amorphous TiO 2 sublayer of several monolayers thickness was detected in the interface between the polycrystalline coating and the Ti substrate. In-column energy dispersive X-ray analysis revealed coatings to be substoichiometric with the average Ca/P ratio being 1.5. It is established that it is possible to deposit Zn substituted hydroxyapatite in a form of a well-crystalline coating when the substrate temperature is exceeding 400°C. © Published under licence by IOP Publishing Ltd.
    view abstract10.1088/1742-6596/1115/3/032077
  • Enhanced selective cellular proliferation by multi-biofunctionalization of medical implant surfaces with heterodimeric BMP-2/6, fibronectin, and FGF-2
    Ettelt, V. and Belitsky, A. and Lehnert, M. and Loidl-Stahlhofen, A. and Epple, M. and Veith, M.
    Journal of Biomedical Materials Research - Part A 106 (2018)
    Increasing cell adhesion on implant surfaces is an issue of high biomedical importance. Early colonization with endogenous cells reduces the risk of bacterial contamination and enhances the integration of an implant into the diverse cellular tissues surrounding it. In vivo integration of implants is controlled by a complex spatial and temporal interplay of cytokines and adhesive molecules. The concept of a multi-biofunctionalized TiO2 surface for stimulating bone and soft tissue growth is presented here. All supramolecular architectures were built with a biotin–streptavidin coupling system. Biofunctionalization of TiO2 with immobilized FGF-2 and heparin could be shown to selectively increase the proliferation of fibroblasts while immobilized BMP-2 only stimulated the growth of osteoblasts. Furthermore, TiO2 surfaces biofunctionalized with either the BMP-2 or BMP-2/6 growth factor and the cell adhesion-enhancing protein fibronectin showed higher osteoblast adhesion than a TiO2 surface functionalized with only one of these proteins. In conclusion, the presented immobilization strategy is applicable in vivo for a selective surface coating of implants in both hard and connective tissue. The combined immobilization of different extracellular proteins on implants has the potential to further influence cell-specific reactions. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2910–2922, 2018. © 2018 Wiley Periodicals, Inc.
    view abstract10.1002/jbm.a.36480
  • Erratum to: 3D biodegradable scaffolds of polycaprolactone with silicate-containing hydroxyapatite microparticles for bone tissue engineering: high-resolution tomography and in vitro study (Scientific Reports, (2018), 8, 1, (8907), 10.1038/s41598-018-27097-7)
    Shkarina, S. and Shkarin, R. and Weinhardt, V. and Melnik, E. and Vacun, G. and Kluger, P.J. and Loza, K. and Epple, M. and Ivlev, S.I. and Baumbach, T. and Surmeneva, M.A. and Surmenev, R.A.
    Scientific Reports 8 (2018)
    A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper. © 2018, The Author(s).
    view abstract10.1038/s41598-018-35952-w
  • Immobilization of cesium from aqueous solution using nanoparticles of synthetic calcium phosphates
    Livitska, O. and Strutynska, N. and Loza, K. and Prymak, O. and Prylutskyy, Y. and Livitska, O. and Epple, M. and Slobodyanik, N.
    Chemistry Central Journal 12 (2018)
    The particularities of cesium incorporation into synthetic calcium phosphates with either apatite or whitlockite-type structures were investigated using the sorption process from aqueous solution and further heating to 700 °C. The nanoparticles for sorption were prepared by wet precipitation from aqueous solutions at a fixed molar ratio of Ca/P = 1.67 and two different ratios of CO3 2−/PO4 3− (0 or 1). The obtained substituted calcium phosphates and corresponding samples after the sorption of cesium from solutions with different molar concentrations (c(Cs+) = 0.05, 0.1 and 0.25 mol L−1) were characterized by powder X-ray diffraction, FTIR spectroscopy, scanning electron microscopy and elemental analysis. Based on the combination of X-ray diffraction and elemental analyses data for the powders after sorption, the cesium incorporated in the apatite- or whitlockite-type structures and its amount increased with its concentration in the initial solution. For sodium-containing calcium phosphate even minor content of Cs+ in its composition significantly changed the general principle of its transformation under annealing at 700 °C with the formation of a mixture of α-Ca3(PO4)2 and cesium-containing apatite-related phase. The obtained results indicate the perspective of using of complex substituted calcium phosphates nanoparticles for immobilization of cesium in the stable whitlockite- or apatite-type crystal materials. © 2018, The Author(s).
    view abstract10.1186/s13065-018-0455-9
  • Improved Models for Metallic Nanoparticle Cores from Atomic Pair Distribution Function (PDF) Analysis
    Banerjee, S. and Liu, C.-H. and Lee, J.D. and Kovyakh, A. and Grasmik, V. and Prymak, O. and Koenigsmann, C. and Liu, H. and Wang, L. and Abeykoon, A.M.M. and Wong, S.S. and Epple, M. and Murray, C.B. and Billinge, S.J.L.
    Journal of Physical Chemistry C 122 (2018)
    X-ray atomic pair distribution functions (PDFs) were collected from a range of canonical metallic nanomaterials, both elemental and alloyed, prepared using different synthesis methods and exhibiting drastically different morphological properties. Widely applied shape-tuned attenuated crystal (AC) fcc models proved inadequate, yielding structured, coherent, and correlated fit residuals. However, equally simple discrete cluster models could account for the largest amplitude features in these difference signals. A hypothesis testing based approach to nanoparticle structure modeling systematically ruled out effects from crystallite size, composition, shape, and surface faceting as primary factors contributing to the AC misfit. On the other hand, decahedrally twinned cluster cores were found to be the origin of the AC structure misfits for a majority of the nanomaterials reported here. It is further motivated that the PDF can readily differentiate between the arrangement of domains in these multiply twinned motifs. Most of the nanomaterials surveyed also fall within the sub-5 nm size regime where traditional electron microscopy cannot easily detect and quantify domain structures, with sampling representative of the average nanocrystal synthesized. The results demonstrate that PDF analysis is a powerful method for understanding internal atomic interfaces in small noble metallic nanomaterials. Such core cluster models, easily built algorithmically, should serve as starting structures for more advanced models able to capture atomic positional disorder, ligand induced or otherwise, near nanocrystal surfaces. © 2018 American Chemical Society.
    view abstract10.1021/acs.jpcc.8b05897
  • Induction of type I interferons by therapeutic nanoparticle-based vaccination is indispensable to reinforce cytotoxic CD8+ T cell responses during chronic retroviral infection
    Knuschke, T. and Rotan, O. and Bayer, W. and Kollenda, S. and Dickow, J. and Sutter, K. and Hansen, W. and Dittmer, U. and Lang, K.S. and Epple, M. and Buer, J. and Westendorf, A.M.
    Frontiers in Immunology 9 (2018)
    T cell dysfunction and immunosuppression are characteristic for chronic viral infections and contribute to viral persistence. Overcoming these burdens is the goal of new therapeutic strategies to cure chronic infectious diseases. We recently described that therapeutic vaccination of chronic retrovirus infected mice with a calcium phosphate (CaP) nanoparticle (NP)-based vaccine carrier, functionalized with CpG and viral peptides is able to efficiently reactivate the CD8+ T cell response and improve the eradication of virus infected cells. However, the mechanisms underlying this effect were largely unclear. While type I interferons (IFNs I) are considered to drive T cell exhaustion by persistent immune activation during chronic viral infection, we here describe an indispensable role of IFN I induced by therapeutic vaccination to efficiently reinforce cytotoxic CD8+ T cells (CTL) and improve control of chronic retroviral infection. The induction of IFN I is CpG dependent and leads to significant IFN signaling indicated by upregulation of IFN stimulated genes. By vaccinating chronically retrovirus-infected mice lacking the IFN I receptor (IFNAR-/-) or by blocking IFN I signaling in vivo during therapeutic vaccination, we demonstrate that IFN I signaling is necessary to drive full reactivation of CTLs. Surprisingly, we also identified an impaired suppressive capability of regulatory T cells in the presence of IFNa, which implicates an important role for vaccine-induced IFNa in the regulation of the T cell response during chronic retroviral infection. Our data suggest that inducing IFN I signaling in conjunction with the presentation of viral antigens can reactivate immune functions and reduce viral loads in chronic infections. Therefore, we propose CaP NPs as potential therapeutic tool to treat chronic infections. © 2018 Knuschke, Rotan, Bayer, Kollenda, Dickow, Sutter, Hansen, Dittmer, Lang, Epple, Buer and Westendorf.
    view abstract10.3389/fimmu.2018.00614
  • Modern dental care from the chemical point of view [Moderne Zahnpflege aus chemischer Sicht]
    Epple, M. and Enax, J.
    Chemie in Unserer Zeit 52 (2018)
    In modern societies, oral health is playing a central role. Therefore, a high variety of tooth-care formulations to enhance oral health and to prevent diseases of teeth and gum is available. Apart from the kind of toothbrush (simple mechanic or electric), the toothpaste is of major importance. Fluoride is regarded as central agent, but there are also interesting developments of biomimetic toothcare products based on hydroxyapatite. © 2018, Wiley-VCH Verlag. All rights reserved.
    view abstract10.1002/ciuz.201800796
  • Moderne Zahnpflege aus chemischer Sicht
    Epple, M. and Enax, J.
    Chemie in Unserer Zeit (2018)
    Zusammenfassung: Die Mundgesundheit spielt in unserer Gesellschaft eine zentrale Rolle. Deshalb gibt es eine Vielzahl von wirkungsvollen Zahnpflegepräparaten, die die Mundgesundheit verbessern und Zahn- und Zahnfleischerkrankungen vorbeugen können. Neben der Zahnbürste (Handzahnbürste, elektrische Zahnbürste) ist dabei insbesondere die Zahnpasta von Bedeutung. Als zentraler Wirkstoff wird Fluorid angesehen, wobei es auch interessante Entwicklungen von biomimetischen Ansätzen auf der Basis von Calciumphosphat gibt. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/ciuz.201800796
  • Precipitation with polyethylene glycol followed by washing and pelleting by ultracentrifugation enriches extracellular vesicles from tissue culture supernatants in small and large scales
    Ludwig, A.-K. and De Miroschedji, K. and Doeppner, T.R. and Börger, V. and Ruesing, J. and Rebmann, V. and Durst, S. and Jansen, S. and Bremer, M. and Behrmann, E. and Singer, B.B. and Jastrow, H. and Kuhlmann, J.D. and El Magraoui, F. and Meyer, H.E. and Hermann, D.M. and Opalka, B. and Raunser, S. and Epple, M. and Horn, P.A. and Giebel, B.
    Journal of Extracellular Vesicles 7 (2018)
    Extracellular vesicles (EVs) provide a complex means of intercellular signalling between cells at local and distant sites, both within and between different organs. According to their cell-type specific signatures, EVs can function as a novel class of biomarkers for a variety of diseases, and can be used as drug-delivery vehicles. Furthermore, EVs from certain cell types exert beneficial effects in regenerative medicine and for immune modulation. Several techniques are available to harvest EVs from various body fluids or cell culture supernatants. Classically, differential centrifugation, density gradient centrifugation, size-exclusion chromatography and immunocapturing-based methods are used to harvest EVs from EV-containing liquids. Owing to limitations in the scalability of any of these methods, we designed and optimised a polyethylene glycol (PEG)-based precipitation method to enrich EVs from cell culture supernatants. We demonstrate the reproducibility and scalability of this method and compared its efficacy with more classical EV-harvesting methods. We show that washing of the PEG pellet and the re-precipitation by ultracentrifugation remove a huge proportion of PEG co-precipitated molecules such as bovine serum albumine (BSA). However, supported by the results of the size exclusion chromatography, which revealed a higher purity in terms of particles per milligram protein of the obtained EV samples, PEG-prepared EV samples most likely still contain a certain percentage of other non-EV associated molecules. Since PEG-enriched EVs revealed the same therapeutic activity in an ischemic stroke model than corresponding cells, it is unlikely that such co-purified molecules negatively affect the functional properties of obtained EV samples. In summary, maybe not being the purification method of choice if molecular profiling of pure EV samples is intended, the optimised PEG protocol is a scalable and reproducible method, which can easily be adopted by laboratories equipped with an ultracentrifuge to enrich for functional active EVs. © 2018, © 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles.
    view abstract10.1080/20013078.2018.1528109
  • Prolonged release of bone morphogenetic protein-2 in vivo by gene transfection with DNA-functionalized calcium phosphate nanoparticle-loaded collagen scaffolds
    Tenkumo, T. and Vanegas Sáenz, J.R. and Nakamura, K. and Shimizu, Y. and Sokolova, V. and Epple, M. and Kamano, Y. and Egusa, H. and Sugaya, T. and Sasaki, K.
    Materials Science and Engineering C 92 (2018)
    In the combination of scaffolds immersed in growth factor solutions, the release of growth factors mainly depends on scaffold degradation. However, the release of bone morphogenetic protein (BMP)-2 at an appropriate concentration during the stage of tissue regeneration would enhance bone regeneration. To achieve this condition, the present study was performed to investigate the effects of scaffolds combined with gene transfection using non-viral vectors. Nanohydroxyapatite-collagen (nHAC) scaffolds cross-linked with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) or ascorbic acid/copper chloride, and a collagen scaffold (Terdermis®) were prepared, loaded with BMP-2-encoding plasmid DNA-functionalized calcium phosphate nanoparticles (CaP), naked plasmid DNA, or BMP-2 solution, and implanted in rats. The yield of released BMP-2 and its releasing period, respectively, were larger and longer from the scaffolds loaded with CaP than from those incubated with BMP-2 solution. In addition, the alkaline phosphatase activity induced by the CaP-loaded scaffolds was higher. Histological analysis showed that released BMP-2 could be observed on the macrophages or multinuclear giant cells surrounding the nHAC fragments or collagen fibres. TRAP-positive or OCN-positive sites were observed in all groups and a mineralization area was observed in the Terdermis®/CaP sample. The present study demonstrates that gene transfection by scaffold loaded with CaP gene transfer vectors induces a larger yield of BMP-2 for a longer period than by scaffolds loaded with BMP-2 solution or naked plasmid. © 2018 Elsevier B.V.
    view abstract10.1016/j.msec.2018.06.047
  • Review of potential health risks associated with nanoscopic calcium phosphate
    Epple, M.
    Acta Biomaterialia (2018)
    Calcium phosphate is applied in many products in biomedicine, but also in toothpastes and cosmetics. In some cases, it is present in nanoparticulate form, either on purpose or after degradation or mechanical abrasion. Possible concerns are related to the biological effect of such nanoparticles. A thorough literature review shows that calcium phosphate nanoparticles as such have no inherent toxicity but can lead to an increase of the intracellular calcium concentration after endosomal uptake and lysosomal degradation. However, cells are able to clear the calcium from the cytoplasm within a few hours, unless very high doses of calcium phosphate are applied. The observed cytotoxicity in some cell culture studies, mainly for unfunctionalized particles, is probably due to particle agglomeration and subsequent sedimentation onto the cell layer, leading to a very high local particle concentration, a high particle uptake, and subsequent cell death. There is no risk from an oral uptake of calcium phosphate nanoparticles due to their rapid dissolution in the stomach. The risk from dermal or mucosal uptake is very low. Calcium phosphate nanoparticles can enter the bloodstream by inhalation, but no adverse effects have been observed, except for a prolonged exposition to high particle doses. Calcium phosphate nanoparticles inside the body (e.g. after implantation or due to abrasion) do not pose a risk as they are typically resorbed and dissolved by osteoclasts and macrophages. There is no indication for a significant influence of the calcium phosphate phase or the particle shape (e.g. spherical or rod-like) on the biological response. In summary, the risk associated with an exposition to nanoparticulate calcium phosphate in doses that are usually applied in biomedicine, health care products, and cosmetics is very low and most likely not present at all. Statement of Significance: Calcium phosphate is a well-established biomaterial. However, there are occasions when it occurs in a nanoparticulate form (e.g. as nanoparticle or as nanoparticulate bone substitution material) or after abrasion from a calcium phosphate-coated metal implant. In the light of the current discussion on the safety of nanoparticles, there have been concerns about potential adverse effects of nano-calcium phosphate, e.g. in a statement of a EU study group from 2016 about possible dangers associated with non-spherical nano-hydroxyapatite in cosmetics. In the US, there was a discussion in 2016 about the dangers of nano-calcium phosphate in babyfood. In this review, the potential exposition routes for nano-calcium phosphate are reviewed, with special emphasis on its application as biomaterial. © 2018 Acta Materialia Inc.
    view abstract10.1016/j.actbio.2018.07.036
  • Shape-Dependent Dissolution and Cellular Uptake of Silver Nanoparticles
    Graf, C. and Nordmeyer, D. and Sengstock, C. and Ahlberg, S. and Diendorf, J. and Raabe, J. and Epple, M. and Köller, M. and Lademann, J. and Vogt, A. and Rancan, F. and Rühl, E.
    Langmuir 34 (2018)
    The cellular uptake and dissolution of trigonal silver nanoprisms (edge length 42 ± 15 nm, thickness 8 ± 1 nm) and mostly spherical silver nanoparticles (diameter 70 ± 25 nm) in human mesenchymal stem cells (hMSC's) and human keratinocytes (HaCaT cells) were investigated. Both particles are stabilized by polyvinylpyrrolidone (PVP), with the prisms additionally stabilized by citrate. The nanoprisms dissolved slightly in pure water but strongly in isotonic saline or at pH 4, corresponding to the lowest limit for the pH during cellular uptake. The tips of the prisms became rounded within minutes due to their high surface energy. Afterward, the dissolution process slowed down due to the presence of both PVP stabilizing Ag{100} sites and citrate blocking Ag{111} sites. On the contrary, nanospheres, solely stabilized by PVP, dissolved within 24 h. These results correlate with the finding that particles in both cell types have lost >90% of their volume within 24 h. hMSC's took up significantly more Ag from nanoprisms than from nanospheres, whereas HaCaT cells showed no preference for one particle shape. This can be rationalized by the large cellular interaction area of the plateletlike nanoprisms and the bending stiffness of the cell membranes. hMSC's have a highly flexible cell membrane, resulting in an increased uptake of plateletlike particles. HaCaT cells have a membrane with a 3 orders of magnitude higher Young's modulus than for hMSC. Hence, the energy gain due to the larger interaction area of the nanoprisms is compensated for by the higher energy needed for cell membrane deformation compared to that for spheres, leading to no shape preference. © 2017 American Chemical Society.
    view abstract10.1021/acs.langmuir.7b03126
  • Silver nanoparticles in complex media: An easy procedure to discriminate between metallic silver nanoparticles, reprecipitated silver chloride, and dissolved silver species
    Loza, K. and Epple, M.
    RSC Advances 8 (2018)
    Silver nanoparticles undergo oxidative dissolution in water upon storage. This occurs in pure water as well as in more complex media, including natural environments, biological tissues, and cell culture media. However, the dissolution leads to the reprecipitation of silver chloride as chloride is present in almost all relevant environments. The discrimination between dissolved silver species (ions and silver complexes) and dispersed (solid) species does not take this into account because all solid species (metallic silver and silver chloride) are isolated together. By applying a chemical separation procedure, we show that it is possible to quantify silver, silver chloride, and dissolved silver species after immersion into a typical cell culture medium (DMEM + 10% FCS). During the dissolution of metallic silver nanoparticles, about half of the dissolved silver is reprecipitated as solid silver chloride, i.e. the mere analysis of the soluble silver species does not reflect the true situation. The separation protocol is suitable for all chloride-containing media in the presence or in the absence of biomolecules. © 2018 The Royal Society of Chemistry.
    view abstract10.1039/c8ra04500c
  • SPICES: a particle-based molecular structure line notation and support library for mesoscopic simulation
    van den Broek, K. and Daniel, M. and Epple, M. and Kuhn, H. and Schaub, J. and Zielesny, A.
    Journal of Cheminformatics 10 (2018)
    Simplified Particle Input ConnEction Specification (SPICES) is a particle-based molecular structure representation derived from straightforward simplifications of the atom-based SMILES line notation. It aims at supporting tedious and error-prone molecular structure definitions for particle-based mesoscopic simulation techniques like Dissipative Particle Dynamics by allowing for an interplay of different molecular encoding levels that range from topological line notations and corresponding particle-graph visualizations to 3D structures with support of their spatial mapping into a simulation box. An open Java library for SPICES structure handling and mesoscopic simulation support in combination with an open Java Graphical User Interface viewer application for visual topological inspection of SPICES definitions are provided.[Figure not available: see fulltext.]. © 2018, The Author(s).
    view abstract10.1186/s13321-018-0294-7
  • Streptavidin-coated surfaces suppress bacterial colonization by inhibiting non-specific protein adsorption
    Ettelt, V. and Ekat, K. and Kämmerer, P.W. and Kreikemeyer, B. and Epple, M. and Veith, M.
    Journal of Biomedical Materials Research - Part A 106 (2018)
    Streptavidin is a 58 kDa tetrameric protein with the highest known affinity to biotin with a wide range of applications in bionanotechnology and molecular biology. Dissolved streptavidin is stable at a broad range of temperature, pH, proteolytic enzymes and exhibits low non-specific binding. In this study, a streptavidin monolayer was assembled directly on a biotinylated TiO2-surface to investigate its stability against proteolytic digestion and its suppression of initial bacterial adsorption of Escherichia coli, Bacillus subtilis, and Streptococcus intermedius. In contrast to nonmodified TiO2 surfaces, streptavidin-coated substrates showed only a negligible non-specific protein adsorption at physiological protein concentrations as well as a significantly reduced bacterial adhesion. The antiadhesive properties were demonstrated to be the main reason for the suppression of bacterial adhesion, which makes this approach a promising option for future surface biofunctionalization applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 758–768, 2018. © 2017 Wiley Periodicals, Inc.
    view abstract10.1002/jbm.a.36276
  • Synthesis and biological characterization of alloyed silver-platinum nanoparticles: From compact core-shell nanoparticles to hollow nanoalloys
    Grasmik, V. and Breisch, M. and Loza, K. and Heggen, M. and Köller, M. and Sengstock, C. and Epple, M.
    RSC Advances 8 (2018)
    Bimetallic nanoparticles consisting of silver and platinum were prepared by a modified seeded-growth process in water in the full composition range in steps of 10 mol%. The particles had diameters between 15-25 nm as determined by disc centrifugal sedimentation (DCS) and transmission electron microscopy (TEM). Whereas particles with high platinum content were mostly spherical with a solid silver core/platinum shell structure, mostly hollow alloyed nanoparticles were observed with increasing silver content. The internal structure and the elemental distribution within the particles were elucidated by high-resolution transmission electron microscopy (HRTEM) in combination with energy-dispersive X-ray spectroscopy (EDX). The particles were cytotoxic for human mesenchymal stem cells (hMSC) above 50 mol% silver. This was explained by dissolution experiments where silver was only released at and above 50 mol% silver. In contrast, platinum-rich particles (less than 50 mol% silver) did not release any silver ions. This indicates that the presence of platinum inhibits the oxidative dissolution of silver. © 2018 The Royal Society of Chemistry.
    view abstract10.1039/c8ra06461j
  • Synthetic hydroxyapatite as a biomimetic oral care agent
    Enax, J. and Epple, M.
    Oral Health and Preventive Dentistry 16 (2018)
    Purpose: Human tooth enamel consists mostly of minerals, primarily hydroxyapatite, Ca10(PO4)6(OH)2, and thus synthetic hydroxyapatite can be used as a biomimetic oral care agent. This review describes the synthesis and characterization of hydroxyapatite from a chemist's perspective and provides an overview of its current use in oral care, with a focus on dentin hypersensitivity, caries, biofilm management, erosion, and enamel lesions. Sources: Reviews and original research papers published in English and German were included. Results: The efficiency of synthetic hydroxyapatite in occluding open dentin tubules, resulting in a protection for sensitive teeth, has been well documented in a number of clinical studies. The first corresponding studies on caries, biofilm management and erosion have provided evidence for a positive effect of hydroxyapatite either as a main or synergistic agent in oral care products. However, more in situ and in vivo studies are needed due to the complexity of the oral milieu and to further clarify existing results. Conclusions: Due to its biocompatibility and similarity to biologically formed hydroxyapatite in natural tooth enamel, synthetic hydroxyapatite is a promising biomimetic oral care ingredient that may extend the scope of preventive dentistry. © 2018 Quintessence Publishing Co., Ltd.
    view abstract10.3290/j.ohpd.a39690
  • Targeting sphingosine-1-phosphate lyase as an anabolic therapy for bone loss article
    Weske, S. and Vaidya, M. and Reese, A. and Von Wnuck Lipinski, K. and Keul, P. and Bayer, J.K. and Fischer, J.W. and Flögel, U. and Nelsen, J. and Epple, M. and Scatena, M. and Schwedhelm, E. and Dörr, M. and Völzke, H. and Moritz, E. and Hannemann, A. and Rauch, B.H. and Gräler, M.H. and Heusch, G. and Levkau, B.
    Nature Medicine 24 (2018)
    Sphingosine-1-phosphate (S1P) signaling influences bone metabolism, but its therapeutic potential in bone disorders has remained unexplored. We show that raising S1P levels in adult mice through conditionally deleting or pharmacologically inhibiting S1P lyase, the sole enzyme responsible for irreversibly degrading S1P, markedly increased bone formation, mass and strength and substantially decreased white adipose tissue. S1P signaling through S1P2 potently stimulated osteoblastogenesis at the expense of adipogenesis by inversely regulating osterix and PPAR-γ, and it simultaneously inhibited osteoclastogenesis by inducing osteoprotegerin through newly discovered p38-GSK3β-β-catenin and WNT5A-LRP5 pathways. Accordingly, S1P2-deficient mice were osteopenic and obese. In ovariectomy-induced osteopenia, S1P lyase inhibition was as effective as intermittent parathyroid hormone (iPTH) treatment in increasing bone mass and was superior to iPTH in enhancing bone strength. Furthermore, lyase inhibition in mice successfully corrected severe genetic osteoporosis caused by osteoprotegerin deficiency. Human data from 4,091 participants of the SHIP-Trend population-based study revealed a positive association between serum levels of S1P and bone formation markers, but not resorption markers. Furthermore, serum S1P levels were positively associated with serum calcium, negatively with PTH, and curvilinearly with body mass index. Bone stiffness, as determined through quantitative ultrasound, was inversely related to levels of both S1P and the bone formation marker PINP, suggesting that S1P stimulates osteoanabolic activity to counteract decreasing bone quality. S1P-based drugs should be considered as a promising therapeutic avenue for the treatment of osteoporotic diseases. © 2018 Nature America Inc., part of Springer Nature. All rights reserved.
    view abstract10.1038/s41591-018-0005-y
  • Wet-Chemical Synthesis of Pd-Au Core-Shell Nanoparticles (8 nm): From Nanostructure to Biological Properties
    Rostek, A. and Breisch, M. and Loza, K. and Garcia, P.R.A.F. and Oliveira, C.L.P. and Prymak, O. and Heggen, M. and Köller, M. and Sengstock, C. and Epple, M.
    ChemistrySelect 3 (2018)
    Pd−Au core-shell nanoparticles with a palladium core (diameter about 5.5 nm) and a gold shell (thickness about 1.7 nm) were wet-chemically synthesized in an easy water-based one-pot synthesis by sequential reduction of Pd2+ and Au3+ with glucose in the presence of poly(N-vinylpyrrolidone) (PVP). The metals are present in about equal amounts (molar ratio Pd:Au about 2:1) with a clear separation between core and shell. The reaction was monitored in-situ by small-angle X-ray scattering (SAXS), showing the initial growth of the palladium seeds, followed by the epitactic formation of the gold shell. The core-shell character of the particles was confirmed by high-resolution scanning transmission electron microscopy (STEM) and energy-dispersive X-ray spectroscopy (EDX). However, X-ray powder diffraction with Rietveld analysis indicated a partial alloying, i. e. a gradual border between the two metals. Cell culture experiments showed no adverse effects on human mesenchymal stem cells (hMSCs) with a Pd−Au nanoparticle concentration (computed as total metal) up to 50 μg mL−1 after 24 h incubation, i. e. the particles can be considered as biologically harmless, even after unintended human exposure. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
    view abstract10.1002/slct.201800638
  • Adhesion properties of a three-layer system based on RF-magnetron sputter deposited calcium-phosphate coating and silver nanoparticles
    Tkachev, M.S. and Melnikov, E.S. and Surmeneva, M.A. and Sharonova, A.A. and Surmenev, R.A. and Korneva, O.S. and Shulepov, I.A. and Loza, K. and Epple, M.
    Proceedings of the 11th International Forum on Strategic Technology, IFOST 2016 (2017)
    A three-layer system of hydroxyapatite (HA) coating - Ag nanoparticles - HA coating with an overall thickness of 1.2 μm was prepared. The radio-frequency (RF) magnetron sputtering was used to prepare the first layer of hydroxyapatite coating on titanium. Then electrophoretic deposition of silver nanoparticles on the prepared HA layer was done followed by deposition of the second layer of HA by RFmagnetron sputtering. The adhesion strength was investigated by the scratch test method. Scanning electron microscopy and optical microscopy allowed to qualitatively estimate the deformation mechanisms of the biocomposites after the scratch test. © 2016 IEEE.
    view abstract10.1109/IFOST.2016.7884197
  • Amine-Functionalized Silica Nanoparticles Incorporating Covalently Linked Visible-Light-Excitable Eu3+ Complexes: Synthesis, Characterization, and Cell-Uptake Studies
    Francis, B. and Neuhaus, B. and Reddy, M.L.P. and Epple, M. and Janiak, C.
    European Journal of Inorganic Chemistry 2017 (2017)
    We report the synthesis, characterization, photophysical investigations, and cell-uptake studies of luminescent silica nanoparticles incorporating covalently linked visible-light-excitable Eu3+ complexes. Visible-light excitation was accomplished by using highly conjugated carbazole-based β-diketonate ligands. Covalent incorporation of the Eu3+ complexes into the silica nanoparticles was achieved by modification of the bidentate phosphine oxide 4,6-bis(diphenylphosphoryl)-10H-phenoxazine (DPOXPO), which was used as the neutral donor for the Eu3+ ion. The surface amine functionalization of the nanoparticles was carried out using aminopropyltriethoxysilane (APTES). The prepared nanoparticles (Eu@Si-OH and Eu@Si-NH2) are around 35–40 nm in diameter, monodisperse, stable in aqueous dispersion, and also retain the luminescent properties of the incorporated Eu3+ complex. The synthesized nanoparticles exhibit a promising luminescence quantum yield of 38 % and an excited-state lifetime of 638 µs at physiological pH. The photobleaching experiments revealed that the developed nanoparticles are more photostable than the parent Eu3+ complex 1. In vitro experiments with Eu@Si-NH2 nanoparticles on HeLa cells showed that they are biocompatible and are readily taken up by cells. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
    view abstract10.1002/ejic.201700240
  • Avidin-conjugated calcium phosphate nanoparticles as a modular targeting system for the attachment of biotinylated molecules in vitro and in vivo
    van der Meer, S.B. and Knuschke, T. and Frede, A. and Schulze, N. and Westendorf, A.M. and Epple, M.
    Acta Biomaterialia 57 (2017)
    Avidin was covalently conjugated to the surface of calcium phosphate nanoparticles, coated with a thin silica shell and terminated by sulfhydryl groups (diameter of the solid core about 50 nm), with a bifunctional crosslinker connecting the amino groups of avidin to the sulfhydryl group on the nanoparticle surface. This led to a versatile nanoparticle system where all kinds of biotinylated (bio-)molecules can be easily attached to the surface by the non-covalent avidin-biotin-complex formation. It also permits the attachment of different biomolecules on the same nanoparticle (heteroavidity), creating a modular system for specific applications in medicine and biology. The variability of the binding to the nanoparticle surface of the was demonstrated with various biotinylated molecules, i.e. fluorescent dyes and antibodies. The accessibility of the conjugated avidin was demonstrated by a fluorescence-quenching assay. About 2.6 binding sites for biotin were accessible on each avidin tetramer. Together with a number of about 240 avidin tetramer units per nanoparticle, this offers about 600 binding sites for biotin on each nanoparticle. The uptake of fluorescently labelled avidin-conjugated calcium phosphate nanoparticles by HeLa cells showed the co-localization of fluorescent avidin and fluorescent biotin, indicating the stability of the complex under cell culture conditions. CD11c-antibody functionalized nanoparticles specifically targeted antigen-presenting immune cells (dendritic cells; DCs) in vitro and in vivo (mice) with high efficiency. Statement of significance Calcium phosphate nanoparticles have turned out to be very useful transporters for biomolecules into cells, both in vitro and in vivo. However, their covalent surface functionalization with antibodies, fluorescent dyes, or proteins requires a separate chemical synthesis for each kind of surface molecule. We have therefore developed avidin-terminated calcium phosphate nanoparticles to which all kinds of biotinylated molecules can be easily attached, also as a mixture of two or more molecules. This non-covalent bond is stable both in cell culture and after injection into mice in vivo. Thus, we have created a highly versatile system for many applications, from the delivery of biomolecules over the targeting of cells and tissue to in vivo imaging. © 2017 Acta Materialia Inc.
    view abstract10.1016/j.actbio.2017.05.049
  • Basis material decomposition in spectral CT using a semi-empirical, polychromatic adaption of the Beer-Lambert model
    Ehn, S. and Sellerer, T. and Mechlem, K. and Fehringer, A. and Epple, M. and Herzen, J. and Pfeiffer, F. and Noël, P.B.
    Physics in Medicine and Biology 62 (2017)
    Following the development of energy-sensitive photon-counting detectors using high-Z sensor materials, application of spectral x-ray imaging methods to clinical practice comes into reach. However, these detectors require extensive calibration efforts in order to perform spectral imaging tasks like basis material decomposition. In this paper, we report a novel approach to basis material decomposition that utilizes a semi-empirical estimator for the number of photons registered in distinct energy bins in the presence of beam-hardening effects which can be termed as a polychromatic Beer-Lambert model. A maximum-likelihood estimator is applied to the model in order to obtain estimates of the underlying sample composition. Using a Monte-Carlo simulation of a typical clinical CT acquisition, the performance of the proposed estimator was evaluated. The estimator is shown to be unbiased and efficient according to the Cramér-Rao lower bound. In particular, the estimator is capable of operating with a minimum number of calibration measurements. Good results were obtained after calibration using less than 10 samples of known composition in a two-material attenuation basis. This opens up the possibility for fast re-calibration in the clinical routine which is considered an advantage of the proposed method over other implementations reported in the literature. © 2016 Institute of Physics and Engineering in Medicine.
    view abstract10.1088/1361-6560/aa4e5c
  • Chemical and structural analysis of gallstones from the Indian subcontinent
    Ramana Ramya, J. and Thanigai Arul, K. and Epple, M. and Giebel, U. and Guendel-Graber, J. and Jayanthi, V. and Sharma, M. and Rela, M. and Narayana Kalkura, S.
    Materials Science and Engineering C 78 (2017)
    Representative gallstones from north and southern parts of India were analyzed by a combination of physicochemical methods: X-ray diffraction (XRD), infrared spectroscopy (IR), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), CHNS analysis, thermal analysis and Nuclear Magnetic Resonance (NMR) spectroscopy (1H and 13C). The stones from north Indian were predominantly consisting of cholesterol monohydrate and anhydrous cholesterol which was confirmed by XRD analysis. FTIR spectroscopy confirmed the presence of cholesterol and calcium bilirubinate in the south Indian gallstones. EDX spectroscopy revealed the presence of carbon, nitrogen, oxygen, calcium, sulfur, sodium and magnesium and chloride in both south Indian and north Indian gallstones. FTIR and NMR spectroscopy confirmed the occurrence of cholesterol in north Indian gallstones. The respective colour of the north Indian and south Indian gallstones was yellowish and black. The morphology of the constituent crystals of the north Indian and south Indian gallstones were platy and globular respectively. The appreciable variation in colour, morphology and composition of south and north Indian gallstones may be due to different food habit and habitat. © 2017 Elsevier B.V.
    view abstract10.1016/j.msec.2017.04.004
  • Colloidal Stability and Surface Chemistry Are Key Factors for the Composition of the Protein Corona of Inorganic Gold Nanoparticles
    Johnston, B.D. and Kreyling, W.G. and Pfeiffer, C. and Schäffler, M. and Sarioglu, H. and Ristig, S. and Hirn, S. and Haberl, N. and Thalhammer, S. and Hauck, S.M. and Semmler-Behnke, M. and Epple, M. and Hühn, J. and Del Pino, P. and Parak, W.J.
    Advanced Functional Materials 27 (2017)
    To study the influence of colloidal stability on protein corona formation, gold nanoparticles are synthesized with five distinct surface modifications: coating with citric acid, bis(p-sulfonatophenyl)phenylphosphine dihydrate dipotassium salt, thiol-terminated methoxy-polyethylene glycol, dodecylamine-grafted poly(isobutylene-alt-maleic anhydride), and dodecylamine-grafted poly(isobutylene-alt-maleic anhydride) conjugated with polyethylene glycol. The nanoparticles are incubated with serum or bronchoalveolar lavage fluid from C57BL/6 mice (15 min or 24 h) to assess the effect of differential nanoparticle surface presentation on protein corona formation in the air–blood barrier exposure pathway. Proteomic quantification and nanoparticle size measurements are used to assess protein corona formation. We show that surface modification has a clear effect on the size and the composition of the protein corona that is related to the colloidal stability of the studied nanoparticles. Additionally, differences in the composition and size of the protein corona are shown between biological media and duration of exposure, indicating evolution of the corona through this exposure pathway. Consequently, a major determinant of protein corona formation is the colloidal stability of nanoparticles in biological media and chemical or environmental modification of the nanoparticles alters the surface presentation of the functional epitope in vivo. Therefore, the colloidal stability of nanoparticles has a decisive influence on nano–bio interactions. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/adfm.201701956
  • Comparative evaluation of the sand blasting, acid etching and electron beam surface treatments of titanium for medical application
    Grubova, I. and Chudinova, E. and Surmeneva, M. and Surmenev, R. and Ivanova, A. and Kravchuk, K. and Shugurov, V. and Teresov, A. and Koval, N. and Prymak, O. and Epple, M.
    Proceedings of the 11th International Forum on Strategic Technology, IFOST 2016 (2017)
    Modification of the surface topography and chemistry are commonly used to achieve the desired biological response to the implants. The influence of the different treatment methods on the physicochemical and mechanical properties of titanium is reported. All samples were divided into 2 groups. First group was sandblasted with 250-320 μm Al2O3 at two pressures 0.45 MPa and 0.61 MPa followed by the chemical etching in a fluorine-containing solution. The second group was acid-etched in the same solution followed by electron beam modification with the energy density 8 J/cm2. The samples were investigated by SEM, EDX, XRD, nanoindentation and sessile drop method. The studies revealed that all groups have nano/micro-patterned surfaces. The EDX analysis detected only titanium in all groups. The XRD results revealed the presence of diffraction peaks corresponding to titanium. The nanoindentation studies revealed significant differences in the mechanical properties between group 1 and 2. The elastic strain to failure and plastic deformation resistance of the group 2 were determined to be 0.035 and 5∗10-3, respectively, which were significantly higher than those of group 1. The obtained results of water contact angle for group 1 revealed moderately hydrophilic properties of treated surfaces. The water contact angle was increased up to 80.85 ± 8.3 ° for group 2. © 2016 IEEE.
    view abstract10.1109/IFOST.2016.7884191
  • Delivery of the autofluorescent protein Rphycoerythrin by calcium phosphate nanoparticles into four different eukaryotic cell lines (HeLa, HEK293T, MG-63, MC3T3): Highly efficient, but leading to endolysosomal proteolysis in HeLa and MC3T3 cells
    Kopp, M. and Rotan, O. and Papadopoulos, C. and Schulze, N. and Meyer, H. and Epple, M.
    PLoS ONE 12 (2017)
    Nanoparticles can be used as carriers to transport biomolecules like proteins and synthetic molecules across the cell membrane because many molecules are not able to cross the cell membrane on their own. The uptake of nanoparticles together with their cargo typically occurs via endocytosis, raising concerns about the possible degradation of the cargo in the endolysosomal system. As the tracking of a dye-labelled protein during cellular uptake and processing is not indicative of the presence of the protein itself but only for the fluorescent label, a label-free tracking was performed with the red-fluorescing model protein R-phycoerythrin (R-PE). Four different eukaryotic cell lines were investigated: HeLa, HEK293T, MG-63, and MC3T3. Alone, the protein was not taken up by any cell line; only with the help of calcium phosphate nanoparticles, an efficient uptake occurred. After the uptake into HeLa cells, the protein was found in early endosomes (shown by the marker EEA1) and lysosomes (shown by the marker Lamp1). There, it was still intact and functional (i.e. properly folded) as its red fluorescence was detected. However, a few hours after the uptake, proteolysis started as indicated by the decreasing red fluorescence intensity in the case of HeLa and MC3T3 cells. 12 h after the uptake, the protein was almost completely degraded in HeLa cells and MC3T3 cells. In HEK293T cells and MG-63 cells, no degradation of the protein was observed. In the presence of Bafilomycin A1, an inhibitor of acidification and protein degradation in lysosomes, the fluorescence of R-PE remained intact over the whole observation period in the four cell lines. These results indicate that despite an efficient nanoparticle-mediated uptake of proteins by cells, a rapid endolysosomal degradation may prevent the desired (e.g. therapeutic) effect of a protein inside a cell. © 2017 Kopp et al.This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
    view abstract10.1371/journal.pone.0178260
  • Delivery of the TLR ligand poly(I:C) to liver cells in vitro and in vivo by calcium phosphate nanoparticles leads to a pronounced immunostimulation
    Sokolova, V. and Shi, Z. and Huang, S. and Du, Y. and Kopp, M. and Frede, A. and Knuschke, T. and Buer, J. and Yang, D. and Wu, J. and Westendorf, A.M. and Epple, M.
    Acta Biomaterialia 64 (2017)
    The selective activation of the immune system is a concurrent problem in the treatment of persistent diseases like viral infections (e.g. hepatitis). For the delivery of the toll-like receptor ligand poly(I:C), an immunostimulatory action was discovered earlier by hydrodynamic injection. However, this technique is not clinically transferable to human patients. A modular system where the immunoactive toll-like-receptor ligand 3 (TLR-3) poly(I:C) was incorporated into calcium phosphate nanoparticles was developed. The nanoparticles had a hydrodynamic diameter of 275 nm and a zeta potential of +20 mV, measured by dynamic light scattering. The diameter of the solid core was 120 nm by scanning electron microscopy. In vitro, the nanoparticle uptake was investigated after 1 and 24 h of incubation of THP-1 cells (macrophages) with nanoparticles by fluorescence microscopy. After intravenous injection into BALB/c and C57BL/6J mice, respectively, the in vivo uptake was especially prominent in lung and liver, 1 and 3 h after the injection. Pronounced immunostimulatory effects of the nanoparticles were found in vitro with primary liver cells, i.e. Kupffer cells (KC) and liver sinusoidal endothelial cells (LSEC) from wild-type C57BL/6J mice. Thus, they represent a suitable alternative to hydrodynamic injection treatments for future vaccination concepts. Statement of Significance The selective activation of the immune system is a concurrent problem in the treatment of persistent diseases like viral infections (e.g. hepatitis). For the delivery of the toll-like receptor ligand poly(I:C), an immunostimulatory action has been discovered earlier by hydrodynamic injection. However, this technique is not clinically transferable to human patients. We have developed a modular system where poly(I:C) was incorporated into calcium phosphate nanoparticles. The uptake into relevant liver cells was studied both in vitro and in vivo. After intravenous injection into mice, the in vivo uptake was especially prominent in lung and liver, 1 and 3 h after the injection. The corresponding strong immune reaction proves their high potential to turn up the immune system, e.g. against viral infections, without adverse side reactions. © 2017 Acta Materialia Inc.
    view abstract10.1016/j.actbio.2017.09.037
  • Do we need retarded delivery of bone growth factors in facial bone repair? An experimental study in rats
    Moser, N. and Lohse, N. and Goldstein, J. and Kauffmann, P. and Sven, B. and Epple, M. and Schliephake, H.
    European Cells and Materials 34 (2017)
    The aim of the present study was to evaluate the effect of different dosages of retarded vs. rapid release of bone morphogenic protein 2 (BMP2) at different recipient sites. Porous composite poly(D,L-lactic acid) (PDLLA)/CaCO3 scaffolds were loaded with three different dosages of rhBMP2 (24 µg, 48 µg and 96 µg) and implanted, together with blank controls, both into non-healing defects of the mandibles and into the gluteal muscles of 24 adult male Wistar rats. After 26 weeks, bone formation and expression of bone specific markers [alkaline phosphatase (AP) and Runx2] were evaluated by histomorphometry and immunohistochemistry. Results showed that the mode of delivery had no quantitative effect on bone formation in mandibular sites. Expression of AP and Runx2 showed significant differences among the three dosage groups. There were significant correlations between the expression of both AP and Runx2, as well as, the extent of bone formation, with both retarded and rapid release of rhBMP2. In ectopic sites, retarded release significantly enhanced bone formation in the low and medium dosage groups, compared to rapid release. Expression of AP was significantly higher and Runx2 significantly lower in ectopic sites, compared to mandibular sites. Significant correlations between the expression of bone specific markers and bone formation occurred only in the retarded delivery groups, but not in the rapid release groups. Within the limitations of the experimental model, it was concluded that retarded delivery of BMP2 was effective, preferably in sites with low or nonexisting pristine osteogenic activity. Expression of bone specific markers indicated that osteogenic pathways might be different in mandibular vs. ectopic sites. © 2017, AO Research Institute. All rights reserved.
    view abstract10.22203/eCM.v034a11
  • Experimental variation of the level and the ratio of angiogenic and osteogenic signaling affects the spatiotemporal expression of bone-specific markers and organization of bone formation in ectopic sites
    Moser, N. and Goldstein, J. and Kauffmann, P. and Epple, M. and Schliephake, H.
    Clinical Oral Investigations (2017)
    Objectives: The aim of the present study was to test the hypothesis that the ratio of angiogenic and osteogenic signaling affects ectopic bone formation when delivered in different amounts. Materials and methods: Porous composite PDLLA/CaCO3 scaffolds were loaded with rhBMP2 and rhVEGF in different dosage combinations and implanted into the gluteal muscles of 120 adult male Wistar rats. Bone formation and expression of alkaline phosphatase and Runx2 were quantified by histomorphometry. Spatial distribution across the scaffolds was assessed by using a grid that discriminated between the periphery and center of the scaffolds. Results: The evaluation showed that the combined delivery of bone morphogenetic protein BMP2 and VEGF in different dosage combinations did not enhance the overall quantity of ectopic bone formation compared to the delivery of BMP2 alone. The addition of VEGF generally upregulated Runx2 after 4 weeks, which may have retarded terminal osteogenic differentiation. However, slow combined delivery of 1.5–2.0 μg BMP2 combined with 50 ng VEGF165 over a period of 5 weeks supported a more even distribution of bone formation across the implanted scaffolds whereas higher amounts of VEGF did not elicit this effect. Conclusions: The findings suggest that structural organization rather than the quantity of ectopic bone formation is affected by the dosage and the ratio of BMP2 and VEGF levels at the observed intervals. Clinical relevance: The development of carriers for dual growth factor delivery has to take into account the necessity to carefully balance the ratio of growth release. © 2017 Springer-Verlag GmbH Germany
    view abstract10.1007/s00784-017-2202-3
  • How Size Determines the Value of Gold: Economic Aspects of Wet Chemical and Laser-Based Metal Colloid Synthesis
    Jendrzej, S. and Gökce, B. and Epple, M. and Barcikowski, S.
    ChemPhysChem 18 (2017)
    Gold is one of the most valuable materials, and its monetary value is enhanced by size reduction from bullions to colloidal nanoparticles by a factor of 450. Wet-chemical reduction with subsequent centrifugation and pulsed laser ablation in liquids are frequently used for pure colloidal gold synthesis. Both methods provide similar physicochemical nanoparticle properties, but are very different synthesis techniques. However, the costs inherent to these methods are surprisingly seldom discussed. Both methods have in common that the labor effort poses the majority of synthesis costs. Besides an increase in batch size and mass concentration, especially an increase of the nanoparticle productivity via higher laser power and centrifugation capacity reduces synthesis costs if pilot- or industrial-scale applications are intended. In this case, laser-based synthesis is more economical if its productivity exceeds a break-even value of 550mgh-1, where the costs arising are limited by the metal costs. In contrast to industrial scale production, wet-chemical synthesis is more feasible for laboratory-scale applications, especially if the advantageous nanoparticle properties provided by laser ablation in liquids are not needed. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/cphc.201601139
  • Hybrid biocomposites based on titania nanotubes and a hydroxyapatite coating deposited by RF-magnetron sputtering: Surface topography, structure, and mechanical properties
    Chernozem, R.V. and Surmeneva, M.A. and Krause, B. and Baumbach, T. and Ignatov, V.P. and Tyurin, A.I. and Loza, K. and Epple, M. and Surmenev, R.A.
    Applied Surface Science 426 (2017)
    In this study, biocomposites based on porous titanium oxide structures and a calcium phosphate (CaP) or hydroxyapatite (HA) coating are described and prepared. Nanotubes (NTs) with different pore dimensions were processed using anodic oxidation of Ti substrates in a NH4F-containing electrolyte solution at anodization voltages of 30 and 60 V with a DC power supply. The external diameters of the nanotubes prepared at 30 V and 60 V were 53 ± 10 and 98 ± 16 nm, respectively. RF-magnetron sputtering of the HA target in a single deposition run was performed to prepare a coating on the surface of TiO2 NTs prepared at 30 and 60 V. The thickness of the CaP coating deposited on the mirror-polished Si substrate in the same deposition run with TiO2 NTs was determined by optical ellipsometry (SE) 95 ± 5 nm. Uncoated and CaP-coated NTs were annealed at 500 °C in air. Afterwards, the presence of TiO2 (anatase) was observed. The scanning electron microscopy (SEM), X-ray diffraction (XRD), photoelectron spectroscopy (XPS) and nanoindentation results revealed the influence that the NT dimensions had on the CaP coating deposition process. The tubular surfaces of the NTs were completely coated with the HA coating when prepared at 30 V, and no homogeneous CaP coating was observed when prepared at 60 V. The XRD patterns show peaks assigned to crystalline HA only for the coated TiO2 NTs prepared at 30 V. High-resolution XPS spectra show binding energies (BE) of Ca 2p, P 2p and O 1s core-levels corresponding to HA and amorphous calcium phosphate on TiO2 NTs prepared at 30 V and 60 V, respectively. Fabrication of TiO2 NTs results in a significant decrease to the elastic modulus and nanohardness compared to the Ti substrate. The porous structure of the NTs causes an increase in the elastic strain to failure of the coating (H/E) and the parameter used to describe the resistance of the material to plastic deformation (H3/E2) at the nanoscale level compared to the Ti substrate. Furthermore, only the HA coating on the NTs exhibits a significantly increased H/E ratio and H3/E2 factor compared to the NTs and Ti substrate. Increases in resistance to penetration for the indenter were also observed for HA-coated TiO2 NTs prepared at 30 V compared to uncoated and CaP-coated NTs prepared at 60 V. © 2017 Elsevier B.V.
    view abstract10.1016/j.apsusc.2017.07.199
  • Incorporation of silver nanoparticles into magnetron-sputtered calcium phosphate layers on titanium as an antibacterial coating
    Surmeneva, M.A. and Sharonova, A.A. and Chernousova, S. and Prymak, O. and Loza, K. and Tkachev, M.S. and Shulepov, I.A. and Epple, M. and Surmenev, R.A.
    Colloids and Surfaces B: Biointerfaces 156 (2017)
    A three-layer system of nanocrystalline hydroxyapatite (first layer; 1000 nm thick), silver nanoparticles (second layer; 1.5 μg Ag cm−2) and calcium phosphate (third layer, either 150 or 1000 nm thick) on titanium was prepared by a combination of electrophoretic deposition of silver nanoparticles and the deposition of calcium phosphate by radio frequency magnetron sputtering. Scanning electron microscopy showed that the silver nanoparticles were evenly distributed over the surface. The adhesion of multilayered coating on the substrate was evaluated using the scratch test method. The resistance to cracking and delamination indicated that the multilayered coating has good resistance to contact damage. The release of silver ions from the hydroxyapatite/silver nanoparticle/calcium phosphate system into the phosphate-buffered saline (PBS) solution was measured by atomic absorption spectroscopy (AAS). Approximately one-third of the incorporated silver was released after 3 days immersion into PBS, indicating a total release time of the order of weeks. There were no signs of cracks on the surface of the coating after immersion after various periods, indicating the excellent mechanical stability of the multilayered coating in the physiological environment. An antimicrobial effect against Escherichia coli was found for a 150 nm thick outer layer of the calcium phosphate using a semi-quantitative turbidity test. © 2017 Elsevier B.V.
    view abstract10.1016/j.colsurfb.2017.05.016
  • Intestinal helminth infection drives carcinogenesis in colitis-associated colon cancer
    Pastille, E. and Frede, A. and McSorley, H.J. and Gräb, J. and Adamczyk, A. and Kollenda, S. and Hansen, W. and Epple, M. and Buer, J. and Maizels, R.M. and Klopfleisch, R. and Westendorf, A.M.
    PLoS Pathogens 13 (2017)
    Inflammatory bowel diseases (IBD) are chronic inflammatory disorders of the gastrointestinal tract, strongly associated with an increased risk of colorectal cancer development. Parasitic infections caused by helminths have been shown to modulate the host’s immune response by releasing immunomodulatory molecules and inducing regulatory T cells (Tregs). This immunosuppressive state provoked in the host has been considered as a novel and promising approach to treat IBD patients and alleviate acute intestinal inflammation. On the contrary, specific parasite infections are well known to be directly linked to carcinogenesis. Whether a helminth infection interferes with the development of colitis-associated colon cancer (CAC) is not yet known. In the present study, we demonstrate that the treatment of mice with the intestinal helminth Heligmosomoides polygyrus at the onset of tumor progression in a mouse model of CAC does not alter tumor growth and distribution. In contrast, H. polygyrus infection in the early inflammatory phase of CAC strengthens the inflammatory response and significantly boosts tumor development. Here, H. polygyrus infection was accompanied by long-lasting alterations in the colonic immune cell compartment, with reduced frequencies of colonic CD8+effector T cells. Moreover, H. polygyrus infection in the course of dextran sulfate sodium (DSS) mediated colitis significantly exacerbates intestinal inflammation by amplifying the release of colonic IL-6 and CXCL1. Thus, our findings indicate that the therapeutic application of helminths during CAC might have tumor-promoting effects and therefore should be well-considered. © 2017 Pastille et al.
    view abstract10.1371/journal.ppat.1006649
  • Live-cell imaging to compare the transfection and gene silencing efficiency of calcium phosphate nanoparticles and a liposomal transfection agent
    Chernousova, S. and Epple, M.
    Gene Therapy 24 (2017)
    The processing of DNA (for transfection) and short interfering RNA (siRNA; for gene silencing), introduced into HeLa cells by triple-shell calcium phosphate nanoparticles, was followed by live-cell imaging. For comparison, the commercial liposomal transfection agent Lipofectamine was used. The cells were incubated with these delivery systems, carrying either enhanced green fluorescent protein (eGFP)-encoding DNA or siRNA against eGFP. In the latter case, HeLa cells that stably expressed eGFP were used. The expression of eGFP started after 5 h in the case of nanoparticles and after 4 h in the case of Lipofectamine. The corresponding times for gene silencing were 5 h (nanoparticles) and immediately after incubation (Lipofectamine). The expression of eGFP was notably enhanced 2–3 h after cell division (mitosis). In general, the transfection and gene silencing efficiencies of the nanoparticles were lower than those of Lipofectamime, even at a substantially higher dose (factor 20) of nucleic acids. However, the cytotoxicity of the nanoparticles was lower than that of Lipofectamine, making them suitable vectors for in vivo application.Gene Therapy advance online publication, 9 March 2017; doi:10.1038/gt.2017.13. © 2017 The Author(s)
    view abstract10.1038/gt.2017.13
  • Local delivery of siRNA-loaded calcium phosphate nanoparticles abates pulmonary inflammation
    Frede, A. and Neuhaus, B. and Knuschke, T. and Wadwa, M. and Kollenda, S. and Klopfleisch, R. and Hansen, W. and Buer, J. and Bruder, D. and Epple, M. and Westendorf, A.M.
    Nanomedicine: Nanotechnology, Biology, and Medicine 13 (2017)
    The local interference of cytokine signaling mediated by siRNA-loaded nanoparticles might be a promising new therapeutic approach to dampen inflammation during pulmonary diseases. For the local therapeutic treatment of pulmonary inflammation, we produced multi-shell nanoparticles consisting of a calcium phosphate core, coated with siRNAs directed against pro-inflammatory mediators, encapsulated into poly(lactic-co-glycolic acid), and coated with a final outer layer of polyethylenimine. Nasal instillation of nanoparticles loaded with a mixture of siRNAs directed against different cytokines to mice suffering from TH1 cell-mediated lung inflammation, or of siRNA directed against NS-1 in an influenza infection model led to a significant reduction of target gene expression which was accompanied by distinct amelioration of lung inflammation in both models. Thus, this study provides strong evidence that the specific and local modulation of the inflammatory response by CaP/PLGA nanoparticle-mediated siRNA delivery could be a promising approach for the treatment of inflammatory disorders of the lung. © 2017 Elsevier Inc.
    view abstract10.1016/j.nano.2017.08.001
  • Mechanical induction of bi-directional orientation of primary porcine bladder smooth muscle cells in tubular fibrin-poly(vinylidene fluoride) scaffolds for ureteral and urethral repair using cyclic and focal balloon catheter stimulation
    Seifarth, V. and Grosse, J.O. and Gossmann, M. and Janke, H.P. and Arndt, P. and Koch, S. and Epple, M. and Artmann, G.M. and Artmann, A.T.
    Journal of Biomaterials Applications 32 (2017)
    To restore damaged organ function or to investigate organ mechanisms, it is necessary to prepare replicates that follow the biological role model as faithfully as possible. The interdisciplinary field of tissue engineering has great potential in regenerative medicine and might overcome negative side effects in the replacement of damaged organs. In particular, tubular organ structures of the genitourinary tract, such as the ureter and urethra, are challenging because of their complexity and special milieu that gives rise to incrustation, inflammation and stricture formation. Tubular biohybrids were prepared from primary porcine smooth muscle cells embedded in a fibrin gel with a stabilising poly(vinylidene fluoride) mesh. A mechanotransduction was performed automatically with a balloon kyphoplasty catheter. Diffusion of urea and creatinine, as well as the bursting pressure, were measured. Light and electron microscopy were used to visualise cellular distribution and orientation. Histological evaluation revealed a uniform cellular distribution in the fibrin gel. Mechanical stimulation with a stretch of 20% leads to a circumferential orientation of smooth muscle cells inside the matrix and a longitudinal alignment on the outer surface of the tubular structure. Urea and creatinine permeability and bursting pressure showed a non-statistically significant trend towards stimulated tissue constructs. In this proof of concept study, an innovative technique of intraluminal pressure for mechanical stimulation of tubular biohybrids prepared from autologous cells and a composite material induce bi-directional orientation of smooth muscle cells by locally and cyclically applied mechanical tension. Such geometrically driven patterns of cell growth within a scaffold may represent a key stage in the future tissue engineering of implantable ureter replacements that will allow the active transportation of urine from the renal pelvis into the bladder. © The Author(s) 2017.
    view abstract10.1177/0885328217723178
  • Mineral in skeletal elements of the terrestrial crustacean Porcellio scaber: SRμCT of function related distribution and changes during the moult cycle
    Ziegler, A. and Neues, F. and Janáček, J. and Beckmann, F. and Epple, M.
    Arthropod Structure and Development 46 (2017)
    Terrestrial isopods moult first the posterior and then the anterior half of the body, allowing for storage and recycling of CaCO3. We used synchrotron-radiation microtomography to estimate mineral content within skeletal segments in sequential moulting stages of Porcellio scaber. The results suggest that all examined cuticular segments contribute to storage and recycling, however, to varying extents. The mineral within the hepatopancreas after moult suggests an uptake of mineral from the ingested exuviae. The total maximum loss of mineral was 46% for the anterior and 43% for the posterior cuticle. The time course of resorption of mineral and mineralisation of the new cuticle suggests storage and recycling of mineral in the posterior and anterior cuticle. The mineral in the anterior pereiopods decreases by 25% only. P. scaber has long legs and can run fast; therefore, a less mineralised and thus lightweight cuticle in pereiopods likely serves to lower energy consumption during escape behaviour. Differential demineralisation occurs in the head cuticle, in which the cornea of the complex eyes remains completely mineralised. The partes incisivae of the mandibles are mineralised before the old cuticle is demineralised and shed. Probably, this enables the animal to ingest the old exuviae after each half moult. © 2016 Elsevier Ltd.
    view abstract10.1016/j.asd.2016.05.004
  • Nanoparticle-based B-cell targeting vaccines: Tailoring of humoral immune responses by functionalization with different TLR-ligands
    Zilker, C. and Kozlova, D. and Sokolova, V. and Yan, H. and Epple, M. and Überla, K. and Temchura, V.
    Nanomedicine: Nanotechnology, Biology, and Medicine 13 (2017)
    Induction of an appropriate type of humoral immune response during vaccination is essential for protection against viral and bacterial infections. We recently observed that biodegradable calcium phosphate (CaP) nanoparticles coated with proteins efficiently targeted and activated naïve antigen-specific B-cells in vitro. We now compared different administration routes for CaP-nanoparticles and demonstrated that intramuscular immunization with such CaP-nanoparticles induced stronger immune responses than immunization with monovalent antigen. Additional functionalization of the CaP-nanoparticles with TRL-ligands allowed modulating the IgG subtype response and the level of mucosal IgA antibodies. CpG-containing CaP-nanoparticles were as immunogenic as a virus-like particle vaccine. Functionalization of CaP-nanoparticles with T-helper cell epitopes or CpG also allowed overcoming lack of T-cell help. Thus, our results indicate that CaP-nanoparticle-based B-cell targeting vaccines functionalized with TLR-ligands can serve as a versatile platform for efficient induction and modulation of humoral immune responses in vivo. © 2016 Elsevier Inc.
    view abstract10.1016/j.nano.2016.08.028
  • Nanoparticle-Protein Interactions: Therapeutic Approaches and Supramolecular Chemistry
    Kopp, M. and Kollenda, S. and Epple, M.
    Accounts of Chemical Research 50 (2017)
    ConspectusResearch on nanoparticles has evolved into a major topic in chemistry. Concerning biomedical research, nanoparticles have decisively entered the field, creating the area of nanomedicine where nanoparticles are used for drug delivery, imaging, and tumor targeting. Besides these functions, scientists have addressed the specific ways in which nanoparticles interact with biomolecules, with proteins being the most prominent example. Depending on their size, shape, charge, and surface functionality, specifically designed nanoparticles can interact with proteins in a defined way. Proteins have typical dimensions of 5-20 nm. Ultrasmall nanoparticles (size about 1-2 nm) can address specific epitopes on the surface of a protein, for example, an active center of an enzyme. Medium-sized nanoparticles (size about 5 nm) can interact with proteins on a 1:1 basis. Large nanoparticles (above 20 nm) are big in comparison to many proteins and therefore are at the borderline to a two-dimensional surface onto which a protein will adsorb. This can still lead to irreversible structural changes in a protein and a subsequent loss of function.However, as most cells readily take up nanoparticles of almost any size, it is easily possible to use nanoparticles as transporters for proteins into a cell, for example, to address an internal receptor. Much work has been dedicated to this approach, but it is constrained by two processes that can only be observed in living cells or organisms. First, nanoparticles are usually taken up by endocytosis and are delivered into an intracellular endosome. After fusion with a lysosome, a degradation or denaturation of the protein cargo by the acidic environment or by proteases may occur before it can enter the cytoplasm. Second, nanoparticles are rapidly coated with proteins upon contact with biological media like blood. This so-called protein corona influences the contact with other proteins, cells, or tissue and may prevent the desired interaction. Essentially, these effects cannot be understood in purely chemical approaches but require biological environments and systems because the underlying processes are simply too complicated to be modeled in nonbiological systems.The area of nanoparticle-protein interactions strongly relies on different approaches: Synthetic chemistry is involved to prepare, stabilize, and functionalize nanoparticles. High-end analytical chemistry is required to understand the nature of a nanoparticle surface and the steps of its interaction with proteins. Concepts from supramolecular chemistry help to understand the complex noncovalent interactions between the surfaces of proteins and nanoparticles. Protein chemistry and biophysical chemistry are required to understand the behavior of a protein in contact with a nanoparticle. Finally, all chemical concepts must live up to the "biological reality", first in cell culture experiments in vitro and finally in animal or human experiments in vivo, to open new therapies in the 21st century. This interdisciplinary approach makes the field highly exciting but also highly demanding for chemists who, however, have to learn to understand the language of other areas. © 2017 American Chemical Society.
    view abstract10.1021/acs.accounts.7b00051
  • Nanoparticulate versus ionic silver: Behavior in the tank water, bioaccumulation, elimination and subcellular distribution in the freshwater mussel Dreissena polymorpha
    Zimmermann, S. and Ruchter, N. and Loza, K. and Epple, M. and Sures, B.
    Environmental Pollution 222 (2017)
    Zebra mussels (Dreissena polymorpha) were exposed to polyvinylpyrrolidone (PVP)-coated silver nanoparticles (AgNP; hydrodynamic diameter 80 nm; solid diameter 50 nm) to investigate the behavior of Ag in the tank water with respect to its uptake, bioaccumulation, elimination and subcellular distribution in the mussel soft tissue. Parallel experiments were performed with ionic Ag (AgNO3) to unravel possible differences between the metal forms. The recovery of the applied Ag concentration (500 μg/L) in the tank water was clearly affected by the metal source (AgNP < AgNO3) and water type (reconstituted water < tap water). Filtration (< 0.45 μm) of water samples showed different effects on the quantified metal concentration depending on the water type and Ag form. Ag accumulation in the mussel soft tissue was neither influenced by the metal source nor by the water type. Ag concentrations in the mussel soft tissue did not decrease during 14 days of depuration. For both metal forms the Ag distribution within different subcellular fractions, i.e. metal-rich granules (MRG), cellular debris, organelles, heat-sensitive proteins (HSP) and metallothionein-like proteins (MTLP), revealed time-dependent changes which can be referred to intracellular Ag translocation processes. The results provide clear evidence for the uptake of Ag by the mussel soft tissue in nanoparticulate as well as in ionic form. Thus, zebra mussels could be used as effective accumulation indicators for environmental monitoring of both Ag forms. © 2016 Elsevier Ltd.
    view abstract10.1016/j.envpol.2016.12.048
  • Peculiarities in thermal evolution of precipitated amorphous calcium phosphates with an initial Ca/P ratio of 1:1
    Zyman, Z. and Epple, M. and Goncharenko, A. and Rokhmistrov, D. and Prymak, O. and Loza, K.
    Journal of Materials Science: Materials in Medicine 28 (2017)
    Thermal evolution of amorphous calcium phosphate (ACP) powder from a fast nitrate synthesis with a Ca/P ratio of 1:1 were studied in the range of 20–980 °C. The powder consisted of amorphous dicalcium phosphate anhydrate (CaHPO4) after heating to 200 °C. CaHPO4 gradually condensed to amorphous calcium pyrophosphate Ca2P2O7 (CPP) between 200 to 620 °C. Amorphous CPP crystallized at 620–740 °C to a metastable polymorph α′-CPP of the high-temperature phase α-CPP and β-CPP. The α′-CPP/ β-CPP phase ratio reached a maximum at 800 °C (60 wt% α′-CPP/40 wt% β-CPP), and α′-CPP gradually transformed to β-CPP at a higher temperature. Some β-TCP occurred at 900 °C, so that a three-phasic mixture was obtained in the powder heated to 980 °C. The occurrence of metastable α′-CPP is attributed to Ostwald’s step rule, and a mechanism for β-TCP formation is proposed. The advantages of prospective biomaterials from these powders are discussed. © 2017, Springer Science+Business Media New York.
    view abstract10.1007/s10856-016-5820-4
  • Reply to the 'Comments on "dental lessons from past to present: Ultrastructure and composition of teeth from plesiosaurs, dinosaurs, extinct and recent sharks"' by H. Botella: Et al., RSC Adv., 2016, 6, 74384-74388
    Luebke, A. and Loza, K. and Patnaik, R. and Enax, J. and Raabe, D. and Prymak, O. and Fabritius, H.-O. and Gaengler, P. and Epple, M.
    RSC Advances 7 (2017)
    The structure and composition of 13 fossilized tooth and bone samples aged between 3 and 70 million years were analysed. It was found that they all contained high amounts of fluoroapatite. This indicates that originally present hydroxyapatite had been converted to fluoroapatite during the diagenesis. Thus, the chemical analysis allows no conclusion with respect to the original composition of our fossil samples. Our results indicate that the diagenetic transformation of hydroxyapatite into fluoroapatite is at least partially dependent on microstructural characteristics of the original tissue such as the degree of porosity. © 2017 The Royal Society of Chemistry.
    view abstract10.1039/c6ra27121a
  • RF magnetron sputtering of a hydroxyapatite target: A comparison study on polytetrafluorethylene and titanium substrates
    Surmenev, R.A. and Surmeneva, M.A. and Grubova, I.Y. and Chernozem, R.V. and Krause, B. and Baumbach, T. and Loza, K. and Epple, M.
    Applied Surface Science 414 (2017)
    A pure hydroxyapatite (HA) target was used to prepare the biocompatible coating of HA on the surface of a polytetrafluorethylene (PTFE) substrate, which was placed on the same substrate holder with technically pure titanium (Ti) in the single deposition runs by radio-frequency (RF) magnetron sputtering. The XPS, XRD and FTIR analyses of the obtained surfaces showed that for all substrates, instead of the HA coating deposition, the coating of a mixture of calcium carbonate and calcium fluoride was grown. According to SEM investigations, the surface of PTFE was etched, and the surface topography of uncoated Ti was preserved after the depositions. The FTIR results reveal no phosphate bonds; only calcium tracks were observed in the EDX-spectra on the surface of the coated PTFE substrates. Phosphate oxide (V), which originated from the target, could be removed using a vacuum pump system, or no phosphate-containing bonds could be formed on the substrate surface because of the severe substrate bombardment process, which prevented the HA coating deposition. The observed results may be connected with the surface re-sputtering effect of the growing film by high-energy negatively charged ions (most probably oxygen or fluorine), which are accelerated in the cathode dark sheath. © 2017 Elsevier B.V.
    view abstract10.1016/j.apsusc.2017.04.090
  • Sand-blasting treatment as a way to improve the adhesion strength of hydroxyapatite coating on titanium implant
    Grubova, I. and Priamushko, T. and Surmeneva, M. and Korneva, O. and Epple, M. and Prymak, O. and Surmenev, R.
    Journal of Physics: Conference Series 830 (2017)
    In the current study, the effect of corundum particle sizes (50 and 250-320 μm) used for sand-blasting on the structure, roughness, wettability, mechanical properties, and adhesion of radio frequency magnetron hydroxyapatite coating deposited on treated titanium substrate is studied. Morphology analysis revealed that pretreatment uniformly deforms the surface and induces the formation of pits, which size depends linearly on the grit size. The deposited coatings (Ca/P was in a range of 1.75-1.79) are homogeneous and repeat the relief of the substrate (mean roughness Ra is 1.9±0.1 (250-320 μm) and 0.8±0.1 μm (50 μm)). Texture coefficient calculations revealed the predominant (002) growth texture of hydroxyapatite coatings. The resistance of the coating to plastic deformation and the surface hardening were significantly higher for coatings formed on sand blasted with particle size of 50 μm. Scratch test have shown the significant improvement of wear resistance and lower friction coefficient of coatings for smoother samples. Dynamic contact angle measurements revealed the hydrophilic properties of the hydroxyapatite coating. Thus, sand-blasting of titanium with corundum powder having the size of 50 μm prior to the deposition of RF magnetron coating is recommended for the medical applications intended to improve the bonding between the substrate and coating.
    view abstract10.1088/1742-6596/830/1/012109
  • Uptake of the proteins HTRA1 and HTRA2 by cells mediated by calcium phosphate nanoparticles
    Rotan, O. and Severin, K.N. and Pöpsel, S. and Peetsch, A. and Merdanovic, M. and Ehrmann, M. and Epple, M.
    Beilstein Journal of Nanotechnology 8 (2017)
    The efficient intracellular delivery of (bio)molecules into living cells remains a challenge in biomedicine. Many biomolecules and synthetic drugs are not able to cross the cell membrane, which is a problem if an intracellular mode of action is desired, for example, with a nuclear receptor. Calcium phosphate nanoparticles can serve as carriers for small and large biomolecules as well as for synthetic compounds. The nanoparticles were prepared and colloidally stabilized with either polyethyleneimine (PEI; cationic nanoparticles) or carboxymethyl cellulose (CMC; anionic nanoparticles) and loaded with defined amounts of the fluorescently labelled proteins HTRA1, HTRA2, and BSA. The nanoparticles were purified by ultracentrifugation and characterized by dynamic light scattering and scanning electron microscopy. Various cell types (HeLa, MG-63, THP-1, and hMSC) were incubated with fluorescently labelled proteins alone or with protein-loaded cationic and anionic nanoparticles. The cellular uptake was followed by light and fluorescence microscopy, confocal laser scanning microscopy (CLSM), and flow cytometry. All proteins were readily transported into the cells by cationic calcium phosphate nanoparticles. Notably, only HTRA1 was able to penetrate the cell membrane of MG-63 cells in dissolved form. However, the application of endocytosis inhibitors revealed that the uptake pathway was different for dissolved HTRA1 and HTRA1-loaded nanoparticles. © 2017 Rotan et al.
    view abstract10.3762/bjnano.8.40
  • Water-free synthesis of ZnO quantum dots for application as an electron injection layer in light-emitting electrochemical cells
    Daumann, S. and Andrzejewski, D. and Di Marcantonio, M. and Hagemann, U. and Wepfer, S. and Vollkommer, F. and Bacher, G. and Epple, M. and Nannen, E.
    Journal of Materials Chemistry C 5 (2017)
    Large-area light emitters like organic (OLEDs) or quantum dot light-emitting devices (QLEDs) and light-emitting electrochemical cells (LECs) have gained increasing interest due to their cost-effective fabrication on various even flexible substrates. The implementation of ZnO nanoparticles as an electron injection layer in large-area emitters leads to efficient solution-based devices. However, ZnO support layers are frequently in direct contact with water-sensitive emitter materials, which requires ZnO nanoparticles with minimum water content. A water-free synthesis route (except for the small amount of water formed during the synthesis) of ligand-free ZnO nanoparticles is presented. The spherical ZnO nanoparticles have a diameter of 3.4 nm, possess a high crystallinity, and form stable dispersions in ethanol or 1-hexanol. Their application together with a transition metal complex (iTMC)-LEC as an additional electron injection layer resulted in an increase of the device efficiency from 1.6 to 2.4 lm W−1 as well as the reduction of the run-up time to one fifth, compared to the same system without ZnO nanoparticles. © The Royal Society of Chemistry.
    view abstract10.1039/c6tc05571k
  • Barium sulfate micro- and nanoparticles as bioinert reference material in particle toxicology
    Loza, K. and Föhring, I. and Bünger, J. and Westphal, G.A. and Köller, M. and Epple, M. and Sengstock, C.
    Nanotoxicology 10 (2016)
    The inhalation of particles and their exposure to the bronchi and alveoli constitute a major public health risk. Chemical as well as particle-related properties are important factors for the biological response but are difficult to separate from each other. Barium sulfate is a completely inert chemical compound, therefore it is ideally suited to separate these two factors. The biological response of rat alveolar macrophages (NR8383) was analyzed after exposure to barium sulfate particles with three different diameters (40 nm, 270 nm, and 1.3 μm, respectively) for 24 h in vitro (particle concentrations from 12.5 to 200 μg mL− 1). The particles were colloidally stabilized as well as fluorescently-labeled by carboxymethylcellulose, conjugated with 6-aminofluorescein. All kinds of barium sulfate particles were efficiently taken up by NR8383 cells and found inside endo-lysosomes, but never in the cell nucleus. Neither an inflammatory nor a cytotoxic response was detected by the ability of dHL-60 and NR8383 cells to migrate towards a chemotactic gradient (conditioned media of NR8383 cells) and by the release of inflammatory mediators (CCL2, TNF-α, IL-6). The particles neither caused apoptosis (up to 200 μg mL− 1) nor necrosis (up to 100 μg mL− 1). As only adverse reaction, necrosis was found at a concentration of 200 μg mL− 1 of the largest barium sulfate particles (1.3 μm). Barium sulfate particles are ideally suited as bioinert control to study size-dependent effects such as uptake mechanisms of intracellular distributions of pure particles, especially in nanotoxicology. © 2016 Informa UK Limited, trading as Taylor & Francis Group.
    view abstract10.1080/17435390.2016.1235740
  • Calcium phosphates in biomedical applications: Materials for the future?
    Habraken, W. and Habibovic, P. and Epple, M. and Bohner, M.
    Materials Today 19 (2016)
    Our populations are aging. Some experts predict that 30% of hospital beds will soon be occupied by osteoporosis patients. Statistics show that 20% of patients suffering from an osteoporotic hip fracture do not survive the first year after surgery, all this showing that there is a tremendous need for better therapies for diseased and damaged bone. Human bone consists for about 70% of calcium phosphate (CaP) mineral, therefore CaPs are the materials of choice to repair damaged bone. To do this successfully, the process of CaP biomineralization and the interaction of CaPs and biological environment in the body need to be fully understood. First commercial CaP bone graft substitutes were launched 40 years ago, and they are currently often regarded as 'old biomaterials' or even as an 'obsolete' research topic. Some even talk about 'stones'. The aim of this manuscript is to highlight the tremendous improvements achieved in CaP materials research in the past 15 years, in particular in the field of biomineralization, as carrier for gene or ion delivery, as biologically active agent, and as bone graft substitute. Besides an outstanding biological performance, CaPs are easily and inexpensively produced, are safe, and can be relatively easily certified for clinical use. As such, CaP materials have won their spurs, but they also offer a great promise for the future. © 2015 The Authors.
    view abstract10.1016/j.mattod.2015.10.008
  • Colonic gene silencing using siRNA-loaded calcium phosphate/PLGA nanoparticles ameliorates intestinal inflammation in vivo
    Frede, A. and Neuhaus, B. and Klopfleisch, R. and Walker, C. and Buer, J. and Müller, W. and Epple, M. and Westendorf, A.M.
    Journal of Controlled Release 222 (2016)
    Cytokines and chemokines are predominant players in the progression of inflammatory bowel diseases. While systemic neutralization of these players with antibodies works well in some patients, serious contraindications and side effects have been reported. Therefore, the local interference of cytokine signaling mediated by siRNA-loaded nanoparticles might be a promising new therapeutic approach. In this study, we produced multi-shell nanoparticles consisting of a calcium phosphate (CaP) core coated with siRNA directed against pro-inflammatory mediators, encapsulated into poly(d,l-lactide-co-glycolide acid) (PLGA), and coated with a final outer layer of polyethyleneimine (PEI), for the local therapeutic treatment of colonic inflammation. In cell culture, siRNA-loaded CaP/PLGA nanoparticles exhibited a rapid cellular uptake, almost no toxicity, and an excellent in vitro gene silencing efficiency. Importantly, intrarectal application of these nanoparticles loaded with siRNA directed against TNF-α, KC or IP-10 to mice suffering from dextran sulfate sodium (DSS)-induced colonic inflammation led to a significant decrease of the target genes in colonic biopsies and mesenteric lymph nodes which was accompanied with a distinct amelioration of intestinal inflammation. Thus, this study provides evidence that the specific and local modulation of the inflammatory response by CaP/PLGA nanoparticle-mediated siRNA delivery could be a promising approach for the treatment of intestinal inflammation. © 2015 Elsevier B.V. All rights reserved.
    view abstract10.1016/j.jconrel.2015.12.021
  • Combination of nanoparticle-based therapeutic vaccination and transient ablation of regulatory T cells enhances anti-viral immunity during chronic retroviral infection
    Knuschke, T. and Rotan, O. and Bayer, W. and Sokolova, V. and Hansen, W. and Sparwasser, T. and Dittmer, U. and Epple, M. and Buer, J. and Westendorf, A.M.
    Retrovirology 13 (2016)
    Background: Regulatory T cells (Tregs) have been shown to limit anti-viral immunity during chronic retroviral infection and to restrict vaccine-induced T cell responses. The objective of the study was to assess whether a combinational therapy of nanoparticle-based therapeutic vaccination and concomitant transient ablation of Tregs augments anti-viral immunity and improves virus control in chronically retrovirus-infected mice. Therefore, chronically Friend retrovirus (FV)-infected mice were immunized with calcium phosphate (CaP) nanoparticles functionalized with TLR9 ligand CpG and CD8+ or CD4+ T cell epitope peptides (GagL85-93 or Env gp70123-141) of FV. In addition, Tregs were ablated during the immunization process. Reactivation of CD4+ and CD8+ effector T cells was analysed and the viral loads were determined. Results: Therapeutic vaccination of chronically FV-infected mice with functionalized CaP nanoparticles transiently reactivated cytotoxic CD8+ T cells and significantly reduced the viral loads. Transient ablation of Tregs during nanoparticle-based therapeutic vaccination strongly enhanced anti-viral immunity and further decreased viral burden. Conclusion: Our data illustrate a crucial role for CD4+ Foxp3+ Tregs in the suppression of anti-viral T cell responses during therapeutic vaccination against chronic retroviral infection. Thus, the combination of transient Treg ablation and therapeutic nanoparticle-based vaccination confers robust and sustained anti-viral immunity. © 2016 Knuschke et al.
    view abstract10.1186/s12977-016-0258-9
  • Comparison of different methods to study effects of silver nanoparticles on the pro- and antioxidant status of human keratinocytes and fibroblasts
    Ahlberg, S. and Rancan, F. and Epple, M. and Loza, K. and Höppe, D. and Lademann, J. and Vogt, A. and Kleuser, B. and Gerecke, C. and Meinke, M.C.
    Methods 109 (2016)
    In times of antibiotic-resistant bacteria new strategies to avoid the septic-inducing threat of dangerous microorganisms are needed. Silver ions (Ag+) in the forms of silver nitrate or silver sulfadiazine have been used as antimicrobial agents for years. A step further was the development of micro and silver particles (AgNP). In contrast to other Ag+ ion sources, AgNP allow a sustained release of Ag+ ions, due to their high surface to volume ratio. However, AgNP are also toxic to eukaryotic cells and the mechanisms of cytotoxicity have not yet been fully elucidated. In this study, the impact of different AgNP preparations on a human keratinocyte cell line was investigated. The intracellular radical formation was confirmed by the 2′,7′-dichlorodihydrofluorescein di-acetate (H2DCF-DA) assay on two cell types (HaCaT cells and normal human dermal fibroblasts) as well as by electron paramagnetic resonance (EPR) spectroscopy, which showed comparable results. EPR spectroscopy was performed for the first time for 24 h in experiments using keratinocytes. Drastic changes in the mitochondrial activity were induced in cells incubated with AgNP containing high concentrations of Ag+ ions. It was also possible to show that the quantitative uptake of AgNP was dependent on the AgNP concentration. In addition, the effects of AgNP on the GSH/GSSG system were elucidated. The results showed a batch- and concentration-dependent decrease of the total glutathione concentration which correlated well with the decrease of cell viability. Furthermore, the results suggest a direct reaction of GSH molecules with Ag+ ions. In conclusion, this study proves the efficacy of the H2DCF-DA assay and the EPR spectroscopy. The investigations show that AgNP formulations containing high amounts of released Ag+ ions induce radicals in human keratinocytes and deplete them of their natural anti-oxidative molecules. On the contrary, nanoparticles prepared and stored under argon did not induce significant adverse effects, suggesting that slowing down the release of Ag+ may help to reduce AgNP-related side effects without affecting the antibacterial impact. © 2016 Elsevier Inc.
    view abstract10.1016/j.ymeth.2016.05.015
  • Composites of fluoroapatite and methylmethacrylate-based polymers (PMMA) for biomimetic tooth replacement
    Lübke, A. and Enax, J. and Wey, K. and Fabritius, H.-O. and Raabe, D. and Epple, M.
    Bioinspiration and Biomimetics 11 (2016)
    Synthetic composite materials that mimic the structure and composition of mammalian tooth enamel were prepared by mixing fluoroapatite rods (diameter 2-3 μm, thickness about 0.5 μm) and methylmethacrylate (MMA), followed by polymerization either during or immediately after ultracentrifugation, using either a tertiary amine/radical initiator for polymerization at room temperature or a radical initiator for thermal polymerization. This led to mineral-rich composites (mineral content between 50 and 75 wt%). To enhance the mechanical stability and the interaction between fluoroapatite and polymer matrix, small amounts of differently functionalized MMA monomers were added to the co-monomer mixture. Another approach was the coating of the fluoroapatite rods with silica and the polymerization in the presence of a siloxane-functionalized MMA monomer. The hardness of the composites was about 0.2-0.4 GPa as determined by Vickers indentation tests, about 2 times higher than the polymer matrix alone. The composites had a good resistance against acids (60 min at pH 3, 37 °C). © 2016 IOP Publishing Ltd.
    view abstract10.1088/1748-3190/11/3/035001
  • Conjugation of thiol-terminated molecules to ultrasmall 2 nm-gold nanoparticles leads to remarkably complex 1H-NMR spectra
    Schuetze, B. and Mayer, C. and Loza, K. and Gocyla, M. and Heggen, M. and Epple, M.
    Journal of Materials Chemistry B 4 (2016)
    Gold nanoparticles, functionalized by aliphatic and aromatic mercapto-functionalized carboxylic acids and by two small peptides (CG and CGGRGD), respectively, were synthesized by the reduction of HAuCl4 with NaBH4 in the presence of the above ligands. After purification by centrifugation or filtration and redispersion, the dispersed nanoparticles were analysed by differential centrifugal sedimentation (DCS), high-resolution transmission electron microscopy (HRTEM), and a variety of NMR spectroscopic techniques: 1H-NMR, 1H,1H-COSY and 1H-DOSY. The hydrodynamic diameter of the particles was between 1.8 and 4.4 nm, as determined by DOSY, in good agreement with the DCS and HRTEM results. Diffusion ordered spectroscopy (DOSY) turned out to be a valuable and non-destructive tool to determine the hydrodynamic diameter of dispersed nanoparticles and to control the purity of the final particles. The coordination of the organic molecules to the gold nanoparticles resulted in distinct and complex changes in the 1H-NMR spectra. These were only partially explainable but clearly caused by the vicinity of the molecules to the gold nanoparticle. © The Royal Society of Chemistry 2016.
    view abstract10.1039/c5tb02443a
  • Copper(II), zinc(II) and copper(II)/zinc(II)-containing carbonate-substituted hydroxyapatite: Synthesis, characterization and thermal behaviour
    Livitska, O. and Strutynska, N. and Zatovsky, I. and Nikolenko, I. and Slobodyanik, N. and Prylutskyy, Y. and Epple, M. and Prymak, O. and Byeda, A.
    Materialwissenschaft und Werkstofftechnik 47 (2016)
    A new approach for the preparation of nanoscale copper- and zinc-containing sodium- and carbonate-substituted apatites is presented. The thermal transformations of the samples in the temperature range 80-1000 °C were determined by temperature-programmed desorption mass spectroscopy and thermogravimetry. The chemical and phase compositions of the copper- and zinc-containing sodium- and carbonate-substituted apatites were studied by atomic absorption spectroscopy and X-ray diffraction, respectively. The degree and nature of the carbonate substitution were determined by elemental analysis (C, H, N) and infrared spectroscopy, respectively. In addition, scanning electron microscopy (SEM) showed nanoparticles (about 10-20 nm in diameter) with a stability to aggregation under processes by microwave radiation. Samples annealed at 700 °C were crystalline and had an apatite structure. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/mawe.201600460
  • Effect of annealing temperature on pore formation in preparation of advanced polyethylene battery separator membranes
    Knoche, T. and Lund, R. and Prymak, O. and Epple, M. and Ulbricht, M.
    Materials Today Communications 8 (2016)
    Polymeric separator membranes are a key component in modern lithium ion batteries, as they are placed between anode and cathode to prevent short-circuiting while at the same time allowing an efficient diffusion of Li ions. Manufacturing conditions must be finely tuned to reach the many diverse and conflicting requirements for battery separators used in modern consumer applications as well as in electric vehicles. Little has been published about these proprietary combined extrusion/biaxial stretching processes. This work presents a closer look on one of the key aspects of separator membrane formation. Extruded sheets of high density polyethylene containing a hydrocarbon solvent as plasticizer were stretched in machine direction and annealed at temperatures between 100 °C and 120 °C, i.e., in a range between the onset of melting and actual melting temperature, as deduced from differential scanning calorimetry. The formation of stacked-lamellar morphologies as seen in scanning electron microscopy required a minimum strain and was also influenced by processing temperatures. The increase of the annealing temperature led to a significant increase in crystallinity and chain orientation as revealed by texture analysis, performed using X-ray powder diffraction studies on samples after dedicated preparation. After a second stretching step in transversal direction, a clear correlation to separator membrane permeability and porosity was found, with a higher crystallinity leading to lower Gurley values, i.e. higher permeabilities. The effects of blending high density with ultra-high molecular weight polyethylene of different molecular weight onto structure and morphology were also elucidated in detail. In light of the growing market of electric vehicles, high performance and safety are the main focus during separator production. The influence of important production parameters on final membrane properties is discussed. © 2016 Elsevier Ltd.
    view abstract10.1016/j.mtcomm.2016.04.003
  • Electrochemistry at single bimetallic nanoparticles - using nano impacts for sizing and compositional analysis of individual AgAu alloy nanoparticles
    Saw, E.N. and Grasmik, V. and Rurainsky, C. and Epple, M. and Tschulik, K.
    Faraday Discussions 193 (2016)
    The increasing interest in producing bimetallic nanoparticles and utilizing them in modern technologies sets the demand for fast and affordable characterization of these materials. To date Scanning Transmission Electron Microscopy (STEM) coupled to energy dispersive X-ray spectroscopy is usually used to determine the size and composition of alloy nanoparticles, which is time-consuming and expensive. Here electrochemical single nanoparticle analysis is presented as an alternative approach to infer the particle size and composition of alloy nanoparticles, directly in a dispersion of these particles. As a proof of concept, 14 nm sized Ag0.73Au0.27 alloy nanoparticles are analyzed using a combination of chronoamperometric single nanoparticle analysis and cyclic voltammetry ensemble studies. It is demonstrated that the size, the alloying and the composition can all be inferred using this approach. Thus, the electrochemical characterization of single bimetallic alloy nanoparticles is suggested here as a powerful and convenient complement or alternative to TEM characterization of alloy nanoparticles. © The Royal Society of Chemistry.
    view abstract10.1039/c6fd00112b
  • Estimation of energy of cubic iron-carbon nanoclusters by molecular mechanic method: Berechnung der Energie von kubischen Eisen-Kohlenstoff-Nanoclustern durch molekularmechanische Methoden
    Epple, M. and Prylutskyy, Y. and Nedolya, A.V. and Shapar, D.Y.
    Materialwissenschaft und Werkstofftechnik 47 (2016)
    The energy of cubic iron-carbon nanoclusters was evaluated using the method of molecular mechanics. The focus was on two types of interstitial sites: octahedral and tetrahedral, in which the carbon atoms can be located. The calculation results showed that in the surface layer of the face-centered cubic nanocluster, all of the tetrahedral interstitial sites were energetically equivalent. If a carbon atom changes position between two tetrahedral interstices in the direction of the 111, it can occupy an energetically preferable position in octahedral interstitial space. The comparison of the nanoclusters energy between the cases of surface and subsurface location of the carbon atoms in the octahedral interstice showed that the system has lower energy in the former case. For body-centered cubic nanocluster, octahedral interstitial sites are more energetically favorable for carbon atoms than the tetrahedral interstice, excluding the surface. However, the octahedral interstitial sites on the surface are more preferable than tetrahedral interstice. Based on the calculations it was found that body-centered cubic and face-centered cubic nanoclusters could be unstable by the volumetric concentration of carbon. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/mawe.201600481
  • Fabrication and physico-mechanical properties of thin magnetron sputter deposited silver-containing hydroxyapatite films
    Ivanova, A.A. and Surmeneva, M.A. and Tyurin, A.I. and Pirozhkova, T.S. and Shuvarin, I.A. and Prymak, O. and Epple, M. and Chaikina, M.V. and Surmenev, R.A.
    Applied Surface Science 360 (2016)
    As a measure of the prevention of implant associated infections, a number of strategies have been recently applied. Silver-containing materials possessing antibacterial activity as expected might have wide applications in orthopedics and dentistry. The present work focuses on the physico-chemical characterization of silver-containing hydroxyapatite (Ag-HA) coating obtained by radio frequency (RF) magnetron sputtering. Mechanochemically synthesized Ag-HA powder (Ca10-xAgx(PO4)6(OH)2-x, x = 1.5) was used as a precursor for sputtering target preparation. Morphology, composition, crystallinity, physico-mechanical features (Young's modulus and nanohardness) of the deposited Ag-HA coatings were investigated. The sputtering of the nanostructured multicomponent target at the applied process conditions allowed to deposit crystalline Ag-HA coating which was confirmed by XRD and FTIR data. The SEM results revealed the formation of the coating with the grain morphology and columnar cross-section structure. The EDX analysis confirmed that Ag-HA coating contained Ca, P, O and Ag with the Ca/P ratio of 1.6 ± 0.1. The evolution of the mechanical properties allowed to conclude that addition of silver to HA film caused increase of the coating nanohardness and elastic modulus compared with those of pure HA thin films deposited under the same deposition conditions. © 2015 Elsevier B.V. All rights reserved.
    view abstract10.1016/j.apsusc.2015.11.087
  • Formation and Characterization of Crystalline Hydroxyapatite Coating with the (002) Texture
    Grubova, I. and Priamushko, T. and Chudinova, E. and Surmeneva, M. and Korneva, O. and Epple, M. and Prymak, O. and Shulepov, I. and Surmenev, R.
    IOP Conference Series: Materials Science and Engineering 116 (2016)
    This study reports the effect of titanium (Ti) microstructure on the mechanical properties and surface wettability of thin (< 800 nm) hydroxyapatite (HA) coating deposited via radio-frequency (RF) magnetron sputtering. It was revealed that the sand-blasting (SB) and acid etching (AE) of Ti prior deposition led to a wide range of surface roughness in nano/micro scale. After nanostructured HA coating deposition such physico-mechanical characteristics as nanohardness H, Young's modulus E, H/E ratio and H3/E2 were significantly improved. Moreover, HA coatings exhibited improved wear resistance, lower friction coefficient and ability of the coating to wetting. © Published under licence by IOP Publishing Ltd.
    view abstract10.1088/1757-899X/116/1/012016
  • Gene transfection of human mesenchymal stem cells with a nano-hydroxyapatite–collagen scaffold containing DNA-functionalized calcium phosphate nanoparticles
    Tenkumo, T. and Vanegas Sáenz, J.R. and Takada, Y. and Takahashi, M. and Rotan, O. and Sokolova, V. and Epple, M. and Sasaki, K.
    Genes to Cells 21 (2016)
    This study aimed to fabricate a growth factor-releasing biodegradable scaffold for tissue regeneration. We prepared multishell calcium phosphate (CaP) nanoparticles functionalized with DNA, polyethyleneimine (PEI), protamine and octa-arginine (R8) and compared their respective transfection activity and cell viability measures using human mesenchymal stem cells. DNA–protamine complexes improved the transfection efficiency of CaP nanoparticles with the exception of those functionalized with R8. These complexes also greatly reduced the cytotoxicity of PEI. In addition, we also fabricated DNA–protamine-functionalized CaP nanoparticle-loaded nano-hydroxyapatite–collagen scaffolds and investigated their gene transfection efficiencies. These experiments showed that the scaffolds were associated with moderate hMSC cell viability and were capable of releasing the BMP-2 protein into hMSCs following gene transfection. In particular, the scaffold loaded with protamine-containing CaP nanoparticles showed the highest cell viability and transfection efficiency in hMSCs; thus, it might be suitable to serve as an efficient growth factor-releasing scaffold. © 2016 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd
    view abstract10.1111/gtc.12374
  • Interaction of C60 fullerene complexed to cisplatin with model bilipid membranes and its uptake by HeLa cells: Wechselwirkung von C60-Fulleren mit Cisplatin im Komplex mit Modell-Bilipidmembranen und ihre Aufnahme durch HeLa-Zellen
    Bychko, A. and Sokolova, V. and Prylutska, S. and Bogutska, K. and Rybalchenko, V. and Evstigneev, M. and Prylutskyy, Y. and Epple, M. and Ritter, U. and Scharff, P.
    Materialwissenschaft und Werkstofftechnik 47 (2016)
    With an aim to elucidate the effects of C60 fullerene complexed with anticancer agent cisplatin (Cis) on model bilipid membranes, the investigation of electric properties of bilipid membranes under the action of Cis and C60 fullerene alone and their complex, C60 + Cis, was performed. It was found that the complex itself as well as its components alone exert clearly detected influence on bilipid membranes, which depends on the concentration and the composition of phospholipid. The mechanism of this effect originates either from intermolecular interaction of the drug with fatty-acid residues of phospholipids or from membranotropic effects of the drug-induced lipid peroxidation, or from the sum of these two effects. By means of fluorescence microscopy, the uptake of C60 + Cis complex into HeLa cells has been directly evidenced which stimulates further use of the C60 fullerene-drug mixture in pre-clinical tests. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/mawe.201600474
  • Interaction of C60 fullerene complexed to doxorubicin with model bilipid membranes and its uptake by HeLa cells
    Prylutskyy, Yu. and Bychko, A. and Sokolova, V. and Prylutska, S. and Evstigneev, M. and Rybalchenko, V. and Epple, M. and Scharff, P.
    Materials Science and Engineering C 59 (2016)
    With an aim to elucidate the effects of C60 fullerene complexed with antibiotic doxorubicin (Dox) on model bilipid membranes (BLM), the investigation of the electrical properties of BLM under the action of Dox and C60 fullerene, and of their complex, C60+ Dox,was performed. The complex aswell as its components exert a clearly detectable influence on BLM, which is concentration-dependent and also depends on phospholipid composition. The mechanism of this effect originates either from intermolecular interaction of the drug with fatty-acid residues of phospholipids, or from membranotropic effects of the drug-induced lipid peroxidation, or from the sum of these two effects. By fluorescence microscopy the entering of C60 + Dox complex into HeLa cells was directly shown. © 2015 Elsevier B.V. All rights reserved.
    view abstract10.1016/j.msec.2015.10.049
  • Kinetics of chemotaxis, cytokine, and chemokine release of NR8383 macrophages after exposure to inflammatory and inert granular insoluble particles
    Schremmer, I. and Brik, A. and Weber, D.G. and Rosenkranz, N. and Rostek, A. and Loza, K. and Brüning, T. and Johnen, G. and Epple, M. and Bünger, J. and Westphal, G.A.
    Toxicology Letters 263 (2016)
    Accumulation of macrophages and neutrophil granulocytes in the lung are key events in the inflammatory response to inhaled particles. The present study aims at the time course of chemotaxis in vitro in response to the challenge of various biopersistent particles and its functional relation to the transcription of inflammatory mediators. NR8383 rat alveolar macrophages were challenged with particles of coarse quartz, barium sulfate, and nanosized silica for one, four, and 16 h and with coarse and nanosized titanium dioxide particles (rutile and anatase) for 16 h only. The cell supernatants were used to investigate the chemotaxis of unexposed NR8383 macrophages. The transcription of inflammatory mediators in cells exposed to quartz, silica, and barium sulfate was analyzed by quantitative real-time PCR. Challenge with quartz, silica, and rutile particles induced significant chemotaxis of unexposed NR8383 macrophages. Chemotaxis caused by quartz and silica was accompanied by an elevated transcription of CCL3, CCL4, CXCL1, CXCL3, and TNFα. Quartz exposure showed an earlier onset of both effects compared to the nanosized silica. The strength of this response roughly paralleled the cytotoxic effects. Barium sulfate and anatase did not induce chemotaxis and barium sulfate as well caused no elevated transcription. In conclusion, NR8383 macrophages respond to the challenge with inflammatory particles with the release of chemotactic compounds that act on unexposed macrophages. The kinetics of the response differs between the various particles. © 2016 Elsevier Ireland Ltd
    view abstract10.1016/j.toxlet.2016.08.014
  • Mechano-pharmacological characterization of cardiomyocytes derived from human induced pluripotent stem cells
    Goßmann, M. and Frotscher, R. and Linder, P. and Neumann, S. and Bayer, R. and Epple, M. and Staat, M. and Artmann, A. and Artmann, G.M.
    Cellular Physiology and Biochemistry 38 (2016)
    Background/Aims: Common systems for the quantification of cellular contraction rely on animal-based models, complex experimental setups or indirect approaches. The herein presented CellDrum technology for testing mechanical tension of cellular monolayers and thin tissue constructs has the potential to scale-up mechanical testing towards medium-throughput analyses. Using hiPS-Cardiac Myocytes (hiPS-CMs) it represents a new perspective of drug testing and brings us closer to personalized drug medication. Methods: In the present study, monolayers of self-beating hiPS-CMs were grown on ultra-thin circular silicone membranes and deflect under the weight of the culture medium. Rhythmic contractions of the hiPS-CMs induced variations of the membrane deflection. The recorded contraction-relaxation-cycles were analyzed with respect to their amplitudes, durations, time integrals and frequencies. Besides unstimulated force and tensile stress, we investigated the effects of agonists and antagonists acting on Ca2+ channels (S-Bay K8644/verapamil) and Na+ channels (veratridine/lidocaine). Results: The measured data and simulations for pharmacologically unstimulated contraction resembled findings in native human heart tissue, while the pharmacological dose-response curves were highly accurate and consistent with reference data. Conclusion: We conclude that the combination of the CellDrum with hiPS-CMs offers a fast, facile and precise system for pharmacological, toxicological studies and offers new preclinical basic research potential. © 2016 The Author(s) Published by S. Karger AG, Basel.
    view abstract10.1159/000443124
  • Nanoparticles as transfection agents: A comprehensive study with ten different cell lines
    Neuhaus, B. and Tosun, B. and Rotan, O. and Frede, A. and Westendorf, A.M. and Epple, M.
    RSC Advances 6 (2016)
    The performance of transfection agents to deliver nucleic acids into cells strongly depends on the cell type. In a comprehensive study, nine different cell lines and primary human mesenchymal stem cells were transfected with DNA encoding for enhanced green fluorescent protein (eGFP). As transfection agents, two kinds of cationic multi-shell calcium phosphate nanoparticles and the commercially available transfection agent Lipofectamine were used. The transfection efficiency was measured by fluorescence microscopy by counting the percentage of green fluorescent cells which expressed eGFP as well as qPCR. Furthermore, the uptake of fluorescent calcium phosphate nanoparticles was measured by fluorescence microscopy. The cell viability was measured by the MTT test after incubation with nanoparticles and Lipofectamine. All cell types took up nanoparticles (with different efficiency), but the expression of eGFP was strongly different, demonstrating that the uptake not necessarily leads to processing of a gene. A clear correlation was found between the transfection efficiency and the cell viability that was independent on the transfection agent: a high transfection efficiency was clearly correlated with a low cell viability and vice versa. © The Royal Society of Chemistry 2016.
    view abstract10.1039/c5ra25333k
  • On the Crystallography of Silver Nanoparticles with Different Shapes
    Helmlinger, J. and Prymak, O. and Loza, K. and Gocyla, M. and Heggen, M. and Epple, M.
    Crystal Growth and Design 16 (2016)
    The crystallographic properties of silver nanoparticles with different morphologies (two different kinds of spheres, cubes, platelets, and rods) were derived from X-ray powder diffraction and electron microscopy. The size of the metallic particle core was determined by scanning electron microscopy, and the colloidal stability and the hydrodynamic particle diameter were analyzed by dynamic light scattering. The preferred crystallographic orientation (texture) as obtained by X-ray powder diffraction, including pole figure analysis, and high resolution transmission electron microscopy showed the crystallographic nature of the spheres (almost no texture), the cubes (terminated by {100} faces), the platelets (terminated by {111} faces), and the rods (grown from pentagonal twins along [110] and terminated by {100} faces). The crystallite size was determined by Rietveld refinement of X-ray powder diffraction data and agreed well with the transmission electron microscopic data. © 2016 American Chemical Society.
    view abstract10.1021/acs.cgd.6b00178
  • Oxygen and ammonia plasma treatment of poly(3-hydroxybutyrate) films for controlled surface zeta potential and improved cell compatibility
    Syromotina, D.S. and Surmenev, R.A. and Surmeneva, M.A. and Boyandin, A.N. and Epple, M. and Ulbricht, M. and Oehr, C. and Volova, T.G.
    Materials Letters 163 (2016)
    The oxygen and ammonia radio-frequency (RF) plasma treatment of poly(3-hydroxybutyrate) P3HB films was performed. We revealed significant changes in the topography, a decrease in the surface zeta potential from -63 to -75 mV after the oxygen-plasma treatment and an increase after ammonia plasma treatment from -63 to -45 mV at a pH of 7.4. Investigations into the NIH 3T3 fibroblast adhesion and growth demonstrated the best cell vitality and a higher cell number for the ammonia plasma treatment at 150 W. © 2015 Elsevier B.V.
    view abstract10.1016/j.matlet.2015.10.080
  • Silver nanoparticles with different size and shape: Equal cytotoxicity, but different antibacterial effects
    Helmlinger, J. and Sengstock, C. and Groß-Heitfeld, C. and Mayer, C. and Schildhauer, T.A. and Köller, M. and Epple, M.
    RSC Advances 6 (2016)
    The influence of silver nanoparticle morphology on the dissolution kinetics in ultrapure water as well as the biological effect on eukaryotic and prokaryotic cells was examined. Silver nanoparticles with different shapes but comparable size and identical surface functionalisation were prepared, i.e. spheres (diameter 40-80 and 120-180 nm; two different samples), platelets (20-60 nm), cubes (140-180 nm), and rods (diameter 80-120 nm, length &gt; 1000 nm). All particles were purified by ultracentrifugation and colloidally stabilized with poly(N-vinyl pyrrolidone) (PVP). Their colloidal dispersion in ultrapure water and cell culture medium was demonstrated by dynamic light scattering. Size, shape, and colloidal stability were analysed by scanning electron microscopy, atomic force microscopy, dynamic light scattering, and differential centrifugal sedimentation. The dissolution in ultrapure water was proportional to the specific surface area of the silver nanoparticles. The averaged release rate for all particle morphologies was 30 ± 13 ng s-1 m-2 in ultrapure water (T = 25 ± 1°C; pH 4.8; oxygen saturation 93%), i.e. about 10-20 times larger than the release of silver from a macroscopic silver bar (1 oz), possibly due to the presence of surface defects in the nanoparticulate state. All particles were taken up by human mesenchymal stem cells and were cytotoxic in concentrations of &gt;12.5 μg mL-1, but there was no significant influence of the particle shape on the cytotoxicity towards the cells. Contrary to that, the toxicity towards bacteria increased with a higher dissolution rate, suggesting that the toxic species against bacteria are dissolved silver ions. © The Royal Society of Chemistry 2016.
    view abstract10.1039/c5ra27836h
  • Squamous cell carcinoma in association with a red tattoo
    Schmitz, I. and Prymak, O. and Epple, M. and Ernert, C. and Tannapfel, A.
    JDDG - Journal of the German Society of Dermatology 14 (2016)
    Background and objectives Although tattoos have become exceedingly popular in recent years, only few cases of severe reactions leading to malignant transformation have been reported in the literature. This stands in contrast to the virtually innumerable number of tattoos worldwide. The composition of tattoo dyes is highly variable, and even the same colors may contain different compounds. The objective of our study was to investigate in what way tattoo dyes may potentially trigger skin cancer. Patient and methods We report the rare case of a 24-year-old woman who - seven months after getting a tattoo on the back of her foot - developed a squamous cell carcinoma in close proximity to the red dye used. Complications started in the form of nonspecific swelling. The lesion was histologically examined. The composition of the incorporated dye was analyzed using scanning electron microscopy in combination with energy dispersive element analysis. Thermogravimetry and powder diffraction were used for further characterization. Results and conclusions While the tattoo dye primarily consisted of barium sulfate, traces of Al, S, Ti, P, Mg, and Cl were also detected. The analysis showed pigment granules of varying sizes. In rare cases, tattoo inks may have carcinogenic effects, which appear to be multifactorial. © 2016 Deutsche Dermatologische Gesellschaft (DDG). Published by John Wiley & Sons Ltd.
    view abstract10.1111/ddg.12730
  • Study of biocompatibility effect of nanocarbon particles on various cell types in vitro
    Tolkachov, M. and Sokolova, V. and Loza, K. and Korolovych, V. and Prylutskyy, Y. and Epple, M. and Ritter, U. and Scharff, P.
    Materialwissenschaft und Werkstofftechnik 47 (2016)
    The viability of primary cells (human mesenchymal stem cells, hMSC, as a model for healthy cells) and a cancer cell line (human transformed cervix epithelial cells, HeLa, as a model for cancer cells) was studied with the MTT assay after the incubation with water-soluble C60 fullerenes and multi-walled carbon nanotubes filled by iron, respectively. The size of the particles was determined by dynamic light scattering. The morphology of the cells incubated with nanocarbon particles was studied by scanning electron microscopy. The effect of C60 fullerenes and Fe-multi-walled carbon nanotubes on the cells is depending on the concentration of applied nanoparticles. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/mawe.201600486
  • Surface wettability and energy effects on the biological performance of poly-3-hydroxybutyrate films treated with RF plasma
    Syromotina, D.S. and Surmenev, R.A. and Surmeneva, M.A. and Boyandin, A.N. and Nikolaeva, E.D. and Prymak, O. and Epple, M. and Ulbricht, M. and Oehr, C. and Volova, T.G.
    Materials Science and Engineering C 62 (2016)
    The surface properties of poly-3-hydroxybutyrate (P3HB) membranes were modified using oxygen and an ammonia radio-frequency (RF, 13.56 MHz) plasma. The plasma treatment procedures used in the study only affected the surface properties, including surface topography, without inducing any significant changes in the crystalline structure of the polymer, with the exception being a power level of 250 W. The wettability of the modified P3HB surfaces was significantly increased after the plasma treatment, irrespective of the treatment procedure used. It was revealed that both surface chemistry and surface roughness changes caused by the plasma treatment affected surface wettability. A treatment-induced surface aging effect was observed and resulted in an increase in the water contact angle and a decrease in the surface free energy. However, the difference in the water contact angle between the polymers that had been treated for 4 weeks and the untreated polymer surfaces was still significant. A dependence between cell adhesion and proliferation and the polar component of the surface energy was revealed. The increase in the polar component after the ammonia plasma modification significantly increased cell adhesion and proliferation on biodegradable polymer surfaces compared to the untreated P3HB and the P3HB modified using an oxygen plasma. © 2016 Elsevier B.V.
    view abstract10.1016/j.msec.2016.01.075
  • Synthesis of nanosized hydroxyapatite/agarose powders for bone filler and drug delivery application
    Kolanthai, E. and Ganesan, K. and Epple, M. and Kalkura, S.N.
    Materials Today Communications 8 (2016)
    Drug-loaded bioactive composite powders are used for the treatment of orthopedic diseases and prevention of infection or inflammatory reaction after surgical implantation. Nanosized (80 × 23 nm2) and porous (17 ± 1 nm) hydroxyapatite (HAp)/agarose composite rods were prepared by sol-gel synthesis and subjected to microwave and conventional heating. Microwave heating increased the degree of crystallinity and the thermal stability and produced calcium-deficient HAp/agarose composite powders. There was a considerable reduction (by 39%) in the size of rods on microwave heating whereas the conventional heating at 700 °C rendered the samples porous and agglomerated with a significant decrease in the specific surface area. The agarose contents in as-synthesized and microwave heated samples were ~14% and 4%, respectively. The samples were partially degradable upon immersion in SBF, and later exhibited calcium phosphate deposition which was confirmed by gravimetry. An antibiotic (amoxicillin) and anticancer (5-fluorouracil) drug-loaded microwave-heated nanosized HAp/agarose composite powder gave an extended drug release when compared to the as-synthesized and the conventionally heated samples. The composite powders showed a negative zeta potential, hemocompatibility and better antimicrobial efficacy than pure HAp (conventional heated sample). The microwave heating retained the organic phase (agarose) along with a reduction in particle size. In addition, this technique is simple, fast and cost-effective to produce mesoporous, bioactive and resorbable nanocomposite (HAp/agarose) powders which could find application as bone filling materials and drug delivery systems. © 2016 Elsevier Ltd.
    view abstract10.1016/j.mtcomm.2016.03.008
  • Synthesis of positively and negatively charged silver nanoparticles and their deposition on the surface of titanium
    Sharonova, A. and Loza, K. and Surmeneva, M. and Surmenev, R. and Prymak, O. and Epple, M.
    IOP Conference Series: Materials Science and Engineering 116 (2016)
    Bacterial infections related to dental implants are currently a significant complication. A good way to overcome this challenge is functionalization of implant surface with Ag nanoparticles (NPs) as antibacterial agent. This article aims at review the synthesis routes, size and electrical properties of AgNPs. Polyvinyl pyrrolidone (PVP) and polyethyleneimine (PEI) were used as stabilizers. Dynamic Light Scattering, Nanoparticle Tracking Analysis, X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDX) have been used to characterize the prepared AgNPs. Two types of NPs were synthesized in aqueous solutions: PVP-stabilized NPs with a diameter of the metallic core of 70 ± 20 nm, and negative charge of -20 mV, PEI-stabilized NPs with the size of the metallic core of 50 ± 20 nm and positive charge of +55 mV. According to SEM results, all the NPs have a spherical shape. Functionalization of the titanium substrate surface with PVP and PEI-stabilized AgNPs was carried out by dropping method. XRD patterns revealed that the AgNPs are crystalline with the crystallite size of 14 nm. © Published under licence by IOP Publishing Ltd.
    view abstract10.1088/1757-899X/116/1/012009
  • Thermally induced crystallization and phase evolution in powders derived from amorphous calcium phosphate precipitates with a Ca/P ratio of 1:1
    Zyman, Z. and Epple, M. and Goncharenko, A. and Rokhmistrov, D. and Prymak, O. and Loza, K.
    Journal of Crystal Growth 450 (2016)
    Calcium phosphate powders of calcium pyrophosphate α1-CPP (the metastable phase of the high-temperature polymorph α-CPP) and the polymorph β-CPP (stable in this range), of α1-CPP, β-CPP, α1-TCP (metastable polymorph of the high-temperature phase α-tricalcium phosphate) and β-tricalcium phosphate β-TCP were prepared by heating amorphous calcium phosphate (ACP) precipitates with the nominal Ca/P ratio of 1:1 by nitrate synthesis. α1-CPP/β-CPP resulted from a crystallization at 530–640 °C and subsequent heating to 980 °C of unwashed and lyophilized ACP. α1-CPP/β-CPP/α1-TCP/β-TCP was formed by crystallization at 620–720 °C, followed by heating of six-time washed and lyophilized ACP precipitates from an ultra-short synthesis. The activation energy for the crystallization of ACP to α1-CPP was determined with 165 kJ mol−1. The reason for the occurrence of the TCP phases (Ca/P ratio=1.5) from ACP (Ca/P ratio=1) is discussed. The powders are prospective biomaterials for bone substitution because they combine effective bioactive phases with the metastable polymorphs α1-CPP and α1-TCP. © 2016
    view abstract10.1016/j.jcrysgro.2016.06.014
  • A continuous method to prepare poorly crystalline silver-doped calcium phosphate ceramics with antibacterial properties
    Range, S. and Hagmeyer, D. and Rotan, O. and Sokolova, V. and Verheyen, J. and Siebers, B. and Epple, M.
    RSC Advances 5 (2015)
    Silver-doped calcium phosphate ceramics were prepared in discontinuous and continuous processes with different amounts of incorporated silver (up to 1.8 wt% silver). In particular, the effects of pH, reaction time and light exposure on the incorporation of silver into the calcium phosphate ceramic were investigated. In the dark, silver can be incorporated as colourless silver ions (Ag+) into the apatite lattice, but the integration occurs slowly. Under ambient light, a rapid photoreduction to elemental silver (Ag0) occurs which permits a continuous process to prepare silver-doped calcium phosphate ceramics. The silver-doped calcium phosphate ceramics were characterized by scanning electron microscopy, X-ray powder diffraction, infrared spectroscopy, thermogravimetry, and elemental analysis (Ca, Ag, phosphate). The silver release from the silver-doped calcium phosphate ceramics was measured by a combination of dialysis and atomic absorption spectroscopy. The antimicrobial effect was tested on bacteria (Escherichia coli), and the cytotoxic effect was tested on HeLa cells (human epithelial cervical cancer cells). For comparison, stoichiometric silver phosphate, Ag<inf>3</inf>PO<inf>4</inf>, was prepared. The release of silver from silver phosphate is much faster, leading to pronounced antibacterial but also cytotoxic effects. © The Royal Society of Chemistry 2015.
    view abstract10.1039/c5ra00401b
  • A rapid, high-yield and large-scale synthesis of uniform spherical silver nanoparticles by a microwave-assisted polyol process
    Helmlinger, J. and Heise, M. and Heggen, M. and Ruck, M. and Epple, M.
    RSC Advances 5 (2015)
    Silver nanoparticles are often employed in medical devices and consumer products due to their antibacterial action. For this, reliable syntheses with quantitative yield are required. Uniform spherical silver nanoparticles with a diameter of about 180 nm were synthesized by carrying out the polyol synthesis in a microwave. Silver nitrate was dissolved in ethylene glycol and poly(N-vinyl pyrrolidone) (PVP) was added as capping agent. The particles were characterized by SEM, HRTEM, XRD, and DLS. The results are compared with the classical method where silver nitrate is reduced by glucose in aqueous solution, heated with an oil-bath. The microwave-assisted synthesis leads to an almost quantitative yield of uniform silver nanoparticles after 20 min reaction time and gives exclusively spherical particles without other shapes like triangles, rods or prisms. Diethylene glycol as solvent gave a more homogeneous particle size distribution than ethylene glycol. For both kinds of particles, dissolution in ultrapure water was examined over a period of 29 days in the presence of oxygen. The dissolution was comparable in both cases (about 50% after 4 weeks), indicating the same antibacterial action for particles from the microwave and from the glucose synthesis. © The Royal Society of Chemistry 2015.
    view abstract10.1039/c5ra20446a
  • Bone augmentation using a new injectable bone graft substitute by combining calcium phosphate and bisphosphonate as composite-an animal model
    Schlickewei, C.W. and Laaff, G. and Andresen, A. and Klatte, T.O. and Rueger, J.M. and Ruesing, J. and Epple, M. and Lehmann, W.
    Journal of Orthopaedic Surgery and Research 10 (2015)
    Objective: The aim of this study was to create a new injectable bone graft substitute by combining the features of calcium phosphate and bisphosphonate as a composite bone graft to support bone healing and to evaluate the effect of alendronate to the bone healing process in an animal model. Material and method: In this study, 24 New Zealand white rabbits were randomly divided into two groups: a calcium phosphate alendronate group and a calcium phosphate control group. A defect was created at the proximal medial tibia and filled with the new created injectable bone graft substitute calcium phosphate alendronate or with calcium phosphate. Healing process was documented by fluoroscopy. To evaluate the potential of the bone graft substitute, the proximal tibia was harvested 2, 4, and 12 weeks after operation. Histomorphological analysis was focused on the evaluation of the dynamic bone parameters using the Osteomeasure system. Results: Radiologically, the bone graft materials were equally absorbed. No fracture was documented. The bones healed normally. After 2 weeks, the histological analysis showed an increased new bone formation for both materials. The osteoid volume per bone volume (OV/BV) was significantly higher for the calcium phosphate group. After 4 weeks, the results were almost equal. The trabecular thickness (Tb.Th) increased in comparison to week 2 in both groups with a slight advantage for the calcium phosphate group. The total mass of the bone graft (KEM.Ar) and the bone graft substitute surface density (KEM.Pm) were consistently decreasing. After 12 weeks, the new bone volume per tissue volume (BV/TV) was still constantly growing. Both bone grafts show a good integration. New bone was formed on the surface of both bone grafts. The calcium phosphate as well as the calcium phosphate alendronate paste had been enclosed by the bone. The trabecular thickness was higher in both groups compared to the first time point. Conclusion: Calcium phosphate proved its good potential as a bone graft substitute. Initially, the diagrams seem to show a tendency that alendronate improves the known properties of calcium phosphate as a bone graft substitute. The composite graft induced a good and constant new bone formation. Not only the graft was incorporated into the bone but also a new bone was formed on its surface. But we could not prove a significant difference between the grafts. Both implants proved their function as a bone graft substitute, but the bisphosphonate alendronate does not support the bone healing process sufficiently that the known properties of calcium phosphate as a bone graft substitute were improved in the sense of a composite graft. In this study, alendronate used as a bone graft in a healthy bony environment did not influence the bone healing process in a positive or negative way. © 2015 Schlickewei et al.
    view abstract10.1186/s13018-015-0263-z
  • Calcium phosphate nanoparticles carrying BMP-7 plasmid DNA induce an osteogenic response in MC3T3-E1 pre-osteoblasts
    Hadjicharalambous, C. and Kozlova, D. and Sokolova, V. and Epple, M. and Chatzinikolaidou, M.
    Journal of Biomedical Materials Research - Part A 103 (2015)
    Functionalized calcium phosphate nanoparticles with osteogenic activity were prepared. Polyethyleneimine-stabilized calcium phosphate nanoparticles were coated with a shell of silica and covalently functionalized by silanization with thiol groups. Between the calcium phosphate surface and the outer silica shell, plasmid DNA which encoded either for bone morphogenetic protein 7 (BMP-7) or for enhanced green fluorescent protein was incorporated as cargo. The plasmid DNA-loaded calcium phosphate nanoparticles were used for the transfection of the pre-osteoblastic MC3T3-E1 cells. The cationic nanoparticles showed high transfection efficiency together with a low cytotoxicity. Their potential to induce an osteogenic response by transfection was demonstrated by measuring the alkaline phosphatase (ALP) activity and calcium deposition with alizarin red staining. The expression of the osteogenic markers Alp, Runx2, ColIa1 and Bsp was investigated by means of real-time quantitative polymerase chain reaction. It was shown that phBMP-7-loaded nanoparticles can provide a means of transient transfection and localized production of BMP-7 in MC3T3-E1 cells, with a subsequent increase of two osteogenic markers, specifically ALP activity and calcium accumulation in the extracellular matrix. Future strategies to stimulate bone regeneration focus into enhancing transfection efficiency and achieving higher levels of BMP-7 produced by the transfected cells. © 2015 Wiley Periodicals, Inc.
    view abstract10.1002/jbm.a.35527
  • Continuous delivery of rhBMP2 and rhVEGF165 at a certain ratio enhances bone formation in mandibular defects over the delivery of rhBMP2 alone - An experimental study in rats
    Lohse, N. and Moser, N. and Backhaus, S. and Annen, T. and Epple, M. and Schliephake, H.
    Journal of Controlled Release 220 (2015)
    The aim of the present study was to test the hypothesis that different amounts of vascular endothelial growth factor and bone morphogenic protein differentially affect bone formation when applied for repair of non-healing defects in the rat mandible. Porous composite PDLLA/CaCO3 carriers were fabricated as slow release carriers and loaded with rhBMP2 and rhVEGF165 in 10 different dosage combinations using gas foaming with supercritical carbon dioxide. They were implanted in non-healing defects of the mandibles of 132 adult Wistar rats with additional lateral augmentation. Bone formation was assessed both radiographically (bone volume) and by histomorphometry (bone density). The use of carriers with a ratio of delivery of VEGF/BMP between 0.7 and 1.2 was significantly related to the occurrence of significant increases in radiographic bone volume and/or histologic bone density compared to the use of carriers with a ratio of delivery of ≤ 0.5 when all intervals and all outcome parameters were considered. Moreover, simultaneous delivery at this ratio helped to "save" rhBMP2 as both bone volume and bone density after 13 weeks were reached/surpassed using half the dosage required for rhBMP2 alone. It is concluded, that the combined delivery of rhVEGF165 and rhBMP2 for repair of critical size mandibular defects can significantly enhance volume and density of bone formation over delivery of rhBMP2 alone. It appears from the present results that continuous simultaneous delivery of rhVEGF165 and rhBMP2 at a ratio of approximately 1 is favourable for the enhancement of bone formation. © 2015 Published by Elsevier B.V.
    view abstract10.1016/j.jconrel.2015.10.032
  • Correlation between surface properties and wettability of multi-scale structured biocompatible surfaces
    Gorodzha, S.N. and Surmeneva, M.A. and Prymak, O. and Wittmar, A. and Ulbricht, M. and Epple, M. and Teresov, A. and Koval, N. and Surmenev, R.A.
    IOP Conference Series: Materials Science and Engineering 98 (2015)
    The influence of surface properties of radio-frequency (RF) magnetron deposited hydroxyapatite (HA) and Si-containing HA coatings on wettability was studied. The composition and morphology of the coatings fabricated on titanium (Ti) were characterized using atomic force microscopy (AFM) and X-ray diffraction (XRD). The surface wettability was studied using contact angle analysis. Different geometric parameters of acid-etched (AE) and pulse electron beam (PEB)-treated Ti substrates and silicate content in the HA films resulted in the different morphology of the coatings at micro- and nano- length scales. Water contact angles for the HA coated Ti samples were evaluated as a combined effect of micro roughness of the substrate and nano-roughness of the HA films resulting in higher water contact angles compared with acid-etched (AE) or pulse electron beam (PEB) treated Ti substrates. © Published under licence by IOP Publishing Ltd.
    view abstract10.1088/1757-899X/98/1/012026
  • Dental lessons from past to present: Ultrastructure and composition of teeth from plesiosaurs, dinosaurs, extinct and recent sharks
    Lübke, A. and Enax, J. and Loza, K. and Prymak, O. and Gaengler, P. and Fabritius, H.-O. and Raabe, D. and Epple, M.
    RSC Advances 5 (2015)
    Teeth represent the hardest tissue in vertebrates and appear very early in their evolution as an ancestral character of the Eugnathostomata (true jawed vertebrates). In recent vertebrates, two strategies to form and mineralize the outermost functional layer have persisted. In cartilaginous fish, the enameloid is of ectomesenchymal origin with fluoroapatite as the mineral phase. All other groups form enamel of ectodermal origin using hydroxyapatite as the mineral phase. The high abundance of teeth in the fossil record is ideal to compare structure and composition of teeth from extinct groups with those of their recent successors to elucidate possible evolutionary changes. Here, we studied the chemical composition and the microstructure of the teeth of six extinct shark species, two species of extinct marine reptiles and two dinosaur species using high-resolution chemical and microscopic methods. Although many of the ultrastructural features of fossilized teeth are similar to recent ones (especially for sharks where the ultrastructure basically did not change over millions of years), we found surprising differences in chemical composition. The tooth mineral of all extinct sharks was fluoroapatite in both dentin and enameloid, in sharp contrast to recent sharks where fluoroapatite is only found in enameloid. Unlike extinct sharks, recent sharks use hydroxyapatite as mineral in dentin. Most notably and hitherto unknown, all dinosaur and extinct marine reptile teeth contained fluoroapatite as mineral in dentin and enamel. Our results indicate a drastic change in the tooth mineralization strategy especially for terrestrial vertebrates that must have set in after the cretaceous period. Possibly, this is related to hitherto unconsidered environmental changes that caused unfavourable conditions for the use of fluoroapatite as tooth mineral. © 2015 The Royal Society of Chemistry.
    view abstract10.1039/c5ra11560d
  • Gene silencing of the pro-inflammatory cytokine TNF-α with siRNA delivered by calcium phosphate nanoparticles, quantified by different methods
    Neuhaus, B. and Frede, A. and Westendorf, A.M. and Epple, M.
    Journal of Materials Chemistry B 3 (2015)
    The pro-inflammatory cytokine TNF-α was silenced by treating MODE-K cells with triple-shell calcium phosphate nanoparticles. These consisted of a core of calcium phosphate, followed by a shell of siRNA, then a shell of calcium phosphate to protect the siRNA from nucleases and finally a shell of poly(ethyleneimine) for colloidal stabilization and to give the particles a positive charge. First, the gene silencing efficiency was demonstrated with HeLa-eGFP cells and determined by manually counting the green fluorescent cells, by quantitative FACS analysis of the green fluorescence per cell, and by qPCR at the RNA level. Cell counting gave the highest degrees of eGFP expression, but FACS and qPCR gave more accurate data as they are not probing the cell colour (green or not green) only as yes/no property. This was transposed to the inflammatory relevant mouse cell line MODE-K that was previously stimulated with LPS to induce the expression of TNF-α. By application of the nanoparticles, the TNF-α expression was reduced almost to the original level, as shown by qPCR. Thus, calcium phosphate nanoparticles are well suited to reduce inflammatory reactions by silencing the corresponding cytokines, e.g. TNF-α. © The Royal Society of Chemistry 2015.
    view abstract10.1039/c5tb01377a
  • Hybrid biocomposite with a tunable antibacterial activity and bioactivity based on RF magnetron sputter deposited coating and silver nanoparticles
    Ivanova, A.A. and Surmenev, R.A. and Surmeneva, M.A. and Mukhametkaliyev, T. and Loza, K. and Prymak, O. and Epple, M.
    Applied Surface Science 329 (2015)
    In this work, we describe fabrication techniques used to prepare a multifunctional biocomposite based on a hydroxyapatite (HA) coating and silver nanoparticles (AgNPs). AgNPs synthesized by a wet chemical reduction method were deposited on Ti substrates using a dripping/drying method followed by deposition of calcium phosphate (CaP) coating via radio-frequency (RF) magnetron sputter-deposition. The negatively charged silver nanoparticles (zeta potential -21 mV) have a spherical shape with a metallic core diameter of 50 ± 20 nm. The HA coating was deposited as a dense nanocrystalline film over a surface of AgNPs. The RF-magnetron sputter deposition of HA films on the AgNPs layer did not affect the initial content of AgNPs on the substrate surface as well as NPs size and shape. SEM cross-sectional images taken using the backscattering mode revealed a homogeneous layer of AgNPs under the CaP layer. The diffraction patterns from the coatings revealed reflexes of crystalline HA and silver. The concentration of Ag ions released from the biocomposites after 7 days of immersion in phosphate and acetate buffers was estimated. The obtained results revealed that the amount of silver in the solutions was 0.27 ± 0.02 μg mL-1 and 0.54 ± 0.02 μg mL-1 for the phosphate and acetate buffers, respectively, which corresponded well with the minimum inhibitory concentration range known for silver ions in literature. Thus, this work establishes a new route to prepare a biocompatible layer using embedded AgNPs to achieve a local antibacterial effect. © 2015 Elsevier B.V. All rights reserved.
    view abstract10.1016/j.apsusc.2014.12.153
  • Mesoscopic simulation of phospholipid membranes, peptides, and proteins with molecular fragment dynamics
    Truszkowski, A. and Van Den Broek, K. and Kuhn, H. and Zielesny, A. and Epple, M.
    Journal of Chemical Information and Modeling 55 (2015)
    Molecular fragment dynamics (MFD) is a variant of dissipative particle dynamics (DPD), a coarse-grained mesoscopic simulation technique for isothermal complex fuids and soft matter systems with particles that are chosen to be adequate fluid elements. MFD choses its particles to be small molecules which may be connected by harmonic springs to represent larger molecular entities in order to maintain a comparatively accurate representation of covalent bonding and molecular characteristics. For this study the MFD approach is extended to accomplish long-term simulations (up to the microsecond scale) of large molecular ensembles (representing millions of atoms) containing phospholipid membranes, peptides, and proteins. For peptides and proteins a generally applicable fragmentation scheme is introduced in combination with specific backbone forces that keep native spatial shapes with adequate levels of flexibility or rigidity. The new approach is demonstrated by MFD simulations of the formation and characteristics of phospholipid membranes and vesicles, vesicle-membrane fusion, the backbone force dependency of the overall structural flexibility of dumbbell-shaped Calmodulin, the stability of subunit-aggregation of tetrameric hemoglobin, and the collaborative interaction of Kalata B1 cyclotides with a phospholipid membrane. All findings are in reasonable agreement with experimental as well as alternative simulation results. Thus, the extended MFD approach may become a new tool for biomolecular system studies to allow for comparatively fast simulative investigations in combination with a comparatively high chemical granularity. © 2015 American Chemical Society.
    view abstract10.1021/ci5006096
  • miR-542-3p exerts tumor suppressive functions in neuroblastoma by downregulating survivin
    Althoff, K. and Lindner, S. and Odersky, A. and Mestdagh, P. and Beckers, A. and Karczewski, S. and Molenaar, J.J. and Bohrer, A. and Knauer, S. and Speleman, F. and Epple, M. and Kozlova, D. and Yoon, S. and Baek, K. and Vandesompele, J. and Eggert, A. and Schramm, A. and Schulte, J.H.
    International Journal of Cancer 136 (2015)
    MicroRNAs (miRNAs) are deregulated in a variety of human cancers, including neuroblastoma, the most common extracranial tumor of childhood. We previously reported a signature of 42 miRNAs to be highly predictive of neuroblastoma outcome. One miRNA in this signature, miR-542, was downregulated in tumors from patients with adverse outcome. Reanalysis of quantitative PCR and next-generation sequencing transcript data revealed that miR-542-5p as well as miR-542-3p expression is inversely correlated with poor prognosis in neuroblastoma patients. We, therefore, analyzed the function of miR-542 in neuroblastoma tumor biology. Ectopic expression of miR-542-3p in neuroblastoma cell lines reduced cell viability and proliferation, induced apoptosis and downregulated Survivin. Survivin expression was also inversely correlated with miR-542-3p expression in primary neuroblastomas. Reporter assays confirmed that miR-542-3p directly targeted Survivin. Downregulating Survivin using siRNA copied the phenotype of miR-542-3p expression in neuroblastoma cell lines, while cDNA-mediated ectopic expression of Survivin partially rescued the phenotype induced by miR-542-3p expression. Treating nude mice bearing neuroblastoma xenografts with miR-542-3p-loaded nanoparticles repressed Survivin expression, decreased cell proliferation and induced apoptosis in the respective xenograft tumors. We conclude that miR-542-3p exerts its tumor suppressive function in neuroblastoma, at least in part, by targeting Survivin. Expression of miR-542-3p could be a promising therapeutic strategy for treating aggressive neuroblastoma. © 2014 UICC.
    view abstract10.1002/ijc.29091
  • Multifunctional calcium phosphate nanoparticles for combining near-infrared fluorescence imaging and photodynamic therapy
    Haedicke, K. and Kozlova, D. and Gräfe, S. and Teichgräber, U. and Epple, M. and Hilger, I.
    Acta Biomaterialia 14 (2015)
    Photodynamic therapy (PDT) of tumors causes skin photosensitivity as a result of unspecific accumulation behavior of the photosensitizers. PDT of tumors was improved by calcium phosphate nanoparticles conjugated with (i) Temoporfin as a photosensitizer, (ii) the RGDfK peptide for favored tumor targeting and (iii) the fluorescent dye molecule DY682-NHS for enabling near-infrared fluorescence (NIRF) optical imaging in vivo. The nanoparticles were characterized with regard to size, spectroscopic properties and uptake into CAL-27 cells. The nanoparticles had a hydrodynamic diameter of approximately 200 nm and a zeta potential of around +22 mV. Their biodistribution at 24 h after injection was investigated via NIRF optical imaging. After treating tumor-bearing CAL-27 mice with nanoparticle-PDT, the therapeutic efficacy was assessed by a fluorescent DY-734-annexin V probe at 2 days and 2 weeks after treatment to detect apoptosis. Additionally, the contrast agent IRDye® 800CW RGD was used to assess tumor vascularization (up to 4 weeks after PDT). After nanoparticle-PDT in mice, apoptosis in the tumor was detected after 2 days. Decreases in tumor vascularization and tumor volume were detected in the next few days. Calcium phosphate nanoparticles can be used as multifunctional tools for NIRF optical imaging, PDT and tumor targeting as they exhibited a high therapeutic efficacy, being capable of inducing apoptosis and destroying tumor vascularization. © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
    view abstract10.1016/j.actbio.2014.12.009
  • Nanostructure of wet-chemically prepared, polymer-stabilized silver-gold nanoalloys (6 nm) over the entire composition range
    Ristig, S. and Prymak, O. and Loza, K. and Gocyla, M. and Meyer-Zaika, W. and Heggen, M. and Raabe, D. and Epple, M.
    Journal of Materials Chemistry B 3 (2015)
    Bimetallic silver-gold nanoparticles were prepared by co-reduction using citrate and tannic acid in aqueous solution and colloidally stabilized with poly(N-vinylpyrrolidone) (PVP). The full composition range of silver:gold from 0:100 to 100:0 (n:n) was prepared with steps of 10 mol%. The nanoparticles were spherical, monodispersed, and had a diameter of ∼6 nm, except for Ag:Au 90:10 nanoparticles and pure Ag nanoparticles which were slightly larger. The size of the nanoalloys was determined by differential centrifugal sedimentation (DCS) and transmission electron microscopy (TEM). By means of X-ray powder diffraction (XRD) together with Rietveld refinement, precise lattice parameters, crystallite size and microstrain were determined. Scanning transmission electron microscopy (STEM) combined with energy-dispersive X-ray spectroscopy (EDX) and electron energy loss spectroscopy (EELS) showed that the particles consisted of a gold-rich core and a silver-rich shell. XRD and DCS indicated that the nanoparticles were not twinned, except for pure Ag and Ag:Au 90:10, although different domains were visible in the TEM. A remarkable negative deviation from Vegard's linear rule of alloy mixtures was observed (isotropic contraction of the cubic unit cell with a minimum at a 50:50 composition). This effect was also found for Ag:Au bulk alloys, but it was much more pronounced for the nanoalloys. Notably, it was much less pronounced for pure silver and gold nanoparticles. The microstrain was increased along with the contraction of the unit cell with a broad maximum at a 50:50 composition. The synthesis is based on aqueous solvents and can be easily scaled up to a yield of several mg of a well dispersed nanoalloy with application potential due to its tuneable antibacterial action (silver) and its optical properties for bioimaging. © The Royal Society of Chemistry 2015.
    view abstract10.1039/c5tb00644a
  • Particle-induced cell migration assay (PICMA): A new in vitro assay for inflammatory particle effects based on permanent cell lines
    Westphal, G.A. and Schremmer, I. and Rostek, A. and Loza, K. and Rosenkranz, N. and Brüning, T. and Epple, M. and Bünger, J.
    Toxicology in Vitro 29 (2015)
    Inflammation is a decisive pathophysiologic mechanism of particle toxicity and accumulation of neutrophils in the lung is believed to be a crucial step in this process. This study describes an in vitro model for investigations of the chemotactic attraction of neutrophils in response to particles using permanent cell lines. We challenged NR8383 rat macrophages with particles that were characterized concerning chemical nature, crystallinity, and size distribution in the dry state and in the culture medium. The cell supernatants were used to investigate migration of differentiated human leukemia cells (dHL-60 cells). The dose range for the tests was determined using an impedance-based Real-Time Cell Analyzer. The challenge of NR8383 cells with 32-96μgcm-2 coarse and nanosized particles resulted in cell supernatants which induced strong and dose-dependent migration of dHL-60 cells. Quartz caused the strongest effects - exceeding the positive control "fetal calf serum" (FCS) several-fold, followed by silica, rutile, carbon black, and anatase. BaSO<inf>4</inf> served as inert control and induced no cell migration. Particles caused NR8383 cells to secrete chemotactic compounds. The assay clearly distinguished between the particles of different inflammatory potential in a highly reproducible way. Specificity of the test is suggested by negative results with BaSO<inf>4</inf>. © 2015 The Authors.
    view abstract10.1016/j.tiv.2015.04.005
  • Quantitative replacement of citrate by phosphane on silver nanoparticle surfaces monitored by Surface-Enhanced Raman Spectroscopy (SERS)
    Grass, S. and Diendorf, J. and Gebauer, J.S. and Epple, M. and Treuel, L.
    Journal of Nanoscience and Nanotechnology 15 (2015)
    Chemical approaches to metal NP synthesis commonly use capping agents to achieve a desired NP size and shape. Frequently, such NPs require chemically different surface ligands after synthesis to generate desired NP properties (e.g., charge or hydrophilicity) and to increase their long term colloidal stability. Here, we prepared SERS active citrate-stabilized silver NPs (d = 38 ± 4 nm), purified them from remaining reactants by ultracentrifugation and redispersion, and immersed them into solutions containing different concentrations of Tris(sodium-m-sulfonatophenyl)phosphine (TPPTS), which is often used in such ligand replacement approaches to increase colloidal stability. After equilibration, SERS spectra were acquired, elucidating the concentration dependence of the ligand replacement reaction. SERS data were complemented by concentration dependent size measurements and relations between ligand exchange and colloidal stability are discussed. Copyright © 2015 American Scientific Publishers
    view abstract10.1166/jnn.2015.9143
  • Size matters: An experimental and computational study of the influence of particle size on the lattice energy of NaCl
    Range, S. and Bernardes, C.E.S. and Simões, R.G. and Epple, M. and Da Piedade, M.E.M.
    Journal of Physical Chemistry C 119 (2015)
    One of the most interesting features of nanomaterials is the change in properties that normally accompanies a decrease in particle size. Enthalpy of solution measurements in water, at 298 K, carried out with sodium chloride samples spanning a 500-fold particle size range (120 nm to 60 μm) evidenced the effect of the increase in surface area to volume ratio in the enthalpy of solution and cohesive energy of NaCl. The nanoscopic samples were prepared by a new malonic ester synthesis, which allowed the production of well-formed and approximately cubic crystals. It was found that a very small change in lattice energy (∼0.01%) can be translated into a comparatively much larger change in enthalpy of solution (∼4%) and that the largest changes in properties are expected to occur for particle sizes below ∼100 nm where a steep decrease in lattice energy (spanning a ∼230 kJ·mol-1 range) down to the limit of monomeric NaCl is expected to occur. The experimental findings were corroborated by the results of atom-atom pair potential calculations, which further suggested that the lattice energy within each crystal layer varies from site to site, with the energy differences between adjacent sites decreasing on moving from the periphery to the center of the crystal. The atoms at the outmost surface layer exhibit the lowest lattice energies. Finally the most stable atoms in terms of lattice energy are located in the second layer possibly because repulsive interactions with ions of similar type beyond the crystal surface are absent. © 2015 American Chemical Society.
    view abstract10.1021/jp5124772
  • Solvent free production of porous PDLLA/calcium carbonate composite scaffolds improves the release of bone growth factors
    Schliephake, H. and Vucak, M. and Boven, J. and Backhaus, S. and Annen, T. and Epple, M.
    Oral and Maxillofacial Surgery 19 (2015)
    Purpose: Incorporation of alkaline nano-/microparticles for neutralization of acidic degradation products into degradable polymer foams requires the use of organic solvents, which may compromise biocompatibility and may be associated with biological hazards. The aim of the present study was to develop and validate a solvent-free method to produce porous poly (DL-lactic acid)/calcium carbonate composite scaffolds (PDLLA/CaCO<inf>3</inf>) for controlled release of incorporated osteogenic growth factors. Methods: Composite PDLLA/CaCO<inf>3</inf> granules were produced using a milling process and compared to composite material fabricated through a solution precipitation process using organic solvents. Particle size and mineral content were comparable in both groups. Supercritical carbon dioxide pressure was used to incorporate rhBMP2 into both composites. Results: Gas foaming resulted in comparable pore structures in both groups exhibiting a homogenous distribution of CaCO<inf>3</inf> microparticles in the polymer scaffolds. The elasticity modulus of both types of scaffolds was not significantly different whereas the bending strength of the solvent-free produced scaffolds was significantly lower. The pH values remained constant between 6.90 and 7.25 during degradation of both composites. Release of BMP2 was significantly higher and the induction of alkaline phosphatase was more reliable in the group of scaffolds produced without organic solvents. Conclusion: Solvent-free fabrication of composite PDLLA/CaCO<inf>3</inf> scaffolds for controlled release of bone growth factors through gas foaming significantly enhances the release of growth factors and improves the biological efficacy of the incorporated growth factors. © 2014, Springer-Verlag Berlin Heidelberg.
    view abstract10.1007/s10006-014-0463-4
  • Structure of Biocompatible Coatings Produced from Hydroxyapatite Nanoparticles by Detonation Spraying
    Nosenko, V. and Strutynska, N. and Vorona, I. and Zatovsky, I. and Dzhagan, V. and Lemishko, S. and Epple, M. and Prymak, O. and Baran, N. and Ishchenko, S. and Slobodyanik, N. and Prylutskyy, Y. and Klyui, N. and Temchenko, V.
    Nanoscale Research Letters 10 (2015)
    Detonation-produced hydroxyapatite coatings were studied by scanning electron microscopy (SEM), X-ray powder diffraction (XRD), Raman spectroscopy, and electron paramagnetic resonance (EPR) spectroscopy. The source material for detonation spraying was a B-type carbonated hydroxyapatite powder. The coatings consisted of tetracalcium phosphate and apatite. The ratio depended slightly on the degree of crystallinity of the initial powder and processing parameters of the coating preparation. The tetracalcium phosphate phase was homogeneous; the apatite phase contained defects localized on the sixfold axis and consisted of hydroxyapatite and oxyapatite. Technological factors contributing to the transformation of hydroxyapatite powder structure during coating formation by detonation spraying are discussed. © 2015, Nosenko et al.
    view abstract10.1186/s11671-015-1160-4
  • Synthesis, characterization and in vitro effects of 7 nm alloyed silver-gold nanoparticles
    Ristig, S. and Chernousova, S. and Meyer-Zaika, W. and Epple, M.
    Beilstein Journal of Nanotechnology 6 (2015)
    Alloyed silver-gold nanoparticles were prepared in nine different metal compositions with silver/gold molar ratios of ranging from 90:10 to 10:90. The one-pot synthesis in aqueous medium can easily be modified to gain control over the final particle diameter and the stabilizing agents. The purification of the particles to remove synthesis by-products (which is an important factor for subsequent in vitro experiments) was carried out by multiple ultracentrifugation steps. Characterization by transmission electron microscopy (TEM), differential centrifugal sedimentation (DCS), dynamic light scattering (DLS), UV-vis spectroscopy and atomic absorption spectroscopy (AAS) showed spherical, monodisperse, colloidally stable silver-gold nanoparticles of ≈7 nm diameter with measured molar metal compositions very close to the theoretical values. The examination of the nanoparticle cytotoxicity towards HeLa cells and human mesenchymal stem cells (hMSCs) showed that the toxicity is not proportional to the silver content. Nanoparticles with a silver/gold molar composition of 80:20 showed the highest toxicity. © 2015 Ristig et al.
    view abstract10.3762/bjnano.6.124
  • Synthesis, morphology and structure of the dense (Y1-xEux)2O3 spherical shape particles
    Bezkrovnyi, O.S. and Matveevskaya, N.A. and Yermolayeva, Y.V. and Tolmachev, A.V. and Prymak, O. and Epple, M. and Baumer, V.N.
    Crystal Research and Technology 50 (2015)
    The method to decrease of the porosity (densification) of crystalline spherical particles of the solid substitution solution, obtained by the method of precipitation from aqueous solution followed by low temperature crystallization of the amorphous intermediate product was proposed. The comparative analysis of morphology and structure of the particles before and after densification have been carried. It has been established that porosity of (Y<inf>1-x</inf>Eu<inf>x</inf>)<inf>2</inf>O<inf>3</inf> particles has decreased 5 times compared to their initial state. It has been shown that densification process of the (Y<inf>1-x</inf>Eu<inf>x</inf>)<inf>2</inf>O<inf>3</inf> spherical particles changes their morphology and structure: the size of the crystals doubles, the number and area of crystalline boundaries decrease, the intercrystalline spaces, which forming pores, are almost absent. The dense (Y<inf>1-x</inf>Eu<inf>x</inf>)<inf>2</inf>O<inf>3</inf>(x=0-0,1) isolated spherical particles of 120-300 nm in diameter and dispersion less than 15 % by size, with the porosity 5 times lower, compared to the initial spherical particles of (Y<inf>1-x</inf>Eu<inf>x</inf>)<inf>2</inf>O<inf>3</inf>, were obtained for the first time. It was established that in the process of the densification of porous spherical particles their morphology and structure state are improved: intercrystalline spaces are almost absent, the size of the crystals doubles. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/crat.201500026
  • The effect of patterned titanium substrates on the properties of silver-doped hydroxyapatite coatings
    Grubova, I.Y. and Surmeneva, M.A. and Ivanova, A.A. and Kravchuk, K. and Prymak, O. and Epple, M. and Buck, V. and Surmenev, R.A.
    Surface and Coatings Technology 276 (2015)
    This paper reports the effect of substrate nano/micro-structure design on the grain size, mechanical properties and surface wettability of nanostructured radio frequency (RF) magnetron sputter-deposited silver-containing hydroxyapatite (Ag-HA) coatings containing 0.13-0.36. wt.% silver. The results of this study revealed that the Ag-HA coating microstructure could be designed by controlling the pre-treated surface topography of titanium. The nano/micro-patterned surfaces of titanium were prepared by sand-blasting followed by acid-etching. The size of the nano-patterns on the surface of titanium was also affected by the sand-blasting procedure; namely, the lower the pressure was, the larger the size of the nano-structures and the distance between them. The effect of the coating grain size on the surface wettability and physico-mechanical properties of the biocomposites was revealed. The hydrophobic properties were imparted to the rough titanium by a nanostructured Ag-HA coating. Although according to the XRD patterns the coatings were mainly composed of HA, some differences in the morphology were observed. Therefore, the decreased wettability of the Ag-HA coatings could be explained by taking into account the different grain sizes of the films rather than the changes to the surface chemistry. Nanoindentation studies revealed that in the case of the Ag-HA-coated samples, smaller grains resulted in significantly higher nanohardness and Young's modulus. © 2015 Elsevier B.V..
    view abstract10.1016/j.surfcoat.2015.06.010
  • The potential of nanoparticles for the immunization against viral infections
    Sokolova, V. and Westendorf, A.M. and Buer, J. and Überla, K. and Epple, M.
    Journal of Materials Chemistry B 3 (2015)
    Vaccination has a great impact on the prevention and control of infectious diseases. However, there are still many infectious diseases for which an effective vaccine is missing. Thirty years after the discovery of the AIDS-pathogen (human immunodeficiency virus, HIV) and intensive research, there is still no protective immunity against the HIV infection. Over the past decade, nanoparticulate systems such as virus-like particles, liposomes, polymers and inorganic nanoparticles have received attention as potential delivery vehicles which can be loaded or functionalized with active biomolecules (antigens and adjuvants). Here we compare the properties of different nanoparticulate systems and assess their potential for the development of new vaccines against a range of viral infections. © The Royal Society of Chemistry 2015.
    view abstract10.1039/c5tb00618j
  • Thin hydroxyapatite coating on AZ91D magnesium alloy fabricated via RF-magnetron sputtering
    Mukhametkaliyev, T.M. and Surmeneva, M.A. and Mueller, M. and Prymak, O. and Epple, M. and Surmenev, R.A.
    IOP Conference Series: Materials Science and Engineering 98 (2015)
    In this study the morphology, composition, structure and wettability of radiofrequency (RF) magnetron sputter-deposited hydroxyapatite (HA) coating deposited on the surface of AZ91D magnesium alloy were investigated. The results revealed that the fabricated coating is uniform, homogeneous with the structure of the stoichiometric HA. The deposition of the HA coating did not change significantly the surface wettability of the bare alloy, however water contact angle dynamics in the case of the HA coated substrates revealed a lower rate of a droplet spreading over the surface. © Published under licence by IOP Publishing Ltd.
    view abstract10.1088/1757-899X/98/1/012027
  • A molecular fragment cheminformatics roadmap for mesoscopic simulation
    Truszkowski, A. and Daniel, M. and Kuhn, H. and Neumann, S. and Steinbeck, C. and Zielesny, A. and Epple, M.
    Journal of Cheminformatics 6 (2014)
    Background: Mesoscopic simulation studies the structure, dynamics and properties of large molecular ensembles with millions of atoms: Its basic interacting units (beads) are no longer the nuclei and electrons of quantum chemical ab-initio calculations or the atom types of molecular mechanics but molecular fragments, molecules or even larger molecular entities. For its simulation setup and output a mesoscopic simulation kernel software uses abstract matrix (array) representations for bead topology and connectivity. Therefore a pure kernel-based mesoscopic simulation task is a tedious, time-consuming and error-prone venture that limits its practical use and application. A consequent cheminformatics approach tackles these problems and provides solutions for a considerably enhanced accessibility. This study aims at outlining a complete cheminformatics roadmap that frames a mesoscopic Molecular Fragment Dynamics (MFD) simulation kernel to allow its efficient use and practical application. Results: The molecular fragment cheminformatics roadmap consists of four consecutive building blocks: An adequate fragment structure representation (1), defined operations on these fragment structures (2), the description of compartments with defined compositions and structural alignments (3), and the graphical setup and analysis of a whole simulation box (4). The basis of the cheminformatics approach (i.e. building block 1) is a SMILES-like line notation (denoted fSMILES) with connected molecular fragments to represent a molecular structure. The fSMILES notation and the following concepts and methods for building blocks 2-4 are outlined with examples and practical usage scenarios. It is shown that the requirements of the roadmap may be partly covered by already existing open-source cheminformatics software. Conclusions: Mesoscopic simulation techniques like MFD may be considerably alleviated and broadened for practical use with a consequent cheminformatics layer that successfully tackles its setup subtleties and conceptual usage hurdles. Molecular Fragment Cheminformatics may be regarded as a crucial accelerator to propagate MFD and similar mesoscopic simulation techniques in the molecular sciences. [Figure not available: see fulltext.]. © 2014 Truszkowski et al.; licensee Springer.
    view abstract10.1186/s13321-014-0045-3
  • A pH-sensitive poly(methyl methacrylate) copolymer for efficient drug and gene delivery across the cell membrane
    Doerdelmann, G. and Kozlova, D. and Epple, M.
    Journal of Materials Chemistry B 2 (2014)
    A versatile drug delivery system based on calcium phosphate/Eudragit®-E100 nanoparticles with a diameter below 200 nm was realized by a water-in-oil-in-water (W1/O/W2) emulsion solvent evaporation technique. Hydrophilic drugs (siRNA for gene silencing and bovine serum albumin as model protein) and hydrophobic drugs (5,10,15,20-tetrakis(4-hydroxyphenyl)-21H,23H-porphine, THPP, a photosensitizer for photodynamic therapy) were encapsulated as model drugs. Eudragit®-E100 is a poly(methyl methacrylate) copolymer with a low solubility at neutral pH and a high solubility at low pH. Thus, the particles are stable in cell culture media and rapidly dissolved in the lysosome after cellular uptake and delivery of their cargo into the cell. The particles had a positive charge (zeta potential +49 mV) and were taken up very well by epithelial cells (HeLa) as shown by fluorescence microscopy and confocal laser scanning microscopy. Gene-silencing experiments on HeLa-eGFP cells gave knockdown efficiencies of 39% with no toxic effects. The particles can be freeze-dried without cryoprotectant and easily redispersed in water, thus making their transport and storage convenient. This journal is © the Partner Organisations 2014.
    view abstract10.1039/c4tb01052c
  • An easy synthesis of autofluorescent alloyed silver-gold nanoparticles
    Ristig, S. and Kozlova, D. and Meyer-Zaika, W. and Epple, M.
    Journal of Materials Chemistry B 2 (2014)
    A one-pot synthesis of fluorescent bimetallic silver-gold nanoparticles in aqueous medium is presented. Carboxylic acid-functionalized nanoparticles were prepared with different metal compositions from 9010 to 1090 (nn) for silvergold with a diameter of 1.8 ± 0.4 nm. Pure silver and gold nanoparticles were prepared for comparison. Spectroscopic analyses showed that the ligand, i.e. 11-mercaptoundecanoic acid, binds to the particle surface by the thiol group, leaving the carboxylic acid accessible for further functionalization, e.g. by suitable coupling reactions. Nanoparticles with a silver content up to 6040 showed autofluorescence with a large Stokes shift of about 250-300 nm (maximum wavelength of the emission between 608 nm and 645 nm). The intracellular localization of bimetallic silver-gold nanoparticles was studied in HeLa cells by confocal laser scanning microscopy (CLSM). The alloyed silver-gold nanoparticles showed no significant cytotoxicity at a metal concentration of 5 μg mL-1 for 24 h, but were cytotoxic to some degree at 50 μg mL-1 at higher silver content. This journal is © the Partner Organisations 2014.
    view abstract10.1039/c4tb01010h
  • Antibacterial AgNPs/CaP biocomposites
    Ivanova, A.A. and Surmenev, R.A. and Surmeneva, M.A. and Mukhametkaliyev, T. and Sharonova, A.A. and Grubova, I.Y. and Loza, K. and Chernousova, S. and Prymak, O. and Epple, M.
    2014 9th International Forum on Strategic Technology, IFOST 2014 (2014)
    The modification of implant surface is in the focus of many scientists worldwide. In this study, multifunctional biocomposite on the basis of calcium phosphate coating and silver nanoparticles has been fabricated through the use of nanofabrication techniques. Dense nanocrystalline HA film was deposited over AgNPs. The properties as well as the in vitro behavior of the developed biocomposites have been studied. The diffraction patterns of the biocomposites revealed the peaks of crystalline HA and silver (Ag). The release of Ag from the developed biocomposites was evaluated. The concentration of the released silver ions for 7 days of dissolution was 0.27±0.02 μg/mL and 0.54±0.02 μg/mL for phosphate and acetate buffers, respectively. In order to estimate the cytotoxicity of the samples the functional activity of osteoclasts, in particular, cell morphology, multinuclearity, actin ring and resorption pit on the substrates coated with HA and AgNPs-HA have been evaluated. © 2014 IEEE.
    view abstract10.1109/IFOST.2014.6991166
  • Calcium phosphate increases the encapsulation efficiency of hydrophilic drugs (proteins, nucleic acids) into poly(d,l-lactide-co-glycolide acid) nanoparticles for intracellular delivery
    Dördelmann, G. and Kozlova, D. and Karczewski, S. and Lizio, R. and Knauer, S. and Epple, M.
    Journal of Materials Chemistry B 2 (2014)
    Calcium phosphate/poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles with a diameter below 200 nm, loaded with either nucleic acids or proteins, were synthesized by a water-in-oil-in-water (W1/O/W2) emulsion solvent evaporation technique. The particles were stabilized by polyvinyl alcohol (PVA) and had a negative charge (zeta potential -26 mV). By the addition of calcium phosphate into the inner aqueous phase of the W1/O/W2-emulsion, the encapsulation efficiency of siRNA was increased to 37%, of DNA to 52%, and of bovine serum albumin to 78%, i.e. by a factor of 3 to 10 compared to PLGA nanoparticles without calcium phosphate. Total loadings of 8 μg siRNA, 5 μg DNA and 280 μg fluorescein isothiocyanate-labelled bovine serum albumin (FITC-BSA) per mg of PLGA nanoparticles were achieved by this method. The addition of an outer layer of either chitosan or polyethyleneimine (PEI) reversed the charge of the particles (zeta potential &gt; +30 mV) and improved the cellular uptake as well as the endosomal escape of these particles as demonstrated by confocal laser scanning microscopy. Calcium phosphate-PLGA nanoparticles loaded with DNA encoding for enhanced green fluorescent protein (eGFP-DNA) showed a good transfection efficiency for epithelial cells (HeLa). Gene silencing with HeLa cells expressing eGFP gave knockdown efficiencies of 53% for anionic nanoparticles, of 68% for chitosan-coated cationic nanoparticles, and of 89% for polyethyleneimine-coated cationic nanoparticles. This journal is © the Partner Organisations 2014.
    view abstract10.1039/c4tb00922c
  • Calcium phosphate nanoparticles show an effective activation of the innate immune response in vitro and in vivo after functionalization with flagellin
    Kozlova, D. and Sokolova, V. and Zhong, M. and Zhang, E. and Yang, J. and Li, W. and Yang, Y. and Buer, J. and Westendorf, A.M. and Epple, M. and Yan, H.
    Virologica Sinica 29 (2014)
    For subunit vaccines, adjuvants play a key role in shaping the magnitude, persistence and form of targeted antigen-specific immune response. Flagellin is a potent immune activator by bridging innate inflammatory responses and adaptive immunity and an adjuvant candidate for clinical application. Calcium phosphate nanoparticles are efficient carriers for different biomolecules like DNA, RNA, peptides and proteins. Flagellin-functionalized calcium phosphate nanoparticles were prepared and their immunostimulatory effect on the innate immune system, i.e. the cytokine production, was studied. They induced the production of the proinflammatory cytokines IL-8 (Caco-2 cells) and IL-1β (bone marrow-derived macrophages; BMDM) in vitro and IL-6 in vivo after intraperitoneal injection in mice. The immunostimulation was more pronounced than with free flagellin. © 2014 Wuhan Institute of Virology, CAS and Springer-Verlag Berlin Heidelberg.
    view abstract10.1007/s12250-014-3379-0
  • Comparison of different characterization methods for nanoparticle dispersions before and after aerosolization
    Fissan, H. and Ristig, S. and Kaminski, H. and Asbach, C. and Epple, M.
    Analytical Methods 6 (2014)
    A well-known and accepted aerosol measurement technique, the scanning mobility particle sizer (SMPS), is applied to characterize colloidally dispersed nanoparticles. To achieve a transfer from dispersed particles to aerosolized particles, a newly developed nebulizer (N) is used that, unlike commonly used atomizers, produces significantly smaller droplets and therefore reduces the problem of the formation of residual particles. The capabilities of this new instrument combination (N + SMPS) for the analysis of dispersions were investigated, using three different dispersions, i.e. gold-PVP nanoparticles (∼20 nm), silver-PVP nanoparticles (∼70 nm) and their 1:1 (m:m) mixture. The results are compared to scanning electron microscopy (SEM) measurements and two frequently applied techniques for characterizing colloidal systems: Dynamic light scattering (DLS) and analytical disc centrifugation (ADC). The differences, advantages and disadvantages of each method are discussed, especially with respect to the size resolution of the techniques and their ability to distinguish the particle sizes of the mixed dispersion. While DLS is, as expected, unable to resolve the binary dispersion, SEM, ADC and SMPS are able to give quantitative information on the two particle sizes. However, while the high-resolving ADC is limited due to the dependency on a predefined density of the investigated system, the transfer of dispersed particles into an aerosol and subsequent analysis with SMPS are an adequate way to characterize binary systems, independent of the density of concerned particles, but matching the high resolution of the ADC. We show that it is possible to use the well-established aerosol measurement technique (N + SMPS) in colloid science with all its advantages concerning size resolution and accuracy. © the Partner Organisations 2014.
    view abstract10.1039/c4ay01203h
  • Comparison of silver nanoparticles stored under air or argon with respect to the induction of intracellular free radicals and toxic effects toward keratinocytes
    Ahlberg, S. and Meinke, M.C. and Werner, L. and Epple, M. and Diendorf, J. and Blume-Peytavi, U. and Lademann, J. and Vogt, A. and Rancan, F.
    European Journal of Pharmaceutics and Biopharmaceutics 88 (2014)
    Bacterial infections decreased considerably after the discovery of antibiotics. Nevertheless, because of the rising rate of infections caused by antibiotic-resistant bacteria strains, the search for new bactericidal agents has again become a crucial topic in clinical medicine. Silver nanoparticles (AgNP) have a huge potential in dermatology and wound care management because of their ability to release silver ions (Ag+ ions) in a prolonged and sustained way. However, negative effects of silver on the patient's cells should not be underestimated. Furthermore, it has been controversially discussed whether AgNP are responsible for nanoparticle-specific outcomes or not. In this study, we investigated the effects of AgNP on human skin keratinocytes (HaCaT) in order to better understand the mechanisms of cytotoxicity and to improve the use of this highly reactive biocide in wound healing. We found that most of the cells with internalized AgNP displayed the typical morphological signs of apoptosis. The cell viability assay (XTT) showed concentration-dependent toxic effects of the AgNP toward HaCaT cells. The generation of reactive oxygen species (ROS) induced by AgNP was investigated in cell suspensions by means of electron paramagnetic resonance (EPR) spectroscopy. In order to distinguish between the effects of Ag+ ions released during AgNP storage and those of Ag+ ions released after nanoparticle application, we compared AgNP stored under air (O2) with AgNP stored under argon (Ar). Dispersions of AgNP stored under Ar have a low content of Ag+ ions because of the absence of oxygen which is needed for oxidative dissolution. The results show that Ag+ ions released during particle storage are responsible for most of the ROS produced during 1 h incubation with the cells. AgNP (Ar) also induced intracellular ROS but to a much smaller extent compared to AgNP (O2). These findings highlight the complexity of experiments to assess the toxicity of AgNP and suggest the possibility of reducing AgNP toxic effects by storing AgNP formulations and even silver-containing wound dressing under an inert gas atmosphere. © 2014 Elsevier B.V. All rights reserved.
    view abstract10.1016/j.ejpb.2014.07.012
  • Effect of silver nanoparticles on human mesenchymal stem cell differentiation
    Sengstock, C. and Diendorf, J. and Epple, M. and Schildhauer, T.A. and Köller, M.
    Beilstein Journal of Nanotechnology 5 (2014)
    Background: Silver nanoparticles (Ag-NP) are one of the fastest growing products in nano-medicine due to their enhanced antibacterial activity at the nanoscale level. In biomedicine, hundreds of products have been coated with Ag-NP. For example, various medical devices include silver, such as surgical instruments, bone implants and wound dressings. After the degradation of these materials, or depending on the coating technique, silver in nanoparticle or ion form can be released and may come into close contact with tissues and cells. Despite incorporation of Ag-NP as an antibacterial agent in different products, the toxicological and biological effects of silver in the human body after long-term and low-concentration exposure are not well understood. In the current study, we investigated the effects of both ionic and nanoparticulate silver on the differentiation of human mesenchymal stem cells (hMSCs) into adipogenic, osteogenic and chondrogenic lineages and on the secretion of the respective differentiation markers adiponectin, osteocalcin and aggrecan. Results: As shown through laser scanning microscopy, Ag-NP with a size of 80 nm (hydrodynamic diameter) were taken up into hMSCs as nanoparticulate material. After 24 h of incubation, these Ag-NP were mainly found in the endo-lysosomal cell compartment as agglomerated material. Cytotoxicity was observed for differentiated or undifferentiated hMSCs treated with high silver concentrations (≥20 μg·mL-1 Ag-NP; ≥1.5 μg·mL-1 Ag+ ions) but not with low-concentration treatments (≤10 μg·mL-1 Ag-NP; ≤1.0 μg·mL-1 Ag+ ions). Subtoxic concentrations of Ag-NP and Ag+ ions impaired the adipogenic and osteogenic differentiation of hMSCs in a concentration-dependent manner, whereas chondrogenic differentiation was unaffected after 21 d of incubation. In contrast to aggrecan, the inhibitory effect of adipogenic and osteogenic differentiation was confirmed by a decrease in the secretion of specific biomarkers, including adiponectin (adipocytes) and osteocalcin (osteoblasts). Conclusion: Aside from the well-studied antibacterial effect of silver, little is known about the influence of nano-silver on cell differentiation processes. Our results demonstrate that ionic or nanoparticulate silver attenuates the adipogenic and osteogenic differentiation of hMSCs even at non-toxic concentrations. Therefore, more studies are needed to investigate the effects of silver species on cells at low concentrations during long-term treatment. © 2014 Sengstock et al.
    view abstract10.3762/bjnano.5.214
  • Enthalpies of formation of europium alkoxides: What lessons can be drawn from them
    Branco, J.B. and Carretas, J.M. and Epple, M. and Cruz, A. and Pires De Matos, A. and Leal, J.P.
    Journal of Chemical Thermodynamics 75 (2014)
    The synthesis and characterization of two europium alkoxides, Eu(OCH 3)2 and Eu(OC2H5)2, were described. For the first time the enthalpies of formation of divalent lanthanide alkoxides were determined by using reaction-solution calorimetry. The values obtained are ΔfH0 [Eu(OCH3) 2,cr] = -850.5 ± 5.0 kJ/mol and Δf H0 [Eu(OC2H5)2,cr] = -902.5 ± 5.5 kJ/mol, respectively. Since these compounds have a large use as catalysts or catalysts precursors, the first step of the reaction of them with CO 2 was addressed, which permits to have an idea of the kind of bond involved in those compounds. Moreover, insertion of CO2 in the europium oxygen bond and formation of metal carboxylate complexes, is in both cases presumably bidentate. © 2014 Elsevier Ltd. All rights reserved.
    view abstract10.1016/j.jct.2014.04.009
  • Erratum to: Wettability of Thin Silicate-Containing Hydroxyapatite Films formed by RF-Magnetron Sputtering
    Gorodzha, S.N. and Surmeneva, M.A. and Surmenev, R.A. and Gribennikov, M.V. and Pichugin, V.F. and Sharonova, A.A. and Pustovalova, A.A. and Prymak, O. and Epple, M. and Wittmar, A. and Ulbricht, M. and Gogolinskii, K.V. and Kravchuk, K.S.
    Russian Physics Journal 56 (2014)
    view abstract10.1007/s11182-014-0180-3
  • Fabrication, ultra-structure characterization and in vitro studies of RF magnetron sputter deposited nano-hydroxyapatite thin films for biomedical applications
    Surmeneva, M.A. and Surmenev, R.A. and Nikonova, Y.A. and Selezneva, I.I. and Ivanova, A.A. and Putlyaev, V.I. and Prymak, O. and Epple, M.
    Applied Surface Science 317 (2014)
    A series of nanostructured low-crystalline hydroxyapatite (HA) coatings averaging 170, 250, and 440 nm in thickness were deposited onto previously etched titanium substrates through radio-frequency (RF) magnetron sputtering. The HA coatings were analyzed using infrared spectroscopy (FTIR), X-ray diffraction (XRD), and scanning and transmission electron microscopy (SEM and TEM). Cross sections of the thin specimens were prepared by FIB to study the microstructure of the coatings by TEM. The deposition process formed nano-scale grains, generating an amorphous layer at the substrate/coating interface and inducing the growth of a columnar grain structure perpendicular to the substrate surface. A microstructural analysis of the film confirmed that the grain size and crystallinity increased when increasing the deposition time. The nanostructured HA coatings were not cytotoxic, as proven by in vitro assays using primary dental pulp stem cells and mouse fibroblast NCTC clone L929 cells. Low-crystallinity HA coatings with different thicknesses stimulated cells to attach, proliferate and form mineralized nodules on the surface better than uncoated titanium substrates. © 2014 Elsevier B.V. All rights reserved.
    view abstract10.1016/j.apsusc.2014.08.104
  • Fetuin-A and albumin alter cytotoxic effects of calcium phosphate nanoparticles on human vascular smooth muscle cells
    Dautova, Y. and Kozlova, D. and Skepper, J.N. and Epple, M. and Bootman, M.D. and Proudfoot, D.
    PLoS ONE 9 (2014)
    Calcification is a detrimental process in vascular ageing and in diseases such as atherosclerosis and arthritis. In particular, small calcium phosphate (CaP) crystal deposits are associated with inflammation and atherosclerotic plaque de-stabilisation. We previously reported that CaP particles caused human vascular smooth muscle cell (VSMC) death and that serum reduced the toxic effects of the particles. Here, we found that the serum proteins fetuin-A and albumin (≥1 μM) reduced intracellular Ca2+ elevations and cell death in VSMCs in response to CaP particles. In addition, CaP particles functionalised with fetuin-A, but not albumin, were less toxic than naked CaP particles. Electron microscopic studies revealed that CaP particles were internalised in different ways; via macropinocytosis, membrane invagination or plasma membrane damage, which occurred within 10 minutes of exposure to particles. However, cell death did not occur until approximately 30 minutes, suggesting that plasma membrane repair and survival mechanisms were activated. In the presence of fetuin-A, CaP particle-induced damage was inhibited and CaP/plasma membrane interactions and particle uptake were delayed. Fetuin-A also reduced dissolution of CaP particles under acidic conditions, which may contribute to its cytoprotective effects after CaP particle exposure to VSMCs. These studies are particularly relevant to the calcification observed in blood vessels in patients with kidney disease, where circulating levels of fetuin-A and albumin are low, and in pathological situations where CaP crystal formation outweighs calcification-inhibitory mechanisms. © 2014 Dautova et al.
    view abstract10.1371/journal.pone.0097565
  • Generation of NiTi nanoparticles by femtosecond laser ablation in liquid
    Chakif, M. and Essaidi, A. and Gurevich, E. and Ostendorf, A. and Prymak, O. and Epple, M.
    Journal of Materials Engineering and Performance 23 (2014)
    NiTi was investigated as a model system for a binary alloy where the properties strongly depend on the relative proportion of the two elements and on the grain size. The NiTi nanoparticles were generated by laser ablation in water. For the analysis of the particle size distribution, we used transmission electron microscopy and dynamic light scattering. Here, we found a broad particle size distribution (10-200 nm). Furthermore, the temperature-resolved x-ray powder diffraction and differential scanning calorimetry (DSC) were used to evaluate the phase transition behavior of the generated NiTi nanoparticles. Here, we found an interesting effect. During the heating by DSC, an austenite phase transition and a weak martensite phase transition in the NiTi nanoparticles appeared. Moreover, the phase transformation temperature was about 40 K lower than that of the bulk target. © 2014 ASM International.
    view abstract10.1007/s11665-014-1007-7
  • Impact of solvent mixture on iron nanoparticles generated by laser ablation
    Chakif, M. and Prymak, O. and Slota, M. and Heintze, E. and Gurevich, E.L. and Esen, C. and Bogani, L. and Epple, M. and Ostendorf, A.
    Progress in Biomedical Optics and Imaging - Proceedings of SPIE 8955 (2014)
    The present work reveals the structural and magnetic properties of iron oxide (FexOy) nanoparticles (NPs) prepared by femtosecond laser ablation. The FexOy-NPs were produced in solutions consisting of different ratios of water and acetone. Laser ablation in water yields agglomerates and that in acetone yields chain structures whereas that in water/acetone show a mixture of both. We observe significant fabrication dependent properties such as different crystallinities and magnetic behaviors. The structural characterization shows a change from iron (Fe) to a Fe xOy state of the NPs which depends on the solution composition. Furthermore, transmission electron microscopy measurements exhibit a broad particle size distribution in all samples but with significant differences in the mean sizes. Using magnetic measurements we show that nanoparticles fabricated in pure acetone have lower coercive fields which come along with a smaller mean particle size and therefore increasing superparamagnetic behavior. © 2014 SPIE.
    view abstract10.1117/12.2037682
  • In vitro and in vivo interactions of selected nanoparticles with rodent serum proteins and their consequences in biokinetics
    Kreyling, W.G. and Fertsch-Gapp, S. and Schäffler, M. and Johnston, B.D. and Haberl, N. and Pfeiffer, C. and Diendorf, J. and Schleh, C. and Hirn, S. and Semmler-Behnke, M. and Epple, M. and Parak, W.J.
    Beilstein Journal of Nanotechnology 5 (2014)
    When particles incorporated within a mammalian organism come into contact with body fluids they will bind to soluble proteins or those within cellular membranes forming what is called a protein corona. This binding process is very complex and highly dynamic due to the plethora of proteins with different affinities and fractions in different body fluids and the large variation of compounds and structures of the particle surface. Interestingly, in the case of nanoparticles (NP) this protein corona is well suited to provide a guiding vehicle of translocation within body fluids and across membranes. This NP translocation may subsequently lead to accumulation in various organs and tissues and their respective cell types that are not expected to accumulate such tiny foreign bodies. Because of this unprecedented NP accumulation, potentially adverse biological responses in tissues and cells cannot be neglected a priori but require thorough investigations. Therefore, we studied the interactions and protein binding kinetics of blood serum proteins with a number of engineered NP as a function of their physicochemical properties. Here we show by in vitro incubation tests that the binding capacity of different engineered NP (polystyrene, elemental carbon) for selected serum proteins depends strongly on the NP size and the properties of engineered surface modifications. In the following attempt, we studied systematically the effect of the size (5, 15, 80 nm) of gold spheres (AuNP), surface-modified with the same ionic ligand; as well as 5 nm AuNP with five different surface modifications on the binding to serum proteins by using proteomics analyses. We found that the binding of numerous serum proteins depended strongly on the physicochemical properties of the AuNP. These in vitro results helped us substantially in the interpretation of our numerous in vivo biokinetics studies performed in rodents using the same NP. These had shown that not only the physicochemical properties determined the AuNP translocation from the organ of intake towards blood circulation and subsequent accumulation in secondary organs and tissues but also the the transport across organ membranes depended on the route of AuNP application. Our in vitro protein binding studies support the notion that the observed differences in in vivo biokinetics are mediated by the NP protein corona and its dynamical change during AuNP translocation in fluids and across membranes within the organism. © 2014 Kreyling et al.
    view abstract10.3762/bjnano.5.180
  • Interaction of dermatologically relevant nanoparticles with skin cells and skin
    Vogt, A. and Rancan, F. and Ahlberg, S. and Nazemi, B. and Choe, C.S. and Darvin, M.E. and Hadam, S. and Blume-Peytavi, U. and Loza, K. and Diendorf, J. and Epple, M. and Graf, C. and Ruhl, E. and Meinke, M.C. and Lademann, J.
    Beilstein Journal of Nanotechnology 5 (2014)
    The investigation of nanoparticle interactions with tissues is complex. High levels of standardization, ideally testing of different material types in the same biological model, and combinations of sensitive imaging and detection methods are required. Here, we present our studies on nanoparticle interactions with skin, skin cells, and biological media. Silica, titanium dioxide and silver particles were chosen as representative examples for different types of skin exposure to nanomaterials, e.g., unintended environmental exposure (silica) versus intended exposure through application of sunscreen (titanium dioxide) or antiseptics (silver). Because each particle type exhibits specific physicochemical properties, we were able to apply different combinations of methods to examine skin penetration and cellular uptake, including optical microscopy, electron microscopy, X-ray microscopy on cells and tissue sections, flow cytometry of isolated skin cells as well as Raman microscopy on whole tissue blocks. In order to assess the biological relevance of such findings, cell viability and free radical production were monitored on cells and in whole tissue samples. The combination of technologies and the joint discussion of results enabled us to look at nanoparticle-skin interactions and the biological relevance of our findings from different angles. © 2014 Vogt et al.
    view abstract10.3762/bjnano.5.245
  • Investigation of the morphology and elemental composition of the silicon-containing calcium phosphate coating treated by intensive pulsed electron beam
    Chudinova, E.A. and Surmeneva, M.A. and Grubova, I.Y. and Priamushko, T.S. and Pushilina, N.S. and Teresov, A.D. and Koval, N.N. and Prymak, O. and Epple, M. and Surmenev, R.A.
    2014 9th International Forum on Strategic Technology, IFOST 2014 (2014)
    The surface of titanium substrate with calcium phosphate (CaP) coating prepared by RF magnetron sputtering was treated by a pulsed electron beam with an energy density of 0.8-8 J/cm2. A partial or complete intermixing of the surface layers of the system CaP-titanium was observed. Significant changes in the topography of the formed surfaces were revealed. Treatment conditions with an energy density of 0.8 and 3 J/cm2 led to the thermal annealing of the coating. The use of beam parameters with an energy density of 6.5 and 8 J/cm2 led to the partial vaporization and intermixing of the coating with a titanium matrix. © 2014 IEEE.
    view abstract10.1109/IFOST.2014.6991155
  • Investigation of the structure, elemental and phase composition of coatings on the basis of oxynitride titanium deposited by reactive magnetron sputtering
    Nikitenkov, N.N. and Kiselyova, E.S. and Konischev, M.E. and Sypchenko, V.S. and Nikitenkov, A.N. and Pichugin, V.F. and Shylepov, I.A. and Epple, M.
    Journal of Surface Investigation 8 (2014)
    The structure, morphology and properties of titanium-oxynitride coatings deposited by pulsed reactive magnetron sputtering were investigated. The methods of X-ray diffractometry, Raman spectroscopy, secondary ion mass-spectrometry and scanning electron microscopy are used. It is established that the structure, and the elemental and phase composition of coatings depend on the size of the ratio of oxygen/nitrogen in the composition of the reactive gas, and also on the magnitude of the negative bias applied to the substrate. The increase in the fraction of nitrogen leads to a reduction in the speed of sputtering, to a reduction in the contact angle of wetting, an increase in hardness and a reduction in Young’s modulus when a negative offset was used. © 2014, Pleiades Publishing, Ltd.
    view abstract10.1134/S1027451014060391
  • Magnesiothermic conversion of the silicamineralizing golden algae Mallomonas caudata and Synura petersenii to elemental silicon with high geometric precision
    Petrack, J. and Jost, S. and Boenigk, J. and Epple, M.
    Beilstein Journal of Nanotechnology 5 (2014)
    Chrysophyceae, also known as golden algae, contain characteristic, three-dimensional biomineralized silica structures. Their chemical composition and microscopic structure was studied. By high-temperature conversion of the skeleton of Mallomonas caudata and Synura petersenii into elementary silicon by magnesium vapour, nanostructured defined replicates were produced which were clearly seen after removal of the formed magnesium oxide with acid. © 2014 Petrack et al; licensee Beilstein-Institut.
    view abstract10.3762/bjnano.5.65
  • Mimicking exposures to acute and lifetime concentrations of inhaled silver nanoparticles by two different in vitro approaches
    Herzog, F. and Loza, K. and Balog, S. and Clift, M.J.D. and Epple, M. and Gehr, P. and Petri-Fink, A. and Rothen-Rutishauser, B.
    Beilstein Journal of Nanotechnology 5 (2014)
    In the emerging market of nano-sized products, silver nanoparticles (Ag NPs) are widely used due to their antimicrobial properties. Human interaction with Ag NPs can occur through the lung, skin, gastrointestinal tract, and bloodstream. However, the inhalation of Ag NP aerosols is a primary concern. To study the possible effects of inhaled Ag NPs, an in vitro triple cell co-culture model of the human alveolar/airway barrier (A549 epithelial cells, human peripheral blood monocyte derived dendritic and macrophage cells) together with an air-liquid interface cell exposure (ALICE) system was used in order to reflect a real-life exposure scenario. Cells were exposed at the air-liquid interface (ALI) to 0.03, 0.3, and 3 μg Ag/cm2 of Ag NPs (diameter 100 nm; coated with polyvinylpyrrolidone: PVP). Ag NPs were found to be highly aggregated within ALI exposed cells with no impairment of cell morphology. Furthermore, a significant increase in release of cytotoxic (LDH), oxidative stress (SOD-1, HMOX-1) or pro-inflammatory markers (TNF-α, IL-8) was absent. As a comparison, cells were exposed to Ag NPs in submerged conditions to 10, 20, and 30 μg Ag/mL. The deposited dose per surface area was estimated by using a dosimetry model (ISDD) to directly compare submerged vs ALI exposure concentrations after 4 and 24 h. Unlike ALI exposures, the two highest concentrations under submerged conditions promoted a cytotoxic and pro-inflammatory response after 24 h. Interestingly, when cell cultures were co-incubated with lipopolysaccharide (LPS), no synergistic inflammatory effects were observed. By using two different exposure scenarios it has been shown that the ALI as well as the suspension conditions for the lower concentrations after 4 h, reflecting reallife concentrations of an acute 24 h exposure, did not induce any adverse effects in a complex 3D model mimicking the human alveolar/airway barrier. However, the highest concentrations used in the ALI setup, as well as all concentrations under submerged conditions after 24 h, reflecting more of a chronic lifetime exposure concentration, showed cytotoxic as well as pro-inflammatory effects. In conclusion, more studies need to address long-term and chronic Ag NP exposure effects. © 2014 Herzog et al.
    view abstract10.3762/bjnano.5.149
  • MSC-derived exosomes: A novel tool to treat therapy-refractory graft-versus-host disease
    Kordelas, L. and Rebmann, V. and Ludwig, A.-K. and Radtke, S. and Ruesing, J. and Doeppner, T.R. and Epple, M. and Horn, P.A. and Beelen, D.W. and Giebel, B.
    Leukemia 28 (2014)
    view abstract10.1038/leu.2014.41
  • Nanocapsules of a cationic polyelectrolyte and nucleic acid for efficient cellular uptake and gene transfer
    Ruesing, J. and Rotan, O. and Gross-Heitfeld, C. and Mayer, C. and Epple, M.
    Journal of Materials Chemistry B 2 (2014)
    Polyelectrolyte nanocapsules, consisting of poly(allylamine hydrochloride) (PAH) and a nucleic acid, i.e. either DNA or siRNA, were prepared with calcium phosphate nanoparticles as template. This inorganic core was removed by a combination of acid treatment and dialysis, leading to capsules with a diameter of about 140 nm. These capsules were well taken up by HeLa cells and led to an efficient gene transfer, i.e. transfection by DNA and gene silencing by siRNA. They behaved clearly different from unstructured aggregates of DNA and PAH, i.e. polyplexes, underscoring the effect of their internal structure. © 2014 the Partner Organisations.
    view abstract10.1039/c4tb00392f
  • NR8383 rat macrophages can induce migration of differentiated HL-60 cells following challenge with TIO2, SIO2 or carbon black
    Westphal, G. A. and Schremmer, I. and Rostek, A. and Loza, K. and Rosenkranz, N. and Bruning, T. and Epple, M. and Bunger, J.
    Toxicology Letters 229 (2014)
    view abstract10.1016/j.toxlet.2014.06.678
  • Physical-Mechanical Characteristics of RF Magnetron Sputter-Deposited Coatings Based on Silver-Doped Hydroxyapatite
    Syromotina, D.S. and Surmeneva, M.A. and Gorodzha, S.N. and Pichugin, V.F. and Ivanova, A.A. and Grubova, I.Y. and Kravchuk, K.S. and Gogolinskii, K.V. and Prymak, O. and Epple, M. and Surmenev, R.A.
    Russian Physics Journal 56 (2014)
    Results are presented of studies of the physical-mechanical properties of silver-doped calcium phosphate coatings formed by radio-frequency magnetron sputtering on a surface of technically pure VT1-0 titanium and 12Cr18Ni10Ti stainless chromium-nickel surgical steel. The films have the nanocrystalline structure of hydroxyapatite with mean diameter of the coherently diffracting domains equal to 20-30 nm. It has been established that the configuration of the magnetic field of the magnetron has an effect on the roughness, thickness, and refractive index of the coatings. The thickness of the coatings and the refractive index vary within the ranges 300-700 nm and 1.68-1.80, respectively. The nanohardness of the coatings is within the range 4-7GPa, the modulus of elasticity of the coatings depends on the material of the substrate and is equal to 130-150 GPa for titanium and 170-200 GPa for steel. Sclerometric measurements of the coatings made it possible to establish a high adhesion strength of the coatings. © 2014 Springer Science+Business Media New York.
    view abstract10.1007/s11182-014-0162-5
  • Polymorphic calcium carbonate phases as adsorbents for allergens in natural rubber latex
    Petrack, J. and Vucak, M. and Nover, C. and Epple, M.
    Journal of Applied Polymer Science 132 (2014)
    Natural rubber latex contains different allergenic proteins and peptides that restrict its application in consumer products. Small mineral particles have a comparatively high specific surface area and are therefore well suited to adsorb such biomolecules. The adsorption of such biomolecules onto different polymorphic phases of calcium carbonate, i.e., calcite, aragonite, and vaterite, was quantitatively determined, both from solution as well as from cured natural rubber latex. All phases were able to adsorb the allergens, with slight differences between the different allergens. Desorption experiments showed differences between the allergens of natural rubber latex, but only small differences between the polymorphic phases of calcium carbonate. The release of the allergens from latex objects with incorporated calcium carbonate particles showed that a retention of allergens is possible by adding calcium carbonate as a filler material. © 2014 Wiley Periodicals, Inc.
    view abstract10.1002/app.41271
  • Proinflammatory and cytotoxic response to nanoparticles in precision-cut lung slices
    Hirn, S. and Haberl, N. and Loza, K. and Epple, M. and Kreyling, W.G. and Rothen-Rutishauser, B. and Rehberg, M. and Krombach, F.
    Beilstein Journal of Nanotechnology 5 (2014)
    Precision-cut lung slices (PCLS) are an established ex vivo alternative to in vivo experiments in pharmacotoxicology. The aim of this study was to evaluate the potential of PCLS as a tool in nanotoxicology studies. Silver (Ag-NPs) and zinc oxide (ZnO-NPs) nanoparticles as well as quartz particles were used because these materials have been previously shown in several in vitro and in vivo studies to induce a dose-dependent cytotoxic and inflammatory response. PCLS were exposed to three concentrations of 70 nm monodisperse polyvinylpyrrolidone (PVP)-coated Ag-NPs under submerged culture conditions in vitro. ZnO-NPs (NM110) served as 'soluble' and quartz particles (Min-U-Sil) as 'non-soluble' control particles. After 4 and 24 h, the cell viability and the release of proinflammatory cytokines was measured. In addition, multiphoton microscopy was employed to assess the localization of Ag-NPs in PCLS after 24 h of incubation. Exposure of PCLS to ZnO-NPs for 4 and 24 h resulted in a strong decrease in cell viability, while quartz particles had no cytotoxic effect. Moreover, only a slight cytotoxic response was detected by LDH release after incubation of PCLS with 20 or 30 μg/mL of Ag-NPs. Interestingly, none of the particles tested induced a proinflammatory response in PCLS. Finally, multiphoton microscopy revealed that the Ag-NP were predominantly localized at the cut surface and only to a much lower extent in the deeper layers of the PCLS. In summary, only 'soluble' ZnO-NPs elicited a strong cytotoxic response. Therefore, we suggest that the cytotoxic response in PCLS was caused by released Zn2+ ions rather than by the ZnO-NPs themselves. Moreover, Ag-NPs were predominantly localized at the cut surface of PCLS but not in deeper regions, indicating that the majority of the particles did not have the chance to interact with all cells present in the tissue slice. In conclusion, our findings suggest that PCLS may have some limitations when used for nanotoxicology studies. To strengthen this conclusion, however, other NP types and concentrations need to be tested in further studies. © 2014 Hirn et al.
    view abstract10.3762/bjnano.5.253
  • Prophylactic and therapeutic vaccination with a nanoparticle-based peptide vaccine induces efficient protective immunity during acute and chronic retroviral infection
    Knuschke, T. and Bayer, W. and Rotan, O. and Sokolova, V. and Wadwa, M. and Kirschning, C.J. and Hansen, W. and Dittmer, U. and Epple, M. and Buer, J. and Westendorf, A.M.
    Nanomedicine: Nanotechnology, Biology, and Medicine 10 (2014)
    Retroviral infections e.g. HIV still represent a unique burden in the field of vaccine research. A common challenge in vaccine design is to find formulations that create appropriate immune responses to protect against and/or control the given pathogen. Nanoparticles have been considered to be ideal vaccination vehicles that mimic invading pathogens. In this study, we present biodegradable calcium phosphate (CaP) nanoparticles, functionalized with CpG and retroviral T cell epitopes of Friend virus (FV) as excellent vaccine delivery system. CaP nanoparticles strongly increased antigen delivery to antigen-presenting cells to elicit a highly efficient T cell-mediated immune response against retroviral FV infection. Moreover, single-shot immunization of chronically FV-infected mice with functionalized CaP nanoparticles efficiently reactivated effector T cells which led to a significant decrease in viral loads. Thus, our findings clearly indicate that a nanoparticle-based peptide immunization is a promising approach to improve antiretroviral vaccination. From the Clinical Editor: In this study, biodegradable calcium phosphate nanoparticles were used as a vaccine delivery system after functionalization with CpG and Friend virus-derived T-cell epitopes. This vaccination strategy resulted in increased T-cell mediated immune response even in chronically infected mice, providing a promising approach to the development of clinically useful antiretroviral vaccination strategies. © 2014 Elsevier Inc.
    view abstract10.1016/j.nano.2014.06.014
  • PVP-coated, negatively charged silver nanoparticles: A multi-center study of their physicochemical characteristics, cell culture and in vivo experiments
    Ahlberg, S. and Antonopulos, A. and Diendorf, J. and Dringen, R. and Epple, M. and Flöck, R. and Goedecke, W. and Graf, C. and Haberl, N. and Helmlinger, J. and Herzog, F. and Heuer, F. and Hirn, S. and Johannes, C. and Kittler, S. and Köller, M. and Korn, K. and Kreyling, W.G. and Krombach, F. and Lademann, J. and Loza, K. and Luther, E.M. and Malissek, M. and Meinke, M.C. and Nordmeyer, D. and Pailliart, A. and Raabe, J. and Rancan, F. and Rothen-Rutishauser, B.-R. and Rühl, E. and Schleh, C. and Seibel, A. and Sengstock, C. and Treuel, L. and Vogt, A. and Weber, K. and Zellner, R.
    Beilstein Journal of Nanotechnology 5 (2014)
    PVP-capped silver nanoparticles with a diameter of the metallic core of 70 nm, a hydrodynamic diameter of 120 nm and a zeta potential of -20 mV were prepared and investigated with regard to their biological activity. This review summarizes the physicochemical properties (dissolution, protein adsorption, dispersability) of these nanoparticles and the cellular consequences of the exposure of a broad range of biological test systems to this defined type of silver nanoparticles. Silver nanoparticles dissolve in water in the presence of oxygen. In addition, in biological media (i.e., in the presence of proteins) the surface of silver nanoparticles is rapidly coated by a protein corona that influences their physicochemical and biological properties including cellular uptake. Silver nanoparticles are taken up by cell-type specific endocytosis pathways as demonstrated for hMSC, primary T-cells, primary monocytes, and astrocytes. A visualization of particles inside cells is possible by X-ray microscopy, fluorescence microscopy, and combined FIB/SEM analysis. By staining organelles, their localization inside the cell can be additionally determined. While primary brain astrocytes are shown to be fairly tolerant toward silver nanoparticles, silver nanoparticles induce the formation of DNA double-strand-breaks (DSB) and lead to chromosomal aberrations and sister-chromatid exchanges in Chinese hamster fibroblast cell lines (CHO9, K1, V79B). An exposure of rats to silver nanoparticles in vivo induced a moderate pulmonary toxicity, however, only at rather high concentrations. The same was found in precision-cut lung slices of rats in which silver nanoparticles remained mainly at the tissue surface. In a human 3D triple-cell culture model consisting of three cell types (alveolar epithelial cells, macrophages, and dendritic cells), adverse effects were also only found at high silver concentrations. The silver ions that are released from silver nanoparticles may be harmful to skin with disrupted barrier (e.g., wounds) and induce oxidative stress in skin cells (HaCaT). In conclusion, the data obtained on the effects of this well-defined type of silver nanoparticles on various biological systems clearly demonstrate that cell-type specific properties as well as experimental conditions determine the biocompatibility of and the cellular responses to an exposure with silver nanoparticles. © 2014 Ahlberg et al.
    view abstract10.3762/bjnano.5.205
  • Structural evolution of silver nanoparticles during wet-chemical synthesis
    Banerjee, S. and Loza, K. and Meyer-Zaika, W. and Prymak, O. and Epple, M.
    Chemistry of Materials 26 (2014)
    The formation of silver nanoparticles during the reduction with glucose in the presence of poly(N-vinyl pyrrolidone) as capping agent was followed for more than 3000 min. First, spherical silver nanoparticles are formed, but in later stages, an increasing fraction of nanotriangles and also a few nanorods develop. Both spherical and trigonal nanoparticles grow with time, indicating separate nucleation pathways. The domain size in the spherical nanoparticles increases proportionally to the particle diameter and is always about 1/ 4 of the diameter, indicating that twinned seeds are formed very early in the process and then simply grow by extending their domains. The lattice constant of the nanoparticles is systematically increased in comparison to microcrystalline silver (4.0877 vs 4.08635 Å) but did not change as a function of particle diameter. A thorough analysis of the texture coefficient, supported by transmission electron microscopy data, showed that the apparently spherical particles are in fact flattened pentagonal prisms, which typically lie on their flat pentagonal face. Neither the presence of oxygen nor the presence of ambient light had any influence on the particle properties. © 2014 American Chemical Society.
    view abstract10.1021/cm4025342
  • Structure and Properties of Microarc Calcium Phosphate Coatings on the Surface of Titanium and Zirconium Alloys
    Legostaeva, E.V. and Sharkeev, Y.P. and Epple, M. and Prymak, O.
    Russian Physics Journal 56 (2014)
    Regularities of the formation of calcium phosphate coatings on the surface of titanium and zirconium alloys formed by the method of microarc oxidation are investigated. The scheme of deposition of the calcium phosphate coating in microarc discharges is suggested that explains the difference in the morphology, microstructure, and properties of coatings on the surface of titanium and zirconium alloys. It is demonstrated that coatings on the surface of titanium and zirconium alloys have different structures, phase compositions, and physicomechanical properties due to different electrophysical characteristics of metal (titanium and zirconium) substrates, disperse β-Nb particles, and passivating oxide films on their surfaces. © 2014 Springer Science+Business Media New York.
    view abstract10.1007/s11182-014-0152-7
  • Structure and Properties of Ti-O-N Coatings Produced by Reactive Magnetron Sputtering
    Konischev, M.E. and Kuzmin, O.S. and Pustovalova, A.A. and Morozova, N.S. and Evdokimov, K.E. and Surmenev, R.A. and Pichugin, V.F. and Epple, M.
    Russian Physics Journal 56 (2014)
    Results of an experimental study of the optical characteristics of gas discharges are presented. The study was aimed at optimizing the operating modes of a mid-frequency magnetron sputtering system to efficiently deposit Ti-O-N coatings. The conditions for maintaining the intensity of the chosen spectroscopic lines that ensure synthesis of titanium oxide and titanium oxynitride coatings have been revealed. The morphology, structure, contact angle, and free surface energy of titanium oxide and titanium oxynitride coatings on type 12Kh18N10T stainless steel substrates were examined by using scanning and transmission electron microscopy and infrared spectroscopy, and by measuring the wetting angle. The results of examination of the structure and properties of the synthesized films and their physicomechanical and optical characteristics are given. © 2014 Springer Science+Business Media New York.
    view abstract10.1007/s11182-014-0154-5
  • Synthesis, characterization and EPR investigation of γ-induced defects for nanoparticles of (MI, CO3)-containing (MI - Na, K) apatites
    Strutynska, N. and Slobodyanik, N. and Malyshenko, A. and Zatovsky, I. and Vorona, I. and Epple, M. and Prymak, O. and Baran, N. and Ishchenko, S. and Nosenko, V.
    International Conference on Oxide Materials for Electronic Engineering - Fabrication, Properties and Applications, OMEE 2014 - Book of Conference Proceedings (2014)
    The results of a comparative study of amorphous (Na, CO3)- and (K, CO3)-containing apatites by TPM MS analysis, FTIR, XRD, SEM and EPR are presented. Annealing of amorphous apatite at temperatures below 400°C does not lead to changing of nanoparticle shape and size and an extent of its crystallinity. A significant difference in the formation of γ-induced defects in (Na, CO3)- and (K,CO3)-containing apatites was found. © 2014 IEEE.
    view abstract10.1109/OMEE.2014.6912346
  • Targeting and activation of antigen-specific B-cells by calcium phosphate nanoparticles loaded with protein antigen
    Temchura, V.V. and Kozlova, D. and Sokolova, V. and Überla, K. and Epple, M.
    Biomaterials 35 (2014)
    Cross-linking of the B-cell receptors of an antigen-specific B-cell is the initial signal for B-cell activation, proliferation, and differentiation into antibody secreting plasma cells. Since multivalent particulate structures are efficient activators of antigen-specific B-cells, we developed biodegradable calcium phosphate nanoparticles displaying protein antigens on their surface and explored the efficacy of the B-cell activation after exposure to these nanoparticles. The calcium phosphate nanoparticles were functionalized with the model antigen Hen Egg Lysozyme (HEL) to take advantage of a HEL-specific B-cell receptor transgenic mouse model. The nanoparticles were characterized by scanning electron microscopy and dynamic light scattering. The functionalized calcium phosphate nanoparticles were preferentially bound and internalized by HEL-specific B-cells. Co-cultivation of HEL-specific B-cells with the functionalized nanoparticles also increased surface expression of B-cell activation markers. Functionalized nanoparticles were able to effectively cross-link B-cell receptors at the surface of antigen-matched B-cells and were 100-fold more efficient in the activation of B-cells than soluble HEL. Thus, calcium phosphate nanoparticles coated with protein antigens are promising vaccine candidates for induction humoral immunity. © 2014 Elsevier Ltd.
    view abstract10.1016/j.biomaterials.2014.04.010
  • The dissolution and biological effects of silver nanoparticles in biological media
    Loza, K. and Diendorf, J. and Sengstock, C. and Ruiz-Gonzalez, L. and Gonzalez-Calbet, J.M. and Vallet-Regi, M. and Köller, M. and Epple, M.
    Journal of Materials Chemistry B 2 (2014)
    Silver ions and silver nanoparticles have a well-known biological effect that typically occurs in biological or environmental media of complex composition. Silver nanoparticles release silver ions if oxidizing species like molecular oxygen or hydrogen peroxide are present. The presence of glucose as a model for reducing sugars has only a small effect on the dissolution rate. In the presence of chloride ions, precipitation of silver chloride nanoparticles occurs. At physiological salt concentrations, no precipitation of silver phosphate occurs as the precipitation of silver chloride always occurs first. If the surface of a silver nanoparticle is passivated by cysteine, the dissolution is quantitatively inhibited. Upon immersion of silver nanoparticles in pure water for 8 months, leading to about 50% dissolution, no change in the surface was observed by transmission electron microscopy. A model for the dissolution was derived from immersion and dissolution experiments in different media and from high-resolution transmission electron microscopy. A literature survey on the available data on the dissolution of silver nanoparticles showed that only qualitative trends can be identified as the nature of the nanoparticles and of the immersion medium are practically never comparable. The dissolution effects were confirmed by cell culture experiments (human mesenchymal stem cells and neutrophil granulocytes) where silver nanoparticles that were stored under argon had a clearly lower cytotoxicity than those stored under air. They also led to a less formation of reactive oxygen species (ROS). This underscores that silver ions are the toxic species. © The Royal Society of Chemistry 2014.
    view abstract10.1039/c3tb21569e
  • The predominant species of ionic silver in biological media is colloidally dispersed nanoparticulate silver chloride
    Loza, K. and Sengstock, C. and Chernousova, S. and Köller, M. and Epple, M.
    RSC Advances 4 (2014)
    We have investigated the behaviour of silver ions in biologically relevant concentrations (10 to 100 ppm) in different media, from physiological salt solution over phosphate-buffered saline solution to protein-containing cell culture media. The results show that the initially present silver ions are bound as silver chloride due to the presence of chloride. Only in the absence of chloride, glucose is able to reduce Ag+ to Ag0. The precipitation of silver phosphate was not observed in any case. We conclude that the predominant silver species in biological media is dispersed nanoscopic silver chloride, surrounded by a protein corona which prevents the growth of the crystals and leads to colloidal stabilization. Therefore, in cell culture experiments where dissolved silver ions are studied in the upper ppm range, in fact the effect of colloidally dispersed silver chloride is observed. We have confirmed this by cell culture experiments (human mesenchymal stem cells; T-cells; monocytes) and bacteria (S. aureus) where the cells were incubated with synthetically prepared silver chloride nanoparticles (diameter ca. 100 nm). These were easily taken up by eukaryotic cells and showed the same toxic effect at the same silver concentration as ionic silver (as silver acetate). Therefore, nanoscopic silver chloride and not free ionic silver is the primary toxic species in biological media. © 2014 the Partner Organisations.
    view abstract10.1039/c4ra04764h
  • The relevance of biomaterials to the prevention and treatment of osteoporosis
    Arcos, D. and Boccaccini, A.R. and Bohner, M. and Díez-Pérez, A. and Epple, M. and Gómez-Barrena, E. and Herrera, A. and Planell, J.A. and Rodríguez-Mañas, L. and Vallet-Regí, M.
    Acta Biomaterialia 10 (2014)
    Osteoporosis is a worldwide disease with a very high prevalence in humans older than 50. The main clinical consequences are bone fractures, which often lead to patient disability or even death. A number of commercial biomaterials are currently used to treat osteoporotic bone fractures, but most of these have not been specifically designed for that purpose. Many drug- or cell-loaded biomaterials have been proposed in research laboratories, but very few have received approval for commercial use. In order to analyze this scenario and propose alternatives to overcome it, the Spanish and European Network of Excellence for the Prevention and Treatment of Osteoporotic Fractures, "Ageing", was created. This network integrates three communities, e.g. clinicians, materials scientists and industrial advisors, tackling the same problem from three different points of view. Keeping in mind the premise "living longer, living better", this commentary is the result of the thoughts, proposals and conclusions obtained after one year working in the framework of this network. © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
    view abstract10.1016/j.actbio.2014.01.004
  • The type of adjuvant strongly influences the T-cell response during nanoparticle-based immunization
    Knuschke, T. and Epple, M. and Westendorf, A.M.
    Human Vaccines and Immunotherapeutics 10 (2014)
    Potent vaccines require the ability to effectively induce immune responses. Especially for the control of infectious diseases with intracellular pathogens, like viruses or bacteria, potent T-cell responses are indispensable. Several delivery systems such as nanoparticles have been considered to boost the immunogenicity of pathogen derived peptides or subunits for the induction of potent T-cell responses. Since they can be further functionalized with immunostimulants, like Toll-like receptor (TLR) agonists, they improve the response by enhanced activation of the innate immune system. Currently, TLR agonists like unmethylated CpG oligonucleotides and the synthetic dsRNA derivate polyriboinosinic acid-polyribocytidylic acid (poly[I:C]) are widely used as vaccine adjuvants. CpG and poly(I:C) trigger different TLRs and therefore show differential signal transduction. Recently, we established biodegradable calcium phosphate (CaP) nanoparticles as potent T cell inducing vaccination vehicles. In this commentary we discuss the role of CpG and poly(I:C) for the effective induction of virus-specific T cells during immunization with CaP nanoparticles. The presented results underline the importance of the right formulation of vaccines for specific immunization purpose. © 2014 Landes Bioscience.
    view abstract10.4161/hv.26203
  • Ultrastructural organization and micromechanical properties of shark tooth enameloid
    Enax, J. and Janus, A.M. and Raabe, D. and Epple, M. and Fabritius, H.-O.
    Acta Biomaterialia 10 (2014)
    The outer part of shark teeth is formed by the hard and mineral-rich enameloid that has excellent mechanical properties, which makes it a very interesting model system for the development of new bio-inspired dental materials. We characterized the microstructure, chemical composition and resulting local mechanical properties of the enameloid from teeth of Isurus oxyrinchus (shortfin mako shark) by performing an in-depth analysis using various high-resolution analytical techniques, including scanning electron microscopy, qualitative energy-dispersive X-ray spectroscopy and nanoindentation. Shark tooth enameloid reveals an intricate hierarchical arrangement of thin (50-80 nm) and long (>1 μm) crystallites of fluoroapatite with a high degree of structural anisotropy, which leads to exceptional mechanical properties. Both stiffness and hardness are surprisingly homogeneous in the shiny layer as well as in the enameloid: although both tooth phases differ in structure and composition, they show almost no orientation dependence with respect to the loading direction of the enameloid crystallites. The results were used to determine the structural hierarchy of shark teeth, which can be used as a base for establishing design criteria for synthetic bio-inspired and biomimetic dental composites. © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
    view abstract10.1016/j.actbio.2014.04.028
  • Wettability of Thin Silicate-Containing Hydroxyapatite Films Formed by RF-Magnetron Sputtering
    Gorodzha, S.N. and Surmeneva, M.A. and Surmenev, R.A. and Gribennikov, M.V. and Pichugin, V.F. and Sharonova, A.A. and Pustovalova, A.A. and Prymack, O. and Epple, M. and Wittmar, A. and Ulbricht, M. and Gogolinskii, K.V. and Kravchuk, K.S.
    Russian Physics Journal 56 (2014)
    Using the methods of electron and atomic force microscopy, X-ray structural analysis and measurements of the wetting angle, the features of morphology, structure, contact angle and free surface energy of silicon-containing calcium-phosphate coatings formed on the substrates made from titanium VT1-0 and stainless steel 12Cr18Ni10Ti are investigated. It is shown that the coating - substrate system possesses bimodal roughness formed by the substrate microrelief and coating nanostructure, whose principal crystalline phase is represented by silicon-substituted hydroxiapatite with the size of the coherent scattering region (CSR) 18-26 nm. It is found out that the formation of a nanostructured coating on the surface of rough substrates makes them hydrophilic. The limiting angle of water wetting for the coatings formed on titanium and steel acquires the values in the following ranges: 90-92 and 101-104°, respectively, and decreases with time. © 2014 Springer Science+Business Media New York.
    view abstract10.1007/s11182-014-0157-2
  • X-Ray Powder Diffraction as a Tool to Investigate the Ultrastructure of Nanoparticles
    Prymak, O. and Ristig, S. and Meyer-Zaika, V. and Rostek, A. and Ruiz, L. and González-Calbet, J.M. and Vallet-Regi, M. and Epple, M.
    Russian Physics Journal 56 (2014)
    The potential of x-ray powder diffraction to analyze the size and ultrastructure of metallic nanoparticles (silver, gold, silver-gold alloy, and calcium phosphate) is demonstrated. By the Rietveld analysis, it is possible to estimate the crystallite size for such nanoparticles, even if they are very small (4 nm), using the effect of peak broadening in small crystallites. The results correspond well to crystallite size as determined by transmission electron microscopy (TEM), also confirming the twinned nature of the metallic nanoparticles. For calcium phosphate nanorods which are not twinned, the results by x-ray powder diffraction and TEM are in good agreement. © 2014 Springer Science+Business Media New York.
    view abstract10.1007/s11182-014-0149-2
  • A genetically active nano-calcium phosphate paste for bone substitution, encoding the formation of BMP-7 and VEGF-A
    Chernousova, S. and Klesing, J. and Soklakova, N. and Epple, M.
    RSC Advances 3 (2013)
    Calcium phosphate nanoparticles with different compositions and morphologies loaded with DNA were prepared. Plasmids which encoded either bone morphogenetic protein 7 (BMP-7) or vascular endothelial growth factor A (VEGF-A) were used for transfection in epithelial cells (HeLa), osteoblast-like cells (MG-63), and human mesenchymal stem cells (hMSC). In particular, cationic nanoparticles showed high transfection efficiency together with low cytotoxicity. The nanoparticles can either be used in dispersion or added to a calcium phosphate paste for injection into bone defects. © 2013 The Royal Society of Chemistry.
    view abstract10.1039/c3ra23450a
  • A porous pH-stabilized composite material consisting of poly (D,L-lactide), calcium carbonate and gentamicin for bone substitution
    Backhaus, S. and Annen, T. and Epple, M.
    Materialwissenschaft und Werkstofftechnik 44 (2013)
    Macroporous objects of poly(D,L-lactide), PDLLA, and calcium carbonate were prepared by hot-pressing (0% porosity), gas-foaming (50% porosity), and a combination of gas-foaming and salt leaching (70% porosity). They were loaded with 15 wt% of gentamicin without loss of the mechanical properties. The release of gentamicin occurred over several weeks, making this material suitable as a mechanically stable porous and degradable bone substitution material with antibacterial properties. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/mawe.201300081
  • Adhesion properties of a silicon-containing calcium phosphate coating deposited by RF magnetron sputtering on a heated substrate
    Surmeneva, M.A. and Surmenev, R.A. and Pichugin, V.F. and Koval', N.N. and Teresov, A.D. and Ivanova, A.A. and Grubova, I.Y. and Ignatov, V.P. and Primak, O. and Epple, M.
    Journal of Surface Investigation 7 (2013)
    Silicon-containing hydroxyapatite coatings 400-700 nm in thickness are prepared by means of radio-frequency (RF) magnetron sputtering on a heated (to 200°C) titanium substrate chemically etched and treated with a pulsed electron beam. The morphology and phase composition of the coating are studied. The morphology and roughness of the composite "calcium-phosphate coating-titanium substrate" differ depending on the treatment procedure of the substrate before deposition. The scratch test method is used to assess the adhesion strength of the coatings formed at different values of bias potential applied to the substrate. It is observed that the adhesion strength of the coating changes with decreasing crystallite size. © 2013 Pleiades Publishing, Ltd.
    view abstract10.1134/S102745101305039X
  • Bioactive surfaces for hard tissue regeneration
    Epple, M. and Surmenev, R.A.
    RSC Advances 3 (2013)
    view abstract10.1039/c3ra90065g
  • Calcium phosphate ceramics with sodium-rich calcium phosphate phases at the surface
    Zyman, Z. and Epple, M. and Goncharenko, A. and Rokhmistrov, D.
    Materialwissenschaft und Werkstofftechnik 44 (2013)
    Calcium phosphate was prepared by aqueous wet-chemical precipitation from sodium phosphate and calcium nitrate under standard conditions. By incomplete removal of the mother liquor, the by-product sodium nitrate remained in the dried powder. Upon annealing at 1150°C, the sodium-rich phases β-rhenanite (NaCaPO4) and Na3Ca6(PO 4)5 were formed. If the powder was first compacted and then annealed at 1150°C, the sodium-rich phases were enriched in the sample surface up to a depth of about 140 μm as shown by X-ray powder diffraction. As these phases have a higher water solubility than sintered hydroxyapatite, they will enhance the resorption of such a ceramic in contact with hard tissues. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/mawe.201300095
  • Characterization of crocodile teeth: Correlation of composition, microstructure, and hardness
    Enax, J. and Fabritius, H.-O. and Rack, A. and Prymak, O. and Raabe, D. and Epple, M.
    Journal of Structural Biology 184 (2013)
    Structure and composition of teeth of the saltwater crocodile Crocodylus porosus were characterized by several high-resolution analytical techniques. X-ray diffraction in combination with elemental analysis and infrared spectroscopy showed that the mineral phase of the teeth is a carbonated calcium-deficient nanocrystalline hydroxyapatite in all three tooth-constituting tissues: Dentin, enamel, and cementum. The fluoride content in the three tissues is very low (<0.1. wt.%) and comparable to that in human teeth. The mineral content of dentin, enamel, and cementum as determined by thermogravimetry is 71.3, 80.5, and 66.8. wt.%, respectively. Synchrotron X-ray microtomography showed the internal structure and allowed to visualize the degree of mineralization in dentin, enamel, and cementum. Virtual sections through the tooth and scanning electron micrographs showed that the enamel layer is comparably thin (100-200 μm). The crystallites in the enamel are oriented perpendicularly to the tooth surface. At the dentin-enamel-junction, the packing density of crystallites decreases, and the crystallites do not display an ordered structure as in the enamel. The microhardness was 0.60 ± 0.05. GPa for dentin, 3.15 ± 0.15. GPa for enamel, 0.26 ± 0.08. GPa for cementum close to the crown, and 0.31 ± 0.04. GPa for cementum close to the root margin. This can be explained with the different degree of mineralization of the different tissue types and is comparable with human teeth. © 2013 Elsevier Inc.
    view abstract10.1016/j.jsb.2013.09.018
  • Cytotoxic and proinflammatory effects of PVP-coated silver nanoparticles after intratracheal instillation in rats
    Haberl, N. and Hirn, S. and Wenk, A. and Diendorf, J. and Epple, M. and Johnston, B.D. and Krombach, F. and Kreyling, W.G. and Schleh, C.
    Beilstein Journal of Nanotechnology 4 (2013)
    Silver nanoparticles (AgNP) are among the most promising nanomaterials, and their usage in medical applications and consumer products is growing rapidly. To evaluate possible adverse health effects, especially to the lungs, the current study focused on the cytotoxic and proinflammatory effects of AgNP after the intratracheal instillation in rats. Monodisperse, PVP-coated AgNP (70 nm) showing little agglomeration in aqueous suspension were instilled intratracheally. After 24 hours, the lungs were lavaged, and lactate dehydrogenase (LDH), total protein, and cytokine levels as well as total and differential cell counts were measured in the bronchoalveolar lavage fluid (BALF). Instillation of 50 μg PVP-AgNP did not result in elevated LDH, total protein, or cytokine levels in BALF compared to the control, whereas instillation of 250 μg PVP-AgNP caused a significant increase in LDH (1.9-fold) and total protein (1.3-fold) levels as well as in neutrophil numbers (60-fold) of BALF. Furthermore, while there was no change in BALF cytokine levels after the instillation of 50 μg PVP-AgNP, instillation of 250 μg PVP-AgNP resulted in significantly increased levels of seven out of eleven measured cytokines. These finding suggest that exposure to inhaled AgNP can induce moderate pulmonary toxicity, but only at rather high concentrations. © 2013 Haberl et al.
    view abstract10.3762/bjnano.4.105
  • Facile synthesis of polymer core@silver shell hybrid nanoparticles with super surface enhanced Raman scattering capability
    Huo, D. and He, J. and Yang, S. and Zhou, Z. and Hu, Y. and Epple, M.
    Journal of Colloid and Interface Science 393 (2013)
    Silver nano-shells (SNSs) were synthesized via a two-step seeds-mediated method. Polymer cores were composed of ultrafine gold nanoparticles (NPs) modified chitosan-poly(acrylic acid) nanoparticles (CS-PAA NPs). Then, deposition of silver upon gold nucleus leads to the seed enlargement and finally forms silver shell on the surface of CS-PAA NPs to get SNSs. Transmission electron microscope (TEM) showed SNSs had a discrete silver shell plus some pores and gaps, which could acted as "hot spots" and provided the great potential of these SNSs to be used as SERS substrates with wavelength ranging from visible to infrared region (700-1000. nm) by tuning shell coverage of silver. SERS experiments with dibenzyl disulphide (DBDS) as the indicator showed that the resulting SNSs allowed the production of highly consistent enhancement of the Raman signals down to nM concentrations of DBDS. Considering the excellent biocompatibility of polymer core and their small size, these SNSs are highly desirable candidates as the enhancers for high performance SERS analysis and as SERS optical labels in biomedical imaging. © 2012 Elsevier Inc.
    view abstract10.1016/j.jcis.2012.11.003
  • Immunization with biodegradable nanoparticles efficiently induces cellular immunity and protects against influenza virus infection
    Knuschke, T. and Sokolova, V. and Rotan, O. and Wadwa, M. and Tenbusch, M. and Hansen, W. and Staeheli, P. and Epple, M. and Buer, J. and Westendorf, A.M.
    Journal of Immunology 190 (2013)
    The ability of vaccines to induce T cell responses is crucial for preventing diseases caused by viruses or bacteria. Nanoparticles (NPs) are considered an efficient tool for inducing potent immune responses. In this study, we describe a novel vaccination approach with biodegradable calcium phosphate (CaP) NPs that serve as carrier of immunoactive TLR9 ligand (CpG) combined with a viral Ag from the influenza A virus hemagglutinin. Functionalized CaP NPs were efficiently taken up by dendritic cells in vivo and elicited a potent T cell-mediated immune response in immunized mice with high numbers of IFN-γ-producing CD4+ and CD8+ effector T cells. Most importantly, both i.p. and intranasal immunization with these NPs offered protection in a mouse model of influenza virus infection. This study demonstrates the great potential of CaP NPs as a novel vaccination tool that offers substantial flexibility for several infection models. Copyright © 2013 by The American Association of Immunologists, Inc.
    view abstract10.4049/jimmunol.1202654
  • Influence of the substrate bias on the stoichiometry and structure of RF-magnetron sputter-deposited silver-containing calcium phosphate coatings
    Ivanova, A.A. and Surmeneva, M.A. and Grubova, I.Y. and Sharonova, A.A. and Pichugin, V.F. and Chaikina, M.V. and Buck, V. and Prymak, O. and Epple, M. and Surmenev, R.A.
    Materialwissenschaft und Werkstofftechnik 44 (2013)
    A coating on the basis of silver-containing hydroxyapatite (silver-hydroxyapatite) was deposited by radio frequency (RF) magnetron sputtering. X-ray diffractometry, scanning electron microscopy, energy-dispersive X-ray spectroscopy, and ellipsometry were used to analyse the change in structure and stoichiometry of the coatings upon the change of the negative electrical bias (-50 V and -100 V) on the substrate. The chemical composition of the sputter-deposited coating was identical to the target. However, an increase in the negative electrical bias on the substrate led to a decrease of the coating thickness. In addition, the average size of the grains decreased from 55 ± 15 nm (grounded substrate) up to 30 ± 10 nm when an electrical bias of -100 V was applied. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/mawe.201300101
  • Mechanism of the uptake of cationic and anionic calcium phosphate nanoparticles by cells
    Sokolova, V. and Kozlova, D. and Knuschke, T. and Buer, J. and Westendorf, A.M. and Epple, M.
    Acta Biomaterialia 9 (2013)
    The uptake of calcium phosphate nanoparticles (diameter 120 nm) with different charge by HeLa cells was studied by flow cytometry. The amount of uptaken nanoparticles increased with increasing concentration of nanoparticles in the cell culture medium. Several inhibitors of endocytosis and macropinocytosis were applied to elucidate the uptake mechanism of nanoparticles into HeLa cells: wortmannin, LY294002, nocodazole, chlorpromazine and nystatin. Wortmannin and LY294002 strongly reduced the uptake of anionic nanoparticles, which indicates macropinocytosis as uptake mechanism. For cationic nanoparticles, the uptake was reduced to a lesser extent, indicating a different uptake mechanism. The localization of nanoparticles inside the cells was investigated by conjugating them with the pH-sensitive dye SNARF-1. The nanoparticles were localized in lysosomes after 3 h of incubation. © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
    view abstract10.1016/j.actbio.2013.02.034
  • Microstructure, mechanical and biological properties of zirconium alloyed with niobium after severe plastic deformation
    Sharkeev, Y.P. and Eroshenko, A.Y. and Kulyashova, K.S. and Fortuna, S.V. and Suvorov, K.A. and Epple, M. and Prymak, O. and Sokolova, V. and Chernousova, S.
    Materialwissenschaft und Werkstofftechnik 44 (2013)
    A comparative investigation of microstructure, mechanical and biological properties for zirconium alloyed with niobium in coarse-grained and ultra-fine grained states is presented. The temperature and deformation regimes of multi-stage abc-pressing resulted in ultra-fine grained states with an average size of the structural elements in the range of 0.28-0.55 μm, depending on the accumulated strain during pressing. The increase of the accumulated strain at each stage of pressing increased the uniformity of the structure. The microhardness increased by 50% with increased accumulated strain during the severe plastic deformation. Between the microhardness and the average size of the structural elements, a linear dependence was found, indicating a Hall-Petch relationship. The alloy had a good biocompatibility as shown by an MTT test with osteoblasts (MG-63 cell line). The good mechanical properties (microhardness) of zirconium alloyed with niobium in the ultra-fine grained state make it suitable for medical applications, e. g. as implant material. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/mawe.201300113
  • Mineralization of the vertebral bodies in Atlantic salmon (Salmo salar L.) is initiated segmentally in the form of hydroxyapatite crystal accretions in the notochord sheath
    Wang, S. and Kryvi, H. and Grotmol, S. and Wargelius, A. and Krossoy, C. and Epple, M. and Neues, F. and Furmanek, T. and Totland, G. K.
    Journal of Anatomy 223 (2013)
    We performed a sequential morphological and molecular biological study of the development of the vertebral bodies in Atlantic salmon (Salmo salar L.). Mineralization starts in separate bony elements which fuse to form complete segmental rings within the notochord sheath. The nucleation and growth of hydroxyapatite crystals in both the lamellar type II collagen matrix of the notochord sheath and the lamellar type I collagen matrix derived from the sclerotome, were highly similar. In both matrices the hydroxyapatite crystals nucleate and accrete on the surface of the collagen fibrils rather than inside the fibrils, a process that may be controlled by a template imposed by the collagen fibrils. Apatite crystal growth starts with the formation of small plate-like structures, about 5 nm thick, that gradually grow and aggregate to form extensive multi-branched crystal arborizations, resembling dendritic growth. The hydroxyapatite crystals are always oriented parallel to the long axis of the collagen fibrils, and the lamellar collagen matrices provide oriented support for crystal growth. We demonstrate here for the first time by means of synchroton radiation based on X-ray diffraction that the chordacentra contain hydroxyapatite. We employed quantitative real-time PCR to study the expression of key signalling molecule transcripts expressed in the cellular core of the notochord. The results indicate that the notochord not only produces and maintains the notochord sheath but also expresses factors known to regulate skeletogenesis: sonic hedgehog (shh), indian hedgehog homolog b (ihhb), parathyroid hormone 1 receptor (pth1r) and transforming growth factor beta 1 (tgfb1). In conclusion, our study provides evidence for the process of vertebral body development in teleost fishes, which is initially orchestrated by the notochord.
    view abstract10.1111/joa.12067
  • Molecular fragment dynamics study on the water-air interface behavior of non-ionic polyoxyethylene alkyl ether surfactants
    Truszkowski, A. and Epple, M. and Fiethen, A. and Zielesny, A. and Kuhn, H.
    Journal of Colloid and Interface Science 410 (2013)
    Molecular fragment dynamics (MFD) is a mesoscopic simulation technique based on dissipative particle dynamics (DPD). MFD simulations of the self-aggregation of the polyoxyethylene alkyl ether surfactants C6E6, C10E6, C12E6 and C16E6 at the water-air surface lead to equilibrium nanoscale structures and computationally determined surface tensions which are in agreement with experimental data for different surfactant concentrations. Thus, molecular fragment dynamics is a well-suited predictive technique to study the behavior of new surfactant systems. © 2013 Elsevier Inc.
    view abstract10.1016/j.jcis.2013.07.069
  • Nanoparticle-mediated gene transfer from electrophoretically coated metal surfaces
    Kovtun, A. and Neumann, S. and Neumeier, M. and Urch, H. and Heumann, R. and Gepp, M.M. and Wallat, K. and Koeller, M. and Zimmermann, H. and Epple, M.
    Journal of Physical Chemistry B 117 (2013)
    The transfer of genetic information into living cells is a powerful tool to manipulate their protein expression by the regulation of protein synthesis. This can be used for the treatment of genetically caused diseases (gene therapy). However, the systemic application of genes is associated with a number of problems, such as a targeted gene delivery and potential side effects. Here we present a method for the spatial application of nanoparticle-based gene therapy. Titanium was electrophoretically coated with DNA-functionalized calcium phosphate nanoparticles. NIH3T3 cells and HeLa cells were transfected with pcDNA3-EGFP. We monitored the transfection in vitro by fluorescence microscopy, flow cytometry, and Western Blot analysis. By coating a transparent substrate, i.e., indium tin oxide (ITO), with nanoparticles, we followed the transfection by live cell imaging. © 2012 American Chemical Society.
    view abstract10.1021/jp303448v
  • Nanoparticles for delivery of therapeutical nucleic acids to corneal endothelium
    Czugala, M. and Mykhaylyk, O. and Singer, B. B. and Steuhl, K. P. and Ergun, S. and Wesselborg, S. and Epple, M. and Plank, C. and Fuchsluger, T. A.
    Acta Ophthalmologica 91 (2013)
    view abstract10.1111/j.1755-3768.2013.4234.x
  • Phase and elemental composition of silicon-containing hydroxyapatite-based coatings fabricated by RF-magnetron sputtering for medical implants
    Surmeneva, M.A. and Surmenev, R.A. and Chaikina, M.V. and Kachaev, A.A. and Pichugin, V.F. and Epple, M.
    Inorganic Materials: Applied Research 4 (2013)
    We studied by X-ray diffraction analysis, IR spectroscopy, and scanning electron microscopy (SEM) the phase composition and the structure of coatings based on silicon-containing hydroxyapatite (Si-HA) deposited by RF-magnetron sputtering. The sputtering target contained two phases (apatite and tricalcium phosphate) and was produced by the ceramic technology from a single-phase mechanically activated powder precursor. The structure of the coating deposited by sputtering from the two-phase target was single phase (hydroxyapatite) and textured in the (002) direction. During deposition, silicate anions partially replaced phosphate ions in the apatite lattice. © Pleiades Publishing, Ltd., 2013.
    view abstract10.1134/S2075113313030131
  • Physical, chemical and biological properties of micro-arc deposited calcium phosphate coatings on titanium and zirconium-niobium alloy
    Legostaeva, E.V. and Kulyashova, K.S. and Komarova, E.G. and Epple, M. and Sharkeev, Y.P. and Khlusov, I.A.
    Materialwissenschaft und Werkstofftechnik 44 (2013)
    A comparative investigation of the physical, chemical and biological properties of micro-arc deposited calcium phosphate coatings on titanium and zirconium-niobium substrates was performed. Calcium phosphate coatings on titanium have a higher surface density, porosity and pore size, and a more homogeneous surface topography. Under the same conditions, calcium phosphate coatings on zirconium-niobium have a relief topography, but their surface density, porosity and pore size were all smaller. X-ray diffraction of the coatings showed that the coatings on titanium were X-ray amorphous whereas the coatings on zirconium-niobium consisted of a mixture of crystalline CaZr 4(PO4)6, ZrP2O7, and ZrO2. These differences are due to different electrical and thermophysical characteristics of substrates and passivating films on their surfaces. The coatings were shown to be biocompatible by in-vitro cell culture experiments. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/mawe.201300107
  • Preparation of a silicate-containing hydroxyapatite-based coating by magnetron sputtering: Structure and osteoblast-like MG63 cells in vitro study
    Surmeneva, M.A. and Kovtun, A. and Peetsch, A. and Goroja, S.N. and Sharonova, A.A. and Pichugin, V.F. and Grubova, I.Y. and Ivanova, A.A. and Teresov, A.D. and Koval, N.N. and Buck, V. and Wittmar, A. and Ulbricht, M. and Prymak, O. and Epple, M. and Surmenev, R.A.
    RSC Advances 3 (2013)
    Silicate-containing hydroxyapatite-based coatings with different structure and calcium/phosphate ratios were prepared by radio-frequency magnetron sputtering on silicon and titanium substrates, respectively. Scanning electron microscopy, X-ray diffraction and IR spectroscopy were used to investigate the effect of the substrate bias on the properties of the silicate-containing hydroxyapatite-based coatings. The deposition rate, composition, and microstructure of the deposited coatings were all controlled by changing the bias voltage from grounded (0 V) to -50 and -100 V. The biocompatibility was assessed by cell culture with human osteoblast-like cells (MG-63 cell line), showing a good biocompatibility and cell growth on the substrates. © 2013 The Royal Society of Chemistry.
    view abstract10.1039/c3ra40446c
  • Silver as antibacterial agent: Ion, nanoparticle, and metal
    Chernousova, S. and Epple, M.
    Angewandte Chemie - International Edition 52 (2013)
    The antibacterial action of silver is utilized in numerous consumer products and medical devices. Metallic silver, silver salts, and also silver nanoparticles are used for this purpose. The state of research on the effect of silver on bacteria, cells, and higher organisms is summarized. It can be concluded that the therapeutic window for silver is narrower than often assumed. However, the risks for humans and the environment are probably limited. Silver shield: Silver is used in different forms as an antibacterial agent. Earlier, sparingly soluble silver salts were predominantly used, but today, silver nanoparticles (see picture for an SEM image of cubic silver nanoparticles) are gaining increasing importance. As silver is also toxic towards mammalian cells, there is the question of the therapeutic window in the cases of consumer products and medical devices. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/anie.201205923
  • Silver-doped calcium phosphate nanoparticles: Synthesis, characterization, and toxic effects toward mammalian and prokaryotic cells
    Peetsch, A. and Greulich, C. and Braun, D. and Stroetges, C. and Rehage, H. and Siebers, B. and Köller, M. and Epple, M.
    Colloids and Surfaces B: Biointerfaces 102 (2013)
    Spherical silver-doped calcium phosphate nanoparticles were synthesized in a co-precipitation route from calcium nitrate/silver nitrate and ammonium phosphate in a continuous process and colloidally stabilized by carboxymethyl cellulose. Nanoparticles with 0.39wt% silver content and a diameter of about 50-60nm were obtained. The toxic effects toward mammalian and prokaryotic cells were determined by viability tests and determination of the minimal inhibitory and minimal bactericidal concentrations (MIC and MBC). Three mammalian cells lines, i.e. human mesenchymal stem cells (hMSC) and blood peripheral mononuclear cells (PBMC, monocytes and T-lymphocytes), and two prokaryotic strains, i.e. Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were used. Silver-doped calcium phosphate nanoparticles and silver acetate showed similar effect toward mammalian and prokaryotic cells with toxic silver concentrations in the range of 1-3μgmL-1. © 2012 Elsevier B.V.
    view abstract10.1016/j.colsurfb.2012.09.040
  • The structure of an rf-magnetron sputter-deposited silicate-containinghydroxyapatite-based coating investigated by high-resolution techniques
    Surmeneva, M.A. and Chaikina, M.V. and Zaikovskiy, V.I. and Pichugin, V.F. and Buck, V. and Prymak, O. and Epple, M. and Surmenev, R.A.
    Surface and Coatings Technology 218 (2013)
    A biocompatible nanostructured silicate-containing hydroxyapatite-based (Si-HA) thin coatingwas deposited by radio-frequency (RF) magnetron sputtering on silicon and titanium substrates. The morphology of the Si-HA coating was pore-free, dense and followed the topography of the underlying substrates. Energy-dispersive X-ray spectroscopy (EDX) gave molar Ca/P and Ca/(P+Si) ratios of 1.78 and 1.45, respectively. According to XRD-analysis, the coatingwas nanocrystallinewith a crystallite size in the range of 10-50 nm. The ultrastructure of the coating was analyzed by high-resolution transmission electron spectroscopy (HRTEM) combinedwith fast Fourier transform (FFT) analysis. The average crystallite size calculated by the Rietveld method was in good agreement with the HRTEM results. Moreover, HRTEM-observations indicated the presence of atomic layer misorientations originating from imperfections between the nanocrystals in the coating. The average coating nanohardness (11.6±1.7 GPa) was significantly higher than that of the uncoated Ti substrate (4.0±0.3 GPa), whereas no significant difference between the Young'smodulus of the coating (125±20 GPa) and the substrate (115±10 GPa) was found. Immersion of the coated substrates in simulated body fluid (SBF) led to the deposition of an apatite layer. © 2013 Elsevier B.V.
    view abstract10.1016/j.surfcoat.2012.12.023
  • Transport of supramolecular drugs across the cell membrane by calcium phosphate nanoparticles
    Rotan, O. and Sokolova, V. and Gilles, P. and Hu, W. and Dutt, S. and Schrader, T. and Epple, M.
    Materialwissenschaft und Werkstofftechnik 44 (2013)
    Many target sites of synthetic supramolecular drug molecules are located inside cells. Since larger and highly charged molecules are typically not able to cross the cell membrane on their own, an efficient carrier is needed. Calcium phosphate nanoparticles were loaded with different artificial protein and DNA binders, i. e. a polyfunctional anionic polymer, a cationic calixarene dimer and amphiphilic molecular tweezers. The loading of calcium phosphate nanoparticles with these molecules was quantitatively determined by UV spectroscopy. As visualized by fluorescence microscopy and confocal laser scanning microscopy (CLSM), the functionalized calcium phosphate nanoparticles were easily taken up by HeLa cells together with their cargo. In contrast, the dissolved molecules alone were not able to penetrate the cell membrane. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/mawe.201300085
  • A new tool for the transfection of corneal endothelial cells: Calcium phosphate nanoparticles
    Hu, J. and Kovtun, A. and Tomaszewski, A. and Singer, B.B. and Seitz, B. and Epple, M. and Steuhl, K.-P. and Ergün, S. and Fuchsluger, T.A.
    Acta Biomaterialia 8 (2012)
    Calcium phosphate nanoparticles (CaP-NP) are ideal tools for transfection due to their high biocompatibility and easy biodegradability. After transfection these particles dissociate into calcium and phosphate ions, i.e. physiological components found in every cell, and it has been shown that the small increase in intracellular calcium level does not affect cell viability. CaP-NP functionalized with pcDNA3-EGFP (CaP/DNA/CaP/DNA) and stabilized using different amounts of poly(ethylenimine) (PEI) were prepared. Polyfect®-pcDNA3-EGFP polyplexes served as a positive control. The transfection of human and murine corneal endothelial cells (suspensions and donor tissue) was optimized by varying the concentration of CaP-NP and the duration of transfection. The transfection efficiency was determined as EGFP expression detected by flow cytometry and fluorescence microscopy. To evaluate the toxicity of the system the cell viability was detected by TUNEL staining. Coating with PEI significantly increased the transfection efficiency of CaP-NP but decreased cell viability, due to the cytotoxic nature of PEI. The aim of this study was to develop CaP-NP with the highest possible transfection efficiency accompanied by the least apoptosis in corneal endothelial cells. EGFP expression in the tissues remained stable as corneal endothelial cells exhibit minimal proliferative capacity and very low apoptosis after transfection with CaP-NP. In summary, CaP-NP are suitable tools for the transfection of corneal endothelial cells. As CaP-NP induce little apoptosis these nanoparticles offer a safe alternative to viral transfection agents. © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
    view abstract10.1016/j.actbio.2011.09.013
  • Biocompatible nanostructured coatings based on calcium phosphates prepared by means of rf-magnetron sputtering deposition
    Surmeneva, M. and Surmenev, R. and Pichugin, V. and Ivanova, A. and Grubova, I. and Chaikina, M. and Khlusov, I. and Kovtun, A. and Epple, M.
    Proceedings - 2012 7th International Forum on Strategic Technology, IFOST 2012 (2012)
    The aim of this study was to prevent the problems associated with implants failure. Biocompatible nanostructured thin films of either Si- or Ag-containing non-stoichiometric hydroxyapatite (HA) were deposited by method of radio-frequency (rf) magnetron sputtering. Plates of Ti, Ti6Al4V and 316 L SS were used as substrates. The thin coatings were characterized by EDX, ESEM, XRD, IR spectroscopy, HRTEM, nanoindentation and scratch-test. HRTEM observations of the coatings showed a nanocrystalline structure mixed with amorphous regions. It was found that the morphology, structure and the preferred orientation of the films are greatly affected by the parameters of deposition (rf-power, substrate temperature and voltage bias). The as-deposited modified CaP-based coatings are dense, pore-free and their composition resembles that of the precursor target composition. The Si- and Ag- containing HA coatings had a hardness of 10-12 GPa. A low rf-power (30 W) resulted in amorphous or low crystalline CaP coating structure. An increase in rf-power (> 200 W) induced the coating crystallization. The occurrence of the different structure types is described as function of the bias voltage and temperatures. The negative substrate bias allowed to vary the Ca/P ratio in the range of 1.53 to 4. In vitro biocompatibility assessments of the films using the MG63 osteoblast-like cells indicated excellent cell adherence and surface colonization. Si-containing rf-magnetron films promote osteogenic differentiation of human stromal stem cells in vitro. The coatings are prospective to be used in clinical practice: in stomatology or craniofacial medicine, where the leaching of toxic ions from the substrate is necessary or the initial material surface porosity for a further bone in growth should be preserved. © 2012 IEEE.
    view abstract10.1109/IFOST.2012.6357526
  • Calcium phosphate nanoparticles as versatile carrier for small and large molecules across cell membranes
    Sokolova, V. and Rotan, O. and Klesing, J. and Nalbant, P. and Buer, J. and Knuschke, T. and Westendorf, A.M. and Epple, M.
    Journal of Nanoparticle Research 14 (2012)
    The successful transport of molecules across the cell membrane is a key point in biology and medicine. In most cases, molecules alone cannot penetrate the cell membrane, therefore an efficient carrier is needed. Calcium phosphate nanoparticles (diameter: 100-250 nm, depending on the functionalization) were loaded with fluorescent oligonucleotides, peptide, proteins, antibodies, polymers or porphyrins and characterized by dynamic light scattering, nanoparticle tracking analysis and scanning electron microscopy. Any excess of molecules was removed by ultracentrifugation, and the dissolved molecules at the same concentration were used as control. The uptake of such fluorescence-labeled nanoparticles into HeLa cells was monitored by fluorescence microscopy and confocal laser scanning microscopy. Calcium phosphate nanoparticles were able to transport all molecules across the cell membrane, whereas the dissolved molecules alone were taken up only to a very small extent or even not at all. © 2012 Springer Science+Business Media B.V.
    view abstract10.1007/s11051-012-0910-9
  • Cell targeting by antibody-functionalized calcium phosphate nanoparticles
    Kozlova, D. and Chernousova, S. and Knuschke, T. and Buer, J. and Westendorf, A.M. and Epple, M.
    Journal of Materials Chemistry 22 (2012)
    Calcium phosphate nanoparticles were coated with a shell of silica and covalently functionalized by silanization, either with thiol or with amino groups. This permits the covalent attachment of molecules like dyes or antibodies. Between the calcium phosphate surface and the outer silica shell, biomolecules like nucleic acids (DNA or siRNA) can be incorporated as cargo. This leads to cell-specific carriers of biomolecules into cells, e.g. for transfection, gene silencing or cell activation. The cellular uptake of antibody-coated calcium phosphate nanoparticles was demonstrated on two cell lines: HeLa (epithelial cell line) and MG-63 (osteoblast-like cell line). Furthermore, the functionalization of calcium phosphate nanoparticles with a dendritic cell-specific antibody (CD11c) led to a cell-specific targeting as shown with primary murine splenocytes. Thus, the successful coating of calcium phosphate nanoparticles with cell-specific antibodies makes them suitable for many clinical applications. © 2012 The Royal Society of Chemistry.
    view abstract10.1039/c1jm14683a
  • Cellular reactions toward nanostructured silicon surfaces created by laser ablation
    Wallat, K. and Dörr, D. and Le Harzic, R. and Stracke, F. and Sauer, D. and Neumeier, M. and Kovtun, A. and Zimmermann, H. and Epple, M.
    Journal of Laser Applications 24 (2012)
    Silicon wafers were structured with a femtosecond laser on the cm 2 scale with high spatial frequency laser-induced periodic surface structures. These areas are characterized by regular parallel ripples with a period of the order of 100 nm. The particular ripple spacing is determined by the illumination wavelength of the tunable femtosecond laser. The cellular reaction to the structured silicon wafers and to the same materials, coated with calcium phosphate nanoparticles by electrophoretic deposition, was studied using L929 fibroblasts, human mesenchymal stem cells, and epithelial cells. The cells adhered uniformly to structured and unprocessed areas after seeding but significantly preferred the unstructured silicon after 48 h. This behavior disappeared after coating the structured surface with calcium phosphate nanoparticles. © 2012 Laser Institute of America.
    view abstract10.2351/1.4732594
  • Chlorhexidine-loaded calcium phosphate nanoparticles for dental maintenance treatment: Combination of mineralising and antibacterial effects
    Kovtun, A. and Kozlova, D. and Ganesan, K. and Biewald, C. and Seipold, N. and Gaengler, P. and Arnold, W.H. and Epple, M.
    RSC Advances 2 (2012)
    One of the main problems in dental medicine is the growth of bacterial biofilms on tooth surfaces which cause caries and periodontitis. We have developed a new system for oral hygiene and dental treatment that consists of either a paste or a rinsing solution containing calcium phosphate nanoparticles, functionalized with the antibacterial agent chlorhexidine. As calcium phosphate is the natural component of tooth mineral, it can lead to the remineralization of damaged enamel, while chlorhexidine prevents the colonization of the tooth surface by bacteria. In the form of a paste, a bifunctional system with both mineralizing and antibacterial properties is obtained. The nanoparticles may also stick to open dentin tubules at the root surface due to their coating with carboxymethyl cellulose. In vitro studies on teeth show that the paste sticks well to the root surface and closes dentin tubules. © 2012 The Royal Society of Chemistry.
    view abstract10.1039/c1ra00955a
  • Direct experimental observation of the aggregation of α-amino acids into 100-200 nm clusters in aqueous solution
    Hagmeyer, D. and Ruesing, J. and Fenske, T. and Klein, H.-W. and Schmuck, C. and Schrader, W. and Piedade, M.E.M.D. and Epple, M.
    RSC Advances 2 (2012)
    Spherical supramolecular aggregates of α-amino acids with a typical diameter of 100-200 nm are formed spontaneously after dissolution in water at a concentration of a few mM, i.e. well below the solubility limit. Their presence was shown by nanoparticle tracking analysis (NTA), atomic force microscopy (AFM), and ESI mass spectrometry (ESI-MS). There is a dynamic equilibrium between the aggregates and dissolved individual molecules which allows them to penetrate through dialysis membranes and filters. The same phenomenon was observed for para-amino salicylic acid and two dipeptides. Thermodynamic considerations suggest an entropy-controlled process. © 2012 The Royal Society of Chemistry.
    view abstract10.1039/c2ra01352e
  • Freeze-dried cationic calcium phosphate nanorods as versatile carriers of nucleic acids (DNA, siRNA)
    Klesing, J. and Chernousova, S. and Epple, M.
    Journal of Materials Chemistry 22 (2012)
    Functionalized calcium phosphate (hydroxyapatite) nanorods were freeze-dried in the presence of the cryoprotectant trehalose, giving a storable and easily redispersible system which can adsorb nucleic acids for transfection and gene silencing. The nanorods were first surface-functionalized with a layer of polyethyleneimine (PEI), purified by ultracentrifugation and redispersion, and freeze-dried in the presence of trehalose. The nanorods can be easily redispersed in water. The concentration-dependent adsorption of nucleic acids (DNA and siRNA) onto the surface of the redispersed cationic nanorods was measured by dynamic light scattering (particle diameter and zeta potential). The transfection efficiency on epithelial cells (HeLa) and osteoblasts (MG-63) was systematically determined for increasing amounts of added nucleic acid, up to a charge reversal by the anionic nucleic acids. Both transfection and gene silencing efficiency increased with increasing amount of nucleic acid, but went through minimum around the point of zero charge where the particles agglomerated. The application of an additional outer layer of polyethyleneimine around the hydroxyapatite/PEI/nucleic acid-nanoparticles reversed the charge back to positive, resulting in even higher transfection rates with almost complete cell viability.
    view abstract10.1039/c1jm13502c
  • Gene therapy in ophthalmology: State of the art
    Kovtun, A. and Epple, M. and Steuhl, K.P. and Erguen, S. and Fuchsluger, T.
    Klinische Monatsblatter fur Augenheilkunde 229 (2012)
    The term gene therapyo" denotes the treatment of diseases or gene deficiencies by introduction of genes into cells. To achieve this goal, vectors are used to transfer the genetic information into the cells. Thus, the protein of interest can be overexpressed or silenced. On account of its easy accessibility, the good compartmentalisation and the separation from the main bloodstream by the blood-retina barrier, the eye represents a very attractive target to treat ocular diseases by gene therapy. In this work, we provide an overview of the progress in ocular gene therapy over the last decade and give an outlook on future developments. © Georg Thieme Verlag KG Stuttgart · New York.
    view abstract10.1055/s-0032-1312914
  • Nanoscopic NaCl crystals as water-soluble porogens for polymer membranes
    Range, S. and Epple, M.
    RSC Advances 2 (2012)
    Nanoscopic NaCl was prepared by elimination from sodium malonate and phenacyl chloride with a particle diameter of 100 nm. The NaCl crystals were added to either poly(d,l)lactide or polysulfone (dissolved in dichloromethane) as water-soluble porogens. The dispersion was applied to silicon wafers by dip-coating. After drying in air, the NaCl crystals were removed by washing with water, leaving behind a porous membrane with pore diameters around 100 nm. These free-standing membranes were prepared with a thickness from 1 to 10 μm. They can be used to separate small molecules from larger molecules like proteins. © 2012 The Royal Society of Chemistry.
    view abstract10.1039/c2ra01237e
  • Quantifying the influence of polymer coatings on the serum albumin corona formation around silver and gold nanoparticles
    Treuel, L. and Malissek, M. and Grass, S. and Diendorf, J. and Mahl, D. and Meyer-Zaika, W. and Epple, M.
    Journal of Nanoparticle Research 14 (2012)
    When nanoparticles (NPs) come into contact with biological fluids, proteins, and other biomolecules interact with their surface. Upon exposure to biological fluids a layer of proteins adsorbs onto their surface, the so-called protein corona, and interactions of biological systems with NPs are therefore mediated by this corona. Here, interactions of serum albumin with silver and gold NPs were quantitatively investigated using circular dichroism spectroscopy. Moreover, surface enhanced Raman spectroscopy was used for further elucidation of protein binding to silver surfaces. The decisive role of poly(vinylpyrrolidone), coatings on the protein adsorption was quantitatively described for the first time and the influential role of the polymer coatings is discussed. Research in nanotoxicology may benefit from such molecular scale data as well as scientific approaches seeking to improve nanomedical applications by using a wide range of polymer surface coatings to optimize biological transport and medical action of NPs. © Springer Science+Business Media B.V. 2012.
    view abstract10.1007/s11051-012-1102-3
  • Risks and side effects: Breast implants
    Sokolova, V. and Epple, M.
    Chemie in Unserer Zeit 46 (2012)
    Breast implants are applied in a larger number each year. To prevent an adverse interaction with the body, bioinert materials are used for the fabrication, with silicones playing a major role both for the shell and the filling. Physiological saline solution is also used as biocompativble filling material. As long as biomaterials with a high purity are used, the application of breast implants can be considered as medically safe. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/ciuz.201200584
  • Silver, gold, and alloyed silver-gold nanoparticles: Characterization and comparative cell-biologic action
    Mahl, D. and Diendorf, J. and Ristig, S. and Greulich, C. and Li, Z.A. and Farle, M. and Köller, M. and Epple, M.
    Journal of Nanoparticle Research 14 (2012)
    Silver, gold, and silver-gold-alloy nanoparticles were prepared by citrate reduction modified by the addition of tannin during the synthesis, leading to a reduction in particle size by a factor of three. Nanoparticles can be prepared by this easy waterbased synthesis and subsequently functionalized by the addition of either tris(3-sulfonatophenyl)phosphine or poly(N-vinylpyrrolidone). The resulting nanoparticles of silver (diameter 15-25 nm), gold (5-6 nm), and silver-gold (50:50; 10-12 nm) were easily dispersable in water and also in cell culture media (RPMI + 10 % fetal calf serum), as shown by nanoparticle tracking analysis and differential centrifugal sedimentation. High-resolution transmission electron microscopy showed a polycrystalline nature of all nanoparticles. EDX on single silver-gold nanoparticles indicated that the concentration of gold is higher inside a nanoparticle. The biologic action of the nanoparticles toward human mesenchymal stem cells (hMSC) was different: Silver nanoparticles showed a significant concentration-dependent influence on the viability of hMSC. Gold nanoparticles showed only a small effect on the viability of hMSC after 7 days. Surprisingly, silver-gold nanoparticles had no significant influence on the viability of hMSC despite the silver content. Silver nanoparticles and silver-gold nanoparticles in the concentration range of 5-20 μg mL -1 induced the activation of hMSC as indicated by the release of IL-8. In contrast, gold nanoparticles led to a reduction of the release of IL-6 and IL-8. © Springer Science+Business Media B.V. 2012.
    view abstract10.1007/s11051-012-1153-5
  • Storm in the weather glass: Admiral Fitzroys legendary storm glass
    Kaempfe, P. and Molt, K. and Epple, M.
    Chemie in Unserer Zeit 46 (2012)
    The composition of a weather glass was determined and its capacity to predict the weather analyzed. Whereas the barometric pressure was only of minimal influence, the ambient temperature had a major influence on the state of the weather glass. A cyclic temperature change between two temperatures showed, that the weather glass reversibly formed the same amount of crystals at a given temperature. Chemically, the storm glass is a complex system that is near the temperature-dependent solubility of campher. A prediction of the weather was not possible. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/ciuz.201100563
  • Structure, composition, and mechanical properties of shark teeth
    Enax, J. and Prymak, O. and Raabe, D. and Epple, M.
    Journal of Structural Biology 178 (2012)
    The teeth of two different shark species (Isurus oxyrinchus and Galeocerdo cuvier) and a geological fluoroapatite single crystal were structurally and chemically characterized. In contrast to dentin, enameloid showed sharp diffraction peaks which indicated a high crystallinity of the enameloid. The lattice parameters of enameloid were close to those of the geological fluoroapatite single crystal. The inorganic part of shark teeth consisted of fluoroapatite with a fluoride content in the enameloid of 3.1 wt.%, i.e., close to the fluoride content of the geological fluoroapatite single crystal (3.64 wt.%). Scanning electron micrographs showed that the crystals in enameloid were highly ordered with a special topological orientation (perpendicular towards the outside surface and parallel towards the center). By thermogravimetry, water, organic matrix, and biomineral in dentin and enameloid of both shark species were determined. Dentin had a higher content of water, organic matrix, and carbonate than enameloid but contained less fluoride. Nanoindentation and Vicker's microhardness tests showed that the enameloid of the shark teeth was approximately six times harder than the dentin. The hardness of shark teeth and human teeth was comparable, both for dentin and enamel/enameloid. In contrast, the geological fluoroapatite single crystal was much harder than both kinds of teeth due to the absence of an organic matrix. In summary, the different biological functions of the shark teeth (" tearing" for Isurus and "cutting" for Galeocerdo) are controlled by the different geometry and not by the chemical or crystallographic composition. © 2012 Elsevier Inc.
    view abstract10.1016/j.jsb.2012.03.012
  • The toxic effect of silver ions and silver nanoparticles towards bacteria and human cells occurs in the same concentration range
    Greulich, C. and Braun, D. and Peetsch, A. and Diendorf, J. and Siebers, B. and Epple, M. and Köller, M.
    RSC Advances 2 (2012)
    Silver is commonly used both in ionic form and in nanoparticulate form as a bactericidal agent. This is generally ascribed to a higher toxicity towards prokaryotic cells than towards mammalian cells. Comparative studies with both silver ions (such as silver acetate) and polyvinylpyrrolidone (PVP)-stabilized silver nanoparticles (70 nm) showed that the toxic effect of silver occurs in a similar concentration range for Escherichia coli, Staphylococcus aureus, human mesenchymal stem cells (hMSCs), and peripheral blood mononuclear cells (PBMCs), i.e. 0.5 to 5 ppm for silver ions and 12.5 to 50 ppm for silver nanoparticles. For a better comparison, bacteria were cultivated both in Lysogeny broth medium (LB) and in Roswell Park Memorial Institute medium (RPMI)/10% fetal calf serum (FCS) medium, as the state of silver ions and silver nanoparticles may be different due to the presence of salts, and biomolecules like proteins. The effective toxic concentration of silver towards bacteria and human cells is almost the same. © 2012 The Royal Society of Chemistry.
    view abstract10.1039/c2ra20684f
  • Upregulation of metallothioneins after exposure of cultured primary astrocytes to silver nanoparticles
    Luther, E.M. and Schmidt, M.M. and Diendorf, J. and Epple, M. and Dringen, R.
    Neurochemical Research 37 (2012)
    To test for the prolonged consequences of a short transient exposure of astrocytes to silver nanoparticles (AgNP), cultured primary astrocytes were incubated for 4 h in the presence of AgNP and the cell viability as well as various metabolic parameters were investigated during a subsequent incubation in AgNP-free medium. Acute exposure of astrocytes to AgNP led to a concentration-dependent increase in the specific cellular silver content to up to 46 nmol/mg protein, but did not compromise cell viability. During a subsequent incubation of the cells in AgNP-free medium, the cellular silver content of AgNPtreated astrocytes remained almost constant for up to 7 days. The cellular presence of AgNP did neither induce any delayed cell toxicity nor were alterations in cellular glucose consumption, lactate production or in the cellular ratio of glutathione to glutathione disulfide observed. However, Western blot analysis and immunocytochemical staining revealed that AgNP-treated astrocytes strongly upregulated the expression of metallothioneins. These results demonstrate that a prolonged presence of accumulated AgNP does not compromise the viability and the basal metabolism of cultured astrocytes and suggest that the upregulation of metallothioneins may help to prevent silver-mediated toxicity that could be induced by AgNPderived silver ions. © Springer Science+Business Media, LLC 2012.
    view abstract10.1007/s11064-012-0767-4
  • Accumulation of silver nanoparticles by cultured primary brain astrocytes
    Luther, E.M. and Koehler, Y. and Diendorf, J. and Epple, M. and Dringen, R.
    Nanotechnology 22 (2011)
    Silver nanoparticles (AgNP) are components of various food industry products and are frequently used for medical equipment and materials. Although such particles enter the vertebrate brain, little is known on their biocompatibility for brain cells. To study the consequences of an AgNP exposure of brain cells we have treated astrocyte-rich primary cultures with polyvinylpyrrolidone (PVP)-coated AgNP. The incubation of cultured astrocytes with micromolar concentrations of AgNP for up to 24h resulted in a time-and concentration-dependent accumulation of silver, but did not compromise the cell viability nor lower the cellular glutathione content. In contrast, the incubation of astrocytes for 4h with identical amounts of silver as AgNO 3 already severely compromised the cell viability and completely deprived the cells of glutathione. The accumulation of AgNP by astrocytes was proportional to the concentration of AgNP applied and significantly lowered by about 30% in the presence of the endocytosis inhibitors chloroquine or amiloride. Incubation at 4 °C reduced the accumulation of AgNP by 80% compared to the values obtained for cells that had been exposed to AgNP at 37 °C. These data demonstrate that viable cultured brain astrocytes efficiently accumulate PVP-coated AgNP in a temperature-dependent process that most likely involves endocytotic pathways. © 2011 IOP Publishing Ltd.
    view abstract10.1088/0957-4484/22/37/375101
  • Amorphous and crystalline calcium carbonate distribution in the tergite cuticle of moulting Porcellio scaber (Isopoda, Crustacea)
    Neues, F. and Hild, S. and Epple, M. and Marti, O. and Ziegler, A.
    Journal of Structural Biology 175 (2011)
    The main mineral components of the isopod cuticle consists of crystalline magnesium calcite and amorphous calcium carbonate. During moulting isopods moult first the posterior and then the anterior half of the body. In terrestrial species calcium carbonate is subject to resorption, storage and recycling in order to retain significant fractions of the mineral during the moulting cycle. We used synchrotron X-ray powder diffraction, elemental analysis and Raman spectroscopy to quantify the ACC/calcite ratio, the mineral phase distribution and the composition within the anterior and posterior tergite cuticle during eight different stages of the moulting cycle of Porcellio scaber. The results show that most of the amorphous calcium carbonate (ACC) is resorbed from the cuticle, whereas calcite remains in the old cuticle and is shed during moulting. During premoult resorption of ACC from the posterior cuticle is accompanied by an increase within the anterior tergites, and mineralization of the new posterior cuticle by resorption of mineral from the anterior cuticle. This suggests that one reason for using ACC in cuticle mineralization is to facilitate resorption and recycling of cuticular calcium carbonate. Furthermore we show that ACC precedes the formation of calcite in distal layers of the tergite cuticle. © 2011 Elsevier Inc.
    view abstract10.1016/j.jsb.2011.03.019
  • Cell type-specific responses of peripheral blood mononuclear cells to silver nanoparticles
    Greulich, C. and Diendorf, J. and Geßmann, J. and Simon, T. and Habijan, T. and Eggeler, G. and Schildhauer, T.A. and Epple, M. and Köller, M.
    Acta Biomaterialia 7 (2011)
    Silver nanoparticles (Ag-NP) are increasingly used in biomedical applications because of their remarkable antimicrobial activity. In biomedicine, Ag-NP are coated onto or embedded in wound dressings, surgical instruments and bone substitute biomaterials, such as silver-containing calcium phosphate cements. Free Ag-NP and silver ions are released from these coatings or after the degradation of a biomaterial, and may come into close contact with blood cells. Despite the widespread use of Ag-NP as an antimicrobial agent, there is a serious lack of information on the biological effects of Ag-NP on human blood cells. In this study, the uptake of Ag-NP by peripheral monocytes and lymphocytes (T-cells) was analyzed, and the influence of nanosilver on cell biological functions (proliferation, the expression of adhesion molecules, cytokine release and the generation of reactive oxygen species) was studied. After cell culture in the presence of monodispersed Ag-NP (5-30 μg ml -1 silver concentration), agglomerates of nanoparticles were detected within monocytes (CD14+) but not in T-cells (CD3+) by light microscopy, flow cytometry and combined focused ion beam/scanning electron microscopy. The uptake rate of nanoparticles was concentration dependent, and the silver agglomerates were typically found in the cytoplasm. Furthermore, a concentration-dependent activation (e.g. an increased expression of adhesion molecule CD54) of monocytes at Ag-NP concentrations of 10-15 μg ml -1 was observed, and cytotoxicity of Ag-NP-treated monocytes was observed at Ag-NP levels of 25 μg ml -1 and higher. However, no modulation of T-cell proliferation was observed in the presence of Ag-NP. Taken together, our results provide the first evidence for a cell-type-specific uptake of Ag-NP by peripheral blood mononuclear cells (PBMC) and the resultant cellular responses after exposure. © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
    view abstract10.1016/j.actbio.2011.05.030
  • Characterisation of exosomes derived from human cells by nanoparticle tracking analysis and scanning electron microscopy
    Sokolova, V. and Ludwig, A.-K. and Hornung, S. and Rotan, O. and Horn, P.A. and Epple, M. and Giebel, B.
    Colloids and Surfaces B: Biointerfaces 87 (2011)
    Exosomes from three different cell types (HEK 293T, ECFC, MSC) were characterised by scanning electron microscopy (SEM), dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). The diameter was around 110. nm for the three cell types. The stability of exosomes was examined during storage at -20 °C, 4 °C, and 37 °C. The size of the exosomes decreased at 4 °C and 37 °C, indicating a structural change or degradation. Multiple freezing to -20 °C and thawing did not affect the exosome size. Multiple ultracentrifugation also did not change the exosome size. © 2011 Elsevier B.V.
    view abstract10.1016/j.colsurfb.2011.05.013
  • Characterization of the solid components of three desensitizing toothpastes and a mouth wash
    Peetsch, A. and Epple, M.
    Materialwissenschaft und Werkstofftechnik 42 (2011)
    The solid components of three toothpastes and a mouth wash which are intended to enhance the remineralization of teeth and occlusion of dentinal tubuli were isolated and analyzed. Samples of the toothpaste BioRepair®, the mouth wash BioRepair®, the toothpaste nanosensitive® hca, and the toothpaste Theramed® S.O.S. Sensitive were characterized by dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), X-ray powder diffraction (XRD), atomic absorption spectroscopy (AAS), thermogravimetric analysis (TG), and elemental analysis. All samples contained primary particles in the size range of 30-60 nm, that were all agglomerated in aqueous dispersion to particles in the size range of 200 to 400 nm. BioRepair® contained a zinc-substituted hydroxyapatite and amorphous silica, nanosensitive®hca contained TiO2 (anatase) and an amorphous Na-Ca-Si-P bioglass, and Theramed® S.O.S. Sensitive contained TiO2 (anatase), amorphous silica and traces of a calcium-containing phase. The size of the mineral particles was in all cases suitable to fit into dentinal tubuli, especially after breaking up the agglomerated nanoparticles by mechanical forces, e. g. during tooth brushing. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/mawe.201100744
  • Chemical composition of surface-functionalized gold nanoparticles
    Rostek, A. and Mahl, D. and Epple, M.
    Journal of Nanoparticle Research 13 (2011)
    The composition of surface-functionalized gold nanoparticles (diameter of the metallic core: 17-20 nm) was determined by elemental analysis (C, H, N, S, Au, Na) after preparation of a larger batch. Gold nanoparticles were prepared and functionalized with citrate according to the classical Turkevich method. The citrate-functionalized nanoparticles contained about 3.1 wt% of organic material (135 ng cm-2 or 3.1 molecules nm-2). A partial exchange of citrate was accomplished by tris(sodium-m-sulfonato-phenyl)phosphine (TPPTS) which led to 2.1 wt% of citrate (90 ng cm-2 or 2.1 molecules nm-2) and 1.4 wt% TPPTS (61 ng cm-2 or 0.6 molecules nm-2). The citrate coating was quantitatively exchanged by poly(N-vinyl pyrrolidone) (PVP) after immersion in solutions with concentrations of 33, 66 and 128 mg L-1, respectively, leading to contents of 4 to 6 wt% of PVP (171-271 ng cm-2 or 9-15 PVP monomer units nm -2). © 2011 Springer Science+Business Media B.V.
    view abstract10.1007/s11051-011-0456-2
  • Comparison of the statolith structures of Chironex fleckeri (Cnidaria, Cubozoa) and Periphylla periphylla (Cnidaria, Scyphozoa): A phylogenetic approach
    Sötje, I. and Neues, F. and Epple, M. and Ludwig, W. and Rack, A. and Gordon, M. and Boese, R. and Tiemann, H.
    Marine Biology 158 (2011)
    The rhopalia and statocysts of Periphylla periphylla (Péron and Lesueur in Ann Mus Hist Nat Marseille 14:316-366,1809) and Chironex fleckeri Southcott (Aust J Mar Freshw Res 7(2):254-280 1956) were examined histologically and showed several homologous characteristics. Differences in sensory area distribution could be connected to a slightly different functionality of equilibrium sensing. In P. periphylla, the statoliths (crystals) grow independently of each other; whereas in C. fleckeri, one large crystal covers the smaller ones. The structures of both statoliths were examined in detail with single-crystal diffraction, microtomography and diffraction contrast tomography. The single compact statolith of C. fleckeri consisted of bassanite as was previously known only for other rhopaliophoran medusae. An origin area with several small oligocrystals was located in the centre of the cubozoan statolith. The origin areas and the accretion of statoliths are similar in both species. Our results lead to the assumption that the single bassanite statolith of C. fleckeri (Cnidaria, Cubozoa) is a progression of the scyphozoan multiplex statolith. It is therefore suggested that the Cubozoa are derived from a scyphozoan ancestor and are a highly developed taxa within the Rhopaliophora. © 2011 Springer-Verlag.
    view abstract10.1007/s00227-011-1637-3
  • Electrophoretic deposition of calcium phosphate nanoparticles on a nanostructured silicon surface
    Epple, M. and Neumeier, M. and Dörr, D. and Leharzic, R. and Sauer, D. and Stracke, F. and Zimmermann, H.
    Materialwissenschaft und Werkstofftechnik 42 (2011)
    Nanostructured silicon surfaces were electrophoretically coated with calcium phosphate nanoparticles. Positively charged calcium phosphate nanoparticles were synthesized by precipitation and then functionalized with poly(ethyleneimine). This permits the electrophoretic deposition on a conductive surface from a dispersion in 2-propanol. The following parameters affected the deposition of the nanoparticles: The deposition potential, the deposition time, and the deposition temperature. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/mawe.201100730
  • Formation of periodic nanoripples on silicon and germanium induced by femtosecond laser pulses
    Le Harzic, R. and Dörr, D. and Sauer, D. and Neumeier, M. and Epple, M. and Zimmermann, H. and Stracke, F.
    Physics Procedia 12 (2011)
    We report on the generation of high spatial frequency LIPSS (HSFL) in silicon and germanium induced by nanojoule femtosecond laser pulses at high repetition rate in air. The periodicity of the ripples is found to be of 125 nm for silicon and of 98 nm for germanium. The formation of these HSFL in silicon and germanium seems to be induced by second harmonic generation (SHG) and the periods agree quite well with a λ/2n-law. Reproducible and accurate patterning of uniform HSFL on surface areas up to 100 mm2 can be performed using a versatile laser processing workstation. © 2011 Published by Elsevier Ltd.
    view abstract10.1016/j.phpro.2011.03.102
  • In-vitro investigation of magnetron-sputtered coatings based on silicon-substituted hydroxyapatite
    Surmeneva, M.A. and Surmenev, R.A. and Pichugin, V.F. and Chernousova, S.S. and Epple, M.
    Journal of Surface Investigation 5 (2011)
    Silicon-containing calcium phosphate (Si-CaP) coatings on titanium and austenite steel substrates have been prepared by method of high-frequency magnetron sputtering. The powder of silicon-containing hydroxyapatite Ca 10(PO 4) 6 - x(SiO 4) x(HO) 2 - x (Si-HA), where x = 0. 5 obtained using a mechanochemical technique, was used as a target material. The obtained coatings were X-ray amorphous; the elemental composition of the coatings depended on the composition of the target to be sputtered. The coatings were heated in air for 3 hours to the temperature 700°C with the aim of changing their structure. The bioactivity of the coatings was studied using in-vitro tests. The solution of the simulated body fluid (SBF) oversaturated with respect to HA was used as a model medium. The phase elemental composition and morphology of the deposited and annealed Si-CaP coatings before and after submersion into the solution were controlled using the methods of X-ray diffraction (XRD), energy-dispersive X-ray analysis (EDAX), and scanning electron microscopy (SEM). According to the XFA and IR-spectroscopy data, heat treatment in the air yields the formation of an apatite-like phase in the coating. Thermostating of "metal + coating" specimens in the solution of simulated body fluid revealed that all obtained coatings were biologically active, and a calcium phosphate layer was formed on the coating surface during mineralization. The annealed coatings show a higher chemical stability under physiological conditions as compared to amorphous coatings. © 2011 Pleiades Publishing, Ltd.
    view abstract10.1134/S1027451011120135
  • Large-area, uniform, high-spatial-frequency ripples generated on silicon using a nanojoule-femtosecond laser at high repetition rate
    Le Harzic, R. and Dorr, D. and Sauer, D. and Neumeier, M. and Epple, M. and Zimmermann, H. and Stracke, F.
    Optics Letters 36 (2011)
    Large-area high-spatial-frequency patterns (HSFLs) of lambda/6 periodicity have been generated by a nanojoule-femtosecond laser scanning technique (80 MHz, 170 fs, 700-950 nm) at the silicon-air interface. The excellent large-area uniformity allowed reproducible and accurate measurements of the periodicity. Variation of experimental parameters as illumination geometry, and pulse energy and number showed no influence on the ripple spacing. A wavelength dependence was observed and compared to current models of HSFL formation. A particular second-armonic model was found to match the results best but needs to take into account transient changes in the refractive index under laser exposure. A second-harmonic mechanism is further supported by direct spectroscopic observation. (C) 2011 Optical Society of America
    view abstract10.1364/OL.36.000229
  • Nanocrystalline calcium-deficient hydroxyapatite prepared by a microwave-assisted solvent-free reaction
    Zyman, Z. and Goncharenko, A. and Rokhmistrov, D. and Epple, M.
    Materialwissenschaft und Werkstofftechnik 42 (2011)
    A microwave-assisted solvent-free synthesis of hydroxyapatite from calcium nitrate and sodium phosphate is reported. The product was characterized with respect to the presence of foreign ions and its thermal behavior. An apatite phase was formed in the mixture after irradiation with 2.45 GHz microwaves for 4 min at 350 or 700 W. Structurally, the product was a nanocrystalline calcium-deficient hydroxyapatite (CDHA) with a Ca/P ratio around 1.5, containing some H2O, HPO4 2- and CO3 2-. After heating to 900°C for 1 h, β-tricalcium phosphate (β-TCP) was formed, demonstrating that this microwave-assisted solvent-free synthesis is well suited to prepare a precursor to bioceramics. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    view abstract10.1002/mawe.201100749
  • Possibilities and limitations of different analytical methods for the size determination of a bimodal dispersion of metallic nanoparticles
    Mahl, D. and Diendorf, J. and Meyer-Zaika, W. and Epple, M.
    Colloids and Surfaces A: Physicochemical and Engineering Aspects 377 (2011)
    Silver nanoparticles (about 70. nm) and gold nanoparticles (about 15. nm) were prepared and colloidally stabilized with poly(vinylpyrrolidone) (PVP). The pure nanoparticles as well as a 1:1 mixture (w:w) were analysed with a variety of methods which probe the size distribution: Scanning electron microscopy, transmission electron microscopy, dynamic light scattering, analytical disc centrifugation, and Brownian motion analysis (nanoparticle tracking analysis). The differences between the methods are highlighted and their ability to distinguish between silver and gold nanoparticles in the mixture is demonstrated. The size distribution data from the different methods were clearly different, therefore it is recommended to apply more than one method to characterize the nanoparticle dispersion. In particular, the smaller particles were undetectable by dynamic light scattering and nanoparticle tracking analysis in the presence of the large particles. For the 1:1 mixture, only electron microscopy and analytical disc centrifugation were able to give quantitative data on the size distribution. On the other hand, it is not possible to make statements about an agglomeration in dispersion with electron microscopy because an agglomeration may also have occurred during the drying process. © 2011 Elsevier B.V.
    view abstract10.1016/j.colsurfa.2011.01.031
  • Quantitative determination of the composition of multi-shell calcium phosphate-oligonucleotide nanoparticles and their application for the activation of dendritic cells
    Sokolova, V. and Knuschke, T. and Buer, J. and Westendorf, A.M. and Epple, M.
    Acta Biomaterialia 7 (2011)
    Biodegradable calcium phosphate nanoparticles as carriers for the immunoactive toll-like receptor ligands CpG and polyinosinic-polycytidylic acid for the activation of dendritic cells (DC) combined with the viral antigen hemagglutinin (HA) were prepared. A purification method based on ultracentrifugation and ultrasonication was developed to separate the nanoparticles from dissolved biomolecules. The number of biomolecules, i.e., oligonucleotides and peptide, incorporated into the nanoparticles was quantitatively determined by UV-spectroscopy, using fluorescent derivatives of the biomolecules. The immunostimulatory effects of purified calcium phosphate nanoparticles on DC were studied, i.e., cytokine production and activation of the cells in terms of the upregulation of surface molecules. Purified calcium phosphate nanoparticles, i.e., without dissolved biomolecules, are capable of inducing adaptive immunity by activation of DC. Immunostimulatory effects of purified calcium phosphate nanoparticles on DC were demonstrated by increased expression of co-stimulatory molecules and MHC II and by cytokine secretion. In addition, DC treated with purified functionalized calcium phosphate nanoparticles induced an antigen-specific T-cell response in vitro. © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
    view abstract10.1016/j.actbio.2011.07.010
  • Rational design of β-sheet ligands against Aβ42- induced toxicity
    Hochdörffer, K. and März-Berberich, J. and Nagel-Steger, L. and Epple, M. and Meyer-Zaika, W. and Horn, A.H.C. and Sticht, H. and Sinha, S. and Bitan, G. and Schrader, T.
    Journal of the American Chemical Society 133 (2011)
    A β-sheet-binding scaffold was equipped with long-range chemical groups for tertiary contacts toward specific regions of the Alzheimer's Aβ fibril. The new constructs contain a trimeric aminopyrazole carboxylic acid, elongated with a C-terminal binding site, whose influence on the aggregation behavior of the Aβ42 peptide was studied. MD simulations after trimer docking to the anchor point (F19/F20) suggest distinct groups of complex structures each of which featured additional specific interactions with characteristic Aβ regions. Members of each group also displayed a characteristic pattern in their antiaggregational behavior toward Aβ. Specifically, remote lipophilic moieties such as a dodecyl, cyclohexyl, or LPFFD fragment can form dispersive interactions with the nonpolar cluster of amino acids between I31 and V36. They were shown to strongly reduce Thioflavine T (ThT) fluorescence and protect cells from Aβ lesions (MTT viability assays). Surprisingly, very thick fibrils and a high β-sheet content were detected in transmission electron microscopy (TEM) and CD spectroscopic experiments. On the other hand, distant single or multiple lysines which interact with the ladder of stacked E22 residues found in Aβ fibrils completely dissolve existing β-sheets (ThT, CD) and lead to unstructured, nontoxic material (TEM, MTT). Finally, the triethyleneglycol spacer between heterocyclic β-sheet ligand and appendix was found to play an active role in destabilizing the turn of the U-shaped protofilament. Fluorescence correlation spectroscopy (FCS) and sedimentation velocity analysis (SVA) provided experimental evidence for some smaller benign aggregates of very thin, delicate structure (TEM, MTT). A detailed investigation by dynamic light scattering (DLS) and other methods proved that none of the new ligands acts as a colloid. The evolving picture for the disaggregation mechanism by these new hybrid ligands implies transformation of well-ordered fibrils into less structured aggregates with a high molecular weight. In the few cases where fibrillar components remain, these display a significantly altered morphology and have lost their acute cellular toxicity. © 2011 American Chemical Society.
    view abstract10.1021/ja107675n
  • Self-assembly of calcium phosphate nanoparticles into hollow spheres induced by dissolved amino acids
    Hagmeyer, D. and Ganesan, K. and Ruesing, J. and Schunk, D. and Mayer, C. and Dey, A. and Sommerdijk, N.A.J.M. and Epple, M.
    Journal of Materials Chemistry 21 (2011)
    Nanoparticles of calcium phosphate assemble spontaneously within a few seconds into hollow spheres with a diameter around 200-300 nm in the presence of dissolved amino acids and dipeptides. The process of formation was followed by cryo-transmission electron microscopy (cryoTEM), proving their hollow nature which was also confirmed by nano-indentation by atomic force microscopy (AFM). The hollow spheres were analyzed by scanning electron microscopy (SEM), dynamic light scattering (DLS), nanoparticle tracking analysis (NTA) and elemental analysis. The hollow spheres were moderately stable against heating and ultrasonication. A self-assembly of the primarily formed calcium phosphate nanoparticles around amino acid-rich domains in water is proposed. As this process was observed with different amino acids, it appears to be a more general phenomenon. © 2011 The Royal Society of Chemistry.
    view abstract10.1039/c1jm11316j
  • Study of physicochemical and biological properties of calcium phosphate coatings prepared by RF magnetron sputtering of silicon-substituted hydroxyapatite
    Pichugin, V.F. and Surmeneva, M.A. and Surmenev, R.A. and Khlusov, I.A. and Epple, M.
    Journal of Surface Investigation 5 (2011)
    Coatings based on pure silicon and silicon-substituted hydroxyapatite were grown by RF magnetron sputtering. The coating surface morphology, phase and elemental composition were studied by scanning electron microscopy, energy-dispersive X-ray analysis, and infrared spectroscopy. It was found that coatings are X-ray-amorphous, their elemental composition being controlled by the sputtered target composition. The distribution of elements over the coating surface is homogeneous. Medical and biological properties of coatings were studied in vivo and in vitro. Osteogenic properties of coatings were studied. Coatings grown by sputtering of a stoichiometric hydroxyapatite target are biocompatible without osteoinductive activity. The introduction of silicate ions into the hydroxyapatite structure that forms an electrode target significantly enhances the in vivo effect of CaP magnetron coatings on the osteogenic activity and stromal bone-marrow stem cells. © 2011 Pleiades Publishing, Ltd.
    view abstract10.1134/S1027451011090138
  • Synthesis of fluorescent core-shell hydroxyapatite nanoparticles
    Neumeier, M. and Hails, L.A. and Davis, S.A. and Mann, S. and Epple, M.
    Journal of Materials Chemistry 21 (2011)
    Lanthanide-doped fluorescent hydroxyapatite/silica core-shell nanorods, 50-100 nm in length and 30 nm in width, were prepared by precipitation of calcium phosphate in the presence of Eu3+ and Y3+ ions at 60 °C, followed by hydrothermally enhanced crystallization, stabilization with poly(ethyleneimine), and reaction with tetraethyl orthosilicate. The fluorescence intensity of the Eu3+-doped hydroxyapatite nanorods was enhanced threefold by co-doping with Y3+ and doubled after hydrothermal treatment. Significantly, fluorescence quenching by water was reduced in the presence of the thin silica nanoshell to give a further doubling of the fluorescence intensity compared with lanthanide-doped hydroxyapatite nanoparticles prepared in the absence of tetraethyl orthosilicate. Our results suggest that a combination of lanthanide doping, controlled crystallization and core-shell fabrication is a promising route to the preparation of biocompatible calcium phosphate nanoparticles with enhanced fluorescence for potential use in biomedical applications. © 2011 The Royal Society of Chemistry.
    view abstract10.1039/c0jm02264k
  • Synthetic pathways to make nanoparticles fluorescent
    Sokolova, V. and Epple, M.
    Nanoscale 3 (2011)
    In biosciences, it is often necessary to follow the pathway of nanoparticles within cells or tissues. The nanoparticles can be used as labeled sensors which may, e.g., address functionalities within a cell, carry other specific agents like drugs or be magnetic for tumor thermotherapy. In the context of nanotoxicology, the fate of a given nanoparticle is of interest. As many methods in cell biology are based on fluorescence detection, there is a strong demand to make nanoparticles fluorescent. Different ways to introduce fluorescence are reviewed and exemplified with typical kinds of nanoparticles, i.e. polymers, silica and calcium phosphate. © 2011 The Royal Society of Chemistry.
    view abstract10.1039/c1nr00002k
  • The influence of the deposition parameters on the properties of an rf-magnetron-deposited nanostructured calcium phosphate coating and a possible growth mechanism
    Surmenev, R.A. and Surmeneva, M.A. and Evdokimov, K.E. and Pichugin, V.F. and Peitsch, T. and Epple, M.
    Surface and Coatings Technology 205 (2011)
    The deposition of a biocompatible calcium phosphate coating on the surface of materials for biomedical implants by rf-magnetron sputtering is reported. The deposition parameters to prepare either stoichiometric crystalline hydroxyapatite or amorphous calcium phosphate coating with a molar Ca/P ratio from 1.53 to 3.88 were established. Crystalline hydroxyapatite coating with a Ca/P ratio of 1.60±0.07 can be deposited if the rf-power density is 0.49W cm-2 and if the samples are arranged within the area of the target erosion zone. A thorough investigation of the influence of rf-power, DC-bias on the substrate, deposition time on the properties of the calcium phosphate coating allowed to formulate a mechanism for the film growth. © 2010 Elsevier B.V.
    view abstract10.1016/j.surfcoat.2010.12.039
  • Ultrastructure and mineral distribution in the tergite cuticle of the beach isopod Tylos europaeus Arcangeli, 1938
    Seidl, B. and Huemer, K. and Neues, F. and Hild, S. and Epple, M. and Ziegler, A.
    Journal of Structural Biology 174 (2011)
    The crustacean cuticle is a hierarchically organised material composed of an organic matrix and mineral. It is subdivided into skeletal elements whose physical properties are adapted to their function and the eco-physiological strains of the animal. Using a variety of ultrastructural and analytical techniques we studied the organisation of the tergite cuticle of the sand burrowing beach isopod Tylos europaeus. The surface of the tergites bear epicuticular scales, sensilla and micro-tubercles. A distal layer of the exocuticle is characterised by a low density of organic fibres and the presence of magnesium-calcite. Surprisingly, the mineral forms regions containing polyhedral structures alternating with smooth areas. Between sub-domains within the distal exocuticle calcite varies in its crystallographic orientation. Proximal layers of the exocuticle and the endocuticle are devoid of calcite and the mineral occurs in the form of amorphous calcium carbonate (ACC). Using thin sections of mineralised cuticle we describe for the first time that ACC forms tubes around single protein-chitin fibrils. © 2011 Elsevier Inc.
    view abstract10.1016/j.jsb.2011.03.005
  • Uptake and intracellular distribution of silver nanoparticles in human mesenchymal stem cells
    Greulich, C. and Diendorf, J. and Simon, T. and Eggeler, G. and Epple, M. and Köller, M.
    Acta Biomaterialia 7 (2011)
    Silver nanoparticles (Ag-NP) are widely used due to their well-known antibacterial effects. In medicine Ag-NP have found applications as wound dressings, surgical instruments and bone substitute biomaterials, e.g. silver-containing calcium phosphate cements. Depending on the coating technique, during resorption of a biomaterial Ag-NP may come into close contact with body tissues, including human mesenchymal stem cells (hMSC). Despite the widespread uses of Ag-NP, there is a serious lack of information concerning their biological effects on human cells. In this study the uptake of Ag-NP into hMSC has been analyzed and the intracellular distribution of Ag-NP after exposure determined. Non-agglomerated (dispersed) Ag-NP from the cell culture medium were detected as agglomerates of nanoparticles within the hMSC by combined focused ion beam/scanning electron microscopy. The silver agglomerates were typically located in the perinuclear region, as determined by light microscopy. Specific staining of cellular structures (endo-lysosomes, nuclei, Golgi complex and endoplasmatic reticulum) using fluorescent probes showed that the silver nanoparticles occurred mainly within endo-lysosomal structures, not in the cell nucleus, endoplasmic reticulum or Golgi complex. Quantitative determination of the uptake of Ag-NP by flow cytometry (scattergram analysis) revealed a concentration-dependent uptake of the particles which was significantly inhibited by chlorpromazine and wortmannin but not by nystatin, indicating clathrin-dependent endocytosis and macropinocytosis as the primary uptake mechanisms. © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
    view abstract10.1016/j.actbio.2010.08.003
  • An in vitro crystallization setup to assess the efficiency of different phosphate binders in nephrology: Quantitative analytical considerations
    Peitsch, T. and Matthes, M. and Brandenburg, V. and Epple, M.
    Analytical Methods 2 (2010)
    An increased phosphate level in the blood (hyperphosphatemia) is a severe problem for dialysis patients. Different phosphate binders which are used to prevent hyperphosphatemia were studied in a custom-made in vitro crystallization apparatus which simulated the stomach (pH 2) and the gut (pH 7). The effective phosphate binding capacity was measured and the resulting products were identified. This apparatus permits the quantitative analysis of the phosphate binding effect under the given in vitro conditions. In particular, phosphate binders on the basis of calcium acetate (Calciumacetat Nefro®), calcium carbonate (Calciumcarbonat® Fresenius), aluminium hydroxide (Antiphosphat®), lanthanum carbonate (Fosrenol®), and poly(allylamine hydrochloride) (Renagel®) were studied, and also compared with pure calcium acetate, calcium carbonate, and aluminium sulfate. © 2010 The Royal Society of Chemistry.
    view abstract10.1039/b9ay00325h
  • An injectable paste of calcium phosphate nanorods, functionalized with nucleic acids, for cell transfection and gene silencing
    Klesing, J. and Chernousova, S. and Kovtun, A. and Neumann, S. and Ruiz, L. and Gonzalez-Calbet, J.M. and Vallet-Regi, M. and Heumann, R. and Epple, M.
    Journal of Materials Chemistry 20 (2010)
    Calcium phosphate nanorods which are typically used as paste for bone substitution were functionalized by DNA or siRNA. The structure and morphology of the nanorods did not change by the functionalization as indicated by dynamic light scattering (DLS), scanning electron microscopy (SEM), and high-resolution transmission electron microscopy (HRTEM). With a load of nucleic acids of about 2.7 wt%, the nanorods were used for transfection with HeLa and T24 cells, and for gene silencing with HeLa-EGFP cells. Removal of the water by filtration gave an injectable paste with a content of nucleic acids of about 2 wt%, and a water content of 43 wt%. This leads to a bioactive paste as hard-tissue regeneration material. © 2010 The Royal Society of Chemistry.
    view abstract10.1039/c0jm01130d
  • An outer shell of positively charged poly(ethyleneimine) strongly increases the transfection efficiency of calcium phosphate/DNA nanoparticles
    Sokolova, V. and Neumann, S. and Kovtun, A. and Chernousova, S. and Heumann, R. and Epple, M.
    Journal of Materials Science 45 (2010)
    Nanoparticles with an inner core of calcium phosphate, followed by layers of DNA and calcium phosphate and an outer layer of poly(ethyleneimine) (PEI) were prepared, characterized, and tested on different cell lines (HeLa, T24, and NIH3T3). A considerable increase in transfection efficiency was found for such nanoparticles, compared to the commercial reagent Polyfect® (a cationic dendrimer). The DNA is incorporated into the nanoparticles and protected from the attack by enzymes (nucleases) inside the cytoplasm of cells. The outer layer of PEI leads to electrosteric colloidal stabilization and gives a positive charge to the nanoparticle, which is helpful for the penetration through the negatively charged cell membrane. © 2010 Springer Science+Business Media, LLC.
    view abstract10.1007/s10853-009-4159-3
  • Application of calcium phosphate nanoparticles in biomedicine
    Epple, M. and Ganesan, K. and Heumann, R. and Klesing, J. and Kovtun, A. and Neumann, S. and Sokolova, V.
    Journal of Materials Chemistry 20 (2010)
    Calcium phosphate has excellent biocompatibility due to its chemical similarity to human hard tissue (bone and teeth). In nanoparticulate dispersed form, it can be used as a carrier in biological systems, e.g. to transfer nucleic acids or drugs. If such nanoparticles are suitably functionalized with fluorescing dyes, they can also be used for imaging or for photodynamic therapy. © 2010 The Royal Society of Chemistry.
    view abstract10.1039/b910885h
  • Biomimetic formation of thin, coherent iron oxide films under Langmuir monolayers
    Maas, M. and Degen, P. and Rehage, H. and Nebel, H. and Epple, M.
    Colloids and Surfaces A: Physicochemical and Engineering Aspects 354 (2010)
    This study focuses on the biomimetic formation and growth of thin iron oxide films under Langmuir monolayers. These coherent film structures were formed in the presence of different iron chloride solutions during the addition of an ammonia atmosphere. Stearic acid, stearyl amine and stearyl alcohol were used as film forming surfactants while the subphase contained FeCl2, FeCl3 or a mixture of both salts. The thin, coherent films consisted of X-ray amorphous iron oxide, hydroxide or oxyhydroxide. The films were studied by scanning electron microscopy, atomic force microscopy, X-ray diffraction, dynamic light scattering and surface potential measurements. Based on the experimental results we propose a growth mechanism that is guided by the formation of nanoparticles in the subphase and their assembly and aggregation underneath the Langmuir films. © 2009 Elsevier B.V. All rights reserved.
    view abstract10.1016/j.colsurfa.2009.04.049
  • Effect of hydroxyapatite on the biodegradation and biomechanical stability of polyester nanocomposites for orthopaedic applications
    Jayabalan, M. and Shalumon, K.T. and Mitha, M.K. and Ganesan, K. and Epple, M.
    Acta Biomaterialia 6 (2010)
    The effect of hydroxyapatite (HAP) on the performance of nanocomposites of an unsaturated polyester, i.e., hydroxy-terminated high molecular weight poly(proplyene fumarate) (HT-PPFhm), was investigated. A thermoset nanocomposite was prepared with nanoparticles of calcined HAP (<100 nm, rod-like shape, filler content 30 wt.%), HT-PPFhm and N-vinyl pyrrolidone, dibenzoyl peroxide and N,N-dimethyl aniline. Two more nanocomposites were prepared with precipitated HAP nanoparticles (<100 nm rod-like shape) and commercially available HAP nanoparticles (<200 nm spherical shape), respectively. Calcined HAP nanoparticles resulted in very good crosslinking in the resin matrix with high crosslinking density and interfacial bonding with the polymer, owing to the rod-like shape of the nanoparticles; this gave improved biomechanical strength and modulus and also controlled degradation of the nanocomposite for scaffold formation. The tissue compatibility and osteocompatibility of the nanocomposite containing calcined HAP nanoparticles was evaluated. The tissue compatibility was studied by intramuscular implantation in a rabbit animal model for 3 months as per ISO standard 10993/6. The in vivo femoral bone repair was also carried out in the rabbit animal model as per ISO standard 10993/6. The nanocomposite containing calcined HAP nanoparticles is both biocompatible and osteocompatible. © 2009 Acta Materialia Inc.
    view abstract10.1016/j.actbio.2009.09.015
  • Gold nanoparticles: Dispersibility in biological media and cell-biological effect
    Mahl, D. and Greulich, C. and Meyer-Zaika, W. and Köller, M. and Epple, M.
    Journal of Materials Chemistry 20 (2010)
    Spherical gold nanoparticles with a hydrodynamic diameter between 25 and 37 nm were prepared and stabilised with poly(N-vinylpyrrolidone) (PVP) or tris(sodium-m-sulfonatophenyl)phosphine (TPPTS). They were subjected to different cell culture media, e.g. pure RPMI, RPMI containing up to 10% of fetal calf serum (FCS), and RPMI containing up to 10% of bovine serum albumin (BSA), and the rate of agglomeration was studied by dynamic light scattering. In pure RPMI, a strong agglomeration was observed whereas in the RPMI-FCS and RPMI-BSA mixtures the particles remained well dispersed above 1 wt% protein concentration. The effect of PVP-stabilised gold nanoparticles on human mesenchymal stem cells (hMSC) was studied as well. No significant influence on the viability and chemotaxis was observed after incubation of hMSC with gold nanoparticles. However, gold nanoparticles induced the activation of hMSC as indicated by the release of IL-6 and IL-8. © 2010 The Royal Society of Chemistry.
    view abstract10.1039/c0jm01071e
  • Positively charged calcium phosphate/polymer nanoparticles for photodynamic therapy
    Klesing, J. and Wiehe, A. and Gitter, B. and Gräfe, S. and Epple, M.
    Journal of Materials Science: Materials in Medicine 21 (2010)
    The charge of nanoparticles influences their ability to pass through the cellular membrane, and a positive charge should be beneficial. The negative charge of calcium phosphate nanoparticles with an inner shell of carboxymethyl cellulose (CMC) was reversed by adding an outer shell of poly(ethyleneimine) (PEI) into which the photoactive dye 5,10,15,20-tetrakis(3-hydroxyphenyl)- porphyrin (mTHPP) was loaded. The aqueous dispersion of the nanoparticles was used for photodynamic therapy with HT29 cells (human colon adenocarcinoma cells), HIG-82 cells (rabbit synoviocytes), and J774A.1 cells (murine macrophages). A high photodynamic activity (killing) together with a very low dark toxicity was observed for HIG-82 and for J774.1 cells at 2 μM dye concentration. The killing efficiency was equivalent to the pure photoactive dye that, however, needs to be administered in alcoholic solution. © 2009 Springer Science+Business Media, LLC.
    view abstract10.1007/s10856-009-3934-7
  • SiRNA-loaded multi-shell nanoparticles incorporated into a multilayered film as a reservoir for gene silencing
    Zhang, X. and Kovtun, A. and Mendoza-Palomares, C. and Oulad-Abdelghani, M. and Fioretti, F. and Rinckenbach, S. and Mainard, D. and Epple, M. and Benkirane-Jessel, N.
    Biomaterials 31 (2010)
    In this study, we presented a new type of coating based on polyelectrolyte multilayers containing sequentially adsorbed active shRNA calcium phosphate nanoparticles for locally defined and temporarily variable gene silencing. Therefore, we investigated multi-shell calcium phosphate-shRNA nanoparticles embedded into a polyelectrolyte multilayer for gene silencing. As model system, we synthesized triple-shell calcium phosphate-shRNA nanoparticles (NP) and prepared polyelectrolyte multilayers films made of nanoparticles and poly-(l-lysine) (PLL). The biological activities of these polyelectrolyte multilayers films were tested by the production of osteopontin and osteocalcin in the human osteoblasts (HOb) which were cultivated on the PEM films. This new strategy can be used to efficiently control the bone formation and could be applicable in tissue engineering. © 2010 Elsevier Ltd.
    view abstract10.1016/j.biomaterials.2010.04.024
  • Sulfate-Containing Biominerals
    Bosselmann, F. and Epple, M.
    Biomineralization: From Nature to Application 4 (2010)
    The chapter summarizes the current knowledge about sulfate-containing biominerals which occur almost exclusively in marine species. New experimental results confirmed the existence of calcium sulfate hemihydrate in statoliths of scyphozoan and cubozoan medusae. Strontium sulfate is the biomineral of the skeleton of Acantharia. In algae, the gravity sensors consist of barium sulfate. The literature survey of the last 150 years clearly points out that many early conclusions were based on morphological and optical considerations only. Chemical analyses were often inaccurate or impossible due to the small amount of substance available. Since the advent of modern analytical methods, some of the old conclusions had to be revised. © 2008 John Wiley & Sons Ltd. All rights reserved.
    view abstract10.1002/9780470986325.ch6
  • Synchrotron microcomputer tomography for the non-destructive visualization of the fish skeleton
    Epple, M. and Neues, F.
    Journal of Applied Ichthyology 26 (2010)
    The objective of this study was to elucidate the potential of synchrotron radiation-based microcomputer tomography as a non-destructive method to visualize hard tissue in fish with high resolution. The high X-ray contrast between mineralized tissue (bone and teeth) and soft tissue permits an easy differentiation. The nature of this method avoids preparation artefacts which may occur during the preparation for histology or scanning electron microscopy. The spatial resolution is of the order of a few μm, depending on the sample. Results obtained for the species medaka (Oryzias latipes) and zebrafish (Danio rerio) are presented. © 2010 Blackwell Verlag, Berlin.
    view abstract10.1111/j.1439-0426.2010.01422.x
  • The effect of radiation processing and filler morphology on the biomechanical stability of a thermoset polyester composite
    Jayabalan, M. and Shalumon, K.T. and Mitha, M.K. and Ganesan, K. and Epple, M.
    Biomedical Materials 5 (2010)
    The effect of radiation processing and filler morphology on the biodegradation and biomechanical stability of a poly(propylene fumarate)/hydroxyapatite composite was investigated. Radiation processing influenced both cross-linking and biodegradation of the composites. Irradiation with a dose of 3 Mrad resulted in enhanced cross-linking, mechanical properties and a higher storage modulus which are favourable for dimensional stability of the implant. The particle morphology of the added hydroxyapatite in the highly cross-linked state significantly influenced the biomechanical and interfacial stability of the composites. Reorganization of agglomerated hydroxyapatite occurred in the cross-linked polymeric matrix under dynamic mechanical loading under simulated physiological conditions. Such a reorganization may increase the damping characteristics of the composite. © 2010 IOP Publishing Ltd.
    view abstract10.1088/1748-6041/5/2/025009
  • The influence of proteins on the dispersability and cell-biological activity of silver nanoparticles
    Kittler, S. and Greulich, C. and Gebauer, J.S. and Diendorf, J. and Treuel, L. and Ruiz, L. and Gonzalez-Calbet, J.M. and Vallet-Regi, M. and Zellner, R. and Köller, M. and Epple, M.
    Journal of Materials Chemistry 20 (2010)
    Spherical silver nanoparticles with a diameter of 50 ± 20 nm and stabilized with either poly(N-vinylpyrrolidone) (PVP) or citrate were dispersed in different cell culture media: (i) pure RPMI, (ii) RPMI containing up to 10% of bovine serum albumin (BSA), and (iii) RPMI containing up to 10% of fetal calf serum (FCS). The agglomeration behavior of the nanoparticles was studied with dynamic light scattering and optical microscopy of individually tracked single particles. Whereas strong agglomeration was observed in pure RPMI and in the RPMI-BSA mixture within a few hours, the particles remained well dispersed in RPMI-FCS. In addition, the biological effect of PVP-stabilized silver nanoparticles and of silver ions on human mesenchymal stem cells (hMSCs) was studied in pure RPMI and also in RPMI-BSA and RPMI-FCS mixtures, respectively. Both proteins considerably increased the cell viability in the presence of silver ions and as well as silver nanoparticles, indicating a binding of silver by these proteins. © The Royal Society of Chemistry 2010.
    view abstract10.1039/b914875b
  • The release of nickel from nickel-titanium (NiTi) is strongly reduced by a sub-micrometer thin layer of calcium phosphate deposited by rf-magnetron sputtering
    Surmenev, R.A. and Ryabtseva, M.A. and Shesterikov, E.V. and Pichugin, V.F. and Peitsch, T. and Epple, M.
    Journal of Materials Science: Materials in Medicine 21 (2010)
    Thin calcium phosphate coatings were deposited on NiTi substrates (plates) by rf-magnetron sputtering. The release of nickel upon immersion in water or in saline solution (0.9% NaCl in water) was measured by atomic absorption spectroscopy (AAS) for 42 days. The coating was analyzed before and after immersion by X-ray powder diffraction (XRD), scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX). After an initial burst during the first 7 days that was observed for all samples, the rate of nickel release decreased 0.4-0.5 ng cm-2 d-1 for a 0.5 μm-thick calcium phosphate coating (deposited at 290 W). This was much less than the release from uncoated NiTi (3.4-4.4 ng cm-2 d-1). Notably, the nickel release rate was not significantly different in pure water and in aqueous saline solution. © 2010 Springer Science+Business Media, LLC.
    view abstract10.1007/s10856-010-3989-5
  • The resorption of nanocrystalline calcium phosphates by osteoclast-like cells
    Detsch, R. and Hagmeyer, D. and Neumann, M. and Schaefer, S. and Vortkamp, A. and Wuelling, M. and Ziegler, G. and Epple, M.
    Acta Biomaterialia 6 (2010)
    Nanocrystalline calcium phosphates containing carbonate have a high similarity to bone mineral. The reactions of bone cells (primary osteoblasts and osteoclast-like cells) on these materials as well as on sintered β-tricalcium phosphate and hydroxyapatite (HA) confirmed a good biocompatibility of the nanocrystalline samples. However, osteoclastic differentiation was constrained on the carbonate-rich samples, leading to a small number of osteoclast-like cells on the materials and few resorption pits. The grain size of the calcium phosphate ceramics (nano vs. micro) was less important than expected from to physico-chemical considerations. When comparing the nanocrystalline samples, the highest resorption rate was found for nano-HA with a low carbonate content, which strongly stimulated the differentiation of osteoclast-like cells on its surface. © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
    view abstract10.1016/j.actbio.2010.03.003
  • The use of calcium phosphate nanoparticles encapsulating Toll-like receptor ligands and the antigen hemagglutinin to induce dendritic cell maturation and T cell activation
    Sokolova, V. and Knuschke, T. and Kovtun, A. and Buer, J. and Epple, M. and Westendorf, A.M.
    Biomaterials 31 (2010)
    Dendritic cells (DCs) are potent antigen-presenting cells that possess the ability to stimulate naïve T cells. Antigen presentation by mature (activated) DCs is a prerequisite for the stimulation of antigen-specific T cells, whereas antigen presentation by immature DCs results in the generation of specific tolerance. Our aim was to develop calcium phosphate nanoparticles which can serve as carriers of immunoactive oligonucleotides into dendritic cells for their activation. We analyzed size, surface charge, and morphology of calcium phosphate nanoparticles loaded with the TLR ligands CpG and poly(I:C) and also with the antigen hemagglutinin (HA) by scanning electron microscopy, dynamic light scattering, Brownian motion analysis and ultracentrifugation. The uptake of fluorescence-labeled nanoparticles into dendritic cells was illustrated by confocal laser scanning microscopy. Immunostimulatory effects of these nanoparticles on DCs were studied, i.e., cytokine production and activation of the cells in terms of upregulation of surface molecules. We show that functionalized calcium phosphate nanoparticles are capable to induce both innate and adaptive immunity by activation of DCs. © 2010 Elsevier Ltd.
    view abstract10.1016/j.biomaterials.2010.03.067
  • Thermoanalytical and structural characterization of fluoridated calcium phosphates prepared in anhydrous alcohols
    Simões, R.G. and Aleixo, A.I. and Lagoa, A.L.C. and Minas Da Piedade, M.E. and Leal, J.P. and Peitsch, T. and Epple, M.
    Journal of Thermal Analysis and Calorimetry 100 (2010)
    A thermoanalytical, morphological, and structural study of fluoridated calcium phosphates that were prepared by different variants of a synthesis in anhydrous alcohols is reported. The obtained materials were neither fully amorphous nor single-phased crystalline, and their nature considerably depended on the synthesis conditions. In all cases, the retention of significant amounts of solvent in the solid product was observed. A complete removal of the solvent was only possible by heating to temperatures above ∼573-673 K which resulted in variations in the elemental composition, phase changes, and an increase of the crystallinity. Consequently, this synthesis in anhydrous alcohols is not a viable route to obtain materials with a defined crystallinity and stoichiometry. © 2010 Akadémiai Kiadó, Budapest, Hungary.
    view abstract10.1007/s10973-009-0654-7
  • Toxicity of silver nanoparticles increases during storage because of slow dissolution under release of silver ions
    Kittler, S. and Greulich, C. and Diendorf, J. and Köller, M. and Epple, M.
    Chemistry of Materials 22 (2010)
    The dissolution of citrate-stabilized and poly(vinylpyrrolidone)-stabilized silver nanoparticles in water was studied by dialysis for up to 125 days at 5, 25, and 37 °C. The particles slowly dissolve into ions on a time scale of several days. However, in all cases, a limiting value of the released silver was observed, i.e., the particles did not completely dissolve. In some cases, the nanoparticles released up to 90% of their weight. Formal kinetic data were computed. Rate and degree of dissolution depended on the functionalization as well as on the storage temperature. The release of silver led to a considerably increased toxicity of silver nanoparticles which had been stored in dispersion for several weeks toward human mesenchymal stem cells due to the increased concentration of silver ions. Consequently, "aged" (i.e., immersed) silver nanoparticles are much more toxic to cells than freshly prepared silver nanoparticles. © 2010 American Chemical Society.
    view abstract10.1021/cm100023p
  • catalysis

  • ceramics

  • coatings

  • cytotoxicity

  • nanobiomedicine

  • nanocrystals

  • nanoparticles

  • scanning electron microscopy

  • silver nanoparticles

  • transmission electron microscopy

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