6 results on '"Germershaus, Oliver"'
Search Results
2. Controlled protein delivery from electrospun non-wovens: novel combination of protein crystals and a biodegradable release matrix.
- Author
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Puhl S, Li L, Meinel L, and Germershaus O
- Subjects
- Caproates chemistry, Crystallization methods, Drug Delivery Systems methods, Lactic Acid chemistry, Lactones chemistry, Muramidase chemistry, Particle Size, Polyethylene Glycols chemistry, Polyglycolic Acid chemistry, Polylactic Acid-Polyglycolic Acid Copolymer, Solvents chemistry, Suspensions chemistry, Polymers chemistry, Proteins chemistry
- Abstract
Poly-ε-caprolactone (PCL) is an excellent polymer for electrospinning and matrix-controlled drug delivery combining optimal processability and good biocompatibility. Electrospinning of proteins has been shown to be challenging via the use of organic solvents, frequently resulting in protein unfolding or aggregation. Encapsulation of protein crystals represents an attractive but largely unexplored alternative to established protein encapsulation techniques because of increased thermodynamic stability and improved solvent resistance of the crystalline state. We herein explore the electrospinning of protein crystal suspensions and establish basic design principles for this novel type of protein delivery system. PCL was deployed as a matrix, and lysozyme was used as a crystallizing model protein. By rational combination of lysozyme crystals 0.7 or 2.1 μm in diameter and a PCL fiber diameter between 1.6 and 10 μm, release within the first 24 h could be varied between approximately 10 and 100%. Lysozyme loading of PCL microfibers between 0.5 and 5% was achieved without affecting processability. While relative release was unaffected by loading percentage, the amount of lysozyme released could be tailored. PCL was blended with poly(ethylene glycol) and poly(lactic-co-glycolic acid) to further modify the release rate. Under optimized conditions, an almost constant lysozyme release over 11 weeks was achieved.
- Published
- 2014
- Full Text
- View/download PDF
3. Integrin alphaVbeta3 targeted gene delivery using RGD peptidomimetic conjugates with copolymers of PEGylated poly(ethylene imine).
- Author
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Merkel OM, Germershaus O, Wada CK, Tarcha PJ, Merdan T, and Kissel T
- Subjects
- Biocompatible Materials chemical synthesis, Biocompatible Materials chemistry, Biocompatible Materials metabolism, Biomimetic Materials chemical synthesis, Cell Line, Tumor, DNA metabolism, Gene Expression Regulation, Humans, Ligands, Maleimides chemistry, Propionates chemistry, Sulfhydryl Compounds chemistry, Transfection, Biomimetic Materials chemistry, Biomimetic Materials metabolism, Gene Transfer Techniques, Integrin alphaVbeta3 metabolism, Oligopeptides chemistry, Polyethylene Glycols chemistry, Polyethyleneimine chemistry
- Abstract
This study describes the synthesis and characterization of five conjugates of poly(ethylene glycol) modified polyethylenimine (PEG-PEIs) coupled in two different synthesis routes to a nonpeptidic pentacyclic RDG-mimetic for integrin receptor-targeted gene delivery. Synthesis of this panel of different conjugates allowed for systematic analysis of structure-activity relationships. Conjugates were therefore characterized regarding molecular composition, DNA condensation, size, and zeta potential of self-assembled polyplexes. In vitro characterization included investigation of blood compatibility, binding affinity to receptor-positive and receptor-negative cells measured by flow cytometry, cellular uptake quantified by scintillation counting, and efficiency and specificity of transfection assayed by reporter gene expression. In a first synthetic approach, low molecular weight PEI (LMW-PEI) was PEGylated using a heterobifunctional PEG linker and coupling of the RGD-mimetic was achieved at the distal end of PEG chains. In a second synthesis route, the RGD-mimetic was directly coupled to AB-block-copolymers of PEI (25 kDa) and PEG (30 kDa). Interactions of RGD-PEG-LMW-PEI conjugates with DNA were strongly impaired, whereas PEG-PEI-RGD conjugates were more promising candidates due to their physicochemical properties and higher receptor specificity. The binding, uptake, and transfection efficiency in receptor-positive cells was strongly increased upon conjugation of the RGD-mimetic to AB-block-copolymers of PEG-PEI and depended on the degree of peptide substitution. The conjugates of PEG-PEI AB-block-copolymers with low ligand density of the RGD-mimetic appear to be promising candidates for in vivo cancer gene therapy.
- Published
- 2009
- Full Text
- View/download PDF
4. HER2 targeted polyplexes: the effect of polyplex composition and conjugation chemistry on in vitro and in vivo characteristics.
- Author
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Germershaus O, Neu M, Behe M, and Kissel T
- Subjects
- Animals, Antibodies, Monoclonal chemistry, Antibodies, Monoclonal immunology, Antibodies, Monoclonal pharmacokinetics, Antibodies, Monoclonal, Humanized, Cell Aggregation, Cell Line, Erythrocytes cytology, Flow Cytometry, Hemolysis, Heparin metabolism, Humans, Immunoconjugates pharmacokinetics, Male, Mice, Polyethylene Glycols chemistry, Polyethyleneimine chemistry, Receptor, ErbB-2 deficiency, Transfection, Trastuzumab, Immunoconjugates chemistry, Immunoconjugates immunology, Receptor, ErbB-2 immunology
- Abstract
Knowledge of the influence of targeting ligands on pharmacokinetics and biodistribution of polymeric nonviral vectors is presently limited. We investigated the properties of three structurally different conjugates of polyethylenglycol-modified polyethylenimine coupled to the HER2 specific antibody Trastuzumab. Unlike polyethylenimine, conjugates formed small (100-230 nm) DNA polyplexes with zeta-potentials of +/- 2 mV at a broad range of N/P ratios. Stability as assessed by heparin displacement was slightly improved compared to unmodified copolymers. Erythrocyte aggregation and hemolysis were strongly reduced with conjugates. Conjugate polyplexes showed significant differences in specificity and transfection efficiency in vitro. These could be attributed to differences in cell binding and uptake assessed by flow cytometry. Pharmacokinetics of conjugates in mice revealed significant improvements over free plasmid DNA and polyethylenimine. Area under the plasma level-time curve of conjugates was increased up to 48% or 114% compared to that of polyethylenimine or free plasmid DNA, respectively. Deposition of conjugate polyplexes in lung and spleen was significantly reduced compared to that of polyethylenimine. Differences could be attributed to antibody conjugation since no significant differences in pharmacokinetics and biodistribution were found between conjugates. These findings demonstrate that conjugated antibodies not only confer active targeting but also significantly improve in vivo properties of polyplexes.
- Published
- 2008
- Full Text
- View/download PDF
5. Influence of polyethylene glycol chain length on the physicochemical and biological properties of poly(ethylene imine)-graft-poly(ethylene glycol) block copolymer/SiRNA polyplexes.
- Author
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Mao S, Neu M, Germershaus O, Merkel O, Sitterberg J, Bakowsky U, and Kissel T
- Subjects
- Animals, Fibroblasts cytology, Fibroblasts physiology, Heparin metabolism, Macromolecular Substances, Mice, Microscopy, Atomic Force, NIH 3T3 Cells, Particle Size, Genetic Vectors chemistry, Genetic Vectors metabolism, Imines chemistry, Polyethylene Glycols chemistry, Polyethylenes chemistry, RNA, Small Interfering chemistry, RNA, Small Interfering metabolism
- Abstract
Polyplexes between siRNA and poly(ethylene imine) (PEI) derivatives are promising nonviral carriers for siRNA. The polyplex stability is of critical importance for efficient siRNA delivery to the cytoplasm. Here, we investigate the effect of PEGylation at a constant ratio ( approximately 50%) on the biophysical properties of the polyplexes. Particle size, zeta potential, and stability against heparin as well as RNase digestion and reporter gene knockdown under in vitro conditions of different siRNA polyplexes were characterized. Stability and size of siRNA polyplexes were clearly influenced by PEI-PEG structure, and high degrees of substitution such as PEI(25k)-g-PEG(550)(30) resulted in large (300-400 nm), diffuse complexes (AFM) which showed condensation behavior only at high N/P ratios. All other polyplexes and the PEI control showed similar sizes (150 nm) and compact structures in AFM, with complete condensation reached at N/P ratio of 3. Stability of siRNA polyplexes against heparin displacement and RNase digestion could be modified by PEGylation. Protection against RNase digestion was highest for PEI(25k)-g-PEG(5k)(4) and PEI(25k)-g-PEG(20k)(1), while siRNA/PEI provided insufficient protection. In knockdown experiments using NIH/3T3 fibroblasts stably expressing beta-galactosidase, it was shown that PEG chain length had a significant influence on biological activity of siRNA. Polyplexes with siRNA containing PEI(25k)-g-PEG(5k)(4) and PEI(25k)-g-PEG(20k)(1) yielded similar efficiencies of ca. 70% knockdown as lipofectamine controls. Confocal microscopy demonstrated enhanced cellular uptake of siRNA into cytosol by polyplexes formation with PEI copolymers. In conclusion, both the chain length and graft density of PEG were found to strongly influence siRNA condensation and stability and hence affect the knockdown efficiency of PEI-PEG/siRNA polyplexes.
- Published
- 2006
- Full Text
- View/download PDF
6. Trastuzumab-polyethylenimine-polyethylene glycol conjugates for targeting Her2-expressing tumors.
- Author
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Germershaus O, Merdan T, Bakowsky U, Behe M, and Kissel T
- Subjects
- Antibodies, Monoclonal metabolism, Antibodies, Monoclonal, Humanized, Antineoplastic Agents chemical synthesis, Antineoplastic Agents metabolism, Cell Line, Tumor, Genes, Reporter, Genetic Therapy methods, Humans, Molecular Structure, Neoplasms metabolism, Particle Size, Trastuzumab, Antibodies, Monoclonal chemistry, Antineoplastic Agents chemistry, Gene Transfer Techniques, Polyethylene Glycols chemistry, Polyethyleneimine chemistry, Receptor, ErbB-2 metabolism
- Abstract
In this study, we describe the synthesis and characterization of a conjugate consisting of poly(ethylene glycol 2,000 Da)(10)-graft-poly(ethylene imine 25 kDa) (PEG-PEI) covalently coupled to Trastuzumab (Herceptin) via N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) for specific gene delivery to Her2-expressing cell lines. The efficiency of DNA condensation was studied using an ethidium bromide exclusion assay and demonstrated negligible differences compared to PEG-PEI. Conjugate complex sizes were determined by dynamic light scattering to be in the range 130-180 nm. zeta potentials at different N/P ratios were close to neutral. Flow cytometry and confocal microscopy revealed efficient binding and uptake of Trastuzumab-PEI-PEG complexes using Her2-positive SK-BR-3 cells. In contrast, binding and uptake into Her2-negative OVCAR-3 cells was negligible. In good correlation with these findings, reporter gene expression using targeted complexes in SK-BR-3 cells was up to sevenfold higher than that of unmodified PEG-PEI complexes. With the use OVCAR-3 cells, no significant difference in expression efficiencies could be observed between conjugate and PEG-PEI complexes. Inhibition experiments with free Trastuzumab showed a significant decrease in reporter gene expression using SK-BR-3 cells but no decrease using OVCAR-3 cells, strongly supporting a specific Her2-receptor-mediated uptake mechanism. Our results suggest that Trastuzumab-PEI-PEG might be a promising new bioconjugate for targeted gene transfer to Her2-positive tumor cells in vivo.
- Published
- 2006
- Full Text
- View/download PDF
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