Anreicherung und Charakterisierung von TRIM28-Interaktionspartnern mit massenspektrometrischen Methoden

Die vorliegende Arbeit befasst sich mit der Anreicherung und Charakterisierung von KRAB-Proteinen. Diese binden an die DNA und führen zur Repression der dortigen Gene. Proteinkomplexe aus Zellen wurden extrahiert und affinitätschromatographisch angereichert. Nach massenspektrometrischer Untersuchung konnte die Aminosäuresequenz des TRIM28 abgedeckt und 29 KRAB-Proteine identifiziert werden. Im Zuge der Untersuchungen zur Phosphorylierung des TRIM28 wurde eine verbesserte Methode zur Phosphopeptidanreicherung und eine neuartige MALDI-Matrix-Präparation entwickelt

PI3K/p110α inhibition selectively interferes with arterial thrombosis and neointima formation, but not re-endothelialization: potential implications for drug-eluting stent design

Background Impaired re-endothelialization and stent thrombosis are a safety concern associated with drug-eluting stents (DES). PI3K/p110α controls cellular wound healing pathways, thereby representing an emerging drug target to modulate vascular homoeostasis after injury. Methods and results PI3K/p110α was inhibited by treatment with the small molecule inhibitor PIK75 or a specific siRNA. Arterial thrombosis, neointima formation, and re-endothelialization were studied in a murine carotid artery injury model. Proliferation and migration of human vascular smooth muscle cell (VSMC) and endothelial cell (EC) were assessed by cell number and Boyden chamber, respectively. Endothelial senescence was evaluated by the β-galactosidase assay, endothelial dysfunction by organ chambers for isometric tension. Arterial thrombus formation was delayed in mice treated with PIK75 when compared with controls. PIK75 impaired arterial expression and activity of tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1); in contrast, plasma clotting and platelet aggregation did not differ. In VSMC and EC, PIK75 inhibited expression and activity of TF and PAI-1. These effects occurred at the transcriptional level via the RhoA signalling cascade and the transcription factor NFkB. Furthermore, inhibition of PI3K/p110α with PIK75 or a specific siRNA selectively impaired proliferation and migration of VSMC while sparing EC completely. Treatment with PIK75 did not induce endothelial senescence nor inhibit endothelium-dependent relaxations. In line with this observation, treatment with PIK75 selectively inhibited neointima formation without affecting re-endothelialization following vascular injury. Conclusion Following vascular injury, PI3K/p110α inhibition selectively interferes with arterial thrombosis and neointima formation, but not re-endothelialization. Hence, PI3K/p110α represents an attractive new target in DES desig

Safety evaluation of resveratrol as an active compound for drug-eluting cardiovascular implants

Resveratrol is a member of stilbenoids with promising anti-atherosclerotic properties. This hallmark makes it an extremely interesting candidate for local drug delivery to damaged tissue adjacent to the implant in order to reduce implant-related complications. For the regulatory approval drug-eluting medical devices have to be thoroughly tested for safety, efficacy and interactions with the surrounding tissue, including tests for sensitization among others. Studies for sensitization help to estimate the risk for an allergic reaction upon prolonged exposure to a chemical compound. Due to increased social and regulatory demand for replacement of animal experiments by in vitro approaches a number of reliable predictive non-animal tests have been developed. Here, we assessed the skin sensitization potential of resveratrol by the direct peptide reactivity assay (DPRA), one of the first non-animal tests adopted by the OECD

Water uptake of various electrospun nonwovens

In recent years, nanofiber based materials have emerged as especially interesting for several biomedical applications, regarding their high surface to volume ratio. Due to the superficial nano- and microstructuring and the different wettability compared to nonstructured surfaces, the water absorption is an important parameter with respect to the degradation stability, thermomechanic properties and drug release properties, depending on the type of polymer [1]. In this investigation, the water absorption of different non- and plasma modified biostable nanofiber nonwovens based on polyurethane, polyester and polyamide were analysed and compared. Also, the water absorption by specified water wetting, the contact angle and morphology changes were examined. The results show that the water uptake is highly dependent on the surface modification and the polymer composition itself and can therefore be partially changed

DLP 3D printing of Dexamethasoneincorporated PEGDA-based photopolymers: compressive properties and drug release

Photopolymerizing, high-resolution 3D printing methods such as Stereolithography (SLA) or Digital Light Processing (DLP) are very promising for the manufacturing of drug-incorporated, patient specific implants. However, a drug-load may be limited by adequately solubility of the active pharmaceutical ingredient (API) in the photopolymer. Furthermore, a drug-load may affect the mechanical properties of the material negatively. Here, we investigate the DLP 3D printing of drugincorporated photopolymers. Polyethylene glycol diacrylate (PEGDA, Mn = 700 g/mol) is used as matrix polymer and Dexamethasone (DEX) is used for drug-loading (10 g/L and 20 g/L). Compressive properties, drug release and drug stability of 3D printed test samples were analyzed. DEX was found to be sparingly soluble in the PEGDA-based photopolymer. Not all drug particles can be dissolved at a concentration of 20 g/L and a slurry-like suspension is formed. Drug-incorporated photopolymers of 10 g/L (solution) and 20 g/L (suspension) were processed successfully via DLP. The higher the drug-load, the lower the compressive strength. Mechanical properties can be improved via a post-curing in a UV light curing box. Drug-incorporated 3D printed test samples show burst-release of DEX. The post-curing process does not affect drug release. DEX degrades in 3D-printed test samples significantly (~ 30 %) over a several days time period

Electrospinning of polyimide nanofibres – effects of working parameters on morphology

The use of the electrospinning technique is a promising and versatile method for producing fibrous nonwovens from various polymers. Here we present fibre formation via direct electrospinning of a soluble polyimide, a class of polymers that is typically insoluble. In this study solution parameters as the solvent and the polymer concentration are investigated. Furthermore relevant process parameters are varied for optimization of the performance. The presented data indicate polyimide as a promising material for the fabrication of nanofibrous nonwovens via direct electrospinning

Automated particle analysis by Raman microscopy – a method development

Chemical identification of particles increasingly gains importance concerning regulatory affairs of medical devices. Particle numbers of samples very easily add up to orders larger than 1.000. Therefore an automated particle measurement is absolutely worthwhile. As a method of chemical identification Raman spectroscopy combined with microscopy is commonly used. The challenge is to distinguish small particles in a range of a few microns on a surface from the background. Different specimen holder made of glass, polymer (e.g. polycarbonate) or metal are commonly used. After the choice of suitable background parameters such as magnification, optical resolution, focal plane and brightness have to be optimized. The used software for Raman microscopy offers the possibility to select particles for the subsequent measurement. Therefore the xand y-coordinates of particles have to be added to a list manually and separately. This so called point list can be processed automatically. However, this procedure is very laborious and requires a high degree of user interaction especially in case of large particle numbers. Furthermore the software offers the possibility to perform a so called area scan, where a defined surface of the the specimen will be spectroscopically analyzed. The measurements are performed in defined distances on a grid, e.g. every 10 micrometer in xand y-direction. The choice of this tool leads to a large number of measuring points associated with an unwanted long measuring time. By means of the freely accessible software ImageJ an alternative path was found to relate xand y-coordinates to every particle. These coordinates can be imported into the point list of the software for Raman microscopy. Afterwards a Raman spectrum for every particle can be measured without further high effort in time

Rheological analysis of hybrid hydrogels during polymerization processes

Development of new implant coatings with temperature-controlled drug release to treat infections after device implantation can be triggered by highly elastic hydrogels with adequate stability and adhesive strength in the swollen state. By using an ionic liquid (IL [ViPrIm]+[Br]−) as additive to N-isopropylacrylamide (NIPAAm) unique effects on volumetric changes and mechanical properties as well as thermoresponsive drug release of the obtained hybrid hydrogels were observed. In this context, rheological measurements allow the monitoring of gelation processes as well as chemical, mechanical, and thermal treatments and effects of additives. Hybrid hydrogels of pNIPAAm and poly (ionic liquid) (PIL) were prepared by radical emulsion polymerization with N,N′-methylenebis(acrylamide) as 3D crosslinking agent. By varying monomer, initiator and crosslinker amounts the multi-compound system during polymerization was monitored by oscillatory time sweep experiments. The time dependence of the storage modulus (G′) and the loss modulus (G″) was measured, whereby the intersection of G′ and G″ indicates the sol-gel transition. Viscoelastic behavior and complex viscosity of crosslinked and non-crosslinked hydrogels were obtained. Within material characterization rheology can be used to determine process capability and optimal working conditions. For biomedical applications complete hydrogelation inter-connecting all compounds can be received providing the possibility to process mechanically stable, swellable implant coatings or wound closures

Influence of additives on physico-chemical properties of electrospun poly(L-lactide)

Electrospun poly(L-lactide) (PLLA) nonwoven represent potential options for biodegradable medical implants. They can be manufactured with high reproducibility and do offer the potential for chemical modification to alter matrix properties. In our study, we investigate the mechanical, thermal and morphological properties of PLLA fiber matrices. Fibrous nonwovens were fabricated from polymer solution by needle electrospinning. The polymer solutions were loaded with Triton X-100 (TX- 100), formic acid and tetraethyl ammonium chloride (TEAC) with respect to polymer weight. Morphology of the PLLA nonwoven scaffolds was examined with SEM. We performed uniaxial tensile tests and differential scanning calorimetry (DSC). Different concentrations of the additives TEAC, formic acid and Triton X-100 lead to strong changes regarding mechanical and thermal properties of the electrospun PLLA fiber matrices. In comparison with mechanical properties of established biological tissue materials, the results indicate the suitability for medical applications

Actinomycin D for fibrosis management in ophthalmic implant surgery

Implants that feature a drug delivery system loaded with antifibrotic active drugs provide a promising approach to address postoperative complications caused by fibrosis. This study is intended to clarify whether Actinomycin D has an impact specifically on components of the extracellular matrix (ECM) and its formation in human primary fibroblasts of the Tenon capsule (hTF). Furthermore, the suitability of this agent in poly(N-vinylpyrrolidone)- poly(methylmethacrylate)(PVP-co-PMMA) as a drug delivery model is evaluated in drug incorporation and release studies. RT-qPCR revealed a significant downregulation of the fibrotic marker genes ACTA2, COL1A1 and FN1 in cells stimulated with TGF- β1 and additionally treated with Actinomycin D. However, these findings could only be confirmed on α- SMA protein level. collagen I and Fibronectin synthesis stayed unaffected. The diffusion based incorporation of Actinomycin D into the polymer model proved to be very effective. The release of the agent was retarded with a slightly prolonged kinetic. These findings make Actinomycin D a promising antifibrotic agent in ophthalmic implant surgery