Towards a biofunctionalized vascular prosthesis: immune cell trapping via a growth factor receptor.

To improve the clinical performance of vascular prostheses, which is inacceptably low for implants with small diameters (< 6 mm), biofunctionalization of synthetic implants by endothelialization has become a major, although still unreached, aim. In order to be able to recruit native endothelial progenitor cells (EPCs) to luminal implant surfaces from the blood stream, we generated monoclonal antibodies against the EPC-specific vascular endothelial growth factor receptor 2 (VEGFR-2). Employing the very efficient genetic immunization strategy, > 10 000 hybridoma clones were generated. Screening with various deletion mutants of VEGFR-2, 49 highly-specific monoclonal antibodies (mAbs) covering all seven Ig domains of VEGFR-2 were selected. mAb 9H10 was characterized in detail. Once immobilized on synthetic surfaces, mAb 9H10 allowed, within min, nearly 100-fold enrichment of VEGFR-2-expressing cells under continuous flow conditions. Cell trapping was cell-type specific and essentially not affected by competing VEGFR-2-negative cells. To exclude that the antibody would adversely modify receptor responses, four different in vitro assays were employed. Cell proliferation, angiogenic tube formation, acetylated low-density lipoprotein uptake and VEGFR-2 phosphorylation remained unaffected, suggesting that the antibody did not interfere with the receptor functioning of human umbilical vascular endothelial cells. The molecular and cellular characteristics make the selected monoclonal antibody a very promising tool for the biofunctionalization of vascular implants. Copyright © 2016 John Wiley & Sons, Ltd.

Fine-tuned PEGylation of chitosan to maintain optimal siRNA-nanoplex bioactivity.

Polyethylene glycol (PEG) is a widely used modification for drug delivery systems. It reduces undesired interaction with biological components, aggregation of complexes and serves as a hydrophilic linker of ligands for targeted drug delivery. However, PEGylation can also lead to undesired changes in physicochemical characteristics of chitosan/siRNA nanoplexes and hamper gene silencing. To address this conflicting issue, PEG-chitosan copolymers were synthesized with stepwise increasing degrees of PEG substitution (1.5% to 8.0%). Subsequently formed PEG-chitosan/siRNA nanoplexes were characterized physicochemically and biologically. The results showed that small ratios of chitosan PEGylation did not affect nanoplex stability and density. However, higher PEGylation ratios reduced nanoplex size and charge, as well as cell uptake and final siRNA knockdown efficiency. Therefore, we recommend fine-tuning of PEGylation ratios to generate PEG-chitosan/siRNA delivery systems with maximum bioactivity. The degree of PEGylation for chitosan/siRNA nanoplexes should be kept low in order to maintain optimal nanoplex efficiency.

Immunoliposomes for Targeted Delivery of an Antifibrotic Drug.

Excessive extracellular matrix formation in organs and tissues arises from an imbalance between the synthesis and degradation of matrix proteins, especially collagen. This condition interferes with proper wound healing and regeneration, and to date, no specific treatment is available. In the present study, we propose a targeted drug delivery system consisting of cell-specific immunoliposomes (ILs) loaded with deferoxamine (DFO) as an antifibrotic drug. ILs were functionalized with polyethylene glycol (PEG) to improve the steric stability and prolong their half-life. In addition, a single-chain Fv (scFv) antibody fragment that specifically targets fibroblast activation protein (FAP) was incorporated. An in vitro fibrosis model was employed to test this construct. This model consisted of highly activated pro-fibrotic fibroblasts with 2- to 6-fold induction of selected fibrosis markers: cell/matrix deposited collagen I, total soluble collagen, and α smooth muscle actin. The activation was accompanied by a significant and cell-specific elevation of FAP expression and activity, thereby confirming that FAP is an adequate target for antifibrotic drug delivery. Purified anti-FAP scFv was shown to bind specifically to these cells without influencing the FAP enzymatic activity. DFO was demonstrated to have a dose-dependent antifibrotic activity as quantified by collagen deposition. Specific binding and intracellular uptake of DiI-labeled ILs into the activated fibroblasts were shown by flow cytometry and microscopy. Finally, DFO-loaded ILs targeted to FAP caused a significant reduction in the collagen deposition, whereas no effect was observed using liposomes that lacked the targeting antibody fragment. These results suggest that the FAP-specific scFv-conjugated liposomes have considerable potential for cell-specific targeting applicable as a therapy for excessive collagen deposition during fibrosis. In general, through liposome encapsulation, bioactive molecules, such as DFO, that have broad effects and poor cell penetration can be converted into cell-specific composites for targeted drug delivery.

miR-124 disinhibits neurite outgrowth in an inflammatory environment.

Lesions of the central nervous system elicit inflammatory responses that counteract the regeneration of neurites. Microglia and infiltrating macrophages that were activated by trauma have been identified as cellular sources of inhibitory factors. We examine cultured macrophage (RAW264.7) and neuronal (PC12) cell lines to ascertain the potential modulators of the inflammatory impact on neurons. By exposing quiescent macrophages to lipopolysaccharide (LPS) and interferon γ (IFN-γ), cells can be transformed into an activated M1 phenotype. Neurite extension was induced in PC12 cells by culturing them in the presence of nerve growth factor. Neurite outgrowth was quantified by analyzing immunofluorescence and phase contrast microscopy images. Activated macrophages significantly reduced neurite extension. Macrophage activation by LPS/IFN-γ induced a 1000-fold increase in tumor necrosis factor alpha (TNF-α) secretion, as quantified by enzyme-linked immunosorbent assays (ELISA). Recombinant TNF-α inhibited neurite formation at concentrations as low as 0.016 ng/ml. In contrast, the masking of TNF-α with specific functional antibodies abrogated neurite growth inhibition by activated macrophages. Taken together, these results indicated that TNF-α is a key component of inhibitory macrophage action. The transfection of PC12 neurons with microRNA-124 (miR-124) counteracted the inhibition of neurites mediated by both recombinant TNF-α and macrophages. miR-124 did not stimulate neurite formation per se, nor was cell viability affected. These data suggest that miR-124 might be a valuable tool for desensitizing neurons to a repulsive inflammatory environment.

Cell-type specific four-component hydrogel.

In the field of regenerative medicine we aim to develop implant matrices for specific tissue needs. By combining two per se, cell-permissive gel systems with enzymatic crosslinkers (gelatin/transglutaminase and fibrinogen/thrombin) to generate a blend (technical term: quattroGel), an unexpected cell-selectivity evolved. QuattroGels were porous and formed cavities in the cell diameter range, possessed gelation kinetics in the minute range, viscoelastic properties and a mechanical strength appropriate for general cell adhesion, and restricted diffusion. Cell proliferation of endothelial cells, chondrocytes and fibroblasts was essentially unaffected. In contrast, on quattroGels neither endothelial cells formed vascular tubes nor did primary neurons extend neurites in significant amounts. Only chondrocytes differentiated properly as judged by collagen isoform expression. The biophysical quattroGel characteristics appeared to leave distinct cell processes such as mitosis unaffected and favored differentiation of sessile cells, but hampered differentiation of migratory cells. This cell-type selectivity is of interest e.g. during articular cartilage or invertebral disc repair, where pathological innervation and angiogenesis represent adverse events in tissue engineering.

Hyaluronic acid/chitosan multilayer coatings on neuronal implants for localized delivery of siRNA nanoplexes.

Binding, stabilizing and promoting cellular uptake of siRNA are all critical efforts in creating matrices for the localized delivery of siRNA molecules to target cells. In this study, we describe the generation of chitosan imidazole/siRNA nanoplexes (NPs) embedded in nano scope polyelectrolyte multilayers (PEMs) composed of hyaluronic acid and chitosan for sustained and localized drug delivery. Regular PEM build-up, successful integration of NPs and controlled release under physiological conditions were shown. Biological efficacy was evaluated in neuronal cell culture concerning cell adhesion, viability, NPs uptake and gene silencing. The additionally shown biological functionalization of neuronal implants possesses potential for future applications in the field of regenerative medicine and treatment of spinal cord injuries.

The bispecific SDF1-GPVI fusion protein preserves myocardial function after transient ischemia in mice.

BACKGROUND: CXCR4-positive bone marrow cells (BMCs) are critically involved in cardiac repair mechanisms contributing to preserved cardiac function. Stromal cell-derived factor-1 (SDF-1) is the most prominent BMC homing factor known to augment BMC engraftment, which is a limiting step of stem cell-based therapy. After myocardial infarction, SDF-1 expression is rapidly upregulated and promotes myocardial repair. METHODS AND RESULTS: We have established a bifunctional protein consisting of an SDF-1 domain and a glycoprotein VI (GPVI) domain with high binding affinity to the SDF-1 receptor CXCR4 and extracellular matrix proteins that become exposed after tissue injury. SDF1-GPVI triggers chemotaxis of CXCR4-positive cells, preserves cell survival, enhances endothelial differentiation of BMCs in vitro, and reveals proangiogenic effects in ovo. In a mouse model of myocardial infarction, administration of the bifunctional protein leads to enhanced recruitment of BMCs, increases capillary density, reduces infarct size, and preserves cardiac function. CONCLUSIONS: These results indicate that administration of SDF1-GPVI may be a promising strategy to treat myocardial infarction to promote myocardial repair and to preserve cardiac function.

Telomerase-based immortalization modifies the angiogenic/inflammatory responses of human coronary artery endothelial cells.

Telomerase reverse transcriptase (TERT) is fundamental in determining the life span by regulating telomere length of chromosomes. To address the question whether the enhancement of the proliferative potential hampers cell differentiation, we generated TERT-over-expressing endothelial cells (ECs) and analyzed in vitro their (1) barrier function; (2) low-density lipoprotein uptake; (3) expression pattern of six selected marker proteins; (4) angiogenic potential in four assays; and (5) inflammatory responses. In contrast to investigations with focus on other cell parameters, we demonstrate that immortalization of ECs by over-expression of TERT resulted in different angiogenic and inflammatory behavior in comparison to cells with low native telomerase levels.

Bioartificial reconstruction of peripheral nerves using the rat median nerve model.

Different bioartificial tubes were recommended for peripheral nerve reconstruction in the past. In order to replace autologous nerve grafts this materials are still under review in different animal studies. Most of them are dealing with the rodent peripheral nerves. One very popular animal model to study different materials is the rat median nerve model. With its easy excess, simple behavioral tests and reliable long term results it is attractive to many scientists in this field. This review gives an overview about the past, current and future options in this model for bioartificial nerve tubes. It summarizes the evolution of successful implantation of different materials across short nerve gaps and demonstrates the obstacles arising from long nerve gaps and the problems associated to them.

Neuronal and glial responses to siRNA-coated nerve guide implants in vitro.

The manipulation of gene expression by RNA interference could play a key role in future neurotherapies, for example in the development of biohydrid implants to bridge nerve and spinal cord lesion gaps. Such resorbable biomaterial prostheses could serve as growth substrates together with specific siRNA to foster neuronal regeneration. To the best of our knowledge, we are the first to biofunctionalize neuronal prostheses with siRNA. We analyzed neuronal and Schwann cell responses to scrambled siRNA coated polydioxanone polymer filaments designed to imitate pro-regenerative bands of Büngner for oriented axonal regrowth. With a view to future clinical applications we were especially interested in potentially detrimental side effects. We employed a variety of in vitro methods, including a novel impedance electrode microchamber assay, fluorescence and scanning electron microscopy, metabolic labeling and RT-PCR. We found that the application of chitosan/siRNA nanoparticles (1) did not affect glial cell motility or (2) axonal growth in contrast to other formulations, (3) only slightly reduced proliferation, and (4) did not induce inflammatory responses that might hamper axonal regeneration. The data suggest that chitosan/siRNA nanoparticle-coated polymer filaments are suitable for use in biohybrid implants with no significant side effects on neuronal and glial cells.