The neuropilin-1 (np1) and neuropilin-2 (np2) receptors form complexes with type-A plexins. These complexes serve as signaling receptors for specific class-3 semaphorins. Np1 and np2 function in addition as receptors for heparin-binding forms of vascular endothelial growth factor (VEGF), such as VEGF 165 . Human umbilical vein endothelial cells (HUVEC) express tyrosine-kinase receptors for VEGF and basic fibroblast growth factor (bFGF), as well as np1, np2, and several type-A plexins. We have found that semaphorin-3F (s3f), a semaphorin which signals through the np2 receptor, was able to inhibit VEGF 165 , as well as bFGF-induced proliferation of HUVECs. Furthermore, s3f inhibited VEGF as well as bFGF-induced phosphorylation of extracellular signalregulated kinase-1/2. Our experiments indicate that bFGF does not bind to neuropilins, nor does s3f inhibit the binding of bFGF to FGF receptors. It is therefore possible that s3f inhibits the activity of bFGF by a mechanism that requires active s3f signal transduction rather than by inhibition of bFGF binding to FGF receptors. s3f also inhibited VEGF 165 , as well as bFGF-induced in vivo angiogenesis as determined by the alginate micro-encapsulation and Matrigel plug assays. Overexpression of s3f in tumorigenic human HEK293 cells inhibited their tumor-forming ability but not their proliferation in cell culture. The tumors that did develop from s3f-expressing HEK293 cells developed at a much slower rate and had a significantly lower concentration of tumor-associated blood vessels, indicating that s3f is an inhibitor of tumor angiogenesis.

The ultimate goal in cancer therapy is achieving selective targeting of cancer cells. We report a novel delivery platform, based on nanoghosts (NGs) produced from the membranes of mesenchymal stem cells (MSCs). Encompassing MSC surface molecules, the MSC-NGs retained MSC-specific in vitro and in vivo tumor targeting capabilities and were cleared from blood-filtering organs. MSC-NGs were found to be biocompatible. Systemic administration of drug loaded MSC-NGs demonstrated 80% inhibition of human prostate cancer.

Therapeutic ultrasound (TUS) has the potential of becoming a powerful nonviral method for the delivery of genes into cells and tissues. Understanding the mechanism by which TUS delivers genes, its bioeffects on cells and the kinetic of gene entrances to the nucleus can improve transfection efficiency and allow better control of this modality when bringing it to clinical settings. In the present study, direct evidence for the role and possible mechanism of TUS (with or without Optison) in the in vitro gene-delivery process are presented. Appling a 1 MHz TUS, at 2 W/cm 2 , 30%DC for 30 min was found to achieve the highest transfection level and efficiency while maintaining high cell viability (480%). Adding Optison further increase transfection level and efficiency by 1.5 to three-fold. Confocal microscopy studies indicate that long-term TUS application localizes the DNA in cell and nucleus regardless of Optison addition. Thus, TUS significantly affects transfection efficiency and protein kinetic expression. Using innovative direct microscopy approaches: atomic force microscopy, we demonstrate that TUS exerts bioeffects, which differ from the ones obtained when Optison is used together with TUS. Our data suggest that TUS alone affect the cell membrane in a different mechanism than when Optison is used.

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Cell encapsulation is a promising approach for long-term delivery of therapeutic agents. Nonetheless, this system has failed to reach clinical settings, as the entrapped cells provoke a host immune reaction. Mesenchymal stem cells (MSCs), however, potentially may overcome this impediment and serve as a promising platform for cell-based microencapsulation. They are known to be hypoimmunogenic and can be genetically modified to express a variety of therapeutic factors. We have designed alginate-PLL microcapsules that can encapsulate human MSCs (hMSCs) for extended periods, as demonstrated by fluorescence and H(3)-thymidine assays. The encapsulated hMSCs maintained their mesenchymal surface markers and differentiated to all the typical mesoderm lineages. In vitro and in vivo immunogenicity studies revealed that encapsulated hMSCs were significantly hypoimmunogenic, leading to a 3-fold decrease in cytokine expression compared to entrapped cell lines. The efficacy of such systems was demonstrated by genetically modifying the cells to express the hemopexin-like protein (PEX), an inhibitor of angiogenesis. Live imaging and tumor measurements showed that encapsulated hMSC-PEX injected adjacent to glioblastoma tumors in nude mice led to a significant reduction in tumor volume (87%) and weight (83%). We clearly demonstrate that hMSCs are the cell of choice for microencapsulation cell based-therapy, thus bringing this technology closer to clinical application.

The use of chitosan in complexation with alginate appears to be a promising strategy for cell microencapsulation, due to the biocompatibility of both polymers and the high mechanical properties attributed by the use of chitosan. The present work focuses on the optimization and characterization of the alginate-chitosan system to achieve long-term cell encapsulation. Microcapsules were prepared from four types of chitosan using one- and two-stage encapsulation procedures. The effect of reaction time and pH on long-term cell viability and mechanical properties of the microcapsules was evaluated. Using the single-stage encapsulation procedure led to increase of at least fourfold in viability compared with the two-stage procedure. Among the four types of chitosan, the use of high molecular weight (MW) chitosan glutamate and low MW chitosan chloride provided high viability levels as well as good mechanical properties, i.e., more than 93% intact capsules. The high viability levels were found to be independent of the reaction conditions when using high MW chitosan. However, when using low MW chitosan, better viability levels (195%) were obtained when using a pH of 6 and a reaction time of 30 min. An alginate-chitosan cell encapsulation system was devised to achieve high cell viability levels as well as to improve mechanical properties, thus holding great potential for future clinical application.

Biomimetic scaffolds generally aim at structurally and compositionally imitating native tissue, thus providing a supportive microenvironment to the transplanted or recruited cells in the tissue. Native decellularized porcine extracellular matrix (ECM) is becoming the ultimate bioactive material for the regeneration of different organs. Particularly for cardiac regeneration, ECM is studied as a patch and injectable scaffolds, which improve cardiac function, yet lack reproducibility and are difficult to control or fine‐tune for the desired properties, like most natural materials. Seeking to harness the natural advantages of ECM in a reproducible, scalable, and controllable scaffold, for the first time, a matrix that is produced from whole decellularized porcine cardiac ECM using electrospinning technology, is developed. This unique electrospun cardiac ECM mat preserves the composition of ECM, self‐assembles into the same microstructure of cardiac ECM ,and ,above all, preserves key cardiac mechanical properties. It supports cell growth and function, and demonstrates biocompatibility in vitro and in vivo. Importantly, this work reveals the great potential of electrospun ECM‐based platforms for a wide span of biomedical applications, thus offering the possibility to produce complex natural materials as tailor‐made, well‐defined structures.