M.Sc Thesis


M.Sc StudentEyal Weinstein
SubjectDeveloping Cell-Derived Vesicles as siRNA Delivery Vehicles
for Cancer
DepartmentDepartment of Nanoscience and Nanotechnology
Supervisor Full Professors Machluf Marcelle


Abstract

The main obstacle in cancer therapy is targeting toxic agents to cancer cells and avoid damage to healthy cells. Drug delivery systems, have been receiving growing attention over the last decade in the field of targeted cancer therapy. Some of these systems exploit the enhanced permeability and retention (EPR) effect, characteristic to tumors and tumor microenvironment, achieving passive targeting of the nanoparticles. On the other hand, some systems achieve cancer selectivity by attaching specific ligands, particularly antibodies, to the surface of the nanoparticle. However cancer specific epitopes are scarce.

In this study, we aimed to develop a novel siRNA targeted system that exploits cell-cell interactions as the basis for targeting. The system is based on unique nano vesicles, produced for the first time from the membranes of mesenchymal stem cells (MSC). We have shown that these nano-vesicles can be loaded with various types of molecules such as proteins, synthetic drugs and nucleic acids. We have examined the ability of MSC to target tumors and derive particles that possess similar features. Using our innovative design, we aimed to create a uniform platform for the delivery of siRNA, allowing knock down of different proteins of choice.

In the first set of experiments, we have characterized our cell derived vesicles (CDVs). We have succeeded in producing vesicles made of the MSC, which have a diameter of ~150nm, which is appropriate for intravenous administration. The Zeta potential values of the CDVs (-17.0mV) show that the colloidal system is stable. The CDVs retain MSC surface molecules. We have found that CDV target cancer cell lines. 

We then prepared and characterized PEI/siRNA complexes to be loaded in the CDVs. We have found that different forms of PEI produce PEI/siRNA complexes of different structure and toxicity. siRNA transfected by PEI induced 15% reduction of EGFP. Protein knockdown profile was affected by the PEI/siRNA ratio. We have chosen the optimal PEI/siRNA ratios for subsequent studies.

We have also attempted to load the PEI/siRNA complexes in CDVs, yet obtained low siRNA loading yields. Suspecting that the siRNA content determination protocol used was not accurate enough, we have proceeded and examined the biological activity of PEI/siRNA loaded CDV. We have found that knockdown activity of siRNA is maintained using the CDV formulations, achieving 23% EGFP knockdown. Furthermore, the CDVs modulate the toxicity of PEI.

Collectively, these findings set the base for constructing a siRNA delivery system made of MSC CDV.