Chemically-induced Extracellular Blebs for Versatile Cancer Therapy

Extracellular vesicles (EVs) have recently gained momentum in the field of therapeutic delivery due to their promise as therapeutic carriers. Biomolecules are naturally transported by EVs for cell-to-cell communication, and EV interactions with target cells are thought to be specific to cell type. Naturally produced extracellular blebs (EBs), a category of EVs that are derived specifically from blebbing of the cell membrane, transport cargo to target cells while maintaining the surface properties of the cells from which they were derived. Although EBs have been used for preclinical drug delivery and gene therapy, obtaining sufficient samples of well-characterized blebs for clinical studies is still a major challenge. A major focus of my work has been on developing and characterizing a method for large-scale production of EBs. More specifically, this dissertation explores a method for controlling EB production by sulfhydryl blocking reagents. The optimized formulation of sulfhydryl-blocking reagents described in this dissertation led to an order of magnitude increase in EB production as characterized by protein quantification assay. Furthermore, this increase in EB production was achieved in a fraction of the time required for natural production of EBs. With the aim of utilizing EBs for cancer therapy, the efficacy of the production method was assessed for two applications: delivery of a chemotherapeutic drug, and presentation of a model cancer antigen for immunotherapy. Studies confirmed that in tumor-challenged mice treated with chemotherapeutic-loaded, nano-sized EBs, there was significantly slowed tumor growth and improved survival compared to treatment with free drug or liposomal drug. The chemotherapeutic-loaded, nano-sized EBs demonstrated improved cellular uptake, facilitated intracellular drug release and targeted accumulation in the tumor, and they avoided accumulation in vital organs in comparison to a commercial liposomal formulation. In a separate study, antigen-presenting micro-sized EBs produced by sulfhydryl-blocking expressed H-2Kb bound to SIINFEKL and effectively activated T cells in vitro and cytotoxic T lymphocytes in vivo. Micro-sized EBs performed as well as whole cell therapy in terms of slowing tumor growth and improving survival outcomes while providing a cell-free alternative therapy. EBs have a broad range of potential health applications beyond drug delivery and immunotherapy; these include gene therapy, immune modulation, tissue regeneration, and pathogen suppression. EB production by sulfhydryl blocking is highly promising for overcoming a current technological gap and allowing EB-based therapeutics to progress more rapidly to clinical trials. In summary, this dissertation provides a study of an innovative method of EB production including evaluation of two therapeutic applications