A Combinatorial Approach for Targeted Delivery using Small Molecules and Reversible Masking to Bypass Non-Specific Uptake In Vivo

We have developed a multi-disciplinary approach combining molecular biology, delivery technology, combinatorial chemistry, and reversible masking to create improved systemic, targeted delivery of plasmid DNA while avoiding non-specific uptake in vivo. We initially used a well characterized model targeting the asialolglycoprotein receptor in the liver. Using our bilamellar invaginated vesicle (BIV) liposomal delivery system with reversible masking, we increased expression in the liver by 76-fold, nearly equaling expression in first-pass organs using non-targeted complexes, with no expression in other organs. The same technology was then applied to efficiently target delivery to a human tumor microenvironment model. We achieved efficient, targeted delivery by attachment of specific targeting ligands to the surface of our BIV complexes in conjunction with reversible masking to bypass non-specific tissues and organs. We identified ligands that target a human tumor microenvironment created in vitro by co-culturing primary human endothelial cells with human lung or pancreatic cancer cells. The model was confirmed by increased expression of tumor endothelial phenotypes including CD31 and VEGF-A, and prolonged survival of endothelial capillary-like structures. The co-cultures were used for high-throughput screening of a specialized small-molecule library to identify ligands specific for human tumor-associated endothelial cells in vitro. We identified small molecules that enhanced the transfection efficiency of tumor-associated endothelial cells, but not normal human endothelial cells or cancer cells. Intravenous injection of our targeted, reversibly masked complexes into mice, bearing human pancreatic tumor and endothelial cells, specifically increased transfection to this tumor microenvironment about 200-fold. Efficacy studies using our optimized targeted delivery of a plasmid encoding thrombospondin-1 eliminated tumors completely after five intravenous injections administered once every week.