(16b) ABC Triblock Copolymer Micelleplexes for Potent Gene Silencing and In Vivo Tumor Targeting

Polymers, particularly polycations, are very popular non-viral gene carriers because they can spontaneously associate with the negatively-charged phosphate backbone of nucleic acids to form protective “polyplexes”. However, among the drawbacks of these systems is that under physiological salt conditions, where repulsive forces between like-charged particles are screened and/or nonspecific interactions with negatively-charged blood components may be present, aggregation of cationic polyplexes can result. Conjugation of poly(ethylene glycol), or PEG, to cationic polymers has been shown to confer several advantageous properties including colloidal stability and reduced aggregation of complexes due to steric shielding and increased hydrophilicity/water solubility. Both polycation (poly(2-(dimethylamino)ethyl methacrylate) or PDMAEMA) and PEGylated polycation (PEG-PDMAEMA) formulations were employed in this study as siRNA delivery vehicles. In addition, we are introducing a novel chemistry for triblock copolymer micelles made from poly(ethylene glycol)-poly(n-butyl acrylate)-poly(2-(dimethylamino)ethyl methacrylate) (PEG-PnBA-PDMAEMA). This micelle-based approach is different from the more traditional polymeric carriers being studied in that micelles form gene silencing complexes (“micelleplexes”) based on particles rather than individual polymer chains which consequently make them larger in relative size. We expect this may have important implications in the ultimate efficiency of the systemic delivery process.

In this study, we examined fundamental in vitro properties such as binding strength, enzymatic resistance, cytotoxicity, protein aggregation and gene silencing efficiency across three types of polymeric carrier systems. By comparing PDMAEMA with PEG-PDMAEMA and PEG-PDMAEMA with PEG-PnBA-PDMAEMA, we sought to elucidate the role of extent of PEGylation and size/architecture, respectively, on performance and hypothesize how this may ultimately translate into in vivo efficiency. A series of in vitro/pre-in vivo experiments showed that each of the three systems tested were stably complexed with siRNA, enzyme-resistant, fairly non-cytotoxic and resisted aggregation in the presence of serum levels of protein. Most importantly, the in vitro silencing levels of the exogenous luciferase gene were about 85% for each system, and were on par with a commercial transfection reagent, Lipofectamine2000, which was also tested. Encouraging preliminary in vivo biodistribution studies with fluorescently-tagged PEG-PDMAEMA and PEG-PnBA-PDMAEMA complexes in athymic mice possessing solid tumors indicate that both complexes do accumulate and get retained in tumor tissue. This is a promising result which suggests that our carriers are good candidates for exploiting the Enhanced Permeability and Retention (EPR) effect, responsible for the nonspecific uptake of favorably-sized nanoparticles in tumors. With some further testing and optimization, we believe our systems will prove to be viable options for in vivo tumor-targeted therapies.