(194t) Folate-Conjugated Negatively Charged Ternary Polyplexes for Targeted Gene Delivery

Polymer-mediated delivery of nucleic acids is a promising alternative to the traditional viral delivery methods for treatment of a variety of conditions including cancer. Polymeric vehicles are generally considered safer than viral vectors but have yet to produce results that rival the efficacy of nature’s virus. Polycations are electrostatically complexed with nucleic acids to form polyplexes that typically possess positive surface charge. These polyplexes are effective at cellular internalization and even endosomal escape. However, they are unsuitable carriers in vivo due to agglomeration with negatively charged serum protein, preventing internalization and promoting renal clearance. Even in vitro transfection with conventional polyplexes is greatly inhibited in the presence of serum. Ternary polyplexes can be formed by adding polymers containing negative moieties to polycation/DNA polyplexes at weight ratios that result in negative zeta potential. These polyplexes resist serum agglomeration by charge repulsion but are also repelled by the predominantly negative cell membrane. To overcome this repulsion, targeting ligands may be conjugated to the polyplexes to promote receptor-mediated uptake.

This project evaluates the transfection potential of folate-conjugated polyplexes consisting of 25-kDa polyethylenimine (PEI), 15-kDa poly(glutamic acid) (PGA), and the pGL3 luciferase reporter plasmid. Carbodiimide crosslinker chemistry is used to crosslink the carboxylic acid group in PGA with the alpha carboxylic group on folate at a ratio of 2, 5, and 8 folates per PGA chain (PGA-fol). PGA-fol is then complexed with PEI:pGL3 complexes to form polyplexes of varying negative zeta potential. Cancer cells known to overexpress folate are transfected in the absence and presence of serum with PGA-fol/PEI/pGL3 polyplexes and transgene expression compared to non-targeted polyplexes of equivalent PGA/PEI/pGL3 weight ratios. To verify uptake due to folate interaction with folate receptors, cells are transfected in the presence of increasing amounts of free folate to saturate the folate receptors and decrease folate uptake (verified by cytometry). RNA interference or small molecule inhibition of the caveolin uptake route (pathway of internalization for folate) will also indicate the targeting specificity of polyanionic polyplexes tagged with folate. These results will validate folate targeting as an effective compensation to the repulsion of the cell membrane with anionic polyplexes to produce increased gene delivery.