(628c) Folate Receptor-Targeted Anionic Ternary Polyplexes for Gene Delivery

Nonviral gene delivery has been investigated as a promising alternative to viral vectors due to its decreased immunogenicity and safety concerns. Polymer-mediated delivery has been widely studied due to ease of polymer modification and production, potential for biocompatibility, and lack of immunogenicity, but these systems fall short of recombinant viruses in terms of gene delivery efficiency. Polymeric gene delivery vectors most commonly comprise electrostatic complexation of polycationic polymers and plasmid DNA to form polyplexes. These complexes are effective for in vitro transfection but, due to excess positive charge, often aggregate with serum proteins and interact strongly with cellular membranes leading to cytotoxicity and non-specific interactions that can interfere with cell-specific targeting. To address these problems, ternary polyplexes have recently been developed that incorporate a polyanionic component to inhibit serum agglomeration and reduce cytotoxicity. The negative charge of these polyplexes also allows the opportunity for addition of targeting ligands to promote receptor-mediated uptake, thus increasing gene delivery efficiency and specificity.

We are investigating the transfection and cellular internalization of folate-targeted ternary polymeric gene delivery vectors comprising 25-kDa branched polyethylenimine (PEI), 15-kDa poly(glutamic acid) (PGA), and pGL3 luciferase reporter plasmid. Folate was conjugated to PGA through carbodiimide crosslinking chemistry at ratios of 3, 7, and 10 folates per chain (PGA-Fol(3, 7, 10)). These conjugated polyanions were then electrostatically complexed with PEI/pGL3 binary complexes to form negatively charged ternary polyplexes. Cell lines overexpressing and under-expressing folate receptor (HeLa and HEK 293, respectively) were transfected with these ternary polyplexes and transgene expression and particle uptake were compared to non-targeted ternary polyplexes of equivalent PGA/PEI/DNA weight ratios. Transfection with PGA-Fol(7)/PEI/DNA polyplexes in HeLa cells increased uptake and transgene expression ~9-fold when compared to nontargeted polyplexes of the same weight ratio. To verify folate receptor targeting, expression of the folate receptor was knocked down through RNA interference (RNAi) and uptake was quantified via flow cytometry. The internalization mechanism was also investigated using endocytosis inhibitors and RNAi to verify specificity of folate-receptor targeting. Transfections in the presence of endocytosis inhibitors confirmed caveolin-dependent endocytosis as a primary internalization mechanism, as expected of folate-targeted internalization. Macropinocytosis was also found to be an important pathway, likely due to the efficient, nonspecific uptake of the pathway. Folate-conjugated anionic complexes improved uptake and gene expression over non-targeted particles of the same PGA/PEI/DNA weight ratio. These results suggest folate-receptor targeting as an effective system to improve the gene delivery efficiency of anionic ternary polyplexes.