(628c) Folate Receptor-Targeted Anionic Ternary Polyplexes for Gene Delivery
AIChE Annual Meeting
2019
2019 AIChE Annual Meeting
Nanoscale Science and Engineering Forum
Bionanotechnology for Gene and Drug Delivery I
Thursday, November 14, 2019 - 8:58am to 9:16am
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.