(H-281) Studying Interactions Between PEI-DNA Polyplexes and Polymer Surface Coatings in Different Media for Localized Transfection

Introduction:: While nucleic acid delivery has shown much therapeutic promise, including clinical applications in cancer, vaccines and spinal muscular atrophy, typical systemic administration faces several limitations, including poor targeting efficiency and systemic clearance. Localized delivery of nucleic acid vectors from biomaterial surfaces can overcome many of these difficulties and increase transfection efficiencies by maintaining higher nucleic acid concentrations at the target site and controlling release kinetics. This project aims to characterize the absorption and release interactions between a model polymeric nucleic acid delivery vector, polyethyleneimine (PEI), and surface coatings composed of bio-inspired and naturally derived polymers in different media with the long-term goal of developing a surface coating that enables localized nucleic acid delivery for tissue engineering and regenerative medicine applications.


Materials and Methods:: DNA-PEI polyplexes were formed via complexation of linear PEI (Mn = 20k) and plasmid DNA encoding GFP at several N:P ratios (0, 10, 20) in various media (PBS, DMEM). Resulting polyplex sizes and zeta potentials were characterized via Dynamic Light Scattering and ZetaPlus analysis. Surface coatings were formed at concentrations of 2 mg/mL and 5 mg/mL within tissue culture plates from the following polymers and polymer combinations: polydopamine (pDA); hyaluronic acid (HA); glycol chitosan (GLY-CHI); pDA + HA; and pDA + GLY-CHI. Uncoated wells (NC) were used as a control. Coatings were incubated with DNA-PEI polyplexes at various concentrations and N:P ratios and DNA loading efficiencies and release kinetics from these coatings were analyzed via the PicoGreen assay. Transfection efficiencies of these coatings were evaluated in NIH3T3 and HEK293 cells and cytotoxicity was evaluated via Live/Dead staining.


Results, Conclusions, and Discussions:: Although showing similar size ranges, DNA-PEI polyplexes formed in DMEM exhibited significantly lower zeta potentials than those formed in PBS. Naked plasmid DNA (N/P = 0) showed significantly lower surface loading efficiencies compared to DNA-PEI polyplexes at N/P ratios of 10 and 20 for both PBS and DMEM. Surprisingly, polyplex surface loading efficiency was higher for polyplexes formed in DMEM than PBS. Naked plasmid DNA showed significantly higher amounts of DNA release when deposited in PBS than in DMEM, whereas polyplexes at N/P ratios of 10 and 20 formed in DMEM showed significantly higher amounts of DNA release than those formed in PBS. Surface-based transfection often resulted in similar GFP levels as bolus transfection controls. Although not statistically significant, GC coatings generally exhibited the lowest levels of surface-based transfection and non-coated tissue culture plastic often showed higher levels. Bolus transfection on the same coatings combined with cell viability assays indicated that the observed differences in surface-based transfection were not only due to variations in DNA surface loading. Future work will focus on improving surface-based transfection efficiency and on studying the effects of coating composition on cellular polyplex uptake.


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