Dicationic Amino Substituted Gemini Surfactants and their Nanoplexes: Improved Synthesis and Characterization of Transfection Efficiency and Corneal Penetration In Vitro

Purpose- To formulate and characterize nanoparticles from m-7NH-m gemini surfactants, synthesized by a new improved method, for non-invasive gene delivery including optimization of composition for transfection efficiency and corneal penetration. Methods – A one-pot, solvent-free, DMAP-free method was developed for the synthesis of m-7NH-m (m=12– 18) gemini surfactant series. Lipoplexes (LPXs) and nanoplexes (NPXs) of gemini surfactant-plasmid DNA were formulated with and without DOPE helper lipid, respectively, at various charge ratios and characterized by dynamic light scattering and zeta potential measurements. Transfection efficiency, cellular toxicity, effect of DOPE and gene expression kinetic studies were carried out in A7 astrocytes by flow cytometry and confocal microscopy. Corneal penetration studies of 18-7NH-18 NPXs were carried out using 3D EpiCorneal® tissue model. Results -The new synthesis method provides a two-fold improved yield and the production of a pure species of m7NH-m without DMAP and trimeric m-7N(m)-m surfactants as impurities. Structure and purity was confirmed by ESI-MS, 1H NMR spectroscopy and surface tension measurements. Particle size of 199.80±1.83 nm±S.D. and a zeta potential valueof+30.18±1.17mV±S.D.was obtained for 18-7NH 18 5:1 ratio NPXs showed optimum transfection efficiency (10.97±0.11%) and low toxicity (92.97±0.57% viability) at the48-h peak expression. Inclusion of DOPE at 1:0.5 and 1:1 ratios to gemini surfactant reduced transfection efficiency and increased toxicity. Treatment of EpiCorneal® tissue model showed deep penetration of up to 100 μm with 18-7NH-18 NPXs. Conclusion – Overall, 18-7NH-18 NPXs are potential gene delivery systems for ophthalmic gene delivery and for further in vivo studies.

EpiCorneal (COR-100), nanoparticle, eye drops, nanoplexes, confocal microscopy

Calcein AM, Image IT dead cell stain