Lipid-polymer hybrid nanoplex loaded microneedle patches as a corneal delivery platform for CRISPR/Cas expressing plasmid.

Lipid-polymer hybrid (LPH) systems have evolved into a promising vehicle for delivering therapeutic agents. This study demonstrates cationic LPH nanoplexes composed of a biodegradable cationic mPEG-polycarbonate based cationic copolymer and cholesterol to enhance delivery efficiency of genetic materials. The optimized blank formulation, BNPX-3, exhibited a particle size of 93.60 nm, polydispersity index (PDI) of 0.116, and a zeta potential (ZP) of 21.9 mV. After complexation of pcDNA3-EGFP plasmid and CRISPRi plasmid at N/P ratios of 10 and 20, respectively, the nanoplexes exhibited particle sizes of 123.4 nm and 131.6 nm, with corresponding PDI values of 0.128 and 0.142 and zeta potentials of 12.2 mV and 10.5 mV, respectively. Cytocompatibility studies demonstrated >80% cell viability in HEK293 and SIRC cells over a wide concentration range. Transfection efficiencies using LPH nanoplexes for pcDNA3-EGFP (6159 bp) and CRISPRi plasmids (11,266 bp) were found to 60.09% and 48.02%, respectively, in HEK293 cells, and 56.04% and 40.34%, respectively, in SIRC cells. These efficiencies were comparable to Lipofectamine 3000 and superior to formulations prepared with cationic polymers alone. To enable efficient delivery of the nanoplexes, the developed nanoplexes were incorporated into dissolvable microneedle (MN) patches. The microneedle patch demonstrated successful loading of pDNA-complexed nanoplexes and efficient delivery ex-vivo.