Novel water insoluble lipoparticulates for gene delivery

Purpose. The objective was to design and prepare water insoluble lipoparticulates (ISLPs) for efficient gene delivery to lung tissue. Methods. Nona{(ethylenimine)-co-[(2-aminoethyl)-N-choleseteryl-oxycarbonyl-ethylenimine]} (NEACE-T) was synthesized in both its free-base and chloride salt-forms using linear polyethylenimine (PEI, M-w 423) as a headgroup and cholesteryl chloroformate as a hydrophobic lipid anchor resulting in a T-shaped lipononamer. Semitele- chelic Nalpha-cholesteryloxycarbonyl nona(ethylenimine) (st-NCNEI-L) was synthesized similarly resulting in a linear lipononamer. As confirmed by H-1-NMR, the site of conjugation was either a primary amine resulting in a linear configuration (st-NCNEI-L) or a secondary amine resulting in a T-shaped configuration (NEACE-T). ISLPs were prepared by combining NEACE-T or st-NCNEI-L with a colipid, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) at 1/1, 1/2, and 2/1 molar ratios and the lipoparticulates were hydrated and filtered. ISLP/p2CMVmIL-12 complexes were characterized for particle size, zeta potential, surface morphology, cytotoxicity, and in vitro transfection efficiency. Results. Transgene expression was dependent on the site of cholesterol conjugation, lipononamer:colipid molar ratio, and ISLP/p2CMVmIL-12 charge ratios. ISLP/p2CMVmIL-12 complexes were nontoxic to murine colon adenocarcinoma (CT-26) cells at 9/1 (+/-) or lower, had a mean particle size of 330-400 nm while the zeta potential varied from 36-39 mV. Atomic force microscopy (AFM) showed the surface morphology to be that of an oblate spheroid with a size comparable to that determined by dynamic light scattering. ISLP/p2CMVmIL-12 complexes prepared using free-base NEACE-T:DOPE (1/2) at charge ratios of 3/1 and 5/1 (+/-) provided the highest levels of transgene expression, 18 times more than the levels provided by the salt-form. Secreted levels of mIL-12 p70 were 75 times higher for ISLP/p2CMVmIL-12 complexes than naked p2CMVmIL-12 and nearly 4 times higher than PEI 25 kDa/p2CMVmIL-12 complexes. Conclusions. The transfection efficiency of the ISLPs was dependent on the site of cholesterol conjugation, amount of colipid, and charge ratio. The highest levels of transgene expression were provided by NEACE-T:DOPE (1/2)/p2CMVmIL-12 at a 3/1 (+/-) charge ratio.