Novel Water Insoluble Lipoparticulates for Gene Delivery Darin Y. Furgeson, 1 Richard N. Cohen, 2 Ram I. Mahato, 3 and Sung Wan Kim 1,4 Received December 14, 2001; accepted January 4, 2002 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 hydro- phobic lipid anchor resulting in a T-shaped lipononamer. Semitele- chelic N-cholesteryloxycarbonyl nona(ethylenimine) (st-NCNEI-L) was synthesized similarly resulting in a linear lipononamer. As con- firmed by 1 H-NMR, the site of conjugation was either a primary amine resulting in a linear configuration (st-NCNEI-L) or a second- ary amine resulting in a T-shaped configuration (NEACE-T). ISLPs were prepared by combining NEACE-T or st-NCNEI-L with a co- lipid, 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 par- ticle size, zeta potential, surface morphology, cytotoxicity, and in vitro transfection efficiency. Results. Transgene expression was dependent on the site of choles- terol 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  potential varied from 36–39 mV. Atomic force microscopy (AFM) showed the surface morphology to be that of an oblate spheroid with a size com- parable 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. KEY WORDS: insoluble; lipononamer; lipoparticulates; cytokine; transgene. INTRODUCTION Over the last decade there have been scores of attempts to design a non-viral gene delivery system which could achieve the level of gene expression and specificity offered by viral vectors while maintaining the flexible characteristics of cDNA size, immune response bypass, and safety. One of the most successful nonviral gene delivery systems has been cat- ionic lipids. Early advances in cationic lipid-mediated gene delivery showed that the chemistry of a cationic lipid-based gene carrier is dependent on three areas (1–4). First, a polar cationic headgroup is necessary for condensation of poly- anionic DNA, which is necessary for DNA protection and efficient delivery. In addition, the cationic headgroup is able to interact with the anionic lipids of the cellular membrane, thus enhancing the cellular uptake. A single positive charge per molecule (1) has been transcended by multi-charged cat- ionic headgroups (5–7), which provide superior electrostatic levels for DNA condensation (8,9). The cationic headgroup has also been modified to increase the ability of DNA con- densation. Both branched and linear polyethylenimine (PEI) have been reported to be effective for gene delivery because of enhanced endosomal release of DNA to the cytoplasm due to the proton-sponge effect of PEI (10,11). However, PEI of 25 kDa or above is toxic to cells and PEI/pDNA complexes are prone to aggregation. Unlike high molecular weight branched PEI, linear PEI of 423 Da is nontoxic and its struc- ture is similar to that of spermine (except in the number of secondary amines), which has been used as a cationic head- group in various cationic lipids. Second, it was found that a hydrophobic lipid anchor is essential for enhanced DNA sta- bility in the bloodstream and cellular uptake. Cellular uptake is enhanced by the favorable interaction between the hydro- phobic groups of the gene carrier and the cellular membrane. Cholesterol is a naturally occurring lipid and metabolized in the body, making it highly biocompatible; therefore, choles- terol has been widely used as a hydrophobic lipid anchor (1,12–18). Finally, the linker between the hydrophobic anchor and the polar headgroup determines the chemical stability and biodegradability of cationic lipids. These linker groups should be biodegradable yet strong enough to sustain in vivo (4). Therefore, in this study, we designed novel lipononamers using linear PEI of 423 Da and cholesteryl chloroformate in both a T-shaped (NEACE-T) and linear (st-NCNEI-L) con- figuration, mixed with a colipid to prepare insoluble lipopar- ticulates (ISLPs), formed complexes with p2CMVmIL-12, and characterized in terms of particle size, zeta potential, sur- face morphology, cytotoxicity, and cellular uptake and gene expression. EXPERIMENTAL PROCEDURES Materials Linear polyethylenimine (PEI, M w 423), benzyl chloro- formate (CBZ-Cl), palladium on activated carbon (10% w/w Pd), Celite 521, and cholesteryl chloroformate were pur- chased from Aldrich Chemical Company, Inc. (Milwaukee, WI). 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was purchased from Avanti Polar Lipids (Alabaster, AL). 1 Department of Pharmaceutics and Pharmaceutical Chemistry, Cen- ter for Controlled Chemical Delivery, Biomedical Polymers Re- search Building, 30 S 2000 E Rm 201, Salt Lake City, Utah 84112- 5820. 2 Summer Undergraduate Research Fellow from the Department of Biologic and Environmental Engineering, Cornell University, Ithaca, New York 14853. 3 Department of Pharmaceutical Sciences, University of Tennessee Health Sciences Center, 26 S Dunlap Street, Memphis, Tennessee 38163. 4 To whom correspondence should be addressed. (e-mail: rburns@pharm.utah.edu) Pharmaceutical Research, Vol. 19, No. 4, April 2002 (© 2002) Research Paper 382 0724-8741/02/0400-0382/0 © 2002 Plenum Publishing Corporation