title: Novel water insoluble lipoparticulates for gene delivery.
abstract: OBJECTIVE
  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, Mw 423) as a headgroup and cholesteryl chloroformate as a hydrophobic lipid anchor resulting in a T-shaped lipononamer. Semitelechelic N(alpha)-cholesteryloxycarbonyl nona(ethylenimine) (st-NCNEI-L) was synthesized similarly resulting in a linear lipononamer. As confirmed by 1H-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 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 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.
authors.affiliation: Department of Pharmaceutics and Pharmaceutical Chemistry, Center for Controlled Chemical Delivery, Salt Lake City, Utah 84112-5820, USA.
authors.forename: Darin Y
authors.initials: DY
authors.lastname: Furgeson
authors.forename: Richard N
authors.initials: RN
authors.lastname: Cohen
authors.forename: Ram I
authors.initials: RI
authors.lastname: Mahato
authors.forename: Sung Wan
authors.initials: SW
authors.lastname: Kim
mesh_terms: D000818:Animals; D002784:Cholesterol / Q000008:administration & dosage / Q000737:chemistry; D016503:Drug Delivery Systems / Q000379:methods*; D015316:Genetic Therapy / Q000379:methods*; D008081:Liposomes / Q000008:administration & dosage* / Q000138:chemical synthesis; D010316:Particle Size; D012995:Solubility; D019076:Transgenes / Q000235:genetics; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D014867:Water / Q000737:chemistry*
chemical_list: D008081:Liposomes; D014867:Water; D002784:Cholesterol