ENHANCED GENE DELIVERY AND MECHANISM STUDIES WITH A NOVEL SERIES OF CATIONIC LIPID FORMULATIONS

The application of cationic liposome reagents has advanced DNA and mRN A transfection research in vitro, and data are accumulating which show their utility for in vivo gene transfer. However, chemical structure- activity data leading to a better mechanistic understanding of their b iological activity is still limited. Most of the cationic lipid reagen ts in use today for this application are formulated as liposomes conta ining two lipid species, a cationic amphiphile and a neutral phospholi pid, typically dioleoylphosphatidylethanolamine (DOPE). The studies re ported here examine the effects of some systematic chemical structural changes in both of these lipid components. Cationic and neutral phosp holipids were formulated together as large multilamellar vesicles (MLV ) or small sonicated unilamellar vesicles (SUV) in water, and each for mulation was assayed quantitatively in 96-well microtiter plates under 64 different assay conditions using COS.7 cells and an RSV-beta-galac tosidase expression plasmid. The cationic lipid molecules used for the se studies were derived from a novel series of 2,3-dialkyloxypropyl qu aternary ammonium compounds containing a hydroxyalkyl moiety on the qu aternary amine. A homologous series of dioleylalkyl (C18:1) compounds containing increasing hydroxyalkyl chain lengths on the quaternary ami ne were synthesized, formulated with 50 mol % DOPE, and assayed for tr ansfection activity. The order of efficacy was ethyl > propyl > butyl > pentyl > 2,3-dioleyloxypropyl-1-trimethyl ammonium bromide (DOTMA). DOTMA, which is commercially available under the trademark Lipofectin Reagent, lacks a hydroxyalkyl moiety on the quaternary amine. A homolo gous series of hydroxyethyl quaternary ammonium derivatives with diffe rent alkyl chain substitutions were synthesized, formulated with 50 mo l % DOPE, and assayed in the transfection assay. The order of transfec tion efficacy was dimyristyl (di-C14:0) > dioleyl (di-C18:1) > dipalmi tyl (di-C16:0) > disteryl (di-C18:0). The addition of 100 muM chloroqu ine in the transfection experiment enhanced the activity of the dioley l compound by 4-fold and decreased the activity of the dimyristyl comp ound by 70%. For each of the compounds and formulations examined in th is report, large multilamellar vesicles (MLV; diameter 300-700 nm) wer e more active than small unilamellar vesicles (SUV; diameter 50-100 nm ). The neutral phospholipid requirements for transfection activity in COS.7 cells with these cationic lipid molecules were examined. Cationi c vesicles formulated with 50 mol % dioleoylphosphatidylethanolamine ( DOPE) were 2-5-fold more active than formulations with 50 mol % dioleo ylphosphatidylcholine or formulations without any neutral lipid, and t he level of DOPE required for optimal activity was 50 mol %. The diffe rences between DOPE and dioleoylphosphatidylcholine were associated wi th differences in titratability of the primary ethanolamine and cholin e moieties on these two phospholipid molecules. DOPE analogs were stud ied for their transfection enhancing activity. Analogs with increasing acyl chain saturation were progressively less active than unsaturated analogs; analogs with increasing numbers of methyl or methylene group s added to the primary amine were also progressively less active. The lysophosphatidylethanolamine analogs examined neither enhanced nor inh ibited the activity of these reagents. These results have implications regarding the design of new cationic and neutral lipid molecules for use in the development of improved cationic lipid gene delivery vector s.