PURPOSE: To identify characteristics of lipid-DNA complexes that correlate with in vivo expression data. METHODS: DOTIM:cholesterol liposomes (1:1 mole ratio) and DNA expressing chloramphenicol acetyl transferase (CAT) were complexed at a 4.2:1 mass ratio (cationic lipid:DNA). Complexes were fractionated by density gradient centrifugation. analyzed for particle size and zeta potential and quantitated using HPLC methods. The unfractionated complexes, "purified" fractions of the complexes, and purified complexes supplemented with liposomes were administered to mice by intravenous injection (i.v.) and intratracheal instillation (i.t.) and their ability to express gene product was assessed. RESULTS: Centrifugation separated two distinct populations within complexes one consisting of free liposomes and the other of lipid complexed with DNA. The vesicle diameter and zeta potential among separated fractions varied from 113 to 354 nm. and + 24 to + 34 mV respectively. Re-centrifugation of the 'purified' fractions containing the lipid-DNA population produced a single band. CAT expression in lung tissue 24 hr post-i.v. was observed with the unfractionated complex, but not the purified form. Some activity was 'restored' with the liposome-supplemented complexes. In contrast, the same series of complexes administered by i.t. resulted in no significant difference in lung expression (p=0.16 ANOVA). CONCLUSIONS: An uncomplexed liposome population exists within DOTIM:cholesterol-DNA complexes that influences the expression of complexes administered i.v. but not i.t..
PURPOSE: To identify characteristics of lipid-DNA complexes that correlate with in vivo expression data. METHODS: DOTIM:cholesterol liposomes (1:1 mole ratio) and DNA expressing chloramphenicol acetyl transferase (CAT) were complexed at a 4.2:1 mass ratio (cationic lipid:DNA). Complexes were fractionated by density gradient centrifugation. analyzed for particle size and zeta potential and quantitated using HPLC methods. The unfractionated complexes, "purified" fractions of the complexes, and purified complexes supplemented with liposomes were administered to mice by intravenous injection (i.v.) and intratracheal instillation (i.t.) and their ability to express gene product was assessed. RESULTS: Centrifugation separated two distinct populations within complexes one consisting of free liposomes and the other of lipid complexed with DNA. The vesicle diameter and zeta potential among separated fractions varied from 113 to 354 nm. and + 24 to + 34 mV respectively. Re-centrifugation of the 'purified' fractions containing the lipid-DNA population produced a single band. CAT expression in lung tissue 24 hr post-i.v. was observed with the unfractionated complex, but not the purified form. Some activity was 'restored' with the liposome-supplemented complexes. In contrast, the same series of complexes administered by i.t. resulted in no significant difference in lung expression (p=0.16 ANOVA). CONCLUSIONS: An uncomplexed liposome population exists within DOTIM:cholesterol-DNA complexes that influences the expression of complexes administered i.v. but not i.t..
Authors: M J Stewart; G E Plautz; L Del Buono; Z Y Yang; L Xu; X Gao; L Huang; E G Nabel; G J Nabel Journal: Hum Gene Ther Date: 1992-06 Impact factor: 5.695