Smith J G, Wedeking T, Vernachio J H, Way H, Niven R W
Megabios Corporation, Burlingame, California 94010, USA.
Pharm Res. 1998 Sep;15(9):1356-63. doi: 10.1023/a:1011937218418.
To identify characteristics of lipid-DNA complexes that correlate with in vivo expression data.
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.
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).
An uncomplexed liposome population exists within DOTIM:cholesterol-DNA complexes that influences the expression of complexes administered i.v. but not i.t..
确定与体内表达数据相关的脂质-DNA复合物的特征。
将DOTIM:胆固醇脂质体（摩尔比1:1）与表达氯霉素乙酰转移酶（CAT）的DNA以4.2:1的质量比（阳离子脂质:DNA）复合。通过密度梯度离心对复合物进行分级分离，分析其粒径和zeta电位，并采用高效液相色谱法进行定量。将未分级的复合物、复合物的“纯化”级分以及补充脂质体的纯化复合物通过静脉注射（i.v.）和气管内滴注（i.t.）给予小鼠，并评估它们表达基因产物的能力。
离心分离出复合物中的两个不同群体，一个由游离脂质体组成，另一个由与DNA复合的脂质组成。分离级分中的囊泡直径和zeta电位分别在113至354nm和+24至+34mV之间变化。对含有脂质-DNA群体的“纯化”级分进行再离心产生了一条单一谱带。静脉注射后24小时，在肺组织中观察到未分级复合物有CAT表达，但纯化形式没有。补充脂质体的复合物“恢复”了一些活性。相比之下，通过气管内滴注给予的相同系列复合物在肺组织中的表达没有显著差异（方差分析，p = 0.16）。
在DOTIM:胆固醇-DNA复合物中存在未复合的脂质体群体，其影响静脉注射而非气管内滴注复合物的表达。
