Zelphati O, Wang Y, Kitada S, Reed J C, Felgner P L, Corbeil J
Gene Therapy Systems Inc., San Diego, California 92121, USA.
J Biol Chem. 2001 Sep 14;276(37):35103-10. doi: 10.1074/jbc.M104920200. Epub 2001 Jul 10.
There are many very effective methods to introduce transcriptionally active DNA into viable cells but approaches to deliver functional proteins are limited. We have developed a lipid-mediated delivery system that can deliver functional proteins or other bioactive molecules into living cells. This delivery system is composed of a new trifluoroacetylated lipopolyamine (TFA-DODAPL) and dioleoyl phosphatidylethanolamine (DOPE). This cationic formulation successfully delivered antibodies, dextran sulfates, phycobiliproteins, albumin, and enzymes (beta-galactosidase and proteases) into the cytoplasm of numerous adherent and suspension cells. Two systems were used to demonstrate that the proteins were delivered in a functionally active form. First, intracellular beta-galactosidase activity was clearly demonstrated within X-gal-stained cells after TFA-DODAPL:DOPE-mediated delivery of the enzyme. Second, the delivery system mediated delivery of several caspases (caspase 3, caspase 8, and granzyme B) into cultured cell lines and primary cells triggering apoptosis. Mechanistic studies showed that up to 100% of the protein mixed with the lipid formulation was captured into a lipid-protein complex, and up to 50% of the input protein associated with cells. This lipid-mediated transport system makes protein delivery into cultured cells as convenient, effective, and reliable as DNA transfection.
将转录活性DNA导入活细胞有许多非常有效的方法，但递送功能蛋白的方法却很有限。我们开发了一种脂质介导的递送系统，它可以将功能蛋白或其他生物活性分子递送至活细胞中。该递送系统由一种新型三氟乙酰化脂多胺（TFA - DODAPL）和二油酰磷脂酰乙醇胺（DOPE）组成。这种阳离子制剂成功地将抗体、硫酸葡聚糖、藻胆蛋白、白蛋白和酶（β - 半乳糖苷酶和蛋白酶）递送至众多贴壁细胞和悬浮细胞的细胞质中。使用了两种系统来证明蛋白是以功能活性形式递送的。第一，在TFA - DODAPL:DOPE介导的酶递送后，在经X - gal染色的细胞内清楚地显示出细胞内β - 半乳糖苷酶活性。第二，该递送系统介导几种半胱天冬酶（半胱天冬酶3、半胱天冬酶8和颗粒酶B）递送至培养的细胞系和原代细胞中，从而引发细胞凋亡。机制研究表明，与脂质制剂混合的蛋白中高达100%被捕获到脂质 - 蛋白复合物中，并且与细胞相关的输入蛋白高达50%。这种脂质介导的转运系统使蛋白递送至培养细胞变得像DNA转染一样方便、有效和可靠。
