Slepushkin V A, Simões S, Dazin P, Newman M S, Guo L S, Pedroso de Lima M C, Düzgüneş N
Department of Microbiology, University of the Pacific School of Dentistry, San Francisco, California 94115, USA.
J Biol Chem. 1997 Jan 24;272(4):2382-8. doi: 10.1074/jbc.272.4.2382.
Liposomes that destabilize at mildly acidic pH are efficient tools for delivering water-soluble drugs into the cell cytoplasm. However, their use in vivo is limited because of their rapid uptake from circulation by the reticuloendothelial system. Lipid-anchored polyethylene glycol (PEG-PE) prolongs the circulation time of liposomes by steric stabilization. We have found that addition of PEG-PE to the membrane of pH-sensitive liposomes composed of cholesteryl hemisuccinate (CHEMS) and dioleoylphosphatidylethanolamine (DOPE) confers steric stability to these vesicles. This modification significantly decreases the pH-dependent release of a charged water-soluble fluorophore, calcein, from liposomes suspended in buffer or cell culture medium. However, the ability of such liposomes to release calcein intracellularly, measured by a novel flow cytometry technique involving dual fluorescence labeling, remains unaltered. As expected, the release of calcein from liposomes endocytosed by cells is inhibited upon pretreatment of the cells with NH4Cl, an inhibitor of endosome acidification. The unique properties of these liposomes were also demonstrated in vivo. The distribution kinetics of 111In-containing CHEMS/DOPE/PEG-PE liposomes injected intravenously into rats has pharmacokinetic parameters similar to control, non-pH-sensitive, sterically stabilized CHEMS/distearoylphosphatidylcholine/PEG-PE liposomes. In contrast, regular pH-sensitive liposomes lacking the PEG-PE component are cleared rapidly. Sterically stabilized pH-sensitive liposomes may therefore be useful for the intracellular delivery in vivo of highly negatively charged molecules such as genes, antisense oligonucleotides, and ribozymes for the treatment of various diseases.
在轻度酸性pH值下不稳定的脂质体是将水溶性药物递送至细胞质的有效工具。然而，由于其被网状内皮系统从循环中快速摄取，它们在体内的应用受到限制。脂质锚定的聚乙二醇（PEG-PE）通过空间稳定作用延长脂质体的循环时间。我们发现，向由半琥珀酸胆固醇（CHEMS）和二油酰磷脂酰乙醇胺（DOPE）组成的pH敏感脂质体膜中添加PEG-PE可赋予这些囊泡空间稳定性。这种修饰显著降低了悬浮在缓冲液或细胞培养基中的脂质体中带电荷的水溶性荧光团钙黄绿素的pH依赖性释放。然而，通过涉及双荧光标记的新型流式细胞术技术测量，此类脂质体在细胞内释放钙黄绿素的能力保持不变。正如预期的那样，在用NH4Cl（一种内体酸化抑制剂）预处理细胞后，细胞内吞的脂质体中钙黄绿素的释放受到抑制。这些脂质体的独特性质在体内也得到了证实。静脉注射到大鼠体内的含111In的CHEMS/DOPE/PEG-PE脂质体的分布动力学具有与对照、非pH敏感、空间稳定的CHEMS/二硬脂酰磷脂酰胆碱/PEG-PE脂质体相似的药代动力学参数。相比之下，缺乏PEG-PE成分的常规pH敏感脂质体被迅速清除。因此，空间稳定的pH敏感脂质体可能有助于在体内将高度带负电荷的分子（如基因、反义寡核苷酸和核酶）细胞内递送至体内以治疗各种疾病。
