Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa, Italy.
Mol Pharm. 2012 Feb 6;9(2):334-40. doi: 10.1021/mp200374e. Epub 2012 Jan 9.
Here we investigate the cellular uptake mechanism and final intracellular fate of two cationic liposome formulations characterized by similar physicochemical properties but very different lipid composition and efficiency for intracellular delivery of DNA. The first formulation is made of cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic helper dioleoylphosphocholine (DOPC), while the second one is made of the cationic 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic lipid dioleoylphosphatidylethanolamine (DOPE). Combining pharmacological and imaging approaches we show that both DOTAP-DOPC/DNA and DC-Chol-DOPE/DNA lipoplexes are taken up in Chinese hamster ovary (CHO) living cells mainly through fluid-phase macropinocytosis. Our results also indicate that lipoplex macropinocytosis is a cholesterol-sensitive uptake mechanism. On the other side, both clathrin-mediated and caveolae-mediated endocytosis play a minor role, if any, in the cell uptake. Colocalization of fluorescently tagged lipoplexes and Lysosensor, a primary lysosome marker, reveals that poorly efficient DOTAP-DOPC/DNA lipoplexes are largely degraded in the lysosomes, while efficient DC-Chol-DOPE/DNA systems can efficiently escape from endosomal compartments.
在这里，我们研究了两种阳离子脂质体制剂的细胞摄取机制和最终的细胞内命运，这两种制剂具有相似的物理化学性质，但脂质组成和 DNA 细胞内传递效率非常不同。第一种制剂由阳离子脂质 1,2-二油酰基-3-三甲铵丙烷（DOTAP）和两性离子辅助剂二油酰基磷脂酰胆碱（DOPC）组成，而第二种制剂由阳离子 3β-[N-（N，N-二甲氨基乙烷）-氨基甲酰基]胆固醇（DC-Chol）和两性离子脂质二油酰基磷脂酰乙醇胺（DOPE）组成。结合药理学和成像方法，我们表明 DOTAP-DOPC/DNA 和 DC-Chol-DOPE/DNA 脂质体复合物主要通过液相巨胞饮作用被中国仓鼠卵巢（CHO）活细胞摄取。我们的结果还表明，脂质体巨胞饮作用是一种胆固醇敏感的摄取机制。另一方面，网格蛋白介导和小窝蛋白介导的内吞作用，如果有作用的话，在细胞摄取中作用很小。荧光标记的脂质体与溶酶体的共定位，溶酶体是初级溶酶体的标志物，表明效率较低的 DOTAP-DOPC/DNA 脂质体复合物在溶酶体中大量降解，而高效的 DC-Chol-DOPE/DNA 系统可以有效地从内体区室逃逸。
