Straubinger R M, Papahadjopoulos D, Hong K L
Department of Pharmacology, University of California, San Francisco 94143.
Biochemistry. 1990 May 22;29(20):4929-39. doi: 10.1021/bi00472a025.
Lipid vesicles (liposomes) containing pH-sensitive fluorophores were used as probes for the study of liposome entry and intracellular fate. Pyranine [8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS)] was entrapped in the liposome aqueous core during preparation to provide a means of detecting internalization into living cells. HPTS is highly water soluble and shows a strong pH-dependent shift in its fluorescence excitation spectrum. Fluorescence emission (FEM) is slightly pH dependent with excitation (lambda EX) at 350-415 nm but highly pH dependent with lambda EX at 450 nm. Liposomes bearing a net negative charge bound rapidly to CV-1 cells and underwent endocytosis. One hour after liposome addition, high FEM with lambda EX at 413 nm and low FEM with lambda EX at 450 nm suggest that most cell-associated liposomes had been internalized and resided at a mean pH of approximately 6.6. Collapse of cellular H+ gradients with NH4Cl or monensin treatment rapidly and reversibly increased FEM with lambda EX at 450 nm. Direct examination by fluorescence microscopy corroborates the fluorometric data on internalization; over time, FEM remained high with lambda EX at 350-405 nm but decreased with lambda EX at 450-490 nm, showing that all lipid vesicles were internalized within 40 min at 37 degrees C. Acidification of intracellular liposomes increased over 3 h, reaching a minimum value of approximately pH 5.5. HPTS persisted within acidic cellular vesicles for 2-3 days, and cytoplasmic dye was observed infrequently, suggesting that liposome fusion with cellular membranes seldom occurs. Material delivered to the endocytic pathway via lipid vesicles labeled an assortment of intracellular organelles of varying motility and morphology, including dynamic tubular structures whose lumen is acidic.
含有pH敏感荧光团的脂质囊泡（脂质体）被用作研究脂质体进入细胞及细胞内命运的探针。在制备过程中，将吡喃荧光素[8-羟基-1,3,6-芘三磺酸盐（HPTS）]包裹在脂质体水相中，以提供一种检测其内化进入活细胞的方法。HPTS高度水溶性，其荧光激发光谱呈现强烈的pH依赖性变化。荧光发射（FEM）在350 - 415 nm激发（λEX）时对pH略有依赖性，但在450 nm激发时对pH高度依赖。带净负电荷的脂质体迅速与CV-1细胞结合并发生内吞作用。添加脂质体1小时后，在413 nm激发时FEM高，在450 nm激发时FEM低，这表明大多数与细胞相关的脂质体已被内化，且平均pH约为6.6。用氯化铵或莫能菌素处理使细胞H⁺梯度崩溃，迅速且可逆地增加了在450 nm激发时的FEM。荧光显微镜直接检查证实了关于内化的荧光测定数据；随着时间推移，在350 - 405 nm激发时FEM保持高值，但在450 - 490 nm激发时降低，表明所有脂质囊泡在37℃下40分钟内都被内化。细胞内脂质体的酸化在3小时内增加，达到约pH 5.5的最小值。HPTS在酸性细胞囊泡中持续存在2 - 3天，很少观察到细胞质中有染料，这表明脂质体与细胞膜的融合很少发生。通过脂质囊泡递送至内吞途径的物质标记了各种具有不同运动性和形态的细胞内细胞器，包括管腔呈酸性的动态管状结构。
