Düzgüneş N, Straubinger R M, Baldwin P A, Friend D S, Papahadjopoulos D
Biochemistry. 1985 Jun 18;24(13):3091-8. doi: 10.1021/bi00334a004.
Liposomes composed of oleic acid and phosphatidylethanolamine (3:7 mole ratio) aggregate, become destabilized, and fuse below pH 6.5 in 150 mM NaCl. Fusion is monitored by (i) the intermixing of internal aqueous contents of liposomes, utilizing the quenching of aminonaphthalene-3,6,8-trisulfonic acid (ANTS) by N,N'-p-xylylenebis(pyridinium bromide) (DPX) encapsulated in two separate populations of vesicles, (ii) a resonance energy transfer assay for the dilution of fluorescent phospholipids from labeled to unlabeled liposomes, (iii) irreversible changes in turbidity, and (iv) quick-freezing freeze-fracture electron microscopy. Destabilization is followed by the fluorescence increase caused by the leakage of coencapsulated ANTS/DPX or of calcein. Ca2+ and Mg2+ also induce fusion of these vesicles at 3 and 4 mM, respectively. The threshold for fusion is at a higher pH in the presence of low (subfusogenic) concentrations of these divalent cations. Vesicles composed of phosphatidylserine/phosphatidylethanolamine or of oleic acid/phosphatidylcholine (3:7 mole ratio) do not aggregate, destabilize, or fuse in the pH range 7-4, indicating that phosphatidylserine and phosphatidylcholine cannot be substituted for oleic acid and phosphatidylethanolamine, respectively, for proton-induced membrane fusion. Freeze-fracture replicas of oleic acid/phosphatidylethanolamine liposomes frozen within 1 s of stimulation with pH 5.3 display larger vesicles and vesicles undergoing fusion, with membrane ridges and areas of bilayer continuity between them. The construction of pH-sensitive liposomes is useful as a model for studying the molecular requirements for proton-induced membrane fusion in biological systems and for the cytoplasmic delivery of macromolecules.
由油酸和磷脂酰乙醇胺（摩尔比3:7）组成的脂质体在150 mM氯化钠中，pH值低于6.5时会聚集、变得不稳定并融合。融合情况通过以下方式监测：（i）利用包裹在两个独立囊泡群体中的N,N'-对二甲苯双（溴化吡啶）（DPX）对氨基萘-3,6,8-三磺酸（ANTS）的淬灭作用，监测脂质体内含水内容物的混合；（ii）通过共振能量转移测定法，检测荧光磷脂从标记脂质体稀释到未标记脂质体的情况；（iii）监测浊度的不可逆变化；（iv）快速冷冻冷冻断裂电子显微镜观察。不稳定之后，共包裹的ANTS/DPX或钙黄绿素泄漏会导致荧光增强。Ca2+和Mg2+也分别在3 mM和4 mM时诱导这些囊泡融合。在存在低（亚融合）浓度的这些二价阳离子时，融合阈值处于更高的pH值。由磷脂酰丝氨酸/磷脂酰乙醇胺或油酸/磷脂酰胆碱（摩尔比3:7）组成的囊泡在pH值7 - 4范围内不会聚集、不稳定或融合，这表明磷脂酰丝氨酸和磷脂酰胆碱不能分别替代油酸和磷脂酰乙醇胺用于质子诱导的膜融合。用pH 5.3刺激后1秒内冷冻的油酸/磷脂酰乙醇胺脂质体的冷冻断裂复制品显示出更大的囊泡和正在融合的囊泡，它们之间有膜嵴和双层连续区域。pH敏感脂质体的构建对于研究生物系统中质子诱导膜融合的分子要求以及大分子的细胞质递送是一种有用的模型。
