Entry of cell-penetrating peptides (CPPs) into living cells by translocating across plasma membranes is an important physiological phenomenon. To elucidate the mechanism of the translocation of CPPs across lipid bilayers, it is essential to reveal its elementary processes. For this purpose, here, we have developed a new method for the continuous, quantitative detection of the entry of CPPs into giant unilamellar vesicles (GUVs), where we investigate the interaction of fluorescent probe-labeled CPPs with single GUVs containing large unilamellar vesicles (LUVs) and fluorescent probes in their lumens using confocal microscopy. Using this method, we investigated the interaction of carboxyfluorescein (CF)-labeled transportan 10 (CF-TP10) with single GUVs comprised of dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC) containing LUVs of the same membrane and Alexa Fluor 647 hydrazide (AF647) in their lumens. At low concentrations of CF-TP10, first the fluorescence intensity (FI) of the GUV membrane increased with time, and then after some lag time the FI of the GUV lumen due to CF-TP10 increased continuously with time without leakage of AF647. At higher concentrations of CF-TP10, after the FI of the GUV lumen due to CF-TP10 increased significantly, leakage of AF647 started. These results indicate that CF-TP10 entered the GUV lumen by translocating across the GUV membrane and then bound to the LUVs there without pore formation and that CF-TP10 concentration in the lumen increased with time. The rate of entry of CF-TP10 into GUV lumen increased with CF-TP10 concentration. We discussed the kinetics of entry of CF-TP10 into single GUVs.

通过跨质膜转运而使细胞穿透肽（CPP）进入活细胞是重要的生理现象。为了阐明CPP跨脂质双层的转运机制，必须揭示其基本过程。为此，在这里，我们开发了一种连续定量检测CPP进入巨大单层囊泡（GUV）的新方法，在该方法中，我们研究了荧光探针标记的CPP与包含大单层囊泡的单个GUV的相互作用（使用共聚焦显微镜检查其腔内的LUV）和荧光探针。使用这种方法 我们研究了羧基荧光素（CF）标记的转运蛋白10（CF-TP10）与单个GUV的相互作用，该GUV包含相同膜的LUV和包含在其腔内的Alexa Fluor 647酰肼（AF647）的单油酰磷脂酰甘油（DOPG）和二油酰磷脂酰胆碱（DOPC）。在低浓度的CF-TP10下，首先GUV膜的荧光强度（FI）随时间增加，然后经过一段滞后时间，由于CF-TP10而导致的GUV管腔的FI随时间连续增加，而没有AF647的泄漏。在较高浓度的CF-TP10下，由于CF-TP10引起的GUV腔内FI显着增加后，AF647开始泄漏。这些结果表明，CF-TP10通过跨过GUV膜而进入GUV内腔，然后结合到那里的LUV上而没有孔的形成，并且内腔中CF-TP10的浓度随时间增加。CF-TP10进入GUV管腔的速率随CF-TP10浓度的增加而增加。我们讨论了CF-TP10进入单个GUV的动力学。