Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, Shizuoka 422-8529, Japan.
Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, Shizuoka 422-8529, Japan; Theoretical Problem Center of Physico-Chemical Pharmacology, Russian Academy of Sciences, Коsugina, 4, 117977, Moscow, Russia.
Chem Phys Lipids. 2018 May;212:120-129. doi: 10.1016/j.chemphyslip.2018.02.001. Epub 2018 Feb 6.
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.
细胞穿透肽（CPPs）通过跨膜转运进入活细胞是一种重要的生理现象。为了阐明 CPP 穿过脂质双层的转运机制，揭示其基本过程至关重要。为此，我们在这里开发了一种新的方法，用于连续、定量检测 CPP 进入巨大单层囊泡（GUVs）的情况，我们使用共焦显微镜研究了荧光探针标记的 CPP 与含有大单层囊泡（LUVs）和腔内荧光探针的单个 GUV 的相互作用。使用这种方法，我们研究了羧基荧光素（CF）标记的转运肽 10（CF-TP10）与由二油酰基磷脂酰甘油（DOPG）和二油酰基磷脂酰胆碱（DOPC）组成的单个 GUV 的相互作用，这些 GUV 包含相同膜的 LUVs 和腔内的 Alexa Fluor 647 酰肼（AF647）。在 CF-TP10 的低浓度下，首先 GUV 膜的荧光强度（FI）随时间增加，然后在一些滞后时间后，由于 CF-TP10 的 GUV 腔 FI 连续随时间增加而增加，而没有 AF647 的泄漏。在较高浓度的 CF-TP10 下，由于 CF-TP10 的 GUV 腔 FI 显著增加后，开始泄漏 AF647。这些结果表明，CF-TP10 通过跨 GUV 膜转运进入 GUV 腔，然后与那里的 LUV 结合，而没有形成孔，并且腔中 CF-TP10 的浓度随时间增加。CF-TP10 进入 GUV 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。
