Integrated Bioscience Section, Graduate School of Science and Technology, Shizuoka University, Shizuoka 422-8529, Japan.
Nanomaterials Research Division, Research Institute of Electronics, Shizuoka University, Shizuoka 422-8529, Japan.
Biochemistry. 2020 May 12;59(18):1780-1790. doi: 10.1021/acs.biochem.0c00102. Epub 2020 Apr 20.
The entry of cell-penetrating peptides (CPPs) into live cells and lipid vesicles has been monitored using probe (e.g., fluorescent dye)-labeled CPPs. However, probe labeling may alter the interaction of CPPs with membranes. We have developed a new method to detect the entry of nonlabeled CPPs into the lumens of single giant unilamellar vesicles (GUVs) without pore formation in the GUV membrane. The GUVs contain large unilamellar vesicles (LUVs) whose lumens contain a high (self-quenching) concentration of the fluorescent dye calcein. If the CPPs enter the GUV lumen and interact with these LUVs to induce calcein leakage, the fluorescence intensity (FI) due to calcein in the GUV lumen increases. The lipid compositions of the LUVs and GUVs allow leakage from LUVs but not from the GUVs. We applied this method to detect the entry of transportan 10 (TP10) into single GUVs comprising dioleoylphosphatidylglycerol and dioleoylphosphatidylcholine and examined the interaction of low concentrations of nonlabeled TP10 with single GUVs whose lumens contain Alexa Fluor 647 hydrazide (AF647) and the LUVs mentioned above. The FI of the GUV lumen due to calcein increased continuously with time without leakage of AF647, indicating that TP10 entered the GUV without pore formation in the GUV membrane. The lumen intensity due to calcein increased with TP10 concentration, indicating that the rate of entry of TP10 into the GUV lumen increased. We estimated the minimum TP10 concentration in a GUV lumen detected by this method. We discuss the entry of nonlabeled TP10 and the characteristics of this method.
细胞穿透肽（CPPs）进入活细胞和脂质体已通过探针（例如荧光染料）标记的 CPPs 来监测。然而，探针标记可能会改变 CPPs 与膜的相互作用。我们开发了一种新方法，可在不形成 GUV 膜孔的情况下，检测非标记 CPP 进入单个大单层囊泡（GUV）腔室的情况。GUV 含有大单层囊泡（LUV），其腔室含有高浓度（自猝灭）荧光染料 calcein。如果 CPP 进入 GUV 腔室并与这些 LUV 相互作用以诱导 calcein 泄漏，则 GUV 腔室中 calcein 的荧光强度（FI）会增加。LUV 和 GUV 的脂质组成允许 LUV 泄漏，但不允许 GUV 泄漏。我们应用此方法检测转运蛋白 10（TP10）进入由二油酰基磷脂酰甘油和二油酰基磷脂酰胆碱组成的单个 GUV，并检查了低浓度非标记 TP10 与含有 Alexa Fluor 647 酰肼（AF647）和上述 LUV 的单个 GUV 相互作用。FI 由于 calcein 的 GUV 腔室随时间连续增加而没有 AF647 的泄漏，表明 TP10 进入 GUV 而没有 GUV 膜的孔形成。由于 calcein 的腔室强度随 TP10 浓度的增加而增加，表明 TP10 进入 GUV 腔室的速率增加。我们估计了该方法检测到的 GUV 腔室中最小的 TP10 浓度。我们讨论了非标记 TP10 的进入以及该方法的特点。
