Watkins Catherine L, Schmaljohann Dirk, Futaki Shiroh, Jones Arwyn T
Welsh School of Pharmacy, Cardiff University, Wales, UK.
Biochem J. 2009 May 13;420(2):179-89. doi: 10.1042/BJ20090042.
The exact mechanisms by which cell-penetrating peptides such as oligo-arginines and penetratin cross biological membranes has yet to be elucidated, but this is required if they are to reach their full potential as cellular delivery vectors. In the present study, qualitative and quantitative analysis of the influence of temperature, peptide concentration and plasma membrane cholesterol on the uptake and subcellular distribution of the model cell-penetrating peptide octa-arginine was performed in a number of suspension and adherent cell lines. When experiments were performed on ice, the peptide at 2 microM extracellular concentration efficiently entered and uniformly labelled the cytoplasm of all the suspension cells studied, but a 10-fold higher concentration was required to observe similar results in adherent cells. At 37 degrees C and at higher peptide concentrations, time-lapse microscopy experiments showed that the peptide rapidly penetrated the entire plasma membrane of suspension cells, with no evidence of a requirement for nucleation zones to promote this effect. Cholesterol depletion with methyl-beta-cyclodextrin enhanced translocation of octa-arginine across the plasma membrane of suspension cells at 37 degrees C, but decreased overall peptide accumulation. Under the same conditions in adherent cells this agent had no effect on peptide uptake or distribution. Cholesterol depletion increased the overall accumulation of the peptide at 4 degrees C in KG1a cells, but this effect could be reversed by re-addition of cholesterol as methyl-beta-cyclodextrin-cholesterol complexes. The results highlight the relatively high porosity of the plasma membrane of suspension cells to this peptide, especially at low temperatures, suggesting that this feature could be exploited for delivering bioactive entities.
诸如寡聚精氨酸和穿膜肽等细胞穿透肽穿过生物膜的确切机制尚未阐明，但如果它们要充分发挥作为细胞递送载体的潜力，就需要阐明这一机制。在本研究中，我们在多种悬浮细胞系和贴壁细胞系中，对温度、肽浓度和质膜胆固醇对模型细胞穿透肽八聚精氨酸的摄取和亚细胞分布的影响进行了定性和定量分析。当在冰上进行实验时，细胞外浓度为2微摩尔的该肽能有效地进入并均匀标记所有所研究悬浮细胞的细胞质，但在贴壁细胞中需要10倍更高的浓度才能观察到类似结果。在37℃及更高的肽浓度下，延时显微镜实验表明该肽能迅速穿透悬浮细胞的整个质膜，没有证据表明需要成核区来促进这种作用。用甲基-β-环糊精消耗胆固醇可增强八聚精氨酸在37℃时跨悬浮细胞质膜的转位，但会降低肽的总体积累。在相同条件下，该试剂对贴壁细胞中的肽摄取或分布没有影响。在4℃时，消耗胆固醇可增加KG1a细胞中肽的总体积累，但通过重新添加胆固醇作为甲基-β-环糊精-胆固醇复合物，这种作用可以逆转。结果突出了悬浮细胞质膜对该肽具有相对较高的孔隙率，尤其是在低温下，这表明这一特性可用于递送生物活性物质。
