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[摘要]：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 tire 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 oil ice, the peptide at 2 mu M extracellular concentration efficiently entered and uniformly labelled the cytoplasm oh all the suspension cells studied. but it 10-fold higher concentration was required to observe similar results ill 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 it requirement for nucleation zones to promote this effect. Cholesterol depiction 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 ion. Under the same conditions in adherent cells this went had no effect on peptide uptake or distribution. Cholesterol depletion increased the overall accumulation of the peptide tat 4 C in KG la cells, but this effect could be reversed by re-addition of cholesterol its methyl-beta-cyclodextrin-cholesterol complexes. The results highlight the relatively high porosity of the plasma membrane of suspension cells to this peptide, especially its low temperatures. suggesting thin this feature could be exploited for delivering bioactive entities. |
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