Cellular Internalization Kinetics of (Luciferin-)Cell-Penetrating Peptide Conjugates
Cellular Internalization Kinetics of (Luciferin-)Cell-Penetrating Peptide Conjugates
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DOI：
10.1021/bc100174y
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发表时间：
2010-09-01
影响因子：
4.7
通讯作者：
Langel, Ulo
中科院分区：
文献类型：
--
作者：
Eiriksdottir, Emelia;Mager, Imre;Langel, Ulo
细胞穿透性多肽(CPPs)是一类广泛应用于多种不透膜大分子的细胞转运载体。然而，由于实验设置的不同，从不同实验室获得的关于CPP细胞内化效率的结果有时难以比较。在这里，首次使用最近发表的可释放的荧光素分析比较了HeLa pLuc 705细胞中八种已建立的CPP的细胞摄取动力学。利用这一检测方法，这些荧光素-CPP结合物的胞浆进入动力学行为被实时记录下来。我们的数据显示，CPP的摄取率在几秒钟或几十分钟内达到最大值，这取决于所使用的CPP。TAT和较高浓度的MAP和TP10表现出类似于膜通透性游离荧光素的动力学曲线的快速内化曲线。PVEC、穿透蛋白、M918和EB I对其他多肽的吸收要慢得多，这与报道的内吞作用是主要内化机制的观察结果一致。此外，在某种程度上，后一种CPP可以根据观察到最显著摄取率的时间点分成不同的亚组。这可能表明不同的(浓度依赖)机制再次参与了CPPs的摄取。总之，CPP内化谱的差异表明了测量动力学而不是仅仅依赖简单的终点研究的重要性，并且利用荧光素CPP试验，可以在研究CPP的内化机制时检索到更清晰的信息。
Cell-penetrating peptides (CPPs) belong to a class of delivery vectors that have been extensively used for the cellular delivery of various, otherwise impermeable, macromolecules. However, results on the cellular internalization efficacy of CPPs obtained from various laboratories are sometimes challenging to compare because of differences in the experimental setups. Here, for the first time, the cellular uptake kinetics of eight well-established CPPs is compared in HeLa pLuc 705 cells using a recently published releasable luciferin assay. Using this assay, the kinetic behavior of cytosolic entry of these luciferin-CPP conjugates are registered in real time. Our data reveal that the uptake rate of CPPs reaches its maximum either in seconds or in tens of minutes, depending on the CPP used. Tat and higher concentrations of MAP and TP10 display fast internalization profiles that resemble the kinetic profile of membrane-permeable free luciferin. The uptake of the other peptides, pVec, penetratin, M918, and EB I, is much slower and is consistent with the reported observations of endocytosis being the predominant internalization mechanism. Additionally, to some extent, the latter CPPs can be clustered into subgroups which are based on time points when the most pronounced uptake rates are observed. This may indicate once more involvement of various (concentration dependent) mechanisms in the uptake of CPPs. In summary, the variances in the internalization profiles for the CPPs demonstrate the importance of measuring kinetics instead of only relying on simple end-point studies, and with the luciferin CPP assay, more lucid information can be retrieved when studying the internalization mechanisms of CPPs.