Tünnemann Gisela, Ter-Avetisyan Gohar, Martin Robert M, Stöckl Martin, Herrmann Andreas, Cardoso M Cristina
Max Delbrueck Center for Molecular Medicine, D-13125 Berlin, Germany.
J Pept Sci. 2008 Apr;14(4):469-76. doi: 10.1002/psc.968.
Cell penetrating peptides (CPPs) are useful tools to deliver low-molecular-weight cargoes into cells; however, their mode of uptake is still controversial. The most efficient CPPs belong to the group of arginine-rich peptides, but a systematic assessment of their potential toxicity is lacking. In this study we combined data on the membrane translocation abilities of oligo-arginines in living cells as a function of their chain length, concentration, stability and toxicity. Using confocal microscopy analysis of living cells we evaluated the transduction frequency of the L-isoforms of oligo-arginines and lysines and then monitored their associated toxicity by concomitant addition of propidium iodide. Whereas lysines showed virtually no transduction, the transduction ability of arginines increased with the number of consecutive residues and the peptide concentration, with L-R9 and L-R10 performing overall best. We further compared the L- and D-R9 isomers and found that the D-isoform always showed a higher transduction as compared to the L-counterpart in all cell types. Notably, the transduction difference between D- and L-forms was highly variable between cell types, emphasizing the need for protease-resistant peptides as vectors for drug delivery. Real-time kinetic analysis of the D- and L-isomers applied simultaneously to the cells revealed a much faster transduction for the D-variant. The latter underlies the fact that the isomers do not mix, and penetration of one peptide does not perturb the membrane in a way that gives access to the other peptide. Finally, we performed short- and long-term cell viability and cell cycle progression analyses with the protease-resistant D-R9. Altogether, our results identified concentration windows with low toxicity and high transduction efficiency, resulting in fully bioavailable intracellular peptides.
细胞穿透肽（CPPs）是将低分子量物质递送至细胞内的有用工具；然而，其摄取方式仍存在争议。最有效的CPPs属于富含精氨酸的肽类，但对其潜在毒性缺乏系统评估。在本研究中，我们结合了关于寡聚精氨酸在活细胞中的膜转位能力与其链长、浓度、稳定性和毒性之间关系的数据。通过对活细胞进行共聚焦显微镜分析，我们评估了寡聚精氨酸和赖氨酸L-异构体的转导频率，然后通过同时添加碘化丙啶监测其相关毒性。赖氨酸几乎没有转导现象，而精氨酸的转导能力随着连续残基数量和肽浓度的增加而增强，其中L-R9和L-R10总体表现最佳。我们进一步比较了L-和D-R9异构体，发现D-异构体在所有细胞类型中总是比L-对应物表现出更高的转导效率。值得注意的是，D-和L-形式之间的转导差异在不同细胞类型中变化很大，这强调了需要使用抗蛋白酶肽作为药物递送载体。对同时应用于细胞的D-和L-异构体进行实时动力学分析，结果显示D-变体的转导速度要快得多。这一现象背后的原因是异构体不会混合，一种肽的穿透不会以让另一种肽进入的方式干扰细胞膜。最后，我们用抗蛋白酶的D-R9进行了短期和长期的细胞活力及细胞周期进程分析。总之，我们的结果确定了具有低毒性和高转导效率的浓度范围，从而产生了完全可生物利用的细胞内肽。
