Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery Systems, School of Pharmaceutical Sciences, Peking University, Beijing 100083, China; Tianjin Key Laboratory on Technologies Enabling Development of Clinical Therapeutics and Diagnostics (Theranostics), School of Pharmacy, Tianjin Medical University, Tianjin 300070, China.
Beijing Key Laboratory of Molecular Pharmaceutics and New Drug Delivery Systems, School of Pharmaceutical Sciences, Peking University, Beijing 100083, China.
J Control Release. 2017 Sep 10;261:174-186. doi: 10.1016/j.jconrel.2017.06.018. Epub 2017 Jun 27.
Cell-penetrating peptides (CPPs) are increasingly important in transporting macromolecules across cell membranes, but their use remains confined to narrow clinical applications due to the systemic toxicity induced by their positive charges. Several newly discovered electronic neutral penetrating peptides are not attracting much attention because their penetrating capacity is normally far less powerful than cationic or amphiphilic CPPs. In this study, we found the electronic neutral cyclic peptide cyclosporin A (CsA) exhibited 5.6-fold and 19.1-fold stronger penetrating capacity, respectively, than two reported electronic neutral peptides PFVYLI (PFV) and pentapeptide VPTLQ (VPT) in MCF-7 human breast cancer cells. To systematically evaluate the efficiency and toxicity of CsA, we utilized CsA to deliver a membrane-impenetrable pro-apoptotic peptide (PAD) and compared this to the well-established cationic penetrating peptide TAT (RKKRRQRRR). By conjugating CsA to PAD, the internalization of PAD increased 2.2- to 4.7-fold in four different tumor cell lines, and that of CsA-PAD conjugate was significantly higher than TAT-PAD conjugate in MCF-7 and HeLa human cervical cancer cells. Cytotoxicity studies demonstrated that CsA-PAD exhibited a large increase in cell cytotoxicity compared to PAD in four different tumor cell lines, with the effect being similar or greater than the effect of TAT-PAD, depending upon the cell type. The mechanistic studies demonstrated that modifying CsA or TAT did not change the cytotoxicity mechanism of PAD, which occurred via mitochondrial membrane damage related to apoptosis. In vivo studies showed that CsA-PAD could achieve similar anti-tumor efficacy to TAT-PAD but with much lower systemic toxicity, especially to the heart and liver. In conclusion, our study demonstrates for the first time that the electronic-neutral penetrating peptide CsA can be used as a powerful tool to deliver peptide drugs with similar efficiency and less toxicity than the positively charged TAT peptide.
细胞穿透肽（CPPs）在将大分子穿过细胞膜方面的作用越来越重要，但由于其正电荷引起的全身毒性，其应用仍然局限于狭窄的临床应用。一些新发现的电子中性穿透肽并没有引起太多关注，因为它们的穿透能力通常远远低于阳离子或两亲性 CPPs。在这项研究中，我们发现电子中性环肽环孢菌素 A（CsA）在 MCF-7 人乳腺癌细胞中的穿透能力分别比两种报道的电子中性肽 PFVYLI（PFV）和五肽 VPTLQ（VPT）强 5.6 倍和 19.1 倍。为了系统地评估 CsA 的效率和毒性，我们利用 CsA 递送一种膜不可渗透的促凋亡肽（PAD），并将其与成熟的阳离子穿透肽 TAT（RKKRRQRRR）进行比较。通过将 CsA 与 PAD 缀合，在四种不同的肿瘤细胞系中，PAD 的内化增加了 2.2-4.7 倍，而 CsA-PAD 缀合物在 MCF-7 和 HeLa 人宫颈癌细胞中的内化明显高于 TAT-PAD 缀合物。细胞毒性研究表明，与 PAD 相比，CsA-PAD 在四种不同的肿瘤细胞系中表现出更大的细胞毒性增加，其效果与 TAT-PAD 相似或更大，具体取决于细胞类型。机制研究表明，修饰 CsA 或 TAT 不会改变 PAD 的细胞毒性机制，该机制与凋亡相关的线粒体膜损伤有关。体内研究表明，CsA-PAD 可以达到与 TAT-PAD 相似的抗肿瘤疗效，但全身毒性要低得多，尤其是对心脏和肝脏。总之，我们的研究首次表明，电子中性穿透肽 CsA 可以用作一种强大的工具，用于递送具有类似效率和更低毒性的肽药物，其效率和毒性均低于正电荷 TAT 肽。
