Cellular uptake and in vivo distribution of polyhistidine peptides
Cellular uptake and in vivo distribution of polyhistidine peptides
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DOI：
10.1016/j.jconrel.2015.05.268
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发表时间：
2015-07-28
影响因子：
10.8
通讯作者：
Nakayama, Yuji
中科院分区：
文献类型：
--
作者：
Iwasaki, Takashi;Tokuda, Yoshihisa;Nakayama, Yuji
细胞穿透肽（CPP）是富含精氨酸/赖氨酸的序列，并且它们被有效地内化到细胞中。在这个过程中，正电荷至关重要。在本研究中，我们发现了多聚组氨酸肽（PHPs），作为一种新的CPP，这是有效的内化到细胞中的正电荷不依赖的方式。有趣的是，PHPs的细胞摄取随着链长的增加而增加，在H16（HHHHHHHHHHHHHHHH-NH 2）达到最大摄取。这种H16肽对HT 1080人纤维肉瘤细胞的细胞穿透能力比主要CPP八精氨酸（RRRRRRRR-NH 2）肽高14.6倍。H16肽的细胞摄取主要是由于巨胞饮作用，并且大多数H16肽定位于溶酶体和高尔基体中。然而，与H16肽缀合的细胞质促凋亡结构域（KLAKLAKLAKLAK-NH 2）显示细胞毒性作用。这表明一部分H16肽从大胞饮体逃逸到细胞质中。在蛋白质转导研究中，通过Ni-NTA层析纯化与H16肽融合的绿色荧光蛋白（GFP-H16），使用抗His标签抗体检测并内化到细胞中。这一系列过程揭示了H16作为His标签和蛋白质转导结构域的功能。此外，体内分布分析表明，H16肽立即在肿瘤组织中积累，并在注射到荷瘤小鼠（HT 1080人纤维肉瘤）后保留长达132小时。这是第一次观察到His-聚合物被有效地内化到细胞中。研究结果表明，PHPs是新的CPP。特别地，H16肽代表了在医学和生物技术领域中有前途的药物递送载体候选者。(C)2015爱思唯尔B. V.保留所有权利。
Cell-penetrating peptides (CPPs) are arginine/lysine-rich sequences, and they are effectively internalized into cells. In this process, positive charge is crucial. In the present study, we found polyhistidine peptides (PHPs), as the novel CPP, which are efficiently internalized into cells in a positive charge-independentmanner. Interestingly, cellular uptake of the PHPs increased as the chain length increased, reaching a maximum uptake at H16 (HHHHHHHHHHHHHHHH-NH2). This H16 peptide showed up to 14.6-fold higher cell-penetrating capacity against HT1080 human fibrosarcoma cells relative to a major CPP, the octa-arginine (RRRRRRRR-NH2) peptide. Cellular uptake of the H16 peptide is mainly due to macropinocytosis and most of the H16 peptide localizes in the lysosome and Golgi apparatus. However, a cytoplasmic pro-apoptotic domain (KLAKLAKKLAKLAK-NH2) conjugated to the H16 peptide showed cytotoxic effects. This indicates that a proportion of the H16 peptide escapes from the macropinosome to the cytoplasm. In a protein transduction study, green fluorescence protein fused to the H16 peptide (GFP-H16) was purified by Ni-NTA chromatography, detected using an anti-His-tag antibody and internalized into cells. This serial process reveals thatH16 functions as a His-tag and protein transduction domain. Furthermore, in vivo distribution analysis showed that the H16 peptide accumulates immediately in tumor tissue and is retained up to 132 h following injection into the tumor (HT1080 human fibrosarcoma)-bearing mice. This is the first observation of a His-polymer being internalize into cells efficiently. The findings suggest that PHPs are novel CPPs. In particular, the H16 peptide represents a promising drug delivery carrier candidate in medical and biotechnological fields. (C) 2015 Elsevier B.V. All rights reserved.