Cellular Reprogramming and Embryo Biotechnology Laboratory, Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Korea.
Fermentation Science Program, School of Agriculture, College of Basic and Applied Sciences, Middle Tennessee State University, Murfreesboro, Tennessee, USA.
Biotechnol Prog. 2020 Sep;36(5):e3014. doi: 10.1002/btpr.3014. Epub 2020 Jun 11.
Cell-penetrating peptides (CPPs) are short amino acid sequences known to act as a vehicle for enhancing the intracellular translocating efficiency of extracellular molecules. Although many groups have attempted to develop peptides with high cell-penetrating efficiencies, very few have demonstrated efficient cellular uptake of CPPs at low concentrations. Here, we describe a newly synthesized peptide derived from Arabidopsis, Ara-27, which exhibits significant improvement in cell-penetrating efficiency compared to existing CPPs. The cell-penetrating efficiency of Ara-27 was compared with the commonly used Tat-protein transduction domain (Tat-PTD) and membrane translocating sequence (MTS) in human dermal fibroblast (HDF) and human dental pulp stem cells (hDPSC). Cell-penetrating efficiency of fluorescein isothiocyanate (FITC)-labeled CPPs were assessed by flow cytometry and visualized by confocal microscopy. Flow cytometric analysis revealed >99% cell-penetrating efficiency for 2 μM Ara-27 in both HDF and hDPSC. In contrast, 2 μM Tat-PTD and MTS showed <10% cell-penetrating efficiency in both cells. In support, relative fluorescence intensities of FITC-labeled Ara-27 were around 8 to 22 times higher than those of Tat-PTD and MTS in both cells. Confocal analysis revealed internalization of 0.2 and 2 μM Ara-27 in both human cells, which was not observed for Tat-PTD and MTS at either concentration. In conclusion, this study describes a novel CPP, Ara-27, which exhibit significant improvement in intracellular uptake compared to conventional CPPs, without affecting cell viability. Thus, development of Ara-27 based peptides may lead to improved delivery of functional cargo such as small molecules, siRNA, and drugs for in vivo studies.
细胞穿透肽（CPPs）是众所周知的短氨基酸序列，可作为增强细胞外分子细胞内转运效率的载体。尽管许多研究小组试图开发具有高细胞穿透效率的肽，但很少有研究表明 CPP 能够以低浓度有效地进入细胞。在这里，我们描述了一种新合成的来自拟南芥的肽，Ara-27，与现有的 CPP 相比，它在细胞穿透效率方面有显著提高。在人皮肤成纤维细胞（HDF）和人牙髓干细胞（hDPSC）中，将 Ara-27 的细胞穿透效率与常用的 Tat 蛋白转导结构域（Tat-PTD）和膜转位序列（MTS）进行了比较。通过流式细胞术评估荧光素异硫氰酸酯（FITC）标记的 CPP 的细胞穿透效率，并通过共聚焦显微镜进行可视化。流式细胞术分析显示，2μM 的 Ara-27 在 HDF 和 hDPSC 中均具有>99%的细胞穿透效率。相比之下，2μM 的 Tat-PTD 和 MTS 在两种细胞中的细胞穿透效率均<10%。支持这一结果的是，FITC 标记的 Ara-27 的相对荧光强度在两种细胞中均比 Tat-PTD 和 MTS 高 8 到 22 倍。共聚焦分析显示，0.2 和 2μM 的 Ara-27 均能在两种人类细胞中内化，而在这两种浓度下均未观察到 Tat-PTD 和 MTS 的内化。总之，本研究描述了一种新型 CPP，Ara-27，与传统 CPP 相比，其细胞内摄取效率有显著提高，而对细胞活力没有影响。因此，基于 Ara-27 的肽的开发可能会提高小分子、siRNA 和药物等功能货物的递药效率，从而促进体内研究。
