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.

中文翻译：

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与传统肽相比，新合成的肽 Ara-27 在细胞穿透能力方面表现出显着改善。

细胞穿透肽 (CPP) 是一种短氨基酸序列，已知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，但很少有人证明在低浓度下 CPP 的细胞有效吸收。在这里，我们描述了一种源自拟南芥的新合成肽, Ara-27，与现有的 CPP 相比，其细胞穿透效率有显着提高。将 Ara-27 的细胞穿透效率与人真皮成纤维细胞 (HDF) 和人牙髓干细胞 (hDPSC) 中常用的 Tat 蛋白转导结构域 (Tat-PTD) 和膜转位序列 (MTS) 进行比较。通过流式细胞术评估异硫氰酸荧光素 (FITC) 标记的 CPP 的细胞穿透效率，并通过共聚焦显微镜观察。流式细胞术分析显示，2 μM Ara-27 在 HDF 和 hDPSC 中的细胞穿透效率 >99%。相比之下，2 μM Tat-PTD 和 MTS 在两种细胞中均显示 <10% 的细胞穿透效率。作为支持，在两种细胞中，FITC 标记的 Ara-27 的相对荧光强度比 Tat-PTD 和 MTS 的相对荧光强度高约 8 到 22 倍。共聚焦分析揭示了两种人细胞中 0.2 和 2 μM Ara-27 的内化，这在任一浓度的 Tat-PTD 和 MTS 中​​均未观察到。总之，本研究描述了一种新型 CPP，Ara-27，与传统 CPP 相比，它在细胞内摄取方面表现出显着改善，且不影响细胞活力。因此，基于 Ara-27 的肽的开发可能会改善功能性货物的递送，例如小分子、siRNA 和用于体内研究的药物。