Macchi Sara, Signore Giovanni, Boccardi Claudia, Di Rienzo Carmine, Beltram Fabio, Cardarelli Francesco
NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, Piazza San Silvestro 12-56127 Pisa, Italy.
Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12-56127 Pisa, Italy.
Sci Rep. 2015 Nov 16;5:16914. doi: 10.1038/srep16914.
Peptides that translocate spontaneously across cell membranes could transform the field of drug delivery by enabling the transport of otherwise membrane-impermeant molecules into cells. In this regard, a 9-aminoacid-long motif (representative sequence: PLIYLRLLR, hereafter Translocating Motif 9, TM9) that spontaneously translocates across membranes while carrying a polar dye was recently identified by high-throughput screening. Here we investigate its transport properties by a combination of in cuvette physico-chemical assays, rational mutagenesis, live-cell confocal imaging and fluorescence correlation spectroscopy measurements. We unveil TM9 ability to self-aggregate in a concentration-dependent manner and demonstrate that peptide self-aggregation is a necessary--yet not sufficient--step for effective membrane translocation. Furthermore we show that membrane crossing can occur with apolar payloads while it is completely inhibited by polar ones. These findings are discussed and compared to previous reports. The present results impose a careful rethinking of this class of sequences as direct-translocation vectors suitable for delivery purposes.
能够自发跨细胞膜转运的肽，可通过将原本不能透过膜的分子转运到细胞内，从而改变药物递送领域。在这方面，最近通过高通量筛选鉴定出了一种9个氨基酸长的基序（代表性序列：PLIYLRLLR，以下简称转运基序9，TM9），它在携带极性染料时能自发跨膜转运。在此，我们通过比色管中的物理化学分析、合理诱变、活细胞共聚焦成像和荧光相关光谱测量等方法相结合，研究了其转运特性。我们揭示了TM9以浓度依赖方式自我聚集的能力，并证明肽的自我聚集是有效膜转运的必要但不充分步骤。此外，我们表明非极性载荷可以发生膜穿越，而极性载荷则完全抑制膜穿越。我们对这些发现进行了讨论，并与之前的报告进行了比较。目前的结果促使人们重新仔细思考这类序列作为适用于递送目的的直接转运载体的问题。
