Improvement of cell-penetrating peptides utilizing high speednano-bioimaging

细胞穿透肽(CPP)/蛋白质转导结构域（PTD）被认为是基因、蛋白质和药物治疗的潜在载体。然而，基于tatp的技术有一些重要的局限性。例如，转导效率受靶分子特性的影响很大，在很多情况下，即使在体外应用，转导效率也不足。因此，在实际应用中需要显著提高转导效率。我们开发了一种量子点（QDs）标记的PTD单分子成像（SMI）技术，用于检测PTD在进入活细胞之前、开始时和进入活细胞后的动力学。因此，利用SMI，我们成功开发了超级PTD (SPTD)，其量子点（QD）转导效率比原始PTD （Tat-PTD）提高了1000倍。在本研究中，我们使用SMI，将SPTD在量子点上进行空间排列，以检验空间排列是否能显著提高转导效率。为此，我们发现增加PTD在纳米颗粒上的价可以使它们表现得像货物运输的纳米机器。因此，我们将（Gly-Pro-Pro） 8序列添加到SPTD （GPP-SPTD）中以进行修饰。我们发现GPP-SPTD使QDs的转导效率比原SPTD提高了10倍。同样，一项可转导的ttp -Cre重组酶报告基因实验显示，GPP-SPTD融合Cre提高了对SPTD-Cre和Tat-PTD-Cre的转导效率，分别提高了30倍和10倍。因此，这些结果表明，SPTD的多聚可以提高转导效率，通过诱导SPTD分子的多聚可以实现超高效转导。

Cell penetrating peptides (CPP)/protein transduction domain (PTD) are envisaged as potentially attractive vectors for gene, protein, and drug therapy. However, TatP-based technologies have some important limitations. For example, the transduction efficiency is greatly influenced by the characteristics of the target molecules, and in many cases, the efficacy has been insufficient even in in vitro applications. Therefore, prominent improvement of the transduction efficiency is required for the practical usages. We developed a single-molecule imaging (SMI) for PTD labeled with quantum dots (QDs) to examine the kinetics of PTD initially and immediately before, at the beginning of, and immediately after entry into living cells. As a result, utilizing of SMI, we have successfully developed super-PTD (SPTD), with its quantum dot (QD) transduction efficacy improve for 1,000 times as compared tooriginal PTD (Tat-PTD). In this study, using the SMI, we spatial arranged SPTD on QDs to examined whether spatial arrangement can dramatically improved the transduction efficacy. To this end we found that increasing the valence of PTD on nanoparticles allows them to behave as cargo-delivery nanomachines. Thus, we added (Gly-Pro-Pro) 8 sequence to the SPTD (GPP-SPTD) to from trimmer. We found that GPP-SPTD increase the transduction efficacy of QDs for >10 times to the original SPTD. Similarly, a transducible TatP-Cre recombinase reporter assay showed GPP-SPTD fusion Cre improved transduction efficiency to SPTD-Cre and Tat-PTD-Cre, for 30 times and 10 times, respectively. Thus, these results suggested that multimerization of SPTD enable to improve the transduction efficacy, and super-efficienent transduction could be realized by inducing multimerization ofthe ,molecules.