细胞穿膜肽（CPPs）常用于递送生物大分子入胞，具有其它手段无法比拟的优势。但是，CPPs的递送效率，特别是从内吞小泡逃逸的效率亟待提高，同时还面临体内应用时血清耐受性差的问题。pH敏感肽的酸化变构可破坏小泡囊膜，已被用于提升CPPs的小泡逃逸效率，但由于CPPs、pH敏感肽与囊膜之间较强的粘附作用，被转运的生物大分子大部分仍无法从小泡中成功逃逸。在前期工作中，我们发现内吞小泡特异性蛋白酶酶切位点和多聚化结构域的引入可显著提升CPPs递送的小泡逃逸效率及其血清耐受性。本项目将在前期工作的基础上，进一步从酶切位点、多聚化结构域以及不同功能肽的相对位置等不同方面进行全面优化；通过筛选合适的异源二聚化配体，建立通用型高效递送系统；在体内外开展初步应用研究。本项目的开展不仅能够为生物大分子的胞内递送提供新的手段，还能为针对胞内靶标的生物大分子药物的基础研究和临床应用奠定基础。

As a commonly used method to deliver biomacromolecules (cargo) into cytosol, cell-penetrating peptides (CPPs) have many incomparable advantages over other transduction methods. However, the efficiency of CPP-mediated delivery, especially the efficiency of escaping from endosome, needs to be improved urgently. In addition, the poor serum tolerance of CPP-mediated delivery is another problem which should be solved if it is used in vivo. pH-dependent membrane active peptides (PMAPs) are often used to improve the endosomal release of CPP-cargo, because that PMAPs undergo dramatic conformational change to disrupt the endosomal membrane as endosomes acidify during maturation from early endosomes to late endosomes and lysosomes. But, due to the strong interaction between CPPs/PMAPs and endosomal membrane, most of the cargo delivered by CPPs/PMAPs will still be tethered to the membrane even if the endosome is disrupted, and cannot successfully escape from the endosome. In the previous works, we found that the endosomal escape and serum tolerance of CPPs/PMAPs-based delivery system can be significantly improved by endosome-specific protease cleavage sites (PCSs) and homo-polymer forming domains (HPFDs). Based on the above findings, in this project, we will further optimize the sequences of PCSs and HFPFs as well as the relative positions of different functional peptides to establish finally the CPP-based intracellular delivery system of biomacromolecules, screen suitable adaptors with the ability of hetero-dimerization to establish a universal delivery system, and carry out preliminary applications of the final system or universal system in vitro and in vivo. The enhanced CPP-based delivery system developed in this project will not only provide a new means for intracellular delivery of biomacromolecules, but also lay a foundation for basic research and clinical application of biomacromolecular drugs targeting intracellular targets.