提高纳米制剂细胞内递送效率是其发展过程中亟待解决的关键问题。采用细胞穿膜肽修饰纳米制剂能够增强其胞内递送效率。我们之前研究首次发现采用低毒性的疏水穿膜肽PFVYLI修饰脂质体，能够通过其与细胞膜之间的疏水作用增加脂质体向不同细胞内的递送效率。而将PFVYLI改造成FFFF (F4)肽，具有比其母体更强的疏水性和胞内递送效率。基于此，我们首次构建F4修饰的紫杉醇纳米递药系统。并用组织蛋白酶B敏感肽GFLG修饰该递药系统，以提高其对肿瘤的特异性靶向。运用体外流式细胞仪、激光共聚焦显微镜和SRB细胞毒技术，以及体内活体成像、药效及毒性评价，从体外和体内两方面对酶敏感肽GFLG和疏水穿膜肽F4修饰的紫杉醇纳米递药系统的抗乳腺癌活性及相关机理进行研究。本研究能够提高紫杉醇抗肿瘤疗效，扩大疏水穿膜肽应用范围。为发展不同于阳离子穿膜肽的疏水穿膜肽及其相关药物递送系统提供了一个全新的视角。

There is one key issue need to be overcame during the study of nano drug delivery systems (NDDS), that is how to increase the intracellular delivery of NDDS. It is indicated that modification with cell penetrating peptides (CPPs) could highly enhance the intracellular delivery of NDDS. We first demonstrated that modification with hydrophobic CPPs PFVYLI can increase the intracellular delivery of liposomes in our previous research. And if the PFVYLI is transformed into FFFF, this new hydrophobic CPPs will possess more stronger hydrophobicity and higher intracellular delivery efficiency. Based on this theory, FFFF will be initially introduced to modify paclitaxel-loaded NDDS against breast cancer. At the same time, a cathepsin B sensitive peptide GFLG is further used to modified this FFFF modified NDDS to improve its specific target. The anti-breast cancer activity and the enhancing intracellular delivery mechanism of GFLG and FFFF modified paclitaxel-loaded NDDS was evaluated in vitro and in vivo through the flow cytometry, confocal microscopy and SRB cytotoxicity testing technology, as well as small animal in vivo imaging, therapeutic efficacy and toxicity evaluation experiments. This study could improves the therapeutic efficacy of paclitaxel against breast cancer and expands the application scope of hydrophobic CPPs. This provides us a new perspective on developing hydrophobic CPPs and its related drug delivery systems that are different to the classic cationic CPPs.

本研究利用低毒性的F4疏水穿膜肽修饰脂质体以便增加化疗药物向癌细胞内的递送效率，增强其对肿瘤疗效。并用组织蛋白酶B敏感肽GFLG修饰该递药系统，以提高其对肿瘤的特异性靶向。首先利用化学合成制备导向化合物DSPE-PEG2000-F4与保护化合物DSPE-GFLG-PEG5000。采用薄膜分散-水化法制备载紫杉醇的GFLG-F4-SSLs-PTX脂质体。脂质体为粒径在140 nm左右的类圆形粒子，PDI在0.25以下，表面带弱的负电，对紫杉醇包封率大于90%，而且药物从脂质体中释放的速率较慢。以二种乳腺癌细胞MCF-和 4T1作为肿瘤细胞模型，运用流式细胞和激光共聚焦显微技术对脂质体进行了体外细胞摄取研究，结果表明F4修饰的隐形脂质体能够促进香豆素向肿瘤细胞内传递，而DSPE-GFLG-PEG5000修饰后细胞的摄取又会降低。SRB比色法评价SSLs-PTX及F4-SSLs-PTX细胞毒性，表明F4修饰的隐形脂质体能够显著增强紫杉醇对细胞的毒性。通过在不同温度条件下4T1细胞对PFV-SSLs内吞量的差异，证明F4修饰的脂质体经4T1细胞的内吞为能量依赖性。通过不同的内吞相关抑制剂对4T1细胞摄取的影响，表明F4修饰的脂质体内吞作用不是是以脂筏微囊和网格蛋白共同参与介导的联合作用。比较了PFV与TAT对正常J774A.1与Eahy.926细胞的毒性，结果表明，与传统的阳离子穿膜肽TAT相比，无论是LDH释放还是共聚焦显微镜实验，PFV都具有更小的细胞毒性。本研究的成功实施为发展不同于传统阳离子细胞穿膜肽的疏水穿膜肽及其相关药物递送系统提供了一个全新的视角。同时，为具有广泛靶向作用的新型靶向纳米药物递送系统的开发起到一定的指导作用。