RGD修饰的线粒体靶向长循环脂质体的制备及初步体外评价

Preparation and Preliminary in vitro Evaluation of RGD Modified Mitochondrial Targeting Long-circulating Liposomes

以前药三苯基膦(TPP)-白藜芦醇(1)为模型药,采用薄膜分散法制备RGD修饰的线粒体靶向长循环脂质体(RLP-TPP-1脂质体)。首先将TPP与抗肿瘤药1共价结合得到线粒体靶向前药TPP-1,然后将其包载于脂质双分子层中,最后通过后插法在脂质体表面连接长循环的聚乙二醇(PEG)和细胞靶向肽RGD。所得RLP-TPP-1脂质体呈球形,分布均匀,平均粒径(128.1±3.4)nm,ζ电位(-20.28±1.68)mV,包封率(75.6±0.8)%。体外细胞毒性试验表明,空白脂质体对表面过表达anb3受体的MDA-MB-231细胞无明显毒性,但载药脂质体对MDA-MB-231细胞的增殖具有明显抑制作用,且呈浓度依赖性。其中,RLP-TPP-1组IC50值最低(4.95mmol/L)。细胞摄取试验表明,孵育4 h后,肿瘤细胞对RGD修饰脂质体的摄取显著高于未修饰脂质体组。采用JC-1染色法考察制品的线粒体靶向作用,结果显示RLP-TPP-1脂质体组线粒体靶向效果最佳,凋亡细胞增多。

As one of the most important organelles in most eukaryotic cells, mitochondria not only supply cellular energy, but also involve in a range of other processes, such as cellular differentiation, cell death, as well as the control of the cell cycle and cell growth. Besides, cancer cell mitochondria exhibit an extensive metabolic activity and are more susceptible to mitochondrial perturbation than normal cell mitochondria. Consequently, induction of apoptosis through targeting of mitochondria of cancer cells can be an effective therapy strategy. Here, we conducted a hierarchical targeted delivery system that could deliver the chemotherapeutic agents, resveratrol （1）, sequentially to tumor cells and mitochondria. In detail, the mitochondrial targeting prodrug triphenylphosphine-resveratrol （TPP-1） was constructed by conjugation. Then, the TPP-1 was entrapped in the lipid bilayer of liposomes. Followed, the liposomes were decorated with polyethylene glycol （PEG） and the cell targeting peptide RGD to form the hierarchical targeted delivery system （RLP-TPP-1 liposomes）. The prepared RLP-TPP-1 liposomes were regularly spherical and well-distributed, the averageparticle size, ζ potential and encapsulation efficiency were（128.1±3.4）nm, （-20.28±1.68）mV and （75.6±0.8）%,respectively. The results of in vitro cytotoxicity test showed that the blank liposomes were nontoxic against MDA-MB-231 cells overexpressing ανβ3 receptors. However, the drug-loaded liposomes presented a significant inhibitory effect on MDA-MB-231 cells in a dose-dependent manner. In addition, the IC50 value （4.95 μmol/L） of the RLP-TPP-1 liposomes was the lowest among the tested liposomes. Cellular uptake experiment revealed that the uptake of RGD decorated liposomes were significantly higher than that of the undecorated liposomes. The results of JC-1 staining test showed that the RLP-TPP-1 liposomes performed the best mitochondrial targeting effect and increased apoptosis distinctively. In summary, RLP-TPP-1 liposomes could target to tumor cells and deliver 1 to mitochondria, resulting in the mitochondrial dysfunction and apoptosis of cancer cells.