Key Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, Sichuan, PR China.
Mol Pharm. 2010 Oct 4;7(5):1816-26. doi: 10.1021/mp100171c. Epub 2010 Aug 11.
Recently, PEGylation has been extensively employed to increase the circulation time of liposomes and enhance their accumulation in tumor tissue via the enhanced permeability and retention (EPR) effect; however, poly(ethylene glycol) (PEG) is unfavorable for the uptake of liposomes by tumor cells because of its steric hindrance. In this study, thiolytic cleavable PEG modified liposomes were used to solve this dilemma. Before arrival at the tumor tissue, PEG presents on the surface of liposomes, which is useful for passive accumulation in tumor tissue. Upon reaching the tumor tissues, the PEG chain could be removed by a safe cleaving reagent l-cysteine (l-Cys), and thus, the steric hindrance of PEG could be overcome conveniently. To further improve the uptake of liposomes, a "functional molecule" cell-penetrating peptide TAT was attached to the distal end of a shorter PEG spacer anchored to the surface of the liposomes, which could be shielded by cleavable PEG during circulation; upon arriving at tumor tissue, PEG was removed and thus the "functional molecule" TAT was exposed, and then TAT could mediate the uptake of the liposomes with high efficiency. In this study, thiolytic cleavable PEG was synthesized via a disulfide bridge, DOPE-PEG(1600)-TAT was synthesized by sulfhydryl-maleimide reaction, and then Rh-PE labeled liposomes composed of 2% DOPE-PEG(1600)-TAT and various amounts of cleavable PEG(5000) (2%, 4%, and 8%) were prepared, with particle size around 100 nm and slightly negative charge. These liposomes showed good stability in the presence of 10% serum. Their uptake by tumor cells HepG2 in vitro was assessed qualitatively and quantitatively. Liposomes modified with 2% DOPE-PEG(1600)-TAT and 8% DOPE-S-S-mPEG(5000) were regarded as the optimal formulation. In this preparation, nearly no uptake could be observed before addition of l-Cys, which meant undesired uptake during circulation could be avoided, while the uptake upon addition of l-Cys was 4 times as high as that in the absence of l-Cys. For the uptake in vivo, calcein loaded and Rh-PE labeled 8% cleavable PEG + 2% TAT modified liposomes were injected intratumorally into H22 tumor bearing mice. Confocal laser scanning microscopy (CLSM) showed that the uptake of 8% cleavable PEG + 2% TAT modified liposomes was much higher than that of 8% noncleavable PEG + 2% TAT modified liposomes in the presence of l-Cys. Thus, tumor targeted delivery could be achieved efficiently by the liposomal drug delivery system developed here in a controlled manner.
最近，聚乙二醇（PEG）化被广泛用于增加脂质体的循环时间，并通过增强通透性和滞留（EPR）效应增强其在肿瘤组织中的积累；然而，由于空间位阻，PEG 不利于肿瘤细胞摄取脂质体。在本研究中，使用硫解可切割的 PEG 修饰的脂质体来解决这一难题。在到达肿瘤组织之前，脂质体表面的 PEG 有利于被动积累在肿瘤组织中。到达肿瘤组织后，可使用安全的裂解试剂 l-半胱氨酸（l-Cys）去除 PEG 链，从而方便地克服 PEG 的空间位阻。为了进一步提高脂质体的摄取效率，将“功能分子”穿膜肽 TAT 连接到锚定在脂质体表面的较短 PEG 间隔臂的末端，该间隔臂在循环过程中可被可切割的 PEG 屏蔽；到达肿瘤组织后，去除 PEG，暴露出“功能分子”TAT，然后 TAT 可以介导脂质体的高效摄取。在本研究中，通过二硫键合成了硫解可切割的 PEG，通过巯基-马来酰亚胺反应合成了 DOPE-PEG(1600)-TAT，然后制备了由 2% DOPE-PEG(1600)-TAT 和不同量的可切割 PEG(5000)(2%、4%和 8%)组成的 Rh-PE 标记脂质体，粒径约为 100nm，带轻微负电荷。这些脂质体在 10%血清存在下表现出良好的稳定性。体外评估了肿瘤细胞 HepG2 对这些脂质体的摄取，定性和定量。被 2% DOPE-PEG(1600)-TAT 和 8% DOPE-S-S-mPEG(5000)修饰的脂质体被认为是最佳配方。在该制剂中，在加入 l-Cys 之前几乎观察不到摄取，这意味着可以避免循环过程中不需要的摄取，而加入 l-Cys 后的摄取是未加入 l-Cys 时的 4 倍。对于体内摄取，将 calcein 负载和 Rh-PE 标记的 8%可切割 PEG+2%TAT 修饰的脂质体瘤内注射到荷瘤 H22 小鼠中。共聚焦激光扫描显微镜（CLSM）显示，在存在 l-Cys 的情况下，8%可切割 PEG+2%TAT 修饰的脂质体的摄取明显高于 8%不可切割 PEG+2%TAT 修饰的脂质体的摄取。因此，通过本研究所开发的脂质体药物递送系统可以以可控的方式高效实现肿瘤靶向递送。
