Efficient Delivery of Payload into Tumor Cells in a Controlled Manner by TAT and Thiolytic Cleavable PEG Co-Modified Liposomes
Efficient Delivery of Payload into Tumor Cells in a Controlled Manner by TAT and Thiolytic Cleavable PEG Co-Modified Liposomes
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TAT 和硫解可裂解 PEG 共修饰脂质体以受控方式将有效负载有效递送至肿瘤细胞
DOI：
10.1021/mp100171c
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发表时间：
2010-09-01
影响因子：
4.9
通讯作者：
He, Qin
中科院分区：
文献类型：
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作者：
Kuai, Rui;Yuan, Wenmin;He, Qin
近年来，聚乙二醇化已被广泛应用于通过增强渗透性和滞留性（EPA）效应来增加脂质体的循环时间和促进其在肿瘤组织中的积累；然而，聚乙二醇（PEG）由于其空间位阻，不利于肿瘤细胞对脂质体的摄取。在本研究中，硫溶可切割PEG修饰脂质体解决了这一难题。PEG在到达肿瘤组织之前就存在于脂质体表面，有利于在肿瘤组织中被动积累。到达肿瘤组织后，PEG链可以被一种安全的切割试剂l -半胱氨酸（L-Cys）去除，从而方便地克服PEG的位阻。为了进一步提高脂质体的吸收，将一个“功能分子”细胞穿透肽TAT附着在锚定在脂质体表面的较短PEG间隔物的远端，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“功能分子”TAT， TAT可以高效介导脂质体的摄取。本研究通过二硫桥合成硫解可切割的PEG，通过巯基-马来酰亚胺反应合成DOPEPEG(1600)-TAT，然后制备由2% DOPEPEG(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，粒径在100 nm左右，带微负电荷。这些脂质体在10%的血清中表现出良好的稳定性。定性和定量地评价了它们在体外被肿瘤细胞HepG2摄取的情况。以2% dopes - peg (1600)-TAT和8% DOPES-S-mPEG（5000）改性脂质体为最佳配方。在该制剂中，添加L-Cys之前几乎没有观察到摄取，这意味着可以避免循环过程中不希望的摄取，而添加L-Cys后的摄取是不添加L-Cys时的4倍。为了在体内摄取，将钙黄蛋白负载和Rh-PE标记的8%可切割PEG +2% TAT修饰脂质体注射到H22荷瘤小鼠瘤内。共聚焦激光扫描显微镜（CLSM）显示，在L-Cys存在的情况下，8%可切割的PEG + 2% TAT修饰脂质体的吸收率远高于8%不可切割的PEG + 2% TAT修饰脂质体。因此，本文开发的脂质体给药系统可以有效地以可控的方式实现肿瘤靶向递送。
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 (EPA) 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% DOPEPEG(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% DOPES-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.