State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Haidian District, Beijing 100191, PR China.
Biomaterials. 2013 Sep;34(28):6976-91. doi: 10.1016/j.biomaterials.2013.05.055. Epub 2013 Jun 15.
In the hormone-refractory stage of prostate cancer (PC), the expression of prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) often remains highly active. Accumulating studies have demonstrated that these two proteins are attractive targets for specific delivery of functional molecules to advanced PC, not merely as potential sensitive markers for PC detection. In this study, we constructed a dual-modified liposome that incorporated PSA-responsive and PSMA-mediated liposomes and potentially offers double selectivity for PC. The folate moiety binds quickly to PSMA-positive tumors, and the PSA-responsive moiety is cleaved by PSA that was enriched in tumor tissues. The activated liposomes (folate and cell-penetrating peptides dual-modifications) are subsequently taken up by the tumor cells via polyarginine's penetrating effects and receptor-mediated endocytosis. To corroborate these assumptions, a series of experiments were conducted, including PSA-responsive peptide hydrolysis kinetics, cellular uptake, internalization mechanism and escape from endosomes in PC-3 and/or 22Rv1 cells, biodistribution and antitumor activity of siRNA-loaded liposomes after systemic administration, gene silencing and cell apoptosis in vitro and in vivo. The results reveal that multivalent interactions play a key role in enhancing PC cell recognition and uptake while reducing nonspecific uptake. The dual-modified liposomes carrying small interfering RNA (siRNA) have significant advantages over the control liposomes, including single-modified (folate, CPP, PSA-responsive only) and non-modified liposomes. The dual-modified liposomes elevated cellular uptake, downregulated expression of polo-like kinase 1 (PLK-1) and augmented cell apoptosis in prostate tumor cells. The entry of the dual-modified liposomes into 22Rv1 cells occurred via multiple endocytic pathways, including clathrin-mediated endocytosis and macropinocytosis, followed by an effective endosomal escape of the entrapped siRNA into the cytoplasm. In vivo studies conducted on a 22Rv1 xenograft murine model demonstrated that the dual-modified liposomes demonstrated the maximized accumulation, retention and knockdown of PLK-1 in tumor cells, as well as the strongest inhibition of tumor growth and induction of tumor cell apoptosis. In terms of targeting capacity and therapeutic potency, the combination of a PSA-responsive and PSMA-mediated liposome presents a promising platform for therapy and diagnosis of PSMA/PSA-positive PC.
在前列腺癌（PC）的激素难治阶段，前列腺特异性抗原（PSA）和前列腺特异性膜抗原（PSMA）的表达通常仍然非常活跃。越来越多的研究表明，这两种蛋白质是将功能性分子特异性递送至晚期 PC 的有吸引力的靶标，而不仅仅是作为 PC 检测的潜在敏感标志物。在这项研究中，我们构建了一种双重修饰的脂质体，该脂质体包含 PSA 响应和 PSMA 介导的脂质体，并为 PC 提供了双重选择性。叶酸部分与 PSMA 阳性肿瘤快速结合，而 PSA 响应部分被富集在肿瘤组织中的 PSA 切割。激活的脂质体（叶酸和穿透肽双重修饰）通过多精氨酸的穿透作用和受体介导的内吞作用被肿瘤细胞摄取。为了证实这些假设，进行了一系列实验，包括 PSA 响应肽水解动力学、细胞摄取、内化机制和 PC-3 和/或 22Rv1 细胞中的内体逃逸、系统给药后负载 siRNA 的脂质体的生物分布和抗肿瘤活性、体外和体内的基因沉默和细胞凋亡。结果表明，多价相互作用在增强 PC 细胞识别和摄取的同时减少非特异性摄取方面发挥了关键作用。携带小干扰 RNA（siRNA）的双重修饰脂质体与对照脂质体相比具有显著优势，包括单修饰（叶酸、CPP、仅 PSA 响应）和非修饰脂质体。双重修饰的脂质体增加了细胞摄取，下调了 polo 样激酶 1（PLK-1）的表达，并增强了前列腺肿瘤细胞的细胞凋亡。双重修饰的脂质体进入 22Rv1 细胞的途径包括网格蛋白介导的内吞作用和巨胞饮作用，随后被包裹的 siRNA 有效地从内体逃逸到细胞质中。在 22Rv1 异种移植鼠模型的体内研究表明，双重修饰的脂质体在肿瘤细胞中最大程度地积累、保留和下调 PLK-1，并最强抑制肿瘤生长和诱导肿瘤细胞凋亡。就靶向能力和治疗效力而言，PSA 响应和 PSMA 介导的脂质体的组合为 PSMA/PSA 阳性 PC 的治疗和诊断提供了有前途的平台。
