纳米递送系统表面修饰靶向促凋亡配体Z-TRAIL共递送双硫仑的协同抗肿瘤作用和机制研究

Exhibition of superior and tumor-specific cytotoxicity is an ideal goal for anticancer therapy. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) belongs to tumor necrosis factor superfamily. TRAIL is able to induce apoptosis in tumor cells by engagement with its cognate death receptors while sparing normal cells. However, the lack of the stability as well as the tumor-homing ability of the recombinant soluble TRAIL impairs the antitumor effects in vivo. Importantly, it is well established that many tumor cells especially primary tumors are resistant towards TRAIL-induced apoptosis. Combining TRAIL with standard chemotherapeutic agents can bypass TRAIL resistance and sensitize tumor cells to TRAIL-induced apoptosis, but also lead to potential systematic toxicity.. Disulfiram (DSF) is a broad spectrum of cancer-killing agent with high therapeutic tolerability. Recently, a study released by Nature has confirmed up the drugs’ anticancer effects and also revealed the mechanism. The researchers found the metabolites of DSF preferentially accumulated in xenografted tumors and blocked the machinery for protein degradation and turnover inside tumor cells. This sparked our interest in comparison of the mechanism with that of TRAIL. Consequently, we presume that DSF can yield synergistic anticancer activity with TRAIL. To prove this hypothesis, we constructed and produced a tumor-homing TRAIL variant, designated Z-TRAIL, by fusion of TRAIL with an affibody recognizing PDGFRβ on pericytes of tumor vessels. We found co-treatment with Z-TRAIL and DSF rendered DSF able to synergize with this ligand and selectively enhanced cytotoxicity in tumor cells. Meanwhile, covalently attaching Z-TRAIL on the surface of PLGA nanoparticles not only facilitated particles’ active tumor-targeting ability, but also exhibited synergistic cytotoxicity in tumor cells by co-delivery of surface-displayed Z-TRAIL with DSF. Therefore, in this project, we will further investigate the reinforcing anticancer activity and the mechanism when co-delivery of Z-TRAIL with DSF to TRAIL-resistant tumor cells. Subsequently, we will optimize the drug dose and ratio of Z-TRAIL to DSF in nanoparticles and evaluate their pharmacokinetics and pharmacodynamics in vivo. We believe that the co-delivery nanoparticles will yield superior and long-lasting antitumor effects as well as good safety profile in vivo, and overcome TRAIL resistance to tumor cells.

凋亡诱导配体TRAIL能特异性清除肿瘤细胞，但其重组蛋白稳定性差，靶向性弱，且肿瘤常抵抗TRAIL的凋亡诱导。利用阿霉素等药物能致敏肿瘤细胞，但也可导致非特异毒性。双硫仑DSF具有良好的安全性和高效的抗肿瘤活性，其在体内还能靶向瘤体富集。申请人前期以特异性识别PDGFRβ的人工抗体Z与TRAIL重组，制备了新型融合蛋白Z-TRAIL。通过靶向肿瘤新生血管周细胞，显著增强了对瘤体的药物递送。令人振奋的是DSF能协同增强Z-TRAIL的活性，且具有肿瘤细胞特异性。不仅如此，Z-TRAIL修饰PLGA纳米载体，能增强载体对肿瘤的主动靶向，并可共递送DSF协同杀伤肿瘤细胞。本项目拟进一步将二者联合，明确DSF协同Z-TRAIL的作用和机制；优化Z-TRAIL/DSF共递送纳米体系；评估其药代动力学和药效学。若获实施，将有望推出高效、安全和长效的共递送靶向纳米药物组合，克服肿瘤对TRAIL的抵抗。

肿瘤凋亡诱导配体TRAIL的半衰期短、靶向性弱，且对凋亡抗性细胞效果不佳。基于双硫仑的良好安全性和抗肿瘤作用，本项目研究了肿瘤靶向Z-TRAIL及其长效剂型Z-ABD-TRAIL与DSF的协同抗肿瘤作用，并构建了系列共递送纳米药物。DSF通过其活性组分CuET发挥抗肿瘤作用，通过上调肿瘤细胞死亡受体DR4和DR5，选择性致敏肿瘤细胞，增强Z-TRAIL的抗肿瘤作用。将DSF载入PLGA，并将Z-TRAIL共价偶联至PLGA纳米颗粒表面，项目构建了Z-TRAIL与DSF的共递送纳米药物。Z-TRAIL偶联的PLGA其靶向性较好，但PLGA对载入药物的缓释效果不佳，且偶联Z-TRAIL后纳米药物稳定性不足。改用为脂质体后，其能较好的提升药物的缓释能力，但仍需偶联Z-TRAIL至脂质体表面，大规模稳定制备不易。项目研究发现DSF的活性组分CuET与HSA结合能形成粒径约50-100 nm的纳米复合物CuET-NPs，其大小均匀可控，且稳定性十分良好。CuET-NPs与Z-ABD-TRAIL联合对结肠癌、乳腺癌和肺癌等多种肿瘤细胞具有显著的协同杀伤作用，且具有肿瘤细胞选择性。为此，项目最终选用了HSA作为共递送载体，巧妙地通过其不同结构域对Z-ABD-TRAIL与CuET-NPs的高亲和作用，构建了共递送纳米药物Z-ABD-T/CuET-NPs。该共递送系统的制备较为简便，载药量易于调整，且在体内外显著增强了两种药物的协同抗肿瘤效果。该共递送纳米药物有望克服肿瘤对TRAIL的抵抗，促进其向临床转化，同时也为基于HSA的药物共递送提供了新的思路。