Endoglin单抗靶向纳米PEG脂质体细胞松弛素D治疗肿瘤研究

Recently, our research group isolated an acetyl cytochalasin D (aCD) from a tropical plant Cephalotaxus hainanensis Li, which possess better cytotoxic effects than the conventional cytochalasin D. Cytochalasin D targets actin that is ubiquitous in eukaryotic cells. When cytochalasin D is used as a cytotoxic agent in cancer therapy, it causes significant side effects. Study results have demonstrated that endoglin is overexpressed and up-regulated in tumor-associated angiogenic vasculature.Passive immunizations with anti-endoglin monoclonal antibody and active immunization with endoglin vaccine are capable of inhibiting tumor growth through suppression of growth of tumor endothelial cells in previous studies. In addition, liposomes are good carriers for the delivery of cytotoxic agents, which have been shown to reduce side effects and promote targeted tumor-cell action by accumulating at tumor sites because they can leak through pores and defects in the capillary endothelium. Recent studies have indicated that sterically- stabilized liposomes that are coated with inert biocompatible polymers, such as polyethylene glycol (PEG), are protected from being recognized by the immune system. To prevent CD's side effects, we encapsulate aCD in the nonaqueous interior of PEG liposomes and conjugate with a monoclonal antibody (mAb) against endoglin (anti-END/CDPL) as a model anticancer drug. Because the anti-endolgin mAb on the anti-END/CDPL can target the endoglin expressed on the tumor endothelial cells, causing inhibition of tumor angiogenesis and inducing specific tumor cell apoptosis, anti-END/CDPL thus possess "double functions" inducing both anti-angiogenesis and cytotoxic to tumor cells. In this study, we will apply in vitro tests such as MTT and TUNEL based immunological assays to acknowledge the capabilities of anti-END/CDPL in inhibition of tumor cell proliferation and induction of tumor cell apoptosis in comparison with the control liposomes.High-performance liquid chromatography will be sued to observe the biodistribution of the anti-END/CDPL in tumor-bearing mice. The antitumor activities of the anti-END/CDPL will be investigated in several murine tumor models to observe the tumor volume and survival time. Tumor cell apoptosis and angiogenesis will be observed in tumor tissues and mice treated with anti-END/CDPL by immunohistological and alginate-encapsulated tumor-cell assays. In addition, PCR Array will be used to screen the differential expression of molecules involving in the tumor apoptosis and tumor angiogenesis. Thereafter, the signaling pathway and epigenetics of the differential molecule will be chosen to study the molecular mechanisms of antitumor activities induced by anti-END/CDPL. Our study will facilitate the widely acceptation of aCD isolated from Hainan tropical medicinal-plants as cytotoxic agents for tumor therapy.

从海南粗榧内生真菌提取的乙酰基细胞松素D（aCD）较现有的CD有更好的细胞毒作用,但CD靶分子为肌动蛋白，作为细胞毒药物难于应用。本项目将aCD包裹于空间结构稳定脂质体中，并与endoglin（END）单抗共轭偶合获得免疫脂质体aCD（anti-END/CDPL）。END是肿瘤血管生成的标志性分子，肿瘤血管有大于正常血管的"孔隙"，anti-END/CDPL能特异靶向结合于表达END的血管内皮细胞，并通过"孔隙"进入肿瘤组织，具有靶向抑制肿瘤血管生成和诱导肿瘤细胞凋亡的"双功能"作用。本项目设计体内外实验了解anti-END/CDPL的靶向特异性，了解抑制内皮和肿瘤细胞增殖和诱导凋亡的能力；用小鼠肿瘤模型了解药物体内分布及抗肿瘤作用效果，了解抑制肿瘤血管生成和肿瘤细胞凋亡的能力；用PCR芯片筛选凋亡和血管生成差异表达分子，从信号和表观遗传学二个角度研究anti-END/CDPL的作用机理。

从海南粗榧内生真菌提取的乙酰基细胞松素D（aCD）较现有的细胞松弛素D（CD）有更好的细胞毒作用,CD靶分子为肌动蛋白，作为细胞毒药物难于应用。在本研究中，我们首先体外培养分泌aCD的内生真菌并分离提取促够的aCD。同时，按照我们以往的方法制备了具有纳米性质的抗endoglin细胞松弛素脂质体（anti-END/CDPL），然后采用免疫荧光技术检测anti-END/CDPL是否具有靶向性，同时用TUNEL和流式细胞术检测是否能够有效诱导细胞凋亡。结果发现，anti-END/CDPL可以高效结合体外培养的内皮细胞表面的靶向分子endoglin，在肿瘤血管内也可以有效结合血管表面的靶标分子，并且可以诱导血管内皮细胞和肿瘤细胞凋亡的能力和天然CD（nCD）相似，二者之间没有统计学差异（P < 0.001）。此外，用高效液相色谱进行药代动力学检测发现，Anti-END/CDPL在黑色素B16荷瘤小鼠体内的半衰期达4小时零10分钟，但nCD只有10分钟，可见用脂质体包裹nCD制成Anti-END/CDPL后明显延长了体内药物滞留时间，Anti-END/CDPL在体内滞留时间是天然aCD的13倍。在Anti-END/CDPL和nCD注入体内后，观察血液中CD达到最高浓度（峰浓度）的时间，发现Anti-END/CDPL达到峰浓度的时间明显慢于nCD，Anti-END/CDPL注射后1h 才达到高峰，而nCytD在5 min后就达高峰；Anti-END/CDPL的峰浓度为116.5±15.2，而nCytD为132.5±18.3，尽管Anti-END/CDPL的峰浓度较nCytD低约16 nmol/ml，但二者之间没有明显的统计学差异（P ＝ 0.12）。以上结果表明，本项目资助制备的endoglin抗体细胞松弛素脂质体Anti-END/CDPL具有肿瘤血管靶向分布的特异性，能够特异地与肿瘤血管新表达的ednoglin相结合，是一种有望成为肿瘤血管靶向的特异肿瘤治疗药物。希望基金委能够继续资助，进行后续的治疗效果及相关机理研究，最终结果可以申请技术专利并发表高水平的文章。

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