隐形纳米粒子90Y-DOTA-mFA-PEG-PLGA介导叶酸受体靶向治疗卵巢癌及淋巴转移的实验研究

Ovarian cancer is the second highest fatal disease in woman because of high rate of recurrence, lymph metastasis and multi-drug resistance. More than 90% of ovarian cancer overexpress a high-affinity folate receptor (FR), involved in cellular folate accumulation by receptor-mediated endocytosis, that is absent from most normal tissues. Despite the excellent tumor targeting ability of radiofolates, the rapid clearance from blood circulation and relatively low FR density have limited the amount of radiofolate in ovarian cancer. A substantial fraction of radioactivity was remained in the kidneys because of re-absorption of radiofolate in the proximal tubule cells and it will increase the risk of damage to the radiosensitive kidneys. It was urgent to increase tumor targeting aggregation and retention in tumor site and reduce its accumulation in kidneys for the development of folate-targeted radionuclide therapy. The increased microvascular permeability of a tumor, characterized by the absence of a basal membrane and the presence of gaps between the endothelial cells, allows nanoparticles to extravasate from the general circulation. Moreover, the absence of lymphatic vessels promotes accumulation within the tumor environment, causing so-called "passive targeting." The drawback of the common nanoparticles was the abnormal increased distribution in RES-rich organs such as liver, spleen, bone marrow which will reduce its blood circulation time and the lack of cell-specific interaction, which reduces the internalization of the NP and its encapsulated drugs. Stealth nanoparticles was prepared with the addition of polyethylene glycol(PEG) in form of physical adhesion or covalent linkage to modify the surface of the nanoparticle. Polyethylene glycol (PEG) improves the pharmacokinetics of NPs by imparting a steric barrier to the surface, minimizing opsonization, and thus increasing its blood circulation time. Radiation was found to modulate the biodegradation of nanoparticles, release of encapsulating drugs and upregulate the folate receptor expression in our studies funded by previous NSFC (NO. 81071185). It was assumed to increase tumor-targeted accumulation and retention of radiofolate and significantly reduce its distribution in kidneys, liver, spleen and bone marrow with PEGylated stealth PLGA nanoparticles. In this study, we plan to prepare DOTA-folate-PEG-PLGA stealth nanoparticle and have it labelled with radionuclide 90Y with one-step method. Physico-chemical properties of 90Y-DOTA-folate-PEG-PLGA stealth particles were studied in vivo. The 90Y radiation regulation to the releasement of 90Y-DOTA-FA from the stealth particle and folate receptor expression changes on ovarian cancer cells were investigated. Further studies were mainly focus on its pharmacokinetics, Toxicology and pharmacodynamics on ovarian cancer bearing nude mice with or without lymph metastasis.

卵巢癌易多发耐药致死率高，90%高表达叶酸受体（FR，密度略低），而放射性标记叶酸及类似物（RFA）迅速经肾排泄并被重吸收而限制其应用。如何增加RFA在卵巢癌的聚集和滞留，减少其经肾排泄和再吸收亟待解决。 肿瘤毛细血管无基底膜、内皮细胞间隙宽且无淋巴系统，纳米粒子外渗入肿瘤聚集且不经肾排泄，但肝脾摄取多，聚乙二醇修饰形成的隐形纳米粒子（SNP）具有抗调理素化作用，在血液中稳定，前期研究（NSFC：81071185）发现辐射上调卵巢癌FR表达和SNP降解，因此，构建叶酸受体靶向放射性SNP，预期可增加RFA卵巢癌靶向聚集，并明显降低其在肾肝脾分布。本课题拟通过共价结合制备DOTA-mFA-PEG-PLGA的SNP，一步法完成90Y标记，体外考察其理化性质及90Y辐射对SNP负载药物释放和卵巢癌FR表达的调节，体内考察其药代动力学、毒理学、药效学，为FR靶向放射治疗卵巢癌及淋巴转移提供依据。

卵巢癌是女性常见恶性肿瘤，细胞表面高表达的叶酸受体为其靶向治疗提供了新思路。本课题首先完成放射性核素标记叶酸单体、二聚体的合成及其体内外药代动力学实验，结果显示尾静脉注射2h时，放射性核素标记的叶酸二聚体在卵巢癌部位的分布（%ID/g:11.07±1.83）明显高于90Y标记的叶酸单体(7.45±1.72)；通过beta射线发射体如32P、177Lu、90Y等轫致辐射显像和生物学分布的对比实验研究，结果显示轫致辐射显像可有效监测其在体内的生物学分布；课题组有效制备了体内外稳定性好的DOTA-FA-PEG-PLGA纳米粒子并通过DOTA完成放射性核素的偶联，体内药代动力学实验结果显示叶酸受体靶向特异性好，肿瘤放射性摄取高达（%ID/g：13.66±1.68），血液循环滞留时间长，从而通过GPR效应和叶酸靶向性结合，增加肿瘤部位放射性摄取、滞留时间和生物学作用，而在肾、肝脾分布少，毒副作用低；首次注射90Y-DOTA-FA-PEG-PLGA通过辐射可增加卵巢癌细胞表面FR表达水平和再循环代谢速率，一定剂量范围内呈量效关系，其中辐射早期引起胞内放射性标记配体增加，晚期可见胞内和细胞表面均明显增加，结果显示辐射可增加90Y-DOTA-FA-PEG-PLGA在肿瘤部位的聚集的效果，对临床静脉系统给药方案采用单次大剂量和分次序贯应用具有指导价值。药效学实验可见卵巢癌肿瘤坏死及新生血管增生受抑制有关，并可见淋巴结转移灶明显的放射性摄取，对发挥卵巢癌淋巴结转移灶具有一定作用。荷瘤鼠在给药途径的研究中发现，间质注射通过联合被动靶向的优势，使药物在局部的剂量达到峰值，纳米粒子缓慢经血液和淋巴系统引流，但明显增加肿瘤通过靶点对药物的有效结合，对治疗具有显著的应用优势。在本课题的研究基础上，开始采用生物源性材料（如黑色素）为载体的叶酸修饰纳米粒子的合成，其优势在于能物理吸附放射性核素并在体内外具有较好的稳定性，为本课题延伸项目的开展提供新的思路。