构建核素标记靶向VEGFR2的多功能载药外泌体的策略优化研究

Some patients with differentiated thyroid cancer may be dedifferentiated in the course of 131I treatment and further developed as radioiodine refractory differentiated thyroid cancer, however, molecular targeted drugs have obvious side effects and might be prone to drug resistance. Our previous studies showed that 17-AAG, the prototypical HSP90 inhibitor, could inhibit the proliferation and induce apoptosis of anaplastic thyroid cancer (ATC) cells; VEGFR2 was highly expressed in the ATC tissues and the modification of anti-VEGFR2 could significantly enhance its targeting effects. Exosomes are nontoxic and have been the current research hotspots in the delivery of anti-tumor drugs or tumor suppressor genes for the treatment of cancer. Based on these results, we plan to construct a novel multifunctional exosome, whose surface is modificated with anti-VEGFR2, labeled via 131I and 17-AAG loaded inside. In vitro and in vivo experiments, we will explore the drug loading efficiency, targeting characteristics and histological distribution of the novel exosome, and observe its effect on the dedifferentiated thyroid cancer. Our study will combine the molecular targeted drug delivery of exosome with the classical 131I treatment to explore the feasibility and efficiency of the combined therapy, and may provide a new direction and target for preventing the progress of dedifferentiated thyroid cancer.

部分甲状腺癌患者在治疗过程中发生失分化而发展为131I难治性甲状腺癌，分子靶向治疗存在毒副作用大及肿瘤耐药等问题。我们的前期研究表明，HSP90抑制剂17-AAG可明显抑制未分化甲状腺癌（ATC）增殖，并可诱导其凋亡；VEGFR2在裸鼠ATC组织中明显高表达，纳米载体经anti-VEGFR2修饰后可明显增强其靶向效果。外泌体安全无毒，用于包载、运输抗肿瘤药物或小分子抑癌基因来治疗肿瘤已成为当前研究的热点。基于现有研究，我们拟构建一种表面anti-VEGFR2修饰、内部包裹17-AAG并携带131I的新型多功能外泌体；通过体内和体外实验，分析外泌体的载药量、靶向性及组织学分布特征，观察其对失分化甲状腺癌细胞的毒性作用及抑瘤效果。本研究将外泌体的药物运输与经典的131I治疗相结合，拟探讨其介导的分子靶向药物与核素联合治疗的可行性和高效性，为失分化甲状腺癌的临床治疗提供新思路。

部分甲状腺癌患者在治疗过程中发生失分化而发展为131I难治性甲状腺癌，未分化甲状腺癌(ATC)病灶亦不摄取131I，1年生存率低于20%，而化疗存在毒副作用大及肿瘤耐药等问题。外泌体(exosomes, Exos)是细胞分泌的天然小囊泡，目前被作为递送化疗药物或核酸药物的新型载体用于多种恶性肿瘤的靶向治疗中。基于此，我们构建了一种 iRGD修饰的、装载盐酸阿霉素(Dox)并标记131I的新型多功能外泌体(Dox@iRGD-Exos-131I)，解决了ATC不摄取131I的问题，同时提高了肿瘤局部的药物浓度，并通过一系列体内、体外研究加以证实。WB示整合素αvβ3在 ATC患者癌组织中表达明显高于癌旁组织；且整合素αvβ3 在4株 ATC细胞（Hth7、Cal-62、THJ16T、8505C）的表达水平明显高于人正常甲状腺细胞。Dox@iRGD-Exos-131I的形貌为一侧凹陷的半球形，粒径范围为144.6 ± 53.7nm；131I的标记率约为 50.16%-60.21%，放化纯为 97.89%-100%，外泌体对Dox的装载效率约为11.73%。共聚焦显微镜及流式细胞术实验显示靶向（iRGD-Exos）和 无靶向（blank-Exos）外泌体均可明显被Hth7及8505C细胞摄取，且外泌体经 iRGD多肽修饰后可显著增强ATC细胞对外泌体的摄取能力。CCK-8实验显示 iRGD-Exos无明显细胞毒性；小动物活体成像及SPECT/CT显像证实 iRGD-Exos具有优良的体内靶向ATC的能力。Dox@iRGD-Exos-131I在体外及体内水平实验中均显示出优异的抑制8505C细胞及荷瘤裸鼠肿瘤生长的能力。病理切片证实肿瘤经 Dox@iRGD-Exos-131I治疗后，荷瘤裸鼠的主要器官未发生明显组织损伤。本研究为ATC的治疗提供了一个新方法，通过主动靶向的方法（iRGD修饰）将核素内照射治疗（131I）与化学治疗（Dox）相联合，提高了新型多功能外泌体（Dox@iRGD-Exos-131I）抑制肿瘤生长的能力，同时也为其他整合素αvβ3高表达的肿瘤提供了新的可能治疗方案。

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