C60-IONP-PEG-GE11多功能MRI对比剂精准靶向肿瘤显像及治疗实验研究

The high expression of the epidermal growth factor receptor EGFR on the epithelium-derived tumor cytomembrane is closely associated with the proliferation and differentiation of tumor cells, the epidermal growth factor receptor is the ideal target spot for anti tumor therapy, and micro-molecule polypeptide GE11 can be targeted to combine the EGFR receptor. In the earlier-stage experiment, we labeled the superparamagnetic iron oxide living body by using Aptamer to carry out targeting MRI development on tumors; fullerene C60 was further connected with ferroferric oxide with smaller particle sizes after carboxyl was modified on its surface to construct C60-IONP-PEG multifunctional nanoparticles, and in vitro had the PDT photodynamics treatment effect on tumor cells. Hence, the construction of the MRI contrast agent taking C60 as a carrier by using GE11 as targeting molecules is expected to realize accurate development and treatment on epithelium-derived tumors. In the study, C60-IONP-PEG-GE11 MRI multifunctional nanoparticles were subjected to chemical synthesis on the basis of the earlier-stage experiment. The cytological experiment was carried out to observe the toxicity, MRI development and PDT treatment effect of in vitro on melanoma cells; and the in vivo experiment was carried out to observe the MRI imaging and PDT+magnetic hyperthermia effect of the living body. The performance of accurately positioning tumors and the tumor treatment effect were analyzed from molecules, cells, tissue and organ level and its mechanism was discussed to establish the experimental and theoretical basis for the design of the multifunctional MR contrast agent.

表皮生长因子受体EGFR高表达于上皮源性肿瘤细胞膜，与肿瘤细胞增殖、分化密切相关，是抗肿瘤治疗理想的靶点，小分子多肽GE11可靶向结合EGFR受体。前期实验我们用Aptamer标记超顺磁性氧化铁活体可靶向肿瘤MRI显像；富勒烯C60表面修饰羧基后进一步连接粒径更小的四氧化三铁构建C60-IONP-PEG多功能纳米粒，体外对肿瘤细胞有PDT光动力学治疗效果；因此用GE11作为靶向分子构建C60为载体的MRI对比剂有望对上皮源性肿瘤精准显像及治疗。本研究在前期实验基础上化学合成C60-IONP-PEG-GE11 MRI多功能纳米粒，先行细胞学实验，观察其体外对黑色素瘤细胞的毒性、MRI显像及PDT治疗效果；再体内实验观察其活体MRI成像及PDT+磁热疗效果。从分子、细胞、组织和器官水平分析其精准定位肿瘤的性能及肿瘤治疗效果，探讨其机制，为新型多功能MR对比剂设计奠定实验及理论基础。

构建C60-IONP-PEG-GE11 MRI多功能对比剂，观察其体内外MD-MBA-231乳腺癌靶向MRI成像及治疗效果，毒性情况。免疫荧光实验结果证实 MD-MBA-231 细胞膜大量表达 EGFR受体；C60-IONP-GE11 体外 MRI 靶向实验可见 T2WI 呈负性强化，且 T2WI 信号随着靶向对比剂浓度增加而降低； 普鲁士蓝染色发现 C60-IONP-GE11 实验组可使 MD-MBA-231 细胞膜区明显蓝染，而非靶向对照组不能；体外毒性实验发现 C60-IONP-GE11 无明显细胞毒性； C60-IONP-GE11 体外光动力学杀伤乳腺癌细胞，细胞存活率仅为（3.74±1.06）%， 并可见细胞内大量活性氧生成，活性氧随 C60-IONP-GE11 浓度增加而增多。实验组C60-IONP-GE11尾静脉给药4小时，腋下肿瘤T2WI明显减低，出现负性靶向强化，8小时候强化消失，对照组未见明显强化。初步结论：C60-IONP-GE11 多功能 MR 对比剂具有体外靶向乳腺癌细胞 MR 成像功能，几乎无毒，外加激光治疗可靶向杀伤肿瘤细胞，同时具有 MRI 成像及 PDT 治疗作用。体内可对乳腺癌肿瘤MR 靶向成像。

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