肿瘤血管靶向肽GX1修饰的载α-亚麻酸-阿霉素腙键接合物纳米给药系统的设计和评价

Tumor vasculature targeted therapy is a new therapeutic strategy for cancer. The key of development is to find tumor vasculature targeted ligands with high specificity. In previous work, the GX1 peptide was screened by phage display peptide library, in vitro and in vivo studies confirmed that GX1 has good targeting on gastric cancer vascular endothelial cells. Moreover, α-linolenic acid-doxorubicin conjugate with a hydrazone bond (L-DOX), a pH sensitive and tumor cell targeting drug, was previously synthesized. It is remains to be confirmed whether the combination of tumor vasculature and tumor cell targeted could further improve tumor targeted therapy. In this present work, we prepared GX1 modified nanostructured nipid carriers loaded with L-DOX (L-DOX-NLCs-GX1) using polyoxyethylene (40) stearate modified with GX1 via the double emulsion method, established nanoparticulate drug delivery systems for combining tumor vasculature and tumor cell targeting. In vitro cell studies were carried out to confirm that whether the L-DOX-NLCs-GX1 have better specific targeted antiproliferation effect and better cellular uptake on gastric cancer cells (SGC7901) and simulated gastric cancer vascular endothelial cells (Co-HUVEC), while low cytotoxicity on gastric epithelial cells (GES) and vascular endothelial cells (HUVEC) compared with the unmodified NLCs. The investigation of in vivo pharmacokinetics, tissue distribution, antitumor activity and toxicity were carried out using nude mice bearing tumor xenografts of human gastric carcinoma. In summary, our study aims to design and evaluate the strategy encompassing both tumor vasculature and tumor cell targeting for anticancer therapy, improve anticancer efficacy, reduce adverse reactions and provide a theoretical basis for the in-depth investigation of tumor targeted drug delivery systems.

血管靶向治疗为肿瘤治疗提供新思路，其发展关键是寻找高特异性肿瘤血管靶向配体。课题组前期通过噬菌体筛选技术得到具良好胃癌血管内皮细胞靶向性的GX1短肽，合成了兼有肿瘤细胞靶向性及酸敏性的药物（α-亚麻酸-阿霉素腙键接合物，L-DOX）。联合血管靶向和细胞靶向能否进一步提高肿瘤靶向治疗效果有待证实。本课题拟在现有研究基础之上，采用GX1修饰聚乙二醇单硬脂酸甘油酯，以L-DOX为药物，制备GX1修饰的载α-亚麻酸-阿霉素腙键接合物纳米脂质载体，构建肿瘤血管和肿瘤细胞双重靶向纳米给药系统，通过体外细胞实验证实GX1修饰纳米粒具有更好的胃癌细胞和胃癌血管内皮细胞特异性靶向抑制作用，且对正常胃粘膜上皮细胞和血管内皮细胞低毒性；建立荷人胃癌裸鼠模型，研究其体内药代学、组织分布、抗肿瘤活性和毒性等，为肿瘤靶向治疗深入研究提供依据。

癌症是严重威胁人类生命健康的高危疾病，肿瘤血管靶向为肿瘤治疗提供新思路，其发展关键是寻找高特异性肿瘤血管靶向配体，幸运的是课题组前期通过噬菌体筛选技术得到胃癌血管内皮细胞靶向肽GX1（CGNSNPKSC）。本课题基于前期研究基础，首先合成了聚乙二醇单硬脂酸酯SA-PEG2000和SA-PEG2000羧基衍生物，进而合成得到GX1修饰的聚乙二醇单硬脂酸酯SA-PEG2000-GX1。接着在α-亚麻酸-阿霉素腙键接合物结构鉴定时发现，接合物极不稳定，无法用其进行后续实验，故尝试以阿霉素替换原计划中α-亚麻酸-阿霉素腙键接合物，完成了阿霉素固体脂质纳米粒DOX·HCl-SLNs制备、制剂学性质、体外释放及稳定性研究，实验过程中发现阿霉素纳米粒制剂工艺重现性不好，粒径偏大且稳定性不好，考虑美国NCCN临床实践指南胃癌治疗药物推荐紫杉醇而非阿霉素，因此我们制备了紫杉醇纳米脂质载体PTX-NLCs，并对其制剂学性质、体外释放及稳定性进行了考察，结果证实相比阿霉素纳米粒，紫杉醇纳米粒具有更小的粒径和更好的稳定性。继而采用SA-PEG2000-GX1制备了GX1修饰的紫杉醇纳米粒GX1-PTX-NLCs，体外细胞实验表明GX1-PTX-NLCs对Co-HUVEC细胞的抑制率最高（31.59±0.30%）、对GES-1细胞毒性最小（18.54±0.64%）。最后建立了荷人胃癌移植瘤裸鼠模型，进行了肿瘤抑制效果、生存曲线、裸鼠体重及裸鼠肿瘤组织H&E染色考察，完成了GX1-PTX-NLCs体内药效学评价，结果显示GX1-PTX-NLCs较PTX和PTX-NLCs，对荷人胃癌裸鼠具有更显著的肿瘤抑制率69.70±4.37%和更高程度的肿瘤组织损伤，证实肿瘤血管靶向肽GX1修饰的紫杉醇纳米粒可以靶向传递药物到肿瘤组织，提高药物疗效，减少毒副反应，为肿瘤血管靶向治疗深入研究提供依据。