具有体内长循环性能的pH及还原双重敏感核酸载体的制备及其在肿瘤治疗中的应用

Aiming at highly efficient and safe transportation of genetic medicine(gene and siRNA), we will develop pH and reduction dual-sensitive nucleic carriers with long circulation property. The internal core of the polyplex is formed by complexation of polyethylenimine(PEI) and nucleic acid. By adjusting the feed ratio (i.e. N/P ratio) of polymer and nucleic acid in polyplex preparation, the polymeric vector efficiently complex nucleic acid to obtain polyplex with weak positive charge. In order to improve the biodegradability of the vector, poly(aspartic acid)s grafted with pH-sensitive groups such as 2-(diisopropylamino)ethylamine(DIP), 2-(diethylamino)ethylamine(DIE), 2-(dimethylamino)ethylamine(DIM) were employed to replace PEI for nucleic acid complexing. The interlayer of the nanocarrier is consisted of 2-mercaptoethylamine (MEA)-grafted poly(aspartic acid), which ties the polyplex up by forming reduction-sensitive disulfide bond. When the solution is adjusted to neutral pH, the pH-sensitive amino groups of the carrier will be further deprotonated to transfer the surface charge of disulfide-packed polyplex from positive to negative, favoring polyplex long circulation and reducing its side effect on normal tissues. Disulfide package not only decrease the polyplex size and stabilize it in blood, but also prevent the genetic medicine from nuclease degradation. When the polyplex enters the acidic tumor tissue, its surface will be transformed from negetive to positive again, enhancing the polyplex endocytosis by cancer cells. In the cancer cells lysosomes with further decreased pH and high concentration of reducing agent glutathione，the complexed nucleic acid will be rapidly release upon disulfide breakage and proton buffering effect of the polymeric carrier.

拟制备具有pH和还原双重敏感的长体内循环核酸传输载体，实现核酸药物（siRNA和基因）的高效、安全传输。粒子内核由聚乙烯亚胺与核酸复合而成，通过调控聚合物和核酸比例，使聚合物在酸性条件下复合核酸后带弱正电；为了提高生物降解性，还将采用不同酸敏基团（二异丙基乙二胺、二甲基乙二胺、二乙基乙二胺）接枝的聚天冬氨酸替代聚乙烯亚胺来构筑核酸载体。复合物粒子中间层由巯基乙胺接枝的聚天冬氨酸组成，可形成还原敏感的二硫键交联捆绑。在pH中性时，载体聚合物的更多酸敏氨基脱质子化，使粒子表面由弱正电转换为负电, 有利于粒子长循环并减少对正常组织的副作用。二硫键捆绑不仅降低复合物粒径并使其在血液中保持稳定，且防止基因药物被核酸酶降解。当复合物进入酸性肿瘤组织后，粒子表面重新由负电转换为正电，促进肿瘤细胞对粒子的内吞；在具有更低pH及高谷胱甘肽浓度的癌细胞溶酶体内，通过载体二硫键断裂和质子缓冲使核酸快速释放。

利用还原敏感和酸敏感提高核酸传输载体安全性和输送能力，设计并制备几种具有血液长循环和高癌细胞转染效率的核酸载体。通过还原敏感二硫键将低分子量的线性聚乙烯亚胺（PEI）接枝到可降解的聚赖氨酸（PLL）上，实现了PEI和PLL载体的优势互补，不仅可高效负载siRNA，而且可响应肿瘤细胞内高还原剂谷胱甘肽浓度（比血液中浓度高约1000倍）而快速释放siRNA，有效诱导肿瘤细胞的调亡。用不同酸敏基团（二异丙基乙二胺、二甲基乙二胺、二乙基乙二胺）接枝的聚天冬氨酸构筑核酸载体，并在中间亚表层引入巯基，两者协同作用，赋予载体血液长循环和癌细胞高转染效率的优势。其中，接枝酸敏基团的聚天冬氨酸嵌段，在酸性条件下高度质子化可高效负载核酸，在pH7.4的生理中性条件下则脱质子化，使核酸/聚合物纳米粒子表面电位由正变负；含有巯基的中间层则形成二硫键捆绑核酸载体，使负电纳米粒子在血液中不解体，赋予核酸纳米粒子体内长循环的优点；当纳米粒子进入肿瘤组织后，酸敏基团再次质子化，正电性增加，从而使纳米粒子表现为正电位，便于被癌细胞内吞，我们也在粒子表面引入肿瘤细胞的靶向分子和肿瘤组织渗透肽，进一步提高载核酸纳米粒子向肿瘤组织深部渗透和转染肿瘤细胞的能力。因此，课题通过制备具有pH和还原双重敏感的核酸输送载体，响应体内药物传输不同阶段生理环境变化，从而调控纳米粒子表面电位的可逆转换、体内血液长循环及高效细胞内吞和药物释放，并在动物模型上取得了良好的抗肿瘤治疗效果。研究成果发表于Biomaterials、Nanoscale、Chemical Communication和Polymer Chemistry等SCI期刊上，合计发表标注论文13篇，申请两项发明专利，授权1项发明专利，培养了8名博士毕业生和硕士毕业生。

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