烷基糖苷介导的肾小管上皮细胞靶向新型纳米递送系统用于治疗糖尿病肾小管间质纤维化的研究

Toward the success drug and gene therapy for diabetic renal tubulointerstitial fibrosis, fabrication of nanovectors that can target renal tubular epithelial cell (RTEC) is of great importance. However, current strategies employed to prepare nanovectors are primarily limited to the tissue level targeting. Even though some nanovectors can achieve cell-level targeting, they cannot efficiently release drug or gene inside the cells, and therefore, only limited therapeutic efficacy has been realized. On the basis of our previous work, a novel polymer, alkyl glycoside-PEG-chitosan-metformin (Glc-S-C8-PEG-CS-MET), is designed and complexed with shRNA SREBP-1c (shSREBP-1c) to form a pH sensitive nanovector (Glc-S-C8-PEG-CS-MET/shSREBP-1c). The proposed nanovector may penetrate through the glomerular filtration barrier owing to its small particle size. The surface presented Glc-S-C8 is anticipated to facilitate the whole nanovector to enter RTEC through megalin receptor-mediated internalization. The uptaken nanovector will escape from endosome via the proton sponge effect. Meanwhile, the nanovectors will be decomposed by breaking the pH-responsive imine bonds, which can accelerate the release of payloads, metformin and shSREBP-1c. Ultimately, the therapy of diabetic tubulointerstitial fibrosis can be successfully achieved by the hypoglycemic and lipid-lowering effects offered by the payloads. The efficacy will be confirmed in vitro and in vivo. If the result is as our suspection, our nanovector will provide both the theoretical guidance and experimental basis for establishment of a new high-efficiency RTEC targeting carrier, which can co-deliver drug and gene. Our project can also provide new ideas for the therapy of diabetic renal tubulointerstitial fibrosis.

构建肾小管上皮细胞（RTEC）靶向载体是实现糖尿病肾小管间质纤维化（TIF）药物和基因治疗的关键。目前的肾靶向策略还局限于肾组织靶向水平，即使实现RTEC水平，也不能在胞内有效释放药物或基因，大大降低治疗效果。本课题在前期研究基础上设计烷基糖苷-聚乙二醇-壳聚糖-二甲双胍（Glc-S-C8-PEG-CS-MET）新颖载体材料，制备pH敏感型纳米复合物Glc-S-C8-PEG-CS-MET/shSREBP-1c。该纳米复合物粒径可控，有望透过肾小球滤过屏障，经由Glc-S-C8结合RTEC高表达的兆蛋白受体入胞，通过质子海绵作用溶酶体逃逸和pH响应性断裂亚胺键释放二甲双胍和shSREBP-1c，发挥降糖降脂双重药效实现抑制糖尿病TIF作用。本项目拟通过离体和在体实验证实，如获成功，可为构建新型高效低毒的RTEC靶向药物和基因共传递系统提供理论指导和实验依据，并为糖尿病TIF的治疗提供新思路。

构建肾小管上皮细胞（RTEC）靶向载体是实现糖尿病肾小管间质纤维化（TIF）药物和基因治疗的关键。本课题制备的pH敏感型纳米复合物粒径可控，可透过肾小球滤过屏障，在HK-2细胞中的转染效率与PEI 25K阳性对照组相当，比游离GFP阴性对照组相比转染效率显著提升，经过EDTA预处理后，该纳米粒在HK-2细胞中的摄取显著降低。TUNEL法检测结果显示该纳米粒可以明显降低TGF-β刺激后纤维化细胞的凋亡。WB检测结果显示游离二甲双胍与该纳米粒均可以有效抗纤维化细胞凋亡，无显著性差异。该纳米粒体外抗纤维化细胞炎症与纤维化的作用显著优于游离二甲双胍。此纳米粒具有较好的肾靶向作用，注射后0.25 h开始出现富集现象并持续至少12 h，注射后4 h达到顶峰，相较于其他器官，该纳米粒在左侧梗阻肾脏的富集最多。H&E结果显示，UUO小鼠肾脏出现肾小球肥大、系膜基质增生、肾小管扩张及炎性细胞浸润，并可见肾小管上皮细胞空泡变性，间质增宽及严重纤维化；分别用游离二甲双胍，空白载体和构建的纳米粒处理后上述现象有不同程度的减轻，其中该纳米粒的治疗效果最显著。免疫组化结果显示，UUO造模小鼠阻塞肾脏的纤维化标志物Collagen I及α-SMA表达明显升高，游离二甲双胍治疗后表达没有降低，空白载体和构建的纳米粒治疗后表达下降，且该纳米粒Collagen I及α-SMA表达抑制作用更明显。免疫印迹结果显示，游离二甲双胍或空白载体的处理对UUO小鼠阻塞肾脏凋亡的影响相对较小，该纳米粒给药后明显降低了Bax并提高了Bcl-2蛋白的表达 (P < 0.05)；与游离二甲双胍和空白载体相比，该纳米粒的治疗导致炎症相关蛋白表达的最大抑制，包括TNF-α和IL-6 (P < 0.05)；此纳米粒对于 TGF-β与Collagen I 的表达，相较游离二甲双胍和空白载体降低效果最明显 (P < 0.05)。该纳米粒治疗细胞凋亡效果与游离二甲双胍类似，但是抗炎与抗纤维化作用显著优于游离二甲双胍。此纳米粒具有肾保护作用，通过降低UUO模型小鼠肾内的凋亡与炎症水平从而起到抑制纤维化的作用。本课题可为构建新型高效低毒的RTEC靶向药物和基因共传递系统提供理论指导和实验依据，并为糖尿病TIF的治疗提供新思路。