基因口服高效递送系统及其作用机制

Despite the easiness of oral administration, it is a challenging task for genes to remain stable in the gastrointestinal (GI) tract and arrive at the target tissues and cells owing to multiple obstacles including dramatic pH change in the GI tract, abundant nuclease, and mucous epithelia, which called for safe and efficient gene delivery vehicles. Consequently, comprehensive investigations on the rational design and mechanisms of gene delivery systems are significant for improved in vivo therapeutic efficacy after oral administration. Based on our previous studies, in the current project, chitosan-based nanoparticles with simultaneously controllable particle size, potential, hydrophobicity/hydrophilicity, and mucoadhesive property were designed, screened, and synthesized as oral gene delivery vectors. The impact and mechanisms of physiochemical properties of nanoparticles exerted on the encapsulation efficiency toward plasmid DNA (pDNA), release profile, gene stability, overcoming multiple delivery barriers, intestinal adsorption, and efficiently arriving at target tissues and cells were investigated. These studies would favor revealing how the physiochemical properties of nanocarriers influence the efficiency of oral gene delivery. Optimal delivery vehicle, which was rationally designed and synthesized according to the resultant mechanisms, was employed to encapsulate Survivin shRNA expressing pDNA (shSur-pDNA). In vitro and in vivo transfection efficiency and in vivo therapeutic efficacy were studied for further verification of the mechanisms. These results would shed light on the rational design of oral gene delivery systems with maximized therapeutic efficacy.

口服给药安全方便，但基因口服给药面临胃肠道剧变的pH、多种核酸酶、黏膜上皮等多重屏障，使其稳定通过胃肠道并高效递送至靶组织及靶细胞是极具挑战性的难点，必须借助安全有效的递送载体。故需对基因口服高效递送载体的合理设计及作用机制进行系统深入研究。根据我们已有的工作基础，设计、筛选和制备可同时调节纳米粒粒径、表面电荷、亲疏水性和黏膜黏附性的壳聚糖类聚合物纳米粒作为递送载体。系统研究载体理化性质对其包载基因（pDNA）及释放、保持基因在递送过程中的稳定性、克服多重递送屏障、肠道吸收并高效转运至靶组织和靶细胞的影响及机制。全面解析纳米递送载体的理化性质对基因口服高效递送的影响规律。根据机理研究结果合理设计基因口服递送载体，包载Survivin shRNA表达pDNA（shSur-pDNA），进行体内外转染效果与治疗功效研究及机制验证。为基因口服高效递送系统的合理设计提供理论和方法指导。

基因口服须借助安全有效的递送载体，以突破胃肠道剧变的pH、多种核酸酶、黏膜上皮等多重屏障，阐明其作用机制为基因口服高效递送系统合理设计提供理论和方法指导。项目制备了壳聚糖季铵盐(TMC)模型纳米粒并进行多种功能化修饰，研究并验证其主要性质对载基因纳米粒口服后突破胃肠道多重屏障递送至作用靶点并发挥治疗功效的影响和机制。结果表明，较大分子量和较高巯基化度及适中季铵化度和表面正电荷的巯基化壳聚糖季铵盐(TC)纳米粒可有效包载及细胞内GSH响应性释放shSur-pDNA，胃肠道及血液中稳定性好，细胞摄取、细胞转染、肠道转运及吸收等效率高，H22 肝癌模型小鼠口服给药后抗肿瘤作用强，抑瘤率达91.5%。制备甘氨胆酸和甘露糖双修饰的TMC(MTG)，包载Sirpα siRNA和肿瘤抗原MUC1表达pDNA，结果表明，纳米粒胃肠道及血液中稳定性均较好，Caco-2细胞摄取及转运效率高，纳米粒入胞后主要经内质网、高尔基体运输，可避开溶酶体途径，小肠上皮转运显著提高且小肠转运后依然保持纳米粒完整性，Raw264.7中以小窝蛋白介导的内吞为主，24 h和48 h基因沉默效率分别达81.7%和89.5%，4T1原位乳腺癌小鼠模型口服给药抑瘤率达92.1%，表明共载Sirpα siRNA与MUC1 pDNA 的MTG口服给药可协同提高抗肿瘤功效。制备共载阿托伐他汀(AVS)和pAnti-miR-33的半乳糖修饰的TMC纳米粒(GTANPs/pAnti-miR-33)，研究其口服抗动脉粥样硬化功效及机制，结果表明，纳米粒在胃肠道及血液中稳定，在Caco-2细胞、离体小肠及C57BL/6小鼠口服给药中，可显著提高pDNA及AVS口服给药后肠道吸收和转运，促进Raw 264.7细胞摄取，有效递送pAnti-miR-33至巨噬细胞，上调ABCA1的表达，促进巨噬细胞胆固醇外流，减少胞内胆固醇积累，促进巨噬细胞表达IL-10，协同AVS和pAnti-miR-33抑制巨噬细胞MCP-1和TNF-α的表达及巨噬细胞向M1型极化，促进巨噬细胞M2型极化，动脉粥样硬化小鼠口服给药后，纳米粒可将pAnti-miR-33递送至肝脏及动脉粥样硬化斑块处并转录anti-miR-33，减少腹部及肠系膜脂肪堆积及脂肪细胞大小，减小动脉粥样斑块面积，显著降低血液中TCh及LDL-C含量，增加HDL-C含量，增强抗炎作用。