【作者】 张丹丹；

【导师】 李孝红；

【作者基本信息】 西南交通大学 ， 生物化学与分子生物学， 2021， 硕士

【摘要】 幽门螺杆菌（Helicobacter pylori,Hp）感染难以治疗的原因包括:细菌耐药性、胃酸阻碍、胃粘液和生物被膜屏障等。常规抗生素在胃酸环境中会被强酸和酶破坏,导致药物活性降低甚至完全失活;且由于胃排空机制,导致药物在胃中的停留时间非常短暂。同时,粘液层的快速清除机制阻止药物或药物递送制剂到达上皮表面,并且粘液层还能通过对物质的空间位阻和相互作用力阻碍物质通过。抗生素在胃酸中停留时间短、胃酸的破坏以及粘液层的阻碍,导致到达Hp定植的胃粘膜层时的浓度不足。同时,致密的细胞外聚合物存在于生物被膜上,其表面限制了药物的进入。这就需要加大抗生素的用量,可能会导致细菌产生耐药性,因此迫切需要设计一种能保护药物免受胃酸破坏的同时,又能增强药物在胃粘液和生物被膜中渗透的制剂。微纳马达引起了许多领域的研究和应用兴趣,其自推进特性能穿透胃酸、胃粘液和生物被膜等障碍。本论文设计了一种以过氧化钙（Ca O₂）为动力源,聚多巴胺（Polydopamine,PDA）作为表面修饰,通过氢键相互作用将克拉霉素（Clarithromycin,CLR）包裹在PDA层形成载药纳米马达。最终,确定PDA包覆核的面积大约为一半的Janus纳米马达（J-Ca/Si@PDA-CLR）用作后续实验。扫描电子显微镜验证了纳米马达的Janus结构,其粒径大约为235 nm。利用MTT法测试了细胞毒性,实验结果表明该纳米马达对AGS细胞无明显的细胞毒性,体外药物释放实验结果显示CLR能有效地负载和释放。纳米马达在胃酸、胃粘液和生物被膜模拟液中均具有增强的运动效果,且能在长时间内维持动能。渗透结果显示该纳米马达具有出色的生物被膜穿透特性,能进入生物被膜的深层,并均匀分布在其中。体外抗生物被膜实验表明,该纳米马达能较好地破坏生物被膜的结构,治疗效果呈现出浓度依赖性,且生物被膜内的大多数细菌被有效杀死,细菌完整性遭到破坏。建立了Hp感染的小鼠模型,体内实验表明,此纳米马达可以携带药物穿过胃粘液,这种自主推动增强了药物在胃中的渗透和滞留,进而增加了其在生物被膜中的积累,从而更好地清除生物被膜深层的细菌。这项工作代表了在粘液相关的生物被膜感染中使用纳米马达增强渗透的治疗策略,为微纳马达的实际生物医学应用提供了新思路。更多还原

【Abstract】 Helicobacter pylori（Hp） infection is difficult to treat due to gastric acid,gastric mucus,biofilm barrier and drug resistance.Conventional antibiotics would be destroyed by strong acid and enzymes in the gastric environment,resulting in reduced drug activity or even complete inactivation,and the gastric emptying mechanism results in a very short residence time of the drug in the stomach.The rapid clearance mechanism of the mucus layer prevents drugs and drug delivery agents from reaching the epithelial surface.The mucus layer could hinder the passage of substances through steric hindrance and interaction forces.The short residence time and destruction in gastric acid,and the obstruction of the mucous layer of antibiotic result in insufficient concentration to reach the gastric mucosal layer where the Hp colonizes.At the same time,dense extracellular polymeric substances（EPS）of biofilm are present and drug access is restricted.Therefore,the dosage of antibiotics needs to be increased,which may lead to the development of bacterial resistance.So,it is urgent to design a preparation that can protect the drug from the destruction of gastric acid while enhancing the penetration of the drug in the gastric mucus and biofilm.Micro-nanomotors have attracted research interest in many fields,and the self-propelled properties can enhance penetration in gastric acid,gastric mucus,biofilm and other barriers.In the current study,Janus nanomotors were designed with Ca O₂ as the power source,and polydopamine（PDA）as the surface modification.And clarithromycin（CLR）is wrapped in the PDA layer through hydrogen bond interaction to form drug-loaded nanomotors.Eventually,about half of the cores surface coated with PDA to forming Janus nanomotors（J-Ca/Si@PDA-CLR）were identified for subsequent experiments.Scanning electron microscope showed the Janus structure of the nanomotors,and the size of J-Ca/Si@PDA-CLR was approximately 235nm.The experimental results showed that the nanomotor had no obvious cytotoxicity to AGS cells.And the in vitro drug release experiment results showed that CLR could be effectively loaded and released.The nanomotors had enhanced motion effects in gastric acid,gastric mucus and biofilms simulations,and maintained kinetic energy for a long time.The results of biofilm penetration showed that the nanomotors had excellent biofilm penetration characteristics,which could penetrate and distribute in the deep biofilm.In vitro anti-biofilm experiments showed that the structure of the biofilm was well destroyed by the nanomotors,and the therapeutic effect was concentration-dependent,and most bacteria in the biofilm were effectively killed with the integrity of the bacteria destroyed.The model of Hp infection was established in vivo,the results showed that the nanomotors could carry drugs through gastric mucus.The self-propulsion increased the penetration and retention in stomach,and resulted in the accumulation of drugs in biofilm,thereby better killed the bacteria in the deep biofilm.The nanomotors showed improved anti-biofilm effects in both in vitro and in vivo models with no significant toxicity.This work represents an enhenced penetration therapeutic strategy by nanomotors in mucus-associated biofilm infections and provides new ideas for practical biomedical applications of micro/nanomotors.更多还原