靶向降解、清除幽门螺杆菌菌膜 (biofilm)的多功能脂质-聚合物杂化纳米粒的制备、作用评价及相关机制研究

Recent research reveals that the biofilm of Helicobacter pylori (H.pylori) principally accounts for the drug resistance, low clearance of H.pylori and relapse of gastrointestinal related disease. Biofilm is a community of bacteria embedded in extracellular polymeric substance (EPS), which is mainly composed of polysaccharides, proteins, lipids and DNA. The growth rates of the biofilm-bacteria are lower than the planktonic counterparts and it's quite difficult for antibiotics to penetrate into the biofilm due to the compact structure. Previously, our group has successfully synthesized polysaccharide sulfates featured by amazing ability off inhibiting adhesion of H.pylori. Therefore, we attempt to design a kind of mutlifunctional lipid-polymer hybrid nanoparticles for targeted disruption and eradication of H.pylori biofilm. The inner cores of the hybrid nanoparticles are aforementioned polysaccharide sulfate nanoparticles, loading N-acetylcysteine (NAC, a biofilm-disrupting agent) and amoxicillin (a BCSIII antibiotic). The outer layer of the hybrid nanoparticles consists of rhamnolipid, which will interact with the polysaccharides of the biofilm, conferring the hybrid nanoparticles an active targeting effect. The interaction between the rhamnolipid and the biofilm will result in peptization, destroying the out layer of the hybrid nanoparticles. The exposed polysaccharide sulfate nanoparticles will start to sustainedly release NAC and amoxicillin. NAC will disrupt the configuration of the biofilm, favoring the penetration of amoxicillin into the biofilm. Meanwhile, the free-floating bacteria from the disrupted biofilm will be adhered by the polysaccharide sulfate nanoparticles and/or killed by amoxicillin. In sum, our project is to engineer mutlifunctional lipid-polymer hybrid nanoparticles for targeted disruption and eradication of H. pylori biofilm, hopefully offering a new therapeutic approach for the treatment of H. pylori biofilm-related diseases.

幽门螺杆菌（H.pylori）形成的菌膜（biofilm）是H. pylori耐药、低清除率、高复发率的原因。本课题组合成了具H. pylori粘附抑制作用的多糖硫酸酯（见工作基础）。本项目拟在已有工作基础上，构建靶向降解、清除H. pylori biofilm的多功能脂质-聚合物杂化纳米粒。果胶硫酸酯纳米粒组成内核并载药，外层包被鼠李糖脂，形成脂质-聚合物杂化纳米粒。纳米粒表面糖结构和biofilm表面聚糖发生相互作用使纳米粒和biofilm发生融合（靶向）。杂化纳米粒内核果胶硫酸酯暴露，biofilm解体（降解）。果胶硫酸酯遇水溶胀，释放药物N-乙酰半胱氨酸（NAC）和阿莫西林。NAC进一步降解biofilm（双重降解），H. pylori从破裂的biofilm中游离，被内核果糖硫酸酯粘附及阿莫西林杀灭。并进行相关体内外评价及机制研究，为临床治疗H. pylori导致的疾病提供新的思路

幽门螺杆菌（Helicobacter pylori, H. pylori）是一种能在人消化道定植的革兰氏阴性菌，可引发急慢性胃炎、胃溃疡甚至胃癌等消化道疾病。H. pylori感染导致的相关疾病具有低清除率、高复发率的特点，其耐药性的产生与菌膜（biofilm）的形成密切相关。H. pylori形成biofilm后，biofilm表面的胞外多聚物可阻碍抗菌药物渗透，膜内细菌代谢减慢，表型改变进一步降低了细菌对抗菌药物的敏感性。本课题组首先从生物表面活性剂鼠李糖脂、表面活性剂十二烷基硫酸钠、粘液溶解剂N-乙酰半胱氨酸中筛选出鼠李糖脂能有效清除幽门螺杆菌、大肠杆菌、金黄色葡萄球菌、铜绿假单胞菌、变异链球菌的biofilm。同时，我们发现，与临床治疗H. pylori经典三联用药（阿莫西林、克拉霉素、质子泵抑制剂）进行比较，鼠李糖脂能增强H. pylori biofilm对抗生素的敏感性。于是，我们采用鼠李糖脂与磷脂混合脂质作为外壳，果胶硫酸酯聚合物材料作为内核，形成特殊的壳-核结构，并载药阿莫西林，构建具有清除biofilm、抑制游离菌黏附定植的脂质聚合物纳米粒。进一步的研究发现，覆盖于胃粘膜上皮细胞表面的黏液层阻碍了抗菌药物到达H. pylori biofilm的定植部位，于是我们设计了一种能穿透黏液层同时抑制biofilm形成并清除成熟biofilm的纳米递药系统，以壳聚糖纳米粒为内核，鼠李糖脂/磷脂、胆固醇为脂质外层，包载抗H. pylori感染一线药物克拉霉素，构建脂质聚合物纳米粒，并在表面修饰亲水性聚乙二醇，达到显著提高H. pylori清除率的目的。我们采取完全不同于传统抗菌策略的、能明显提高H. pylori清除率及抗菌素敏感性的方案，为H. pylori相关疾病的临床治疗提供新的思路。