聚糖定位接枝氨基糖苷类抗生素治疗胞内菌感染的研究

The relapse and protracted course of intracellular bacterial infections frequently occur in clinical cases, in which low doses of antibiotics will readily lead to bacterial drug-resistance and high doses will increase their side effects,threatening health of animals and patients. Up to date, the aminoglycoside antibiotics are one of the most important drugs to treat bacterial infections, however, these antibiotics are not effective in treating infectious diseases caused by intracellular pathogens. Preliminary studies indicated that the polysaccharide grafting aminoglycoside antibiotics were effective in killing listeria monocytogenes in RAW 264.7 macrophages. The present proposal is to figure out the reliability, structure-activity relationship and underlying mechanism how these conjugates kill intracellular bacteria. In detail, we synthesize series of conjugates by conjugation of polycationic chitosan or CD44 ligand, hyaluronic acid with aminoglycoside antibiotics through reductive amination and ‘click’ chemistry respectively. We will test the bactericidal activity of these conjugates against four common pathogens and one drug-resistant organism in five different cell types. The structure-activity relationship is also explored. The underlying mechanism will be uncovered based on the entry of conjugates, the inducible free radical and the visualization of pathogens and conjugates in infected cells. Finally the bactericidal activity of conjugates will be evaluated in vivo by use of normal and immune-deficient mice infected with intracellular organisms. The strategy through conjugation between polysaccharides and aminoglycoside antibiotics will be useful to treat chronic intracellular infections with high efficacy, which will decrease the side effects of antibiotics and prevent bacterial resistance. In addition, this proposal will extend the application of functional polysaccharides in the field of infectious diseases.

细菌胞内感染通常会反复发作与迁延不愈，低剂量抗生素易诱发耐药性，而高剂量会增加毒副反应，严重影响动物和人类的健康。氨基糖苷类抗生素为临床重要的抗感染药，但对胞内感染的疗效差。前期研究表明聚糖接枝氨基糖苷类抗生素能有效地清除胞内菌。本申请拟系统地阐明该策略的可靠性、构效关系和分子机制：利用具有阳离子特性的壳聚糖和特定细胞受体CD44的透明质酸，通过还原胺化和点击化学反应合成系列聚糖-抗生素复合物，采用四种常见病原菌和一种耐药菌侵染五种不同细胞建立胞内感染模型，明确复合物的杀菌能力，阐明构效关系；通过弄清楚细胞摄入复合物的途径、复合物诱导的活泼自由基情况、复合物与细菌在细胞内的分布，来确定其分子机制；采用正常或免疫缺陷小鼠感染模型来明确复合物的体内疗效。本申请通过多糖接枝糖苷类抗生素来实现胞内菌感染的有效治疗，对降低药物副反应及阻止耐药性的产生、拓展功能性聚糖应用于细菌感染领域具有重要意义。

细菌胞内感染通常会反复发作与迁延不愈，低剂量抗生素易诱发耐药性，而高剂量会增加毒副反应，严重影响动物和人类的健康。氨基糖苷类抗生素为临床重要的抗感染药，但对胞内感染的疗效差。前期研究表明聚糖接枝氨基糖苷类抗生素能有效地清除胞内菌。本项目通过还原胺化反应分别合成了系列壳聚糖-链霉素复合物、透明质酸-链霉素复合物，利用点击化学合成了透明质酸-阿米卡星复合物；上述复合物能有效清除多种宿主细胞（巨噬细胞、上皮细胞、内皮细胞）内的多种病原菌（单增李斯特菌、金色葡萄球菌、鼠伤寒沙门氏菌、铜绿假单胞菌），阐明了其构效关系：分子机制研究表明上述复合物分别利用壳聚糖的阳离子特性和透明质酸作为特定细胞受体CD44的配体，使得负载的抗生素更容易进入宿主细胞；利用正常或免疫缺陷小鼠感染模型证明了这些复合物的体内疗效。在该项目的资助下，以通讯作者在糖生物学领域top期刊Glycobiology发表文章2篇，在糖领域期刊Carbohydrate polymers等专业期刊发表文章8篇,获批专利2项，培养了3名博士、4名硕士。参加国内学术交流7次，并做了大会学术报告。该项目通过多糖接枝糖苷类抗生素来实现胞内菌感染的有效治疗，对降低药物副反应及阻止耐药性的产生、拓展功能性聚糖应用于细菌感染领域具有重要意义。