防治动物细菌性感染的壳聚糖/溶菌酶纳米制剂制备及抑菌机理研究

As the increase of bacterial infection diseases in livestock and the risk of long-term abuse of antibiotics in the current culture environment, the efficient broad-spectrum drugs alternative to antibiotics are in great demand. Based on the cationic and amphiphilic structure of antibacterial peptide, this project designed the antibacterial nanoparticles with modified chitosan and lysozyme. It is supposed that the modified chitosan as the carrier could penetrate the outer membrane of gram-negative bacteria (G-) and release lysozyme at the cell wall, which combined the cell-penetrating ability of chitosan and the specific cell wall degrading ability of lysozyme, cooperatively enhanced G- inhibition. Herein, guanidino modification could strengthen the cell membrane penetration of chitosan; polyethylene glycol (PEG) and histidine modification enabled the formation of the amphiphilic nanoparticles under the physiological environment (pH 7.4), conjugating lysozyme with hydrophobic force, and then delivery to the target site. The relationship between the molecular weight / the modification group ratio and the antibacterial properties / cytotoxicity of chitosan is conducted. The mechanism of synergistic antibacterial effect with modified chitosan and lysozyme is also studied. The dosage and safety of the nanoparticles administration to the animals are preliminary explored, as the foundation for the development of novel antibacterial animal drugs, which has important scientific value and social significance.

当前养殖环境下，畜禽细菌感染性疾病的频发及抗生素长期滥用的风险，使得高效广谱的抗生素替代药物研究迫在眉睫。本项目参考抗菌肽活性基团阳离子性和两亲性的结构特点，设计了改性壳聚糖包裹溶菌酶的纳米抑菌制剂，拟通过改性壳聚糖为载体穿透革兰氏阴性菌（G-）的外膜，递送溶菌酶至细胞壁释放，协同发挥改性壳聚糖的细胞膜作用能力及溶菌酶的专一细胞壁分解能力，增强对G-的抑制作用。其中，胍基改性可加强壳聚糖的G-细胞膜穿透能力；聚乙二醇与组氨酸改性使得生理环境（pH 7.4）下两亲性纳米胶束形成，以疏水力结合溶菌酶至胶束内部，递送到靶部位。本项目研究壳聚糖的分子量、改性基团比例与其抑菌性能、细胞毒性之间的关系；研究改性壳聚糖结构对细菌细胞膜的作用机制，探索其与溶菌酶的协同抑菌机理；初步探索该纳米制剂在动物体内的给药剂量和安全性，为开发替代抗生素作用的新型兽用抗菌药物奠定基础，具有重要的科学意义和社会价值。

当前养殖环境下，畜禽细菌感染性疾病的频发及抗生素长期滥用的风险，使得高效广谱的抗生素替代药物研究迫在眉睫。本项目采用双氧水法降解得到了系列分子量在8.4-52.2kDa之间，脱乙酰度>85%的壳聚糖。然后采用精氨酸法改性壳聚糖，得到了胍基接枝率在17.8%-37.2%的胍基化壳聚糖。以硫酸葡聚糖为交联剂，制备了系列包裹溶菌酶的胍基化壳聚糖微球，水合粒径在100-250nm之间，溶菌酶的载药率在16.73%-38.91%之间。微球的体外释放时间可达7天以上，其中1天以内为突释过程，1-7天为缓释阶段。分别以革兰氏阳性菌金黄色葡萄球菌（S.aureus）和革兰氏阴性菌大肠杆菌（E.coli）为细菌模型，本项目制备的胍基化壳聚糖/溶菌酶纳米微球表现出了有效的抑菌作用，24h抑菌效率均在50%以上，最高可达80%左右。项目通过该纳米制剂对E.coli细胞壁及细胞膜的作用研究，总结其抑菌机理为: 胍基改性的中低分子量壳聚糖可穿透革兰氏阴性菌（G-）的外膜，递送溶菌酶至细胞壁释放，溶菌酶分解细菌细胞壁，引起胞内物质外泄，抑制细菌增殖。该纳米微球的细胞相容性良好，目前正在探索其作为生猪生产过程中饲用添加剂的应用效果，后续可进一步研究其在畜禽生产过程中部分替代抗生素的作用，以期减少抗生素滥用导致的耐药性及食品安全问题。