山嵛酸固体脂质纳米对胞内金黄色葡萄球菌的靶向及调控机制研究

The applicant previous work demonstrated that docosanoic acid solid lipid nanoparticles(SLN) could enhance enrofloxacin concentration and residence time in cells, providing a new breakthrough to solve the problem of cow staphylococcus aureus mastitis due to its intracellular residence. The intracellular antimicrobial efficacy depend on more target selectivity at target subcellular compartments than intracellular delivery efficacy. In this work, the following reasearch will be done by using murine macrophages RAW264.7 and murine mammary epithelial cells as model cells. First, the cellular uptake, trafficking, drug release and exocytosis kinetics,and endocytosis, trafficking and exocytosis pathways of enrofloxacin-loaded docosanoic acid SLN will be investigated using fluorescence labeling, confocal microscopy, förster resonance energy transfer(FRET), specific inhibition intervention of endocytosis, endocellular and efflux transporters and others. Secondly, the influence of particle size, zeta potential, surface hydrophobicity and functional modification on the intracellular dynamic process of enrofloxacin-loaded docosanoic acid SLN will be further researched. Given the above experiments, the efficiency, speed, biological and regulation mechanisms of enrofloxacin-loaded docosanoic acid SLN for subcellular delivery will be clarified and the optimum docosanoic acid SLN for targeting delivery to the intracellular niches where staphylococcus aureus reside were selected. The co-localization between optimum SLN and staphylococcus aureus were determined using fluorescence labeling and confocal microscopy. The efficacy of the SLN against intracellular staphylococcus aureus were studied using intracelular infection and mice experimental mastitis.This study will help to achieve an efficient treatment for staphylococcus aureus mastitis and provide a new way for overcoming the treatment difficulty of cow staphylococcus aureus mastitis.

申请人前期构建的山嵛酸固体脂质纳米可提高恩诺沙星在胞内的浓度和停留时间，为解决金黄色葡萄球菌胞内寄生导致的奶牛乳腺炎治疗难题提供了契机。纳米药效学不仅取决于胞内传递效率，更与其在胞内的靶向选择性密切相关。本研究拟选用小鼠巨噬细胞RAW264.7和奶牛乳腺上皮细胞，通过转运特异性抑制剂、荧光标记、共聚焦和荧光共振能量转移等技术研究山嵛酸纳米入胞、分布、释药和消除等胞内转运动力学以及入胞方式、转运途径和外排方式等胞内转运机制，揭示其胞内转运效率和生物学机制；研究粒径、电荷、亲疏水性和功能修饰对其胞内转运动力学和转运机制的影响，阐明其胞内靶向转运的调控方式和机制。在此基础上，优化出对胞内金葡菌靶向传递的山嵛酸固体脂质纳米，利用荧光标记和共聚焦验证其与金葡菌的胞内共定位，建立细胞感染和小鼠乳腺炎模型进行药效学评价，实现金葡菌胞内感染的靶向和高效治疗，为解决奶牛金葡菌乳腺炎治疗难题提供新模式及理论。

为解决奶牛金黄色葡萄球菌乳腺炎的治疗难题提供新模式，本项目实现了恩诺沙星-山嵛酸固体脂质纳米的可控制备。山嵛酸固体脂质纳米荷载的恩诺沙星与RAW264.7细胞孵育0.5h时在胞内的浓度达到平衡，而卟啉修饰的山嵛酸固体脂质纳米荷载的恩诺沙星与细胞孵育3h时在胞内的浓度达到平衡。粒径、电荷和功能修饰对固体脂质纳米的胞内转运动力学有显著的影响。与细胞孵育0.5h时，300nm固体脂质纳米荷载的恩诺沙星在胞内的浓度为0.486μg/mg，而800nm固体脂质纳米荷载的恩诺沙星在胞内的浓度达1.608μg/mg。当二甲基双十八烷基氯化铵修饰的固体脂质纳米电荷从-8.1增加-24.9 mv时，胞内恩诺沙星的浓度从0.960μg/mg增加至1.147μg/mg。胞内恩诺沙星的浓度不受电荷正负的影响，与电荷绝对值的高低有关。300nm固体脂质纳米经卟啉修饰后，胞内恩诺沙星的平衡浓度达0.865μg/mg，是未修饰纳米粒摄入效率的1.80倍。.入胞机制研究表明，山嵛酸固体脂质纳米通过能量依赖的方式被细胞摄取，受理化特征的调控。300nm和500nm的粒子主要通过小窝蛋白介导的内吞和巨胞饮进入RAW264.7细胞；而800nm山嵛酸固体脂质纳米主要通过网格和小窝蛋白介导的内吞以及巨胞饮摄入细胞。负电荷的山嵛酸固体脂质纳米相比于正电荷的纳米更多的利用窝蛋白介导的内吞进入细胞。卟啉修饰的山嵛酸固体脂质纳米主要利用网格蛋白介导的内吞和巨胞饮摄入细胞。胞内分布研究表明，与细胞孵育10min后，纳米粒子开始与溶酶体共定位，20min后与溶酶体共定位接近峰值，40min后无共定位。未功能化修饰的纳米对金黄色葡萄菌无明显的靶向作用，而卟啉修饰的纳米对金黄色葡萄球菌具有显著的靶向性。卟啉修饰的纳米优先向胞内寄生的金黄色葡萄球菌精准递呈，随后逐渐在胞质内蓄积。卟啉修饰的山嵛酸固体脂质纳米经静脉注射给小鼠2h后开始在感染部位聚集，持续至36h。小鼠乳腺炎感染治疗试验表明山嵛酸固体脂质纳米治疗效果显著强于恩诺沙星溶液。.综上所述，本研究为胞内金黄色葡萄球菌靶向的山嵛酸固体脂质纳米的设计提供指导，将为解决金黄色葡萄球菌感染的治疗难题提供新的策略。