固体脂质纳米抗菌药物抗胞内沙门氏菌感染及机理研究

The treatment of Salmonella infections in veterinary clinic is very difficulty since the facultative intracellular infection. The applicant previous work demonstrated that solid lipid nanoparticles(SLN) could significantly enhance the antibiotics concentration and residence time in cellular, providing a new breakthrough to solve the problem of intracellular Salmonella infections. While up to now, there are no available report about the efficacy and mechanism of antibiotics-loaded SLN against intracellular Salmonella infections. In this work,the following reasearch will be done using the previous prepared enrofloxacin-loaded SLN and murine macrophages RAW264.7 as model nanoparticle delivery system and cell line. First, the adhesion, endocytosis pathways and pharmacokinetics of nanoaprticles will be investigated using fluorescence labeling， confocal microscopy, flow cytometry,inhibition intervention of endocytosis and other technologies.Second, the pharmacokinetics and efficacy of enrofloxacin-loaded SLN will be studied in RAW264.7 macrophages infected with Salmonella. Finally, the effect of lipid matrix, particle size and zeta potential of nanoparticles on the pharmacokinetics and pharmacodynamics of enrofloxacin-loaded and unloaded SLN will be further studied. Given the above experiments,the pharmacodynamics, structure-activity relationships and biological mechanism of enrofloxacin-loaded SLN against intracellular Salmonella infections will be elucidated. The strategies in the design of SLN were explored in this work for solving the difficulty in treatment of intracellular Salmonella infections, meanwhile providing new ideas and theoretical basis for treatment of other intracellular infection diseases.

沙门氏菌的兼性细胞内感染导致临床治疗困难，申请人研究表明固体脂质纳米能显著提高抗菌药物在胞内的浓度和停留时间，为解决这一难题提供了新的突破口。但是固体脂质纳米抗菌药物抗胞内沙门氏菌感染的药效学及其机理研究还未见报道。本项目拟以前期研制的恩诺沙星-固体脂质纳米为基础，选用小鼠巨噬细胞RAW264.7，通过荧光标记、共聚焦、流式细胞术和内吞特异性抑制干预等技术，研究固体脂质纳米粒的细胞粘附性，内吞途径和胞内动力学过程以及脂质基质、粒径和电荷对其动力学的影响；研究恩诺沙星-固体脂质纳米的胞内动力学和对细胞内沙门氏菌感染的药效学；研究脂质基质、粒径和电荷对恩诺沙星-固体脂质纳米的胞内动力学和药效学的影响。通过以上研究，揭示恩诺沙星-固体脂质纳米抗胞内沙门氏菌感染的药效学、构效关系和生物学机制，建立基于固体脂质纳米技术解决沙门氏菌胞内感染治疗难题的新模式和理论，也为其它胞内菌感染性疾病的治疗提供新的策略。

本项目通过处方筛选和制备工艺的优化实现了四种恩诺沙星-脂肪酸固体脂质纳的可控制备，在此基础上研究了其胞内传递性能及其影响因素。研究了细胞摄取优化筛选的恩诺沙星-固体脂质纳米的胞内沙门氏菌感染治疗效率和给猪体不同给药途径的药动学，以探索其解决胞内感染的可行性和优效性。试验结果表明，研制的豆蔻酸、棕榈酸、脂肪酸和山嵛酸等四种脂肪酸固体脂质纳米在不同的孵育时间点使荷载的恩诺沙星在RAW.264.7细胞内的浓度提高4.34-5.05，4.47-5.25，10.27-12.71和27.06-37.71倍，并延长恩诺沙星在细胞内停留时间。山嵛酸固体脂质纳米向细胞内传递恩诺沙星的效率著强于其它三种脂肪酸固体脂质纳米，电荷和粒径对其摄入RAW.264.7细胞内的恩诺沙星具有显著的影响。当山嵛酸固体脂质纳米的粒径从150nm增加到605nm，胞内恩诺沙星的浓度在孵育0.5h时从0.336增加到1.147μg/mg蛋白；当山嵛酸固体脂质纳米电荷从-8.1增加 -24.9 mv，胞内浓度从0.960 增长到1.147μg/mg细胞蛋白。山嵛酸固体脂质纳米胞内传递恩诺沙星的效率受粒径的影响比电荷更加显著，且与表面电荷的正负无关。利用沙门氏菌CVCC541建立的细胞感染模型治疗试验表明，在与感染细胞孵育期间，0.24μg/mL山嵛酸固体脂质纳米包装的恩诺沙星的抑菌作用显著强于0.6μg/mL恩诺沙星溶液；当孵育至48h时，0.06 μg/mL山嵛酸固体脂质纳米包装的恩诺沙星的抑菌效果强于0.6μg/mL恩诺沙星溶液。0.6μg/mL山嵛酸固体脂质纳米包装的恩诺沙星能到胞内杀菌浓度，而0.6μg/mL恩诺沙星溶液却不能抑制住胞内细菌生长。优化的恩诺沙星山嵛酸固体脂质纳米给猪肌肉注射(2.5mg/kg)的生物利用度提高到163.62%，在120h后能检测到药物，而对照制剂拜有利在48h已低于检测限。与可溶性粉相比，猪灌服(5mg/kg)山嵛酸固体脂质纳米后的平均停留时间(MRT)和消除半衰期(T1/2ke)分别从12.33和5.58h延长到35.15和12.05h，生物利用度提高到了238.13%。综上所述，本课题研制的恩诺沙星-山嵛酸固体脂质纳米能显著提高细胞内恩诺沙星的浓度、停留时间和抗菌活性，在猪体内具有显著缓释和促进吸收的作用，为解决沙门氏菌等胞内菌感染的治疗难题提供新的治疗策略。