超声介导缓释型载药纳米粒促进抗结核药物增效杀菌的实验研究  

Experimental study on ultrasound-mediated sustained-release drug-loaded nanoparticles to promote synergistic bactericidal effect of anti-tuberculosis drugs

作　　者：张志飞 胡璨 杜永洪[1] 杨敏 ZHANG Zhifei;HU Can;DU Yonghong;YANG Min(College of Biomedical Engineering,Chongqing Medical University,Chongqing 400016,China)

机构地区：[1]重庆医科大学生物医学工程学院超声医学工程国家重点实验室,重庆市400016

出　　处：《临床超声医学杂志》2022年第10期721-726,共6页Journal of Clinical Ultrasound in Medicine

基　　金：重庆市研究生科研创新项目(CYS21240);贵州省卫生健康委科学技术基金项目(gzwjkj2020-1-042)。

摘　　要：目的探讨超声介导缓释型载药聚乳酸-羟基乙酸(PLGA)共聚物纳米粒对减毒牛结核分枝杆菌的体内外增效杀菌作用。方法采用双乳化法制备PLGA载左氧氟沙星(LEV)缓释纳米粒(LEV-NPs),并检测其物理特性和药物释放情况。将卡介苗(BCG)菌液随机分为LEV组(仅加入LEV)、超声联合LEV组(加入LEV后立即经超声辐照)、超声联合LEV-NPs组(加入LEV-NPs后立即经超声辐照)及对照组(加入等量PBS缓冲液),实验处理24 h后检测各组活菌落数,观察各组BCG活死菌变化及其表面结构。建立SD大鼠皮下BCG结核肉芽肿模型,将20只大鼠分为LEV组(仅皮下注射LEV)、超声联合LEV组(皮下注射LEV后立即经超声辐照)、超声联合LEV-NPs组(皮下注射LEV-NPs后立即经超声辐照)及对照组(皮下注射等量生理盐水),于治疗后第3、7、14天分别测量各组大鼠皮下BCG结核肉芽肿体积,计算并比较各组治疗效果。结果制备的LEV-NPs呈大小均一的球形,分散性良好,粒径为(282.42±3.55)nm,Zeta电位为-(20.40±0.63)mV,载药率、包封率分别为6.21%、65.30%;LEV-NPs在24、48、72 h自然条件下LEV累计释放率分别为31.2%、45.7%、46.9%。体外实验显示,超声联合LEV-NPs组细菌活性下降最明显,菌落存活率约26%,与其余各组比较差异均有统计学意义(均P<0.01);激光共聚焦显微镜下示超声联合LEV-NPs组可见视野下呈明亮的红色荧光,以死菌为主,仅见少量活菌落;场发射扫描电镜下示超声联合LEV-NPs组细菌数量明显减少,菌体伸展、断裂,细菌损伤情况最严重。体内实验显示,超声联合LEV-NPs组大鼠皮下BCG结核肉芽肿体积较其余各组明显减小(均P<0.05)。结论超声介导LEV-NPs可显著提高LEV对减毒牛结核分枝杆菌的体内外杀菌效果,具有协同增效作用。Objective To investigate the synergistic bactericidal effect of ultrasound-mediated sustained-release drug-loaded poly lactic-co-glycolic acid(PLGA)copolymer nanoparticles on attenuated bovine mycobacterium tuberculosis in vitro and in vivo.Methods PLGA-loaded levofloxacin(LEV)sustained-release nanoparticles(LEV-NPs)were prepared by double emulsification method,and their physical characteristics and drug release were detected.Bacillus Calmette-Guerin(BCG)bacterial solution was randomly divided into LEV group(only added LEV),ultrasound combined with LEV group(added LEV and irradiated by ultrasound immediately),ultrasound combined with LEV-NPs group(added LEV-NPs and irradiated by ultrasound immediately),and control group(added the same dose of PBS buffer).After 24 h of experimental treatment,the number of viable colonies in each group was detected.The changes of live and dead bacteria and the surface structure of BCG in each group were observed.A model of subcutaneous BCG tuberculosis granuloma was established in SD rats,20 rats were divided into LEV group(only subcutaneous injection of LEV),ultrasound combined with LEV group(subcutaneous injection of LEV and irradiated by ultrasound immediately),ultrasound combined with LEV-NPs group(subcutaneous injection of LEV-NPs and irradiated by ultrasound immediately)and control group(subcutaneous injection of the same dose of normal saline).The volume of subcutaneous BCG tuberculosis granuloma was measured on the 3rd,7th,and 14th day after treatment,and the therapeutic effect of each group was calculated and compared.Results LEV-NPs were spherical with uniform size and good dispersion,with a particle size of(282.42±3.55)nm and a Zeta potential of-(20.4±0.63)mV.The drug loading rate and encapsulation rate were 6.21%and 65.3%,respectively.The cumulative drug release rate of LEV-NPs at 24,48 and 72 h were 31.2%,45.7%and 46.90%,respectively.In vitro experiments showed that the bacterial activity in the ultrasound combined with LEV-NPs group decreased most significantly,and

关 键 词：超声辐照 聚乳酸-羟基乙酸共聚物纳米粒 左氧氟沙星 增效抗菌 减毒牛结核分枝杆菌 

分 类 号：R445.1[医药卫生—影像医学与核医学]