低频低强度超声联合载左氧氟沙星纳米粒对卡介苗生物被膜的损伤效果评价

Damage effect of low frequency and low intensity ultrasound combined with levofloxacin-loaded nanoparticles on Bacille Calmette-Guerin biofilm

目的探究低频低强度超声(low frequency and low intensity ultrasound,LFLIU)联合载左氧氟沙星(levofloxacin,LVFX)PLGA-PEG纳米粒对卡介苗(Bacille Calmette-Guerin,BCG)生物被膜的损伤效果。方法采用双乳化法制备载左氧氟沙星PLGA-PEG纳米粒(LVFX-NPs),检测其物理特性。运用XTT法及结晶紫染色分别检测超声联合LVFX-NPs作用后生物被膜的活性及生物量变化等,SYTO9/PI染色定性观察生物被膜损伤效果,流式细胞仪检测BCG内ROS含量变化,激光共聚焦显微镜观察生物被膜的渗透性改变。结果成功制备LVFX-NPs纳米粒,镜下观察呈均匀球形,大小均一,分散度良好,粒径(254.90±9.78)nm,电位(-17.83±0.74)mV,载药率及包封率分别为(3.50±0.51)%、(67.00±2.69)%。XTT及结晶紫染色结果显示,与对照组相比,超声联合LVFX-NPs能显著降低生物被膜活性,差异具有统计学意义(P<0.05)。流式细胞仪结果显示超声联合LVFX-NPs可产生大量ROS物质。激光共聚焦显微镜结果显示超声可以促进纳米粒进入生物被膜内部。结论成功制备的LVFX-NPs在超声的作用下,能渗透进入BCG生物被膜内部,并产生ROS,达到有效杀菌作用。

Objective To explore the damage effect of low frequency and low intensity ultrasound(LFLIU)combined with levofloxacin-loaded PLGA-PEG nanoparticles(LVFX-NPs)on Bacille Calmette-Guerin(BCG)biofilm.Methods Double-emulsification was used to prepare LVFX-NPs,and the physical features of the nanoparticles were detected.XTT assay and crystal violet staining were performed to detect the biofilm activity and biomass changes after treatment of ultrasound combined with LVFX-NPs.SYTO9/PI staining was carried out to qualitatively observe the biofilm damage.Flow cytometry was employed to detect the production of reactive oxygen species(ROS)in BCG,and laser confocal microscopy was adopted to observe the permeability changes of the biofilm.Results LVFX-NPs were successfully prepared,in spherical shape and uniform size,good dispersion,particle size of 254.90±9.78 nm,potential-17.83±0.74 mV,and with drug loading content and encapsulation efficiency of(3.50±0.51)% and(67.00±2.69)%,respectively.The biofilm activity was statistically lower in the ultrasound combined with LVFX-NPs group than the control group(P<0.05).Flow cytometry results showed that ultrasound combined with LVFX-NPs could produce a large amount of ROS.The results of laser confocal microscopy indicated that ultrasound could promote nanoparticles to enter the biofilm.Conclusion LVFX-NPs are successfully prepared,and can penetrate into the BCG biofilm under the action of ultrasound and produce ROS to achieve effective sterilization.