Damage Effects Of Lfliu Combined With Nanoparticles Loaded With Different Anti-tuberculosis Drugs On Mycobacterium Smegmatis And Biofilms

Tuberculosis(TB)is a worldwide infectious disease caused by the pathogen of Mycobacterium tuberculosis(MTB).MTB is an intracellular pathogen with a thick cell wall of poor permeability,which makes it difficult for anti-tuberculosis drugs to penetrate into the MTB organism,leading to the emergence of a large number of drug-resistant strains.The ease of biofilm formation further exacerbates the refractory nature of Mycobacterium tuberculosis.The aim of this study was to prepare nanoparticles of poly(lactic-co-glycolic acid)-polyethylene glycol(PLGA-PEG)containing Rifampicin,a first-line anti-tuberculosis drug,and levofloxacin,a second-line antituberculosis drug.The drug release was controlled by combining the radiation and acoustic flow effects of low frequency and low intensity ultrasound(LFLIU)to comparatively study the damage of synergistic therapy on Mycobacterium smegmatis(MS),the model strain of MTB,and its biofilm and its bactericidal mechanism.ObjectiveTo investigate the effect of LFLIU combined with different anti-tuberculosis drug nanoparticles on promoting the efficient entry of nanoparticles into MS bacteria and killing MS under the sonodynamic force,and its damage of LFLIU combined with drug-loaded nanoparticles on MS biofilm.Methods1.Preparation and characterization of rifampicin-loaded PLGA-PEG nanoparticles(RIF-NPs)and levofloxacin-loaded PLGA-PEG nanoparticles(LEV-NPs):Rifampicin-loaded PLGA-PEG nanoparticles(RIF-NPs)and levofloxacin-loaded PLGA-PEG nanoparticles(LEV-NPs)were prepared by double emulsification method.RIF-NPs)and levofloxacin-loaded PLGA-PEG nanoparticles(LEV-NPs)were prepared by double emulsification.The nanoparticle encapsulation rate and drug loading rate were measured by electron microscopy,and the safety of the nanoparticles was verified by hemolysis assay.2.Bactericidal effect of LFLIU combined with RIF-NPs and LEV-NPs on MS in vitro:LFLIU ultrasound equipment(42 k Hz fixed frequency)with continuously adjustable sound intensity of 0.60 W/cm~2was used.Sound intensity 0.45W/cm~2and irradiation time 15min were selected.Plate counting,XTT and LSCM were used to explore the effect of ultrasound combined with RIF-NPs and LEV-NPs on the activity of MS bacteria in vitro,and to explore the mechanism of LFLIU combined with two kinds of drug-loaded nanoparticles on MS.Reactive oxygen species kit staining was used to observe the production of reactive oxygen species(ROS)under laser confocal scanning microscope(LSCM).3.Bactericidal effect of LFLIU combined with drug-loaded nanoparticles on MS biofilm in vitro:the in vitro MS biofilm model was established by cell culture plate method.After MS biofilm was treated with LFLIU and drug-loaded nanoparticles,the biofilm activity was detected by crystal violet staining.In order to explore the damage effect of LFLIU combined with drug-loaded nanoparticles on MS biofilm in vitro,laser confocal microscopy was used to detect the survival/death of bacteria in the biofilm after ultrasound irradiation.Results1.Levofloxacin-loadednanoparticles(LEV-NPs)and rifampicin-loaded nanoparticles(RIF-NPs)with good physical properties were succ-essfully prepared:(1)they were spherical in shape with homogeneous morphology and good dispersion under scanning electron microscopy.(2)The particle size of LEV-NPs and RIF-NPs were 298.267±12.413 nm and 293.167±11.554 nm,respectively,with negative ZETA pote-ntial.The encapsulation rates were 66.021±2.363%and 65.740±1.094%,and the drug loading rates were 3.317±0.140%and 3.042±0.257%.2.The bactericidal efficacy of LFLIU combined with two kinds of drug-loaded nanoparticles on planktonic MS:(1)the bactericidal efficacy of MS suspension irradiated with LFLIU combined with LEV-NPs and RIF-NPs for 15 minutes was significantly lower than that of other experimental groups 24 hours after exposure to LFLIU combined with two kinds of drug-loaded nanoparticles and MS suspension,and the difference was statistically significant(P<0.05).(2)LSCM observation after STYO9/PI fluorescence staining:the number of living cells(green fluorescence)decreased significantly and the number of dead cells(red fluorescence)increased significantly after the treatment of LFLIU combined with LEV-NPs,indicating that LFLIU combined with LEV-NPs could significantly reduce the activity of bacteria.(3)The transmission electron microscope observation showed that there was no obvious damage to the bacterial cell wall in the control group and non-ultrasound group,but the bacterial cell wall was broken with incomplete bacterial structure,and MS was seriously damaged in the US+LEV-NPs group.(4)After MS biofilm was irradiated by LFLIU combined with LEV-NPs,the results of confocal microscope and flow cytometry showed that the production of reactive oxygen species in LFLIU combined with LEV-NPs group was significantly higher than that in other groups.3.LFLIU combined with drug-loaded nanoparticles has bactericidal effect on MS biofilm in vitro:(1)The mature biofilm model of MS in vitro was established by the in vitro cell culture plate method.(2)the in vitro mature biofilm model was irradiated by LFLIU combined with drug-loaded nanoparticles,and the results of crystal violet staining showed that the activity of MS biofilm decreased significantly(P<0.01).(3)the survival rate of bacteria in MS biofilm was observed by laser confocal scanning microscope,and it could be found that the survival rate of bacteria in biofilm after LFLIU combined with LEV-NPs irradiation was significantly lower than that of other experimental groups.Quantitative analysis of fluorescence intensity by Image J showed that the fluorescence intensity in LEV-NPs group was significantly higher than that in other groups(P<0.001).ConclusionsLFLIU combined with two drug-loaded nanoparticles has a signif icant synergistic antibacterial effect on MS,especially the combination of LEV with sonosensitizer properties and LFLIU can significantly r educe the survival rate of MS.Such sonodynamically triggered therap eutic drug-loaded nanoparticles show great potential and can provide a promising strategy for the treatment of drug-resistant tuberculosis.