| BackgroundTuberculosis(TB)is the second largest infectious disease in the world caused by Mycobacterium tuberculosis(MTB)infection.China is a highly endemic country of tuberculosis,and the number of new cases is far higher than the international average level.The prevention and control of tuberculosis is facing severe challenges[1-2].The cell wall of MTB is very thick,contains a large number of lipids,can prevent the drug from entering the body through the cell wall space,causing a variety of antibiotics to be resistant to its natural resistance [3-4].This helpful to explain why the clinical effect is not significant,the treatment cycle is long,and the number of drug-resistant strains is increasing.Poly(lactic acid)hydroxyacetic acid [poly(Plactic-co-glycolic acid),PLGA] is a polymer made from lactic acid and hydroxy acetic acid.It has many characteristics,such as biocompatibility,biodegradability,and so on.It has been widely used in the biomedical field [5],and the ultrasoniccombined drug microbubbles have been demonstrated in the anti infection and so on.Mycobacteria smegmatis(MS)is similar to MTB in genetic characteristics and has no biological hazards.Therefore,MS is chosen as the research object.The levofloxacin was slected,was prepared PLGA nanoparticles carrying levofloxacin,and irradiated MS suspension in vitro with low frequency and low intensity ultrasound(Low frequency and low intensity ultrasound,LFLIU)to explore its synergistic effect.It is expected to lay a potential for the study of tuberculosis with PLGA nanoparticles for the treatment of tuberculosis.Objective The synergistic bactericidal effect of LFLIU combined with levofloxacin PLGA nanoparticles on MS was explored in order to provide a new therapeutic method for the treatment of anti tuberculosis in clinic.Method1.The preparation and characterization of 1.loads of levofloxacin PLGA nanoparticles: 3 kinds of PLGA nanoparticles were prepared by double emulsification.That is,levofloxacin nanoparticles,empty nanoparticles and green fluorescent glucan nanoparticles.(1)the morphology of nanoparticles was observed under light and electron microscopy.The particle size and potential of nanoparticles were detected by Malvin laser particle size analyzer;(2)laser confocal microscopy was used to observe the fluorescence package of green fluorescent glucan nanoparticles;(3)spectrophotometer was used to detect the automatic release efficiency of the drug in the 12 h nanoparticles loaded with levofloxacin and the release efficiency of the drug after sound irradiation;(4)The drug release efficiency of levofloxacin PLGA nanoparticles under 0.15W/cm2 and 0.3W/cm2 ultrasound dose was detected by spectrophotometer.2.Synergistic germicidal efficacy of MS with different ultrasonic irradiation doses and different concentrations of drugs.A low frequency and low-intensity ultrasonic device with frequency 42 KHz,sound intensity 0.15-0.6W/cm2 and 1.5cm transducer diameter is selected.(1)to determine the minimum inhibitory concentration of levofloxacin against Mycobacterium foul,and to determine the concentration of experimental drugs for reference.(2)the MS suspension 5min and 10 min were irradiated by ultrasonic intensity 0.15W/cm2,0.3W/cm2,levofloxacin concentration 0.125ug/ml,0.25ug/ml(with the drug concentration in the nanoparticles),and calculated by plate colony counting method.The synergistic bactericidal effect of ultrasound combined with loaded nanoparticles on MS was evaluated.(3)The ultrasonic irradiation time was 5min,and the drug concentration was0.125ug/ml and 0.25ug/ml respectively.With different ultrasonic intensity 0.15W/cm2,0.3W/cm2 LILFU combined with levofloxacin and levofloxacin PLGA nanoparticles,the bactericidal activity of 36 h was calculated by plate colony counting method,and the synergistic bactericidal effect of different intensity ultrasound combined with levofloxacin PLGA nanoparticles on MS was evaluated.(4)the intensity of ultrasound was 0.15W/cm2,and the concentration of drugs was 0.125ug/ml and 0.25ug/ml respectively.At different irradiation time 5min,10 min,using LILFU combined with levofloxacin and levofloxacin PLGA nanoparticles,the bactericidal activity of 36 h was calculated by plate colony counting method,and the synergistic bactericidal effect of LFLIU combined with levofloxacin PLGA nanoparticles on MS was evaluated at different irradiation times.Result1.The physical properties and release efficiency of three kinds of PLGA nanoparticles:(1)under light and electron microscopy,three kinds of PLGA nanoparticles have uniform size,sphericity and good dispersivity.Malvin laser particle size analyzer was used to detect levofloxacin PLGA nanoparticles with a diameter of 282.5 ± 2.5nm and a surface charge of-18.5 ± 0.8.Green fluorescent dextran PLGA nanoparticles showed green fluorescence under confocal laser scanning microscope,indicating that green fluorescent dextran successfully loaded PLGA nanoparticles.(2)the encapsulation efficiency and drug loading rate of levofloxacin PLGA nanoparticles were 1.04%,21.40%,respectively,but the drug carrying rate of green fluorescent glucan PLGA nanoparticles was higher than that of levofloxacin PLGA nanoparticles,which was 2.70%,which may be related to the polarity of different substances.(3)the drug release rate within 12 h of levofloxacin PLGA nanoparticles showed that the cumulative release rate was 22.87% in the initial release process of 2H.After 8h,the drug release tended to be stable.After 12 h,the amount of drug release was 57.64% of the total.(4)when LILFU was irradiated with levofloxacin PLGA nanoparticles,the drug release rate increased significantly.When the LILFU sound intensity is 0.15W/cm2 and0.3W/cm2 irradiated 10 min,the drug release amount is more than 50% in short time,and the 10 min release rate of the two irradiation is not very different.It can be seen that LILFU can promote the rupture of nanoparticles and increase the drug release rate.2.LFLIU combined drug loaded nanoparticles had synergistic antibacterial effect:(1)levofloxacin showed a minimum inhibitory concentration of 0.25μg/ml MS.(2)under the same condition of ultrasonic intensity and time,the bactericidal effect of different concentrations of drugs and PLGA loaded levofloxacin nanoparticles combined with LFLIU on Mycobacterium foul showed that the bactericidal effect would be better with the increase of drug concentration.The bactericidal effect of ultrasonic combined drug group is better than that of free drug group,but the bactericidal effect of levofloxacin PLGA nanoparticles combined with LFLIU group is better than that of LFLIU combined with free drug group,and the bactericidal effect of levofloxacin PLGA nanoparticles combined with LFLIU group is remarkable.(3)with the prolongation of ultrasonic irradiation time,the germicidal efficacy of levofloxacin PLGA nanoparticles combined with LFLIU was more obvious at different times of ultrasonic irradiation.The bactericidal effect of ultrasonic combined drug group is better than that of the single ultrasound group,but the bactericidal effect of the drug loaded nanoparticles group is better than that of the free drug group,and the bactericidal effect of the ultrasound combined drug loading microsphere group is the best.(4)the higher the sound intensity is,the more obvious the bactericidal effect is under different ultrasonic intensity.The sterilization effect of ultrasound combined with drug group was better than that of ultrasonic group alone,while the levofloxacin PLGA nanoparticles group had better germicidal efficacy than the drug group.Moreover,the combination of levofloxacin nanoparticles and ultrasound has the best germicidal efficacy.ConclusionThe double emulsification method was used to successfully prepare levofloxacin PLGA nanoparticles with homogeneous size,high encapsulation efficiency and low biotoxicity,and combined LFLIU irradiation with high efficiency and synergistic bactericidal effect on MS. |
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