Experimental Study Of Low-frequency And Low-intensity Ultrasound Combined With Levofloxacin Nanoparticles For Targeted Inhibition Of BCG And Its Biofilm Infection

Tuberculosis(Tuberculosis,TB)is still one of the top ten causes of death worldwide,and it is also a major infectious disease caused by a single pathogen.Tuberculosis is a highly contagious chronic disease caused by Mycobacterium tuberculosis(Mycobacterium tuberculosis,MTB),which global treatment success rate is only 55%.The increasing number of drug-resistant strains and the formation of bacterial biofilm are the main reasons for the failure of clinical treatment.There are similar growth cycle and the same cell wall component-fatty arabinomannan(Mannose-capped lipoarabinomannan,Man LAM)in Bacillus Calmette-Guerin and MTB,in this paper,levofloxacin nanoparticles modified by BM2 aptamers were prepared,which could specifically recognize Man LAM in BCG cell membrane.Under the action of Low-frequency and Low-intensity Ultrasound(LFLIU),it is released at a fixed point,which synergistically enhances the concentration of the drug to reach the bacteria and the biofilm,and explores a new anti-TB infection strategy.ObjectiveIn this study,levofloxacin,which has the duality of anti-tuberculosis drugs and sound-sensitive agents,was selected to study the targeted germicidal efficacy and mechanism of LFLIU combined with levofloxacin-loaded nanoparticles on BCG and its biofilm infection,which is expected to provide a new idea for clinical non-invasive treatment of subcutaneous solid tuberculosis.Methods1.Preparation and characterization of levofloxacin-loaded PLGA-PEG nanoparticles modified by BM2 aptamer(BM2-LVFX-NPs):Levofloxacin-loaded PLGA-PEG nanoparticles(LVFX-NPs)were prepared by double emulsification method,and BM2 aptamers were modified on the surface of LVFX-NPs by carbodiimide method.The particle size,Zeta potential and dispersion of nanoparticles were measured by Malvern laser particle size analyzer and the morphological size and internal structure of BM2-LVFX-NPs were observed by electron microscope.The production of reactive oxygen species(ROS)of nanoparticles irradiated by ultrasound was detected respectively.The confocal microscope and flow cytometry were used to detect the targeting ability of aptamer modified nanoparticles to BCG bacteria.2.The bactericidal effect and mechanism of LFLIU combined with BM2-LVFX-NPs on BCG in vitro:The continuous adjustable ultrasound equipment with 42 k Hz,output sound intensity of 0-1.0 W/cm~2was selected,and the ultrasonic irradiation parameter of the follow-up experiment was 0.67 W/cm~2,5 min.The bactericidal effect of LFLIU combined with BM2-LVFX-NPs on BCG in vitro was observed by plate counting and confocal microscope.The production of ROS in BCG and the phagocytosis of nanoparticles before and after ultrasound irradiation were detected by confocal microscope and flow cytometry,respectively.3.The therapeutic effect of LFLIU combined with BM2-LVFX-NPs on BCG infection in vivo:The rat model of subcutaneous infection of BCG in SD rats was established,and verified by H&E and acid-fast staining.The targeting ability of BM2 aptamer modified nanoparticles in vivo was verified by small animal in vivo imager.After 14 days of treatment,the therapeutic effect in vivo was explored by measuring the volume of abscess,bacterial loading,pathological sections and other indicators.4.The damage effect of LFLIU combined with BM2-LVFX-NPs on BCG biofilm in vitro:The mature BCG biofilm model was established,the growth cycle was calculated,and the morphological changes were observed by crystal violet staining.The targeted binding effect of BM2aptamer modified nanoparticles on BCG biofilm was observed by confocal microscope.The activity of biofilm was detected by XTT and crystal violet staining.In order to explore the damage mechanism of LFLIU combined with BM2-LVFX-NPs on BCG biofilm in vitro,the permeability of BM2 aptamer modified nanoparticles before and after ultrasound irradiation in biofilm was detected by confocal microscope.Results1.The characterization of BM2-LVFX-NPs:The particle size of BM2-LVFX-NPs was 273.9±1.14 nm,Zeta potential was-14.6±0.778m V.Under scanning electron microscope,the nanoparticles were regular spherical and uniformly distributed.Under transmission electron microscope,the drug was encapsulated in the nanoparticles.Reactive oxygen species were produced after ultrasonic irradiation of BM2-LVFX-NPs,confirming the characteristics of LVFX as a sonosensitizer.The confocal microscope and flow cytometry showed that the BM2 aptamer modified nanoparticles had good binding ability to BCG,and could specifically recognize BCG,without targeted ligation with other bacteria(Escherichia coli,Mycobacterium smegmatis).2.The bactericidal effect and mechanism of LFLIU combined with BM2-LVFX-NPs on BCG in vitro:The plate colony counts showed that the survival rate of BCG treated by LFLIU combined with BM2-LVFX-NPs decreased significantly.At the same time,the results of confocal microscope also showed that the number of living bacteria(green fluorescence)in LFLIU combined with BM2-LVFX-NPs group was less,and a large number of dead bacteria(red fluorescence)gathered.After irradiated with LFLIU(0.67 W/cm~2,5 min),the absorptivity of BCG to nanoparticles increased to 39.44±5.13%,and that of BM2aptamer modified nanoparticles increased to 97.15±2.55%,indicating that LFLIU could promote the entry of nanoparticles into BCG bacteria.When BCG was treated with LFLIU and BM2-LVFX-NPs,the results of confocal microscope and flow cytometry showed that the production of ROS in BCG was significantly higher than that in other groups.3.The therapeutic effect of LFLIU combined with BM2-LVFX-NPs on BCG infection in vivo:After subcutaneous injection of BCG solution into SD rats for 14 days,the granuloma structure was found in H&E,and acid-fast staining showed that the abscess contained BCG bacteria.The results of in vivo imaging of small animals showed that the BM2 aptamer modified nanoparticles could successfully identify and target the infected site in vivo.At the same time,the aggregation of targeted nanoparticles reached the peak at the infected site at 24 hours after tail vein injection.After treated for 14 days,the volume of the infected site was significantly reduced,the bacterial load of the infected tissue was significantly reduced,and the pathological sections showed that the necrotic area and inflammatory cells decreased.4.The damage effect of LFLIU combined with BM2-LVFX-NPs on BCG biofilm in vitro:The crystal violet staining showed that with the increase of time,the matrix of BCG biofilm gradually thickened and the lumen-like substances used to transport nutrients became thicker.The confocal microscope showed that the targeted nanoparticles adhered tightly to the surface of the biofilm and had a good connection effect on the BCG biofilm.After treated with LFLIU and BM2-LVFX-NPs,the XTT and crystal violet staining revealed that the activity of BCG biofilm decreased significantly.Three-dimensional reconstruction of BCG biofilm by confocal microscope displayed that only a small number of non-targeted nanoparticles gathered in the biofilm after ultrasound irradiation,while most of the targeted nanoparticles gathered on the surface of the biofilm without ultrasound irradiation.After ultrasound irradiation,a large number of targeted nanoparticles gathered in the biofilm,indicating that ultrasound can promote targeted nanoparticles to enter the BCG biofilm.ConclusionsWesuccessfullypreparedBM2aptamer-modified levofloxacin-loaded PLGA-PEG nanoparticles,which can specifically recognize BCG and its biofilm.And LFLIU combined with BM2-LVFX-NPs showed significant therapeutic effects on BCG and its biofilm infections in vivo and in vitro.