| ObjectiveC.albicans is the most common conditional pathogen in humans.The specificity and refractory nature of the infection is that C.albicans is easy to form a biofilm,and the biofilms greatly increases the resistance of the fungus,and the infection is not cured for a long time.In this experiment,the antifungal drug amphotericin B(AmB)was pre-loaded intoPLGA nanoparticles,combinedwithLow-frequencyand Low-intensity Ultrasound(LFLIU)irradiation to explore the synergistic antibacterial effect on C.albicans biofilm in order to achieve drug synergy and targeted release,providing a new idea for the treatment of clinical fungal biofilm infection.Methods1.Preparation and characterization of amphotericin B-loaded-PLGA nanoparticles(AmB-NPs):PLGA nanoparticles were prepared by double emulsification method.The particle size,potential and dispersion of PLGA nanoparticles were measured by Malvine laser particle size analyzer.The surface and internal morphology of PLGA nanoparticles were observed under electron microscopy.The drug release rate within 48hours and the drug release efficiency after ultrasound irradiation were measured and the toxicity of AmB-NPs to normal cells was compared with that of free AmB.2.The synergistic fungicidal effect of ultrasound combined with AmB-NPs on plankton of C.albicans:C.albicans standard strain ATCC10231 was cultured to logarithmic phase and drug sensitivity test was carried out.Ultrasound instruments are equipped with fixed frequency of 42 kHz,transducer diameter of about 1.0 cm and output sound intensity of 0.15-0.60 W/cm~2 continuously adjustable.The effects of different ultrasound intensity(0.15,0.30 and 0.60 W/cm~2)and irradiation time(5,15 and 30 min)on plankton of C.albicans were investigated to screen suitable ultrasound dosage.Then,low-frequency ultrasound combined with different concentration of AmB-NPs was used to act on planktonic fungal of C.albicans to explore its effect on the activity and structure of planktonic fungal.3.In vitro fungicidal effect of ultrasound combined with AmB-NPs on C.albicans biofilm:cell culture plate method was used to establish an in vitro biofilm model and the minimum inhibitory concentration(SMIC)of AmB and AmB-NPs on C.albicans biofilm was determined.The activity and total amount of biofilm were detected and CLSM was used to analyze the biofilm qualitatively and quantitativel and the activities of protease and phospholipase secreted by biofilm were detected.4.In vivo fungicidal effect of ultrasound combined with AmB-NPs on C.albicans biofilm:the polyurethane catheter immersed in C.albicans liquid were implanted into SD rat back subcutaneously to establish the infected model of C.albicans biofilm in vivo.Before treatment,LFLIU was used to verify the safety of percutaneous irradiation in rats.After 3days and 7 days of continuous treatment with ultrasound combined with AmB-NPs,the fungal colonies on the surface of catheters were measured.The changes of biofilm structure on the surface of catheters were observed by CLSM and SEM.Results1.AmB-NPs characteristics:AmB-NPs were spherical,the particle size was about 300 nm and the surface was negatively charged.The drug loading rate and encapsulation rate of AmB-NPs were 5.7%and 85%respectively.The results of drug release rate showed that ultrasound could significantly promote drug release.After 48 hours,the drug release was about twice as much as that of natural release,which improved the therapeutic effect of follow-up ultrasound combined with AmB-NPs.2.The fungicidal effect of ultrasound combined with AmB-NPs on plankton of C.albicans:After different doses of ultrasound,the cell wall barrier of C.albicans was effectively destroyed and cell permeability was increased when the ultrasound irradiation condition was 0.30W/cm~2 for15 minutes,according to the detection of the activity of C.albicans and TEM observation.However,the activity of C.albicans did not decrease significantly.Therefore,ultrasound intensity of 0.30W/cm~2 and irradiation time of 15 minutes provide the best conditions for the subsequent drug delivery of nanoparticles into cells.Plate culture colony count showed that the survival rate of C.albicans decreased significantly(91±0.12)%after ultrasound combined with AmB-NPs treatment.3.The fungicidal effect of ultrasound combined with AmB-NPs on C.albicans biofilm in vitro:After 48 hours of continuous culture,the growth curve of C.albicans biofilm became flat and the absorbance value tends to be stable and gradually becomes mature.After ultrasound combined with AmB-NPs treatment on mature C.albicans biofilm,the activity and total amount of biofilm decreased significantly,up to 74.64%and 31.26%(P<0.01)compared with the control group.At the same time,CLSM observed that the biofilm structure was sparse,the thickness became thinner,and the number of dead bacteria increased.In addition,ultrasound combined with AmB-NPs can significantly reduce the activity of protease and phospholipase.4.In vivo fungicidal effect of ultrasound combined with AmB-NPs on C.albicans biofilm:After 72 hours of catheter implantation,the colonization of fungi on catheter tended to be stable,and crystal violet staining and scanning electron microscopy showed that the obvious biofilm structure attached to the catheter surface.Histopathological results of skin tissues of rats after different doses of ultrasound irradiation showed that the safety threshold of ultrasound irradiation was 0.3W/cm~2irradiation time for 15 minutes.After 3 or 7 days of treatment with ultrasound combined with AmB-NPs,the fungal load of rats treated with ultrasound combined with AmB-NPs was significantly lower than that of rats treated with free AmB or rats treated with other treatments(P<0.01),and the effect was more obvious after 7 consecutive days of treatment.At the same time,CLSM and SEM observation showed that after the combined action of ultrasound and AmB-NPs,the structure of biofilm on the surface of catheter was destroyed,and the extracellular matrix decreased or disappeared,leaving only a single fungal colony,basically eliminating the biofilm.ConclusionsThis study demonstrated that LFLIU combined with AmB-NPs had synergistic antimicrobial effect on C.albicans biofilm in vitro and in vivo,which could significantly improve the antimicrobial effect.It provided a new effective method for clinical treatment of C.albicans biofilm infections and improved the cure rate of clinical fungal infections. |
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