Aptamer-modified Amphotericin B-loaded PLGA-PEG Nanoparticles For A Targetingtherapeutic Effect On C.albicans Infection Under Low-frequency And Low-intensityultrasound

The fungal species Candida albicans(C.albicans)is an opportunistic pathogen that can cause severe infections in humans,especially in immunocompromised patients and in vivo implantation of various biological materials.It easily forms a biofilm and adheres to the surface of the implant.Recently,C.albicans biofilm-associated infection has posed an increasing threat to public health.Moreover,traditional medicine treatment has poor treatment effects and large side effects.There is an urgent need to achieve a productive,targeted therapeutic efficacy on C.albicans biofilms.Therefore,in this study,we have developed AD1 aptamer-modified amphotericin B(AmB)-loaded Poly(lactic-co-glycolic acid)-Polyethylene glycol(PLGA-PEG)nanoparticles(AD1-AmB-NPs),which enhanced its ability to recognize 1,3-?-glucan on C.albicans wall.Then combined with low frequency and low intensity ultrasound(LFLIU)for controlled release,loosens the biofilm structure and promotes the drug to penetrate deep into the biofilm,as a novel effective antifungal biofilm infection strategy.ObjectiveTo investigate the in vitro and in vivo synergistic antibacterial effect of LFLIU combined with AD1-AmB-NPs on C.albicans biofilm,which is expected to provide new ideas for clinical fungal infection treatment.Methods1.Preparation and characterization of AD1-AmB-NPs:AmB-loaded PLGA-PEG nanoparticles(AmB-NPs)were prepared by double emulsification method,and the aptamer AD1 was modified on the surface of the nanoparticles by carbodiimide method(AD1-AmB-NPs).Proton nuclear magnetic resonance(~1H-NMR)was used to verify whether the aptamer was successfully modified.The particle size,Zeta potential and dispersibility of nanoparticles were detected by malvin laser particle sizer.The surface morphology and internal drug loading of the nanoparticles were observed under scanning electron microscopy(SEM)and transmission electron microscopy(TEM)respectively,and the serum nuclease resistance of the aptamer modified nanoparticles was detected by polyacrylamide gel electrophoresis.Ultraviolet spectrophotometry was used to measure the natural cumulative release rate of drug-loaded nanoparticles and the cumulative release rate after certain ultrasonic irradiation(42 kHz,at the intensity of 0.30 W/cm~2 for 15 min).The toxicity of AmB-NPs,AD1-AmB-NPs and free AmB to normal cells cytotoxicity and side effects of drugs in animals were compared by the XTT method and blood biochemical analysis.2.Specificity identification and binding evaluation in vitro:The connection rates of different fluorescent dye-loaded DiI nanoparticles(DiI-NPs/AD1-DiI-NPs)with planktonic C.albicans,M.smegmatis and E.coli were quantitatively detected by flow cytometry.Then,the attachment of different functional nanoparticle formulations to plankton and biofilms were compared by laser scanning confocal microscope(LSCM),and the in vitro specific recognition ability of the aptamer modified nanoparticles to plankton and biofilms were compared and analyzed.3.The bactericidal experiment in vitro:The following experimental parameters were sound intensity of 0.30 W/cm~2 for 15 min.The effects of ultrasound combined with AD1-AmB-NPs on C.albicans and biofilms activity in vitro were investigated by plate counting method,XTT method and LSCM observation after Calcein-Am/PI staining.Then,in order to explore the mechanism of this method,the penetration degree of non-targeted/targeted nanoparticles in biofilms after ultrasonic irradiation was analyzed by LSCM.4.In vivo targeted therapy evaluation:A biofilm infection model of subcutaneous C.albicans was established by local injection of biofilms suspension into the buttocks of BALB/c mice.The model was successfully established by fungal load measurement,LSCM and pathological section observation.And the aptamer modified nanoparticles in vivo targeting and distribution were observed at different time points by in vivo fluorescence imaging system and LSCM.Then the infected mice were treated in different groups for 7 days,and the local synergistic antifungal effect of ultrasound combined with AD1-AmB-NPs on the subcutaneous C.albicans infection model of mice was evaluated through fungal load measurement and pathological section observation.Results1.Design and characterization of AD1-AmB-NPs:~1H-NMR confirmed that the aptamer AD1 was successfully combined with PLGA-PEG nanoparticles.AD1-AmB-NPs is regularly spherical under the scanning electron microscope,with uniform particle size and good dispersibility.The particle size is 273.9±1.1 nm,and the surface shows a negative potential lower than-20 mV.Under the transmission electron microscope,the drug was encapsulated inside the nanoparticles.The drug loading content was5.3%and the entrapment efficiency was 83%.The aptamer modified nanoparticles have certain resistance to serum nuclease,i.e.the aptamer has not been degraded after incubation in mouse serum for 72 h.The cumulative drug release of drug-loaded nanoparticles was continuously monitored within 98 h.The results showed that ultrasound could promote the drug release of drug-loaded nanoparticles.After 48 h,the drug release amount was about twice that of drug natural release,and the release situation of AD1-AmB-NPs and AmB-NPs was the same.The nanoparticle surface modification aptamer did not affect the drug release.After incubation of macrophages with free AmB for 24 h,the activity of macrophages decreased significantly to 72%compared with the control group(P<0.01).However,at the same concentration of AmB,the macrophage activity in the empty nanoparticles,AmB-NPs and AD1-AmB-NPs groups was not significantly decreased compared with the control group(P>0.05).The levels of BUN and SCr induced by normal mice treated with AmB-NPs and AD1-AmB-NPs were significantly lower than that of free AmB group(P<0.05),and there was no significant difference compared with the control group(P>0.05),indicating that encapsulated AmB can reduce renal toxicity of drugs.2.Highly specific targeting of aptamer nanoparticles to C.albicans and biofilms in vitro:Flow cytometry showed that the binding rate of AD1-DiI-NPs to C.albicans increased to 87%with the extension of co-culture time,but the binding rate to M.smegmatis/E.coli were always less than 10%.LSCM observation showed that AD1-DiI-NPs tightly adhered to C.albicans and biofilms,in sharp contrast to the DiI-NPs group(no nanoparticle adhesion),and had no targeting effect on M.smegmatis/E.coli.The ability of AD1-DiI-NPs to target biofilms was quantified by software analysis,the results showed that the binding force of the targeted nanoparticles group was much higher than that of non-targeted nanoparticles group(P<0.05).3.Synergistic antifungal effects on C.albicans and biofilms in vitro:The plate colony count showed that after ultrasound combined with AD1-AmB-NPs,the survival rate of Candida albicans decreased to 37.17%compared with the control group(P<0.01),while LSCM observed a significant decrease in the number of living cells(green fluorescence)and a significant increase in the number of dead cells(red fluorescence).After ultrasound combined with AD1-AmB-NPs acted on C.albicans biofilms,compared with the control group,biofilms activity decreased 62%(P<0.01),while LSCM observed a decrease in the extracellular matrix of biofilms,loose structure,and a large number of dead bacterias(red fluorescence)residues in biofilms.Three-dimensional reconstructed structural images of biofilms after different treatments were observed by LSCM.The results showed that there were almost no residual nanoparticles on the surface/middle/bottom layer of the non-targeted biofilms,and a large number of nanoparticles were observed on the surface of the targeted biofilms,especially the number of nanoparticles in the inner layer of the biofilms increased significantly after ultrasonic irradiation(P<0.05).This indicates that ultrasound significantly promotes the penetration of nanoparticles into biofilms.4.Targeted synergistic antifungal activity in vivo:After 24 h of subcutaneous local injection of biofilm suspension in mice,a large number of C.albicans and hyphae formed biofilm structures in infected tissue sites,and the main organs(heart,liver,spleen,lung,kidney)were not infected.The BALB/c mice subcutaneous C.albicans biofilm infection model was successfully established.The in vivo fluorescence imaging and LSCM observation showed that the aptamer modified nanoparticles still have high efficiency and specificity in vivo,can target the infection site of C.albicans biofilms,and reach the peak value 24 h after the nanoparticle preparation is injected.After the infected mice were treated with ultrasound combined with AD1-AmB-NPs,the swelling of the infected site gradually disappeared,and the fungal colony-forming unit in the infected tissue was significantly reduced(P<0.05).The pathological section of the infected tissue showed normal subcutaneous tissue,no inflammatory cells and fungal colony aggregation,and the fungal infection was significantly improved.ConclusionsWe successfully prepared aptamer AD1 modified amphotericin B-loaded PLGA-PEG nanoparticles,which confirmed that low-frequency low-intensity ultrasound combined with AD1-AmB-NPs had synergistic antibacterial effect on C.albicans biofilmsin vitro and in vivo,further confirming the feasibility of targeted antibacterial strategy.