| ObjectiveBacterial infectious disease present an increasing threat to global healthcare and are the leading cause of mortality and morbidity worldwide.Antibiotics are the most common means of preventing and treating bacterial infections.However,the abuse of antibiotics and their residues in the environment can lead to the emergence and prevalence of drug-resistant bacteria.Therefore,alternative therapeutic strategies targeting bacterial infections and biofilms are urgently needed to avoid the emergence of drug resistance.The rapid development of nanomaterials,especially the controllable drug delivery system based on intelligent nanoparticles,provides a new strategy for antibacterial therapy.Among them,photothermal therapy(PTT)is a non-antibiotic sterilization method that has attracted much attention in recent years.Zeolitic imidazolate frameworks-8(ZIF-8)has good biocompatibility,high porosity and adjustable surface function,and has become a kind of important nanomaterials for drug delivery applications.In this study,a p H/NIR dual stimuli-responsive antibacterial nanomaterial based on ZIF-8 was designed to combine the thermotherapy of PTT and enhanced the delivery and release of antibiotics,which can not only significantly improve the antibacterial efficiency of drug-resistant bacteria,but also reduce the amount of antibiotics,enhance the antibacterial effect,avoid the occurrence of drug resistance,and exert the synergistic antibacterial effect of PTT and drug therapy.Methods1.Synthesis and characterization of Van@ZIF-8@PDA: Vancomycin(Van)was encapsulated in ZIF-8 by one-step synthesis,and its surface was further modified by polydopamine(PDA)to construct a new nano-antibacterial platform(Van@ZIF-8@PDA),and Van@ZIF-8@PDA was characterized by scanning electron microscope(SEM),dynamic light scattering(DLS),X-ray diffraction(XRD)and other instruments.2.Determination of photothermal conversion ability and drug release in vitro: the photothermal conversion ability and drug release rate of Van@ZIF-8@PDA were measured by 808 nm near infrared laser and UV spectrophotometer.3.Degradation behavior of nanomaterials in vitro: the morphological changes of Van@ZIF-8@PDA nanoparticles under different p H with NIR were observed by transmission electron microscope(TEM).4.Antibacterial activity assay in vitro: the experiment was divided into 10 groups:(1)PBS,(2)Van,(3)Van@ZIF-8,(4)ZIF-8@PDA,(5)Van@ZIF-8@PDA,(6)PBS+NIR,(7)Van+NIR,(8)Van@ZIF-8+NIR,(9)ZIF-8@PDA+NIR,(10)Van@ZIF-8@PDA+NIR,to evaluate the photothermal antibacterial effect of Van@ZIF-8@PDA by observing the number of viable bacteria.5.Anti-biofilm activity in vitro: the ability of Van@ZIF-8@PDA to eradicate established biofilm and inhibit biofilm formation after NIR irradiation for 10 min was studied by crystal violet staining and confocal laser scanning microscopy(CLSM).6.Antibacterial mechanism: the morphological changes of bacterial cell membrane under different conditions were observed by scanning electron microscope,and the integrity of DNA from bacteria was further evaluated by agarose gel electrophoresis.7.Photothermal antibacterial assay in vivo: the mouse model of subcutaneous abscess infection was established.Afterwards,50 μL Van,Van@ZIF-8,ZIF-8@PDA,or Van@ZIF-8@PDA,was injected subcutaneously into the abscess site respectively.The abscess areas were exposed to NIR or not.The therapeutic photothermal effect of Van@ZIF-8@PDA on subcutaneous abscess infection was evaluated by observing the wound changes of mice.8.Biocompatibility in vitro and in vivo: the toxicity of synthetic Van@ZIF-8@PDA in vitro was evaluated by MTT test and hemolysis assay.The toxicity of Van@ZIF-8@PDA or PBS in vivo was evaluated by analyzing the changes of biochemical indexes of liver and kidney function(blood urea nitrogen,creatinine,aspartate aminotransferase,alanine aminotransferase and albumin),pathological changes of important organs and tissues(heart,liver,spleen,lung,kidney)and daily body weight changes during treatment.Results1.Synthesis and characterization of Van@ZIF-8@PDA: p H/NIR daul stimuliresponsive nanosystem Van@ZIF-8@PDA synthesized by a simple chemical method have small particle size suitable for biological applications.The encapsulation of vancomycin does not affect the crystal structure of ZIF-8.PDA modification can improve the biocompatibility and stability of nanoparticles.2.Determination of photothermal conversion ability and drug release in vitro: after808 nm NIR irradiation of 10 min,the temperature of Van@ZIF-8@PDA(100 μg/m L)can be rapidly heated to about 50 ℃.The results show that Van@ZIF-8@PDA has a high photothermal conversion efficiency(η = 37.7 %).The results of vancomycin and zinc ion release showed that Van@ZIF-8@PDA had high drug release rate under the acid condition and NIR irradiation.3.Degradation behavior of nanoparticles in vitro: the results showed that the shape and structure of Van@ZIF-8@PDA was obviously damaged under the dual stimulation of NIR irradiation and acid condition.4.Antibacterial activity in vitro : the majority of the bacteria treated with Van@ZIF-8@PDA+NIR were killed,which proved that Van@ZIF-8@PDA had strong antibacterial ability in vitro,but Van@ZIF-8@PDA or NIR alone could not exert strong antibacterial activity.And in our Van@ZIF-8@PDA formula,the working concentration of vancomycin is significantly lower(3.62 μg/m L),indicating that the combination of photothermal and drug therapy can reduce the effective concentration of antibiotics.5.Anti-biofilm activity in vitro: Van@ZIF-8@PDA+NIR inhibited the formation of 85.6% Mu50 biofilm,and the eradicated rate of Mu50 established biofilm was75.5%,indicating that Van@ZIF-8@PDA could strongly inhibit the growth of phytoplankton bacteria before biofilm formation and eradicate the established biofilm.6.Antibacterial mechanism: the growth inhibition of Escherichia coli(E.coli)in Van@ZIF-8@PDA+NIR treatment group was mediated by destroying the cell membrane,while the growth of intermediate vancomycin-resistant Staphylococcus aureus(Mu50)was not inhibited in this way.Further research results show that Van@ZIF-8@PDA nanomaterials under NIR irradiation destroy genomic DNA,through the synergistic action of PTT and drugs,and finally kill bacteria.7.Antibacterial activity in vivo: Van@ZIF-8@PDA+NIR showed strong antibacterial effect for mice infected with subcutaneous abscess in vivo,and reduced the effective dose of antibiotics,with no obvious damage to the surrounding tissue;and the antibacterial effect was better than that of laser treatment group and antibiotics alone.8.Biocompatibility in vivo and in vitro: the survival rate of cells co-cultured with Van@ZIF-8@PDA in vitro was high,and there was no obvious hemolysis after co-culture of RBC with Van@ZIF-8@PDA,indicating that Van@ZIF-8@PDA has low toxicity in vitro.Compared with PBS group and Van@ZIF-8@PDA group,there was no significant difference in liver and kidney blood biochemical indexes,H&E staining of important organs and daily body weight changes in mice,which proved that Van@ZIF-8@PDA had lower toxicity in vivo.ConclusionThe synthesis process of the nanomaterial Van@ZIF-8@PDA is simple,with small particle size,good biocompatibility,low cytotoxicity,high stability and photothermal conversion ability,and Van@ZIF-8@PDA can release drugs in response to the dual stimulation of p H and NIR,and has significant antibacterial properties against planktonic Gram-positive and Gram-negative bacteria before biofilm formation and the established biofilm.Under the irradiation of NIR,the high temperature mediated by the photothermal effect of PDA and antibiotics play a synergistic germicidal effect.At the same time,compared with MIC of vancomycin,the combination of PTT and drug therapy also significantly reduced the effective concentration of antibiotics.The study shows that this p H/NIR dual stimuli-responsive formulation provides a promising potential strategy for clinical application to avoid infection of antibiotic-resistant bacteria. |
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