Preparation Of Nanomedicines For Multivariate Synergistic Therapy And Its Application In Bacterial Skin Abscesses

Diseases induced by pathogenic bacteria account for one-third of global mortality.Staphylococcus aureus is a versatile pathogen that causes diseases,from skin and soft tissue infections to severe systemic illnesses.Currently,vancomycin is the treatment of choice for pathogen infections,but misuse of antibiotics has led to the emergence and spread of resistant strain.Thus,it is critical in clinic to develop novelty antimicrobial strategies that have strong antimicrobial efficacy.In recent years,photothermal therapy(PTT)is an emerging alternative to treat diseases by using materials with higher photothermal conversion efficiency to convert light energy into heat under near-infrared(NIR)laser irradiation.PTT,which has the advantages of deep tissue penetration and less side effects,has been widely studied.However,it is difficult to accurately monitor the temperature inside the target during photothermal therapy.On the one hand,excessively high temperature can easily cause damage to the surrounding normal tissues.On the other hand,the intensity of light decreases when it penetrates the target,which may cause insufficient temperature to rise in the depths.Nanozymes,nanomaterials which mimic the activities of natural enzymes,are attractive for applications in the biomedicine because of unique advantages like low cost,high stability,and good biocompatibility.Among them,nanozymes that mimic peroxidase(POD)activity can convert H₂O₂ into toxic free radicals(·OH),which can damage tumor cells or bacteria;this is referred to as chemodynamic therapy(CDT).Because of the catalytic reaction capacity of multivalent metal ions,some nanoparticles based on metal elements,like iron oxide nanoparticles,copper/carbon hybrid nanospheres,Cu-TCPP nanosheets,vanadium oxide nanodots,and nano Mo S₂,have been harnessed for their POD-like activities for antibacterial therapies.However,the high level of glutathione(GSH)in the microenvironments of infection sites greatly reduce CDT efficacy.In chapter 1 of this project,we designed and prepared liposome nanoparticles with near-infrared-controlled release of tungsten sulfide quantum dots(WS₂QDs)and vancomycin(named WS₂QDs-Van@lipo)and studied its performance.In chapter 2 of this project,we studied the antibacterial effect and mechanism of WS₂QDs-Van@lipo nanoparticles.In chapter 3 of this project,we conducted a preliminary assessment of the safety of WS₂QDs-Van@lipo nanoparticles.The research results of this subject not only provide new theoretical explorations of the effects and mechanisms of near-infrared-controlled release of nanoparticles in bacterial infections,but more importantly,lay the foundation for the development of new anti-infective drugs with high biocompatibility.OBJECTIVE:This project intends to select vancomycin-intermediate S.aureus(VISA)standard strain Mu50 and E.coli standard strain ATCC 25922 as the research objects,design and prepare liposome nanoparticles with near-infrared-controlled release of WS₂QDs and vancomycin,observe their antibacterial and anti-biofilm effects through the combination of PTT,CDT and drug therapy,clarify their specific mechanisms and evaluate their biological safety.METHODS:(1)Synthesis and characterization of WS₂QDs-Van@lipo:WS₂QDs-Van@lipo were prepared using a thin lipid filmhydration method.The transmission electron microscopy image shows that the morphology and size of WS₂QDs-Van@lipo.The hydrodynamic particle size and the Zeta potentials of WS₂QDs-Van@lipo were evaluated by dynamic light scattering.(2)Performance evaluation of WS₂QDs-Van@lipo:NIR-triggered release of WS₂QDs-Van@lipo,in vitro catalytic performance(including simulated POD enzyme activity detection,·OH generation detection and GSH oxidation ability detection),photothermal effect evaluation(including evaluation of light-heat stability and calculation of photothermal conversion efficiency).(3)In vitro antibacterial activity test:The vancomycin MIC for Mu50 was determined by modified broth microdilution assays.The antibacterial ability in vitro was determined by plate colony detection and live/dead bacterial staining assays.In order to characterize the morphology of the bacteria treated under various conditions,we use electron microscope.We also used DCFH-DA assay to detect changes in the level of reactive oxygen species(ROS).(4)In vivo experiments:A subcutaneous abscess was obtained by local injection of Mu50on the back of the mice.The abscesses were photographed,and the mouse weights were measured every 2 days.After 12 days of treatment,the mice were sacrificed.All the skin tissues and major organs were excised and stained with H?E and Masson staining for the histology analysis.The bacterial suspensions from the skin tissues were spread on solid media and cultured in a 37°C incubator for 24 h.The relative bacterial viability was obtained based on colony counts.(5)Preliminary assessment of safety:The blood serum of all mice was collected to evaluate the liver and kidney according to different indicators.MTT assay on human keratinocyte cell line cells was used to evaluate the cytotoxicity of WS₂QDs-Van@lipo.RESULTS:(1)The transmission electron microscopy(TEM)image shows that the size of WS₂QDs was less than 10 nm.The hydrodynamic particle size of WS₂QDs was about 11.25±1.22 nm.The X-ray diffraction(XRD)pattern result shows that the WS₂QDs had characteristic peaks similar to those of the previously published WS₂QDs.Both cryo-electron microscopy images and field-emission scanning electron microscopy(SEM)images showed that WS₂QDs-Van@lipo were spherical,with uniform morphology and an average diameter of less than 100 nm.Elemental mapping analysis with TEM showed a homogeneous distribution of O and S in WS₂QDs-Van@lipo.The hydrodynamic particle size of WS₂QDs-Van@lipo were about 146.37±0.67 nm,with PDI values of 0.15±0.01 measured by dynamic light scattering(DLS).The Zeta potentials of WS₂QDs-Van@lipo were approximately-35.4 m V.(2)WS₂QDs-Van@lipo has strong absorption in the NIR region.The temperature of the WS₂QDs-Van@lipo solution increased instantly upon exposure to NIR irradiation.The final temperature reached 47°C within 10 min.In addition,WS₂QDs-Van@lipo solution exhibited laser density-dependent photothermal conversion.The photothermal conversion efficiency was 43.67%.No significant temperature change was observed in a recycling heating procedure we performed with the WS₂QDs Van@lipo over a course of six on/off cycles.Liposomes loaded with fluorescein and WS₂QDs(denoted as FITC-WS₂QDs@lipo)were also prepared to investigate the release behavior of fluorescein isothiocyanate(FITC)under NIR irradiation.Upon addition of H₂O₂ to the WS₂QDs,the characteristic UV-vis absorption at 652 nm for a typical TMB radical was readily observed,indicating POD-like activity.We explored this with assays monitoring the degradation of methylene blue(MB)as induced by·OH:WS₂QDs catalyzed the H₂O₂to·OH,doing so in a WS₂QDs concentration dependent manner.The degradation of MB increased along with increasing temperature.In addition,WS₂QDs still have high reaction rates at p H 6-7.The degradation of MB increased with the increase of GSH concentration,which we speculate may result from GSH's stimulation of tungsten ion release from WS₂QDs,demonstrating that WS₂QDs exhibit OXD-like activity.(3)We conducted agar plate counting assays and live/dead bacterial staining assays.The experimental results show that the WS₂QDs Van@lipo+H₂O₂+NIR treatment induced the best bactericidal effects.We used SEM to study the morphological changes of the two bacterial strains upon treatment.We observed small ruptures of the Mu50 cell wall upon WS₂QDs-Van@lipo+H₂O₂+NIR treatment and the morphology of E.coli became rough,exhibiting clear lesions and holes.In addition,DCFH-DA probes were used to detect the ROS level in bacteria.Many green fluorescence spots in the bacteria were observed in the WS₂QDs-Van@lipo+H₂O₂+NIR treatment group,indicating the production of ROS.Confocal laser scanning microscopy and the crystal violet staining assay showed the largest extent of biofilm destruction resulted from the WS₂QDs-Van@lipo+H₂O₂+NIR treatment.The potential biofilm penetration ability of antibiotics was evaluated by FITC-WS₂QDs@lipo.A strong green fluorescence was observed after the biofilms were treated with the FITC-WS₂QDs@lipo+H₂O₂+NIR,suggesting an effective penetration of therapeutic agents inside the biofilms.(4)WS₂QDs-Van@lipo+H₂O₂+NIR treatments exhibited improved recovery that their scar sizes gradually decreased with increasing time.Almost no bacterial growth was observed for the suspensions taken from the mice that were given the WS₂QDs-Van@lipo+H₂O₂+NIR treatment by spreading bacterial suspensions extracted from deep within the abscess sites.The recovery of the infected skin tissues was further evaluated by histological analysis using hematoxylin and eosin(H?E)staining.Masson staining results also indicate that collagen fiber(blue)deposition is the highest in the WS₂QDs-Van@lipo+H₂O₂+NIR treatment.(5)MTT assays indicated that there was no obvious cytotoxicity,even at a WS₂QDs-Van@lipo concentration of 200?g/m L.Furthermore,no appreciable hemolysis was observed in a hemolysis assay,indicating that WS₂QDs-Van@lipo exerts negligible,if any,damage to membranes of red blood cells(RBCs).Blood from the abscess model mice of the different treatment groups was collected on day 12,and we found that none of the treatments caused obvious liver toxicity and nephrotoxicity.Significant increase in WBC and PLT counts were observed in abscess model mice compared with the healthy mice.Those blood indicators partially recovered in the mice that were given the WS₂QDs-Van@lipo+H₂O₂+NIR treatment,indicating that this treatment can effectively eliminate infection and inflammation.CONCLUSION:Our work illustrates that the photothermal and nanozyme properties of WS₂QDs can be deployed alongside a conventional therapeutic to achieve synergistic PTT/CDT/pharmaco therapy for powerful antibacterial effects.