Preparation Of Near-infrared Triggered Nanoparticles For The Treatment Of MRSA Infection And Transcriptomic Analysis

Background:Bacterial infections,especially drug-resistant bacterial infections,seriously affect the healthy development of human society.Especially for drug-resistant bacteria represented by Methicillin-resistant Staphylococcus aureus（MRSA）,due to the abuse of antibiotics,the cost of clinical treatment is getting higher and higher,and traditional antibacterial methods are gradually limited.New antibacterial methods have become a new research direction.Nanotechnology has developed rapidly in the past two decades,and many nanomaterials with special physical and chemical properties have been gradually applied to wider fields.Among them,emerging treatment methods represented by photothermal therapy（PTT）and photodynamic therapy（PDT）have become alternative treatment options that may replace traditional antibiotics.PTT can convert light energy into thermal energy under near-infrared（NIR）excitation conditions by taking advantage of the excellent photothermal conversion capabilities of some nanomaterials.PDT is that some nanomaterials generate active oxygen under the excitation of NIR light,both of which have good application prospects for killing bacteria.Among them,metal sulfides and upconversion nanomaterials have become one of the ideal research directions of antibacterial nanomaterials because of their unique photothermal conversion capabilities,as well as the ability to generate active oxygen and ideal biological safety.Aims:To investigate the killing effect and mechanism of a core-shell nanospherical structure（UC@ABS）formed by coating metal sulfur bismuth silver compound on the surface of upconverting nanoparticles on MRSA bacteria.Methods:（1）Prepare nanoparticles and characterize their morphology:prepare UC@ABS nanoparticles by ion exchange method and hydrothermal assisted template method;Use transmission electron microscopy（TEM）to study the morphology and size of UC@ABS;Use High Resolution Transmission Electron Microscope（HRTEM）was used to observe the lattice size of nanoparticles;Dynamic Light Scattering（DLS） was used to detect its hydrated particle size and zeta potential;（2）Research on the properties of UC@ABS nanoparticles:UV-Visible spectrophotometer（UV-Visible spectrophotometer,UV-vis）to observe the absorbance range of different concentrations;Detect its photothermal conversion performance combined with photothermal conversion rate,Photothermal stability and active oxygen generation ability;（3）To explore the in vitro antibacterial ability of the nanoparticles:the minimum inhibitory concentration and minimum bactericidal concentration were studied by gradient broth dilution method;the in vitro antibacterial ability of the nanoparticles was detected by dilution coating plate method and bacterial viability staining method;Scanning electron microscope（SEM）morphological changes of MRSA bacteria treated with nanoparticles;active oxygen probes were used to detect changes in the level of reactive oxygen species inside the bacteria under the treatment of UC@ABS nanoparticles;（4）Research on antibacterial ability of UC@ABS nanoparticles in vivo:MRSA bacteria wound infection model and subcutaneous MRSA bacteria abscess model were constructed in female BALB/c mice.Hematoxylin-eosin staining（hematoxylin-eosin,H&E staining）and collagen fiber staining（Masson staining）at the abscess site,as well as the bacterial spread plate counting method at the infected tissue site,were used to explore their effects after treatment in different groups;（5）Biosafety exploration:explore and analyze the cytotoxicity of nanoparticles in mouse mammary epithelial cells（HC11）and human embryonic kidney cells 293（HEK-293t）by CCK8 and MTT methods;Tissue sections of heart,liver,spleen,lung,and kidney were used to explore the toxicity of UC@ABS nanoparticles in mice;UC@ABS nanoparticles were evaluated by collecting serum from the treatment group and analyzing the blood routine and blood biochemical values of the mice Effects on mouse liver and kidney function;（6）Research on the antibacterial mechanism of UC@ABS nanoparticles:by determining the growth curve of MRSA bacteria under the influence of different concentrations of UC@ABS nanoparticles,in order to sequence the transcriptome of MRSA bacteria;Verify the credibility of the sequencing results by real-time quantitative PCR.Results:（1）Prepare nanoparticles and characterize their morphology:TEM shows that the average size of UC@ABS nanoparticles is about 90nm,DLS shows that the hydrated particle size of UC@ABS is about 100nm,the shape of nanospheres is uniform,and the overall appearance is relatively Good dispersion.HRTEM results show that the lattice spacing of hexagonal Na YF4 and cubic Ag Bi S2 are 2.91（?）（110）and 2.77（?）（200）,respectively;Element mapping analysis results show that Na,Er,Ag,Bi,S,Y,F,Yb,Nd elements are evenly distributed;（2）Research on the performance of UC@ABS nanoparticles:UV-vis results show that UC@ABS solution has obvious light absorption in the NIR region;Thermal imager shows that the temperature of UC@ABS solution irradiated by NIR increases with concentration and power dependence,excellent light-to-heat conversion efficiency, the temperature change law of UC@ABS solution is basically consistent in 5 cold/heat cycles,and the change of its ultraviolet absorbance before and after light is not obvious;use TMB to verify UC@ABS has ROS generation under the excitation of 808 nm;（3）To explore the in vitro antibacterial ability of the nanoparticles:the optimal laser power is 1.0 W·cm^-2through colony count analysis;The MIC value of the nanoparticles is 20μg m L^-1;The MBC value is 40μg·m L^-1;Bacterial live/dead staining was used to detect that when only NIR light or only UC@ABS nanoparticles were used,the bacterial liquid emitted green fluorescence,and when the two were combined,the bacterial liquid emitted red fluorescence;SEM images showed that the structure of the bacteria in the UC@ABS combined with 808 nm NIR light treatment group was obviously damaged,and the shrinkage and damage appeared;（4）Study on the antibacterial ability of UC@ABS nanoparticles in vivo:The size of the back scars of mice in the UC@ABS+NIR treatment group gradually decreased with the increase of days,and the wounds healed up to 99%by the 14th day;UC@ABS nanoparticles combined with 808 nm NIR light,the number of remaining colonies on the wound tissue of the mice was the least,and the colony viability decreased by more than 98%;After the 14th day of treatment,the tissue of the skin infection site of the mice basically returned to normal and there were new hair follicles.The appearance of inflammatory cell infiltration was significantly improved,and it can be found from the results of Masson staining that collagen fibers were produced the most in the combined treatment group of UC@ABS+NIR;（5）Exploration of biological safety:UC@ABS solution has no obvious cytotoxicity to HC11 and HEK-293t cells when the concentration of UC@ABS solution is increased to 160μg·m L^-1;By collecting the blood around the eyes of mice,it is found that there is no significant cytotoxicity in each treatment group.Obvious liver and kidney toxicity,pathological sections of each organ showed obvious organ damage;（6）Research on the antibacterial mechanism of UC@ABS nanoparticles:A total of2613 genes were detected,including 710 DEGs with statistically significant changes（DESeq2 at FC>2 and FDR adjusted p<0.05）,of which 518 genes were upregulated and 192 genes were downregulated;Pathways regulated by these genes include galactose metabolism,riboflavin metabolism,ATP-binding cassette（ABC） transporter,phosphotransferase system（PTS）,β-lactam resistance,aminoacyl- t RNA biosynthesis,quorum sensing,and biofilm formation,among others;For all identified genes,an overall correlation value of 0.98 was obtained between RNA-seq and q RT-PCR.These show that the RNA-seq and q RT-PCR results are in good agreement,with the q RT-PCR results matching the RNA-seq data.Conclusion:Through a series of studies on this subject,it has been shown that metal sulfur bismuth silver compound is coated on the surface of upconversion nanoparticles to form a core-shell nanospherical structure（UC@ABS）,which can interfere with galactose metabolism,riboflavin metabolism,ATP binding Cassette（ABC）transporter,phosphotransferase system（PTS）,quorum sensing and biofilm formation and other pathways have excellent killing effects on MRSA bacteria.