Research On The Sterilization Effect And Mechanism Of Water-soluble Carboxymethyl Chitosan-curcumin Nanomaterials Combined With PACT On S.aureus And MDR S.aureus

Staphylococcus aureus and its resistant strains MDR Staphylococcus aureus are common pathogens causing suppurative infection,they are also one of the high-incidence strains of clinical infection,causing great economic losses.Although traditional antibacterial methods such as antibiotic treatment have certain curative effects,they also cause many problems.Therefore,finding a way to treat bacterial infections effectively and improving the life quality of patients has gradually become a research hotspot in the treatment of bacterial infectious diseases.In this study,PDT(photodynamic therapy)was introduced to the treatment of bacterial infection,common clinical pathogens S.aureus and MDR S.aureus were used as research objects,and mice burned with infection were used as animal models.The photosensitizer curcumin was linked with water soluble polymer carboxymethyl chitosan through ester bond to prepare photoresponsive water-soluble carboxymethyl chitosan-curcumin nanomaterial Cmcs-Cur.The aim was to study the antimicrobial effect of this nanomaterials combined with PACT(photodynamic antimicrobial chemotherapy)on Staphylococcus aureus and its resistant strains,and explore its bactericidal mechanism,which could provide theoretical and practical basis for the clinical application of PACT.The contents of this study1.Synthesis and characterization of water-soluble photoresponsive nanomaterial Cmcs-C urThe water-soluble photosensitizing material Cmcs-Cur was prepared by linking the carboxymethyl chitosan with curcumin with ester bond in a water bath at 60? in the dark,using DCC and DMAP as catalysts.The connection ratio of Cur was detected by fluorescence spectrophotometry to be 0.7%.The particle size of Cmcs-Cur detected by laser particle size analyzer and transmission electron microscope was 110±20 nm with good dispersion in water,and the spectral properties of curcumin did not change significantly in the product.2.In vitro antibacterial effect evaluationThe antibacterial effect of Cur,Cmcs-Cur,light illumination and Cur-PACT or Cmcs-Cur-PACT was detected by plate counting method.The results showed that free Cur,Cmcs-Cur and illumination alone had no obvious sterilization effect on S.aureus and MDR S.aureus,while PACT treatment could effectively kill the bacterium.Under the same concentration of Cur,the bactericidal effect of Cmcs-Cur combined with PACT was more obvious.When the Cur concentration was 6 ?g/ml and the light dose was 150 J/cm2,more than 99%bacterlum were killed in planktonic cultures.The results of crystal violet staining showed that Cmcs-Cur combined with PACT also had good bactericidal effect on S.aureus and MDR S.aureus biofilms.Flow cytometry and fluorescence microscope were used to detect cell membrane permeability and DNA damage after Cmcs-Cur treatment combined with PACT.The results showed that after illumination,cell membrane permeability increased significantly,resulting cellular contents extravasated.Also,PACT treatment can significantly damage bacterial DNA,too.All of the above effects were exhibited in dose-dependent manner.3.Sterilization mechanism study of Cmcs-Cur combined with PACTFlow cytometry analysis of ROS production indicated that PACT treatment can stimulate the production of ROS,and the effect was exhibited in light-dose dependent manner.The expression of reactive oxygen target gene Sta and SigB after PACT treatment was analyzed by qRT-PCR.The results showed that the expression levels of the two genes were significantly decreased after illumination,and the expression of Toxin,a gene related to the pathogenicity of S.aureus was also significantly downgraded.After the addition of reactive oxygen species inhibitor NAC,the expression levels of the three genes were up-regulated in different extent,showed that ROS could indeed regulate the expression of these three genes.The yield of Sta after PACT treatment was extracted with organic solvent under in vitro conditions and analyzed by microplate reader.It was confirmed that the combined treatment could effectively inhibit the production of Sta.4.In vivo antibacterial effect of Cmcs-Cur combined with PACTMale Balb/c mice aged 5-8 weeks and weighing 18-22 g were selected as experimental animals to establish a model of burned infection.The PACT treatment was started on the third day after infection and the treatment was repeated twice once a day during the experiment process.The mice were killed at different periods and the skins of the infected site were taken for test.The homogenate was ground for bacterial counting,and the healing was determined by photographing the wound and detecting the cytokine.Finally,weight monitoring,H&E staining,hemolysis activity and skin toxicity were peformed for safety evaluation.The results showed that Cmcs-Cur combined with PACT could effectively sterilize and promote wound healing at the infected site,and the treatment method was safe,non-toxic and had no side effects.Conclusion:Compared with free Cur,the Cmcs-Cur nanomaterial prepared in this experiment possessed good water solubility and good photosensitivity.It showed obvious bactericidal effect under both in vitro and in vivo conditions,and the treatment scheme was safe,non-toxic and had no side effects,which provided data support and experimental basis for clinical application of PACT.