Preparation And Antibacterial Activity Of Covalent Organic Framework Molecules Of Porphyrins

Bacterial infections pose a great threat to human life and health.The clinical treatment of bacterial infection mainly relies on antibiotics.However,the overuse of antibiotics not only has obvious side effects,but also has serious drug resistance.In particular,the drug resistance of Gram-positive bacteria such as S.aureus and E.faecalis poses a serious threat to public health.If not effectively controlled,bacterial infections will cause a serious global health crisis.Therefore,exploring effective treatments that do not cause bacterial resistance has become an urgent issue to be addressed.Antimicrobial photodynamic therapy（a PDT）is a new treatment method,which has attracted more and more attention due to non-drug resistance,spatial and temporal control and high efficiency.The elements of photodynamic therapy are photosensitizer,oxygen and light,PDT can rapidly kill bacteria by stimulating photosensitizer to cytotoxic produce reactive oxygen species（ROS）.The generation mechanisms of ROS are mainly divided into type I（electron transfer）and type II（energy transfer）.Photosensitizers play a crucial role in a PDT.Traditional small molecule photosensitizers（such as porphyrin and phthalocyanine,etc.）can aggregate to cause fluorescence quenching（ACQ）phenomenon,affecting its photosensitive activity.Compared with small-molecule antimicrobial photosensitizers,covalent organic frameworks（COFs）attenuate the ACQ phenomenon by arranging the structural units in an ordered periodic arrangement through covalent bonding.Porphyrins COFs are widely used,the rich pores of COFs make them easy to transfer oxygen and ROS,and the large conjugated structure gives them a narrow band gap and wide absorption,which make porphyrins COFs have good photodynamic properties and facilitate optical visual diagnosis.The current porphyrin-based COFs mainly produce type II ROS.The activity of these antimicrobial photosensitizers is strongly influenced by the surrounding oxygen content,and their application is limited in bacterial infection sites with hypoxia or low oxygen content.On the contrary,the antibacterial photosensitizer produced type I ROS has very few dependencies on oxygen,which is suitable for the hypoxic microenvironment of the infected site..In view of this,a porphyrin-based COF produced type I ROS is prepared by the solvothermal method,followed by the preparation of soluble COF nanoparticles.its antibacterial activity in vitro is analyzed.The specific contents and results are as follows:1.Synthesis of porphyrin-based COF molecule,preparation and characterization of nanoparticles.Firstly,monomer 1 and monomer 2 were prepared by multi-step organic reaction using pyrrole and guanidine hydrochloride.The monomers were dehydrated by solvothermal method to obtain metal Zn porphyrin-based COF(BPTG_Cl)materials.The structure of the intermediate compound and the target product were characterized by H NMR,¹³C NMR,¹³C CP/MAS NMR,XRD,XPS,FTIR.Polyethylene glycol（PEG）-wrapped BPTG_Cl nanoparticles(BPTG_Cl@DSPE-PEG)were prepared by nanoprecipitation method.The particle size and zeta potential result showed that the potential of nanoparticles with a size of about 80 nm was-12.03±0.50 m V,suggesting good dispersibility and stability.The photophysical property of BPTG_Cl@DSPE-PEG NPs exhibited that the nanoparticles had a maximum absorption of 430 nm and a weak absorption at 500-625 nm,The maximum emission wavelength was 720 nm,and the strongest emission peak was located at 657 nm,The peak was located in the red light emission region,which is suitable for biofluorescence imaging.The electrochemical measurement and the species of ROS demonstrated BPTG_Cl@DSPE-PEG NPs can produce more photogenerated electrons to produce type I reactive oxygen species including hydroxyl radicals（·OH）、superoxide anion（·O₂^-）、hydrogen peroxide（H₂O₂）.Additionally,the biocompatibility test with L929 revealed that BG NPs had good biocompatibility.2.The specific recognition and antibacterial performance analysis of porphyrin-based COF.Based on the preliminary performance measurement of BPTG_Cl@DSPE-PEG NPs,we focused on the recognition imaging and in vitro antibacterial effect of BPTG_Cl@DSPE-PEG NPs against Gram-positive and Gram-negative bacteria.The Confocal laser scanning microscope（CLSM）assay revealed that BG NPs could specifically light up Gram-positive bacteria,and the spread plate results revealed that 0.2μg/m L of BPTG_Cl@DSPE-PEG NPs could kill nearly 100%of Gram-positive bacteria within 20 min under white light（20.0m W/cm²）irradiation.However,BPTG_Cl@DSPE-PEG NPs have no significant effect on the imaging and killing of gram-negative bacteria.The antibacterial mechanism of BPTG_Cl@DSPE-PEG NPs was explored by isothermal titration calorimetry（ITC）,competitive binding assay,bacterial morphology,bacterial fluorescence staining,etc.The results showed that BPTG_Cl@DSPE-PEG NPs killed bacteria by specifically combinding with lipoteichoic acid（LTA）on the cell wall of Gram-positive bacteria and producing type I ROS under light.Moreover,the recognition and killing effect of BPTG_Cl@DSPE-PEG NPs on Staphylococcus aureus hosted in macrophages was deeply explored.The results showed that BPTG_Cl@DSPE-PEG NPs could also specifically recognize imaging and eliminate intracellular bacteria.This dissertation not only provides a drug molecule that specifically recognizes and photodynamically kills Gram-positive bacteria,but also expands the application of covalent organic backbone COF molecules in the biomedical field.