The Study Of Photodynamic Antimicrobial Activity And Mechanism Of Cationic Porphyrin Photosensitizer

This paper involves four parts:Part 1 Computational chemistry study of active compounds of PACT.In order to explore the quantitative structure-property relationships(QSPR) of photosensitizers and active compounds of PACT. Based upon computational chemistry method, we analyzed 150 photosensitizers and 202 photodynamic antimicrobial activity compounds. The multi-dimensional chemical space was mapped to two-dimensional space by principal component analytical method, the aim was to reveal the chemical space distributions of photodynamic antimicrobial descriptors. Results indicated that most active compounds of PACT did not follow the five rules of Lipinski, so we preliminary summarized the principles of similarity evaluation which could be applied to photosensitizers were the following 5 items:the molecular weight is less than 1500; the number of Hbond donor is less than 10; the number of Hbond acceptor is less than 12; Alop is less than 8; the number of rotatable bond is less than 15. According to the principal component analysis method, photosensitizing agents and active compounds of PACT had some parallel chemical spaces and also some differences in physical and chemical properties.Part 2 Content determination of cationic porphyrin photosensitizer LD4First RP-HPLC method was established to determined the content of LD4, results showed that the linear range was 1.55-95μg/ml with the correlation coefficient of 0.9994. The RSDs of precision, reproducibility and stability tests were no more than 1.0%. The limit of detection and quantitation were 1.1 and 3.8 ng, respectively. The recoveries were in the range of 99.00%～101.60%(RSD=0.89%, n=9). The established methods are simple, rapid, accurate, specific and can be used for the content determination of LD4.Part 3 The PACT of LD4 on Pseudomonas aeruginosa in vitro.To investigate the photodynamic antimicrobial activity and mechanism of cationic porphyrin photosensitizer on P. aeruginosa., surrounding PACT of LD4 on Pseudomonas aeruginosa in vitro, the following factors were studied:the impacts of culture environments such as bacterial inoculum, pH of medium and serum protein content on minimum inhibitory concentration (MIC) of LD4 When the bacteria inoculum size was less than 106 CFU/mL, the value of MIC kept no change in light and dark reaction, when the bacteria inoculum size was more than 106 CFU/mL, the value of MIC became greater. Increasing the pH of culture medium would decrease the activity of antimicrobial effect of LD4-PACT. The influence of serum protein was different between light and dark reaction, increasing the concentration of serum protein would decrease the LD4-PACT, while enhancing the dark reaction. The effect of laser energy density had a great effect on the LD4-PACT, the increasing laser energy density had high inactivation effect of P. aeruginosa at the same LD4 concentration. Post-antibiotic effect of LD4-PACT was dose-dependent.2h after LD4-PACT, the survival P. aeruginosa will start to proliferate, going through the exponential phase to the stationary phase. Bacterial viability was evaluated by the flow cytometry method via using the LIVE/DEAD BacLighTM bacterial viability kit. The proportion of viable bacteria is decreased with the photosensitizer dose-dependent. The morphology and microstructure of P. aeruginosa after LD4-PACT was demonstrated by microscope including laser scanning confocal microscope, atomic force microscope and scanning electron microscope. Results showed that LD4-PACT destroyed the bacterial membrance and causing the leakage of cell components. The photo-toxicty of LD4 among normal cells was evaluated on cell line L929 (Mice fibroblast cells) by MTT assay. The IC50 is 7.08μM, while the viability of L929 cell was more than 80% at 3.9μM.Part 4 The PACT of LD4 on Pseudomonas aeruginosa in vivo.To study the effect of photodynamic antimicrobial of wound infection in rats. Experiment in vivo was conducted with the wound rats infected with P. aeruginosa as model, bacterial counts in wounds were determined by the colony counting method, the dimensions of wounds were measured by vernier caliper, based on which the wounds area and wound healing rate were calculated. Inflammatory cytokines IL-6 and TNF-a in the tissue were measured by Elisa assay. Results showed that colony counts of wound decreased and wound healing accelerated in treatment groups, compared with control group. The expression of the inflammatory cytokines IL-6 and TNF-a was down-regulated, reducing the inflammation of the wound and accelerating angiogenesis and wound healing. The effect of PACT treated with high dose photosensitizer was notable and better than antibiotic treatment group.