The Study Of Antibacterial Activity And Cytotoxicity Of The Novel Antibacterial Amino Acid-Porphyrins Photosensitizer 4I In Vitro

Objective(1)To evaluate the antibacterial activity of novel amino acid-porphyrin photosensitizer against eleven kinds of clinical isolated sensitive and multi-resistance strainsrandomly; comparing and analysing the difference antibacterial activity of 4I against the same kind of bacterial between sensitive bacterial and resistance bacterial.(2)To test cytotoxicity to Hacat and RAW264.7 cells for providing basis in vivo experiment.Methods(1) antibacterial activity of 4I test according to the clinical medicine sensitive test result,picking up single bacterial colony to make bacterial suspension,divided into four groups:PDT group(4I+ bacterial suspension+irradiation),non-PDT group(4I+ bacterial suspension),negative control group(bacterial suspension+ water+ irradiation),blank control group(medium+ water+irradiation),8 difference concentration groups,the maximum concentration is 62.5?M,the minimum concentration is 0.49?M,two-fold dilution methods to co-culture the difference concentration of photosensitizer with bacterial suspension for 30 minites in the dark,and then irradiated by red laser(650nm,6J/cm2) for 30 minites immediately,after irradiated,the MIC and MBC value of 4I to difference strains were evaluated using tablet coated plate method,calculating the ED50 and ED90 concentration; comparing and analyzing the difference results between sensitive and resistance bacterial.(2) cytotoxicity of 4I test culture and collect Hacat and RAW264.7 cells,seeding the cells into 96 wells plant,setting two groups a PDT(4I+medium+cells+ irradiation),non-PDT group(4I+medium+cells),every group is divided into control group(medium+cells + irradiation /non-irradation),blank group(medium+CCK-8) and 7 defference concentration groups,the maximum concentration is 62.5?M,the minimum concentration is 0.98?M,after adherent cultured 24 h,exchanging the cell medium with defference concentration 4I solutions,then co-cultured 30 minutes in the dark,a PDT group was irradiated 30 minutes immediately,the parameter is the same to antibacterial activity test,on the contrary,non-PDT group without irradiation.discarded the solution after irradiation,insteaded of CCK-8 assay 100?l per well.microplate system 450 nm measure OD value,recorded the results.Results(1)In this test,excepted Proteus mirabilis and Morganella strains,4I is efficient to all of the rest strains.Its antibacterial activity against Gram-positive bacteria is superior to Gram-negative bacteria(P<0.01).there is no variability of the effectiveness between sensitive and multi-resistance strains(P>0.05).the concentration of MIC50 and MIC90 value are respectively 0.822?M and 1.947?M;photosensitizer or irradiated by red laser alone showed no efficient to the bacterium.(2)irradiated by red laser alone showed promoting growth toward Hacat cells(P<0.001),and no effect on RAW264.7 cell(P=1.00).co-cultured with photosensitizer alone 30 minutes suggested mildly growth toward Hacat cells(P<0.05),but there is no significant difference between difference concentration groups.the higher concentration groups from 62.5?M to 15.6?M have a little inhibition to RAW264.7 cell(P<0.001),the others have no impact on growing.after PDT,Hacat and RAW264.7 cell survival rate dropped sharply,there is no significant difference between difference concentration groups.the IC50 value that 4I against Hacat and RAW264.7 cell after PDT are 1.846?M and 7.447?M respectively.Conclusions(1)the novel amino acid-porphyrin antibacterial photosensitizer 4I has high efficiency and broad spectrum antimicrobial properties,no cytotoxicity,low-cytotoxicity effectivenesseven after PDT,it is a promising antimicrobial photosensitizer.(2)in addition,it has the effect of promoting cell growth and the antimicrobial therapy of photosensitizer with red lower power laser has more broad clinical application prospect.