Antibacterial Effect Of Polylysine On Streptococcus Mutans

Background:Dental caries,among the most ubiquitous chronic diseases worldwide,is a complex biofilm-mediated bacterial infection.Streptococcus mutans（S.mutans）is one of the primary causative pathogens of caries.Traditional chemical antibacterial agents cause side effects.Antimicrobial peptides（AMPs）have great potential in the prevention of caries due to the advantages of not easily inducing drug resistance and good antibacterial activity.However,natural AMPs face problems such as high-cost production,low bioavailability,instability to proteases,and toxicity when systemically administered.In recent years,antibacterial polypeptides that mimic AMPs have attracted widespread attention due to the advantages of easy synthesis and modification,low-cost production,high stability to proteases,and good biocompatibility.In this study,we synthesized 5 homopolymeric polylysines with different hydrophobic end groups and degrees of polymerization to investigate their antibacterial effects on S.mutans and explore their potential in caries prevention.Objectives:In this study,5 polylysines(C_x-PLL_n:C₆-PLL₁₅,C₁₂-PLL₁₅,C₁₂-PLL₂₆,C₁₆-PLL₁₅and C₁₈-PLL₁₁)were synthesized,and their antibacterial effects,antibacterial mechanisms,inhibition of biofilm formation and reduction of cariogenicity were investigated to explore the effect of the polylysine structure on the antibacterial properties of S.mutans and screen the structure with the best antibacterial properties.Methods:A set of homopolymeric polylysines with different hydrophobic end groups and degrees of polymerization,C₆-PLL₁₅,C₁₂-PLL₁₅,C₁₂-PLL₂₆,C₁₆-PLL₁₅ and C₁₈-PLL₁₁were synthesized using primary amine initiators with different carbon chain lengths to initiate the ring-opening polymerization（ROP）ofε-carbobenzyloxy-l-lysine N-carboxyanhydride（ZLL-NCA）.After deprotection,the polylysines were obtained,which were characterized by proton nuclear magnetic resonance（~1H NMR）and gel permeation chromatography（GPC）.The minimum inhibitory concentration（MIC）and minimum bactericidal concentration（MBC）of polylysines were determined to evaluate their in vitro antibacterial properties.The colony-forming units（CFU）counting method was applied to evaluate the bactericidal kill-kinetics activity.The antibacterial mechanism was investigated using fluorescent probes on the inner and outer membranes of cells and transmission electron microscopy（TEM）.Through crystal violet（CV）staining experiment,scanning electron microscopy（SEM）experiment,live and dead staining of bacteria experiment and methyl thiazolyl tetrazolium（MTT）assay,the effect of synthetic polylysines on the formation of S.mutans biofilm was studied.The effect of polylysine on the cariogenic ability of S.mutans was investigated by measuring the acid and exopolysaccharides（EPS）production.The cytotoxicity of polylysines was evaluated by a cell counting kit-8（CCK-8）assay.Results:1.Antibacterial effect of C_x-PLL_n:1.1.MIC and MBC of polylysines:The MIC values of C₆-PLL₁₅,C₁₂-PLL₁₅,C₁₂-PLL₂₆ and C₁₆-PLL₁₅ were all 7.8μg/m L,and the MIC value of C₁₈-PLL₁₁ was 15.6μg/m L.The MBC values of each group was:C₆-PLL₁₅:15.6μg/m L;C₁₂-PLL₁₅:7.8μg/m L;C₁₂-PLL₂₆:31.2μg/m L;C₁₆-PLL₁₅:15.6μg/m L;C₁₈-PLL₁₁:15.6μg/m L.1.2.Bactericidal kill-kinetics activity of C_x-PLL_n on S.mutans:C₆-PLL₁₅ at the concentration of 15.6μg/m L exerted bactericidal effect within 3 to 4 h,killing all viable bacteria within 4 h.C₁₂-PLL₁₅ at the concentration of 7.8μg/m L exerted bactericidal effect within 2 to 3 h,killing all viable bacteria within 3 h.31.2μg/m L C₁₂-PLL₂₆ and 15.6μg/m L C₁₆-PLL₁₅/C₁₈-PLL₁₁ exerted bactericidal effect within 1 to 2 h,killing all viable bacteria within 2 h.2.Antibacterial mechanism of C₁₂-PLL₁₅:2.1.Effects of C₁₂-PLL₁₅ on outer membrane permeability of S.mutans:Since C₁₂-PLL₁₅ showed the best antibacterial activity,it was selected as a representative material for the study of antibacterial mechanism.The C₁₂-PLL₁₅-treated S.mutans solution displayed enhanced fluorescence of 8-anilino-1-naphthalenesulfonic acid（ANS）dye,indicating an increase in the outer membrane permeation.2.2.Effects of C₁₂-PLL₁₅ on depolarization of the inner membrane of S.mutans:The C₁₂-PLL₁₅-treated S.mutans solution displayed enhanced fluorescence of 3,3′-dipropylthiadicarbocyanine iodide[Di SC3（5）]dye,indicating the inner membrane depolarization.2.3.Effects of C₁₂-PLL₁₅ on the cell structure of S.mutans:The cell wall and membrane of C₁₂-PLL₁₅-treated S.mutans became blurry and disrupted,leaking cytoplasmic material spewed from the holes of membranes.3.Inhibitory effect of C_x-PLL_n on S.mutans biofilm formation:3.1.Biofilm biomass:C₆-PLL₁₅,C₁₂-PLL₁₅ and C₁₈-PLL₁₁ inhibited the formation of S.mutans biofilm at the concentration of 1/2 MIC,and C₆-PLL₁₅ exerted a better inhibitory effect on biofilm formation.The minimum biofilm inhibition concentrations（MBIC）that showed 90%or higher inhibition of biofilm formation(MBIC₉₀)of C_x-PLL_n were:C₆-PLL₁₅,C₁₂-PLL₁₅ and C₁₂-PLL₂₆:7.8μg/m L;C₁₆-PLL₁₅,C₁₈-PLL₁₁:15.6μg/m L.3.2.Morphology of biofilms and bacterial viability:Under the treatment of C_x-PLL_n at MBIC concentrations,biofilm formation was greatly reduced,and the biofilm structure was not visible,with a small number of planktonic dead bacteria and other substances still visible.3.3.Metabolism of biofilms:Low concentrations of C_x-PLL_n（lower than 3.91μg/m L）failed to reduce the biofilm viability and metabolic activity,but increased the biofilm metabolic rate instead.4.The effect of C_x-PLL_n on cariogenic ability of S.mutans:4.1.Effects of C_x-PLL_n on acid production by S.mutans:The p H values of planktonic S.mutans treated with 3.9μg/m L of C₆-PLL₁₅/C₁₂-PLL₁₅ remained neutral within 24 h.In the first 12 h,the p H values of planktonic S.mutans treated with 1.95μg/m L C₆-PLL₁₅/C₁₂-PLL₁₅could be maintained at about 7,and the acid-suppressing ability decreased after 12 h,reaching a p H similar to the control group after 24 h.The other group of C_x-PLL_n could only reduce the acid production rate of S.mutans to a certain extent.4.2.Effects of C_x-PLL_n on the ability of S.mutans to produce EPS:C₆-PLL₁₅,C₁₂-PLL₁₅ and C₁₆-PLL₁₅ significantly inhibited the ability of S.mutans to produce EPS when the concentration was higher than 3.91μg/m L.5.Cytotoxicity of C_x-PLL_n:When the concentration was lower than 62.5μg/m L,C₆-PLL₁₅ had no obvious cytotoxicity;when the concentration was lower than 31.2μg/m L,C₁₂-PLL₁₅,and C₁₂-PLL₂₆ had no obvious cytotoxicity;when the concentration was lower than 15.6μg/m L,C₁₆-PLL₁₅ had no obvious cytotoxicity;when the concentration was higher than 3.91μg/m L,C₁₈-PLL₁₁exhibited cytotoxicity.The 50%inhibitory concentrations(IC₅₀)of C₆-PLL₁₅on MC-3T3 ranged from 62.5 to 125μg/m L;the IC₅₀ values of C₁₂-PLL₁₅and C₁₂-PLL₂₆ were 125μg/m L;the IC₅₀ values of C₁₆-PLL₁₅ and C₁₈-PLL₁₁were 31.2μg/m L.Conclusions:1.Polylysines,C₆-PLL₁₅,C₁₂-PLL₁₅,C₁₂-PLL₂₆,C₁₆-PLL₁₅ and C₁₈-PLL₁₁ exhibit bactericidal effects on S.mutans,among which C₁₂-PLL₁₅ is most effective（MBC:7.8μg/m L）.2.C₆-PLL₁₅ exhibits the best effect on inhibiting the biofilm formation of S.mutans(MBIC₅₀:3.9μg/m L).3.C₆-PLL₁₅ and C₁₂-PLL₁₅ significantly inhibit the ability of S.mutans to produce acid and synthesize EPS.4.C₆-PLL₁₅ and C₁₂-PLL₁₅ inhibit S.mutans effectively with good biocompatibility under certain concentration.