Engineered Chimeric Peptides With Antimicrobial And Titanium-Binding Functions To Inhibit Biofilm Formation On Ti Implants

Objective:The biofilm formation on the surface of oral implants can easily lead to the occurrence of diseases around the implant,which can eventually lead to the loss of implants and the failure of implants.The early attachment of streptococci on the surface of the implant is the basis of biofilm formation,thus inhibiting the initial adhesion of streptococci on the surface of the implant and interfering with the subsequent biofilm formation,which is conducive to the formation of osseointegration at the initial stage of implant and the initial stability of the implant.Human beta-defensin-3(h BD-3)is a human-made cationic antimicrobial peptide with a broad spectrum of antibacterial properties.Peptide: RKLPDAGPMHTW(Ti-binding peptide-1,TBP-1)can specifically recognize and stably bind to titanium surface.To realize the simple process of environmental-friendly implant surface modification,here,chimeric peptides(TBP-1-GGG-h BD3-1、TBP-1-GGG-h BD3-2、TBP-1-GGG-h BD3-3)were designed by connecting three fragments(h BD-3-1/2/3)derived from human β-defensin-3(h BD-3)with TBP-l via a triple glycine(G)linker.The synthesized peptides can not only adsorb on the surface of titanium plate,but also can effectively exert antibacterial effect,and it is not easy to produce drug resistance.Using the synthesized peptides to modify Ti surface and then to characterize the surfaces and evaluate its the cytology and bacteriology effect in vitro.Methods:1.The structures and basic properties of the peptides were predicted using PSIPRED software and the peptide property calculator.In addition,circular dichroism(CD)and Raman spectroscopy experiments were conducted to further analyze the secondary structure of the peptides.The MIC,MBC and biofilm susceptibility test experiments were performed to investigate antibacterial activity of the chimeric peptide against Streptococcus oralis(S.oralis),Streptococcus gordonii(S.gordonii),Streptococcus sanguinis(S.sanguinis).2.Using X-ray photoelectron spectroscopy(XPS),the properties of individual domains of the chimeric peptides were evaluated for their binding activity toward the Ti surface.The antimicrobial and anti-biofilm efficacy of the peptides against initial settlers,S.oralis,S.gordonii and S.sanguinis,was evaluated with confocal laser scanning microscopy(CLSM),antibacterial efficiency experiments,and scanning electron microscopy(SEM).Electron microscopy(TEM)was used to observe the changes of bacterial membranes,and real-time fluorescence quantitative PCR(q RT-PCR)experiments were used to explore the underlying antibacterial mechanism of chimeric peptides.3.MC3T3-E1 cells were inoculated on the surfaces of chimeric peptide-modified titanium plate samples after culture for 1 d,3 d,5 d,and 7 d,and then CCK-8 and AO/EB fluorescence staining was used to detect cell proliferation and evaluate the biocompatibility of chimeric peptides.Results:1.Software predictions,CD spectra and Raman spectra showed that TBP-1-GGG-h BD3-1,TBP-1-GGG-h BD3-2,TBP-1-GGG-h BD3-3 all had hydrophilic,hydrophobic groups and good solubility,secondary structures related to antibacterial properties(alpha helices,beta sheets)and positive charge.Among them TBP-1-GGG-h BD3-3 had more hydrophilic groups and higher positive charge(+8.9).MIC,MBC,and biofilm susceptibility results showed that TBP-1-GGG-h BD3-1,TBP-1-GGG-h BD3-2,TBP-1-GGG-h BD3-3 had antibacterial effects on the growth of TBP-1-GGG-h BD3-3 performed the strongest antibacterial properties against S.oralis,S.gordonii and S.sanguinis in planktonic and biofilm state than other peptides.2.XPS results showed that TBP-1-GGG-h BD3-1,TBP-1-GGG-h BD3-2,and TBP-1-GGG-h BD3-3 all adsorbed on the surface.The results of CLSM and antibacterial efficiency(R)on the surface of titanium film showed that TBP-1-GGG-h BD3-3 could still be stably modified on the surface of titanium plate in the presence of serum or saliva.SEM results showed the decrease of biomass and bacterial morphology destruction.TEM results showed TBP-1-GGG-h BD3-3 disrupted the plasma membrane and formed pore-like channels with overflowing of cell contents.Which suggested chimeric peptides might through act on cell membranes to present antibacterial activity.q RT-PCR results showed TBP-1-GGG-h BD3-3 could inhibit the expression of ssp A and ssp B genes and thus affected the S.gordonii adhesion on the surface of titanium samples.3.The results of CCK-8 and CLSM showed that the proliferation of MC3T3-E1 cells on the surface of the chimeric peptide-modified titanium samples was not significantly different from that of the control group,which indicating that TBP-1-GGG-h BD3-3 had good biocompatibility.Conclusion:The study showed the chimeric peptides TBP-1-GGG-h BD3-1,TBP-1-GGG-h BD3-2 and TBP-1-GGG-h BD3-3 all had bifunctional properties that could specifically adsorb on titanium and exert its antibacterial activity and had good biocompatibility.Therefore,engineered chimeric peptides with antimicrobial activity provide a potential solution for inhibiting the growth of initial adherent streptococci and biofilm formation on Ti surfaces to reduce or prevent the occurrence of peri-implant diseases and thus increasing the success rate of implants.