Mitigation of peri-implantitis by rational design of bifunctional peptides with antimicrobial properties

Abstract

The integration of molecular and cell biology with materials science has led to strategies to improve the interface between dental implants with the surrounding soft and hard tissues in order to replace missing teeth and restore mastication. More than 3 million implants have been placed in the US alone and this number is rising by 500,000/year. Peri-implantitis, an inflammatory response to oral pathogens growing on the implant surface threatens to reduce service life leading to eventual implant failure, and such an outcome will have adverse impact on public health and create significant health care costs. Here we report a predictive approach to peptide design, which enabled us to engineer a bifunctional peptide to combat bacterial colonization and biofilm formation, reducing the adverse host inflammatory immune response that destroys the tissue surrounding implants and shortens their lifespans. This bifunctional peptide contains a titanium-binding domain that recognizes and binds with high affinity to titanium implant surfaces, fused through a rigid spacer domain with an antimicrobial domain. By varying the antimicrobial peptide domain, we were able to predict the properties of the resulting bifunctional peptides in their entirety by analyzing the sequence-structure-function relationship. These bifunctional peptides achieve: 1) nearly 100% surface coverage within minutes, a timeframe suitable for their clinical application to existing implants; 2) nearly 100% binding to a titanium surface even in the presence of contaminating serum protein; 3) durability to brushing with a commercially available electric toothbrush; and 4) retention of antimicrobial activity on the implant surface following bacterial challenge. A bifunctional peptide film can be applied to both new implants and/or repeatedly applied to previously placed implants to control bacterial colonization mitigating peri-implant disease that threatens dental implant longevity.

Keywords: Antimicrobial; Bifunctional Peptides; Dental Implant; Function; Peptide Film; Peri-implant disease; Stability; Structure; Titanium/Ti Alloy Implant.

Figure 1:. Helical wheel predictions of bifunctional peptides.

Hydrophilic amino acid residues are represented as circles, hydrophobic amino acid residues as diamonds, potentially negatively charged residues as triangles, and potentially positively charged residues as pentagons. The most hydrophobic amino acid residue is shown in green with the chroma intensity decreasing proportionally to hydrophobicity, with zero hydrophobicity coded as yellow. Hydrophilic residues are coded red, with intense red chroma being the most hydrophilic (uncharged) residue, and the chroma decreasing proportionally to the hydrophilicity. Potentially charged residues are shown as blue.

Figure 2:. DynaMine classification for backbone dynamics of amino acids comprising each bifunctional peptide.

The AMP domains located on the C’-terminus represent a more ordered region relative to the TiBP binding domain located on the N’-terminus. The TiBP domain is an intrinsically disordered peptide. AMPA has more order than GL13K, which could contribute to its greater predicted antimicrobial function.

Figure 3:. Secondary structure models and structural similarity analysis.

Each TiBP domain is colored purple, the AMPA domain is colored orange, the GL13K domain is colored red, and the spacer domain linking the antimicrobial and binding domain is colored black. The chart depicts the structural similarity determined by superimposing the domain model over the bifunctional model and calculating the percent identity.

Figure 4:. Theoretical and experimental CD spectra with deconvolution using Beta Sheet Selection (BeStSel).

Experimental CD spectra were collected in aqueous environment and with increasing concentrations of 2,2,2-trifluoroethanol. The pie charts represent BeStSel’s deconvolution of the CD spectra.

Figure 5:. Fluorescent microscopy images of bifunctional peptide binding to titanium implant discs, binding with competition from BSA, and durability following 1 minute of brushing with an electric toothbrush.

The chart depicts the means and standard deviations of three replicate experiments for each bifunctional peptide in each condition. TiBP-AMPA binding was statistically significant compared to binding in competition with BSA and durability after 1-minute of brushing (p < 0.05). Statistical significance was determined for all conditions of TiBP-GL13K bifunctional peptide (p < 0.05). Statistical analysis was conducted using a one-way ANOVA.

Figure 6:

Visualization of FITC labeled bifunctional peptides using fluorescence microscopy after challenge by S. mutans for 24 hours. The percentage of peptide coverage was determined by evaluating images with a MATLAB script. The chart represents results obtained during three replicate experiments, of which, the fluorescence images are selected as representative of the whole. A statistically significant difference (p < 0.05) was found between the means for TiBP-AMPA and TiBP-GL13K coverage using a one-way ANOVA.

Figure 7:. Fluorescence microscopy images and quantification of propidium iodide (PI) staining of dead S. mutans bacteria on implant discs after challenge for 24 hours.

Dead bacteria appear with red fluorescence. The means and standard deviations are depicted in the chart for bare, sterilized titanium discs and discs functionalized by 2 minutes of bifunctional peptide binding at 37°C prior to bacterial challenge. Three replicate experiments were performed and a statistically significant difference (p < 0.05) was observed between means using ANOVA.

Scheme 1.

Our approach includes an antimicrobial peptide film based upon an engineered bifunctional peptide composed of peptide domains for implant binding and antimicrobial activity separated by a spacer. The peptide was tested using a variety of in vitro assays to demonstrate its suitability.