# Peptide Mediated Antimicrobial Dental Adhesive System
## 3 Results and Discussion
### Bacterial Viability on Treated Discs  
We first explored if the resin formulations have any antibacterial effects, we prepared discs using both commercial analogue and \(\varepsilon\)-pL polymerized discs. The discs were soaked in water overnight for a baseline control. Neither formulation exhibited any innate antibacterial activity against _S. mutans_. We next tested the antibacterial activity on the discs soaked in antimicrobial peptide solutions across a range of concentrations up to 50\(\times\) the minimum inhibitory concentration (MIC) (1560 \(\mathrm{\SIUnitSymbolMicro mL}\)). The \(\varepsilon\)-pL discs soaked with peptide solutions exhibited significant antibacterial activity against _S. mutans_. Complete bactericidal efficacy was observed for \(\varepsilon\)-pL discs soaked in GH12-M2 peptide at 25\(\times\) MIC value (780 \(\mathrm{\SIUnitSymbolMicro mL}\)). Near complete reduction in _S. mutans_ growth was observed at 12.5\(\times\) MIC (390 \(\mathrm{\SIUnitSymbolMicro g/mL}\)), however antibacterial activity was not statistically significant at concentrations of 6.25\(\times\) MIC (195 \(\mathrm{\SIUnitSymbolMicro g/mL}\)) and below for the GH12-M2 peptide. The positive control studies included discs that are soaked in chlorhexidine (CHX), where robust antimicrobial property was observed for both discs, but the CHX showed higher growth inhibition in the commercial analogue discs as compared to the \(\varepsilon\)-pL formulation discs. CHX-soaked discs exhibited complete bactericidal efficacy against _S. mutans_ at 25\(\times\) and 12.5\(\times\) MIC concentration for each disc formulation. While, the CHX was effective in both disc formulations at different levels, GH12-M2 peptide demonstrated antibacterial activity in disc formulations featuring \(\varepsilon\)-pL. These results indicate a critical dependence on the adhesive formulation to incorporate functional peptides. Though the \(\varepsilon\)-pL discs coupled with GH12-M2 peptide demonstrated bactericidal efficacy at concentrations of 390 \(\mathrm{\SIUnitSymbolMicro g/mL}\) and 780 \(\mathrm{\SIUnitSymbolMicro g/mL}\), commercial analogue discs coupled with peptide at these concentrations did not show any significant bactericidal efficacy. Engineering the peptide to the adhesive formulation has a synergetic antibacterial effect when incorporated into \(\varepsilon\)-PL adhesive system, while commercial analogue for the same peptide concentrations did not show similar synergetic effect (Figure 5). The results demonstrate the importance of biocompatible adhesive system to induce antimicrobial effects through novel antimicrobial agents. An increased occurrence of affinity and non-specific interactions of the GH12-M2 peptide with commercial analogue resin discs was noted based upon the observed reduction in bacterial viability at peptide concentrations of 390 \(\upmu\)g/mL and 780 \(\upmu\)g/mL, as seen in Figure 5b. The hydropathy of the resin with \(\varepsilon\)-polyllysine is hydrophilic and highly positively charged, unlike bacterial membranes. Since the commercial analogue is more hydrophobic, the peptide is likely adsorbing to the resin with the similar hydrophobic interactions, such as hydrogen bonding and van der Waal forces, that occur in the bacterial membrane [92]. The delivery mechanism of the antimicrobial agents, either the peptide or the CHX, is based on the non-specific adsorption of the agent to the resin. The antimicrobial agents are coupled to the resin through adsorption overnight before the bacteria are added to the resin material. Unbound agent is also rinsed off before bacteria are added. Therefore, only the adsorbed agent is available for any bactericidal effect. However, the resin must act as a delivery system so that the adsorbed  
Figure 5: **(a)** Schematic of adhesive system samples starting with commercial analogue. Experiments included the addition of GH12-M2 antimicrobial peptide onto the discs with \(\varepsilon\)-polyllysine to see bacterial inhibition.