# Peptide Mediated Antimicrobial Dental Adhesive System
## 3 Results and Discussion  
### Antibacterial Activity of the Peptide  
Table 2 and Figure 1 provide the antimicrobial efficacy of the peptides explored in this study. In addition to the GH12 peptide sequence we explored the sequence modifications for this peptide for effective delivery into the adhesive formulations [67]. We first synthesized a sequence featuringa lysine residue, attached via a spacer sequence [43] then we changed the C-terminal of the peptide to COOH since this would replace the adhesive's lost carboxy functional groups due to the peptide conjugation. The next design choice was aimed to select the peptide terminus to attach the spacer sequence that would allow for optimal antimicrobial activity. As the control, we used the GH12-NH\({}_{2}\) (with an C-terminal amide), the reported MIC value of GH12-NH\({}_{2}\) was about 6.7 \(\upmu\)g/mL against _S. mutans_[67]. The GH12 peptide was synthesized to feature a carboxyl group to allow for further sequence modifications involving additional amine-containing moieties such as polypeptide chains or monomers. Next, we designed GH12-M1 to include a spacer domain featuring a single lysine residue at the N-terminal for coupling to the polymer. Additionally, a GH12-M2 peptide was created with the same spacer sequence and single lysine residue as GH12-M1, but placed at the C-terminus of the peptide.  
The activity of each peptide was evaluated against _S. mutans_ UA159 using an inhibitory concentration (IC\({}_{50}\)) assay. The minimum inhibitory concentration (MIC) values for all synthesized peptides were calculated at 31.7 \(\upmu\)g/mL (Figures S1-S5). CHX was used as a positive control and exhibited an IC\({}_{50}\) value of 0.37 \(\upmu\)g/mL. The IC\({}_{50}\) of GH12, GH12-M2, and GH12-M1 were found 11.6, 12.8, and 17.1 \(\upmu\)g/mL, respectively. We next investigated the addition of \(\upvarepsilon\)-PL to our formulation which resulted in the IC\({}_{50}\) value about 71.3 \(\upmu\)g/mL. The N-terminal modification (GH12-M1) demonstrated a larger IC\({}_{50}\) value indicating reduced activity when compared to the unmodified peptide (See Table 2). The spacer modification carried out at the C-terminal (GH12-M2) resulted in a slight increase in the IC\({}_{50}\) value as compared to the unmodified form. GH12-M2 was therefore selected for further coupling studies due to its superior antimicrobial efficacy as well as flexibility allowing for more direct coupling to an adhesive formulation.  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Peptide** & **Sequence** & \
\begin{tabular}{c} _S. mutans_ \\ **IC\({}_{50}\)** (\(\upmu\)g/mL)** \\ \end{tabular} &
\begin{tabular}{c} _S. Mutans_ \\ **MIC (\(\upmu\)g/mL)** \\ \end{tabular} \\ \hline GH12-NH\({}_{2}\) & GLLWHLHHLLH (-NH\({}_{2}\) ) & - & 6.7 \({}^{*}\) \\ GH12 & GLLWHLLHHLH (-COOH) & 11.6 & 31.3 \\ GH12-M1 & K\_GGGSG\_GLLWHLHHLLH & 17.1 & 31.3 \\ GH12-M2 & GLLWHLHLHLH\_GSGGG\_K & 12.8 & 31.3 \\ \(\upvarepsilon\)-Polyllysine & (\(\upvarepsilon\)-Lysine)\({}_{n}\) & 71.3 & 125 \\ \hline \hline \end{tabular} \({}^{*}\) As reported by [PERSON], et al., in a 2016 study.  
\end{table}
Table 2: Properties of antimicrobial peptides, i.e., GH12, and its modifications, and \(\upvarepsilon\)-polyllysine.  
Figure 1: Antibacterial activity of chlorhexidine (CHX), antibacterial peptides and \(\upvarepsilon\)-polyllysine against _S. mutans_.