Development of a Novel Peptide with Antimicrobial and Mineralising Properties for Caries Management

Abstract

The purpose of the study is to develop a novel peptide for caries management. Gallic-Acid-Polyphemusin-I (GAPI) was synthesised by grafting Polyphemusin I (PI) and gallic acid (GA). Biocompatibility was evaluated using a Cell Counting Kit-8 Assay. Antimicrobial properties were assessed using minimum inhibitory concentration (MIC) and minimum bactericidal/fungicidal concentration (MBC/MFC). The bacterial and fungal morphology after GAPI treatment was investigated using transmission electron microscopy (TEM). The architecture of a consortium biofilm consisting of Streptococcus mutans, Lacticaseibacillus casei and Candida albicans was evaluated using scanning electron microscopy (SEM) and confocal laser scanning microscopy. The growth kinetics of the biofilm was examined using a propidium monoazide-quantitative polymerase chain reaction. The surface and calcium-to-phosphorus molar ratio of GAPI-treated enamel after pH cycling were examined with SEM and energy-dispersive X-ray spectroscopy. Enamel crystal characteristics were analysed using X-ray diffraction. Lesion depths representing the enamel's mineral loss were assessed using micro-computed tomography. The MIC of GAPI against S. mutans, L. casei and C. albicans were 40 μM, 40 μM and 20 μM, respectively. GAPI destroyed the biofilm's three-dimensional structure and inhibited the growth of the biofilm. SEM showed that enamel treated with GAPI had a relatively smooth surface compared to that treated with water. The calcium-to-phosphorus molar ratio of enamel treated with GAPI was higher than that of the control. The lesion depths and mineral loss of the GAPI-treated enamel were less than the control. The crystallinity of the GAPI-treated enamel was higher than the control. This study developed a biocompatible, mineralising and antimicrobial peptide GAPI, which may have potential as an anti-caries agent.

Keywords: antimicrobial; caries; mineralisation; peptides; prevention.

Figure 1

TEM micrographs of the bacteria and the fungus before and after GAPI treatment. Abnormal cell morphology; ↖ cytoplasmic clear zone; disrupted membrane/cell wall; and cytoplasmic content leakage.

Figure 2

Three-species biofilm with or without GAPI peptide treatment. (A) Scanning electron micrographs of the biofilm with or without GAPI treatment. GAPI treatment inhibited biofilm formation and altered cell morphology. (B) Confocal laser scanning micrographs of the biofilm with or without GAPI treatment. GAPI reduced the microorganisms. C. albicans (green) could be distinguished from bacterial cells (pink). (C) Log10 cell counts/biofilm of the bacteria/fungus in a three-species biofilm with or without GAPI peptide (n = 6 per group).

Figure 3

Enamel treated with 160 μM GAPI, 160 μM GA, 160 μM PI or deionised water (Water). (A) Scanning electron micrographs of the enamel in four groups. (B) Microcomputed tomographs of the enamel in four groups. (C) X-ray diffraction patterns of the enamel in four groups.