Antimicrobial peptide-loaded liquid crystalline precursor bioadhesive system for the prevention of dental caries

Abstract

Background: Anticaries agents must interfere with the adhesion of Streptococcus mutans and its proliferation in dental biofilm, without causing host toxicity and bacterial resistance. Natural substances, including cationic antimicrobial peptides (CAMPs) and their fragments, such as β-defensin-3 peptide fragment (D1-23), have been widely studied. However, the chemical and physical stability of CAMPs may be compromised by external factors, such as temperature and pH, reducing the period of antimicrobial activity.

Methods: To overcome the aforementioned disadvantage, this study developed and character-ized a drug delivery system and evaluated the cytotoxicity and effect against S. mutans biofilm of a D1-23-loaded bioadhesive liquid crystalline system (LCS). LCS was composed of oleic acid, polyoxypropylene-(5)-polyoxyethylene-(20)-cetyl alcohol, Carbopol^® 974P and Carbopol^® 971P. LCS was analyzed by polarized light microscopy (PLM), rheology (viscoelasticity and flow properties) and in vitro bioadhesion. The viability of epithelial cells was evaluated. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against S. mutans were determined for D1-23 for further evaluation of the effect against S. mutans biofilm after 4 and 24 h of exposure to treatments.

Results: PLM, rheology, and in vitro bioadhesion tests showed that both viscosity and bioadhesion of LCS increased after it was diluted with artificial saliva. D1-23-loaded LCS system presented better activity against S. mutans biofilm after 24 h when compared to 4 h of treatment, showing a cumulative effect. Neither LCS nor D1-23-loaded LCS presented toxicity on human epithelial cells.

Conclusion: D1-23-loaded LCS is a promising drug delivery system for the prevention of dental caries.

Keywords: Streptococcus mutans; biofilm; dental caries; drug delivery system; peptide fragments.

Figure 1

Photomicrographs representing the effect of artificial saliva in the structure of F (liquid crystal system) by light polarized microscopy at 20% magnification. Notes: F is the liquid crystal system; F10 is a 0.1:1 (wt/wt) dilution of F containing 10% artificial saliva; F30 is a 0.3:1 (wt/wt) dilution of F containing 30% artificial saliva; F50 is a 0.5:1 (wt/wt) dilution of F containing 50% artificial saliva; and F100 is a 1:1 (wt/wt) dilution of F containing 100% artificial saliva.

Figure 2

Rheograms of formulations F (squares) and F100 (triangles). Notes: Ascendant curve, filled symbols; descendent curve, empty symbols. F is the liquid crystal system; F100 is a 1:1 (wt/wt) dilution of F containing 100% artificial saliva.

Figure 3

Variation of the storage module (G′, filled symbols) and loss module (G″, empty symbols) as a function of the frequency to F and F100. Notes: F is the liquid crystal system; F100 is a 1:1 (wt/wt) dilution of F containing 100% artificial saliva.

Figure 4

Boxplot of the percentage of biofilm biomass reduction after 4 and 24 h of the treatment. Notes: Bars indicate minimum and maximum values. Boxes indicate lower and upper quartiles. Line in the middle of boxes is median. ^A–DDifferent letters show statistical difference among the groups, according to Kruskal–Wallis/Mann–Whitney tests (p<0.05). F, liquid crystalline formulation; D1–23, peptide D1–23; F+D1–23, formulation+peptide D1–23; F+CHX, formulation+CHX. Abbreviation: CHX, chlorhexidine diacetate.

Figure 5

Mean (SD) of the percentage of epithelial cell viability after 24 h of the treatment. Notes: ^A–DDifferent uppercase letters show statistical difference among the concentrations of the same agent, according to ANOVA/Tukey tests (p,0.05). ^a,bDifferent lowercase letters show statistical difference among CHX and D1–23 or F+CHX and F+D1–23, according to Student’s t-test (p<0.05). F, liquid crystalline formulation. Abbreviations: ANOVA, analysis of variance; CHX, chlorhexidine diacetate.