Cationic Amphiphilic Polymers with Antimicrobial Activity for Oral Care Applications: Eradication of S. mutans Biofilm

Abstract

The antibacterial and antibiofilm activities of cationic amphiphilic methacrylate polymers against cariogenic bacterium S. mutans were investigated. Cationic homopolymer PE₀ and copolymer PE₃₁ containing 31 mol % of ethyl methacrylate were synthesized by reversible addition-fragmentation chain transfer polymerization. These polymers displayed bactericidal activity toward S. mutans and prevented biofilm formation by killing the planktonic bacteria. At a concentration of 1000 μg/mL when incubated for 2 h the polymers reduced >80% of biofilm biomass. When the polymer assay solution with the biofilm was vigorously mixed using a pipet for 30 s, >50% of biofilm mass was removed at a polymer concentration of 250 μg/mL. Chlorhexidine and a cationic surfactant failed to reduce the biofilm mass at the same concentration. PE₀ was the most effective in removing biofilm and did not show any significant cytotoxicity to human gingival fibroblast and periodontal ligament stem cells when incubated for 10 min.

Figure 1

Antimicrobial peptide-mimetic methacrylate polymers. (A) Synthesis of cationic amphiphilic methacrylate random copolymer (PE₃₁) by reversible addition fragmentation chain transfer (RAFT) polymerization. (B) Chemical structure of cationic homopolymer PE₀.

Figure 2

Bactericidal kinetics of polymers, chlorhexidine (CHX), and vancomycin (VAN) against S. mutans at twice MIC. The bacterial cell counts below 1000 cfu/mL are presented by open markers at 1000 cfu/mL. 0.001% acetic acid was tested as solvent control. [PE₀]= 125 μg/mL, [PE₃₁]= 15.6 μg/mL, [CHX]= 0.8 μg/mL and [VAN]= 1.6 μg/mL.

Figure 3

Prevention of S. mutans biofilm formation by antimicrobial polymers. After 24 h incubation with PE₀ (A), PE₃₁ (B) or chlorhexidine (CHX) (C), the formation of biofilm was determined by CV staining (filled markers), and planktonic bacterial growth in solution was determined by OD₆₀₀ (opened markers), relative to solvents as 100% (0.001% acetic acid for PE₀ and PE₃₁ and water for CHX).

Figure 4

Eradication of pre-formed S. mutans biofilm by antimicrobial polymers. One-day matured S. mutans biofilm was incubated with the polymers PE₀ and PE₃₁ or chlorhexidine (CHX), and the mass of remaining biofilm was determined by CV staining after 2- (A) or 24- (B) hour incubation, relative to solvent treatment as 100% (0.001% acetic acid for PE₀ and PE₃₁ and water for CHX).

Figure 5

Eradiation of S. mutans biofilm by swishing treatment with antimicrobial polymers for 30 seconds. (A) Schematic presentation of swishing treatment of biofilm by vigorously pipetting for 30 seconds. (B) The effect of solvents (0.001% acetic acid and water) on biofilm eradication. (C) The biomass of remaining biofilm after 30 seconds treatment of polymers, chlorhexidine (CHX) and surfactant cetyltrimethylammonium bromide (CTAB) measured by CV staining. [Polymer, CHX, or CTAB] = 62.5, 125 and 250 μg/mL. *p< 0.05.

Figure 6

S. mutans biofilm images after treatment with antimicrobial polymers and chlorhexidine (CHX). The biofilms after antimicrobial treatment were stained by a LIVE/DEAD® staining kit and imaged by confocal scanning laser microscopy. Green color (SYTO® 9) showed live bacterial cells, and red/orange color (propidium iodide) showed dead bacterial cells. 0.001% acetic acid was tested as solvent control. [Polymer or CHX] = 125 μg/mL. Bar = 30 μm.

Figure 7

Cytotoxicity of antimicrobial polymers and chlorhexidine (CHX) against human gingival fibroblasts (hGFs) (A) and periodontal ligament stem cells (PDLSCs) (B). Cell viability was determined against hGFs and PDLSCs after incubation with the polymers or chlorhexidine for 10 min, relative to solvents as 100% cell viability (0.001% acetic acid for PE₀ and PE₃₁ and water for CHX).