Harnessing the antibacterial properties of chitosan to tackle dental biofilms

Abstract

There are 2.3 billion people suffering from dental caries worldwide with 530 million children affected as well. Dental caries is a preventable disease but still highly prevalent, above all in developing countries. A strategy to prevent the formation of dental caries is maintaining daily oral hygiene including brushing teeth twice a day with fluoridated toothpaste and using dental floss and mouthwash to reach small gaps between teeth. The gold standard mouthwash used to prevent and treat dental caries contains chlorhexidine, an effective antiseptic but that shows toxicity to epithelial cells in acute and long-term use. Therefore, we developed a new natural and sustainable polymer, chitosan which has good antibacterial activity, low cost production, and low toxicity to prevent and treat the dental biofilms.
The antibacterial activity of chitosan is influenced by its molecular weight as chitosan consists of long polymeric chains which cause its poor solubility at physiological pH. Production of chitosan low molecular weight will increase its solubility and also its antibacterial activity. The protonated amino groups in the chitosan backbone are also responsible for the antibacterial activity. By conjugating ethylenimine and lysine moieties to the free amine groups of chitosan, these will remain protonated at physiological pH and have enhanced antibacterial properties against Streptococcus mutans, a caries-causing bacterium. Prepared polymers are able to bind high concentrations of fluoride ions that assist the anti demineralisation process and when formulated with peppermint oil into low hydroalcoholic concentration mouthwashes possess enhanced ability to inhibit formation of dental biofilms.
Successful modification of chitosan polymers was confirmed by NMR and infrared spectroscopy; modification led to changes in molecular weight that was quantified by GPC. The increased antibacterial activity of the polymer was demonstrated against Staphylococcus aureus and S. mutans. Fluoride concentration was determined by ion-selective electrode while the positive effect of fluoride in inhibiting the acid attack was demonstrated by vanadomolybdate reagent. In the second part of this study, the modified chitosan was formulated into a mouthwash, which was tested against S. mutans biofilm, and evaluated in terms of stability and cytotoxicity.
Results showed that the ethylenamine and lysine moiety were successfully incorporated into chitosan (CS3H EtNH₂ and CS3H Lys, respectively). However, the antibacterial activity of CS3H Lys showed higher activity compared to CS3H EtNH₂. The degree of substitution of lysine was 0.18 and this afforded a 30% enhancement in antibacterial activity based on MIC values. This modified chitosan was also successfully formulated into a mouthwash solution with effective anti-biofilm activity. The additional fluoride and peppermint oil did not enhance the anti-biofilm efficacy against S. mutans confirming the key role of our newly developed polymer. Cytotoxicity of chitosan polymers and chlorhexidine were investigated using human gingival fibroblast cells and the results showed that chlorhexidine showed toxicity even after dilution with artificial saliva, while all chitosan-based mouthwashes were cytocompatible. Novel chitosan-based mouthwashes (containing CS3H Lys and CS3H Lys F) are a safe and effective anti-biofilm product with ability to protect teeth from acid-caused demineralisation, have good stability at least up to 6 months of storage, and lower cytotoxicity compared to CHX mouthwash.

Date of Award	2021
Original language	English
Supervisor	Aikaterini Lalatsa (Supervisor) & Marta Roldo (Supervisor)