Chitosan is a linear biopolymer which has been extensively studied in recent years for its inherent properties like biocompatibility, non-toxicity, biodegradability and antimicrobial activity. Previous studies have reported the synthetic modification of chitosan either at the amino group or at the C-3 and C-6 hydroxyl groups to enhance their solubility and bioactivity. However, most studies reported the chemical modification on native chitosan in aqueous medium resulting in heterogeneous products. Therefore, in our study, we aim to utilize 3,6-di-O-TBDMS protected chitosan as a precursor for selective modification of the 2-amino group in organic medium.
The aim of this study was to introduce specific functionalities at different degrees of substitution at the 2-amino position to obtain derivatives having improved solubility and antimicrobial activity. To introduce permanent positive charge on the polymer backbone, we quaternized the amino group using trimethylation and introduced cationic moieties like guanidine, pyridiniumyl or trimethylammoniumyl groups. We inserted the hydrophobic group in two ways- either within the quaternary ammoniumyl (cationic) group itself or distributed randomly along the polymer chain. We also varied the cationic/hydrophobic balance in the polymer by using different chain lengths and ratio of the hydrophobic functionality to optimize the antimicrobial activity. This gave us derivatives having tuneable antimicrobial properties. Additionally, we also investigated the effect of the polymer chain length on antimicrobial activity to obtain a critical molecular weight for optimum activity. The structural modification of the chitosan derivatives was confirmed using FT-IR, 1H-NMR and 2D-NMR spectroscopy, and their average molecular weight (Mw) was measured by size exclusion chromatography.
Most of the derivatives showed high activity against a panel of clinically important bacterial strains like Gram positive Staphylococcus aureus and Enterococcus faecalis and Gram negative Escherichia coli and Pseudomonas aeruginosa. We investigated the effect of four parameters on antimicrobial activity of the derivatives: the degree of substitution (ratio) of the functional group, the positioning of the functional group, presence of hydrophobicity and chain length or Mw. The results were combined to obtain a structure-activity relationship for these materials. For further applications of these materials as antibacterial agents, we investigated their preliminary toxicity towards human red blood cells. Through this study, we identified compounds possessing high selectivity towards the bacterial cells. We then utilized the highly selective compounds to investigate the mechanism of antibacterial action.
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