Synthesis and Toxicity Assay of Trimethyl Chitosan Chloride as a Carrierfor Vaccines and Drugs

Tri-methyl chitosan is used to deliver genes, peptide drugs and vaccines through nasal and oral mucosal routes. This polymer is a chitosan derivative that the solubility in water has been enhanced by methylation of primary amine group and its convertion to quaternary amonium. During this process, O-methylation also occurs, reducing the water solubility of the product. The purpose of this study is to evaluate the toxicity of trimethyl chitosan with high molecular weight and high degree of quaternary amine. Thus, high molecular weight chitosan was used for the synthesis of dimethyl chitosan through the Eschweiler-Clarke methylation method. Then, to avoid the O-methylation, methylation was continued by Menshutkin reaction using iodomethane as alkylating agent in NMP solvent. The quaternized chitosan was characterized by NMR and FTIR spectroscopy methods. The cytotoxicity of the product was investigated on Caco-2 cells via MTT assay. Hemolytic activity of the synthesized polymer was evaluated using haemoglobin release test. The data were statistically analyzed by ANOVA and Tukey's multiple comparisons test. The results showed that the synthesized polymer had no toxicity on Caco2 cells (up to the concentration of 1 mg/ml) (p<0.001). Haemoglobin release test further confirmed the lack of toxicity of the polymer up to the mentioned concentration. In conclusion, the polymer can be envisaged as a carrier in biomaterials delivery.