In vitro evaluation of the toxicity profile of N-trimethyl chitosan chloride

Abstract

The pharmaceutical scientist 1s confronted by numerous challenges in the area of developing new and effective delivery systems for new classes of drugs such as peptide and protein drugs. Due to the size and polarity of these drugs they are excluded from the normal transcellular transport pathway. The co-administration of absorption enhancers has proven to be an effective way of increasing the delivery of peptide and protein drugs. However, they have also been shown to cause significant cell damage and their use are considered rather impractical due to the fact that their toxicity was directly related to the mechanism of enhanced absorption. Chitosan opens the paracellular pathway by acting on the tight junctions but is only effective in acidic environments. N-trimethyl chitosan chloride (TMC), a partially quaternised derivative of chitosan, has proven to be effective in neutral and basic environments for opening of tight junctions to increase paracellular transport. However, limited toxicity data is available for this absorption enhancing polymer. In this study TMC polymers with different degrees of quaternisation were synthesised (12 - 53 %) and evaluated for their possible toxic effects. In vitro cytotoxicity assays performed on human intestinal epithelial cell cultures (Caco-2) included the MTT and the LDH assay. Propidium iodide staining of Caco-2 cell monolayers was used to visualise possible cell membrane damage caused by the polymer. Results of these assays indicated significant toxic effects for the synthesised polymers. A concentration dependent decrease in cell metabolic activity was seen with the MTT assay while the LDH assay and propidium iodide staining of the Caco-2 cell monolayers indicated that these polymers cause significant cell membrane damage. However, no direct correlation could be seen between the different toxicity assays. TMC-12 ( degree of quaternisation = 12 %), which was not synthesised in our laboratory, showed no toxic effects. It was concluded that the polymers synthesised in our laboratory were not safe but should be investigated further to determine the cause of the toxicity.