Amphiphilic chitosan derivatives as antimicrobial agents: study of the activity and mechanism of action

This study describes the synthesis, characterization and the study of mechanism and antimicrobial activity of chitosan derivatives against the fungus Aspergillus flavus. Chitosan was obtained by the heterogeneous deacetylation of commercial chitosan. The hydrophilic derivative was obtained by reaction of chitosan with 2-chloro-N, N-diethylaminoethyl (DEAE) chloride. The degree of substitution (GS) of DEAE groups was determined using the techniques of Nuclear Magnetic Resonance of Hydrogen (NMR¹H) and Absorption Spectroscopy in the Infrared region (FTIV), obtaining 40% of substitution. Afterwards, samples of the obtained derivative were degraded with sodium nitrite to obtain polymers with different molecular weights (Mw). Molecular weights were determined by Gel Permeation Chromatography (GPC), and the obtained values were 8 - 25 - 60 - 116 and 136 kDa respectively. Subsequently, chitosan with different molecular masses were alkylated with dodecyl aldehyde followed by reduction with sodium borohydride to provide ~ 20% hydrophobic content that was characterized by NMR¹H. The solution behavior of the amphiphilic derivatives was studied using Fluorescence Spectroscopy, and the results showed that at low concentrations, chitosan derivatives form self-assembled particles with hydrophobic domains. Derivatives self-aggregate at the critical aggregation concentrations (CAC), which were shown to be dependent on the molecular mass and degree of substitution by hydrophobic groups present in the polymeric structure. The results of the microbiological assays showed a greater inhibition for derivatives having low molecular weights which may be related to a better interaction between the polymer chain and the cell wall and cell membrane of the A. flavus, which can also be associated to the smaller size of the chain polymer of such derivatives. To investigate the mechanism of interaction of the macromolecule with the fungus, the study of the different amphiphilic polymers with model membranes prepared with the phospholipids L-alpha-phosphatidylcholine and L-alpha-phosphatidyl-DL-glycerol was carried out using measurements of Dynamic Light Scattering (DLS) and zeta potential. The results allowed to postulate an interaction model based on the strength of interaction, which depends mainly of the molecular weight and the surface charge of the vesicle. For the zwitterionic vesicles the interaction with the surface depends more critically on the molecular mass, for the amphiphiles of low Mw (Mw 25 and 8 kDa) the interaction leads to the formation of aggregates of vesicles. For the derivatives with higher Mw the interactions are stronger and the abrupt increase of the zeta potential leads to the disintegration of the aggregates. The images obtained by Transmission Electron Microscopy showed that the polymer acts in the disassembly of the cell wall by the association with its constituents. By the correlation of the images obtained by the confocal microscopy, it can be affirmed that the greater interaction observed with the conidia is due to the presence of a greater concentration of hydrophobic proteins in their surface, strengthening the interactions between the polymer and the constituents of the cell wall. (author)