Synthesis and Study of the Properties of Dual-Substituted Amphiphilic Low-Molecular-Weight Chitosan Derivatives

A series of low-molecular-weight chitosan derivatives with varying degrees of hydrophobic and cationic substitution was synthesized using a reductive alkylation method. At the first stage, a hydrophobic C14-alkyloxybenzyl moiety was introduced into the macromolecule; at the second stage, a cationic quaternary ammonium substituent was introduced. The structure and degree of substitution of the resulting compounds were confirmed by 1H NMR and IR spectroscopy. The actual degrees of substitution ranged from 0.5 to 4.1% for the hydrophobic substituent and 6 to 18% for the cationic substituent. We investigated the pH-dependent aggregation of the derivatives, the sizes and ζ-potential of the nanoaggregates in acidic and physiological media (pH 7.4), as well as their cytotoxic activity against the SH-SY5Y, BT474, and Skov3 cell lines. It was shown that the introduction of hydrophobic moieties induces self-association of macromolecules, whereas cationic groups increase hydrophilicity and ensure that the aggregates retain a positive charge at physiological pH. Turbidimetric titration and dynamic light scattering revealed a nonlinear dependence of colloidal properties on the ratio of hydrophobic to cationic substituents. The most stable systems were formed at moderate hydrophobicity and a low degree of quaternization, whereas an increase in the content of hydrophobic groups enhanced aggregation. Cytotoxic activity of the derivatives depended on their substitution pattern, polymer concentration, and cell line. Although higher degrees of substitution are associated with increased cytotoxicity, this dependence is not universal and depends on the balance between the two types of substituents. The results obtained demonstrate the potential of the synthesized amphiphilic chitosan derivatives as a platform for developing nanoscale delivery systems for biologically active substances.