Effects of the hydrophobization on chitosan-insulin nanoparticles obtained by an alkylation reaction on chitosan

In this work, we investigate the influence of chitosan hydrophobization on the formation, physicochemical properties, solubilization, and release profiles of chitosan-based nanoparticles (NPs) complexed with the protein insulin, used as a protein model. We use an alkylation procedure to insert 8, 10, and 12 carbon chains along the chitosan macromolecule with a final 5, 10, or 50% substitution degree. Nuclear magnetic resonance (NMR) and infrared spectroscopes (IR) were used to evaluate the success and extent of the hydrophobization procedure. The size, shape, and charge of bare polymer and polymer-insulin NPs were evaluated by dynamic light scattering (DLS), transmission electron (TEM), and atomic force (AFM) microscopes, and zeta potential, respectively. DLS and zeta potential data demonstrated that polymeric NPs made with hydrophobized chitosans possess smaller sizes and higher positive charges than NPs obtained with unmodified chitosan. Also, TEM and AFM images showed that modified chitosan-made NPs have more elongated structures. Isothermal titration calorimetry (ITC) was used to determine the type and extent of the existing interactions between the different constituting components of complexed insulin-hydrophobized chitosan nanoparticles. The association efficiency and loading capacity of insulin into the polymeric nanoparticles were also investigated under different solution conditions. Our results showed that hydrophobized chitosan-based NPs possess both higher association efficiencies and protein loading capacities at pH 6 in comparison with unmodified chitosan-based ones. In vitro protein release studies at pH 5.3, 6, and 7.4 demonstrated that insulin is released more slowly from hydrophobized chitosan NPs, which would favor a more sustained protein release.