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. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013

In this study, we investigated the influence of hydrophobized chitosan on the formation and thermodynamic and surface tension properties of insulin-chitosan (I-Ch) polyelectrolyte complexes (PECs). We used an alkylation procedure to insert 12 carbon chains along the chitosan macromolecule with final substitution degrees of 5, 10, and 50%. NMR and IR spectroscopy were used to evaluate the success and extent of the hydrophobization procedure. Isothermal titration calorimetry (ITC) was used to determine the type and extent of the existing intermolecular interactions between the different constituting components of the insulin-hydrophobized chitosan PECs. Through the surface tension and diffusion coefficients at the air-water interface and ITC experiments with different I-Ch proportions, we demonstrated that around 34, 24, 25, and 60-80 insulin molecules saturated 0, 5, 10, and 50% hydrophobized chitosans, respectively. Surface tension experiments at the air-water interface demonstrated that the interaction of insulin molecules on the unmodified chitosan increased the hydrophobicity; this was mainly due to electrostatic interaction. On the contrary, insulin-hydrophobized chitosan interaction lowered the PEC hydrophobicity because of insulin alkyl chain interaction, and therefore, the hydrophilic insulin groups at the PEC surface contributed to a higher surface tension.

In this work, we investigate the effect of chitosan hydrophobization on the internalization and cytotoxic effect of chitosan-based nanoparticles (NPs) on breast cancer cells (MDA-MB-231), cervical cancer cells (HeLa) and noncancer cells (Arpe-19). We also analyzed the interaction of NPs with a phospholipid (DPPC) membrane model at the airwater interface. An alkylation procedure to insert 8 carbon chains along the chitosan macromolecule with final 10 and 30 % substitution degrees was used. Nuclear magnetic resonance (NMR) and infrared spectroscopes (IR) were used to evaluate the success and extent of the hydrophobization procedure. Size, shape, and charge of NPs were evaluated by dynamic light scattering (DLS), atomic force microscope (AFM), and zeta potential, respectively. The effect of hydrophobicity on NPs was the reduction of the NPs average size, the formation of slightly elongated structures and the enhancing of the interaction of NPs with a DPPC monolayer at the air-water interface. By using fluorescence images on fluorescein-chitosan NPs, we observed a higher internalization of hydrophobic chitosan NPs in cancer cells in comparison with a low internalization of these NPs in normal cells. Even when non modified chitosan NPs were highly internalized in all cell lines, hydrophobized chitosan NPs showed a significantly higher cytotoxic effect on cancer cells in comparison with a lower effect showed by non-modified chitosan NPs on these cells. The cytotoxic effect on the normal cell line used was low for native chitosan NPs and negligible for hydrophobized chitosan NPs.