Effects of the Hydrophobization on Chitosan–Insulin Nanoparticles Obtained by an Alkylation Reaction on Chitosan Emmanuel Robles, 1 Eva Villar, 2 Manuel Alatorre-Meda, 2 Marı ´a. G. Burboa, 3 Miguel A. Valdez, 4 Pablo Taboada, 2 Vı ´ctor Mosquera 2 1 Departamento de Investigaci  on en Polı ´meros y Materiales, Universidad de Sonora, Rosales y Transversal, 83000 Hermosillo, Sonora, M exico 2 Laboratorio de Fı ´sica de Coloides y Polı ´meros, Grupo de Sistemas Complejos, Departamento de Fı ´sica de la Materia Condensada, Facultad de Fı ´sica, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain 3 Departamento de Investigaciones Cientı ´ficas y Tecnol ogicas, Universidad de Sonora, Rosales y Transversal, 83000 Hermosillo, Sonora, M exico 4 Departamento de Fı ´sica, Universidad de Sonora, Rosales y Transversal, 83000 Hermosillo, Sonora, M exico Correspondence to: M. A. Valdez (E-mail: mvaldez@correo.fisica.uson.mx) ABSTRACT: In this work, we investigate the influence of chitosan hydrophobization on the formation, physicochemical properties, sol- ubilization, 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 interac- tions between the different constituting components of complexed insulin-hydrophobized chitosan nanoparticles. The association effi- ciency 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. V C 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 129: 822–834, 2013 KEYWORDS: biopolymers and renewable polymers; proteins; drug delivery systems; nanostructured polymers; surfaces and interfaces Received 8 August 2012; accepted 24 November 2012; published online 15 December 2012 DOI: 10.1002/app.38870 INTRODUCTION Although their possible safety has not yet been completely proved, nanoparticles, and special polymeric NPs, have been tested as potential vehicles for drug, protein, and peptide deliv- ery. They demonstrated to be an exciting alternative to solve some of the difficulties associated to direct drug administration by modulating their physicochemical characteristics. 1–3 For example, NP polymeric formulations have been used to increase the concentration of solubilized hydrophobic drugs in aqueous solution, which is thermodynamically limited by their aqueous solubility, 4,5 by favoring their internalization in the hydrophobic core of the NPs, hence, allowing the increase of their bioavaila- bility 5 and prolonging their circulation time. Their submicron size and large specific surface area also favor their absorption compared with larger particles. 6 Also, NPs can provide a greater stability and protection of the cargo in biologic fluids, a more suitable biodistribution, a sustained release pattern, and a reduction in systemic side effects. 7 In recent years, a number of potential oral insulin-loaded poly- meric nanoparticles have been developed. 8–10 However, most of Additional Supporting Information may be found in the online version of this article. V C 2012 Wiley Periodicals, Inc. 822 J. APPL. POLYM. SCI. 2013, DOI: 10.1002/APP.38870 WILEYONLINELIBRARY.COM/APP