Chitosan–caseinate nanoparticles were synthesized by polyelectrolyte complex (PEC) formation. Caseinate is an anionic micellar nanocolloid in aqueous solutions, which association with the polycationic chitosan yielded polyelectrolyte complexes with caseinate cores surrounded by a chitosan corona. The pre-structuration of caseinate micelles facilitates the formation of natural polyelectrolyte nanoparticles with good stability and sizes around 200 nm. Such natural nanoparticles can be loaded with molecules for applications in drug-controlled release. In the nanoparticles processing, parameters such as the chitosan degree of acetylation (DA) and molecular weight, order of addition of the polyelectrolytes chitosan (polycation) and caseinate (polyanion), and added weight ratio of polycation:polyanion were varied, which were shown to influence the structure of the polyelectrolyte association, the nanoparticle size and zeta potential. Attenuated total reflection-Fourier transform infrared (ATR-FTIR) analyses revealed the chemical structure of hydrogel colloidal systems consisting of nanoparticles that contain chitosan and caseinate. Transmission electron microscopy (TEM) allowed further characterization of the spherical morphology of the nanoparticles. Furtherly, insulin was chosen as a model drug to study the application of the nanoparticles as a safe biodegradable nanocarrier system for drug-controlled release. An insulin entrapment efficiency of 75% was achieved in the chitosan-caseinate nanoparticles.
The 3D printing of a multifunctional hydrogel biomaterial with bioactivity for tissue engineering, good mechanical properties and a biodegradability mediated by free and encapsulated cellulase was proposed. Bioinks of cellulase-laden and cellulose nanofiber filled chitosan viscous suspensions were used to 3D print enzymatic biodegradable and biocompatible cellulose nanofiber (CNF) reinforced chitosan (CHI) hydrogels. The study of the kinetics of CNF enzymatic degradation was studied in situ in fibroblast cell culture. To preserve enzyme stability as well as to guarantee its sustained release, the cellulase was preliminarily encapsulated in chitosan–caseinate nanoparticles, which were further incorporated in the CNF/CHI viscous suspension before the 3D printing of the ink. The incorporation of the enzyme within the CHI/CNF hydrogel contributed to control the decrease of the CNF mechanical reinforcement in the long term while keeping the cell growth-promoting property of chitosan. The hydrolysis kinetics of cellulose in the 3D printed scaffolds showed a slow but sustained degradation of the CNFs with enzyme, with approximately 65% and 55% relative activities still obtained after 14 days of incubation for the encapsulated and free enzyme, respectively. The 3D printed composite hydrogels showed excellent cytocompatibility supporting fibroblast cell attachment, proliferation and growth. Ultimately, the concomitant cell growth and biodegradation of CNFs within the 3D printed CHI/CNF scaffolds highlights the remarkable potential of CHI/CNF composites in the design of tissue models for the development of 3D constructs with tailored in vitro/in vivo degradability for biomedical applications.
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