Molecular properties of chitosan and its derivatives and their potential for biomedical applications

Macro- and micro/nano-hydrogels are highly hydrated networks consisting of polymers and solvents. They are widely studied for different applications, especially for biomedical ones. One of the most challenging uses of hydrogels regards cartilage regeneration. Indeed, cartilage degeneration represents a severe burden for the healthcare worldwide. None of the currently proposed treatments is able to fully satisfy patients’ needs, probably since most of treatments lack of peculiar spatial complexity and mechanical properties of native tissues, i.e. a non-linear response to stress (strain hardening effect). One of the emerging approaches complies with the use of nano-composite networks consisting of hydrogels and nanoparticles embedding drugs. The main goals of the present thesis have tackled the fabrication and characterization - in terms of physical-chemical and biological properties - of hydrogels, nanoparticles and nanocomposite networks based on polysaccharides, i.e. chitosan, derivatives thereof and hyaluronan. To this aim, new approaches mimicking cartilage features have been devised. In Chapter I is described the extensive characterization of macromolecular association and macromolecular re-arrangement between a lactose-modified chitosan, i.e. CTL, in the presence of a crosslinking agent, i.e. boric acid, in dilute conditions. Furthermore, the interaction sites between CTL and boric acid were identified. Switching to semi-dilute polymer samples, mechanical properties were deeply investigated. Peculiar mechanical properties of native tissues, i.e. a non-linear response to stress and strain hardening, were detected. This system displayed some features akin to natural occurring molecular motors, and in this case, energy can be provided by stress or heat in order to foster the reorganization of the network. In Chapter II are reported two different strategies, which were developed to reduce the very fast kinetics of CTL/boron self-assembly, i.e. a pH- and a competitor-assisted gelation. Both strategies allowed forming homogeneous networks. Resulting matrices displayed self-healing ability, non-linear response to stress (strain-hardening effect), and were responsive towards different stimuli. Furthermore, the possibility to embed different cell types in such networks was studied. Chapter III deals with the improvement of stability and performance of CTL-based networks by using a second stable covalent crosslinking agent, i.e. genipin. The binding between polymer and genipin and mechanical properties were monitored. The kinetics of genipin crosslinking resulted to be directly proportional to the time and temperature. Genipin, also at low concentrations, promoted the gelation of CTL. Resulting gels displayed a strain hardening behavior, which was preliminary attributed to formation and reorganization of intermolecular low-energy bonds (e.g. hydrophobic interactions and hydrogen bounds). In Chapter IV is described the characterization of a broad library of chitosan/hyaluronan nanoparticles (NPs). NPs were fabricated by using chitosans with different molecular properties, i.e. fraction of acetylated units and molecular weight. Stability and shape of NPs were investigated, and the best performing formulation was identified, namely the one fabricated with chitosan with a medium molecular weight and partial acetylation. Stable NPs displayed spherical shape and a diameter close to 220 nm. When diluted in media with physiological pH and osmolarity, osmotic forces promoted the increase of size and porosity of NPs. These NPs avoided engulfment during the early stage of incubation with macrophages and did not influence the response of neutrophils and of macrophages. This behavior was attributed to their peculiar physical-chemical properties. Stable NPs were also able to encapsulate different payloads. Finally, NPs were embedded in the CTL-based networks and resulting networks were able to promote a sustained release of drugs.