Design, synthesis, characterization and in vitro evaluation of new cross-linked Hyaluronic acids for Pharmaceutical, Nutraceutical and Cosmetic applications

Hyaluronic acid (HA) is an excellent biomaterial which thanks to its peculiar properties, such as biocompatibility, biodegradability, mucoadhesiveness, hydration and viscoelasticity is currently one of the most attractive polymers for many biomedical applications. Unfortunately, many of its potential applications are limited due to its short half-life as it is rapidly degraded by the hyaluronidase enzyme. In order to improve its half-life and consequently increase its performance, in this PhD thesis, native HA was modified through cross-linking reactions in which the polymer chains are stabilized by covalent bonds using natural and biocompatible aminoacids as cross-linkers agents, such as Arginine and Ornithine to overcome the potential toxicity of commonly used synthetic molecules. CDMT / NMM was used as activating agent and the structure of the new products was characterized by 1H NMR and FT-IR to confirm the occurence of the chemical modification. The morphology of the compacted and interconnected structure characterized by cages of variable size was studied by SEM. The thermal behaviour (TGA and DSC) and the rheological properties were analized and the results had shown a stable thermal profile with a rheological behaviour liquid-like and a weak-gel profile for HA-Arg and HA-Orn respectively. Swelling studies suggested a dependence on the degree of modification and pH value. Finally, the Gel Permeation Chromatography data did not reveal significant changes in molecular weight but a lower recovery value indicated that the cross-linking process has occurred. It was also demonstrated by an in vitro degradation test that the products presented an enhanced resistance profile towards enzymatic digestions therefore the cross-linking process has reduced the susceptibility of HA to the digestive action of enzymes. An evaluation of the biological profile of HA-Arg and HA-Orn was studied as possible co-adjuvants therapy for the treatment of lung inflammation. In particular, in vitro studies were performed to evaluate the release of IL-6 and IL-8, antioxidant capacity, cytotoxicity by MTS assay, and evaluation of the integrity of the epithelial barrier. The studies were performed on Calu-3 and H441 cells evaluating the treatment of HA-Arg and HA-Orn individually and in combination with sodium ascorbyl phosphate (SAP). None of the investigated treatments appeared cytotoxic and the inflammatory and antioxidant activity was promising and different based on the cell line tested. HA-Arg and HA-Orn were employed for the production of microspheres by emulsification-solvent evaporation, as potential carrier of SAP: optimization studies led to the to the realization of MSs with spherical morphology and smooth surface. An in vitro release study have demostrated that the drug was released from microspheres in controlled manner. This work has been object of the patent application N° 102019000024117 of the 16th December 2019.