Structure and dynamics of bio-hydrogels investigated by neutron and X-ray scattering techniques

Hyaluronic acid (HYA) is a natural polysaccharide, belonging to the family of glycosaminoglycans, characterized by the repetition of a disaccharide unit of glucuronic acid and N-acetylglucosamine. HYA is present throughout the animal kingdom, from the extracellular matrices in which most tissues differentiate, to the vitreous of the human eye and the synovial joint fluid. Thanks to its ability to form viscous solutions in water, HYA is widely used to treat inflammatory and degenerative joint diseases, a group of pathologies with a high impact in society since they contribute heavily to the rise of health costs and they affect life quality. Beside HYA, we investigated a chemically modified form of hyaluronic acid, HYADD, obtained derivatizing the polysaccharide backbone with hexadecylic (C-16) side chains, through amide bonds, with a 1-3 mol-% degree of substitution of repeating units. Even if the modification is very small, it alters dramatically some macroscopic properties such as the elastic behavior and the rheological response. The resulting system is a relatively stable hydrogel at polymer concentrations higher than 0.3% (weight of polymer/total volume), whereas native hyaluronic acid forms highly viscous solutions only at concentrations ten times higher. HYADD, however, does not differ from HYA in other relevant features such as charge density along the chain and swelling ability, as well as in its interaction behavior with metabolites and cellular material in the synovial matrix. On the basis of the beneficial effects noticed in recent tests performed on animal models of osteoarthritis, HYADD is expected to have a positive clinical effect in joint mobility and function. The aim of this PhD thesis is to compare the natural gel and the chemically modified one focusing on microscopic structural and dynamic properties such as the structure and organization of the polymer chains, the dynamics of the polymer and the self-diffusivity of water in the gels. We have performed several experiments using X-ray and neutron scattering to probe gels at different polymer concentrations and temperatures. From the structural point of view we found that HYADD has a structure more compact than HYA owing to the hexadecylic branches added along the chain. The characteristic lengths derived from neutron and X-ray experiments (~ 150 à...) are in fair agreement with those resulting from dynamics light scattering measurements. The small angle diffraction patterns show, in addition, the presence of some degree of order in the organization of the natural saccharide chain in the most concentrated gels. The dynamics of HYA and HYADD shows some small differences in the 100-1000 ps timescale (mostly in the low-Q region) that can be related to the presence of the chemical modification. On the other hand the diffusive dynamics of gel water is similar to that of bulk water for both HYA and HYADD gels at least up to 10% gel concentration. In the frame of a collaboration with the group of Prof. Santi at the Department of Pharmacy of Parma University, we contributed to the characterization of a novel platform called Patch-non Patch®, developed for transdermal drug delivery. In particular, using quasielastic neutron scattering we investigated the diffusivity of a model drug (lidocaine) through the patch. The obtained data indicate that the diffusion of lidocaine is triggered by that of hydration water present in the patch. In the framework of a CNR PhD grant, I spent 18 months at the Institut Laue Langevin in Grenoble, where I joined the group of Italian and French researchers in charge of the backscattering spectrometer IN13. Under the supervision of Dr. Francesca Natali, I took part to the group activities, contributing to the technical developments and to the assistance to users during their experiments.