DEVELOPMENT OF NOVEL HYALURONIC ACID BASED SYSTEMS TOWARDS REGENERATIVE MEDICINE AND DRUG DELIVERY

Hyaluronic acid (HA) is a natural linear glycosaminoglycan that, thanks to its peculiar properties such as biocompatibility, resorbability and the possibility for an easy chemical functionalization, currently represents one of the most attractive building block for the preparation of advanced biomaterials for many biomedical applications. The overall aim of this thesis was to design, prepare and characterize advanced HA based systems for applications in regenerative medicine and drug delivery. However although HA is an excellent biomaterial, due to its hydrophilic nature, native HA is unsuitable to encapsulate hydrophobic drugs. Furthermore native HA exhibits physico-chemical properties incompatible to form stable structures to be used in vivo for regenerative medicine and drug delivery applications. Infact when HA is injected in physiological environment, it is subjected to a degradation process due to its high hydrophilicity and to the action of an enzyme known as hyaluronidase. For these reasons in this study in order to obtain HA stable nanostructures, able to incorporate hydrophobic drugs, two strategies were developed. The first strategy was to realize nanostructured systems in which HA was anchored onto nanoparticles, based on hydrophobic polymers, containing drugs; the second strategy was to modify HA molecules with hydrophobic groups. Moreover to obtain a HA stable structure at macroscale for regenerative medicine application, HA molecules were modified by a crosslinking reaction. In particular in the first part of the thesis the design, the preparation and the characterization of HA-coated biodegradable nanoparticles (NPs) as new drug carriers for tumor targeting were reported. In particular the idea was to bind a HA shell to a biodegradable core (Polylactic-coglycolic acid (PLGA) particle) by means of a physical binding using an anphiphilic polymer, known as Pluronic ®, that acts as a bridge between the hydrophobic PLGA and the hydrophilic HA. One of the most challenge in the design of nanoparticles is an efficient targeting. NPs can passively accumulate into tumors, taking advantage of enhanced permeation and retention (EPR) effect. However, NPs in vivo efficacy can be hampered by lack of cell internalization and/or by the fact that the loaded drugs may be released before nanoparticles uptake. HA is an attractive material for tumor targeting delivery since it can specifically bind to the cancer cells overexpressing at their surface CD44, an HA binding receptor. Thanks to this specific interaction, HA binding to the tumor cells and its subsequently internalization are strongly enhanced. In light of this the HA based nanoparticles realized in this project can be efficiently internalized by the tumors cells by means of both a passive and an active targeting strategy. NPs were prepared by a single emulsion technique and characterized for their morphology, size, and surface charge. HA based nanoparticles shown a spherical shape and a size ranging from 170 to 300 nm. Bare PLGA particles shown immediate aggregation phenomena; HA addition allowed to obtain stable NPs size for more than 10 days. Furthermore Irinotecan, a widely employed chemotherapeutic drug, was chosen to load NPs, and its in vitro release kinetics were assessed. The results demonstrated that nanoparticles were able to sustain Irinotecan release for at least 24 days. The second part of the thesis deals with the development of amphiphilic hyaluronic acid derivative towards the design of micelles for the sustained delivery of hydrophobic drugs and for the viscosupplementation. The new syntetized amphiphilic HA derivative is an octenyl succinic anhydride (OSA) modified HA, obtained through a simple reaction in an aqueous medium involving exclusively HA hydroxyl groups. In this way it was possible to overcome the problems related to the HA modifications that involve carboxylic groups, which result in an alteration of the distribution of negative charges along the polymer backbone at physiological pH and probably affect fundamental biological and pharmacological HA properties. A morphological, dimensional, calorimetric and rheological studies of this novel HA derivatives were conducted. Furthermore the ability of this novel amphiphilic HA derivative to self-assemble into micelles and to act as a solubility enhancer and as a modulator of release kinetics of a hydrophobic anti-inflammatory drug was demonstrated. In particular from morphological analysis it resulted that micelles are spherical objects with diameters around 100 nm. Differential scanning calorimetric (DSC) analysis revealed that the ability of HA to sequester water seems to be enhanced by the introduction of lipophilic functions within HA molecules, resulting in a further decrease of the fraction of free water able to freeze compared to the unmodified HA. Moreover in the perspective of using the novel OSA derivatives as viscosupplementation product, the rheological features assume a crucial role since they must properly restore the biomechanical functions of the normal synovial fluid. From a rheological point of view OSA-HA solutions appeared to be an appropriate tool to be used in viscosupplentation therapy owing to their suitable viscoelastic features; infact the OSA-HA solutions exhibit a rheological behavior similar to the human synovial fluid, that is viscous at low frequencies and prevalently elastic at high frequencies and characterized by the presence of crossover frequency. Concerning release studies, the results indicated that OSA-HA is able to self-assemble into micelles, load a hydrophobic drug and release the active molecule with controlled kinetics. In particular, the analysis of release profiles shown that drug diffusion into the gel is faster compared to gel/drug dissolution with the dissolution contribution becoming more and more relevant as the OSA-HA concentration increases. In the third part of this thesis the optimization and the characterization of HA hydrogels for regenerative medicine were reported. HA hydrogels, produced crosslinking HA molecules with divinyl sulfone (DVS) and based on a simple, reproducible and safe process that does not employ any organic solvents, were developed. Owing to an effective purification step, the resulting homogeneous hydrogels do not contain any detectable residual crosslinking agent and are easier to inject through a fine needle. HA hydrogels were characterized in terms of their viscoelastic and network structural properties. They exhibit a rheological behavior typical of a strong gel and show improved viscoelastic properties by increasing HA concentration and decreasing HA/DVS weight ratio. Furthermore it was demonstrated that processes such as sterilization and extrusion through clinical needles do not imply significant alteration of viscoelastic properties. Moreover the crosslinks appear to compact the network, being a reduction of the mesh size by increasing the crosslinker amount. In vitro and in vivo HA hydrogel degradation tests demonstrated that these novel hydrogels show a good stability against enzymatic degradation, that increases by increasing HA concentration and decreasing HA/DVS weight ratio. Finally the hydrogels show a good biocompatibility confirmed by in vitro and in vivo tests. In conclusion, HA coated nanoparticles composed of a hydrophilic shell and hydrophobic inner core were developed. These nano-sized particles could be suitable tools for applications in drug delivery and in particular for cancer therapy, taking advantage of both passive accumulation in tumor tissues via the EPR effect and active targeting by the strong receptor-binding affinity of HA to CD44 receptor. Furthermore soft nanostructures, based on an amphiphilic HA derivative, were developed. These HA derivatives represent novel and promising biomaterials for the realization of systems able to self-assemble into micelles, sustain the delivery of an anti-inflammatory hydrophobic drug, release the active molecule with a controlled kinetic and at the same time able to act as a viscosuplementation agent for the treatment of joints affected by osteoarthritis. Moreover HA crosslinked hydrogels were developed; these systems represent promising injectable biomaterials for application in regenerative medicine