Hydrogel based on chemically modified polysaccharides for protein delivery

Hydrogels have become one of the most researched drug delivery systems, applied in many medical fields and for treating many diseases from diabetes to cancer. In recent years, more and more injectable and biodegradable hydrogels have been created using natural polymers. Through these delivery systems, drugs can be administered with minimally invasive procedures and be released sustainably at the target site increasing efficiency and duration of the treatment and lowering side effects1,2. In this thesis project, we created hydrogels designed for the delivery of proteins, in particular of the neurotoxin BoNTA (botulinum neurotoxin type A). This metalloprotease produced by the bacterium Clostridium botulinum is one of the most potent and lethal toxins known to men. BoNTA inhibits the release of acetylcholine, a neurotransmitter released in cholinergic nerve terminals that allows the contraction of mussels. Its action therefore causes flaccid paralysis. It has been used in aesthetic medicine and as a therapeutic agent in diseases that present muscles with spasticity and hyperactivity such as hyperhidrosis, strabismus, dystonia, etc. The commercially available formulations for BoNTA are simple saline solutions of the toxin mixed with albumin. The lasting power of these products is limited, ranging from three to four months and the dose administered is very low due to its very low LD50 (1-2 ng/kg in humans). This means painful injections must be repeated frequently to treat the disease. This thesis project aimed to create hydrogels in which BoNTA could be loaded in higher amounts and then be slowly released at the target site, lowering the number of administrations and improving patients’ compliance. Two hyaluronic acid-based hydrogels were designed. One based on chemical crosslinks and one on supramolecular interactions/physical crosslinks. Both were designed to be biodegradable so that no polymer would accumulate in the patient’s body causing side effects or toxicity. The chemically crosslinked hydrogel was created using the crosslinking agent adipic acid di-hydrazide (ADH). After oxidating the vicinal diols of HA to aldehydes by using sodium periodate, ADH was added creating Shiff Bases. The hydrazone groups obtained are more stable at pH 7.4 while at pH 5 are less stable, therefore all crosslinks created are reversible. The yield of oxidation, hence the amount of ADH to be added, was assessed using 1H NMR spectroscopy. The hydrogel was made by adding ADH in a molar ratio of 1:2 ADH: HA aldehydes to the oxidized HA and then diluting it to the lowest HA concentration possible without losing its structure. The best formulation created was the one made using 50% NaIO4 in relation to the moles of HA, using phosphate buffer 63mM with 25% ADH and at a final concentration of 2% HA w/V. The macroscopic structure and the pH (7.2) of the hydrogel were kept at room temperature for 15 days. The hydrogel was also characterized by studying its shear thinning properties by using the Rheometer Physica MCR-101 from Anton Paar and studying its release profile by doing Release Studies using marked antibodies and Fluorescence Microplate Reader. The physically crosslinked hydrogel was based on the synthesis of two different polymers, an adamantane-HA polymer and a β-cyclodextrin-HA polymer. By chemically modifying HA adding guest-host molecules such as adamantane and β-cyclodextrin the crosslinking was created through the formation of inclusion complexes based on hydrophobic interaction. Future studies will focus on the completion of the characterization of the chemically crosslinked hydrogel and the formulation and characterization of the physically crosslinked one.