Dextran Nanogels for the Delivery of Granzyme B

Medulloblastoma is the most common malignant brain tumor in children. Although advancements in survival rates over the past decades, conventional therapies still significantly compromise the lives of patients. Radiation, surgery, and chemotherapy have dramatic consequences on the development of patients, impacting their lives with severe, dramatic long-term side effects. Recognizing these problems, research is exploring alternative therapeutical valuable approaches in order to mitigate the pronounced collateral toxicities associated with traditional treatment approaches. The immune system represents a useful tool in the fight against cancer; accordingly, several immunotherapeutic approaches have been exploited for potentially instructing the immune system to selectively kill cancer cells. However, in some tumors the presence of immune cells is known to be locally low, thus an anticancer approach in these forms of neoplasia could be addressing the physiologically released immune system proapoptotic intermediates at the tumoral site. Granzyme B is a cytotoxic protein released by Cytotoxic T cells and Natural Killers that is known to be able, once inside of the cell, to induce different pathways of apoptosis. This thesis project aims to design and produce a drug delivery system to vehiculate Granzyme B to tumoral cells. Nanogels are nanosized hydrogels that, when designed properly, can be injected intravenously or subcutaneously into the body and then enter cells. Their potential relies also on the possibility of tuning their features depending on the application. Thanks to their hydrophilic internal core they can create the perfect environment for proteins, providing protection against denaturation and degradation. The project mainly focused on designing a nanogel-based Granzyme B delivery system that could exploit the cytosol-reducing conditions, known to have higher concentrations of Glutathione with respect to the extracellular environment, to prompt the protein release from the carrier after internalization by cancer cells. Granzyme B is known to have an isoelectric point between 9-10, meaning that at pH 7.4 the total charge of the protein is positive. Hence, a negative monomer was inserted in the nanogel during its formulation to drive the process of protein post-loading towards high yields through electrostatic interactions after the gel formation. To ensure a reduction-sensitive release, a pyridine-disulfide-containing linker was synthesized and inserted into the formulation to covalently bind the protein after the initial loading. This was thought to limit the protein leakage in the bloodstream before the arrival at the targeted site, thus allowing a release triggered by the cytosol-reducing conditions. To conjugate the protein to the linker, we chemically modified the protein by inserting a protected thiol group able to react with the pyridine-disulfide linker after deprotection, to enable a covalent binding of the protein to the nanogel. In this project, we were able to synthesize the protein-thiol derivative and the pyridine-disulfide-linker and successfully load them into our nanogels. Nanogels loaded with Lysozyme as model protein and Granzyme B were obtained and fully characterized for size and zeta-potential. Moreover, some preliminary release studies and in vitro cell association studies were carried out with Granzyme B-loaded nanogels, showing promising results.