Mesoporous Silica Nanoparticle as advanced drug delivery system: the effect of biomimetic lipid shielding

In the last years, nanomedicine has gained a growing attention by the research community, thanks to the small size of the used devices, which allows an easy interaction with cells and biomolecules. For this purpose, in this Master Thesis Mesoporous Silica has a starring role, thanks to its well-known properties like biocompatibility, resistance to biodegradation, and drug uploading capacity. It has been used to produce Mesoporous Silica Nanoparticles (MSNs) of 40-50 nm in diameter and showing mesopores with uniform size (2-3 nm) through a template-assisted sol-gel self-assembly process. As MSNs do not have intrinsic imaging properties, their surface has been functionalized with amino-groups (-NH2) in order to be labelled with fluorescent dyes, while the highly porous structure was used to upload the nanoparticles with a therapeutic cargo. Two different drugs against Multiple Myeloma were used: an inhibitor of the Ubiquitin Proteasome Pathway (Carfilzomib, CFZ) and the drug AGI-6780, which is an inhibitor of the isocitrate dehydrogenase 2 (IDH2), both FDA approved. The choice of these two drugs is not accidental: previous studies revealed that the combination of these two drugs can induce a synergistic cell death even in those cells that developed drug resistance if treated only with CFZ. However, the drug AGI-6780 has at present failed the translation to clinical studies due to its strong hydrophobicity, which makes impossible to administer it through an oral or intravenous way. This obstacle can be overcome by the strategy reported in this Master Thesis, i.e. by incapsulating AGI-6780 in MSNs that can be uploaded with a consistent amount of drug thanks to their huge surface area (~1000 m2/g). Also CFZ was incapsulated in MSNs, as the two drugs have to be used in concomitance. Both drugs were uploaded by mixing MSNs and the drug in a dynamic condition. However, nude MSNs are not colloidally stable in an aqueous environment like the human body. Therefore, another aim of this thesis was to try different lipid bilayer shielding formulation in order to optimize the colloidal stability, dispersibility, biocompatibility of the final lipid-coated MSNs. Firstly, synthetic lipid formulations were tested. One was composed by DOPC, the second one was formulated by mixing DOPC and DOTAP at a molar ratio of 2:1 and the third one was DOPC-CHOL-DSPE-PEG2000, prepared mixing DOPC, DSPE-PEG2000 and cholesterol at a molar ratio of 55:44:2. A further and more advanced biomimetics approach explored in this Master Thesis was to make the coating of MSNs with exosomes, that are extracellular vescicles of 30-200 nm in size extracted from living cells. Here in particular, two different biological sources were tested: KB cancer cell and B lymphoblasts. Exosomes have been revealed to play an important role in cells communication with other cells from the same tissue or different tissues, and if of autologous origin they are not subjectd to immune adverse response. For this reason, in this Master Thesis it was proposed to include MSN into exosomes biovesicles in order to carry the inorganic nanoparticles straight to the target cancer cells. The experimental focus was here on the coupling between MSNs and exosomes, to obtain an efficient enclosure of the MSN inside the lipid biovesicles. Cellular tests were also carried out, using Human Multiple Myeloma cell line KMS-28, to evaluate the cell-uptake of covered MSNs and the synergistic effect of CFZ and AGI-6780.