Metal-Organic Frameworks as a Platform for Therapeutic Delivery

Abstract

Metal-organic frameworks (MOFs) are porous self-assembling materials composed of inorganic nodes and organic linkers, and are currently used for catalysis, gas separation, and gas storage. They have recently been applied to drug delivery because of their large pore sizes and highly modifiable surface properties and functionalities. MOFs can lower the required amount of active pharmaceutical ingredient (API), and can provide a more efficacious therapy through their ability to shuttle insoluble or unstable molecules into cells. This dissertation demonstrates the benefits of using MOFs as a delivery platform, due to their ability to 1) extend a small molecule chemotherapy drug’s release time and minimise the “burst release effect,” 2) protect a fragile biomacromolecule from degradation and deliver it successfully intracellularly, and 3) exhibit high levels of biocompatibility. Two zirconium-based MOFs (called NU-1000 and NU-901) that exhibit exceptional pore dimensions and chemical stability, developed by collaborators at Northwestern University, were utilised in a novel temperature-based material treatment after being loaded with two different molecules. This treatment entraps the payloads, successfully delaying the release of the model drug calcein from both MOFs’ porosity for up to 30-49 days. The integrity of the payload was maintained; this temperature treatment offers an improvement of release compared to the crystalline MOF, lessening the “burst release” effect. A super-resolution microscopy technique confirms visualisation of NU-901 and NU-1000 uptake into HeLa cells, and characterisation on endocytosis pathways reveals the active mechanism for uptake of both MOFs. When NU-1000 and NU-901 are loaded with a clinically relevant small molecule chemotherapy drug (α-CHC) and treated following this method, the drug can effectively kill HeLa cells at lower delivery concentrations compared to the crystalline systems. NU-1000 was specifically selected for entrapping and delivering the biomacromolecule – small interfering ribonucleic acids (siRNA). The pore dimensions of NU-1000 made it feasible to internally retain siRNA, and modelling supported a thermodynamic preference for siRNA’s presence within the pore. The siRNA was successfully delivered by the MOF into modified HEK 293 cells, where up to a 27% knockdown of expression is quantified. Characterisation of the system, including fluorescence-lifetime imaging microscopy (FLIM) and enzyme degradation studies, reaffirm the protection of the payload after enzymatic attack. In vitro studies, after complexation with endosomal release cofactors, including proton sponges and cell penetrating peptides, demonstrated consistent levels of knockdown. The mechanism of these cofactors was explored through FLIM by assessing pH changes in HEK 293 intracellular vesicles. Biocompatibility of the MOF systems was assessed through two different in vitro assays, both indicating that the system is not cytotoxic at employed concentrations. Determination of the NU-1000 and NU-901 linker as the limiting factor for biocompatibility led to the testing of other linkers. These additional linkers were highly biocompatible and syntheses of other MOFs (MOF-808 and PCN-777) using these ligands were explored to generate particles with optimal nano-sizes for this biological application. Modifications in temperature, reaction time, heating method, and preformation of component parts were all evaluated to successfully create particles below 200 nm with narrow size distributions. These studies demonstrate the advantages of MOFs as delivery systems, specifically through their ability to accomplish three main objectives: extension of therapeutic release, protection from enzymatic degradation and avoidance of endosomal retention, and high levels of biocompatibility. This work shows that MOFs are exciting possibilities for the future of therapeutic delivery, and can be used as a platform material.