Designed synthesis of silica based nanocarriers for mRNA delivery

Abstract

In recent decades, introducing messenger RNA (mRNA) into cells has garnered intense interest for diverse applications. Limited by the short half-life and cell-membrane impermeability of mRNA, the success requires delivery solutions. With extensive efforts devoted in the nanomaterials based mRNA delivery platform, current design strategies for mRNA delivery systems primarily focus on mRNA binding, intracellular delivery and protection from degradation, but few provide the functionality to directly regulate mRNA translation. Besides, current delivery technology has limited delivery efficiency in hard-to-transfect cells (e.g., macrophages). Thus, new strategies are highly desired to exploit the multifunctional nanoparticles as both transfection agents and translation regulators for enhanced mRNA delivery with improved performance.Inspired by our recent breakthroughs in mesoporous silica nanoparticles (MSNs) synthesis, combined with the fundamental regulatory mechanism and intracellular environment of specific cells, this thesis focuses on the synthesis of functional dendritic mesoporous silica nanoparticles (DMSNs) designed for enhanced mRNA delivery. A facile, water phase based synthetic system was developed for the fabrication of large pore DMSNs with controlled structure and composition. Through the systematic study of the structural evolution of dahlia-like DMSNs to pomegranate-like MSNs via a micelle filling mechanism, the heterogeneous porous structure of DMSNs was reported for the first time for delivery of two positively charged cargoes. By comparing tetrasulfide bond bridged dendritic mesoporous organosilica nanoparticles (DMONs) with inorganic DMSNs with keeping other parameters the same, the composition contribution on intracellular glutathione (GSH) depletion, translation pathway regulation and mRNA delivery performance in antigen presenting cells were studied for the first time. Moreover, zeolitic imidazolate framework-8 (ZIF-8) was introduced to grow inside the mesopores of DMONs. The combined effect of ZIF-8 and tetrasulfide bond on the intracellular regulation of GSH and mRNA delivery was studied, providing new understandings in controllable synthesis of advanced materials as mRNA delivery vehicles.In the first experimental chapter, we report a unique dynamic structural transition from large pore sized DMSNs with the dahlia-like morphology to pomegranate-like MSNs with small mesopores. The silica-coated micelle composites were revealed to contribute to the kinetic structural evolution and heterogeneity of DMSNs. The structural evolution window was finely controlled by several parameters, including reaction time, reaction temperature, molar ratio of anionic micelle penetration agents to cationic surfactant, type of anions and amount of silica precursor. The advantage of the heterogeneous DMSNs with dual mesopores was further demonstrated for the co-delivery of two positively charge molecules, ribonuclease (RNase A, pKa= 9.6, Mw = 13.7 kDa, a model therapeutic protein) and doxorubicin (DOX, pKa = 8.3, Mw = 580, an anticancer drug). DMSNs with partially filled micelles exhibited higher loading capacities of RNase A and DOX than DMSNs with less micelles filling or DMSNs with complete micelle filling. It is suggested that the partially filled micelles formed small mesopores to entrap DOX, leaving a negative surface charge for the following RNase A loading. The successful cellular uptake and better cytotoxicity of DOX and RNase A co-loaded, dual pore sized DMSNs compared to free drugs were demonstrated in 4T1 murine breast cancer cells. Our study provides fundamental understanding in structural control of MSNs. The heterogeneity of DMSNs with partially filled micelle composites has been applied for intracellular delivery of two positively charged anticancer drugs, which gives the guideline for the rational design of silica based nano-carriers for improved drug delivery performance in bioapplications.In the second experimental chapter, the composition of DMSNs was controlled to incorporate tetrasulfide bond to regulate mRNA translation and enhance delivery efficiency for the first time. The impacts of nanoparticles chemistry and GSH level on mRNA translation and delivery were studied in several cell lines with various GSH levels. Compared with polyethylenimine (PEI) modified inorganic DMSNs (DMSNs-PEI) with similar structure, PEI modified DMONs (DMONs-PEI) with tetrasulfide bond composition were proved to consume intracellular GSH, deactivate glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and activate mammalian target of rapamycin complex 1 (mTORC1), consequently enhancing mRNA translation activity and delivery efficiency in macrophages, a typical type of hard-to-transfect cells. Similar GSH depletion, GAPDH deactivation, mTORC1 activation and enhanced mRNA delivery efficacy of DMONs-PEI were also observed in cancer cells with a relatively high GSH level compared to DMSNs. No significant difference was observed between DMSNs and DMONs in normal cells with a low GSH level. The in vivo mRNA delivery also suggested the potential of DMONs-PEI enhanced delivery in immune cells, indicated by the strong signal existed in lymph nodes compared to DMSNs-PEI and commercial product, in vivo-jetPEI. The in-depth understanding of composition effect on mRNA translation provides insights into the rational design of nanovectors acting as both transfection agents but also mRNA translation regulators.In the third experimental chapter, we propose a creative strategy to confine the growth of zeolitic imidazolate framework-8 (ZIF-8) for the first time by taking the advantage of the tetrasulfide composition in the framework of DMONs. The prepared DMONs-ZIF8 exhibited several advantages: (1) high loading capacity of mRNA enabled by the retained large mesopores; (2) promoted endosomal escape contributed by the imidazole group from ZIF-8 upon the acidic breakage; (3) long-term GSH depletion as a result of the tetrasulfide bond mediated GSH oxidation and zinc ions inhibited glutathione reductase (GR) catalysed GSH reduction; (4) GSH depletion enabled GAPDH deactivation and mTORC1 activation for enhanced translation; (5) improved biocompatibility without the use of toxic PEI modification. The concept gained from this work will advance more structural controls in material synthesis and provide novel delivery platforms with good delivery performance in vitro and in vivo over commercial products for mRNA delivery. The nanochemistry regulated mRNA translation is expected to be broadened in other translation stages, such as translation elongation and termination, and integrated functions of nanomaterials will be investigated to specific applications such as antibacterial applications to address antibiotic resistance challenge in my future research.