Functionalization of mesoporous silica nanoparticles as advanced vehicles for biomacromolecule delivery

Abstract

Biomacromolecules, such as miRNA/siRNA and proteins, have been drawing increasing attention because of their powerful application in treating disease. These biomolecules open new treatment opportunities for diseases which were previously considered as untreatable by conventional approaches. However, the broad clinical utilization of biomacromolecules is hindered by inefficient cellular uptake. Therefore, a variety of materials have been developed as carriers for biomacromolecule delivery. These materials include gold nanoparticles, lipid-based carriers, polymer spheres and silica nanoparticles. Among these carriers, mesoporous silica nanoparticles (MSNs) are particularly attractive due to their large surface area for cargo adsorption, tuneable pore size to encapsulate large molecules, and adjustable particle size to facilitate cellular uptake. In order to efficiently load cargos, a surface functionalization of MSN is often necessary to introduce functional groups. The covalent chemical functionalization of MSN usually requires multiple complicated chemical reactions and introduces extra cytotoxicity to the carrier. In some cases, the MSN modified by this approach cannot release the cargo efficiently. To address these challenges, this PhD thesis has developed two strategies to functionalize MSN for biomacromolecule delivery. These strategies are: i) developing and optimizing covalent chemical functionalization to enable the controlled release of miRNA, and; ii) developing non-covalent biological functionalization of MSN by using binding peptides/proteins as linkers to conjugate carriers and cargos through non-covalent binding. In the first approach, a cleavable S-S linker was introduced to enable a controlled release of miRNA. The density of functional groups (amine groups) was optimized on the surface of a large pore MSN (LP-MSN) with a pore size of 17 nm and a particle size of 200 nm. Three molecular layers were covalently attached on the surface of LP-MSN sequentially, including: i) a layer of oligo-lysine that controls the density of amine groups; ii) a layer containing the S-S linker that enables the release of miRNA, and; iii) a final polylysine layer that can efficiently bind miRNAs. From 5 different surface structures, LP-MSN-Epoxy-Trilysine-DSP-PLL has been identified as the optimal carrier that shows minimum cytotoxicity to normal rat kidney (NRK) cells while possessing a relatively high loading capacity and a controlled release for miRNA delivery. In the second approach, a non-covalent biological functionalization of MSN was developed based on self-assembly of the silica binding peptides/protein. Both the bi-functional peptides and the silica binding protein (the L2 protein) have been evaluated for functionalization of LP-MSN as delivery vehicles of biomacromolecules. Three bi-functional peptides that can bind both silica and nucleic acids have been designed, and it was found that the LP-MSN-RGRRRRLSCRLLK₈ complex is most effective in delivering a Cy3 labelled miRNA into NRK cells, while no cytotoxicity was observed at the working concentration. The function of miR-29B delivered by LP-MSN-RGRRRRLSCRLLK₈ was demonstrated by determining the down-regulated protein genes using a quantitative polymerase chain reaction (qPCR). The potential of the silica binding protein, the L2 protein, for functionalization of silica particles, was confirmed by the successful uptake of the LP-MSN-L2 complex into NRK cells. To further explore the influence of MSN properties on the delivery system, a dendritic mesoporous silica nanosphere (DMSN) modified by bi-functional peptides was evaluated for miRNA delivery. Although the fabricated DMSN-peptide complex shows relatively higher cytotoxicity to NRK cells compared with the LP-MSN-peptide complex, Alexa647-labelled miRNA was successfully delivered by DMSN-RGRRRRLSCRLLK₈ into NRK cells. Fundamental studies were carried out on the non-covalent biological functionalization system. Quartz crystal microbalance (QCM) results have quantified the binding amounts of bi-functional peptides on silica surfaces and confirmed rapid adsorption of these peptides. Small angel neutron scattering characterization of the L2 protein on a silica nanoparticle has shown that the affinity of L2 with silica is not affected by a high ionic strength up to 2 M MgCl₂, suggesting that the interaction between the L2 protein and silica particle is not just a charge interaction. Live cell imaging on the cellular uptake of DMSNS-RGRRRRLSCRLLK₈-Alexa647 labelled miRNA into NRK cells was performed, and the results suggest a phagocytotic pathway for the cluster of carrier-cargo complexes entering NRK cells. In summary, this PhD research has demonstrated two complementary approaches for functionalization of MSN as advanced delivery vehicles of macromolecules. The non-covalent biological functionalization approach, in particular, can serve as a powerful tool to modify silica surfaces by the self-assembly of designed bi-functional peptides. The method offers a simple fabrication process while decreasing cytotoxicity. The evaluation and fundamental study of these delivery systems provides valuable information to guide the functionalization of MSN as next generation of delivery vehicles for biomacromolecules (e.g. miRNA and proteins).