Development of silica based nanoparticles for plasmid DNA delivery

Abstract

Gene therapy is the targeted insertion of missing or absent genetic materials into the cells with aims of preventing or curing disease. As such, the successful delivery of site and pathology specific therapeutic genetic materials has gained increasing attention in biomedical applications. Because of their relative versatility and biocompatibility, silica nanoparticles (SNPs) have become highly sought- after carrier vehicles for the cellular delivery of sensitive bare genetic materials. The physicochemical properties of SNPs such as particle size, pore size, nanotopography and surface chemistry play an important role in regulating their intracellular delivery efficacy of genetic molecules. Control over these parameters is expected to significantly advance the development of silica nanocarriers for gene delivery applications.Recently there have been reports of a new class of SNPs designed with biomimetic rough surfaces that offer enhanced cellular uptake and efficient payload delivery. However, several research questions remain in using these particles in intracellular gene delivery, such as the effect of particle size and structure on pDNA transfection efficiency. Furthermore, previous studies looked at model gene molecules, further insight into the functional outcomes. The focus of this thesis is to develop a nanocarrier with the potential of increasing targeted gene expression through the use of these new class of SNPs as gene therapy carrier vehicles and evaluate their functional in an osteogenic model.In the first part, novel silica nanocarriers engineered with uniquely rough surfaces will be studied to describe the impact of particle size on pDNA binding and delivery performance. Compared to particles with larger size, rough SNPs with smaller size demonstrate enhanced cellular uptake promoting the effective delivery of pDNA into the cells for expression. Cellular uptake and payload delivery are dose dependent and moderated by general and dynamin dependent endocytosis pathways. These findings provide guidance for the rational design of silica-based non-viral vectors for efficient gene delivery.In the second part, nanocarriers engineered with rough outer surface have been designed to compare the impact of their internal core structures (hollow vs. solid) on pDNA binding and delivery performance. Additionally, the functional downstream effects were studied in cells of osteoblastic origin, mouse calvaria cells (MC3T3-E1) and bone marrow mesenchymal stem cells (BMSCs). Compared to hollow core SNPs, solid core SNPs, characterised by increased specific surface areas and particle density, preferentially bind more cationic polymer polyethyleneimine (PEI) and as a result demonstrate a higher affinity for pDNA. Furthermore, solid SNPs exhibited increased transfection performance and in turn added upregulation of genes associated with osteogenesis. Over a period of 14 days solid SNP demonstrate enhanced mineralization in osteoblastic cell lines. The outstanding performance of solid core rough surfaced SNPs shows great promise for their practical application in gene therapies.This work demonstrates that rough surfaced silica nanoparticle with smaller size have significantly higher pDNA cellular uptake and delivery when compared to their larger counterparts. Furthermore, rough silica nanoparticles designed with solid core structures show enhance pDNA binding capacity and cellular uptake thereby enhancing their genetic payload in osteogenic models. These findings provide a new understanding of how to fine tune the design of highly efficient silica gene delivery vectors.