Small interfering RNA (siRNA), a double-stranded RNA consisting of between 19 and 23 nucleotides in length, holds great potential in the cure of various human diseases, including cancers and infectious diseases. siRNA silences specific genes with high efficiency by interfering with mRNA translation via the RNA interference (RNAi) process. The clinical use of siRNA, however, has been hampered by multiple biological barriers, some of which are nuclease stability, pharmacokinetics, biodistribution, clearance via reticuloendothelial system, endothelial barrier, cellular uptake and subcellular distribution. To overcome these issues, siRNA requires suitable delivery systems. Viral delivery carriers, although are effective at gene expression knockdown, can induce insertional activation or interruption of genes as well as immunologic responses, making them a safety concern. In contrast, nonviral delivery carriers such as polymers are believed to be safer and cheaper. To overcome the biological barriers, a polymeric carrier must have many functions. They must allow gene packing, serum stability, specific targeting, cell uptake, endolysosomal escape and gene releasing as well as suitable size, shape and surface chemistry. The main focus of this thesis was to design and synthesize polymers as a novel class of 3D nanostructured material having many of these functions, with particular attention on endosome escape and release of siRNA.
Initially, a low cytotoxic polycation that maintains its cationic strength for well over a few hours then degrades into a benign polymer with nontoxic byproducts was prepared using reversible addition fragmentation chain transfer (RAFT) “living” radical polymerization. Well-defined poly(2-dimethylaminoethyl acrylate) (PDMAEA) of five different molecular weights degrades slowly over 200 h (~8 days). As this degradation is independent of both the polymer molecular weight and solution pH, it is consistent with a self-catalyzed hydrolysis process without the need for an internal or external degradation trigger. In addition, the polymer shows little or no cytotoxicity to HeLa cells for the molecular weights of 5600 and below, even at very high polymer concentrations (equivalent to a nitrogen/phosphorus ratio of 200). These attributes make PDMAEA a promising candidate as a component in the design of a gene delivery carrier without the concern about accumulated toxicity of nanoparticles in the human body after multiadministration, an issue that has become increasingly more important.
The next step was to evaluate the use of the self-catalyzed degradable PDMAEA to strongly bind, protect, and then release oligo DNA (a mimic for siRNA) without the need for a cellular or external trigger. The PDMAEA formed large nanoparticle complexes with oligo DNA of~400 nm that protected the oligo DNA from DNase in serum. PDMAEA was found to be an ideal component of a ii delivery carrier by protecting the oligo DNA for a sufficiently long period of time to transfect most cells (80% transfection after 4 h) and then the capacity to release the DNA inside the cells after ~10 h. However, no gene knockdown was observed by using PDMAEA as a carrier due to the endosome barrier.
The next generation of polymeric carrier was designed to mimic influenza virus mechanism to escape from the endosome. It consisted of a diblock copolymer with a first block of PDMAEA. A second block consisting of P(N-(3-(1H-imidazol-1-yl)propyl) acrylamide (PImPAA) and poly(butyl acrylate) (PBA) was designed to induce fusion with the endosome membrane (and act in a similar way to the fusion peptide HA2) that resulted in the escape of polymer/siRNA complex to the cytosol where release of the siRNA can occur after degradation to PAA. Single electron transfer-living radical polymerization (SET-LRP) was utilized to synthesize a range of block copolymers as the RAFT technique could not control the polymerization of the second block. To examine the mode of delivery and release, an Osteosarcoma cell line was then used to test siRNA delivery through siRNA knockdown of the polo-like kinase 1 (PLK1) pathway. The best polymer carrier from this study was then trialed in vitro to silence the MAPK–ERK1/2 pathway in primary chondrocytes.
Although the combination of three functional polymers including PDMAEA, PImPAA and PBA showed high gene knockdown efficiency (up to 80%), the size, shape and surface characteristics of the polymer/siRNA complexes were not able to be controlled. Therefore, a new class of 3D nanostructured material for siRNA delivery with different shapes (e.g. cylinder and sphere) and tuneable surface compositions was synthesized using the temperature directed morphology transformation (TDMT) process. The rods surfaces were functionalised with an alkyne functional group that could be further coupled to β-Cyclodextrin (β-CD) by Cu-catalyzed azide/alkyne cycloaddition (CuAAC) “click” reaction. The functional rods were able to be converted to functional spherical particles. PDMAEA conjugated with pyrene (i.e. a florescence compound) was then used as a model polymer to decorate on the rods and spheres surfaces by using inclusion chemistry of pyrene in β-CD cavity. The ability of the novel 3D nano-rods and nano-spheres to bind and release oligo DNA (a model of siRNA) suggested their promising potential use as templates in gene delivery systems.