Integrated genetic analysis systems

The overall objective of this thesis is to integrate a number of micro/nanotechnologies into integrated cartridge type systems to implement such biochemical protocols. Instrumentation and systems were developed to interface such cartridge systems: (i) implementing microfluidic handling, (ii) executing thermal control during biochemical protocols and (iii) detection of biomolecules associated with inherited or infectious disease. This system implements biochemical protocols for DNA extraction, amplification and detection. A digital microfluidic chip (ElectroWetting on Dielectric) manipulated droplets of sample and reagent implementing sample preparation protocols. The cartridge system also integrated a planar magnetic microcoil device to generate local magnetic field gradients, manipulating magnetic beads. For hybridisation detection a fluorescence microarray, screening for mutations associated with CFTR gene is printed on a waveguide surface and integrated within the cartridge. A second cartridge system was developed to implement amplification and detection screening for DNA associated with disease-causing pathogens e.g. Escherichia coli. This system incorporates (i) elastomeric pinch valves isolating liquids during biochemical protocols and (ii) a silver nanoparticle microarray for fluorescent signal enhancement, using localized surface plasmon resonance. The microfluidic structures facilitated the sample and reagent to be loaded and moved between chambers with external heaters implementing thermal steps for nucleic acid amplification and detection. In a technique allowing probe DNA to be immobilised within a microfluidic system using (3D) hydrogel structures a prepolymer solution containing probe DNA was formulated and introduced into the microfluidic channel. Photo-polymerisation was undertaken forming 3D hydrogel structures attached to the microfluidic channel surface. The prepolymer material, poly-ethyleneglycol (PEG), was used to form hydrogel structures containing probe DNA. This hydrogel formulation process was fast compared to conventional biomolecule immobilization techniques and was also biocompatible with the immobilised biomolecules, as verified by on-chip hybridisation assays. This process allowed control over hydrogel height growth at the micron scale.

Highly stable PEGylated gold nanoparticles in water: applications in biology and catalysis

Gold nanoparticles (Au NPs) with diameters ranging between 5-60 nm have been synthesised in water, and further stabilized with polyethylene glycol-based thiol polymers (mPEG-SH). Successful PEGylation of the Au NPs was confirmed by Dynamic Light scattering (DLS) and Zeta potential measurements. PEG coating of the Au NPs is the key of their colloidal stabilty, and its successful applications. Catalytic efficiency testing of the PEG-AuNPs were carried out on homocoupling of boronic acid. PEG-Au NPs with AuNps diameter < 30 nm were useful as catalyst in water. Finally, the PEG-Au NPs were also shown to be stable in biological fluid and not cytotoxic on B16.F10 cell line, making them attractive for further studies.

Gene delivery for the treatment of prostate cancer

Prostate cancer is one of the most common cancers diagnosed in men. Whilst treatments for early-stage disease are largely effective, current therapies for metastatic prostate cancer, particularly for bone metastasis, offer only a few months increased lifespan at best. Hence new treatments are urgently required. Small interfering RNA (siRNA) has been investigated for the treatment of prostate cancer where it can ‘silence’ specific cancer-related genes. However the clinical application of siRNA-based gene therapy is limited due to the absence of an optimised gene delivery vector. The optimisation of such gene delivery vectors is routinely undertaken in vitro using 2D cell culture on plastic dishes which does not accurately simulate the in vivo bone cancer metastasis microenvironment. The goal of this thesis was to assess the potential of two different targeted delivery vectors (gold or modified β-cyclodextrin derivatives) to facilitate siRNA receptor-mediated uptake into prostate cancer cells. Furthermore, this project aimed to develop a more physiologically relevant 3D in vitro cell culture model, to mimic prostate cancer bone metastasis, which is suitable for evaluating the delivery of nanoparticulate gene therapeutics. In the first instance, cationic derivatives of gold and β-cyclodextrin were synthesized to complex anionic siRNA. The delivery vectors were targeted to prostate cancer cells using the anisamide ligand which has high affinity for the sigma receptor that is overexpressed by prostate cancer cells. The gold nanoparticle demonstrated high levels of uptake into prostate cancer PC3 cells and efficient gene silencing when transfection was performed in serum-free media. However, due to the absence of a poly(ethylene glycol) (PEG) stabilising group, the formulation was unsuitable for use in serum-containing conditions. Conversely, the modified β-cyclodextrin formulation demonstrated enhanced stability in the presence of serum due to the inclusion of a PEG chain onto which the anisamide ligand was conjugated. However, the maximum level of gene silencing efficacy from three different prostate cancer cell lines (DU145, VCaP and PC3 cells) was 30 %, suggesting that further optimisation of the formulation would be required prior to application in vivo. In order to develop a more physiologically-relevant in vitro model of prostate cancer bone metastasis, prostate cancer cells (PC3 and LNCaP cells) were cultured in 3D on collagenbased scaffolds engineered to mimic the bone microenvironment. While the model was suitable for assessing nanoparticle-mediated gene knockdown, prostate cancer cells demonstrated a phenotype with lower invasive potential when grown on the scaffolds relative to standard 2D cell culture. Hence, prostate cancer cells (PC3 and LNCaP cells) were subsequently co-cultured with bone osteoblast cells (hFOB 1.19 cells) to enhance the physiological relevance of the model. Co-cultures secreted elevated levels of the MMP9 enzyme, a marker of prostate cancer metastasis, relative to prostate cancer cell monocultures (2D and 3D) indicating enhanced physiological relevance of the model. Furthermore, the coculture model proved suitable for investigating nanoparticle-mediated gene silencing. In conclusion, the work outlined in this thesis identified two different sigma receptor-targeted gene delivery vectors with potential for the treatment of prostate cancer. In addition, a more physiologically relevant model of prostate cancer bone metastasis was developed with the capacity to help optimise gene delivery vectors for the treatment of prostate cancer.