Massey Documents by Type

Permanent URI for this communityhttps://mro.massey.ac.nz/handle/10179/294

Browse

Subject: search.filters.subject.Nanobiotechnology

Search Results

Now showing 1 - 3 of 3
  • Thumbnail Image
    Item
    Development and applications of filamentous phage-derived particles in immunotherapy and diagnostics : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University (Manawatū), New Zealand
    (Massey University, 2020) Rajič, Marina
    Most vaccines that are currently in clinical use induce antibody-mediated responses. However, for many infectious diseases, T cells are an essential part of naturally acquired protective immune responses. T cell-inducing vaccines, such as the one developed in this research, could additionally be used for treatments in cancer or chronic viral infections. One way to target the immune cells and stimulate their responses is to use filamentous phage particles. Filamentous phages (Ff) are ssDNA viruses that infect Escherichia coli, which have been adapted and used extensively in phage display technology and nanotechnology. In this research, a filamentous phage (Ff)-based vaccine carrier was constructed to allow the tuneable display of non-protein immune adjuvant molecules (BODIPY-α-GalCer) and antigenic peptides (OVA; MHC I + MHC II) on the same particle. For the first time, azide groups were incorporated into the recombinant pVIII phage coat proteins that expressed recombinant peptides. Azide groups were subsequently used to attach fluorescently labelled adjuvant molecules, which were successfully presented to NKT cells in vivo. Additionally, high induction of in vitro proliferation of OVA-specific CD8+ T-cells was achieved. However, the Ff-derived particle use outside the laboratory is hindered because they are genetically modified viruses, which in addition, carry antibiotic resistance genes that can be horizontally transferred to gut bacteria. These limitations were overcome by developing a system for efficient production of non-replicating, controllable-length protein-DNA nanorods, derived from Ff, named BSFnano (~100 × 6 nm). In this research, functionalised BSFnano particles were constructed, and their application in diagnostics was tested in a proof of concept dipstick assay for detection of a soluble analyte (fibronectin). For the first time, an ultrasensitive dipstick assay was achieved with Ff-derived nanorods, detecting the test-analyte at a concentration of as low as 0.04 pg/μL, equivalent to only 100,000 molecules/μL. Overall, while the phage-based vaccine produced in this research elicited CD8+ T-cell responses in vitro, but not in vivo, the Ff-derived nanorods were successfully functionalised and tested in lateral flow immunoassay, with promising implications for use in point of care diagnostics.
  • Thumbnail Image
    Item
    Filamentous phage-derived nano-rods for applications in diagnostics and vaccines : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Palmerston North, New Zealand
    (Massey University, 2013) Sattar, Sadia
    Filamentous bacteriophage, as their name indicates are filament-like bacterial viruses. The F-pilus-specific filamentous phage of Escherichia coli, Ff (f1, M13 and fd) are resistant to heat, pH extremes and detergents. Their structural properties and amenability to engineering using recombinant DNA technology have enabled their extensive use in modern biotechnology. For example, Ff can be functionalized by displaying up to five different proteins and peptides on their surface. Ff phage have been successfully employed in diagnostic devices. Moreover, direct use as antigen-carriers is also a subject of interest in vaccine development. However, use of Ff-phage vaccines and in the at-home diagnostic devices is controversial, mainly because of their ability to replicate in gut E. coli, and possibility of mobilization and horizontal gene transfer of antibiotic resistance or virulence factor-encoding genes transfer among the gut and environmental bacteria. Moreover, the large length-to-diameter ratio of the virion (1000 nm x 6 nm) impairs diffusion of filamentous phage through complex matrices and could restrict use of filamentous phage in lateral flow diagnostic devices. To overcome both of these problems we have constructed much shorter, rod-like functionalized particles (50 nm x 6 nm), named “Ff-nano”, which do not carry any genes. The properties of these short particles were investigated, showing that they have superior resistance to heating in the presence of ionic detergent sodium dodecyl sulphate (SDS) in comparison to the full-length phage of the same virion composition. The Ff-nano particles displaying a bacterial Fibronectin-Binding (FnB) protein as fusion to virion protein pIII, localized in five copies at one of the two ends of the virion, were produced and purified. These functionalized nanorods were tested in two applications: as detector particles in a dip-stick-type lateral flow device and as antigen carrier in a vaccine trial. The FnB-displaying nanorods were able to quantitatively detect fibronectin in solution. In the vaccine trial, the Ff-nano particles elicited a weak response to the FnB displayed at a low-copy-number at the nanorod end. In contrast, the response to the major protein pVIII was strong, indicating that the multi-copy display of antigenic peptides along the rod, as fusion to the major coat protein pVIII, is required for using the Ff-nano effectively as vaccine carriers.
  • Thumbnail Image
    Item
    Supramolecular helical arrangement of porphyrins along DNA : a thesis submitted in the partial fulfilment of the requirements for the degree of Doctor of Philosophy in Chemistry, Massey University, Palmerston North, New Zealand
    (Massey University, 2010) Stephenson, Adam Wayne Ian
    Porphyrins are useful chromophores and have been used in numerous biological applications including light harvesting, oxygen transport and energy transfer. DNA is a perfect template for the controlled assembly of organic chromophores. By combining DNA and porphyrins in a controlled manner we have developed a novel range of porphyrin-DNA supramolecular constructs for future applications in nanobiotechnology. A number of β-pyrrolic functionalised porphyrin precursors were synthesised to be used as building blocks in the construction of both covalently and non-covalently modified DNAs. Using these porphyrins we have created several lipophilic porphyrin-DNA complexes through non-covalent attachment methods. Using a CuI catalysed azide alkyne cycloaddition (CuAAC) reaction of azido functionalised porphyrins we have developed a versatile approach for the covalent, site specific internal porphyrin insertion into oligonucleotides in a post-synthetic manner. We have investigated a number of duplex structures where porphyrins were located in the major or minor grooves of the duplex. Additionally, porphyrins were studied as intercalating moieties when they were inserted as a bulge in the middle of the duplexes or parallel triplexes. Additionally, when porphyrins were placed in both strands of the duplex they formed a zipper type structure in the minor groove. This resulted in a significant increase in the duplex thermal stability due to the formation of porphyrin H-aggregates. UV-Vis and CD spectroscopy as well as molecular modelling were used to help understand the interactions between porphyrins in the duplex. These findings lay the foundation for the future design of artificial DNA-chromophore supramolecular architectures and for their applications in material science and nanotechnology.