M.Sc. (Biochemistry) / Peptides have become useful therapeutic and targeting molecules in the treatment of various diseases. In nano-medicine, gold nanoparticles (AuNPs) are potential carrier systems of various targeting and therapeutic molecules including peptides. This study investigated the ability of 14 nm AuNPs as intracellular carriers of therapeutic and targeting peptides by assessing the toxic effects of these peptides when conjugated to AuNPs, on U937 monocyte-derived macrophages. These peptides include the proapoptotic peptide (klaklak)2, the targeting Glucose-Regulated Protein 78 (GRP-78) binding peptide and the carrier Trans-Activating Transcriptional (TAT) cell penetrating peptide (CPP). These peptides were conjugated to the AuNPs via poly(ethylene glycol) (PEG) polymers. The size, morphology, aggregation state and surface charge of citrate-stabilized, PEGylated, as well as peptide-conjugated PEG-AuNPs were determined using Transmission Electron Microscopy (TEM), Ultraviolet-Visible Spectroscopy (UV-vis), Dynamic Light Scattering (DLS) and Zeta Potential (ζ-Potential). Intracellular uptake of the tested AuNPs was investigated using the Cytoviva® dark-field hyperspectral imaging system. The toxicity was assessed using the conventional toxicity assay systems including adenosine triphosphate (ATP) and lactate dehydrogenase (LDH) assays, as well as the impedance based technology, xCELLigence real time cell analysis (RTCA) single plate (SP) system. The genotoxicity was investigated with the alkaline comet assay and the mechanisms of toxicity were investigated using western blotting through the ability of the AuNPs to activate the oxidative stress pathways, namely, the nuclear factor erythroid 2-related factor 2 (Nrf2) and factor kappa B (Nf-κB) pathways. Characterisation of the AuNPs revealed that the physicochemical properties of the particles were altered when suspended in culture medium. All the AuNPs tested have shown increase in size through aggregation. Although they all kept their negative charge, this charge was increased, with the greatest increase in charge shown for PEGylated AuNPs (OHPEG-AuNPs). Intracellular uptake was confirmed with 14 nm citrate stabilized AuNPs, TAT and GRP PEG-AuNPs. Some degree of uptake was also observed with (klaklak)2 PEG-AuNPs but not with OHPEG-AuNPs which was generally inaccessible to cells.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:uj/uj:11604 |
| Date | 26 June 2014 |
| Creators | Boodhia, Kailen |
| Source Sets | South African National ETD Portal |
| Detected Language | English |
| Type | Thesis |
| Rights | University of Johannesburg |
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