Philosophiae Doctor - PhD / Obesity is a complex metabolic disease of excessive fat accumulation. It is a worldwide epidemic affecting billions of people and its pharmacological management is hampered by drug toxicity and undesirable side effects. Therefore, a need still exists for the development of safe medication for treatment of obesity. Nanotechnology involves the use of small particles at atomic and molecular scale. It has application in medical diagnostics, drug delivery and molecular imaging. Various nanoparticles (NPs) functionalized with different biomolecules have been successfully used in many therapeutic and research applications due to their versatility, ease of chemical synthesis, low toxicity and unique properties. Examples of NPs used in this study are Gold nanoparticles (GNPs) and Quantum dots (QDs). GNPs and QDs are extensively used as drug delivery, labelling and imaging tools in biomedical research. Nanotechnology offers a new potential useful avenue for solving the problem of toxicity of anti-obesity drugs. This could be achieved through targeted drug delivery. In this study, rats were fed a high fed diet (HFD) to induce obesity. The streptavidin conjugated GNPs and QDs were functionalized with biotinylated adipose-homingpeptide (AHP) and/or anti-obesity drug (Gallic acid). Functionalization was characterized using agarose gel electrophoresis, UV-vis spectroscopy and transmission electron microscopy. The binding-specificity and targeting ability of AHP was evaluated in vitro and in vivo. The apoptotic effect of AHP functionalized-drug loaded GNPs (AHP-GA-GNPs) was tested in vitro using APOPercentage TM and Caspase-3 activation assays. The in vitro data indicated that the binding was specific to prohibitin (PHB) expressing cells (MCF-7 and Caco-2), and that the binding was temperature dependent. PHB was confirmed as a target for AHP after overlaying AHP-FITC and anti-prohibitin antibody staining. Cellular uptake was detected on the cells treated with AHP-functionalized NPs as compared to unfunctionalized NPs. The GA and AHP-GA-GNPs reduced cellular viability and induced apoptosis through activation of Caspase-3. The Ex-vivo studies using primary endothelial cells (ECs) isolated from the WAT of lean and obese Wistar rats showed that the binding of AHP was receptor mediated, and specific to receptors differentially expressed in ECs from obese WAT. The in vivo studies showed that, treatment of obese rats with AHP-functionalized NPs resulted in targeted delivery of the NPs to the WAT as compared to those treated with unfunctionalized NPs. Qualitative analysis using fluorescence microscopy and IVIS Luminar XR, live-imaging system showed that the unfunctionalized NPs accumulated mostly in the organs of the reticuloendothelial system, namely: liver, spleen, lungs and kidneys. In contrast, AHP-functionalized NPs accumulated mostly in the WATs as compared to the rest of the organs of the obese rats. Uptake and binding of the NPs to the tissues was quantitatively confirmed by the inductive coupled plasma-optical emission spectroscopy (ICP-OES). In conclusion, this study reports the 1) successful functionalization of GNPs and QDs with AHP, 2) use of AHP-functionalized GNPs and QDs as delivery and imaging agents to the WAT, and 3) potential use of AHP-functionalized drug-loaded GNPs in the treatment of obesity.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:uwc/oai:etd.uwc.ac.za:11394/4753 |
| Date | January 2013 |
| Creators | Thovhogi, Ntevheleni |
| Contributors | Madiehe, Abram M., Meyer, Mervin M. |
| Publisher | University of the Western Cape |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Rights | University of the Western Cape |
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