Browsing by Author "Meyer, Mervin M."

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    Development of nanotechnology-based drug delivery and imaging system to the white adipose tissue vasculature using Wistar Rat Model
    (University of the Western Cape, 2013) Thovhogi, Ntevheleni; Madiehe, Abram M.; Meyer, Mervin M.
    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.
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    Evaluating the specancer cell targeting peptides for applications in cancer diagnostics
    (University of the Western Cape, 2013) Mazyambe, Margaret Kena; Meyer, Mervin M.
    Cancer is a disease most often associated with poor prognosis. During the development of the disease, cells acquire genetic mutations which result in changes in bio-molecules (DNA and protein), thus altering normal functioning of cells. These bio-molecules can thus serve as biomarkers for the diagnosis of cancer and can also facilitate the early detection of cancer. Antibodies labelled with organic fluorophores are typically used in immunohistochemistry techniques to screen cancerous tissue for the presence of biomarkers. More recently, researchers started to use cancer specific peptides (e.g LYP-1, RGD,) rather than antibodies for this purpose. Advantages of peptides include high affinity to their binding target, rapid accumulation at target sites and the ability to evade the immune system. Fluorescent nanocrystals or quantum dots are emerging as nanoparticles that can replace organic fluorophores. Several properties of quantum dots make these nanoparticles an ideal application in the detection of cancer related biomarkers. These include size tunable fluorescence emission, resistance to photobleaching as well as high quantum yields that result in bright emission of fluorescence. The aim of this research project was to investigate the specific binding of selected peptides to cancer cells using functionalized quantum dots. Since the cost of synthetic peptides are so high, the aim of this study was also to express these peptides in E.coli bacterial cells. Cancer targeting peptides were identified from literature and oligonucleotides with sequences encoding these peptides were designed. Four oligonucleotides encoding the peptides p6.1, p.L, MV and NL1.1 were successfully cloned using the pET21b plasmid vector. However, the peptides were not successfully expressed in E.coli. Cancer targeting peptides namely p.C, p.H, p.L, p6.1 and Frop-1 were chemically synthesized and obtained from GL biochem (Shanghai). These peptides were conjugated to quantum dots (Qdot 525) using 1-ethyl-3-(3-dimethylamino) carbodiimide HCl (EDC) chemistry. The peptidequantum dot conjugates were applied to cancer cells to achieve specific binding. The Kmst-6 noncancerous cell line served as a control. The binding of the peptide-quantum dot conjugates was analyzed using flow cytometry and fluorescence microscopy. The p.H peptide revealed the highest binding affinity to cancer cells as indicated by fluorescence intensity. This was followed by the p.C peptide which showed differential binding amongst the cancer cell lines. The Frop-1 peptide displayed the lowest binding affinity, while the binding affinity of the peptides to Kmst-6 cell lines was very low. This study demonstrated that the cancer targeting peptides used in this study bind to cancer cells and that the specificity with which these peptides bind to the cells depends on the cell types and the peptide
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    Targeted delivery of embelin to cancer cells
    (University of the Western Cape, 2013) Emjedi, Zaakiyah Z.; Meyer, Mervin M.
    Apoptosis or programmed cell death is vital to the development of organisms as they maintain the balance between cell death and cell growth. Failure to activate apoptosis has been implicated in carcinogenesis and often results from the over expression of anti–cancer proteins such as the X–linked inhibitor of apoptosis protein (XIAP). XIAP is over expresses in certain cancers and is a potent inhibitor of the initiator caspase 9 and effector caspases 3 and 7. The increased expression of XIAP in cancer cells result in the resistance to apoptosis. The control of XIAP is therefore considered as a target for anti–cancer drug development. Embelin or 2,5–dihydroxy–3–undecyl–1,4–benzoquinoine is a dihydroxyquinone compound that was previously shown to inhibit XIAP. This drug was discovered by structure based computational screening. The binding of embelin to XIAP displaces XIAP from caspases, consequently eliminating the inhibitory effect of XIAP on apoptosis. The objective of this study was to develop a gold nanoparticle that can be used for the targeted delivery of embelin to cancer cells thereby enhancing pro–apoptotic effects of the pro–apoptotic drug, ceramide. XIAP expression levels were investigated by Western blot analysis in a panel of human cancer cell lines available in the laboratory to identify two cell lines that can be used as low and high XIAP expression controls. Gold nanoparticles were synthesized and conjugated with embelin and a cancer targeting peptide with the amino acid sequence LTVSPWY. The biconjugated nanoparticles were used to co–treat MCF7 and HepG2 cells with ceramide. Apoptosis was quantified using flow cytometry. The uptake of gold nanoparticles was investigated using HR–TEM and ICP–OES. This study showed that gold nanoparticles conjugated with the LTVSPWY peptide is specifically targeted to and taken up by cancer cells. Gold nanoparticles conjugated with embelin promoted ceramide induced apoptotic cell death of cancer cells. However, it was observed that gold nanoparticles biconjugated with the LTVSPWY peptide and embelin failed to enhance the pro–apoptotic effects of ceramide. iii This study successfully demonstrated that gold nanoparticles conjugated with embelin could be used to enhance the effects of anti–cancer drugs using ceramide as an example.