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Theses and dissertations (Applied Sciences)

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    In vitro toxicity testing of phthalocyanines on different cell lines using a continuous laser source
    (2010) Maduray, Kaminee; Odhav, Bharti; Karsten, Aletta
    Photodynamic therapy is a promising treatment for cancer. It involves the combination of a photosensitizer and light of an appropriate wavelength (laser source) to cause the destruction of cancer cells. Phthalocynanines are second–generation photosensitizers with enhanced photophysical and photochemical properties. In this in vitro study the effect of aluminium (AlTSPc) or zinc (ZnTSPc) tetrasulfophthalocyanines in its inactive and active state (laser induced) on melanoma (skin cancer cells), fibroblast (healthy normal skin cells) and keratinocyte (healthy normal skin cells) cells was evaluated. For each of the cell lines approximately 3 x 104 cells/ml were seeded onto 24-well cell culture plates and allowed to attach overnight, after which cells were treated with different concentrations of AlTSPc or ZnTSPc. The photosensitizers were synthesized at Rhodes University. After 2 hrs, cells were irradiated with a diode laser at a wavelength of 672 nm and a beam diameter of 1 cm. The laser power varied between 20-30 mW and the irradiation time was calculated to deliver a light dose of 4.5 J/cm2. Post-irradiated cells were incubated for 24 hrs before cell viability was measured using the CellTiter-BlueTM Viability Assay. Also, the efficacy of the light dose and laser source used for the killing of approximately 50% of the melanoma cancer cells were investigated. AlTSPc and ZnTSPc decreased cell viability of melanoma cancer cells to approximately 50% with photosensitizer concentrations of 40 μg/ml and 50 μg/ml respectively. These photosensitizer concentrations caused a slight decrease in the percentage cell viability of fibroblast and keratinocyte cells. Results for the dark toxicity assay showed that iii both photosensitizers in the presence of high concentrations (60 μg/ml – 100 μg/ml) showed cytotoxicity effects on melanoma cancer cells in their inactive state. This was not observed in fibroblast and keratinocyte cells treated under the same experimental conditions. The optimal AlTSPc and ZnTSPc concentrations in combination with the light dose of 4.5 J/cm2 was the most efficient in killing the melanoma cancer cells with reduced killing effects on healthy normal fibroblast and keratinocyte cells when compared to other light doses (2.5 J/cm2, 7.5 J/cm2 and 10 J/cm2). The irradiation of cells photosensitized with the optimal photosensitizer concentrations with a femtosecond laser using similar laser parameters to continuous wave laser experiments resulted in a reduction in the cell viability of healthy normal fibroblast and keratinocyte cells compared to melanoma cancer cells. The presence of DNA degradation on agarose gel, morphological changes like blebbing and ultrastructural changes like nucleus condensation indicated that photodynamic therapy treated melanoma cancer cells with the optimal concentrations of AlTSPc and ZnTSPc induced cell death via apoptosis. This concludes that low concentrations of AlTSPc and ZnTSPc activated with an appropriate laser source can be used to induce cell death in melanoma cancer cells. Both AlTSPc and ZnTSPc exhibit the potential to be used as a photosensitizer in photodynamic therapy for the treatment of melanoma cancer with the occurrence of minimal damage to surrounding healthy tissue.
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    Expression of anti-HIV peptides in tobacco cell culture systems
    (2009) Moodley, Nadine; Odhav, Bharti; Chikwamba, Rachel
    Nearly half of all individuals living with HIV worldwide at present are woman and the best current strategy to prevent sexually transmitted HIV is antiretrovirals (ARVs). Microbicides are ARV’s which directly target viral entry and avert infection at mucosal surfaces. However, most promising ARV entry inhibitors are biologicals which are costly to manufacture and deliver to resource-poor areas. Microbicides formulated as simple gels, which are currently not commonly used in ARV therapy, show immense potential for use in prevention and treatment of multidrug-resistant viral infections in developing countries. Among the most potent HIV entry inhibitory molecules are lectins, which target the high mannose N-linked glycans which are displayed on the surface of HIV envelope glycoproteins. Of the microbicides, the red algal protein griffithsin (GRFT) has potent anti-HIV inhibitory activity and is active by targeting the terminal mannose residues on high mannose oligosaccharides. It has a total of 6 carbohydrate binding sites per homodimer, which likely accounts for its unparalleled potency. The antiviral potency of GRFT, coupled with its lack of cellular toxicity and exceptional environmental stability make it an ideal active ingredient of a topical HIV microbicide. v Scytovirin (SVN) is an equally potent anti-HIV protein, isolated from aqueous extracts of the cyanbacterium, Scytonema varium. Low, nanomolar concentrations of SVN have been reported to inactivate laboratory strains and primary isolates of HIV- 1. The inhibition of HIV by SVN involves interactions between the protein and HIV-1 envelope glycoproteins gp120, gp160 and gp41. Current recombinant production methods for GRFT and SVN molecules are unfortunately hampered by inadequate production capacities. This project therefore aimed to determine if these molecules can be produced in plant cell culture systems. The transgenic tobacco cell culture system was evaluated to determine if it can be an alternative, cost effective production system for these molecules. Results of the study show that the microbicide genes can be cloned into plant transformation vectors, used to successfully transform SR1 tobacco cell lines and adequately produce 3.38ng and 10.5ng of GRFT and SVN protein respectively, per gram of SR1 tobacco callus fresh weight. The promising results attained in this study form the basis for further work in optimising plant cell based production systems for producing valuable anti-HIV microbicides, a possible means to curbing the elevated HIV infection rates worldwide.