Browsing by Author "Lee, Nadine"

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    Engineering Brome mosaic virus as a potential drug delivery nanoparticle for prostate cancer
    (Stellenbosch : Stellenbosch University, 2017-03) Lee, Nadine; Burger, Johan T.; Maree, H. J.; Stellenbosch University. Faculty of AgriScience. Dept. of Genetics.
    ENGLISH ABSTRACT: Cancer is a leading cause of annual mortality worldwide. Prostate cancer is the second most prevalent type of cancer after breast cancer. The conventional treatment options that are currently available are not optimal due to their non-specificity as well as treatment often failing. Recent advances have turned to nanotechnology as the future of cancer therapy, and viral nanoparticles (VNPs) in particular are promising delivery vehicles. VNPs provide a protein scaffold that is relatively easy to modify while being biodegradable. Plant viruses specifically can be purified with ease at high concentrations and are safe for use in humans. Brome mosaic virus (BMV) is an icosahedral virus selected for this study due to its stability under a range of experimental conditions, such as pH and temperature, and its robustness in chemical conjugation experiments. BMV was also selected for the availability of modifiable amino acids on its exterior surface. The goal of this project was to engineer BMV as a potential delivery nanoparticle for prostate cancer treatments. Wild type BMV was purified from Nicotiana benthamiana and particles quantified using transmission electron microscopy as well as dot blots. The virus particles were modified by conjugation of two fluorescent molecules, Alexa Fluor-647 and Cy5, to the glutamic acid residues on the exterior surface of the BMV capsid. Two peptides, PKRGFQD-C and SNTRVAP-C, were conjugated to the solvent-exposed lysine residues using three SM(PEG)n crosslinkers of different lengths. These peptides respectively target the receptors α-2-macroglobulin and GRP78, which are found on the cell surface of androgen-independent prostate cells. The SM(PEG)24 crosslinker could successfully conjugate the peptides. When the fluorescent labeling was performed first, the peptide conjugation was unsuccessful. As an alternative, the fluorescent molecule and peptides were both conjugated to the lysine residues. The VNPs were assessed in normal and cancerous prostate cell lines for non-specific and targeted uptake. This was assessed using fluorescence microscopy and flow cytometry. The uptake of the VNPs was 75% for PKRGFQD-C and 95% for SNTRVAP-C in the PC3 cell line, which is indicative of late-stage androgen-independent cancer. The uptake in VCaP (early stage androgen-dependent cancer) was lower than for PNT2 (normal prostate cells). We consider these results positive as the VNPs will most likely target the androgen-independent cells. This study demonstrated that BMV, as a candidate VNP, can successfully be modified with a fluorescent molecule and targeting peptide in order to specifically target prostate cancer cells.