Engineering Brome mosaic virus as a potential drug delivery nanoparticle for prostate cancer

Lee, Nadine (2017-03)

Thesis (MSc)--Stellenbosch University, 2017.

Thesis

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.

AFRIKAANSE OPSOMMING: Kanker is 'n beduidende oorsaak van die jaarlikse sterftesyfer wêreldwyd. Prostaatkanker is die tweede mees algemene vorm van kanker, na borskanker. Die konvensionele behandeling opsies wat tans beskikbaar is, is nie optimaal weens hul nie-spesifisiteit en die behandelings wat dikwels oneffektief is. Onlangse vooruitgang kyk na nanotegnologie as die toekoms van kankerterapie, en virale nanopartikels (VNPs) word gesien as ‘n belowende afleweringsmiddel. VNPs bied 'n proteïensteier wat relatief maklik is om te modifiseer terwyl dit ook bio-afbreekbaar is. Plantvirusse spesifiek kan met gemak gesuiwer word teen 'n hoë konsentrasie en is veilig vir menslike gebruik. Brome mosaic virus (BMV) is 'n twintigvlak-virus wat gekies is vir hierdie studie as gevolg van sy stabiliteit onder 'n verskeidenheid van eksperimentele toestande, soos pH en temperatuur, en sy stabiliteit in chemiese konjugasie eksperimente. BMV is ook gekies vir die beskikbaarheid van veranderbare aminosure op sy buitenste oppervlakte. Die doel van hierdie projek was om BMV te modifiseer as 'n potensiële afleweringsnanopartikel vir prostaatkankerbehandelings. Wilde tipe BMV is gesuiwer van Nicotiana benthamiana en die partikels is gekwantifiseer met behulp van transmissie-elektronmikroskopie asook immunoklad-tegnieke. Die virusdeeltjies is gewysig deur konjugasie van twee fluoresserende molekules, Alexa Fluor-647 en Cy5, aan die glutamiensuur aminosuur op die buitenste oppervlak van die BMV kapsied. Twee peptiede, PKRGFQD-C en SNTRVAP-C, is gekonjugeer aan die lisien aminosure met behulp van drie koppelstukke van verskillende lengtes. Hierdie peptiede teiken onderskeidelik die reseptore α-2-macroglobulin en GRP78 wat gevind word op die seloppervlak van androgeen-onafhanklike prostaatselle. Die SM(PEG)24 koppelstuk het die peptiedes sukkesvol gekonjugeer. Alternatiewelik is beide die fluoresserende molekule en peptiede ook gekonjugeer aan die lisien aminosure. Wanneer die fluoresserende aanhegting eerste uitgevoer was, was die peptied konjugasie onsuksesvol. Beide die fluoresserende molekule en peptiede is suksesvol gekonjugeer aan die lisien aminosure. Die VNPs is getoets in normale en kanker prostaat sellyne vir nie-spesifieke en geteikende opname met behulp van fluoresserende mikroskopie en vloeisitometrie. Die opname van die VNPs was 75% vir PKRGFQD-C en 95% vir SNTRVAP-C in die PC3 sellyn, wat 'n voorbeeld van 'n laat-stadium androgeen-onafhanklike kanker is. Die opname in VCaP, 'n voorbeeld van die vroeë stadium androgeen-afhanklike kanker, was minder beduidend as vir PNT2, wat normale prostaat selle is. Ons is van mening dat hierdie resultate positief is, want die VNPs sal waarskynlik die androgeen-onafhanklike selle teiken. Hierdie studie het getoon dat BMV, as 'n kandidaat VNP, suksesvol gemodifiseer kan word met 'n fluoresserende molekule en teikenpeptied om spesifiek prostaatkankerselle te teiken.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/100930
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