Evaluating the specancer cell targeting peptides for applications in cancer diagnostics
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Date
2013
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Publisher
University of the Western Cape
Abstract
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
Description
>Magister Scientiae - MSc
Keywords
Cancer, Immunohistochemistry, Early detection, Diagnosis