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Kean, Thomas
Languages: English
Types: Doctoral thesis
Subjects: RS
Preliminary flow cytometry experiments studied the ability of the peptides, CLNGGTC (u7) and GVSNKYFSNIHWG (Gu11G), derived from the binding region of uPA to inhibit uPA-FITC binding to U937 cells. The optimised trimethylated chitosan oligomer (TMO) was functionalised with chloroacetic acid to give 6-O-carboxymethyl N,N,N-trimethyl chitosan (CMTMO). Two peptides derived from the binding region of uPA, u7 and Gu11G were conjugated to the 6-O-carboxymethyl functionality using 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide methiodide and hydroxy-2,5-dioxopyrrolidine-3-sulfonicated. Fluorescent derivatives of u7-CMTMO and Gu11G-CMTMO were then produced by reaction with the succinimidyl ester of 5-(and-6)-carboxyfluorescein and Oregon Green RTM 488-X, succinimidyl ester. The cell-association of the fluorescent u7-CMTMO and Gu11G-CMTMO was assessed in U937 cells (a histocytic lymphoma known to over-express uPAR) and DU145 cells (a human prostate carcinoma) over time at 37°C and 4°C. The development of an improved gene delivery system using Gu11G-CMTMO was then investigated in COS-7, DU145 and MCF-7 cells. It was shown that chitosan could be trimethylated in a controllable and repeatable manner by increasing reaction time. Increasing degree of trimethylation decreased cell viability, an effect more pronounced in polymeric (TMC) over oligomeric (TMO) derivatives. The Gu11G-CMTMO-FAM showed an increase in cell-associated fluorescence (>5 fold) compared to CMTMO-FAM on U937 cells. u7-CMTMO-FAM showed decreased cell association compared to CMTMO. When Gu11G-CMTMO was mixed with TMO/pDNA or PEI/pDNA in the transfection mixture the transfection efficiency, as measured by the luciferase expression, was equal to that of the transfection achieved with uncoated polyplexes. Although Gu11G was shown to bind uPAR selectively, it was not possible to translate into an improved conjugate.
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