2011 YÜksek lisans tez özetleri

Cell Transfectıon And Cytotoxıcıty Studıes Of Newly Synthesızed Dna Nanocarrıers For Gene Delıvery

Gene therapy is based on transferring therapeutic genes into the targeted cells via DNA carriers called as vector and expression of these genes to cure genetic and acquired diseases. During this process vector encounters cellular barriers which effects transfection efficiency. Vector planned to use for gene delivery is desired to overcome cellular barriers and to achieve transfection of cells with high efficiency and low cytotoxicity. As a result, selection of appropriate vector is highly important. Although there are two major class of vectors such as viral and non viral vectors in gene delivery; non-viral, newly synthesized oligoelectrolyte based copolymer which is called as BG-2 was chosen since non-viral vectors have many advantages over viral-vectors.

In this thesis study it was aimed to improve potency of gene therapy to cure especially cancer and other genetic or acquired diseases by studying the ability of newly synthesized BG-2 oligoelectrolyte to overcome cellular barriers, and to determine efficiency of transfection and cytotoxic effects of oligoelectrolyte on three different cell lines, human cervix epiteloid carcinoma (HeLa), human neuroblastoma SH-SY5Y and rat C6 glioma. Moreover, it was objected to enlighten the transfection steps and to fulfill the missing parts in literature dealing with this subject.

Critical micellar concentration (CMC) of BG-2 oligoelectrolyte was determined by 9-diethylamino-5H-benzo[a]phenoxazine-5-one, (nile red) fluorescence characteristics and complex formation of DNA with BG-2 was determined by monitoring the changes on 1,1'-(4,4,8,8-tetramethyl-,8-diazaundecamethylene)bis-4-[(3-methylbenz-1,3-oxazol-2-yl)methylidine]-1,4-dihydroquinolinium] tetraiodide (YOYO-1) fluorescence characteristics with the help of flourescence spectrometer. Size of DNA/BG-2 complex was measured by Nanosizer. Interaction between anionic 1,2- dimyristoyl L- α-phosphatidyl-DL-glycerol (DMPG) or neutral 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) model membranes and DNA/BG-2 complex was studied by monitoring the changes on ethidium bromide (EtBr) fluorescence characteristics by using flourescence spectrometer. Sensitivity of DNA molecule, complexed with BG-2, against deoxyribonuclease I (DNase I) and serum nucleases was determined by agarose gel electrophoresis. HeLa, SH-SY5Y and C6 glioma cells were transfected with green fluorescent protein (GFP) encoding pEGFP plasmid and luciferase enzyme encoding pGL4.51 plasmid. Transfection efficiency of BG-2 oligoelectrolyte was determined by monitoring GFP expression by fluorescence invert microscope and measuring the luciferase gene expression by luminometer. Besides the transfection efficiency of BG-2, the toxicity of oligoelectrolyte on cells was also examined. Cytotoxicity was assessed by 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide MTT ve Bicinchoninic acid (BCA) method.

Transfection efficiency of BG-2 oligoelectrolyte on three different cell lines was analysed and it was observed that BG-2 transfected HeLa cells with highest rate, transfection of C6 glioma cells was not achieved with BG-2 oligoelectrolyte. Although BG-2 showed some toxic effect on the cells, it was also observed that BG-2 oligoelectrolyte was successful to overcome cellular barriers by forming stable and small sized complexes with DNA, interacting with model membranes in a desirable manner and protecting DNA from nuclease activity. As a result, with small modifications on BG-2 oligoelectrolyte structure, it can be suitable for gene delivery applications in the future.