2011 YÜksek lisans tez özetleri

Characterization of Interactions Between Newly Synthesized Nanocarriers and DNA for Gene Delivery

Gene delivery approaches intend to carry the therapeutic gene to targeted tissues and cells in treatment of cardiovascular system diseases, monogenic diseases, neurological and immunological diseases. Use of proper vector while carrying the therapeutic gene to targeted sites is crucial. Gene delivery vectors are divided into two classes as viral and non-viral. By carrying DNA to the cell, viral vectors have been used earlier than non-viral vectors, but due to disadvantages of viral vectors, such as inducing immune response, recombination of host genome, lack of DNA carrying capacity, natural and synthetic DNA carriers have been already started to design instead of viral carriers. These vectors that are designed at nanosize must overcome cellular barriers such as cellular uptake, trafficking within cell, protection of DNA from degradation against endosomal/lysosomal enzymes by releasing at right time and reaching of the therapeutic gene to nucleus, also they must be cheap, easy to produce, biocompatible, less cytotoxic and less immunogenic and must remain long period of time in circulation by staying stable in vivo.

In this thesis, physicochemical and biophysical characterization of newly synthesized BG-2 oligoelectrolyte copolymer has been performed for gene delivery. The transfection conditions were optimized based on these characterizations and transfection and cytotoxicity studies in cell culture were performed.

The critical micelle concentration have been determined by fluorescence spectroscopy method to understand how BG-2 oligoelectrolyte molecule acts in physiological environment before interaction with DNA. Afterwards, the measurement of BG-2/pDNA complexes in terms of physicochemical characteristics; size and zeta potential were performed by laser technique and demonstration of stable complex formation agarose gel electrophoresis technique was used. DNA condensation of BG-2/DNA complex and the interaction with anionic and neutral model membranes have been examined by fluorescence spectroscopy. GFP expression has been imaged in fluorescence microscope to determine the transfection efficiency of the molecule of HeLa cell in in vitro. Finally, the cytotoxic effects of BG-2 oligoelectrolyte on HeLa cell were studied by MTT and BCA tests based on spectrophotometric methods.

Depending on these studies, it has been stated that BG-2 oligoelectrolyte molecule forms micelle-like structure according to critical micelle concentration experiment results. In size measurement, complex size being under 150 nm has been evaluated quite prospective in terms of cellular uptake and transfection efficiency. Zeta potential measurement results showed that (+) charged BG-2 molecule neutralizes and condensates the (-) charged pDNA. At complex formation experiment by electrophoresis, determining no free pDNA bands in agarose gel lanes indicated that the complex is at stable structure. According to DNA condensation experiment results, it has been indicated that molecule condensates bovine DNA and pDNA and at certain concentrations are more successful than commercial jetPEI. As for that demonstration of interaction between DNA and anionic and neutral model membranes, positive charged complex has interacted with anionic model membrane, but did not interact with neutral model membrane as expected. Even if in transfection much efficient results could not be obtained, images with little transfection were considered as promising. Examining toxicity profile, BG-2 toxicity profile on HeLa cell was evaluated as reasonable. Considering all these experimental results, BG-2 oligoelectrolyte molecule could be used for gene delivery but to improve transfection efficiency some modifications should be made on the molecule. Despite the molecule was newly synthesized, it is considered to be used in clinical implementations because of yielding significant results, after in vivo studies in the future.