Finiuk N. Application of novel efficient nanoscale polymer carriers for nucleic acids delivery into cells

Gene delivery into specific target cells is a key research tool in modern molecular and cellular biology, as well as in gene therapy and biotechnology. The thesis is devoted to the study of novel comb-like polyampholytic carriers for nucleic acids delivery into cells. Formation of the complexes of plasmid DNA and the carriers was studied by agarose gel electrophoresis. Since the complex formation led to increase of molecular size and decrease of the net negative charge, polyampholyte/DNA complex was seen stuck in the wells of the gel, confirming that carriers condensate plasmid DNA. Optimal conditions for the formation of polyampholyte/DNA polyplexes were determined. It was found that polyampholyte possessing quaternized amino-containing side chains is capable of forming the most stable polyplexes with plasmid DNA. Turbidimetry study also shows that the polymers can form stable polyplexes with plasmid DNA in a wide range of polyampholyte/DNA ratio. Moreover, the polyampholytic carriers under study protected DNA against DNase I cleavage, which is one of the crucial factors for efficient gene delivery. It was found the increase in the surface activity of the polyampholyte/DNA complexes compared to the solution of native plasmid DNA. Polyampholytes form small sized complexes (around 100 nm) with plasmid DNA. SEM images of BG-2/DNA complexes indicated that size distribution of complexes was homogenous. According to zeta potential measurements, BG-2/pDNA complexes carry positive (+) surface charge. The surface charge of the polyplexes was around +40.8 mV, the positive surface charge allows an electrostatic interaction between negatively charged cellular membranes and the positively charged complexes. We develop new method enabling easy and effective delivery of DNA into several industrially important yeast species, including Saccharomyces cerevisiae. The method is based on using novel polyampholyte of comb-like structure. Application of this method provides twice more transformants of Hansenula polymorpha NCYC 495 than the electroporation, and enabled 15 times more transformants than the best practice lithium acetate method. By using our method, we also obtained five times more transformants of Pichia pastoris GS115, in contrast to electroporation, and 79 times more transformants than with lithium acetate method. Similar results were obtained for DNA delivery into cells of Saccharomyces cerevisiae yeast. DNA delivery method into Escherichia coli has been successfully developed using novel carriers. Comparison Streptomyces lividans transformation by PEG-dependent protoplast method and transformation that was carried out by using BG-2 carrier, showed five times higher transformation efficiency in case of the nanocarrier application. It was demonstrated efficient DNA delivery into mammalian cells of 7 lines in vitro using polyampholytic carriers. It is important to note the low toxicity of polyampholytic carriers. It was shown that polyampholytic carrier/DNA complexes exhibited higher gene expression of p53 and p21 proteins in HEK293T and MCF-7 cells. Our results demonstrated that polyampholytic carrier/DNA complexes could successfully transfect cancer cells and be used as a potential delivery system for DNA in cancer gene therapy. The carriers were not capable of triggering gene mutations in the Ames test (at the absence or presence of metabolic activation) and ana-telophase test. Thus, novel polyampholytes are perspective carriers for DNA delivery to the recipient cells.