A. S. Zaichenko’s research while affiliated with Lviv Polytechnic National University and other places

The development of a new anticancer drugs targeted at energy metabolism of tumor cells is a promising­ approach for cancer treatment. The aim of our study was to investigate the action of thiazole derivative N-(5-benzyl-1,3-thiazol-2-yl)-3,5-dimethyl-1-benzofuran-2-carboxamide (BF1) and its complex with PEG based polymeric nanoparticle (PEG-PN) on respiration and mitochondrial membrane potential in murine NK/Ly tumor cells. The rate of oxygen uptake in NK/Ly cells was recorded by a polarographic method using a Clark electrode. The mitochondrial potential relative values were registered using fluorescence TMRM dye. No changes in glucose-fuelled basal respiration or maximal FCCP-stimulated respiration was detected after 15-min incubation of cells with BF1 (10 µM), PEG-PN or BF1 + PEG-PN complex Fluorescent microscopy data showed that BF1 or PEG-PN separately had no effect on the value of mitochondrial membrane potential, while BF1 + PEG-PN complex caused a significant decrease in mitochondrial membrane potential, indicating­ on the decrease of NK/Ly cells viability. Keywords: cell respiration, mitochondrial membrane potential, NK/Ly tumor cells, PEG, polymeric nanoparticles, thiazole derivative

Background. Various cationic polymers have been proposed for use as carriers of genetic materials in biotechnology and biomedicine. The poly(2-dimethylamino)ethyl-methacrylate (poly(DMAEMA)) demonstrated high transfection efficiency and low cytotoxicity when used as a gene delivery system. The aim of the present study was to evaluate the ability of poly(DMAEMA)-block-poly(N-vinylpyrrolidone)-co-(butyl-acrylate)-co-2-aminoethyl methacrylate carrier, BP83-1, to deliver p21 gene into human cervix carcinoma HeLa cells and to define its effects on the viability of tumor cells in vitro. Methods. Transfection assay, Western-blot analysis, MTT test, DNA comet analysis in alkaline conditions, diphenylamine assay for DNA fragmentation (Barton’s assay), FACS analysis of cell cycling. Results. The BP83-1 polymer effectively transferred pFlag-P21WT plasmid DNA containing p21 gene into human cervix carcinoma HeLa cells. The level of BP83-1-facili­tated delivery of p21 into HeLa cells was significantly higher than the level achieved with linear polyethyleneimine (PEI). A significant (26.1 % and 40.1 %) reduction in the viability of HeLa cells transfected with pDNA/BP83-1 and pDNA/PEI polyplexes was detected compared to non-transfected cells. The reverse dependence between the elevated amount of p21 and the reduced amount of Cdk2 was observed in the transfected HeLa cells. The number of cells in G1 phase of the cell cycle in HeLa cells increased from 54.9 % to 65.8 % and to 64.9 % after their transfection with pFlag-P21WT/BP83-1 and pFlag-P21WT/PEI polyplexes, correspondingly. Besides, an increased number of single-strand breaks in DNA and content of the fragmented DNA was detected in HeLa cells transfected with pDNA/BP83-1 and pDNA/PEI polyplexes. The DNA dama­ging effects of the BP83-1 carrier and pDNA/BP83-1 polyplex were less pronounced in treated HeLa cells, compared with such effects of PEI and pDNA/PEI polyplex. Conclusion. An effective transfer of p21 gene with BP83-1 carrier into human cervix carcinoma HeLa cells was demonstrated. The overexpression of p21 gene led to inhibition of viability of HeLa cells, DNA damage, and blocking of cell cycle progression from G1 phase to S phase via a reduction of the amount of cyclin-dependent kinase 2 (Cdk2) and accumulation of cells in G1 phase.

Background. Many types of tumors are sensitive to changes in prooxidant-antioxidant balance. Thus, further studies on reactive oxygen species inducing antitumor drugs that generate oxidative stress-dependent cytotoxic effects are promising. Our previous works showed that thiazole derivatives in combination with polymeric carriers have a pronounced cytotoxic effect on tumor, while not being cytotoxic against pseudo-normal cells in vitro. It was found that thiazole derivatives in complex with PEG-based polymeric carriers affected the antioxidant system of lymphoma cells in vitro. The aim of this work was to study the in vitro effect of the complex of thiazole derivative N-(5-benzyl-1,3-thiazol-2-yl)-3,5-dimethyl-1-benzofuran-2-carboxamide (BF1) in combination with polymeric carriers poly(VEP-co-GMA)-graft-mPEG (Th1), poly(PEGMA) (Th3) and poly(PEGMA-co-DMM) (Th5) on the level of lipid peroxidation products in NK/Ly cells. Materials and Methods. The experiments were conducted on white wild-type male mice with a grafted NK/Ly lymphoma. Ascites tumor cells were inoculated into mice intraperitoneally. Abdominal drainage with ascites of anesthetized mice was performed with a sterile syringe on the 7th–10th days after inoculation. Investigated compounds BF1, polymeric carriers Th1, Th3, Th5 and combination of BF1 + Th1 (Th2), BF1 + Th3 (Th4) and BF1 + Th5 (Th6) at a final concentration of 10 μM were added to the lymphoma samples and incubated for 10 minutes. The level of lipid peroxidation products, such as lipid hydroperoxides and thiobarbituric acid-positive products) were determined according to the techniques described below. Results. All applied complexes based on thiazole derivative BF1 and PEG-based polymeric carriers at a concentration of 10 μM increased the activity of lipid hydroperoxi­des in the lymphoma cells by 29–36% compared to control. Complexes Th2 and Th6 increased the significance of BF1 influence on lymphoma cells from P

Introduction. Genetic engineering in plants is of great importance for agriculture, biotechnology and medicine, and nanomaterials are widely used for genetic engineering. The aim of present study was to evaluate the potential of poly(2-dimethylamino)ethyl methacrylate (DMAEMA)-based comb-like polymers as gene delivery systems in moss Ceratodon purpureus (Hedw.) Brid. protoplasts and determine the level of phytotoxicity of these polymers. Materials and Methods. In order to confirm the formation of complex of poly-DMAEMA carrier with plasmid DNA pSF3, gel retardation assay was used. The PEG-mediated transformation protocol was adapted to transform the protoplasts of C. purpureus moss with poly-DMAEMA carriers. Light microscopy was used to study a toxicity of polymers for moss protoplasts. The level of the polymers toxicity was estimated as IC50 value. Results and Discussion. The formation of pDNA complex with DMAEMA-based carriers took place at 0.03% concentration of the polymers BGA-21, BGA-22(2ph), BG-24, BG-25, BG-26 or 0.1% concentration of the BGA-22 polymer. Poly-DMAEMA carriers were able to deliver plasmid DNA pSF3 into protoplasts of C. purpureus moss. Three stable transformants of C. purpureus were obtained at using BGA-22 polymer, 2 clones – at using BGA-21 carrier, and 1 clone – at using BGA-22(2ph), BG-24, BG-25, BG-26 polymers. The poly-DMAEMA carriers at the working 0.0025% dose were relatively non-toxic for protoplasts of C. purpureus moss. 83.1-88.4% of viable protoplasts of C. purpureus moss were detected after treatment with studied carriers at 0.0025% dose. A survival ratio of protoplasts reached 66.7-72.9% under the effect of these polymers at 0.025% dose, which is 10 times higher than their working concentration. The IC50 value of poly-DMAEMA carriers was in the range of 0.113-0.164%, that was approximately 10 times higher than that of the PEG-6000 used for gene delivery in plants. Conclusion. Novel synthetic poly-DMAEMA carriers delivered the gene of interest into moss C. purpureus protoplasts and possessed a low phytotoxicity. Thus, these carriers can be useful for gene delivery into plant cells.