An influence of complexes of therapeutic antisense oligodeoxynucleotides with cationic polymers on cell respiration

Antisense-DNA technologies are new strategy for the treatment of prion infections. This stra-te gy requires prolonged administrations of the drugs, which are likely to alter cell redox processes. Aim. The evaluation of cell survival and intensity of oxidative processes in vitro under the influence of antisense-oligodeoxynucleotides (asODNs) as cell prion inhibitors (PrP C ) complexed with cationic polyelectrolyte. Methods. Free diffusion in agarose gel, study of cytotoxic action on model cells (bull semen), polarography and potentiometric measurement of oxygen uptake, statistical analysis. Results. Poly(dimethylaminoethyl methacrylate)[-]based surfactants form complexes with asODNs. Polyethylene glycol containing surfactants increase oxygen uptake by cells: by 18 % ( VI ), by 37 % ( IV ) and 2.6-fold for V . An addition of the IV -asODNs complex into [the] cell medium did not affect the oxygen absorption; however, it increased reduction processes. Interpolyelectrolyte complex V -asODNs increased the cell respiration by 1.95 times. VI separately increased the cell absorption of oxygen by 18 % and in the complex with asODNs — by 36 %. VI possessed the lowest cytotoxicity. Conclusions. New cationic polyelectrolytes form complexes with asODNs. VI causes the smallest effect on the RedOx processes of model cells and possesses the lowest cytotoxic effect.


Introduction
The pathogenesis of prion infections is associated with the synthesis and aggregation of the cellular form of prion (PrP C ). Therefore, it is assumed that the elimination or reduction of PrP C synthesis in the body will prevent the development and manifestation of transmissible spongiform encephalopathy [1][2].
The possible ways to inhibit the protein synthesis include the use of antisense oligodeoxynucleotides (asODNs), which are complementary to a specific region of mRNA [2][3][4]. Antisense technology allows "turning off" the gene temporarily, which is useful for the development of an adequate strategy for the prevention and treatment of prion infections. Noteworthy, the nucleic acids due to the nucleases degradation activity and poor cellular delivery does not penetrate the cell membrane, which imposes serious restrictions on their use. The success of the use of antisense technology largely depends on the availability of appropriate carriers for asODNs. Cationic synthetic and natural polymers can be carriers for the delivery of genetic material to cells because through electrostatic interactions they effectively bind negatively charged DNA molecules [5]. Thus, they shield DNA from the enzyme degradation, prolong the residence time of genetic constructs in the body and facilitate their penetration into the cells [4]. This invented scheme for inhibition of the development and prevention of prion infections also requires constant drug administration [2,4,6]. Prolonged administration of the active substances likely alters the processes associated with ATP resynthesis and protein phosphorylation in cells. The alteration of redox homeostasis is recog-nized as a significant cause of male factor infertility. Many factors can deregulate this complex network in animals, including exposure to chemicals, toxins and diseases [7].
The aim of the study was to evaluate qualitatively the ability of polymeric carriers to bind antisense oligodeoxynucleotides, and to investigate their effect on the cell oxidative processes in vitro.
Synthesis of poly(DMAEM)-MP (I). AIBN (2 mmol) was dissolved in ethyl acetate (15 mL). DMAEM (112 mmol) and MP (30 mmol) were added, and the mixture was purged with Argon. Polymerization was carried out at 70 °C for 6 h. The conversion was 65-70 %, as determined from dilatometer and gravimetric measurements [9]. The target product was precipitated from acetone into hexane three times and dried in vacuum.
Synthesis of poly(DMAEM-co-BAC)-MP (II). AIBN (2 mmol) was dissolved in ethyl acetate (15 mL). DMAEM (112 mmol), BAC (10 mmol) and MP (30 mmol) were added, and the mixture was purged with Argon. Polymerization was carried out at 70 °C for 6 h. The conversion was 65-70 %. The target product was precipitated from acetone into hexane three times and dried in vacuum.

MP-poly(DMAEM)-PEG-poly(DMAEM)-MP (IV-VI).
Tri-block-copolymers IV-VI were synthesized via polymerization in water in the flask protected from light at 291 K. DMAEM (0.45 mol/L), 0.08 mol/L PEG, MP and concentrated nitric acid were dissolved in 20 mL of solvent. The mixture was purged by argon. The reaction was started by injecting the Ce(IV) salt (0.076 mol/L) dissolved in 5 mL solvent. Polymerization was carried out for 4 h. The polymer was washed with acetone and dried under vacuum. Polymer V was not purified from Ce ions. 1 HNMR of polymers: PEG fragments are confirmed by signals at 3.51 and 3.44 ppm corresponding to protons of CH 2 -groups of the main chain. DMAEM link: the protons of the methyl groups -CH 2 -N(CH 3 ) 2 appear as an intense peak at 2.79 ppm; a peak at 4.23 ppm corresponds to methylene groups -CH 2 -CH 2 N(CH 3 ) 2 ; the signals of the protons of the side methyl group -CH 2 -C(CH 3 ) -appear in the region of 1.18 ppm, and the protons of the methylene group of the main chain -at 1.91 ppm. The presence of the peroxide group is confirmed by signals of the benzene ring (7.22 and 7.14 ppm), tert-butyl C(CH 3 ) 2 OOC(CH 3 ) 3 (1.1 ppm) and С(СН 3 ) 2 ООС(СН 3 ) 3 (1.50 ppm).
The asODNs-polymer complexes were synthesized by the method [10]. Polymer (8 mg) was dissolved in 0.01M HCl, pH of the solution 7.4 and volume 10 ml. 0.5 mL of polymer solution were mixed with 0.5 mL of aqua solu-tion of asODNs (C = 20 nmol/mL). Mixtures were incubated for 30 min (298 K).
Polymer cytotoxicity. The effect of polymers and their complexes with asODNs on the cell survival and oxidative processes was studied in vitro on sperm. The ejaculates were collected from bulls: V = 2-4 mL; C = 0.8-1.3•10 9 cells/mL; vitality = 70 % or more. To the sperm suspension (200 μL) 10 μL of polymers in concentrations of 0.05 %, 0.5 % and 1 % were added. Sperm survival (h) was assessed under a microscope (×200) until the cessation of rectilinear translational motion [5]. Detection of complexes formation as ODNspolymers. The ability of polymers to bind oligonucleotides was determined by the method of free diffusion in agarose gel [5,11].
Cellular metabolic activity was determined for the cells after 96 hours of the cultivation. The intensity of oxygen consumption by cells was established polarographically (ng-atoms O 2 •min -1 •mL -1 cell suspension) using the Clark electrode, which was mounted in a thermostated container (t = 38.5 °C). The cell respiration was measured using standard polarographic techniques with a Clark-type electrode. Cell reduction processes were measured potentiometrically (mV/min/10 6 cells) [12] using open system of microelectrodes [13]. The fraction of oxygen consumed by aerobic glycolysis was determined using an inhibitor of the specified metabolic pathway (NaF; 10 -3 M). The fraction of oxygen consumed by the NAD-dependent chain of the electron transport chain was detected using amytal (5•10 -3 M) and sodium azide NaN 3 (5•10 -2 M). The intensity of free radical oxidation of unsaturated fatty acids was determined using Na 2 EDTA (0.6•10 -3 М). The study of the specific oxygen uptake rate of cells was carried out at 38.5 °C in phosphatebuffered saline (PBS: NaCl -0.8 g; KCl -0.02 g; Na 2 HPO 4 -0.11 g; KH 2 PO 4 -0.02 g; MgCl 2 -0.01 g; Н 2 О to 100 mL; рН -7.4). 0.9 mL of PBS and 0.1 mL of cell suspension were introduced into the thermostatic container.

Result and Discussion
The free diffusion in agarose gel is a rapid and useful method for the screening of suitable transporter of therapeutic nucleic acid compounds, including asODNs [5,11]. It was es-tablished that the polymers based on DMAEM, which are characterized by a positive charge, form complexes with asODNs. The formed complexes are visible as characteristic precipitation arcs (Fig. 2).
Polymer V has slow diffusion, which indicates its higher molecular weight, apparently due to the presence of cerium ions.
Polymers that showed the ability to bind asODNs were tested for their cytotoxic effects on cells. The cell survival was 72 hours for polymers I, II and III, and 96 hours for polymers IV and V. Polymer VI was characterized by the lowest cytotoxicity. Cell survival under VI exposure was 120 hours indicating its low toxicity.
We measured the redox processes caused by the polymers and their complexes with therapeutic asODNs, which decrease the PrP C level [5]. The prion-infected transgenic mice overexpressing the hamster prion protein (Tg7 mice) suffer from mitochondrial respiratory deficits [14]. Mitochondrial respiration of cells/tissues is a significant proportion of total oxygen uptake. The cell respiration involves oxidation and reduction processes (redox). It was found that the polymers increase cel-  Table 1).
In order to establish the effect of active substances on cell metabolism and ATP resynthesis, the intensity of substrate use and electron transport in the mitochondrial respiratory chain was assessed. In particular, NaF inhibits the conversion of phosphoglyceric acid into phosphopyruvic acid and therefore arrests glycolysis [15], amital inhibits the I complex of mitochondrial electron transport chain [16,17], NaN 3 is an inhibitor of cytochrome oxidase [18,19]. The inhibitors cause a gradual decrease of oxygen uptake. At the same time, depending on the intensity of electron trans-port in the respiratory chain, there are different magnitudes of the flow of electrons/protons into the extracellular space and, therefore, different reduction processes. During addition of inhibitors, the polymers depress both the use of substrates and the transport of electrons in the mitochondrial respiratory chain to a greater extent than in control. In particular, NaF together with polymer I reduce respiratory activity by 2.2 times (p <0.05), and by 40.4 % (p <0.001) in control (without polymers). Sequential addition of inhibitors: amytal, NaN 3 and Na 2 EDTA gradually reduces the oxygen consumption by cells, respectively, by 44.5 %, 33.3 % and 43.0 %. Similarly, the reduction ability of cells decreases: by 32.0 % under NaF influence, by 19.2 % -under amytal and by 51.0 %under NaN 3 , but adding Na 2 EDTA causes an increase by 14.8 % (Fig. 3). The effect of NaF under the action of II is manifested by 42.4 % decrease of the oxidation process, whereas the sequential addition of amytal, NaN 3 and Na 2 EDTA inhibits this process by 12.7 times. The reduction processes of cells with the ad- dition of II and the use of inhibitors of both glycolysis and mitochondrial respiratory chain were almost unchanged. The range was from 3.7 to 5.3 mV/min/10 6 cells. Na 2 EDTA decreased oxygen uptake by 49.1 % (p <0.05) compared to the initial value (Fig. 3). Polymer III reduced redox potentials (Fig. 3). The analysis of the effect of polymers on the respiratory activity of cells in vitro shows that the polymer I causes the accumulation of protons in the extracellular space, but the penetration into the cells has low impact on the oxidation processes. Polymer II apparently penetrates the membranes and activates oxygen consumption and almost does not change the outflow of protons from cells. Polymer III stimulates both oxidation processes and the flow of electrons into the extracellular space. Polymer I inhibits the aerobic glycolysis and increases the sensitivity of this process to the influence of the inhibitor. Inhibition of aerobic glycolysis is accompanied by a double accumulation of protons in the extracellular space. By adding polymer II, the effect of inhibitors on the ATP resynthesis is similar to the control. However, during the inhibition of the NAD-dependent link in the mitochondrial respiratory chain, the flow of electrons increases in contrast to the control, where an increase of the space potential was found. Polymer III, after activation of oxidation processes, reduces the sensitivity to the inhibitors of both aerobic glycolysis and electron transport links in the mitochondrial respiratory chain.
Polymers IV and VI, which are derived from polymer I and contain PEG, reduce the cell oxygen uptake by 53.2 % and 34.6 %, respectively. Polymer V increases the cell oxygen uptake by 23.9 % (Fig. 4 A). The presence of cerium ions in the V affects the membrane structure of cells and modifies them [20,21], which provides penetration and use of exogenous substrates. The addition of IV to the sperm suspension stimulated (or caused) the flow of positively charged particles (protons) into the extracellular environment and, conversely, the additions of VI and V were characterized by the changes in cell metabolism, accompanied by the formation of negatively charged particles and their accumulation in the extracellular space.
IV-asODNs and VI-asODNs сomplexes do not change the intensity of cellular respiration, compared with separately added polymers. We suggest that the reducing of the im- pact on cells by these polymers in combination with asODNs happens due to already bound active groups of the polymers. The cell membranes of mammalian are negatively charged [22]. The free active groups of cationic polymers obviously are able to interact with the components of negatively charged cell membranes, change their structure, disrupt integrity and, accordingly, have a stronger effect on redox processes in cells. However, the V-asODNs complex, apparently due to the modification of membranes, penetrates into the cells and reduces the redox processes. With the addition of IV the potential of cell medium increased by 2.5 times, and with V and VI decreased equally by 30.3 %, compared to the control. IV-asODNs complex does not change the cell oxidation, compared to IV without asODNs, but it increases the reduction processes ( fig. 4 A). Similarly, the cell oxidation processes were at the same level during addition of VI-asODNs complex or polymer VI (0.5 ng-atom O 2 /min/10 6 cell), and the reduction processes increased to 3.0 mV/min/10 6 cell (Fig. 4 C). V-asODNs complex reduces both cell oxygen uptakes by 45.2 % and cell reduction processes by 96.7 %, compared to separately added V (Fig. 4 B).
After the addition of NaF to cultural mediums with IV and IV-asODNs, the absorption of oxygen decreased by 2.1 and 8.2 times respectively. The addition of NaF also reduces the respiratory activity together with the influence of other polymeric compounds and their complexes with asODNs. During the addition of amital to the cell medium with IV or with IV-asODNs complex, the formation of oxygen in a polarographic container was detected. At the same time, in the presence of IV the flow of charged particles is almost equal to 0 (0.001 mV/min/10 6 cell). Complexes IV-asODNs and VI-asODNs stimulate an increase of space potential, respectively, by 29.4 and 80.0 %. During the test of VI, the reduction processes do not change (2.0 mV/min/10 6 cells). Under the action of V and its complex with asODNs the reduction processes decrease by 33.4 and 99.9 %, respectively.
Inhibition of the terminal link of the mitochondrial respiratory chain by NaN 3 in the presence of IV-asODNs and V led to the release of oxygen into the cell environment. At the same time, the addition of amytal to the cell with IV and VI almost completely inhibits the flow (outflow) of charged particles (protons and electrons) from the NAD-dependent path of electron transport in the mitochondrial electron transport chain, which is manifested by a decrease of the potential to zero. The addition of amytal to the cell with V does not change the potential (2.0 mV/min/10 6 cells), and in the presence of both IV-asODNs and VI-asODNs decreases it. The addition of Na 2 EDTA to cells with polymers both alone and in complexes with asODNs leads to an inhibition of reduction processes. It was detected that IV and IV-asODNs mostly inhibit aerobic glycolysis and stimulate the flow of protons into the extracellular space. These compounds together with amytal stimulate the generation of oxygen in a polarographic container. It is known that the blocking of the electron transport can lead to the formation of О 2 •− [23][24][25]. Probably, the polymer IV and the complex IV-asODNs change the structure of membranes, penetrate into cells and enhance the inhibitory action of amytal. Generation of the active form of oxygen (О 2 •− ) by the mito-chondrial respiratory chain is one of the causes of membrane destructions [26]. Therefore, the use of IV and V, both alone and in combination with asODNs, can cause changes in cell structure and stimulate the processes of free radical oxidation and O 2 •formation. However, the effect of these compounds on the structures and metabolism is "mild", as their action ensures the survival of cells for 96 hours. The least toxic for cells is the polymer VI and its complex with asODNs. These compounds support aerobic glycolysis, with a weak effect on individual links of electron transport in the mitochondrial respiratory chain. Obviously, after penetration through the membrane into the cell, the polymer VI and VI-asODNs complex are able to normalize the use of endogenous substrates and provide resynthesis of ATP, high maintaining mobility and survival for 120 hours.

Conclusions
Polycationic polymers, which are based on DMAEM, form the complexes with prion inhibitors -antisense oligodeoxynucleotides. Polymers differed in their effect on REDOX processes and cytotoxicity. Polymer VI is characterized by the lowest cytotoxicity and minimal effect on redox processes in cells. Polymer VI is the most suitable transporter of asODNs for inhibition of the synthesis of physiological prion in animal cells.