CC BY
24
УДК 615
O. V. Alekseeva, O. G. Sitnikova, N. A. Bagrovskaya, A. V. Noskov, G. E. Zaikov, O. Yu. Emelina
POLYSTYRENE/FULLERENE COMPOSITES AS ANTIOXIDATIVE AGENTS
Keywords: antioxidant activity; free-radical processes; fullerene-containing nanocomposites; chemiluminescence; malonic dialdehyde.
Effect of polystyrene/fullerene composite on free-radical processes in blood serum has been investigated in vitro. The parameters of lipid peroxidation in native serum after adding of the composite films were determined by chemiluminescent analysis and spectrophotometry. It was revealed that fullerene-containing nanocomposites can manifest antioxidant activity in blood serum.
Ключевые слова: антиоксидантная активность; свободно-радикальные процессы; фуллеренсодержащие нанокомпозиты;
хемилюминесценция; малоновый диальдегид.
Было исследовано влияние полистирол-фуллереного композита на свободно-радикальные процессы в сыворотке крови. Параметры перекисного окисления липидов в природной сыворотке после добавления композитных пленок определяли с помощью хемилюминесцентного анализа и спектрофотометрии. Было выявлено, что фуллеренсодержащие нанокомпозиты могут проявлять антиоксидантную активность в сыворотке крови.
Introduction
Nanocomposite materials are promising field of advanced material sciences with scope of using in biology, medicine, pharmacology, etc. Nanomaterials are based on nanoparticles with unique characteristics resulting from their microscopic size [1].
The discovery of soccer-ball shaped Buckmin-ster fullerene in 1985 [2] was an exciting and unexpected discovery that established an entirely new branch of chemistry. Fullerene is molecular compound belonging to the class of carbon allotropes. Fullerene molecule is a closed convex polyhedron made of carbon atoms that arrange at the vertices of regular hexagons and pentagons.
Fullerene is possessed the unusual physical and chemical properties. Based on these properties the new materials were obtained, such as superconducting "full-erenes/alkali metal atoms" compounds, thin films and solutions of fullerenes with nonlinear optical properties, new superhard materials based on fullerenes [3].
Currently, various derivatives of fullerenes exhibit a broad spectrum of biological activity: anticancer, antiviral, antibacterial, neuroprotective, and antioxidant activities [4]. Biological abilities of fullerenes are due to lipophilic properties which facilitate cell penetration and lack of electrons, helping to react with free radicals and generate active oxygen species [5].
Interaction mechanisms of fullerenes with cell are still poorly understood. To research these processes, various methods have been used, such as molecular dynamics simulations [6], radioactivity method [7], etc. It was demonstrated in ref. [7] that fullerene derivative C61(CO2H)2 could cross the external cellular membrane, and it localized preferentially to the mitochondria. Formation of oxygen free radicals occur due to the electrons leakage in the mitochondrial electron transport chain. Therefore, the localization of fullerenes near derivatives mitochondrions may contribute to their antioxidant action.
The mechanism of biological role of fullerene is shown to depend on its aggregate form: crystalline, colloid or soluble organic complex [8, 9]. Soluble or-
ganic fullerene complex has highest bioactivity [8]. The authors of ref. [8] explained this effect by low association of nanocarbonic particles.
A new direction of fullerene science has been developed, and the related studies in this direction have concerned the preparation of fullerene-containing polymers, which would combine the unique characteristics of fullerene with the useful properties of matrix polymers [10].
One of polymers able to complex with nanoparticles is polystyrene (PS), which is widely spread in industry. Also polystyrene is used, for example, for the manufacture of surgical instruments, as well as an arterial embolization to stop bleeding. Replacement of pure polymer by fullerene-containing one will impart it antibacterial and antimicrobial properties. Therefore, poly-styrene/fullerene composites are the subject of numerous studies [11-13]. It is considered that integration of fullerenes into polymer matrix can produce biocomposites which have medical potential as drug transporters, antiseptics, and antioxidants. Polysty-rene/fullerene composite materials may be used to manufacture containers for blood storing too.
Early in ref. [14, 15] we reported on studies of the polystyrene/fullerene composites by scanning electron microscopy, infrared spectroscopy, and X-ray diffraction. In scanning electron microscopy images (Fig. 1) it can be observed, the surface is composed of rounded particles with irregular relief, the size of irregularities being comparable with the polystyrene molecule size (10 nm).
The above listed methods and mathematical modeling have been used to study the influence of the fullerene additives on structure of polystyrene [14, 15]. We have concluded that during the solvent evaporation and film formation the individual polymer molecules collide and stick together into aggregates. In films without fullerenes, packing of straightened chains parallel to each other predominated. This shows that a polystyrene molecule, which had the shape of a coil in solution, straightened and stretched itself along the aggregate surface when attached to it. In polystyrene/fullerene composite, the intermolecular interactions between pol-
ystyrene and fullerene are appreciable, and under the influence of fullerene, polystyrene molecules straightened with the formation of ordering elements in the arrangement of chains.
Fig. 1 - Electron microscopic image of the surface of the film: a) polystyrene film; b) polystyrene film filled with fullerene (0.035 wt % of C60+C70). (This figure was presented in our paper [14]).
So the fullerene additives effect on structure of polystyrene. Such modification may result to occurrence of new properties of polymer, for example, biological activity.
Bioactivity of material may be evaluated by its effect on the free radical processes in biologic fluid. Currently regulation of free-radical processes is adjusted by both natural and synthetic pharmaceutical compositions [16]. As any other medicine some antioxidants may produce adverse events. Thus, finding of safe preparations with high antioxidant activity is still actual.
We have analyzed the literature on studies of the polystyrene/fullerene composites, and have concluded that the antioxidant activity of these materials was not researched enough, although for fullerenes it was known. For our opinion, it is necessary to fill this gap. Therefore the goal of present research was to investigate the influence of polystyrene/fullerene nanocomposites on free-radical processes in biologic fluid (blood serum) in vitro.
Experimental
Materials and Subjects
We have chosen polystyrene (Aldrich, Germany, Mn=1.4T05, Mw/Mn=1.64) as a matrix for fabrication of fullerene-containing nanocomposites, because it has high solubility in aromatic hydrocarbons like fuller-ene itself. C60+C70 fullerene mix (Fullerene Technologies Ltd., Russia) was preliminary purified [17].
Fullerene-polystyrene composition films were produced as follows. Batches of polymer and C60+C70 were solved separately in o-xylene (or toluene). Concentrations of fullerenes in o-xylene solutions were equal to 0.018, 0.054, 0.18, and 1.8 g/l. Concentrations of fullerenes in toluene solutions were equal to 0.18, and 0.9 g/l. Polystyrene batches were dissolved in respective solvents (179.38 g/l of PS) too. Then, fuller-ene/o-xylene solutions and polystyrene/o-xylene solutions were mixed together, so that weight fractions of fullerene were equal to 0.01, 0.03, 0.1, and 1 wt %. The mixed solutions were stirred for about 1 day before being cast into thin films. Similar actions were carried out with toluene solutions. After casting the solvent was slowly evaporated over several days to produce the pol-ystyrene/fullerene composite films.
Subject of research was native blood serum of 10 patients managed in V.N. Gorodkov Research Institute of Maternity and Childhood (Ivanovo, Russia). Specimen of pure PS or composite film (size 1.5 cm2, weight 5 mg) was put into blood serum (1 ml). System has been incubated for 1 hour at 4oC.
Methods
After contact of blood with pure polystyrene films or fullerene-containing polystyrene films, we have studied the free radical processes in blood. Also we have studied the free radical processes in native blood serum without preliminary contact, choosing as control. The parameters of lipid peroxidation have been determined by chemiluminescent analysis and spectropho-tometry.
Chemiluminescence
Induction of chemiluminescence (ChL) by hydrogen peroxide and iron sulfate is based on Fenton reaction: at mixing the components the catalytic decomposition of hydrogen peroxide takes place by divalent iron ions. Thus formed free radicals oxidize the lipopro-teins of blood serum in the test samples, leading to the formation of new free radicals. At the recombination of radicals, unstable products are formed and decomposed with the release of photons.
Generally, a chain reaction in which formation of radicals leads to chemiluminescence, may be represented by the scheme [18]:
->R<
^ P + photon
where R • - the free radicals; P* - the radical decay products in excited state; P - ones in ground state.
The intensity of chemiluminescence, I, is proportional to the rate reaction, v:
I = rv, (1)
where n - quantum efficiency of the chemiluminescent reaction.
For the process, the ChL curve dips because of antioxidant agents. The decay rate constant of free radicals, k, is defined by the dip rate of ChL curve. Therefore, the main indicator of the antioxidant activity of the system is tangent of maximum slope angle of ChL curve towards time axis, tana.
Also, we used following parameters: Im is maximum intensity of ChL during the experiment. Value of Im quantifies the level of free radicals, i.e. gives an idea of the potential ability of the blood serum to free radical lipid peroxidation; S is an area covered by intensity curve or total light sum. Value of S is inversely proportional to the antioxi-dant activity of the sample;
Sn=S/Im is normalized light sum. The value of Sn evaluates antioxidant activity more correctly than the value of S, because the total area covered by ChL curve depends on value of Im.
The induced chemiluminescence tests have been performed by BChL-07 luminometer (Medozons, Russia). We have used hydrogen peroxide and ferric sulfate as inductors of ChL. 0.1 ml of serum, 0.4 ml of phosphate buffer (pH 7.5), 0.4 ml of 0.01M ferric sulfate and 0.2 ml of 2 % hydrogen peroxide have been put into cuvette. Luminescence has been registered for 40 s.
Free radical processes in serum have been studied after contact with original polystyrene films and fullerene-containing polystyrene films. The mean values
of ChL parameters in native serum without addition of the film have been used as controls. The results have been expressed as percentages relative to controls and have been given as mean values ± standard deviations. Level of significance, p, was 0.05.
Spectrophotometry
Also we have defined the malonic dialdehyde (MDA) concentration in blood samples after contact with PS and PS/C60+C70 films. This indicator reflects the amount of the lipid peroxidation products. MDA concentration has been determined by the SF-46 spec-trophotometer (Russia) at wave length of 532 nm. This method based on the formation of complex MDA with 2-thiobarbituric acid [19]. The concentration of MDA has been calculated as follows:
c = D,
MDA ,
is
(2)
where D is absorbance; l is the large of the cuvette; e is the molar extinction coefficient.
Value of total antioxidant reactivity, TAR, has been evaluated by measuring the absorbance before and after incubation of samples by equation [20]:
TAR = 1 D "D
D - D
^t ^0
(3)
where D0 and Dt are values of absorbance for blood samples before and after incubation, respectively;
D0 and Dst are the same quantities for solution of linolenic acid choosing as standard.
Results and discussion
Using above described technique and o-xylene as solvent, we have prepared one sample of polystyrene film and four samples of polystyrene/fullerene composite films with various C60+C70 percentages. Unmodified polystyrene samples were colorless, whereas the poly-styrene/fullerene composite films were light purple. The intensity of color depended on the content of C60+C70 in the composite.
Fig. 2 - Kinetic chemiluminescence profiles of native blood serum (1) and after contact of it with studied materials: 2 - PS; 3 - PS/C60+C70 (1.0 wt %); 4 -PS/C60+C70 (0.03 wt %). Solvent: o-xylene
Fig. 2 shows kinetics of chemiluminescence in blood serum after contact with original polystyrene and nanocomposite films. Peak of chemiluminescence due to free radical production was in 2 s of reaction. This can be explained by production of active oxygen species (HO2*, O2*, O2-, OH-). The highest intensity, Im, was registered in case of nanocomposites with 0.01 and 0.03 wt % of fullerenes.
In Table 1 we have represented the main ChL parameters for films prepared by casting of o-xylene solution. It can be seen in case of original polystyrene film the ChL parameters were approximate to controls. Value of Im for the PS/C60+C70 composites is higher than for control serum samples. A light sum, S, was significantly increased only for films with 0.01 and 0.03 wt % of fullerenes (p<0.05). For nanocomposite containing 1 wt % of C60+C70, no significant change in value of S was revealed. In addition we found both significant growth in value of tana and reduction in value of Sn for all fullerene-containing films (Table 1). So regardless of the fullerenes content, the antioxidant activity of PS/C60+C70 composites is higher than for original polystyrene. It seems nanocomposites containing fullerenes were easy to react with oxygen species, preventing lipid peroxidation.
Table 1 - Chemiluminescence parameters in blood serum after contact with original polystyrene film and fullerene-containing nanocomposites (solvent: o-xylene)
Fullerene content, wt % S, % Im, % tana, % Sn, %
Controls 100.0 100.0 100.0 100.0
0 96.0±9.0 97.0±7.0 97.5±10.5 99.0±11.5
0.01 121.5±10.5* 131.5± 11.5* 139.0±27.0* 92.0±6.5 *
0.03 115.0±8.0* 125.0±11.0* 146.0±27.0* 92.0±7.0 *
0.1 105.0±3.0 110.0±6.0* 110.0±8.0 * 95.5±5.0
1.0 96.0±12.0 111.0±6.0* 121.0±7.0 * 86.5±7.0 *
* - significant differences compared to control (p<0.05)
Also intensity of lipid peroxidation has been estimated by malodic dialdehyde concentration and total antioxidant reactivity assessed by spectrophotometry (Table 2). We have obtained that nanocomposite with 0.03 wt % of C60+C70 increased MDA level in blood serum (p<0.05). The increasing of MDA level indicates the activation of lipid peroxidation and the accumulation of free radicals in the blood serum. But the film specimens with higher fullerene content decreased this parameter. Note the similar trend correlates with dependence of Im value on fullerene concentration in film (Table 1).
It can be seen in Table 2 that total antioxidant reactivity was increased in serum samples after contact with nanocomposites containing 0.03, 0.10, and 1.00 wt % of fullerenes (p<0.05). This proved antioxidant effect of experimental materials. It appears the amount of active centers, which able to effectively capture and inactivate the free radicals, increases with concentrations of fullerenes in composite material.
It is interesting to reveal the effect of the medium in which the films have been fabricated. For this we have performed experiments for films prepared by casting of other aromatic compound - toluene.
The main ChL parameters for "toluene" films are given in Table 3. It can be seen that in case of original polystyrene film all values were approximate to controls. But using nanocomposites (0.5 wt % of C60+C70) we have found significant increase in value of tana. It demonstrates the antioxidant activity of researched PS/C60+C70 composites.
Table 2 - Lipid peroxidation parameters (MDA, TAR) in blood serum after contact with original polystyrene film and fullerene-containing nanocomposites (solvent: o-xylene)
Fullerene content, wt % МЭА TAR
Controls 100.0 100.0
0 103.0±23.0 108.0±4.0
0.03 113.0±5.0 * 116.0±5.0 *
0.1 94.0±8.0 110.0±4.5 *
1.0 96.5±10.5 107.0±4.0 *
* - significant differences compared to control (p<0.05)
Table 3 - Chemiluminescence parameters in blood serum after contact with original polystyrene film and fullerene-containing nanocomposites (solvent: toluene)
Fullerene content, wt % S, % Im, % tana, % Sn, %
Controls 100.0 100.0 100.0 100.0
0 98.0±7.5 99.0±7.0 98.5±7.5 100.0±7.0
0.1 101.5±10.5 120.5±14.0* 100.5±10.5 84.0±8.5*
0.5 103.0±7.5 101.0±7.5 130.0±14.0* 102.0±5.0
* - significant differences compared to control (p<0.05)
In addition, it can be seen in Tables 1 and 3 that at the same concentration of fullerene (0.1 wt %), the value of tana is higher for film formed of o-xylene than for film formed of toluene. This can be explained by that the solubility of fullerenes in o-xylene is higher than in toluene [21]. It appears that in toluene solution the fullerene molecules are in the form of clusters, which does not ensure uniform distribution of the nano-particles in the composite film during its formation of solution.
Note also we have performed the preliminary experiments with PS films containing fullerene that have been fabricated by casting of aliphatic compound -chloroform. The results of chemiluminescent analysis and spectrophotometry for serum samples after contact with both original polystyrene film and composite films regardless of the fullerenes content were approximate to controls. This again emphasizes the significance of the medium in which the films were fabricated.
In conclusion, our investigation proved that polystyrene/fullerene nanocomposites have ability to inhibit the lipid peroxidation in blood serum. Furthermore, possibility of such inhibition depends on conditions for forming composite.
Acknowledgments
The study was supported by the Russian Foundation for Basic Research (project no. 12-03-97528-a).
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© O. M. Alekseeva - PhD in biology, G.A. Krestov Institute of Solution Chemistry, RAS, Ivanovo, olgavek@yandex.ru; O. G. Sitnikova - V.N. Gorodkov Research Institute of Maternity and Childhood, Ivanovo; N. A. Bagrovskaya -G.A. Krestov Institute of Solution Chemistry, RAS, Ivanovo; A. V. Noskov - G.A. Krestov Institute of Solution Chemistry, RAS, Ivanovo, G. E. Zaikov -professor, Department of technology of plastic materials, KNRTU, Kazan, chembio@sky.chph.ras.ru; O. Yu. Emelina - assistant, Department of technology of plastic materials, KNRTU, Kazan, emelina_oy@mail.ru.
© О. В. Алексеева - канд. биол. наук, Институт химии растворов имени Г.А. Крестов РАН, olgavek@yandex.ru; О. Г. Ситни-кова - сотр., Ивановский научно-исслед. институт материнства и детства имени В.Н. Городкова; Н. А. Барговская - сотр., Институт химии растворов имени Г.А. Крестов РАН, Иваново; А. В. Носков - сотр., Институт химии растворов имени Г.А. Крестов РАН, Иваново; Г. Е. Заиков - профессор, кафедры технологии пластических масс КНИТУ, Казань; О. Ю. Емелина - ассистент кафедры технологии пластических масс КНИТУ, emelina_oy@mail.ru.
