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PVP-stabilized tungsten oxide nanoparticles (WO3) nanoparticles cause hemolysis of human erythrocytes in a dose-dependent manner
A. L. Popov1, I. V. Savintseva1, N. R. Popova1, T. O. Shekunova2'3, O. S. Ivanova3, A. B. Shcherbakov4, D. A. Kozlov2'3, V.K. Ivanov2'3*
1 Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow region, 142290, Russia 2Lomonosov Moscow State University, Moscow, 119991, Russia 3Kurnakov Institute of General and Inorganic Chemistry, Russian Academy of Sciences, Moscow, 119991, Russia 4Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine,
Kyiv, D0368, Ukraine
antonpopovleonid@gmail.com, savintseva_irina@maiLru, nellipopovaran@gmail.com, taisia.shekunova@yandex.ru, runetta05@mail.ru, carotene@igic.ras.ru, kozlov@inorg.chem.msu.ru,
*van@igic.ras.ru
PACS 68.65.k, 81.20.n, 82.70.Dd, 87.17.Ee DOI 10.17586/2220-8054-2019-10-2-199-205
Tungsten oxide nanoparticles (WO3 NPs) are increasingly being considered as a promising material for biomedical applications. However, toxicological information on their effect on red blood cells (RBCs) remains very scarce. In this study, we examined the toxicity of PVP-stabilized tungsten oxide nanoparticles against human RBCs. Optical microscopy and spectrophotometry data showed that WO3 NPs induce hemolytic activity. This effect is probably attributed to the direct interaction of the nanoparticles with the RBCs, resulting in the oxidative stress, membrane injury, and subsequent hemolysis.
Keywords: tungsten oxide nanoparticles, human erythrocytes, hemolysis.
Received: 10 February 2019 Revised: 21 February 2019
1. Introduction
Tungsten oxide nanoparticles (WO3 NPs) are considered as a promising nanomaterial for biomedical applications due to their multifunctionality and therapeutic importance. In recent years, WO3 NPs have been employed
in advanced biomedical applications as antibacterial coatings, contrast agents for X-ray computed tomography or biosensors [1-8]. However, a comprehensive multi-faceted study of their cytotoxicity, in particular hemotoxicity, is still missing. Meanwhile, hemolytic analysis is mandatory for all types of nanomaterials, since their hemolytic activity depends strongly on the size, shape and charge of the nanoparticles, as well as synthesis approaches. Earlier, Chen et al. examined the size-dependent cytotoxicity of silver nanoparticles (Ag NPs) against fish RBCs using three different preparations with characteristic size of nanoparticles of 15 nm, 50 nm, or 100 nm. Data obtained showed that Ag NPs exhibited size effect on their adsorption and uptake by RBCs: the smaller particles possess higher hemolytic activity than that of the larger particles [9]. Aisaka et al. demonstrated hemoglobin release from human erythrocytes upon incubation with TiO2 nanoparticles. However, the hemolysis was abolished by plasma, and so physical (mechanical) factors are the most important in TiO2-induced hemolysis [10]. Vinardell et al. compared the hemolytic behavior of bulk aluminum oxide and aluminum oxide nanoparticles on erythrocytes from humans, rats and rabbits. Aluminum oxide nanoparticles are less hemolytic than bulk aluminum oxide and aluminum oxide nanowires, and behave differently depending on the size and shape of the particles [11]. Babu et al. investigated the size-dependent interaction of zinc oxide nanoparticles (ZnO NPs) with RBCs, and their impact on cell viability, DNA damage, reactive oxygen species (ROS) generation. Results obtained showed that ZnO NPs exhibited a size dependent effect on RBCs and hemoglobin (Hb), particularly those NPs with size less than 50 nm [12].
Considering WO3 nanoparticles as a promising contrast agent for X-ray computed tomography, one should analyze their effect on human blood cells. Here, we evaluated the hemolytic activity of PVP-stabilized WO3 nanoparticles and suggested possible WO3 nanoparticles cytotoxicity mechanisms.
2. Materials and methods
2.1. Synthesis and characterization of tungsten oxide nanoparticles (WO3 NPs)
Ultrasmall hydrated tungsten oxide nanoparticles were synthesized by hydrothermal processing of tungstic acid in the presence of polyvinylpyrrolidone (PVP K-30, average mol. wt. 40,000) as template, stabilizer and growth regulator. Tungstic acid was prepared by ion-exchange method using sodium tungstate (Na2WO4) solution and strongly acidic cation exchange resin (Amberlite® IR120). Briefly, ion exchange resin (in a hydrogen form) was swelled in water and loaded into the glass column (filling volume 200 ml). Then, 100 ml of 0.05 M sodium tungstate solution was passed through the column dropwise; 4 g of PVP was added to the obtained eluent; solution was transferred to the flask and stirred for 4 h at reflux. During heating a clear sol of hydrated WO3 was formed, as evidenced by the appearance of UV-absorption band at 325 nm and Tyndall cone. For cell experiments, sol obtained was diluted to prepare 0.1 - 25.0 mg/ml WO3 colloid solutions.
In order to determine the possible influence of polyvinylpyrrolidone stabilizer (PVP) on RBCs, we also prepared individual PVP solutions in a similar way.
High-resolution transmission electron microscopy (HR-TEM) analysis was performed using a Libra 200 MC microscope (Zeiss, Germany). TEM images were recorded using a CCD camera (Gatan, USA) with a matrix size of 4096 x 4096 pixels.
X-ray diffraction (XRD) patterns were collected using a Rigaku D/MAX 2500 diffractometer (Bragg-Brentano reflection geometry) with a scintillation counter. All measurements were performed with CuK«i,2 radiation generated on a rotating Cu anode (50 kV, 250 mA) and monochromatized by a curved graphite [0 0 2] monochromator. XRD patterns were obtained in the 26 range 5 - 80° at a 26 step of 0.02° and a counting time at least of 10 s per step. Before the measurements the WO3 sols were applied to an ITO substrate and dried.
The FTIR spectra of the samples were recorded on a Bruker ALPHA spectrometer, in a range of 400 -4000 cm-1, in attenuated total reflectance mode. To avoid solvent effect WO3 sol and PVP solution were dried at 50 °C for 1 h.
2.2. Analysis of hemolytic activity
The analysis of hemolytic activity was performed on human blood collected from a healthy patient. The method for hemolysis assay was reported earlier [13]. Prior to WO3 nanoparticles exposure, the absorbance spectrum of the positive control supernatant was checked and used only when the optical density was in the range of 0.50 - 0.55. Red blood cells (RBCs) were then incubated with WO3 nanoparticles for 2 h and further centrifuged to isolate the cells. After that, 100 ^L of supernatant for each sample was transferred to a 96-well plate. The absorbance values of the supernatant at 570 nm were determined by using a microplate reader. The percent hemolysis of RBCs was calculated according to the equation: percent hemolysis = ((sample absorbance -negative control absorbance)/(positive control absorbance - negative control absorbance)) x 100.
2.3. Optical microscopy of RBCs
Optical microscopy images of RBCs incubated with WO3 nanoparticles were taken using a Carl Zeiss Axiovert 200 fluorescence-light microscope and recorded by a Canon A620 digital camera.
2.4. Statistical analysis
The experiments were conducted in 3 - 4 repetitions, with analytical estimations done for each WO3 NPs concentration in three repetitions. Experimental results were compared with the control. Statistical analysis was performed using the methods of variation statistics. We determined the mean values and the standard deviation of the mean. The significance of differences between the groups was determined by Student t-test. The obtained data were processed using GraphPad 6.0 and Microsoft Excel 2007 software.
3. Results and discussion
According to HR-TEM (Fig. 1), WO3 nanoparticles are ultra small and about 1 nm in size. Obviously, the growth of WO3 nanoparticles was effectively suppressed by the presence of PVP surfactant.
The XRD data are presented in Fig. 2. The XRD pattern of a dried PVP solution (Fig. 2(a)) contains two broad maxima at 12.1° and 19.9°26 which are characteristic for pure PVP. These data are in a good agreement with previously reported results [14,15]. The sharp peaks are corresponding to ITO substrate signal. The XRD pattern of the dried WO3 sol (Fig. 2(b)) is mostly X-ray amorphous and partially similar to PVP XRD pattern. A significant increase in intensity at 26 < 10° may be due to X-ray scattering on ultra small WO3 nanoparticles.
Fig. 1. HR-TEM image of PVP-stabilized WO3 nanoparticles
Fig. 2. X-ray diffraction patterns of dried PVP solution (a) and dried WO3 sol (b). ITO substrate diffraction maxima are marked with *
The FTIR spectra of dried PVP solution and WO3 sol are shown in Fig. 3. The spectrum for dried PVP solution is similar to the spectra of individual PVP given in literature [16-18]. The FTIR spectrum of dried WO3 sol is identical to dried PVP solution excepting the ranges of 795 - 995 cm-1 and 420 - 435 cm-1. Absorbance in these ranges is typical for tungsten oxide [19-22]. Note that FTIR spectrum of dried WO3 sol after UV irradiation (A = 365 nm, exposure time - 1 min) is similar to dried WO3 sol kept in dark, while a slight difference in splitting of the absorption band at 430 cm-1 is observed. Such a difference can be caused by distortions of [WO6] octahedra upon changes in tungsten oxidation state.
The appearance of the test tubes with RBCs upon exposure to WO3 NPs for 2 h is shown in Fig. 4(a). It can be seen that hemolytic activity of WO3 NPs is dose-dependent. High concentrations of WO3 NPs lead to the aggregation of the particles, which increases hemolytic activity. It is also well known that tungsten oxide nanoparticles possess enormous redox activity, which can lead to oxidative damage of red blood cell membranes [6].
The results of the spectrophotometric analysis of supernatants confirm the trend revealed by the appearance of the RBCs (Fig. 4(b)). Optical microphotographs of RBCs without WO3 NPs (Fig. 4(c)) showed cells with
-I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I—I
3600 3200 2800 2400 2000 1600 1200 800 400
v, cm
n—1—i—1—i—1—i—1—i—1—i—1—i—1—i—1—i—1—i—1—i—1—I
150014001300120011001000 900 800 700 600 500 400
v, cm"1
Fig. 3. Survey IR spectrum (above), and its fragment (below): dried PVP solution (a), dried WO3 sol (b), dried WO3 sol after UV irradiation (c)
undamaged membranes, however treatment with WO3 NPs (12.5 mg/ml) for 2 h caused damage to 100 % of RBCs with hemoglobin release and cell lysis leading to formation erythrocyte membrane ghosts (Fig. 4(d)).
Surface functionality of nanoparticles is one of the key factors determining their possible uses in therapeutic applications, imparting functional properties and dictating their circulation profile in the blood stream [23,24]. For example, the nanoparticles' surface hydrophobicity has a critical role in the cellular uptake, toxicity, and immune responses of nanomaterials [25-27]. Meanwhile, when entering the bloodstream, nanoparticles interact with blood proteins to form a protein corona, which changes their functional characteristics thus affecting final physiological effect [28-32]. It was previously shown that the preincubation of nanoparticles with plasma proteins can give rise to hemolytic activity of nanomaterials [33]. In our experiments, we also simulated the conditions of the microenvironment in the bloodstream by preincubating nanoparticles in a solution of serum albumin, the main protein of the blood plasma, and evaluated their hemolytic activity upon this treatment (Fig. 5).
(c)
(d)
Fig. 4. Hemolysis of human red blood cells upon incubation with WO3 nanoparticles. Appearance of the test tubes containing RBCs upon exposure to WO3 NPs for 2 h (a). The hemolysis of WO3 nanoparticles measured spectrophotometrically at 540 nm (b). Optical microscopy images of RBCs without WO3 NPs (c) and RBCs exposed to WO3 NPs (12.5 mg/ml) for 2 h (d)
Fig. 5. Hemolytic activity of WO3 NPs after preincubation with serum albumin. The rate of hemolysis was calculated using water (Milli Q) as the positive control. Error bars represent standard deviations (n = 3)
Meanwhile, no significant changes in hemolytic activity of WO3 NPs were observed in the presence of serum albumin.
Thus, the toxic action of tungsten oxide nanoparticles on human blood cells is probably realized via molecular mechanisms. Further research is required to clarify the nature of this toxic action.
4. Conclusions
Ultra small tungsten oxide nanoparticles were synthesized using polyvinylpyrrolidone as the growth regulator. Tungsten oxide nanoparticles were comprehensively studied using HR-TEM, XRD and FTIR techniques.
PVP-stabilized tungsten oxide nanoparticles were shown to exhibit notable hemolytic activity in a dose-dependent manner. The reasons for WO3 NPs' toxic action were clarified.
Acknowledgements
The work supported by Russian Science Foundation (project 18-73-10150).
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