Journal of Nanoparticle Research

, 15:1507

Cytotoxicity of cuprous oxide nanoparticles to fish blood cells: hemolysis and internalization

Authors

    • Asian International Rivers Center, Yunnan Key Laboratory of International Rivers and Trans-boundary Eco-securityYunnan University
  • Bin Kang
    • Asian International Rivers Center, Yunnan Key Laboratory of International Rivers and Trans-boundary Eco-securityYunnan University
  • Jian Ling
    • College of Chemistry and Chemical EngineeringYunnan University
Research Paper

DOI: 10.1007/s11051-013-1507-7

Cite this article as:
Chen, L.Q., Kang, B. & Ling, J. J Nanopart Res (2013) 15: 1507. doi:10.1007/s11051-013-1507-7

Abstract

Cuprous oxide nanoparticles (Cu2O NPs) possess unique physical and chemical properties which are employed in a broad variety of applications. However, little is known about the adverse effects of Cu2O NPs on organisms. In the current study, in vitro cytotoxicity of Cu2O NPs (ca. 60 nm in diameter) to the blood cells of freshwater fish Carassius auratus was evaluated. A concentration-dependent hemolytic activity of Cu2O NPs to red blood cells (RBCs) and the phagocytosis of Cu2O NPs by leukocytes were revealed. The results showed that dosages of Cu2O NPs greater than 40 μg/mL were toxic to blood cells, and could cause serious membrane damage to RBCs. The EC50 value of Cu2O NPs as obtained from RBCs and whole blood exposure was 26 and 63 μg/mL, respectively. The generation of reactive oxygen species and the direct interaction between Cu2O NPs and the cell membrane were suggested as the possible mechanism for cytotoxicity, and the intrinsic hemolytic active of Cu2O NPs was the main contributor to the toxicity rather than solubilized copper ions. The adsorption of plasma proteins on the surfaces of Cu2O NPs led to their aggregation in whole blood, and aggregate formation can significantly alleviate the hemolytic effect and subsequently mediate the phagocytosis of Cu2O NPs by leukocytes.

Keywords

Cuprous oxide nanoparticlesToxicityHemolytic behaviorPhagocytosisBlood cells

Introduction

Copper oxide nanoparticles (NPs) possess unique physical and chemical properties, which are employed in a broad variety of applications, such as semiconductors, gas sensors, imaging contrasts agents, and photovoltaic cells (Jiang et al. 2002; Qi et al. 2010; Zhang et al. 2006). These unique properties may also incite toxicity, causing damage to organisms and posing risks to human health and the ecological environment (Karlsson et al. 2008). There are two major forms of copper oxide nanostructures, cupric (CuO NPs), and cuprous (Cu2O NPs). While the toxic effects and mechanisms of CuO NPs related to the production of reactive oxygen species (ROS), lipid peroxidation, and DNA damage have been fully investigated (Hanagata et al. 2011; Mortimer et al. 2011; Wang et al. 2011; Baek and An 2011; Ivask et al. 2010), little is known about the toxic effects of Cu2O NPs to organisms other than one literature report on the toxic effects of Cu2O NPs to zebrafish (Chen et al. 2011). As a result of the growing application of Cu2O NPs in solar energy conversion, electronics, cell imaging, and gas sensors (Zhang et al. 2006; Qi et al. 2010), there is an increasing risk of their accidental release to the environment leading to possible adsorption to aquatic organisms and uptake by phagocytotic organisms or fish (Farre et al. 2009). Therefore, it is important to address the toxicity of Cu2O NPs.

Characterization of in vitro hemolytic activity of the NPs is especially important because in vivo biomedical applications require the NPs to be delivered into the circulatory system via intravenous injection. Silica-based nanomaterials are widely known to cause the hemolysis of mammalian RBCs. Various explanations for such hemolytic effects have been proposed, including the generation of ROS, denaturation of membrane proteins, and the high affinity of silicate for binding with the tetra-alkyl ammonium groups on membranes (Zhao et al. 2011; Thomassen et al. 2011). In addition to silica-based nanomaterials, the in vitro hemolytic potential of other types of nanomaterials, including silver NPs, graphene nanomaterials, and hyperbranched polyglycerol-based nanoparticles, in human RBCs has also been investigated (Kainthan et al. 2006; Choi et al. 2011; Liao et al. 2011). However, to the best of our knowledge, no prior study on the hemolytic interaction between copper oxide NPs and RBCs has been reported.

In our previous report, we developed a facile solution-phase method for synthesis of uniform Cu2O NPs and revealed that Cu2O NPs not only can be internalized by human cells but also can lead to the local structural alteration of proteins (Qi et al. 2010). In the current study, we describe an investigation of the hemolytic properties of Cu2O NPs with RBCs of silver carp (Carassius auratus). CuSO4 was served as a control for the effect of dissolved copper ions (Cu2+) since dissolved cuprous ions (Cu+) are unstable in environmental conditions, and Cetyltrimethylammonium Bromide (CTAB) was used as a control for known toxic action because Cu2O NPs were capped by a CTAB layer to prevent oxidization to Cu2+ during preparation. A concentration-dependent hemolysis of Cu2O NPs with fish RBCs was revealed by the means of both direct visual colorimetric assay and flow cytometric analysis, and the phagocytosis of Cu2O NPs by leukocytes was observed using dark-field light scattering microscopic imaging. This is the first work that evaluates the hemolytic behavior of Cu2O NPs on RBCs and the results provide the first evidence of the phagocytosis of NPs by leukocytes.

Experimental

Materials

Sodium borohydride (NaBH4) and copper (II) sulfate (CuSO4·5H20) were purchased from Huanwei Fine Chemial Co. Ld. (Tianjin, China) and Chujiang Chemical Co. Ld. (Wuhan, China), respectively. CTAB was commercially obtained from Sinopharm Group Chemical Regent Co., Ltd. (Shanghai, China). Both heparin and propidium iodide (PI) dye were purchased from Sigma-Aldrich Co., Ltd. (Shanghai, China). Catalase analysis, Superoxidase dismutase analysis, and Bradford protein assay kits were purchased from Beyotime Biotechnology Inc. (Beijing, China) and used as received. All reagents were prepared by dissolving their commercial products in doubly distilled water.

Synthesis and characterization of Cu2O NPs

The details relating to the synthesis and property characterization of Cu2O NPs have been described by us previously (Qi et al. 2010). Scanning electron microscopy (SEM) images were taken using a Hitachi S-4800 microscope (Tokyo, Japan) operating at an accelerating voltage of 20 kV and a working current of 10.0 μA. The absorption and light scattering spectra were measured using a Hitachi U-3010 spectrophotometer and a Hitachi F-4500 fluorescence spectrophotometer (Hitachi Ltd., Tokyo, Janpan), respectively.

Hemolysis

Heparin-stabilized fish blood was freshly collected according to the literature a slightly modified method (Lin and Haynes 2010). Briefly, a 4 mL sample of whole blood was added to 8 mL of fish phosphate-buffered saline (FPBS) and the RBCs were isolated from serum by centrifugation at 10,000×g for 5 min. The RBCs were further washed three times with FPBS solution. Following the last wash, the RBCs were diluted to 40 mL with FPBS. Then, 0.5 mL of the diluted RBC suspension was added to 0.5 mL of the Cu2O NPs suspension in FPBS at concentrations of 16, 40, 80, 320, and 640 μg/mL to make the final NPs concentration of 8, 20, 40, 60, 80, 160, and 320 μg/mL. All samples were prepared in triplicate and the suspension was briefly vortexes before leaving at static conditions at room temperature for 3 h. After that, the mixture was subjected to further analysis for flow cytometry (FCM) or dark-field imaging.

Flow cytometry analysis

The cytotoxicity of Cu2O NPs on fish RBCs was evaluated by flow cytometry (FCM, Beckman Counter Epics XL, USA). In a typical experiment, a certain amount of Cu2O NPs were co-incubated fish RBCs at room temperature for 3 h. Then, the mixture was centrifuged at 10000 g for 3 min at 4 °C, after staining with 5 μL PI in the dark at 4 °C for 10 min. The supernatant was decanted and the precipitated RBCs were dispersed. After rinsing twice with FPBS, the RBCs were suspended in 1.0 mL FPBS. The percentage of PI fluorescence was measured with the FCM.

Dark-field imaging

The diluted RBC suspension was mixed with Cu2O NP suspensions in FPBS at a concentration of 120 μg/mL and incubated at room temperature for 3 h. For controls, whole blood with FPBS was used instead of RBC suspension with Cu2O NPs. After incubation, the mixture was washed three times with FPBS and centrifuged to remove unreacted nanoparticles. The cell suspensions were then dropped onto glass coverslips and examined using a dark-field light microscope. Dark-field light scattering imaging of the interaction between Cu2O NPs and blood cells was obtained using an Olympus BX-51 microscope (Tokyo, Japan) equipped with a highly numerical dark-field condenser (U-DCW) and the images were captured using an Olympus DP72 digital camera.

Measurements of ROS

Both CAT and SOD activities were measured according to the literature (Huang et al. 2010) with minor modification. Briefly, the diluted RBC suspension was mixed with Cu2O NP suspensions in FPBS at concentrations of 0, 10, 20, 40, and 80 μg/mL and incubated at room temperature for 3 h. Then, the cell lysis buffer was added to the mixture for free hemolysis. After centrifugation, the supernatant was collected for measurements of SOD and CAT activity. The enzymatic activity of SOD was measured as inhibition of reduction of nitrotetrazolium blue in the system xantine-xantinoxidase, and CAT activity was determined spectrophotometrically by measuring the decomposition of hydrogen peroxide. The protein content in preparations was measured using a Bradford protein assay kit (Beyotime Biotechnology Inc.).

Results and discussion

As shown in Fig. 1a, b, a representative extinction spectrum of the Cu2O NP suspension was characterized by a broad absorption band below 450 nm, and the scattering spectrum was characterized at a localized surface plasmon scattering peak of 475 nm. The light scattering property of Cu2O NPs was in line with previous report (Qi et al. 2010). The photograph in Fig. 1a shows that Cu2O NP solution exhibited yellow-green color under visible light. The morphology and particle size distribution of Cu2O NPs were evaluated via SEM as shown in Fig. 1c, d. The available Cu2O NPs are dispersed in solution showing highly uniform spheres with the size ranging from 40 to 80 nm, and the average diameter was 60 ± 20 nm by counting 300 particles at random.
https://static-content.springer.com/image/art%3A10.1007%2Fs11051-013-1507-7/MediaObjects/11051_2013_1507_Fig1_HTML.gif
Fig. 1

Characterization of the physical and chemical properties of Cu2O NPs. a Extinction spectrum, photograph and b scattering spectrum of Cu2O NP suspension; c SEM image and d diameter distribution of Cu2O NPs

To investigate the hemolytic behavior of Cu2O NPs, RBCs were isolated from freshly obtained heparin-stabilized fish blood by centrifugation, and purified by two successive washes with fish isotonic phosphate-buffered saline solution (FPBS). Then, the RBCs suspensions were diluted with FPBS and mixed with Cu2O NPs for several toxicity assays according to the literature (Slowing et al. 2009). We found that the hemolytic activity of Cu2O NPs accorded a dose-dependent manner. The photographs of fish RBCs after exposure to Cu2O NPs, Cu2+ (1.6 μg/mL), and CTAB (4.7 μg/mL) for 3 h are shown in Fig. 2a. It is noteworthy that the dosages of Cu2+ (1.6 μg/mL) and CTAB (4.7 μg/mL) used here were the highest concentrations attained by equivalent the corresponding dosage of Cu2O NPs with FPBS (Griffitt et al. 2007; Qi et al. 2010). Figure 2a clearly shows that Cu2O NPs caused observable release of hemoglobin from damaged RBCs at concentrations of NPs greater than 40 μg/mL. To attain rough estimate of the hemolytic activity, a direct visual colorimetric assay was used based on the degree of red color saturation in the photo-picture. It was found that the intensity value of the RBC suspension can reach 240 after the highest concentration (160 μg/mL) of Cu2O NPs exposure for 3 h. In contrast to the pronounced hemolytic activity of Cu2O NPs, no obvious hemolysis of RBCs was observed in the samples of Cu2+ or CTAB, which exhibited lower colorimetric values ranging from 80 to 100 (Fig. 2a).
https://static-content.springer.com/image/art%3A10.1007%2Fs11051-013-1507-7/MediaObjects/11051_2013_1507_Fig2_HTML.gif
Fig. 2

Toxicity assays of Cu2O NPs to RBCs. a Direct visual colorimetric assay. b Flow cytometric analysis. The concentrations of CTAB and Cu were 4.7 and 1.6 μg/mL, respectively. c Exposure of whole blood cells to Cu2O NPs resulted in lower percent of dead cells than that of RBCs in buffer. d Photo pictures show the different hemolytic effects after exposure of RBCs in suspension or whole blood to 40 μg/mL Cu2O NPs

The concentration-dependent hemolytic activity of Cu2O NPs was further confirmed by a FCM cell count performed after 3 h of mixing fish RBCs with a series of concentrations of Cu2O NP suspensions followed by staining with PI. When the exposure concentration was lower than 8 μg/mL, Cu2O NPs exhibited minor toxic effects to RBCs (PI % < 10 %). However, the PI % dramatically increased from 6.78 to 99.03 % when the corresponding doses of Cu2O NPs increased from 8 to 60 μg/mL (Fig. 2b). This result clearly demonstrated that cytotoxicity of Cu2O NPs to RBCs accorded a dose-dependent manner. In addition, Cu2+ exhibited slight toxic effect on RBCs, resulting in PI % ≤ 10 % at a concentration of 3.2 μg/mL (equal to the highest concentration of Cu2+ dissolved from 320 μg/mL Cu2O NPs). The time-dependent toxic effects of Cu2O NPs on fish RBCs at different concentrations were also investigated (Figure S1 in the supporting information).

Previous work has demonstrated that exposure to high levels of soluble copper (100 μg/L) is associated with proliferation of chloride cells (Pelgrom et al. 1995). Therefore, it is critical to ascertain if toxicity is due to dissolution or to the particles themselves. Our data demonstrate that Cu2+ will cause a sight toxic to blood cells if dissolution does actually occur, but will not result in hemolysis of RBCs, suggesting that dissolution causes toxicity by a route different from that of particle. This result is consistent with a previous report demonstrating that aquatic exposure to copper NPs causes toxicity in zebrafish or bacteria (Griffitt et al. 2007; Baek and An 2011). Therefore, this result suggests that particle is the main contributor to the toxic effects of Cu2O NPs in the present work.

Interestingly, it was found that the hemolytic activity of Cu2O NPs could be alleviated when whole blood was exposed to Cu2O NPs instead of RBCs in buffer. For each Cu2O NP concentration, exposure of whole blood resulted in lower PI % values than exposed of RBCs in buffer, as shown in Fig. 2c. This phenomenon was quite obvious when the concentration of Cu2O NPs increased from 40 to 80 μg/mL. For instance, 40 μg/mL Cu2O NPs led to PI % over 81 % when exposure of RBCs in buffer, but in whole blood the PI % was only 34 %, lower than half the value in buffer. At the same time, the EC50 values of Cu2O NPs t RBCs in buffer, and in whole blood exposure were 26 and 63 μg/mL, respectively (Figure S2 in the supporting information). Figure 2d shows that RBCs exhibit slight hemolysis after expose to 40 μg/mL Cu2O NPs for 3 h, while no hemolytic effect was observed in the whole blood exposure group. This result further confirmed that RBCs in whole blood have stronger resistance to the hemolytic activity of Cu2O NPs compared to RBCs in buffer. We speculated that the alleviation of hemolytic effect could be attributed to the adsorption of plasma proteins on the surfaces of Cu2O NPs. Upon entrance of the Cu2O NPs into the whole blood, their surfaces will be rapidly covered by selective sets of blood plasma proteins to forming protein corona (Cedervall et al. 2007). This process will lead to changes in the physicochemical properties of the surface of Cu2O NPs or result in NP aggregation (Lundqvist et al. 2008), which may effectively reduce their cytotoxicity (Ge et al. 2011).

Assaying the hemolytic effect of Cu2O NPs may provide a straightforward approach to study the mechanism of toxicity of NPs on biologic membranes, which are important target sites of cytotoxicity. It is well known that the hemolysis of silica nanoparticles is closely related to the number of silanol groups accessible to the RBC membranes (Slowing et al. 2009), as well as the physicochemical properties of the NPs, such as the surface charge, particle size, and mesoporous structure (Lin and Haynes 2010; Yu et al. 2011). Considering that the common toxicity mechanism of CuO NPs is the generation of ROS (Mortimer et al. 2011), we hypothesized that damage to fish RBC membranes induced by Cu2O NPs was likely mediated by particle generated ROS since the same metallic elements complement and the similar spherical structure of these two types of copper NPs possessed. To verify the hypotheses, we determined the CAT and SOD activity of RBCs and whole blood after exposure to Cu2O NPs at concentrations of 0, 10, 20, 40, and 80 μg/mL. As shown in Fig. 3, both CAT and SOD activities are enhanced with increasing concentrations of Cu2O NPs, demonstrating that RBC membrane damage is closely related to the generation of ROS. These results provide evidence that the production of ROS is responsible for the cytotoxic effects of Cu2O NPs.
https://static-content.springer.com/image/art%3A10.1007%2Fs11051-013-1507-7/MediaObjects/11051_2013_1507_Fig3_HTML.gif
Fig. 3

a CAT and b SOD activity of RBCs and whole blood after exposure to Cu2O NPs at concentration of 0, 10, 20, 40, and 80 μg/mL. Data are expressed as mean ±SEM. The ordinate value represents the amount of enzyme that reduced the absorbance change by 50 % normalized per milligram of total protein content (U/mg protein)

In addition to the ROS-based mechanism of toxicity, the modes of interaction between Cu2O NPs and RBC cell membranes should also be considered. These may include specific or nonspecific forces, receptor-ligand binding interactions or membrane wrapping of the NPs (Nel et al. 2009). Through these interactions, Cu2O NPs may have a direct damaging effect on the RBC membrane compositions similar to that of CuO NPs, which change the membrane fatty acid composition of freshwater ciliated protozoa (Chen et al. 2011).

Dark-field imaging was used to monitor the uptake of Cu2O NPs by blood cells. As shown in Fig. 4a, many oval RBCs and fragments of RBCs can be clearly observed after exposure of RBCs to 120 μg/mL Cu2O NPs for 3 h. However, the morphology of RBCs in the control group (only FPBS added) was intact showing normal oval shapes. Compared to RBCs in buffer, exposure of whole blood to Cu2O NPs caused only minor damage (Fig. 4b). Many dispersed Cu2O NPs have been either located on the surfaces of RBCs or entered into the cytoplasm of leukocytes after incubation with blood cells for 3 h (Fig. 4c). In such case, most of the Cu2O NPs that adsorbed on the RBCs can be found in two forms: dispersed or aggregated. Many dispersed Cu2O NPs scattered the blue light, and only a small number of Cu2O NPs aggregates that were adsorbed on the fragments of RBCs exhibiting a white light block. Nevertheless, no dispersed or aggregated Cu2O NPs, which were absorbed or internalized by blood cells, have been observed in the control experiment (Fig. 4d). These results are in line with previous reports that other NPs can adsorb to the surface of RBC membranes (Zhao et al. 2011) or penetrate the RBC membrane by a still unknown mechanism different from phagocytosis and endocytosis (Rothen-Rutishauser et al. 2006). In addition, it is worth noting that the multicolor exhibited from Cu2O NPs under dark-field microscopy is closely related to their photo-physical properties (Qi et al. 2010), since the light scattering properties of metal NPs are dependent on the composition, size, and shape of NPs, as well as their surrounding medium, according to the Rayleigh and Mie theories (Yguerabide and Yguerabide 1998).
https://static-content.springer.com/image/art%3A10.1007%2Fs11051-013-1507-7/MediaObjects/11051_2013_1507_Fig4_HTML.jpg
Fig. 4

Dark-field light scattering images of the interaction between Cu2O NPs and RBCs. Pictures show the morphology of RBCs after exposure to a Cu2O NPs or b FPBS for 3 h, respectively. c, e Many dispersed or aggregated Cu2O NPs were located on RBCs or internalized by leukocytes after these blood cells were incubated with Cu2O NPs for 3 h. d, f No analogous phenomenon was observed in the control. Bar was 10 μm

Phagocytosis is the cellular process of engulfing solid particle by the cell membrane to form an internal phagosome by phagocytes, therefore, it is a major mechanism used to remove pathogens or foreign particles. Previous research has clearly demonstrated that CTAB-coated gold nanorods can be rapidly internalized by human blood phagocytes (Bartneck et al. 2010). The present work show that large numbers of single or aggregated Cu2O NPs were located in the cytoplasm of leukocytes (Fig. 4e, f), demonstrating leukocyte phagocytosis occurred after Cu2O NP exposure. Since nanoparticle size and surface chemistry determine plasma serum protein adsorption and subsequent uptake by macrophages (Walkey et al. 2011), we speculated that the phagocytosis of Cu2O NPs by leukocytes is mediated mainly by the adsorption of plasma serum proteins on the surface of Cu2O NPs. However, after internalization, further interaction of blood phagocytes with Cu2O NPs, as well as the mechanisms involved in cellular aggregation, transportation, and toxic effects of Cu2O NPs need further investigation.

Conclusions

This work is the first study to examine the hemolytic behavior of Cu2O NPs with blood cells. A concentration-dependent hemolytic activity of Cu2O NPs to fish RBCs was observed. We speculated that the intrinsic toxic properties of the nanoparticles are mainly responsible for their cytotoxicity, rather than dissolved Cu2+. Although further studies are needed to determine the specific mechanism of Cu2O NPs toxicity to blood cells, these initial lines of evidence indicate that (1) higher dose of Cu2O NPs (≥40 μg/mL) are toxic to fish blood cells and can cause serious damage RBCs membrane, eventually leading to hemolysis; (2) the production of ROS is responsible for the cytotoxic effects of Cu2O NPs; and (3) the adsorption of plasma proteins on the surfaces of Cu2O NPs leads to the changes in their physicochemical properties and eventually causes the aggregation of Cu2O NPs in whole blood, can significantly alleviating the hemolytic effect and subsequently mediating the phagocytosis.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (U0936602), the Doctoral Program Foundation of Institutions of Higher Education of China (20115301120002), the National Key Technology R&D Program of China (2011BAC09B07), and the Yunnan provincial Foundation for Basic Science (2011FB007) and Education (2011Y107) of China.

Supplementary material

11051_2013_1507_MOESM1_ESM.doc (331 kb)
Supplementary material 1 (DOC 331 kb)

Copyright information

© Springer Science+Business Media Dordrecht 2013