Article pubs.acs.org/crt
Nanotoxicity of Silver Nanoparticles to Red Blood Cells: Size Dependent Adsorption, Uptake, and Hemolytic Activity Li Qiang Chen,*,† Li Fang,† Jian Ling,‡ Cheng Zhi Ding,† Bin Kang,† and Cheng Zhi Huang*,§ Asian International Rivers Center, Yunnan Key Laboratory of International Rivers and Trans-boundary Eco-Security, and ‡College of Chemical Science and Technology, Yunnan University, Kunming, 650091, People’s Republic of China § Key Laboratory of Luminescence and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, P. R. China
Chem. Res. Toxicol. 2015.28:501-509. Downloaded from pubs.acs.org by IOWA STATE UNIV on 01/29/19. For personal use only.
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ABSTRACT: Silver nanoparticles (AgNPs) are increasingly being used as antimicrobial agents and drug carriers in biomedical fields. However, toxicological information on their effects on red blood cells (RBCs) and the mechanisms involved remain sparse. In this article, we examined the size dependent nanotoxicity of AgNPs using three different characteristic sizes of 15 nm (AgNPs15), 50 nm (AgNPs50), and 100 nm (AgNPs100) against fish RBCs. Optical microscopy and transmission electron microscopy observations showed that AgNPs exhibited a size effect on their adsorption and uptake by RBCs. The middle sized AgNPs50, compared with the smaller or bigger ones, showed the highest level of adsorption and uptake by the RBCs, suggesting an optimal size of ∼50 nm for passive uptake by RBCs. The toxic effects determined based on the hemolysis, membrane injury, lipid peroxidation, and antioxidant enzyme production were fairly size and dose dependent. In particular, the smallest sized AgNPs15 displayed a greater ability to induce hemolysis and membrane damage than AgNPs50 and AgNPs100. Such cytotoxicity induced by AgNPs should be attributed to the direct interaction of the nanoparticle with the RBCs, resulting in the production of oxidative stress, membrane injury, and subsequently hemolysis. Overall, the results suggest that particle size is a critical factor influencing the interaction between AgNPs and the RBCs.
1. INTRODUCTION Silver nanoparticles (AgNPs) are the most commonly used engineered nanoparticles in commercialized products previously due to their outstanding antimicrobial properties. They are now widely used for biomedical purposes such as antimicrobial agents, drug delivery, molecular imaging, biomedical sensing, and even cancer photodynamic therapy.1 Along with the development of AgNPs with the potential for biomedical applications, the question of their possible toxicity has increasingly gained attention. In vitro and in vivo studies have already revealed the significant toxic effects associated with AgNPs exposure, including cell membrane injury, deoxyribonucleicacid (DNA) damage,2 embryo development malformations,3,4 inhalation, and cardiovascular system diseases.5,6 Blood, which directly or indirectly encounters any hazardous nanoparticles, is able to transport the foreign substances to cells, tissues, and organs. In this case, it is highly required to study the nanotoxicity on blood, particularly erythrocytes.7 Accidental exposure by inhalation or ingestion, via the skin or mucosa, can potentially lead to the translocation of AgNPs into the bloodstream.8 More importantly, many of the proposed biomedical applications will require direct injection into the body via different routes of administration, which will most probably also lead to direct contact of the nanoparticles with blood components. The evaluation of AgNPs’ blood compat© 2015 American Chemical Society
ibility is therefore considered of great importance given their interactions with blood components and the cardiovascular system. To date, the hemotoxicity and heamocompatibility of AgNPs remains largely unknown. Few toxicity studies showed that erythrocytes exhibited significant lysis, heamagglutinatin, membrane damage, detrimental morphological variation, and cytoskeletal distortions following exposure to AgNPs at high concentration.9,10 Nanosize particles compared with microsize particles have more potential for hemolysis because their greater surface area would facilitate the release of more silver ions.11 Both the silver ions released from particles and the direct interaction of AgNPs with RBCs were proposed to be responsible for the toxicity.11 The size of AgNPs has a strong effect on their interactions with living cells, influencing uptake efficiency,12,13 production of reactive oxygen species,14,15 and cytotoxicity.13,16−19 However, the effects of particle size on the toxicity of AgNPs to RBCs has not been clarified and the membrane injury mechanism involving the interaction among AgNPs and the membrane, internalization, cellular uptake, and oxide stress Special Issue: Chemical Toxicology in China Received: November 21, 2014 Published: January 20, 2015 501
DOI: 10.1021/tx500479m Chem. Res. Toxicol. 2015, 28, 501−509
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Chemical Research in Toxicology
Figure 1. Physicochemical characterization of AgNPs. (a−c) TEM images of the AgNPs15, AgNPs50, and AgNPs100, respectively. (d−f) The size distribution of AgNPs15, AgNPs50, and AgNPs100, respectively. (g) The extinction spectrum of the AgNPs with three diameters. (h) DLS data shows the hydro diameters distribution of the three sizes of AgNPs. JEM-2100 microscope (JEOL Inc., USA) operating at an accelerating voltage of 200 kV. 2.3. RBCs Collection and Handing. Heparin-stabilized fish RBCs (Carassius auratus) were freshly collected according to our previous method21 with a slight modification. Briefly, a 4 mL sample of whole blood was added to 8 mL of fish phosphate-buffered saline (PBS), and the RBCs were isolated from serum by centrifugation at 1500 rmp for 10 min. The RBCs were further washed twice with PBS solution. Following the last wash, the RBCs were diluted to 8 mL with PBS solution. Then, 0.2 mL of the diluted RBC suspension was added to 0.8 mL of the AgNP suspension in PBS to make the final AgNP concentration to 1.25, 2.5, 5, 10, and 20 μg/mL. We chose the highest dose of 20 μg/mL because this concentration is very close to the serum silver concentration after AgNP exposure.8 Deionized water and PBS were added to RBC suspensions as the positive and negative controls, respectively. Considering the possible toxic effects of silver ions on RBCs, the concentration for silver ion exposure was chosen as 2 μg/mL (equal to the highest dose of release from 20 μg/mL AgNPs). All samples were prepared in triplicate, and the suspension was briefly vortexed before leaving at static conditions at 24 °C for 2 h. Finally, the mixtures were centrifuged at 1500 rmp for 10 min, and the RBCs were isolated and washed twice with PBS. After that, the RBCs were subjected to further analysis with optical microscopy, TEM, and flow cytometry (FCM). 2.4. Hemolysis Assay. The method for the hemolysis assay of AgNPs was referred to the literature.22,23 Prior to AgNP exposure, the absorbance spectrum of the positive control supernatant was checked and used only if it was in the range of 0.50−0.55 optical density units
responses remains unexplored. The aim of this study is to investigate the size-dependent adsorption, uptake, and hemolysis of AgNPs to fish RBCs and to explore the mechanisms of toxicological effects. The results suggest that particle size plays a critical role in the interaction between AgNPs and the RBCs.
2. MATERIALS AND METHODS 2.1. Reagents. Both heparin and propidium iodide (PI) dye were purchased from Sigma−Aldrich Co., Ltd. (Shanghai, China). WrightGiemsa dye, lipid peroxidation assay kit, superoxide dismutase assay kit, and Bradford protein assay kit were purchased from Nanjing Jiancheng Bioengineering Institute, Nanjing, China. Other commercial reagents such as silver nitrate (AgNO3) and trisodium citrate are of analytical reagent grade and were used without further purification. All reagents were prepared by dissolving their commercial products in doubly distilled water. 2.2. Synthesis and Characterization of AgNPs. The AgNPs with different diameters were prepared according to the modified LeeMeisel’s method.20 Different sizes of AgNPs were synthesized by reducing AgNO3 solution (1 nM) using trisodium citrate of various quality and different control times. The extinction spectra and hydrodynamic diameter of the AgNP suspension were measured with a Hitachi U-2010 spectrophotometer (Hitachi Ltd., Tokyo, Japan) and a N5 Submicron Particle Size Analyzer (Beckman, USA), respectively. Transmission electron microscopy (TEM) images were taken with a 502
DOI: 10.1021/tx500479m Chem. Res. Toxicol. 2015, 28, 501−509
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Figure 2. Optical microscopic images of AgNP incubated RBCs. (a−c) Normal morphology of RBCs in the control. (d−f) AgNPs15 treated RBCs. (d−f) AgNPs50 treated RBCs. (d−f) AgNPs100 treated RBCs. Blood cells were stained with Wright-Giemsa dye. The left column, transmitted light imaging; the middle column, dark-field imaging; and the right column, the magnification image of the white box in the middle column. to reduce sample difference from different donors.23 RBCs were then incubated with AgNPs for 2 h and centrifuged to isolate the RBCs as described above. After that, 100 μL of the supernatant of all samples 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)) × 100. 2.5. Optical Microscopy and TEM Oberservations. RBCs were incubated with AgNPs for 2 h and washed three times to remove excessive particles. The RBCs were then dropped and tiled on the slide glass. After drying, the blood smears were stained with Wright-Giemsa dye according to the manufacturer’s instructions ((Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and examined with an Olympus BX-51 microscope (Tokyo, Japan). The images were captured with an Olympus DP72 digital camera. For TEM imaging, RBCs were incubated with AgNPs for 2 h at 24 °C. Then, the mixtures were centrifuged at 1500 rmp for 10 min and washed twice with PBS buffer. After that, RBCs were fixed in 2.5% glutaraldehyde (0.2 M, phosphate buffer solution, pH 7.4) overnight at 4 °C. RBCs were further subjected to post-fixing, dehydration, mounting, and sectioning according to the standard protocol. The samples were finally examined and photographed on a JEM-2100 TEM (JEOL Inc., USA) at an accelerating voltage of 20 kV.
2.6. Dark-Field Imaging and Single Particle Microspectrum Analysis. Dark-field light scattering imaging of the interaction between AgNPs and RBCs was obtained with an Olympus BX-51 microscope (Tokyo, Japan) equipped with a highly numerical dark field condenser (U-DCW), and the images were captured with an Olympus DP72 digital camera. The light scattering spectra of single particles and the cell membrane were carried out through the Olympus BX-51 dark-field system integrated with an Acton Research MicroSpec 2300i monochromator and a Princeton Instruments PI-MAX intensified charge coupled device (ICCD) (Trenton, USA), as we reported previously.24 2.7. Flow Cytometry Analysis. Membrane injury induced by AgNPs on RBCs was evaluated by flow cytometry (FCM, Beckman Counter Epics XL, USA). In a typical experiment, RBCs were incubated with AgNPs for 2 h and isolated from the mixture by centrifugation at 1500 rmp for 10 min. The RBCs were then stained with 5 μL of PI in the dark at 4 °C for 10 min. The supernatant was decanted, and the precipitated RBCs were dispersed. After being rinsed twice with PBS, the RBCs were suspended in 1.0 mL of PBS solution. Finally, the percentage of PI fluorescence was measured with the FCM. 2.8. Assay of Superoxide Dismutase (SOD) Activity. SOD activity was measured according to our previously reported method.21 Briefly, the diluted RBC suspension was mixed with AgNPs in PBS solution and incubated at 24 °C for 2 h. Then, the cells were lysed, and 503
DOI: 10.1021/tx500479m Chem. Res. Toxicol. 2015, 28, 501−509
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Figure 3. Dark-field imaging and single particle spectrum analysis of AgNPs. (a) Dark-field image of AgNPs50. (b) The corresponding single particle spectrum of the different colored dots. (c,d) Image shows the red and green membranes of the RBC and blood cell, as well as their corresponding microspectra. (e) Representative image of RBC uptake of AgNPs50. (f) Single particle spectrum of the AgNPs50 located in the RBCs. Red circles and white arrows indicate the position for acquiring the single particle spectrum. the supernatant was collected for the measurement of SOD activity. The enzymatic activity of SOD was assayed using the xanthine/ xanthine oxidase method based on the production of O2− anions. The protein content in preparations was measured using a Bradford protein assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The enzymatic activity of SOD is expressed as units per milligrams of protein (U/mg protein). 2.9. Lipid Peroxidation Measurement. Membrane damage in RBCs was determined by quantifying the release of malondialdehyde (MDA) after AgNP exposure. MDA is a product of lipid peroxidation and its release levels are indicative of membrane damage. The assay was performed according to the manufacturer’s instructions for the lipid peroxidation assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Briefly, RBCs were incubated with AgNPs for 2 h and washed twice with PBS solution to remove extra nanoparticles. Then, the RBCs were sonicated and treated with the reagent containing thibabituric acid, which reacted with MDA to form a stable complex dye with maximum absorbance at 530 nm. The concentrations of MDA in samples were detected by a Microplate Reader through generating a standard curve from MDA standards provided in the assay kit.
AgNP100, respectively (Figure 1d−f). The UV−visible spectrum of the AgNPs shows the absorbance peaks at 390, 410, and 420 nm, (Figure 1g), and the hydrodynamic diameters of AgNPs were 48.5, 264.3, and 362.8 nm for AgNPs15, AgNPs50, and AgNPs100, respectively, based on dynamic light scattering analysis (Figure 1h). 3.2. Optical Imaging of the Adsorption and Uptake of AgNPs in RBCs. Figure 2 shows the adsorption and uptake of the AgNPs with different sizes in fish RBCs. The morphology of untreated RBCs exhibited a normal oval shape with a purple nucleus under common optical microscopy (Figure 2a). Under dark-field microscopy, the membrane and nucleus of RBCs appeared green and deep purple, respectively (Figure 2b and c). Exposure to all three sizes of AgNPs induced the appearance of adsorption and uptake of nanoparticles in RBCs. Under common optical microscopy, no obvious difference in the RBCs after exposure to AgNPs was observed. In contrast, many blue and white dots appeared in the RBCs under dark-field imaging (Figure 2d−i). The presence of the white dots in RBCs indicates the formation of the big aggregates of AgNPs since only big aggregates tend to scatter white light. Among them, AgNPs50 exhibited the highest degree of adsorption and uptake in RBCs. A large number of AgNPs50 have been observed either located on the surface of membrane or entered into the cytoplasm of RBCs (Figure 2g−i). In contrast to AgNPs50 exhibiting a high level of adsorption and uptake, the number of AgNPs15 and AgNPs100 that associated with RBCs decreased
3. RESULTS 3.1. Characterization of AgNPs. Figure 1 showed the morphology and particle size distribution of AgNPs determined by using TEM (Figure 1a−c). The available AgNPs were dispersed in solution with sizes of 14.68 ± 2.3 nm, 51.21 ± 8.2 nm, and 105.52 ± 17.6 nm, by counting 100 particles at random, and they were then named AgNPs15, AgNPs50, and 504
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Figure 4. Quantitative analysis of the AgNP association with RBCs. (a) Number of the dots associated with RBCs. (b) The percentage of the white dots associated with RBCs exhibiting a size dependent increase.
Figure 5. TEM images of the AgNP adsorption and uptake by RBCs. (a) RBCs control; (b) AgNPs15 treated RBCs; (c) AgNPs50 treated RBCs; (d) AgNPs100 treated RBCs. All three sizes of AgNPs were incubated with RBCs for 2 h. Individual AgNPs are indicated by red circles, and the aggregate of AgNPs are indicated by black arrows.
500, 570, and 640 nm corresponding to the blue, cyan, yellow, and red dots, respectively (Figure 3a and b). Most of the AgNPs with spheres appeared blue (size 10−40 nm) in color, while a few of them appeared orange (50−70 nm) and red (80−100 nm).24,25 The membrane of RBC and monocytes exhibited different colors such as green or red, and their LSPR peaks were characterized as 574 and 578 nm, respectively (Figure 3c and d). After incubation, AgNPs50 were taken up by RBCs and exhibited quite a few different colors. Three main colors of the dots including blue, purple, and white in the RBCs
a lot. Treatment of RBCs with all three sizes of AgNPs did not result in any visible changes in morphology. 3.3. Single Particle Spectrum Analysis of AgNPs in RBCs. Since AgNPs50 exhibited the highest level of association with RBCs, they were thus chosen as the representative AgNPs for microspectrum analysis. Figure 3 showed the representative dark-field images and the corresponding single particle microspectrum of AgNPs50 in RBCs. The AgNPs50 with different sizes were observed as many multicolor dots, and their localized surface plasmon resonance (LSPR) peaks were 450, 505
DOI: 10.1021/tx500479m Chem. Res. Toxicol. 2015, 28, 501−509
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Chemical Research in Toxicology
Figure 6. Size dependent hemolytic activities of AgNPs to RBCs. (a) Photographs of RBCs after exposure to three sizes of AgNPs for 2 h. (b) Percentage of hemolysis measured by a spectrophotometer.
Figure 7. AgNP size and concentration dependencies of RBC membrane injury measured by a flow cytometric assay. (a) Membrane injury induced by AgNPs15 exposure in a series of dosages. (b) A comparison of the biocompatibility of AgNPs15, AgNPs 50, and AgNPs100.
RBCs. Notably, smaller particles (AgNPs15 and AgNPs50) were scattered in the cytoplasm of RBCs (Figure 5 b, c), while bigger particles (AgNPs100) tended to form big aggregates (Figure 5d). The numbers of AgNPs15 and AgNPs50 in RBCs were significantly more than AgNPs100, indicating that the internalization into RBCs of small size particles through passive uptake is easier compared to that of big ones. 3.6. Size Dependent Hemolytic Activity. The photograph of RBCs after exposure to three diameters of AgNPs for 2 h is shown in Figure 6a. It was apparent that smaller sized AgNPs15 caused observable release of hemoglobin from damaged RBCs at lower nanoparticle exposure concentration. However, the hemoglobin release was not observed when RBCs were incubated with AgNPs50 and AgNPs100 for 2 h. This result demonstrated that the smaller particles possess higher hemolytic activity than that of the larger particles. Spectrophotometric analysis of the supernatants showed similar results as indicated by the photographs. Among three sizes of AgNP preparations, AgNPs15 caused a significantly higher level of hemolysis, exceeding 60% hemolysis at 20 μg/mL. In comparison, AgNPs50 and AgNPs100 caused less than 12% hemolysis even at the highest concentration (Figure 6b). According to the criterion in the ASTM E2524-08 standard, percent hemolysis >5% indicates that the test nanoparticle causes damage to RBCs. This criterion was exceeded at the concentration of 20 μg/mL for all three sizes of AgNPs in the present study. 3.7. Size and Concentration Dependent Membrane Injury. By using the flow cytometry technique with PI cell straining procedures, the RBC membrane injuries were
were usually observed. Notably, the corresponding single particle microspectrum exhibited as a composite spectrum combining the spectrum of single AgNPs50 and the RBC membrane, indicating the adhering of the nanoparticle on the membrane. With the color of AgNPs50 changing from blue to white, the LSPR peak at 450 nm decreased, while the LSPR peak at 630 nm dramatically increased (Figure 3e and f). 3.4. Quantitative Analysis of the AgNP Association with RBCs. A quantitative analysis revealed that AgNPs with different sizes exhibited a distinct level of adsorption and uptake by RBCs. Among them, AgNPs50 exhibited the highest level of association with RBCs. The amount of both total dots and white dots of AgNPs50 associated with RBCs was much higher than that of AgNPs15 or AgNPs100 exposure group. More than 45 dots per cell on average were counted indicating the intensive adsorption and uptake of AgNPs50 in the RBCs (Figure 4a). It is apparent that the percentage of the white dots uptake by RBCs exhibited a size dependent pattern. With increasing diameter of AgNPs from 15 to 100 nm, the percent of the white dots uptake by RBCs increased accordingly from 22.04 ± 7.1% to 57.73 ± 15.7% (Figure 4b). Since white dots represent the big aggregates of AgNPs, the results demonstrate that the percent of aggregates in RBCs follows a dependency on size. 3.5. TEM Imaging Identified the Internalization of AgNPs in RBCs. TEM images of the cellular uptake of different sizes of AgNPs are shown in Figure 5. No particle was observed in the untreated control RBCs (Figure 5a). In contrast, all three sizes of AgNPs were found not only adhered to the membrane but also internalized into the cytoplasm of the 506
DOI: 10.1021/tx500479m Chem. Res. Toxicol. 2015, 28, 501−509
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Figure 8. Size dependent SOD activity and MDA production in RBCs induced by AgNPs exposure. (a) SOD activity. (b) The production of MDA.
oxidative stress responses induced by AgNP exposure were according to a size dependent pattern. For interaction with RBCs especially, the size of nanoparticle is one of the most important factors that influence their toxicity and uptake efficiency, besides their structure, shape, and surface coating.17 For example, a quantitative analysis revealed that only the size determined the efficiency of polystyrene nanoparticle uptake by RBCs. Only the polystyrene nanoparticles with a size smaller than 0.2 μm can enter RBCs, which indicated that the size is a key factor for nanoparticle internalization by RBCs.26 Recent research on gold, titanium dioxide (TiO2), and hydroxyapatite nanoparticles has clearly demonstrated that small particles have higher hemolytic activity compared to that of large particles, which was in line with our results.7,27,28 In contrast, increased hemolytic activity with increasing particle size was reported for fumed silica, mesoporous silica, and colloidal amorphous silica nanoparticles, which indicated that large silica nanoparticles exhibited greater hemolytic activity than small particles.23,29,30 The hemolytic activity of silica based nanomaterials depends only on the concentration of negatively charged silanol groups that are accessible by the membranes of the RBCs. With increasing size of silica nanopartilces, the amount of silanol groups accessed by the membrane of the RBCs increased accordingly, which resulted in greater hemolytic activity.31 However, the same scenario cannot apply to the hemolytic effect of AgNPs since AgNPs may promote RBC hemolysis through a distinctive mechanism. The mechanism by which AgNPs may induce hemolysis in erythrocytes has not been completely elucidated. It is still unclear whether the hemolytic activity of AgNPs is due to their direct cellular interaction with particles, release of free silver ions, or a combination of both events.12,32−34 Since no significance of hemolytic effects induced by silver ions was observed in the present study, the direct interaction of AgNPs with RBCs is thus suggested to be the main factor that is responsible for hemolysis. It is established that AgNPs are able to bind to thiol groups within biological moieties such as proteins or phospholipids of membrane with high affinity and thereby promote their denaturation.34 In addition, the negatively charged silver surface is expected to strongly interact with the organic cations in the membrane of RBCs, which may also contribute to the hemolysis. Thus, we speculated that the high reactivity and easy transportability of small AgNPs in comparison to those of the bigger particles may lead to more
identified after incubation with AgNPs for 2 h. As shown in Figure 7a, an increase of the PI % indicates that membrane injury was identified for AgNPs15 with an increase of the mass concentration. Obviously, 0.75 μg/mL of AgNPs15 exposure can lead to evident membrane injury of RBCs, and the highest degree of PI % reached 89.2% when the concentration increased to 20 μg/mL. RBC membrane injury caused by AgNPs15 exposure was significantly larger than those promoted by AgNPs50 and AgNPs100 (Figure 7b), and this finding was consistent with the results of hemolysis analysis. 3.8. Size Dependent Oxidative Stress Responses. AgNP induced turbulence in the membrane integrity of RBCs by possible oxidative stress was investigated using the SOD activity and MDA production studies. Figure 8 shows that both the SOD activity and MDA production in RBCs induced by AgNPs followed a size dependent pattern. With decreasing the diameter of AgNPs, the reactive oxygen species (ROS) activity and MDA production of RBCs increased accordingly. At 5 μg/mL concentration, the SOD activity reached a high level with AgNPs15 exposure, while it was lower with AgNPs50 exposure. RBCs treated with AgNPs100 did not induce a significant increase of SOD activity even at the highest concentration of 20 μg/mL (Figure 8a). The tendency of MDA production induced by AgNPs was quite similar to SOD activity. In contrast to low SOD activity, AgNPs100 promoted a significant increase of MDA production at the concentration of 5 μg/mL. These results indicate that the production of oxidative stress induced by AgNP exposure in RBC is size and concentration dependent.
4. DISCUSSION Most nanoparticles are known to induce significant cytotoxicity and cause oxidative stress response in various cell lines. AgNPs, in particular, are regarded as one of the most toxic potential nanoparticles owing to their metallic silver composition and high biological reactivity.2 The effects and mechanism of toxicity have been fully investigated in various cell lines and animal model.5 Nevertheless, a rare study has been conducted in this area that can provide a clear understanding of the toxic effect and mechanism of AgNP exposure to RBCs. Considering that more and more AgNP-based medicine will be introduced into the vascular system via injection, direct interaction between AgNPs and erythrocytes may occur frequently. The present study compared the adsorption, uptake, and hemolytic potentials of different sizes of AgNPs in the RBCs. Our results clearly demonstrated that both the hemolytic activity and 507
DOI: 10.1021/tx500479m Chem. Res. Toxicol. 2015, 28, 501−509
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Chemical Research in Toxicology
Program Foundation of Higher Education of China (20115301120002), and the fund of Chongqing Fundamental and the Cultivation Plan of Chongqing Science & Technology Commission for 100 Outstanding Science and Technology Leading Talents.
severe damage to the membrane of RBCs, resulting in greater hemolytic activity. The adsorption and uptake of AgNPs by RBCs were monitored through dark-field imaging, which showed that all sizes of AgNPs were attached to the RBC surface or internalized into the cytoplasm. Interestingly, a quantitative analysis reveals that not the small or big sized particles but the middle sized particle (AgNPs50) exhibited the highest level of association with RBCs in the present work. This result suggests that there may be an optimal size for the adsorption and uptake of AgNPs by RBCs. In culture cell lines, a size dependent uptake has been observed for gold,35−37 mesoporous silica,38 polystyrene,39 and iron oxide nanoparticles,40 with the maximum cellular uptake at a nanoparticle size in the range of 30−50 nm, which suggests that there is an optimal size for active uptake. Considering that RBCs do not have any phagocytic receptors on their surface and no actin−myosin system, the mechanism involved in the uptake of nanoparticles and their interaction may be greatly different from that of culture cells. Thus, it appears that our results indicate a novel optimal size of ∼50 nm for passive uptake of AgNPs by RBCs. Note that the overall numbers of AgNPs50 adsorption and uptake by RBCs were extremely large, which were more than 45 particles per cell on average. Although 45 particles per cell is not a large number for the active uptake of nanoparticles by culture cells, for AgNPs it is exactly a huge quantity for RBCs to absorb and take up, when compared with polystyrene particles (
