Eryptosis Indices as a Novel Predictive Parameter for Biocompatibility of Fe₃O₄ Magnetic Nanoparticles on Erythrocytes

Abstract

Fe₃O₄ magnetic nanoparticles (Fe₃O₄-MNPs) have been widely used in clinical diagnosis. Hemocompatibility of the nanoparticles is usually evaluated by hemolysis. However, hemolysis assessment does not measure the dysfunctional erythrocytes with pathological changes on the unbroken cellular membrane. The aim of this study is to evaluate the use of suicidal death of erythrocytes (i.e. eryptosis indices) as a novel predictive and prognostic parameter and to determine the impact of Fe₃O₄-MNPs on cellular membrane structure and the rheology properties of blood in circulation. Our results showed that phosphatidylserine externalization assessment was significantly more sensitive than classical hemolysis testing in evaluating hemocompatibility. Although no remarkable changes of histopathology, hematology and serum biochemistry indices were observed in vivo, Fe₃O₄-MNPs significantly affected hemorheology indices including erythrocyte deformation index, erythrocyte rigidity index, red blood cell aggregation index and erythrocyte electrophoresis time, which are related to the mechanical properties of the erythrocytes. Oxidative stress induced calcium influx played a critical role in the eryptotic activity of Fe₃O₄-MNPs. This study demonstrated that Fe₃O₄-MNPs cause eryptosis and changes in flow properties of blood, suggesting that phosphatidylserine externalization can serve as a predictive parameter for hemocompatibility assay.

Introduction

With rapid progress in nanotechnology, many nano-size materials have been extensively used in biomedical and pharmaceutical industry and industrial production¹. Nevertheless, the rapid growth of nanotechnology has raised biological safety concerns because of the unique dimensional and physicochemical properties of the nano-size materials². Although the mechanism of toxicity of nanomaterials is complicated and markedly different from that of traditional biomaterials, current evaluations of nanotoxicology are still confined to testing the compatibility of materials using traditional methodologies. A standard research protocol to evaluate the nanotoxicity is lacking, severely limiting the development and applications of nanoparticles³. Therefore, there is an urgent need to develop a sensitive, accurate and reliable evaluation system to assess nanotoxicity and to understand its mechanisms.

Fe₃O₄ magnetic nanoparticles (Fe₃O₄-MNPs) is the only nanomaterial that has been approved for clinical applications because of their relative safety, unique magnetic responsiveness and their simple and controllable preparation^4,5. Up until now, Fe₃O₄-MNPs have been widely explored in the clinic fields, such as magnetic resonance imaging (MRI) contrast agents, biosensors, tumor-targeting photothermal therapy and regenerative medicine^6,7,8,9. For example, commercially available Fe₃O₄-MNPs (e.g. Feridex) have been used as MRI contrast agent with recommended concentration of 0.56 mg Fe/kg¹⁰. Although studies concerning the potential risks of Fe₃O₄-MNP have been reported¹¹, the biocompatibility evaluation relies mainly on in vitro cytotoxicity such as hemolysis testing, cell viability, oxidative damage, inflammatory reactions and genotoxicity, or on pharmacokinetics and in vivo bio-distribution¹².

Erythrocytes are the main components in the circulation system and are also one of the first components that Fe₃O₄-MNPs contact when the nanoparticles are administered through intravenous injection. Fe₃O₄-MNPs are generally regarded as hemocompatible based on very low hemolytic activity¹³. Hemolysis testing is a well-accepted classical assay for acute toxicity screening in evaluating hemocompatibility and can reflect the breakage to the erythrocyte membrane. However, hemolysis testing cannot indicate the dysfunction of unbroken erythrocytes with pathological changes on cellular membranes. Increased number of erythrocytes with cellular membrane injuries (not broken to cause hemolysis yet) can cause some physiologic derangement or serious health problems, such as anemia, microcirculation dysfunction and thrombogenic activation^14,15,16,17. Moreover, various clinical disorders are associated with excessive injured red blood cells (RBCs) with cellular membrane changes leading to exposure of phosphatidylserine. This process is a contributing factor for several serious diseases, including heart failure-associated anemia¹⁸, chronic renal failure¹⁹, hemolytic uremic syndrome²⁰, Wilson’s disease²¹ and malignancies²². Impairments in erythrocyte deformability have been associated with diabetes mellitus-related renal failure, sepsis and hypertension²³. Therefore, it is important to detect the early process of erythrocyte damage before hemolysis.

On the other hand, accumulated evidence in recent years suggests that some nanoparticles have an adverse effect on erythrocytes pre-hemolysis after interacting with RBC in vitro and in vivo. Owing to the induction of specific structural changes in the lipid bilayer, these nanoparticles caused erythrocyte shape transformation²⁴, decreased the deformability and oxygen-delivering ability²⁵, modified the heme conformation²⁶ and changed hemorheological properties²⁷. Although it has been reported that MgNPs-Fe₃O₄ (100 μg/ml) can cause cellular membrane damage in cultured lung epithelial cells²⁸, the impact of Fe₃O₄-MNPs at safe dose on cellular membrane of erythrocytes and circulatory properties beside hemolysis remains unknown.

Eryptosis, also known as programmed erythrocytes death and defined similarly as nucleated cell apoptosis²⁹, is characterized by cell shrinkage, phosphatidylserine exposure, membrane blebbing, intracellular Ca²⁺ level increase, elevated ROS (reactive oxygen species) levels, as well as the rapid consumption of ATP. Eryptosis has been used as metric to evaluate the effects of drugs³⁰, heavy metal ions³¹, fungi and bacterial toxins^32,33, plant extracts³⁴ and pathological factors³⁵ on the cellular structure and function of erythrocytes. Thus far, eryptosis has not been investigated as a possible detection index to assess nanoparticle-blood compatibility during clinical application of Fe₃O₄-MNP. In nucleated cells, oxidative stress, DNA damage, cell cycle arrest, autophagy and apoptosis are important parameters for evaluation of nanotoxicity³⁶; in RBC, the parameters associated eryptosis also can be used to measure erythrocytes injury that cannot be evaluated by hemolysis analysis.

In this study, we evaluated the use of eryptosis indices as a predictive and prognostic parameter to assess the effect of Fe₃O₄-MNPs on RBCs in the cellular function, the cellular membrane microstructure and flow properties of blood in clinic setting.

Results

Fe₃O₄-MNPs synthesis and characterization

Fe₃O₄-MNPs were prepared by aging ferrous hydroxide gels at elevated temperatures. On the basis of TEM micrographs, Fe₃O₄-MNPs displayed a spheroid-like shape with a relatively uniform size. Meanwhile, its size distribution analysis confirmed that the diameter of Fe₃O₄-MNPs centered around 72.6 ± 0.57 nm (n = 1200), while 58.17% Fe₃O₄-MNPs were between 60 and 90 nm in diameter (Fig. 1A,B). Meanwhile, the average particle size of Fe₃O₄-MNPs was measured using DLS. Fe₃O₄-MNPs dispersed in water had a hydrodynamic size of 88.78 nm (Figure S 1). Additionally, result from UV-vis absorption spectra suggested that Fe₃O₄-MNPs had no characteristic absorption peak (Figure S 2).

Figure 1

The characterization of Fe₃O₄-MNPs.

(A) An image of Fe₃O₄-MNPs morphology obtained by TEM, scale bar = 100 nm. (B) The distribution of particle size of Fe₃O₄-MNPs. The data were quantified from TEM micrographs.

Dose- and time-dependent hemolytic activity of Fe₃O₄-MNPs

As shown in Figure S 3A and S 4, after 3 h incubation the Fe₃O₄-MNPs at the concentration of 100, 800 and 1,600 μg/ml resulted in 1.78%, 4.08% and 7.81% erythrocyte lysis, respectively. When the incubation time was extended to 24 h, the nanoparticles at the concentration of 100, 200 and 400 μg/ml led 4.44%, 6.81% and 12.34% erythrocyte lysis (Figure S 3A,B and S 4). These results indicated that the hemolytic rate was affected by Fe₃O₄-MNPs in a dose- and time-dependent manner. Accordingly, the images of erythrocytes collected after incubation with Fe₃O₄-MNPs for 24 hours showed that hemolysis was visible (seen by unaided eye) when Fe₃O₄-MNPs reached a concentration greater than 400 μg/ml (Figure S 3C).

Moreover, the blood gas analysis showed that the standard bicarbonate, TCO₂ and HCO₃⁻ were increased significantly after incubation with Fe₃O₄-MNPs, suggesting a decreased affinity of CO₂ (Table S1). However, most of parameters, including pH, K⁺, Ca²⁺, Na⁺, Mg²⁺, pO₂ and pCO₂, changed slightly after treating with Fe₃O₄-MNPs at 200 μg/ml for 48 hours.

Impact of Fe₃O₄-MNPs on RBCs morphology

The externalization of phosphatidylserine can be quickly and reliably detected by fluorescent annexin V conjugates³⁷, while no study has reported the use of annexin V for evaluation of Fe₃O₄-MNPs caused eryptosis. To evaluate Fe₃O₄-MNPs induced injury, phosphatidylserine exposure was quantified by flow cytometry and fluorescent imaging. When RBCs were exposed to 200 μg/ml Fe₃O₄-MNPs, the number of annexin V-positive erythrocytes increased gradually after 6, 12, 24 and 48 h incubation (Fig. 2A,C). As shown in Fig. 2A, the percentage of PS-displaying cells almost reached 40% at 48 h. And the percentage of PS-displaying cells was increasing along with the increase of Fe₃O₄-MNPs concentration (Fig. 2B,D). After exposure with Fe₃O₄-MNPs at the concentration of 25 μg/ml for 24 h, 95% of the erythrocytes still had an intact cellular membrane; however, the percentage of phosphatidylserine exposing cells was statistically higher than that of control group.

Figure 2

Eryptotic activity of Fe₃O₄-MNPs.

Percentage of eryptosis for cells incubated with Fe₃O₄-MNPs at (A) different time points (0, 6, 12, 24 and 48 h) and (B) different concentrations (0, 25, 50, 100 and 200 mg) determined by flow cytometry. Annexin V – phosphatidylserine confocal images of erythrocytes incubated with Fe₃O₄-MNPs at (C) different concentrations and (D) time points. Scale bars = 10 μm. The comparison between hemolytic and erypotic RBCs at different concentrations (E) and time points (F). Values represent means ± SEM, n = 9, **p < 0.01, ***p < 0.001.

Additional experiments were performed to elucidate the differences between hemolysis and eryptosis caused by Fe₃O₄-MNP exposure. The hemolytic grade was lower than 5% after incubation with the Fe₃O₄-MNPs at a dose of 100 μg/ml for 24 hours. The corresponding percentage of PS-displaying erythrocytes was 15.1 ± 1.85%. Consistent with the notion of excellent hemocompatibility, Fe₃O₄-MNPs displayed remarkably low hemolytic activity. However, erythrocytes with annexin V positive were three times more than hemolytic erythrocytes (Fig. 2E,F). Annexin V binding is indicative of alterations in the cell surface characteristics, phospholipid organization and membrane integrity. As a result, the injured cells are recognized, phagocytized and degraded by macrophages³⁸.

Eryptosis induced by Fe₃O₄-MNPs

In contrast to the control group (Fig. 3A), the Fe₃O₄-MNPs exposure group showed that Fe₃O₄-MNPs aggregated, agglomerated and adhered to the cellular membrane of RBCs (Fig. 3B, white arrow), resulting in various types of transformations of cellular membrane along with the incubation time. The blebbing on cellular membrane (Fig. 3B–D, black arrow) was more tangible after 12-hour exposure of Fe₃O₄-MNPs, while pores (Fig. 3D, red arrow) were much more apparent after 48-hour exposure. Subsequently, an obvious perturbation and curvature of the cellular membrane occurred after Fe₃O₄-MNPs exposure. These morphological characteristics reflected the changes occurring at metabolic levels, indicating that after incubation with Fe₃O₄-MNPs erythrocytes were more prone to Phorbol 12-myristate 13-acetate (PMA)-induced THP-1 cells phagocytosis (Fig. 3E,F). With phosphatidylserine externalization and membrane blebbing, RBCs deformability (represented by elongation index) decreased in a dose-dependent manner. When the concentration reached 50 μg/ml, the elongation indices in most shear rates were significantly different from those of untreated group (Fig. 3G). Meanwhile, the concentration of cytosolic 2,3-Disphosphoglycerate, an indicator of RBCs oxygen delivering capacity, also showed a reduction in a dose-dependent manner (Fig. 3H).

Figure 3

Impact of Fe₃O₄-MNPs exposure on erythrocyte membrane, deformability, cytosolic 2,3-DPG and THP-1 phagocytosis of erythrocytes.

(A–D) shows time-dependent impact of Fe₃O₄-MNPs on erythrocyte membrane shape and blebbing. White, black and red arrows indicate Fe₃O₄-MNPs, bubbles and pores, respectively. (E,F) shows THP-1 phagocytosis of erythrocytes with or without Fe₃O₄-MNPs incubation, respectively. Scale bars = 5 μm. Red: DiI labeled erythrocytes, blue: DAPI labeled nuclear material. RBCs deformability (G) and cytosolic 2,3-DPG (H) decrease induced by 24 h exposure of 0, 25, 50, 100, 200 μg/ml Fe₃O₄-MNPs. Values represent means ± SEM, n = 9.

Reactive oxygen species (ROS) is known as an important trigger for eryptosis and ROS antagonist N-acetylcysteine (NAC), an antioxidant agent³⁹, can block ROS-mediated eryptosis. Incubation with Fe₃O₄-MNPs resulted in significant increases in ROS production and increases in phosphatidylserine exposure. NAC efficiently inhibited Fe₃O₄-MNPs-induced ROS production and phosphatidylserine externalization (Fig. 4A,B,E). Ca²⁺ influx is an important signaling mechanism leading to eryptosis. Since Fe₃O₄-MNPs exposure also led to high cytosolic Ca²⁺ levels, blocking Ca²⁺ entry by Ca²⁺-free Ringer solution remarkably decreased phosphatidylserine exposure (Fig. 4C–E). It was observed that the inhibitory effect of Ca²⁺-free Ringer solution in combination with NAC was more effective compared to the use of NAC or Ca²⁺-free alone (Fig. 4F).

Figure 4

Fe₃O₄-MNPs –induced ROS production, Ca2 + influx, ATP consumption and eryptosis.

Intracellular ROS concentrations (A) and phosphatidylserine exposure percentages (B) induced by 24 h exposure to Fe₃O₄-MNPs and protected by NAC. Cytosolic Ca²⁺ concentrations (C) and phosphatidylserine exposure percentages (D) induced by 24 h exposure to Fe₃O₄-MNPs and protected by Ca²⁺-free Ringer solution. (E) Annexin V – phosphatidylserine confocal images of erythrocytes incubated with Fe₃O₄-MNPs with NAC-, Ca²⁺-free and NAC- + Ca²⁺-free – Ringer solution. Scale bars = 10 μm. (F) Percentage of phosphatidylserine exposure after 24 h exposure to Fe₃O₄-MNPs in NAC Ringer solution. Forward scatter (G) and cytosolic Ca²⁺ concentration (H) of erythrocytes exposed to Fe₃O₄-MNPs for 24 h in Ringer solution or NAC Ringer solution. (I) Cytosolic ATP concentrations of erythrocytes exposed to Fe₃O₄-MNPs for 24 h. (J) Confocal images of cytosolic calcium stained by Fluo-3/AM in Ringer solution for 6 h, Cr⁶⁺ (20 μM) in Ringer solution for 6 h, Fe₃O₄-MNPs in Ringer solution for 6 h and 12 h, Fe₃O₄-MNPs in N-acetylcysteine added Ringer solution for 12 h. Scale bars = 10 μm, values represent means ± SEM, n = 9, *p < 0.05, **p < 0.01, and***p < 0.001.

Mature erythrocytes lack the capacity to store Ca²⁺ and as a result, the Ca²⁺ entry caused by Fe₃O₄-MNPs exposure can be readily blocked by Ca²⁺-free Ringer solution. However, Ca²⁺-free Ringer solution could not completely block phosphatidylserine externalization. The percentage of phosphatidylserine exposure in cells in Ca²⁺-free Ringer solution was 13.36% and 20.23% in the presence of Ca²⁺ (Fig. 4B). Furthermore, hexavalent (VI) chromium, an eryptotic trigger causing Ca²⁺ influx, resulted in observable Ca²⁺ entry when exposed for 6 h. However, a detectable increase in cytosolic calcium appeared after Fe₃O₄-MNP exposure for more than 12 h, with the cells volume unaltered indicating by forward scatter (Fig. 4G–J). In addition, N-acetylcysteine inhibited ROS production, even with Ca²⁺ entry (Fig. 4H,J). Altogether, these findings indicated that oxidative stress played a pivotal role in Fe₃O₄-MNPs induced eryptosis and the initial process of eryptosis involves calcium-independent phosphatidylserine externalization, while Ca²⁺ entry also played an important role in this programmed cell death. In Ringer solution, ATP was consumed rapidly, regardless of whether erythrocytes were treated with Fe₃O₄-MNP incubation or not (Fig. 4I) and it was consistent with previous report⁴⁰.

Blood compatibility of Fe₃O₄-MNPs and protective effect of NAC in vivo

To investigate pharmacokinetic characteristics of Fe₃O₄-MNPs, blood Fe ions concentration after Fe₃O₄-MNPs injection was measured using inductively coupled plasma optical emission spectrometry (ICP-OES). We found that Fe ion concentrations were higher than that in in vitro experiment until 6 h (Figure S 5). As compared with the control group, the Fe₃O₄-MNPs treated rats showed no obvious damage or inflammation in the major organs (i.e. Liver, spleens, kidneys, hearts and lungs). The aggregation of Fe₃O₄-MNPs was mainly found in the livers and spleens, consistent with the normal distribution pattern of substances injected intravenously⁴¹. NAC administration did not change the distribution or the aggregation of Fe₃O₄-MNPs in the major organs (Fig. 5).

Figure 5

H&E staining on heart, liver, spleen, lung and kidney samples from rats injected with saline, saline with NAC, Fe₃O₄-MNPs and Fe₃O₄-MNPs with NAC.

Rats were sacrificed 24 h after the last injection. Aggregated Fe₃O₄-MNPs were found in Fe₃O₄-MNPs injected groups and indicated by green circles. Scale bars = 50 μm.

Compared with the control groups, Fe₃O₄-MNP injection significantly increased ROS production, consistent with the in vitro results (Fig. 6A,C). The percentage of PS-displaying erythrocytes was less than 1% in the control groups. With Fe₃O₄-MNP injection, this percentage increased to 2.48%, meaning that erythrocyte degradation and regeneration was accelerated roughly 2-fold (Fig. 6B,D). NAC significantly inhibited Fe₃O₄-MNP-induced increases in ROS and phosphatidylserine exposure, although they were still higher than those of the control groups (Fig. 6A–D). These results suggested that Fe₃O₄-MNPs injection in rats induced eryptosis in vivo via ROS production and NAC could alleviate the oxidative stress to some extent.

Figure 6

Cytosolic ROS and phosphatidylserine exposure of erythrocytes from rats.

Cytosolic ROS (A,C) and phosphatidylserine exposure (B,D) of erythrocytes from rats injected with saline, saline with NAC, Fe₃O₄-MNPs and Fe₃O₄-MNPs with NAC. Blood samples collected from tail vein were immediately stained by fluorescent Probe-DCFH-DA and Annexin-V-FLUOS and analyzed by flow cytometry. Values represent means ± SEM, n = 7, *p < 0.05, **p < 0.01 and ***p < 0.001.

The majority of the clinical chemistry parameters were within the normal ranges and showed no difference between treated- and the control groups, suggesting limited systemic toxicity caused by the doses investigated in this study (Figure S 6). Only the value of Total bilirubin (TBIL) in Fe₃O₄-MNP-treated group showed a significant increase compared with that of the control group (p < 0.05) and NAC administration inhibited Fe₃O₄-MNP-induced increase of TBIL, falling down within the normal range (Figure S 6). TBIL level is related to the degradation of senescent or injured erythrocytes and fluctuations in this value suggest that Fe₃O₄-MNP injection can lead to excess erythrocyte degradation that can be alleviated by NAC.

During hematology analysis, the majority of hematology markers relevant to erythrocytes displayed significant difference between the control group and the nanoparticle injected group, such as RBC counts, hemoglobin, mean corpuscular volumes, mean corpuscular hemoglobin and red cell distribution widths (Figure S 7). The values of all of these markers were within the normal range except hemoglobin (Figure S 7). Decreases of RBC counts, hemoglobin and mean corpuscular hemoglobin indicated an excessive degradation of erythrocytes. There was also a decrease in mean corpuscular volumes and red cell distribution widths, which reflected a more uniform size and indicated that there were an enormous number of erythrocytes to be generated. However, NAC administration did not reverse changes of these erythrocyte-relevant markers. These results suggested that Fe₃O₄-MNP injection might lead to a mild to moderate risk for anemia.

The impact of nanoparticles on the flow properties of blood was evaluated by hemorheology. The hematocrit and plasma viscosities were not statistically significant. However, the indices related to the mechanical properties of erythrocytes differed significantly between the control and the Fe₃O₄-MNPs groups, such as erythrocyte electrophoresis time, RBC aggregation index, erythrocyte deformation index, erythrocyte rigidity index and the viscosity of whole blood. The changes in RBC aggregation index and erythrocyte electrophoresis were inhibited by NAC administration (Fig. 7). These findings demonstrated that Fe₃O₄-MNPs treatment led to a decrease in deformability and changes in hemorheology, which would contribute to microcirculation disturbance and tissue damage by directly blocking capillaries and potential thrombogenesis⁴². Altogether, the results in vivo suggested that although there was a risk of anemia or thrombus formation, there was no obvious systemic toxicity risk after the administration of nanoparticles.

Figure 7

Results of hemorheology of rats injected with saline, saline with NAC, Fe₃O₄-MNPs and Fe₃O₄-MNPs with NAC.

Viscosity of whole blood (ηWB), viscosity of plasma (ηp), RBC aggregation index (RAI), erythrocyte deformation index (TK), erythrocyte electrophoresis time (EI) and erythrocyte rigidity index (IR). Values represent means ± SEM, n = 7, *p < 0.05, **p < 0.01.

Discussion

Developing an assessment system, in addition to hemolysis, with sensibility, predictability and reliability is critical to detect the cytotoxicity of Fe₃O₄-MNPs⁴³. Currently, evaluation of the hemocompatibility of nanoparticles mainly relies on hemolysis analysis, which is not delicate enough to reflect the full spectrum of erythrocyte injuries. However, determination of the hematologic toxicity of Fe₃O₄-MNPs on alternations of cellular membrane structure and function of erythrocytes remains unexplored. Thus, a predictive parameter beyond hemolysis testing is highly desirable. In this study, we developed a reliable system with eryptotic indices to evaluate the erythrocyte compatibility with Fe₃O₄-MNPs and to determine hazards (e.g. anemia and thrombogenesis) associated with the MNPs even at safe doses.

In the present study, our data demonstrated that eryptosis analysis was more sensitive in measuring hemocompatibility of nanoparticles for erythrocyte injuries, compared with currently used chemolysis for hemolysis. Currently, the safe and non-cytotoxic concentration is up to 100 μg/ml based on hemolysis standard⁴⁴. Even when the concentration of nanoparticles rises to up to 400 μg/ml, the hemolysis rate of modified magnetic nanoparticles is less than 2%⁴³. However, our data showed that Fe₃O₄-MNP caused phosphatidylserine externalization was statistically higher in the treated group than in the non-treated group when the dosage of nanoparticles is 25 μg/ml in vitro. Previous studies have shown that phosphatidylserine externalization evaluation is more sensitive than hemolysis testing^30,31,32. Eryptosis is presumably a physiological protective mechanism to eliminate injured or defective erythrocytes to forestall hemolysis and the release of hemoglobin^14,45. Phosphatidylserine acts as a key role in cell cycle signaling, specifically in relationship to apoptosis. When exposed to Fe₃O₄-MNP in vitro, the percentage of phosphatidylserine exposed on the cell surface reached over 3-fold greater than the hemolysis rate in this study. Then the phagocytosis and subsequent degradation of cells with exposed phosphatidylserine are inevitable.

In vivo, accelerated eryptosis leads to erythropenia, anemia and impairment of microcirculation. Normally, the loss of eryptotic erythrocytes is compensated by stimulation of erythropoiesis³⁷. However, the results showed that Fe₃O₄-MNPs exposure caused excessive eryptosis could not be fully compensated by erythropoiesis. This is because numbers of erythrocytes and the hemoglobin concentration were significantly lower in the Fe₃O₄-MNP treated groups compared with the non-treated group. Particularly, for hemoglobin concentrations, Fe₃O₄-MNP administration resulted in a sharp decline beyond the normal ranges, suggesting a significant risk for anemia. In hemorheologcal analysis, markers relevant to the mechanical properties of erythrocytes decreased with Fe₃O₄-MNP treatment. These outcomes can lead to a risk of ischemia or hypoxia caused by microcirculation blood hypoperfusion (Fig. 8)⁴⁶. Interestingly, N-acetylcysteine administration could not alleviate the acceleration of eryptosis and erythrocyte degradation, although it may be useful to ameliorate defects in some mechanical properties, such as whole blood viscosity (WBV), RBC aggregation index (RAI) and erythrocyte electrophoresis time (EI).

Figure 8

Schematic illustration of Fe₃O₄-MNPs impacts on erythrocytes and circulation.

Furthermore, evaluations based on eryptosis can better predict the fate of the erythrocyte in circulation and can also reflect the risks posed by damaged erythrocytes that disrupt circulation. Phosphatidylserine is restricted to the cytoplasmic leaflet of the plasma membrane. However, it was shifted to the outer cellular membrane induced by Fe₃O₄-MNPs exposure, where it is recognized by specific phagocytic receptors, such as T-cell immunoglobulin mucin receptor 4 (TIM4). Therefore, cells with exposed PS were rapidly engulfed and degraded by macrophages^47,48. Systematic administration of Fe₃O₄-MNPs induced 2% of erythrocytes’ phosphatidylserine externalization, which was lower that in vitro. It is attributed to one remarkable fact that the Annexin-V-FLUOS labeled erythrocytes represented only a fraction of eryptotic cells, since phosphatidylserine externalization is recognized and rapidly engulfed and degraded by macrophages. In fact, the percentage was over 2-fold greater and statistically higher than those of phosphatidylserine externalization in the control group. Membrane blebbing accounts for nearly 20% loss of membrane surface area, leading to a shape transformation and a reduction in deformability. Excessive blebbing accelerated the aging processes of erythrocytes⁴⁹. ROS production resulted in the degradation of band 3 and spectrin and the opening of cation channels. It is critical for maintaining RBCs morphology, plasticity and osmotic stability⁵⁰. The increased cytosolic Ca²⁺ affected the skeleton flexibility and stability, intracellular ion balance and facilitates phosphatidylserine externalization⁵¹. Membrane blebbing, phosphatidylserine externalization, shape transformation and reduction in deformability contributed to the phagocytic uptake and mechanical properties changes, which were reflected by hematology and hemorheology alternation after the treatment of Fe₃O₄-MNPs. Furthermore, microvesicles generated by blebbing and externalized phosphatidylserine have procoagulant activity suggesting a hazard of thrombin generation^15,52. Therefore, the acceleration of eryptosis induced by Fe₃O₄-MNPs should not be overlooked and warrants further investigation. Overall, some hallmarks of eryptosis may represent more sensitive and predictive parameter of toxicity compared with hemolysis.

Recently, the interaction between nanoparticles and erythrocytes has been investigated^25,53. However, the possible mechanism underlining the Fe₃O₄-MNP induced eryptosis was still not clear. There are reports that the increases of intracellular ROS might cause the potential toxicity in eryptosis^37,54. Oxidative stress induces activation of Ca²⁺-permeable nonselective cation channels. With Ca²⁺ influx, erythrocytes suffered from PS externalization, cellular membrane blebbing, the loss of deformability and clearance by macrophages. Our results confirmed that ROS production was the original and pivotal trigger of eryptosis. N-acetylcysteine, a ROS inhibitor, effectively blocked the eryptotic processes. Ca²⁺ influx is also a powerful trigger of eryptosis. Inhibition of Ca²⁺ entry relieved the phosphatidylserine externalization, consistent with previous studies^14,51,55. However, the loss of membrane phosphatidylserine asymmetry was calcium-independent. The significant increase in cytosolic calcium concentrations occurred after ROS production, while Ca²⁺ influx was remarkably influenced by oxidative stress. Consequently, with ROS generation and Ca²⁺ entry promoted the eryptosis caused by Fe₃O₄-MNPs.

There is mounting evidence that the impacts of nanoparticle exposure on erythrocytes include a series of subtle changes in ionic balance, energy metabolism, physiology and rheological properties, in addition to hemolysis. As a result, there are often conflicting data between hemolysis testing in vitro and blood-compatibility in vivo. The injured erythrocytes progress to eryptosis, lose capability of oxygen delivering, impeding microcirculation and are cleared from circulating blood at last. These changes, as correlated with some specific clinical disorders, can be detected by eryptotic indices in vitro. Consequently, eryptotic indices seem to be reasonable prediction metrics for predicting how nanoparticles impact erythrocytes and blood circulation in vivo (Fig. 8).

Conclusions

In summary, we reported that 25 ug/ml Fe₃O₄-MNPs caused significant damage to erythrocytes in in vitro experiments and 12 mg/kg Fe₃O₄-MNPs lead to apoptosis of circulating erythrocytes in vivo. Erythrocyte injury of Fe₃O₄-MNPs can be divided into the early and later phages, eryptosis and hemolysis. Eryptosis does not induce acute hemolyzation characterized by hemoglobin releasing; it leads pathological alternations on cellular membrane and erythrocyte dysfunction. In this study, we demonstrated that Fe₃O₄-MNPs cause programmed cell death in erythrocytes with pathological changes on cellular membrane, abnormal cytosolic calcium levels and oxidative stress and changes in the mechanical property of erythrocytes in vitro and in vivo. This study indicates that phosphatidylserine exposure, the index of eryptosis, can serve as a sensitive and reliable predictor for erythrocyte injury, while monitoring the nanotoxicity of the nanoparticles when systemically administrated. In addition, these metrics provide potential in determining the hazards of new types of nanoparticles or other biomaterials for clinical applications.

Materials and Methods

Materials

FeSO₄·7H₂O, KNO₃ and KOH were purchased from Alfa Aesar Co. (China). Annexin-V-FLUOS was provided by Roche (Germany). Fluo-3/AM was supplied by Invitrogen (USA). An ATP assay kit and 2′,7′-dichlorofluorescin diacetate (DCFH-DA) were purchased from Beyotime (China). Human 2,3-DPG (2,3-Disphosphoglycerate) ELISA Kit was purchased from USCNLIFE (China). Phorbol 12-myristate 13-acetate (PMA), 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), N-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid (HEPES), N-acetylcysteine (NAC) and 4,6-Diamidino-2-phenylindole dihydrochloride (DAPI) were obtained from Sigma-Aldrich (USA). Fetal bovine serum (FBS) was purchased from Gibco (AU). Penicillin-Streptomycin and L-Glutamine were provided by Gibco (USA). All Ringer solution and other salt solutions were freshly prepared before each experiment.

Preparation and characterization of Fe₃O₄-MNPs

Fe₃O₄-MNPs were prepared according to a previously reported method with some modifications⁵⁶. Briefly, 10 ml of 2 M KNO₃ and 10 ml of 0.5 M KOH were added to flask and nitrogen was bubbled into the flask, then 10 ml of 0.1 M FeSO₄ solution was added. The flask was immersed in an oil bath at 90 °C for 4 h. The precipitate (Fe₃O₄-MNPs) was washed by distilled water, freeze-dried and preserved in a drier. The morphology and diameter distribution were observed by transmission electron microscopy (TEM) using a JEm-1400 (JEOL, Japan) and dynamic light scattering (DLS) using a NanoZS90 Zetasizer (Malvern Instruments Co. Ltd., UK). The UV-vis absorption spectra of Fe₃O₄-MNPs was evaluated using a TU-1901 UV spectrophotometer (BEIJING PUXI General Instruments, China) with a slit dimension of 2.0 nm and a quartz cuvette with inner size at 10 mm x 10 mm and outside size at 12.5 mm x 12.5 mm.

Erythrocyte preparation

This study was approved by the Medical Ethics Committee of The Second Hospital affiliated with The Third Military Medical University. All procedures were conducted in accordance with the approved guidelines of the Medical Ethics Committee of The Second Hospital affiliated with The Third Military Medical University. All human subjects involved gave written informed consent. Leukocyte-free erythrocytes from healthy donors were used shortly after collection and were provided by the Chongqing Blood Centre. Hematocrit was adjusted to 0.4% with Ringer solution (125 mM NaCl, 32 mM HEPES, 5 mM glucose, 5 mM KCl, 1 mM MgSO4, 1 mM CaCl₂, pH7.4) and all incubation condition of erythrocytes were 37 °C, 5% CO₂, with 95% humidity. Calcium-free Ringer solution was made by using 1 mM EGTA as a substitute for 1mM CaCl₂.

Hemolysis measurement and blood gas analysis

To determine the impact of Fe₃O₄-MNPs on hemolysis, RBCs were incubated with Fe₃O₄-MNPs at the concentrations from 3.125 to 1600 μg/ml for 3 h and 24 h. Erythrocytes incubated with deionized water and Ringer solution were used as positive (+) and negative (−) controls, respectively. After centrifuged at 10,016 × g for 3 min, supernatants were collected for spectrophotometry. The absorbance (A) was analyzed at 570 nm with a reference at 655 nm in a Varioskan Flash Multimode Reader (Thermo Fisher, USA). The percentage of hemolysis was calculated as: ⁵⁷. The UV-vis absorption spectra of supernatant were measured with a slit dimension of 5.0 nm. Erythrocytes were incubated with 200 μg/ml Fe₃O₄-MNPs for 48 h. Blood gas detecting indices were analyzed using a Critical Care Xpress Blood Gas Analyzer (Nova, US).

Measurement of phosphatidylserine exposure

To evaluate the effects of Fe₃O₄-MNPs on eryptosis, the externalization of phosphatidylserine was detected by fluorescent Annexin V conjugates. After washed with Ringer solution, erythrocytes were resuspended in 250 μl incubation buffer with 5 μl Annexin-V-FLUOS at 20 °C for 15 min. The forward scatter (FSC) and Annexin-V-FLUOS fluorescence intensities were measured by FACS Calibur (BD, USA). The percentage of phosphatidylserine unmasking in erythrocytes was analyzed using Flowjo (Treestar, USA). Images were obtained using confocal microscopy (Carl Zeiss, 510 meta, Germany).

Scanning electron microscopy (SEM)

To characterize the interaction between Fe₃O₄-MNPs and erythrocytes, SEM was employed to observe the transformation, blubbing and pores of cellular membrane. Erythrocytes were incubated with Fe₃O₄-MNPs (200 μg/ml) for 12, 24 and 48 h. Erythrocytes treated with Ringer solution for 12 h were used as a control. The cells were gold-coated with a JEOL JEC-3000FC and observed using a Hitachi S-3400N.

Cell culture and cell uptake assay

THP-1 cells were obtained from the American Type Culture Collection. 2 × 10⁶ cells were pre-activated by PMA 10 ng/ml for 24 h. Differentiated THP-1 cells were washed by complete RPMI-1640 medium and cultured for another 48 h before phagocytosis⁵⁸. Erythrocytes were incubated with Fe₃O₄-MNPs (200 μg/ml) or with Ringer solution (negative control) for 12 h, then labeled by DiI and washed by PBS. Differentiated THP-1 cells were incubated with labeled erythrocytes for 120 min. After incubation, THP-1 cells were washed and counterstained with DAPI⁵⁹. Finally, the images were obtained by CLSM z-stack scanning and analyzed using 3D reconstruction.

Measurement of intracellular ROS, Ca²⁺, 2,3-DPG, ATP and RBC deformability

Intracellular ROS, Ca²⁺ were monitored using fluorescent probes DCFH-DA and Fluo-3 AM, respectively. The changes in fluorescence were measured using FACS Calibur in fluorescence channel FL-1. The geometric means of fluorescence intensity were analyzed by Flowjo software. Intracellular 2,3-DPG and ATP concentrations were detected using Human 2,3-DPG ELISA Kit and ATP assay kit, respectively. The RBC deformability was monitored using an erythrocyte deformability analyzer (LBY-BX, China).

Toxicity of Fe₃O₄-MNPs and protective effects of N-acetylcysteine in vivo

The toxicity of Fe₃O₄-MNPs and the protective effects of N-acetylcysteine were investigated in a rodent model. The animal received concentration of 12 mg/kg/injection of Fe, a dosage generally considered to be safe¹². All procedures were performed in accordance with protocols approved by the Animal Management Rules of the Ministry of Health of the People’s Republic of China (Document NO. 55, 2001). Female CD^® IGS Rats, aged 8 weeks, weighing 190 ± 10 g were obtained from Beijing Vital River Laboratories (China). Twenty-eight rats were randomly assigned to the following four groups (G) (n = 7/G): G1, the control group in which animals were injected with 1 ml saline; G2, the control + N-acetylcysteine group in which animals were injected with 1 ml saline with N-acetylcysteine (2 g/L) administered in distilled water; G3, the Fe₃O₄-MNPs group in which Fe₃O₄-MNPs were injected at a dosage of 12 mg/kg of Fe by intravenous injection; G4, the Fe₃O₄-MNPs + N-acetylcysteine group in which animals received Fe₃O₄-MNPs at a dosage of 12 mg/kg of Fe by intravenous injection and were administered N-acetylcysteine (2 g/L) in distilled water. Rats were intravenously treated with saline or Fe₃O₄-MNPs every other day for a total of three treatments. N-acetylcysteine was administered during the experimental period.

At day 6, animals were anaesthetized using isoflurane. Blood samples were collected and used to measure ROS, phosphatidylserine exposure, hematology analysis, blood serum biochemistry and hemorheology analysis. The normal ranges of biochemistry and hematology data of healthy female CD^® IGS Rats were obtained from Charles River Laboratories (http://www.criver.com/files/pdfs/rms/cd/rm_rm_r_cd_rat_clinical_pathology_data.aspx). The major organs were stained with hematoxylin and eosin (H&E). The distribution of Fe₃O₄-MNPs and the morphology of organs were observed under a light microscope (Olympus BX63, Japan).

In order to investigate pharmacokinetic characteristics of Fe₃O₄-MNPs, we measured blood Fe ions concentration after Fe₃O₄-MNPs injection using ICP-OES. Eight Rats were randomly assigned to two groups (n = 4/G): G’1, the control group in which animals were injected with 1 ml saline; G’2, the Fe₃O₄-MNPs group in which Fe₃O₄-MNPs were injected intravenously at a dosage of 12 mg/kg of Fe. Blood samples were obtained from G’1 group and G’2 group at 5, 10, 20, 30, 60, 120, 180, 240, 360, 480, 720 and 1440 min after the last injection. Blood Fe ions concentration was measured using iCAP 6000 SERIES (Thermo SCIENTIFIC, USA) after acid hydrolysis.

Statistical analysis

All data were expressed as mean ± SEM (standard error of the mean). The results were analyzed using GraphPad Prism 5 software (GraphPad Software Inc., CA). A p-value < 0.05 was considered statistically significant.