Journal of Nanoparticle Research

, Volume 13, Issue 1, pp 199–212

Systematic evaluation of biocompatibility of magnetic Fe3O4 nanoparticles with six different mammalian cell lines

Authors

  • Yingxun Liu
    • State Key Laboratory of Bioelectronics, School of Biological Science and Medical EngineeringSoutheast University
  • Zhongping Chen
    • State Key Laboratory of Bioelectronics, School of Biological Science and Medical EngineeringSoutheast University
    • State Key Laboratory of Bioelectronics, School of Biological Science and Medical EngineeringSoutheast University
Research Paper

DOI: 10.1007/s11051-010-0019-y

Cite this article as:
Liu, Y., Chen, Z. & Wang, J. J Nanopart Res (2011) 13: 199. doi:10.1007/s11051-010-0019-y

Abstract

This article systematically evaluated the biocompatibility of multiple mammalian cell lines to 11-nm DMSA-coated Fe3O4 magnetic nanoparticles (MNPs). Cells including RAW264.7, THP-1, Hepa1-6, HepG2, HL-7702, and HeLa were incubated with six different concentrations (0, 20, 30, 40, 50, and 100 μg/mL) of MNPs for 48 h, and then the cell labeling, iron loading, cell viability, apoptosis, cycle, and oxidative stress were all quantitatively evaluated. The results revealed that all the cells were effectively labeled by the nanoparticles; however, the iron loading of RAW264.7 was significantly higher than that of other cells at any dose. The proliferations of all the cells were not significantly suppressed by MNPs at the studied dose except HepG2 that was exposed to 100 μg/mL MNPs. The investigation of oxidative stress demonstrated that the levels of total superoxide dismutase and xanthine oxidase had no significant changes in all the cells treated by all the doses of MNPs, while the levels of malonyldialdehyde activity of MNP-treated cells significantly increased. The nanoparticles did not produce any significant effect on cell cycles at any of the doses, but resulted in significant apoptosis of THP-1 and HepG2 cells at the highest concentration of 100 μg/mL. At a concentration of 30 μg/mL which was used in human studies with an intravascular nanoparticle imaging agent (Combidex), the nanoparticles efficiently labeled all the cells studied, but did not produce any significant influence on their viability, oxidative stress, and apoptosis and cycle. Therefore, the nanoparticles were concluded with better biocompatibility, which provided some useful information for its clinical applications.

Keywords

Magnetic nanoparticlesUptakeBiocompatibilityOxidative stressApoptosisHealth and safety

Introduction

With the development of nanotechnology, numerous nanomaterials are created for various applications. Among these nanomaterials, the magnetic Fe3O4 nanoparticles (MNPs) have attracted increasing attention, due to their appropriate surface coatings and promising potential applications in biomedical sciences (Pankhurst et al. 2003; Ito et al. 2005). MNPs are increasingly being used in the fields of biomedicine and biology, such as bioseparation, magnetic resonance imaging (MRI), hyperthermia, and drug delivery (Duguet et al. 2006; Shubayev et al. 2009). In vitro streptavidin-coated magnetic beads are used for phenotypic selection in different cell sorting protocols, including stem cells (Maxwell et al. 2008), sensory neurons (Tucker et al. 2005), and others (Dunning et al. 2004). The antibody-conjugated MNPs were used to decontaminate blood from infective agents (Weber and Falkenhagen 1997). MNPs were also used as contrast agents in MRI. The MNPs coated with a layer of low-molecular weight dextran and covalently conjugated to holo-transferrin, promoted overexpression of engineered transferrin receptor (ETR) to selectively visualize tumors in vivo in real time at exceptionally high spatial resolution (Weissleder et al. 2000). The iron oxide magnetite (Fe3O4) has been approved by the FDA as an MRI contrast agent (Tiefenauer 2007). Targeted drug/gene delivery strategy is used to deliver drugs or genes region-specifically by attaching them to MNPs and locally concentrating the resulting complexes in vivo to the desired locale (Lin et al. 2007). MNPs had been shown to be able to treat cancer by magnetic hyperthermia; additionally, MNPs conjugated with antibodies to cancer-specific antigens improved selectivity of MNP uptake by tumors during hyperthermia therapy (Ito et al. 2005). Currently, the most often used biocompatible material for the preparation of magnetic particles is the iron oxide magnetite (Fe3O4), which is minimally toxic (Bacon et al. 1987; Weissleder et al. 1989).

Magnetic nanoparticles useful for magnetic drug targeting or MRI must be biocompatible, nontoxic, and nonimmunogenic. The mounting evidence suggests that certain properties of nanoparticles (e.g., enhanced reactive area, ability to cross cell and tissue barriers, and resistance to biodegradation) amplify their cytotoxic potential relative to molecular or bulk counterparts (Brayner 2008). In vivo administered MNPs are quickly challenged by macrophages of the reticuloendothelial system (RES), resulting in transient and acute toxicity to unremarkable changes in vivo (Kim et al. 2006; Gajdosíková et al. 2006; Yu et al. 2008; Jain et al. 2008). In vitro experiment has demonstrated that nanoparticles can enter into macrophages, and cause cell viability loss (Häfeli and Pauer 1999), oxidative stress (Stroh et al. 2004; Valko et al. 2006), hyperoxic injury (Wesselius et al. 1999), dose-dependent apoptosis (Muller et al. 2007), inflammation, and mitochondrial injury (Muller et al. 2007). Similarly, MNPs’ cytotoxicity to non-immunogenic cells in vitro, including Glial cell line (Green-Sadan et al. 2005), neuro-2A cells (Jeng and Swanson 2006; Pisanic et al. 2007), BRL 3A rat liver cells (Hussain et al. 2005), dermal fibroblasts (Berry et al. 2004), and stem cells (Bulte et al. 2001) is increasingly being noted.

In fact, MNPs of various compositions, sizes, and even surface modifications can activate cytotoxicity in cultured cells. Bare iron oxide nanoparticles have very low solubility, and have hydrophobic surfaces with large surface area-to-volume ratios and a propensity to agglomerate (Cheng et al. 2005; Liu et al. 2009). A proper surface coating allows being hydrophilic and getting dispersed into homogenous ferrofluids and improving stability of MNPs. In terms of cytotoxicity, uncoated oxide nanoparticles exert some toxic effects, while coated MNPs have been found relatively little cytotoxicity. The reported coatings of iron oxide nanoparticles include polyethylene glycol (PEG), dextran, dendrimers, citrate, or Dimercaptosuccinic acid (DMSA) (Bulte et al. 2001; Gupta and Curtis 2004; Lacava et al. 2004; Berry et al. 2003; Auffan et al. 2006). The iron oxide nanoparticles were also bound to complex biological molecules such as antibodies, peptides, hormones, or drugs (Sadeghiani et al. 2005). These modifications play an important role in internalization efficiency and cytotoxicity (Pisanic et al. 2007; Jain et al. 2005; Wilhelm et al. 2003). For example, the anionic maghemite (Fe2O3) MNPs coated with a negatively charged sulfur-containing chelating agent of DMSA were shown to be able to rapidly, uniformly, and efficiently label a very wide range of cells via adsorptive endocytosis (Wilhelm et al. 2002a, b, 2003; Wilhelm and Gazeau 2008). The DMSA-coated maghemite (Fe2O3) MNPs were also shown to be safe, producing cytotoxic changes at levels of 100 μg/mL or higher (Wilhelm and Gazeau 2008). Owing to the efficient intracellular uptake of iron-based nanoparticles, considerations for functional effects of iron accumulation and concentration-dependent cytotoxicity should be given. The systematic studies are necessary to improve the basis for in vitro assessment of nanoparticle toxicity by advancing the understanding of the cellular particle dose–response assessment in this field.

This study investigated the biocompatibility of multiple mammalian cell lines to a DMSA-coated Fe3O4 MNPs. The cells included mouse macrophage cells (RAW264.7), hepatoma cells (Hepa1-6), and human acute monocytic leukemia cell line (THP-1), hepatoma cells (HepG2), liver cells (HL-7720), and cervical adenocarcinoma cells (HeLa). Cells were incubated with various concentrations of Fe3O4 MNPs for 48 h, and the MNPs’ cell uptake, and MNPs’ effects on viability, apoptosis, cycle, and oxidative stress of different cells were quantitatively evaluated.

Experimental section

Materials and cell lines

HEPES, gluteraldehyde, and paraformaldehyde were purchased from Sigma. The cell culture media of RPMI-1640 and Dulbecco’s modified Eagle medium (DMEM) were purchased from Gibco. Potassium peroxydisulfate (K2S2O8), potassium ferrocyanide (KSCN), Iron chloride hexahydrate (FeCl3), and hydrochloric acid were purchased from Sinopharm Chemical Reagent Co. Ltd, China. The cells including RAW264.7, Hepa1-6, THP-1, HepG2, HL-7720, and HeLa were all purchased from China Center for Type Culture Collection, Chinese Academy of Sciences, Shanghai, China.

The selected cells were often chosen to evaluate the intravascularly administered agents. The macrophage (RAW264.7) and monocyte (THP-1) may ingest many nanoparticle imaging agents, and were mostly used to investigate the phagocytosis of nanoparticles. The hepatocytes (Hepa1-6, HepG2, and HL-7720) were often used to evaluate the hepatotoxicity of novel agents (Shaw et al. 2008).

Characterization of MNPs

Magnetic Fe3O4 nanoparticles coated with 2,3-dimercaptosuccinnic acid (DMSA) were produced by Dr. Chen of the Biological and Biomedical Nanotechnology Group, State Key Lab of Bioelectronics, Southeast University, Nanjing, China (Chen et al. 2008). The prepared particles were dispersed in water and sterilized by filtering with 0.22-μm membrane. The iron content of MNPs solution was measured by a standard colorimetric assay (Fish 1988; Adams 1995; Kalambur et al. 2007; Rad et al. 2007).

The dispersibilities and sizes of MNPs in its prepared water solution and two complete cell culture media of DMEM and RPMI-1640 were investigated by transmission electron microscopy (TEM) as previously described (Chen et al. 2005). The complete cell culture media contained 10% fetal calf serum (FCS). For TEM observation, the particles were added in complete cell culture media at the final concentration of 30 μg/mL and incubated at 37 °C for 48 h. A medium-free control was done by diluting the stored particles to 30 μg/mL with water. After incubation, the particles were collected by centrifugation, washed three times with water, and redispersed in water at the same concentration of 30 μg/mL. The redispersed particles were added to the copper grid and observed using JEM-2100 electron microscope (JEOL, Japan). The size of nanoparticles were measured with Image Origin 6.1.

Cell culture and MNPs treatment

The adherent cells, RAW264.7, Hepa1-6, HepG2, and HeLa, were cultured in DMEM supplemented with 10% FCS, penicillin (100 units/mL), streptomycin (100 μg/mL), and 10 mM HEPES in a humidified 5% CO2 atmosphere at 37 °C in 75 cm2 flasks. The adherent cell HL-7702 was cultured in RPMI-1640 medium, supplemented with 10% FCS, 2 mm l-glutamine, 50 IU/mL penicillin, and 50 mg/mL streptomycin. The suspension cell THP-1 was cultured in RPMI-1640 medium containing 20% FCS. In order to treat adherent cells with MNPs, 100,000 cells per well were seeded into six-well plate for cell apoptosis and cycles, 50,000 cells per well were seeded into 24-well plate for iron content measurement, 15,000 cells per well were seeded into 48-well plate for Prussian blue staining, and 10,000 cells per well were seeded into 96-well plate for MTT assay. After seeding, cells were cultured for 24 h.

In order to treat cells with MNPs at different concentrations in complete cell culture media, the required amount of nanoparticles were transferred in a set of tubes and diluted with sterile water to the volume of the nanoparticles for the highest concentration. Then, the same volume of complete cell culture media was added to each tube. The cell culture media were mixed evenly by gently inverting tubes. The prepared MNPs-containing complete cell culture media were exchanged into wells, and cells were cultured for more 48 h.

In order to treat suspension cell with MNPs, cells were seeded into microwells at a density of 1 × 106 per mL and cultured for 24 h; then the MNPs were directly added into wells, and the cells were continuously cultured for 48 h.

Observation of intracellular MNPs

In order to observe the intracellular MNPs with light microscope, cells were stained with Prussian blue as previously described (Rivière et al. 2005; Ju et al. 2006; Song et al. 2007; Schwarz et al. 2009; Farrell et al. 2009). In brief, cells were washed three times with PBS and then fixed with 4% paraformaldehyde for 30 min. After fixation, cells were washed three times again with PBS, and then stained with Perls Prussian blue (2% potassium ferrocyanide in 6% aqueous hydrochloric acid) for 30 min. After staining, cells were washed again with PBS and counterstained with nuclear fast red. The stained cells were observed with light microscope and photographed.

In order to observe the intracellular MNPs with TEM, cells were incubated with complete culture media containing 30 μg/mL MNPs at 37 °C for 48 h, then collected and incubated overnight at 4 °C with a fixative solution (1.25% gluteraldehyde in 0.1 M phosphate buffer, pH 7.4). Cells were rinsed with PBS and post-fixed with 1% osmium tetroxide for 1 h, dehydrated with a series of ethanol solutions of 50–100%, cleared with propylene oxide, and gradually infiltrated with Epon resin. The samples were arranged in molds filled with Epon, and the resin was allowed to polymerize at 60 °C for 24 h. Then the samples were sectioned with a diamond knife mounted on a RMC MT-7 ultramicrotome. The ultrathin sections were supported on a copper grid and observed using a Hitachi 8100 electron microscope (Tokyo, Japan) at an acceleration voltage of 100 kV.

Quantification of intracellular iron content

After treatment with MNPs, cells were washed three times with PBS, collected in 1.5-mL tube and counted. Cells were precipitated by centrifugation, resuspended in 50 μL of hydrochloric acid, and incubated at 60 °C for 4 h. Cells were precipitated by centrifugation again, and the supernatants were transferred into 96-well plates. Fifty microliters of freshly prepared detection reagent (0.08% K2S2O8, 8% KSCN and 3.6% HCl dissolved in water) was added to each well, and the plates were kept at room temperature for 10 min. The absorbance was measured using an absorption reader (Bio-Tek instruments, Winooski, Vermont) at an excitation wavelength of 490 nm. The iron content was determined by normalizing the obtained absorbance with a standard curve using FeCl3 calibration standards that contained 0, 0.5, 1, 2, 4, 6, 10, 20, 40, 50, and 100 μg/mL of iron. A blank sample of cells was used to confirm the absence of iron contamination in the tested cells. The average iron contents per cell were calculated as mean values divided by the number of cells in each sample. Each experiment was repeated in triplicate wells at least six times.

Assessment of cell viability

The in vitro cytotoxicity of MNPs was tested using a modified cell viability assay (Pieters et al. 1989; Häfeli et al. 2009). After MNPs’ treatment, the media containing MNPs were carefully removed, and 180 μL of serum-free media and 20 μL of a MTT solution (2 mg/mL in PBS) (Sigma) were added and incubated further for 4 h. As a control, 150 μL of PBS at pH 7.4 was added to cells in three of the wells. The supernatant in each well was aspirated, and 150 μL of dimethyl sulfoxide (DMSO) was added to solubilize the cells and MTT crystals. After vigorous stirring using an Eppendorf Thermomixer at 400 rpm at 37 °C for 10 min to dissolve all the crystals, the blue color was read in a multiwell reader (Bio-Tek instruments, Winooski, Vermont) at 570 nm. The cell viability was calculated by comparing the absorption of treated cells to that of control cells, which was defined as 100%. MNPs were considered toxic if the difference between cell growth inhibition of control and exposed cells was statistically significant at the P < 0.05 level, as determined by one-way analysis of variance.

In order to exclude nanoparticles that interfere with the MTT assay, 180 μL of cell culture medium or cell culture medium containing MNPs in different concentrations was added to the wells (without cells). After incubation under the same conditions, 20 μL of MTT solution was added to each well, and the absorbance of each well was measured as described above. The absorbance value of this cell-free control was subtracted from that of the cell groups.

Assay of oxidative stress

The extent of MNP-induced oxidative stress was inferred from the activities of total superoxide dismutase (T-SOD), xanthine oxidase (XOD) and malondialdehyde (MDA). The T-SOD activity was from the ratio of auto-oxidation rates measured in the presence (sample) and in the absence of SOD (blank), which was presented as units per milligram of protein (Beyer and Fridovich 1991; Nebot et al. 1993). The lipid peroxidation indicator MDA was determined using the method of reactive species (TBARs). In brief, the samples were reacted with thiobarbituric acid (TBA) to determine TBARS and the absorbance was extrapolated from a standard curve generated by using pure MDA. The level of MDA was expressed as nmol MDA/mg protein. XOD catalyzed the oxidation of hypoxanthine to xanthine to produce superoxide radical, which eventually results in pink adduct detected at 530 nm with a microplate reader. In order to investigate cellular T-SOD, XOD, and MDA levels, the MNP-treated cells were collected, frozen, and thawed repeatedly three times, and the lysate was centrifuged at 10,000 rpm for 10 min to obtain the supernatants. After protein concentrations were determined using Bradford assay in which bovine serum albumin was used as the standard, the supernatants were subjected to intracellular T-SOD, XOD, and MDA activity assays, which were determined with commercial kits from Jiancheng Bioengineering Institute according to the manufacturer’s instruction. In order to exclude nanoparticles that interfere with the oxidative stress assays, cell-free controls were assayed for each concentration of MNPs, and the corresponding absorbance was subtracted from the absorbance of cell groups.

Analysis of cell apoptosis

Cells were analyzed for annexin V binding and propidium iodide (PI) incorporation to distinguish between apoptotic and necrotic cells, which were performed using the Vybrant® Apoptosis Assay Kit (Molecular Probes) according to the manufacturer’s instructions. Briefly, the cells were seeded into 6-well plates and treated with MNPs as described above. The treated cells were harvested and washed with ice-cold PBS, and then resuspended in 100 μL of 1× annexin-binding buffer (10 mM HEPES (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2) at 1 × 106 cells/mL. Cells were stained for 15 min in the dark at room temperature with 5 μL FITC Annexin V and PI at 1 μg/mL. After incubation, cells were added 400 μL of 1× annexin-binding buffer, mixed gently and kept on ice. The stained cells were analyzed via flow cytometry (Becton Dickinson, San Jose, CA) measuring the fluorescence at an excitation wavelength of 488 nm for FITC fluorescence and 610 nm for PI fluorescence. The percentages of viable (PI negative, Annexin negative), apoptotic (PI negative, Annexin positive) and necrotic cells (PI positive, Annexin positive) were evaluated with the CellQuestPro® software (BD Heidelberg).

Analysis of cell cycle

After MNPs treatment, cells were washed with PBS and harvested with trypsinization. The collected cells were treated with a set of reagents provided by the CycleTEST™ PLUS DNA Reagent Kit (BD) to dissolve the cell membrane lipids, eliminate the cellular proteins and RNA. Nucleus were then stained with a 50 μg/mL PI solution for 30 min at 4 °C in the dark. The cell cycle were analyzed by flow cytometry (Becton Dickinson, San Jose, CA) to estimate the distributions of cell-cycle phases.

Statistical analysis

Each experiment was repeated six times in duplicate. Data were expressed as the mean ± SD, and one-way analysis of variance and the unpaired Student test were used to test for significant differences, and P less than 0.05 was considered to indicate a significant difference.

Results and discussion

Characterization of MNPs

The dispersibilities of particles in its prepared water solution and two complete cell culture media (DMEM and RPMI-1640) were examined by TEM. The results were displayed in Fig. 1. These results demonstrated that the MNPs existed in monodispersed state in three solutions. The average size of the nanoparticles in the three solutions were similar to each other, which averaged 11.0 ± 1.25 nm in diameter. The size of the nanoparticles was not changed by incubation of the complete cell culture media; this result was similar to a cationic lipid-coated MNPs the hydrodynamic size of which was not affected by exposure to serum-contained culture medium (Huang et al. 2009). The MNPs concentration in sterile water solution was 800 μg/mL.
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Fig. 1

TEM examination of dispersibilities of MNPs. a MNPs in prepared water solution; b MNPs in complete cell culture media of DMEM; c MNPs in complete cell culture media of RPMI-1640. Insets are statistical size distributions of MNPs. The complete cell culture media contained 10% fetal calf serum

Internalization of MNPs

Prussian blue staining and TEM had been previously used to visualize the uptake of iron-based MNPs in cells (Rivière et al. 2005; Ju et al. 2006; Song et al. 2007; Schwarz et al. 2009; Farrell et al. 2009). Therefore, we employed Prussian blue staining to investigate larger amounts of cell samples from six different mammalian cells treated with MNPs at six different concentrations (0, 20, 30, 40, 50, and 100 μg/mL) for 48 h, while, TEM was employed to image the subcellular distribution of nanoparticles. After Prussian blue staining, the intracytoplasmatic inclusions containing MNPs presented as dense blue-stained vesicles, which could be clearly seen under light microscope. The typical images of six different cell lines stained with Prussian blue were presented in Fig. 2. Accordingly, TEM observations revealed that Fe3O4 nanoparticles entered into all of the six different cell lines after being labeled with the MNPs in cells. Moreover, the majority of intracellular Fe3O4 nanoparticles were distributed in polydispersed vesicles in cytoplasm (Fig. 3). This phenomenon revealed that the internalization of this MNPs means non-specificity with regard to cell. All the cells studied could be efficiently labeled by these MNPs. This kind of facile cell-nonspecific labeling of variety of mammalian cells by DMSA-coated Fe3O4 MNPs suggested that this MNPs may be a good material for MRI imaging reagent. However, the endocytosis capability of different cells was not the same. It was obvious that the RAW264.7 cells endocytosed many more MNPs than other cells, and the endocytosis rate of RAW264.7 was faster than other cells, which clearly revealed the physiological function of this cell. It could be also found that along with increase of MNPs dose in cell culture media, the amount of the blue-stained vesicles containing MNPs increased in cytoplasm. At high concentration, it seems that the whole cytoplasm of RAW264.7 was filled up with MNPs vesicles.
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Fig. 2

Visualization of MNPs-treated cells by Prussian blue staining. The cells of RAW264.7, Hepa1-6, THP-1, HepG2, HL-7702, and HeLa were incubated with MNPs at the concentration of 0, 20, 30, 40, 50, and 100 μg/mL for 48 h. The cells were stained with Prussian blue and observed with light microscope (original magnification ×400)

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Fig. 3

TEM show uptake of iron oxide nanoparticles in two different cell lines. The cells of RAW264.7 (a) and THP-1 (b) were incubated with MNPs at the concentration of 30 μg/mL after 48 h of incubation at 37 °C. The cellular nanoparticles appeared as high electron density vesicles within the cytoplasm

Intracellular iron contents

The quantitative evaluation of the cellular uptake of MNPs is essential to their clinical applications. The colorimetric assay is widely used to measure the cellular iron content, relying on the spectrophotometric detection of the colored complex formed after reaction of Fe2+ with ferrozine (Fish 1988; Adams 1995; Kalambur et al. 2007; Rad et al. 2007). Therefore, we measured the cellular iron contents (pg Fe/cell) of 216 samples with this method in this study. The results are displayed in Fig. 4. In agreement with that observed with Prussian blue staining, the cellular iron loading increased with the dosage of MNPs in each of cell lines. However, the amount of MNPs taken by cells was closely related to cell types. At each level of MNPs concentration, the iron content of RAW264.7 was significantly higher than that of other cell lines. At the concentration of 100 μg/mL, the iron content of RAW264.7 was about 3–4 times higher than that of THP-1, Hepa1-6, HepG2, HL-7702, and HeLa. The iron content of all the cell lines significantly increased with the concentration of MNPs. Nevertheless, for all the cell lines that we measured, the cellular iron loading was of the same order of magnitude.
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Fig. 4

Cellular iron contents. The cells of RAW264.7, Hepa1-6, THP-1, HepG2, HL-7702, and HeLa were incubated with MNPs at the concentration of 0, 20, 30, 40, 50, and 100 μg/mL for 48 h. The iron content per cell was detected with colorimetric assay. The results are means ± SD from six independent experiments. * P < 0.05, ** P < 0.01, respectively

The hepatocytes are of importance to the evaluation of hepatotoxicity of novel agents. Therefore, we selected three hepatocytes, including mouse hepatoma cells (Hepa1-6) and human hepatoma cells (HepG2), and liver cells (HL-7702) in this study, to investigate their endocytosis to the MNPs in detail. It was found that among the six different mammalian cell lines, both the human hepatoma cells (HepG2) and normal liver cells (HL-7702) showed the similar lowest iron contents (1.89 ± 0.46 pg/cell, 2.22 ± 0.13 pg/cell) at 100 μg/mL.

Cell viability

The effect of MNPs on cell viability was extensively assessed using MTT assay (Häfeli et al. 2009; Pieters et al. 1989). This assay was used to assess the viability of the six cell lines after exposure to MNPs at various concentrations (0, 20, 30, 40, 50, and 100 μg/mL) for 48 h. The results are displayed in Fig. 5. The assay demonstrated that the cell viability of RAW264.7 and THP-1 was not significantly (P > 0.05) affected by the various concentrations of MNPs. It was also shown that the increase of MNPs’ dosage resulted in slight decrease of cell viability of Hepa1-6, HepG2, HL-7702, and HeLa. However, the decrease was not significant (P > 0.05) except in the case of HepG2 treated by MNPs at the highest concentration of 100 μg/mL. These results revealed that the DMSA-coated Fe3O4 MNPs had little toxicity to investigate cells and exerted no significant influence on cell proliferation. In other words, this DMSA-coated Fe3O4 MNPs had better biocompatibility in in vitro cell assays. The results of this study are in agreement with the current fact that the most often used biocompatible material for the preparation of magnetic particles is the iron oxide magnetite (Fe3O4), which is minimally toxic (Bacon et al. 1987; Weissleder et al. 1989) and has also been approved by the FDA as an MRI contrast agent (Tiefenauer 2007). The results of this study suggested that the 11-nm DMSA-coated Fe3O4 MNPs should be a good agent for future medical applications.
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Fig. 5

Cell viability. The cells of RAW264.7, Hepa1-6, THP-1, HepG2, HL-7702, and HeLa were incubated with MNPs at the concentration of 0, 20, 30, 40, 50, and 100 μg/mL for 48 h. The cell viability was measured with MTT assay. The viability of the control cells was defined as 100%. The results are means ± SD from six independent experiments. * P < 0.05

Oxidative stress

The levels of T-SOD, XOD, and MDA were determined after the cells were incubated with MNPs at various concentrations for 48 h. The results are shown by Fig. 6. It demonstrated that there was a slight fluctuation of T-SOD and XOD production in the labeled cells, however, the change was not significant in comparison with blank control cells. Transient increases in XOD production in the HepG2 cells were also observed following the same labeling; however, these increases were not significant in comparison with XOD level of untreated HepG2 cells. Therefore, the treatments of different concentrations of MNPs did not significantly affect the levels of T-SOD and XOD of various cells studied.
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Fig. 6

Oxidative stress. The cells of RAW264.7, Hepa1-6, THP-1, HepG2, HL-7702, and HeLa were incubated with MNPs at the concentration of 0, 20, 30, 40, 50, and 100 μg/mL for 48 h. The T-SOD, XOD and MDA were measured as described in “Assay of oxidative stress” in “Experimental section”. Results are represented as mean ± SD from six independent experiments. * P < 0.05

The MDA level of the labeled cells were expressed as nmol MDA/mg protein, as compared with the activity of the corresponding control cells under different concentration of MNPs. As shown in Fig. 6, the treatment of MNPs resulted in a great difference of MDA level among different cells. THP-1 cells most sensitively responded to the treatment of MNPs; even the lowest concentration of MNPs resulted in significant increase of MDA level. Two medium concentration of 40 and 50 μg/mL and the highest concentration of 100 μg/mL all brought about very significant increase of MDA level in THP-1 cells. The highest concentration of 100 μg/mL resulted in very significant increases of cellular MDA activity in RAW264.7, HepG2, Hepa1-6, and HL-7702 cells. The medium concentration of 50 μg/mL also brought about significant and very significant increases of MDA level in HL-7702 cells. However, the increase of MNPs dose from 20 to 100 μg/mL did not result in any significant increase of MDA level in HeLa cell.

Cell apoptosis

In order to identify apoptosis of the labeled cells, annexin V/PI double staining in cells was performed by flow cytometry. The population of the detected cells separated into three groups in each graph: live cells stained with negative annexin V-FITC and PI fluorescence (bottom left quadrant), apoptotic cells stained with positive annexin V-FITC and negative PI (bottom right quadrant) and advanced apoptotic cells stained with positive annexin V-FITC and PI (upper right quadrant). The results were demonstrated by Fig. 7. It was clear that only the highest concentration of 100 μg/mL resulted in very significant apoptosis in THP-1 and HepG2 cells. The medium concentration of 50 μg/mL also brought about a significant apoptosis in THP-1. Very significantly, the treatment of MNPs at various concentrations from 20 to 100 μg/mL did not produce any significant apoptosis in RAW264.7, Hepa1-6, HL-7702, and HeLa cells. Figure 7 showed two typical flow cytometry plots of cell population, one was RAW264.7 treated with 0, 50, and 100 μg/mL of MNPs, which showed no significant apoptosis; and the other was THP-1 treated with same doses of MNPs, which showed significant and very significant apoptosis under the treatment of MNPs at the concentration of 50 and 100 μg/mL, respectively.
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Fig. 7

Cell apoptosis. The cells of RAW264.7, Hepa1-6, THP-1, HepG2, HL-7702, and HeLa were incubated with MNPs at the concentration of 0, 20, 30, 40, 50, and 100 μg/mL for 48 h. The cell apoptosis was assay with flow cytometry. Results are represented as mean ± SD from six independent experiments. * P < 0.05

Cell cycle

The results of flow cytometric analysis of the cell cycles of MNPs-treated and untreated cells are shown in Fig. 8. It is clear that all the concentrations of MNPs did not affect the cell cycle of six different cells after a 48-h incubation. These results agreed with the non-decreased cell viability in Fig. 4. At the highest concentration of MNPs (100 μg/mL), the percentage of the HepG2 cells in G2/M phase increased to 12.6%, greater than that of control cells (7.6%). This agreed with the significant decrease of cell viability of HepG2 treated by 100 μg/mL MNPs (Fig. 4). These results demonstrated that the MNPs at the studied concentrations had no significant influence on the cell cycles of six different mammalian cells.
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Fig. 8

Cell cycle. The cells of RAW264.7, Hepa1-6, THP-1, HepG2, HL-7702, and HeLa were incubated with MNPs at the concentration of 0, 20, 30, 40, 50, and 100 μg/mL for 48 h. The cell apoptosis was assay with flow cytometry. Results are represented as mean ± SD from six independent experiments

Conclusion

This study investigated the biocompatibility of multiple mammalian cell lines to a 11-nm DMSA-coated Fe3O4 MNPs. The doses (20, 30, 40, 50, and 100 μg/mL) of the nanoparticles used in this study encompassed an intravascular concentration of 0.03 mg/mL Fe which was achieved in human studies (Shaw et al. 2008; Harisinghani et al. 2003). It was found that at this concentration, the nanoparticles efficiently labeled all the cells studied, while they did not produce significant effects on viability, oxidative stress, apoptosis, and cycles of all cells. Because the selected cells were from two species and were commonly used to evaluate the intravascularly administered agents, including macrophage/monocyte and hepatocytes, the Fe3O4 MNPs were confirmed as having better biocompatibility, which provided some useful information for clinical applications of the nanoparticles.

Acknowledgment

This investigation was partially funded by the National Important Science Research Program of China (Nos. 2006CB933205).

Copyright information

© Springer Science+Business Media B.V. 2010