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Toxicity Evaluations of Superparamagnetic Iron Oxide Nanoparticles: Cell “Vision” versus Physicochemical Properties of Nanoparticles )
Morteza Mahmoudi,†,‡,* Sophie Laurent,§ Mohammad A. Shokrgozar,† and Mohsen Hosseinkhani^, ,* National Cell Bank, Pasteur Institute of Iran, Tehran, 1316943551 Iran, ‡Nanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran, §Department of General, Organic, and Biomedical Chemistry, NMR and Molecular Imaging Laboratory, University of Mons, Avenue Maistriau, 19, B-7000 Mons, Belgium, ^Cardiovascular Research Center, Mount Sinai School of Medicine, New York, New York 10029, United States, and National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran )
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uperparamagnetic iron oxide nanoparticles (SPIONs) have great potential for a wide use in various biomedical applications, including magnetic resonance imaging (e.g., as contrast agents),1 drug delivery,2 hyperthermia,3 transfections,4 in vivo cell tracking,1 and tissue repair.5 Research involving the use of SPIONs with various surface chemistries continues to evolve with their growth for human in vivo applications. Although only dextran-coated SPIONs are approved for human in vivo use by the Food and Drug Administration (FDA),2 several SPIONs with various physicochemical properties are in clinical trials; so, one can expect that several SPIONs will be utilized in many more commercial products for various biomedical applications in the not-too-distant future.6 Therefore, biological issues in relation to SPIONs have been increasingly addressed in the past few years, and toxicity has clearly become an important one. In order to achieve deep understanding of the toxicity pathways of various SPIONs, crucial for their safe use in humans, a great amount of reliable research must be conducted at present; a significant knowledge gap exists on a complete toxicological profile of SPIONs, and there is urgent need for risk assessment and safety regulations of SPIONs. A precise analysis of the literature shows conflicting results on the toxicity of nanomaterials and specifically of SPIONs. For instance, Karlsson et al.7 evaluated the toxicity of bare SPIONs on human lung epithelial cell line (i.e., A549) and found that there MAHMOUDI ET AL.
ABSTRACT In the last few decades, nanoparticles (NPs) have been recognized as promising
candidates for starting a new revolution in science and technology due to their unusual properties, attracting the attention of physicists, chemists, biologists, and engineers. The aim of this study is to evaluate the toxicities (at both cellular and molecular levels) of three forms of superparamagnetic iron oxide nanoparticles (SPIONs) of various surface chemistries (COOH, plain, and NH2) through the comparison with gene expression patterns of three cell types (i.e., human heart, brain, and kidney). For this purpose, both an MTT assay and a DNA microarray analysis were applied in three human cell lines;HCM (heart), BE-2-C (brain), and 293T (kidney);under the exposure to SPIONs-COOH, SPIONs-NH2, and bare SPIONs. The specific gene alteration and hierarchical clustering revealed that SPIONs-COOH altered genes associated with cell proliferative responses due to their reactive oxygen species (ROS) properties. It was also found that the cell type can have quite a significant role in the definition of suitable pathways for detoxification of NPs, which has deep implications for the safe and high yield design of NPs for biomedical applications and will require serious consideration in the future. KEYWORDS: toxicity . superparamagnetic iron oxide nanoparticles' cell “vision” . physicochemical properties
was no or low toxicity of SPIONs at the applied concentrations (2080 μg/mL), while in other studies, bare SPIONs showed severe toxicity at the same concentration on human fibroblast.8,9 In addition, there was no trace of bare SPION toxicity on mouse fibroblast cells even at higher concentrations (500 μg/mL).1012 There are plenty of additional examples of contradictory reports on the toxicity of particles when the exact same nanoparticles (SPIONs) were interacted with various cells.1330 We postulate that these significant differences between results may be related to the different detoxification approaches that the cells use for toleration/fight against NPs. VOL. 5
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* Address correspondence to
[email protected],
[email protected], Web: www. biospion.com. Received for review June 8, 2011 and accepted August 14, 2011. Published online August 15, 2011 10.1021/nn2021088 C 2011 American Chemical Society
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Figure 1. TEM images of (a) bare, (b) SPIONs-COOH, and (c) SPIONs-NH2. Bar denotes 50 nm.
TABLE 1. Sizes Determined by DLS and ζ-Potentials in
Water of the Different Surface-Modified SPIONs, Presented as Mean ( SD over Four Samples SPIONs
size (nm)
ζ-potential (mV)
bare CAES-grafted APTES-grafted
13.7 ( 2.1 13.8 ( 2.1 17.8 ( 2.6
þ43.7 ( 1.7 15.4 ( 0.5 þ32.6 ( 0.3
The aim of this study was to investigate and compare the cytotoxicity of SPIONs with various surface chemistries on various cell lines (i.e., brain, heart, and kidney) at both cellular and molecular levels. In this case, MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) and DNA microarray methods were employed. In addition, we aimed to prove that the cell responses to the same amounts of NPs are significantly dependent on the cell types. MAHMOUDI ET AL.
RESULTS AND DISCUSSION Characterization of SPIONs. Transmission electron micrographs of the bare and surface-modified SPIONs are shown in Figure 1, confirming the formation of SPIONs with a very narrow size distribution. More quantitative size determinations are summarized in Table 1, together with the ζ-potentials of the SPIONs in water. There are no significant differences in size of the various SPIONs, whereas ζpotentials in water differ considerably. CAES-grafted particles are negatively charged (SPIONs-COOH), while both bare and APTES-grafted SPIONs are positively charged (SPIONs-NH2). Bare SPIONs have a positive surface charge. This last measurement was done on the suspension at acidic pH. Thus, there are OH2þ on the surface of bare nanoparticles. (At pH 7, we are close to the isoelectric point and the suspension is not really stable.) VOL. 5
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ARTICLE Figure 2. Cell viability of MTT assay results for SPIONs-COOH, SPIONs, and SPIONs-NH2 samples on (a) HCM, (b) BE-2-C, and (c) 293T cell lines.
The number of functional groups on the surface can be estimated at about 2%; the calculation was based on titration as described by Kralj et al.31 Physicochemical Effects of SPIONs on Their Toxicity. MTT Assay. MTT reduction was used to metabolically quantify active cells after exposure to the SPION samples. The effect of direct contact of negative, bare, and positive SPIONs with three cell lines was investigated. It is wellrecognized that not only the efficacy of SPIONs but also their biomedical fate within cells are strongly dependent on their surface charges.2 According to the results (Figure 2), positive SPIONs display lower cell viabilities, in all cell lines, in comparison to negative particles.2 We have previously reported that positive poly(vinyl alcohol)-coated SPIONs with NH2 terminal groups can escape from endosomes and are released into the cytosol.2 It is worth noting that, by introducing the positively charged SPIONs into an acidifying lysosomal compartment, the unsaturated amino groups are capable of sequestering protons that are supplied by the proton pump for the digestion of our positive SPIONs. One Cl ion and one water molecule per proton are diffused in the lysosome vesicle.2 Due to the Coulombic interaction between Hþ and the positively charged SPIONs, more protons will be injected to the lysosome, causing lysosomal swelling and rupture, which leads to nanoparticle deposition in the cytoplasm and the spillage of the lysosomal content. Thus, the reason for the lower cell viability of positive SPIONs may relate to their significant and deep cellular uptake amounts in comparison with negative SPIONs. It is also worth mentioning that the majority of cellular membranes have net-negative charges, causing attractive Coulombic interaction with positively charged SPIONs; this leads to the considerable uptake by cells of positive SPIONs in comparison with negative ones. The above results may explain the observation that the toxicity amounts of bare SPIONs are higher than for other coated SPIONs, due to their tendency to absorb proteins, vitamins, amino acids, and ions causing changes in pH and composition in cells and cell medium.11,32,33 DNA Microarrays. Commonly Altered Genes among Three Model SPIONs. SPIONs-COOH, SPIONs, or SPIONs-NH2 MAHMOUDI ET AL.
treatment altered the expression levels of 167, 328, or 160 genes, respectively. Among these, 64 genes were altered by two or more chemical treatments (Table 2). Particularly noteworthy was the up-regulation of oxidative stress-inducible genes TXNRD1 coding thioredoxin reductase 1 by the three SPION exposures, HMOX1 coding heme oxygenase 1 by SPIONs-COOH and SPIONs-NH2 exposures, and the genes associated with glutathione biosynthesis (GCLC and GSR) by SPION exposure. Identification of Genes Specifically Altered by the SPION Treatments. The genes altered specifically in these cell lines under the exposure of SPIONs-COOH, SPIONs, or SPIONs-NH2 were identified. To further identify genes that would discriminate among the biological actions of SPIONs-COOH, SPIONs, and SPIONs-NH2, the genes overlapping among these chemical treatments were excluded. This resulted in a total of 481 genes consisting of 103 SPIONs-COOH specific, 241 SPIONs specific, and 137 SPIONs-NH2 specific genes, which were used for evaluation of the heavy-metal toxicities. Genes Specifically Altered by SPIONs-COOH Treatment. Of the 103 genes selected to discriminate SPIONs-COOH from SPIONs and SPIONs-NH2, 51 were up-regulated and 52 were down-regulated. For further analyses, these genes were functionally classified on the basis of gene ontology (GO). As shown in Figure 3 (and Figure S1, in Supporting Information), 37 up-regulated genes could be annotated and 21 biological processes containing at least three gene hits were found. A particularly important finding was that the SPIONsCOOH treatment induced the genes classified in “M phase”, “mitotic cell cycle”, “regulation of cell cycle”, and “regulation of cell proliferation”. These classes contained eight genes (UBE2C, CDC25B, CDKN3, KIF22, H2AFX, SMC4L1, RAE1, and CCNB2), most of which have been reported to be up-regulated in association with acceleration of cell division and proliferation.3436 Of these genes, CCNB2, UBE2C, SMC4L1, and CDKN3 exhibited remarkably high fold increases. On the other hand, the functional classification of 40 annotatable genes repressed by SPIONs-COOH exposure revealed 30 biological processes including VOL. 5
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Table 2. Continued (b)
(a)
log2-fold change (p value)
log2-fold change (p value) genes induced
SPIONs-COOH
SPIONs
SPIONs-NH2
FOSL1 TXNRD1 PTTG1 AKAP12 KRT19 TLMP1 RBPMS SPP1 ANXA2 IER2 GLA SQRDL LGALS1 HMGA1 SFN TNFAIP8 TMAP1 GRB10 MAFF HMOX1 TNFAIP3 BHLHB2 VRK2 DUSP1 KLF6 SNFILK SLC12A6 ETS2 UGCG PIM1 PIK3CD KPNA4 TRIB1 GCLC GSR
1.60 (0.0018) 1.04 (0.0025) 3.07 (0.0022) 2.01 (0.0094) 1.86 (0.0016) 1.53 (0.0064) 1.44 (0.0221) 1.43 (0.0334) 1.38 (0.0046) 1.34 (0.0042) 1.32 (0.0021) 1.29 (0.0018) 1.23 (0.0026) 1.21 (0.0206) 1.17 (0.0459) 1.15 (0.0376) 1.13 (0.0098) 1.01 (0.0331) 1.00 (0.046) 1.77 (0.0317) 1.31 (0.0313) 1.11 (0.0001) 0.068 (0.0521) 0.00 (0.9934) 0.96 (0.0005) 0.49 (0.2852) 0.55 (0.1020) 0.41 (0.2006) 0.20 (0.3998) 0.54 (0.4087) 0.52 (0.1532) 0.02 (0.9689) 0.31 (0.1786) 0.31 (0.1002) 0.51 (0.1109)
1.56 (0.0021) 2.22 (0.0001) 1.32 (0.0408) 3.0 (0.0020) 1.79 (0.0015) 4.45 (0.0001) 1.90 (0.0064) 1.89 (0.0136) 2.16 (0.0006) 1.06 (0.0062) 1.07 (0.0045) 1.35 (0.0022) 1.94 (0.0004) 3.04 (0.0009) 1.44 (0.0249) 1.98 (0.0084) 2.10 (0.0007) 1.11 (0.0246) 2.22 (0.0034) 0.28 (0.6343) 0.68 (0.1513) 0.86 (0.0016) 1.07 (0.0049) 1.24 (0.0206) 1.58 (0.0003) 1.44 (0.0192) 1.00 (0.0107) 1.38 (0.0083) 1.22 (0.0064) 2.06 (0.0250) 1.59 (0.0040) 1.31 (90.0387) 1.19 (0.0018) 2.29 (0.0030) 2.21 (0.0020)
1.08 (0.0436) 1.03 (0.0405) 0.77 (0.4891) 0.10 (0.8617) 0.13 (0.6997) 0.34 (0.4900) 0.25 (0.6364) 0.08 (0.9229) 0.27 (0.7398) 0.25 (0.4740) 0.48 (0.2745) 0.24 (0.2590) 0.05 (0.8897) 0.84 (0.0745) 1.4 (0.0413) 0.78 (0.3243) 0.06 (0.9129) 0.10 (0.8948) 0.14 (0.7957) 2.47 (0.0114) 1.97 (0.0129) 1.41 (0.0178) 2.17 (0.0003) 3.58 (0.0005) 1.75 (90.0019) 2.36 (0.0035) 1.19 (0.0068) 1.55 (0.0073) 1.23 (90.0096) 2.62 (0.0124) 1.06 (0.0171) 1.50 (0.03589) 1.31 (0.433) 1.60 (0.0090) 1.01 (0.0081)
(b) log2-fold change (p value) genes repressed
SPIONs-COOH
SPIONs
SPIONs-NH2
MGAT2
1.10 (0.0014)
1.20 (0.0008)
2.10 (
