DMSA-Coated Iron Oxide Nanoparticles Greatly Affect the Expression

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DMSA-coated Iron Oxide Nanoparticle Greatly Affect the Expression of Genes Coding Cysteine-rich Proteins by its DMSA Coating Ling Zhang, Xin Wang, Jinglu Zou, Yingxun Liu, and Jinke Wang Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.5b00161 • Publication Date (Web): 17 Sep 2015 Downloaded from http://pubs.acs.org on September 24, 2015

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Chemical Research in Toxicology

DMSA-coated Iron Oxide Nanoparticle Greatly Affect the Expression of Genes Coding Cysteine-rich Proteins by its DMSA Coating Ling Zhang1,2†, Xin Wang1†, Jinglu Zou1, Yingxun Liu1, Jinke Wang1,* 1 State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China 2 School of Biomedical Engineering, Hubei University of Science and Technology, Xianning 437000, China †

Authors contributed equally to this work.

*Correspondence should be addressed to Jinke Wang: Tel.: +86 25 83793620; fax: +86 25 83793620. E-mail address: [email protected]

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ABSTRACT: The dimercaptosuccinic acid (DMSA) was widely used to coat iron oxide nanoparticles (FeNPs); however, its intracellular cytotoxicity remains to be adequately elucidated. This study analyzed the differentially expressed genes (DEGs) in four mammalian cells treated by a DMSA-coated magnetite FeNP at various doses for different times. The results revealed that about one fourth of DEGs coded cysteine-rich proteins (CRPs) in all cells under each treatment, indicating that the nanoparticles greatly affected the expressions of CRP-coding genes. Additionally, about 26% of CRP-coding DEGs were enzyme genes in all cells, indicating that the nanoparticles greatly affected the expression of enzyme genes. Further experiments with the nanoparticles and a polyethyleneimine (PEI)-coated magnetite FeNP revealed that the effect mainly resulted from DMSA carried into cells by the nanoparticles. This study thus firstly reported the cytotoxicity of DMSA at the gene transcription level as coating molecules of FeNPs. This study provides new insight into the molecular mechanism why the DMSA-coated nanoparticles resulted in the transcriptional changes of many CRP-coding genes in cells. This study evokes attention toward the intracellular cytotoxicity of DMSA as coating molecule of nanoparticles, which has very low toxicity as orally administered antidote due to its extracellular distribution. Key words: magnetite nanoparticles; DMSA; coating; gene transcription; cysteine-rich proteins

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INTRODUCTION Nowadays, the iron oxide nanoparticles (FeNPs) have great potential usage in biomedicine and some have already been applied in clinics, such as magnetic resonance imaging (MRI) contrast enhancement, hyperthermia and drug delivery with tissue specificity.1-3 For these applications, the internalization of FeNPs into specific cells is the critical step.4 The bare FeNPs have hydrophobic surfaces with large surface area-to-volume ratios and a propensity to agglomerate.5 A proper surface modification allows FeNPs being hydrophilic, getting dispersibility and improving the efficiency of internalization. Thus, to improve biocompatibility and biodistribution and to hold the promise for clinical and biological application, FeNPs are often produced with various coating molecules, such as polyethylene glycol (PEG), meso-2,3dimercaptosuccinic acid (DMSA), dextran, dendrimers, polyphosphazene, polypyrrole, lipid, amino acids, and citrate. 6-14 Among multiple surface modifications, DMSA, a small organic molecule, has been widely used to form water-dispersible FeNPs. Besides improving the stability of FeNPs,15 it was reported that DMSA coating could improve the biocompatibility, internalization and biodistribution of FeNPs in vivo and in vitro. The DMSA coating on the surface of FeNPs plays an important role in monodispersibility and internalization efficiency as well as others surface modification.16, 17 It was observed that the coating of FeNPs with DMSA enhanced endocytosis by various cell types.17, 18 The previous studies also reported that the DMSA coating improved uptake efficiency significantly.17 It was reported that the DMSA-coated FeNPs produced relatively weak cytotoxic and no genotoxic effects, compared to uncoated FeNPs.9, 19 The DMSA-coated maghemite FeNPs were also shown to be safe, producing cytotoxic changes at levels of 100 µg/mL or higher, which is much higher than the intravascular concentration of Ferumoxtran-10 imaging agent in human bodies.20 Additionally, the in vitro and in vivo studies have demonstrated that the cell viability, differentiation and proliferation were not affected by the DMSA coating of maghemite or magnetite FeNPs.9, 21-23 Therefore, the DMSA-coated FeNPs are considered to be biocompatible FeNPs and with great potential clinical applications.9, 24 Since the DMSA-coated FeNPs have shown great promise for use as tools in a wide variety of biomedical applications, more and more studies have paid attention to assess the extent of any cytotoxicity related to these FeNPs prior to use in clinical treatments, especially at molecular level.25 The earlier literature and clinical evidence now indicate that DMSA shows the most promise for the better biocompatibility. It was verified that even up to 750 mM net DMSA has little to no measurable effect on cell function.16, 26 In fact, DMSA is used as orally administered metal chelating agent for treating young children and pregnant women with lead intoxication, receiving the approval of Food and Drug Administration of USA.27 DMSA has low toxicity compared to other dithiols due to its extracellular distribution.28 In fact, previous studies reported that DMSA alone showed low toxicity in various biological systems and was therefore considered as a biocompatible agent.29, 30 However, when used as a modification molecule, DMSA can be carried into the cells together with the internalization of FeNPs 9, and thus introduces a number of active thiol groups into cells. After the intracellular introduction, the biological effect of DMSA molecules on cells may be different from its extracellular distribution. The toxicity of FeNPs was found to be dependent on various factors such as types of surface-coatings or the breakdown products of FeNPs.16, 31 Although the previous studies examining the cytotoxicity of a range of DMSA-coated FeNPs generally found low or no cytotoxicity even at high exposure levels (>100 µg/mL),9, 21, 32-34 the influence of FeNPs with a proper surface coating on the cell morphology and function has been reported by previous studies.35, 36 For example, it was observed that the DMSA-coated FeNPs 4


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caused a concentration-dependent decrease in cell adhesion as well as a reduction in the viability of the PC12 pheochromocytoma clonal cell line.16 The DMSA-coated FeNPs also affected the release of inflammatory cytokines, induced cell adhesion molecules, promoted cell migration, and modulated the expression of cell adhesion molecules.21, 37 Nevertheless, as a whole, it is difficult to discriminate the effects of coating, physical morphology, and breakdown products of FeNPs (such as iron ions). Therefore, until now, very little is known about the biological effect of DMSA as a coating molecule of FeNPs, especially at the molecular level. To investigate the biological effects of FeNPs at the molecular level, we recently explored the effects of a DMSA-coated magnetite FeNP on the gene expression profiles of two mouse cell lines (RAW264.7 and Hepa1−6) and two corresponding human cell lines (THP-1 and HepG2) by using the GeneChip microarray. It was found that the nanoparticles changed the expressions of many genes in two mouse cell lines (RAW264.7 and Hepa1−6),38, 39 such as genes responsible for iron and osmosis homeostasis.38, 40 The nanoparticles also induced expressions of many apoptosis-related genes and apoptosis of human THP-1 cells.41 In scrutinized gene analyses, we found that many differentially expressed genes (DEGs) coded the cysteine-rich proteins (CRPs). Because the FeNPs used in these studies were coated with DMSA that contains abundant thiol groups, we reasoned that the expression change of these genes might be related with the DMSA internalized into cells with the FeNPs. In this study, to verify this hypothesis, we thoroughly analyzed the DEGs found in the four cell lines and identified all CRP-coding genes. We then performed functional annotation analysis to these genes for elucidating their molecular functions and the involved biological processes. Finally, we validated the speculation by detecting expressions of some genes in the RAW264.7 cells treated by the nanoparticles and a magnetite FeNPs coated with polyethyleneimine (PEI). This study thus found that the DMSA-coated FeNPs exerted extensive and significant effects upon the transcription of CRP-coding genes by their DMSA coating.

EXPERIMENTAL PROCEDURES Chemicals and Cells Two types of magnetite (Fe3O4) FeNPs, DMSA-coated FeNPs (DMSA-FeNPs) and PEI-coated FeNPs (PEI-FeNPs), were provided by Gu’s Laboratory.15, 42 The FeNPs were dissolved in water and filtered with 0.22-µm membrane for sterilization. The cytotoxicity of the DMSA-FeNPs at various concentrations to the multiple cell lines had been extensively investigated with the MTT assay in our previous study.38, 43 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), DMSA, nuclear fast red, paraformaldehyde, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), osmium tetroxide, propylene oxide, and glutaraldehyde were purchased from Sigma Aldrich (MO, USA). The potassium peroxydisulfate (K2S2O8), potassium ferrocyanide (KSCN), iron chloride hexahydrate (FeCl3), hydrochloric acid, ethanol, and methanol were purchased from Sinopharm Chemical Reagent Co. Ltd, China. Triazol reagent and Dulbecco’s modified Eagle’s medium (DMEM) cell culture medium were purchased from Invitrogen Gibco (Carlsbad, CA, USA). RAW264.7 cells were purchased from the China Center for Type Culture Collection (Shanghai, China). Identification and Functional Annotation of CRP-coding DEGs The gene expression profiles of mouse macrophage RAW264.7 and hepatoma Hepa1 −6 and human monocyteline THP-1 and hepatoma HepG2 treated by DMSA-FeNPs were analyzed with the Affymetrix MouseGenome 430 2.0 GeneChips microarrays and the Human Genome U133 Plus 2.0 GeneChips microarrays (Santa Clara) as previously described 40, 44. The genes with fold change ≥ or ≤ 2.0 were identified as DEGs. The gene annotation was performed by providing the Probeset IDs of the interested 5



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genes to the databases of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway web servers, respectively. Characterization of Iron Oxide Nanoparticles The size of FeNPs was analyzed with a transmission electron microscope (TEM) of JEM-2100. The hydrodynamic size and zeta potential of FeNPs were analyzed with a Zetasizer Nano ZS90 (Malvern, UK). The iron contents of FeNPs were analyzed with the thiocyanate colorimetric assay as previously described.43 A modified Ellman’s method, which was introduced to estimate free thiol groups,45 was used to measure the thiol groups on the surfaces of FeNPs. Briefly, 50 µL of FeNPs (~500 µg Fe/mL) was added 75 µL of Tris-HCl (pH 8.0), 5 µL of 0.01 M DTNB (Elman’s reagent) dissolved in methanol and 170 µL of methanol. After mixing, the mixture was incubated in an Eppendorf Thermomixer at 500 rpm at 37 ℃ for 4 h. The absorbance was read at 412 nm using a microplate reader (Synergy HT). The thiol content was determined according to standard curve prepared with a range of concentrations of DMSA (0.5 to 100 µg/mL). The mol of free thiol groups per gram of Fe (the detected iron content of FeNPs) was calculated by the formula of [(thiol content/iron content)/182.22]×2, in which 182.22 and 2 refer to the molecular weight of DMSA and the number of thiol groups in DMSA molecule, respectively. The unit of thiol and iron contents was microgram/milliliter (µg/mL). Treatment of Cells with Nanoparticles The RAW264.7 cells were cultured in the DMEM supplemented with 10% FCS, 100 units/mL penicillin, 100 µg/mL streptomycin, and 10 mM HEPES in a humidified 5% CO2 atmosphere at 37℃ in 75 cm2 flasks. To treat cells with the magnetite nanoparticles, cells were seeded at the density of 5×104 per mL in culture medium in plates and cultivated overnight. The culture medium was then replaced with fresh medium containing 50 µg/mL of FeNPs or 35.58 µg/mL DMSA. Cells were cultivated for an additional 24 h. Detection of Intracellular Uptake of Nanoparticles The internalization of FeNPs was detected with the Prussian blue staining as previously described.40 Cells treated with FeNPs or DMSA were washed three times with phosphate buffered saline (PBS), trypsinized and collected in 1.5-mL tube. Cells were precipitated by centrifugation and resuspended in 10 mM HEPES (pH7.9). Cells were then thoroughly sonicated with a cell cracker. The intracellular nanoparticles were separated from the cell lysate by using a magnet. The separated nanoparticles were used to measure the cellular iron content. The magnetic precipitate was resuspended with 5 M HCl and the iron content was analyzed using a thiocyanate colorimetric assay.40 Analysis of Gene Expression with Quantitative PCR The total RNA was prepared from cells with a Trizol reagent (Invitrogen) and analyzed with UV spectrophotometer and agarose gel electrophoresis. The total RNA (1 µg) was reversely transcribed into the complementary DNA (cDNA) using the RNA reverse transcriptase kit (AMV) Ver.3.0 (TaKaRa). The cDNA was analyzed with quantitative PCR (qPCR). The qPCR primers were shown in the Table S1 (Supporting Information). The qPCR reaction contained 1 µL of cDNA, 10 µL of the Fast SYBR Master Mix (Applied Biosystems, Foster City, CA, USA), and 200 nM forward and reverse primers in a final volume of 20 µL. The PCR reactions were run on a Step One Plus real-time PCR thermocyler (ABI) by using a program of 95°C for 10 min, 40 cycles of 95 °C for 15 s, and proper annealing temperature for 1 min. The melting curve was performed to confirm the amplification specificity. The fold change of detected gene was calculated by using ∆∆Ct method. 6


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RESULTS Identification of DEGs Coding Cysteine-rich Proteins To find all CRP-coding genes, we searched the NCBI database with the key words including zinc finger (zf), metal binding (mb), cysteine (ct), ring finger (rf), iron sulfur (and Fe-S) (is), metallothionein (mt), disulfide bond (db), and zinc-containing exopeptidases (ze), respectively. As a result, we obtained 3916 zfCRP, 3309 mbCRP, 1296 ctCRP, 632 rfCRP, 138 isCRP, 46 mtCRP, 23 dbCRP, and 19 zeCRP genes (for details see Table S2, Supporting Information). To find all DEGs that code CRPs, we compared these CRP-coding genes with the DEGs identified in the RAW264.7 cells that were treated by the DMSA-FeNPs at three doses (30, 50, and 100 µg/mL) for three times (4, 24, and 48 h).38, 39 As a result, we found that 114 zfCRP DEGs, 270 mbCRP DEGs, 97 ctCRP DEGs, 24 rfCRP DEGs, 7 isCRP DEGs, 6 mtCRP DEGs, 1 dbCRP DEG, and 1 zeCRP DEG (Table 1). We called these DEGs as CRP-DEGs. Removing redundancy, we found 365 CRP-DEGs (Table 1; for details see Table S3, Supporting Information). There were 166, 148 and 153 CRP-DEGs in the cells treated for 4 h, 24 h, and 48 h, respectively (Table 1). In comparison with total DEGs of these times (Table 1), the proportion of CRP-DEGs was 25.3% (4 h), 23.3% (24 h) and 23.9% (48 h), respectively, and averaged 24.2% (Table 1). These data demonstrate that about one fourth of DEGs were CRP-DEGs, indicating that the nanoparticles exerted significant effects upon the transcription of CRP-coding genes. Especially, the expression of 15 CRP-DEGs were commonly regulated in cells treated for various times (Figure 1A and B), which mainly belong to genes coding cysteine, metal binding and zinc finger proteins. Functional Annotation of CRP-DEGs To analyze the biological functions of CRP-DEGs, we first performed functional annotation to all 365 CRP-DEGs. The results revealed that these genes are involved in a total of 404 biological processes and 39 molecular functions with p < 0.05 and represented by at least 2 genes (for details see Table S3, Supporting Information). It was found that 10 biological processes related to metabolic process were highly enriched by the CRP-DEGs (Table 2). The metabolic process was most significantly enriched by as many as 218 CRP-DEGs (about 60% of total CRP-DEGs) (Table 2). These data demonstrate that most CRP-DEGs are related to cellular metabolism, indicating that the cell metabolism was greatly affected by the nanoparticles. The functional annotation also revealed that three biological processes related to cell death and apoptosis were also highly enriched by as many as 40 CRP-DEGs (Table 2). Additionally, the immune system process was highly enriched by the CRP-DEGs (Table 2). The GO analysis revealed that four molecular functions, including ion binding, cation binding, metal ion binding, and transition metal ion binding, were most significantly enriched by the CRP-DEGs (Table 2). The molecular functions of ion binding, cation binding, and metal ion binding were enriched by as many as 70% of CRP-DEGs (256) (Table 2). The molecular function of catalytic activity was enriched by 171 CRP-DEGs (47% of total CRP-DEGs). The hydrolase activity was enriched by 78 CRP-DEGs. These data indicate that the metabolic and apoptosis processes were greatly affected by the nanoparticles, and the molecular functions of most CRP-DEGs were related to metal ion binding and catalytic activity. The functional annotation indicated that the metabolic process, catalytic activity, cysteine-type peptidase activity, hydrolase activity, and ligase activity were significantly enriched by the CRP-DEGs (Table 2). Because these biological processes and molecular functions were mainly enriched by the genes coding enzymes, the enrichment of these GO terms suggests that the expressions of many enzyme-coding genes were significantly affected by the nanoparticles (Table 2). In total of 365 CRP-DEGs, there are as many as 112 enzyme-coding CRP-DEGs (for details see Table S3, Supporting Information). In the CRP-DEGs of 7



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cells treated with 4, 24, and 48 h, there were 54, 41, and 40 enzyme genes, respectively (for details see Table S4, Supporting Information). The functional annotation revealed that each of biological processes and molecular functions enriched by the CRP-DEGs contained many enzyme genes (Table 2). These data suggest that the nanoparticles exerted extensive effects on enzymes in cells. By these enzymes, the nanoparticles affected apoptosis and many metabolic processes. For example, in the top 15 biological processes enriched by the CRP-DEGs (Table 2), the metabolic processes contained 80 enzyme genes (Table 2), and the apoptosis-related processes commonly contained 17 enzyme genes (Table 2). To find the possible treating time-dependent effects of the nanoparticles, we performed functional annotation analyses to the CRP-DEGs of three times (Table S4, Supporting Information). The results revealed that the top enriched biological processes of cells treated for 4 h were greatly different from those of cells treated for two long times (Table 3). In the cells treated for 4 h, the biological possesses related to cell death and apoptosis were highly enriched by the CRP-DEGs; however, in the cells treated for 48 h, the biological possesses related to metabolic and biosynthetic process were highly enriched by the CRP-DEGs (Table 3). The functional annotation also revealed that four molecular functions, including ion binding, cation binding, metal ion binding, and transition metal ion binding, were consistently and most significantly enriched by the CRP-DEGs of cells treated by various times (Table 3). Moreover, the genes involved in these molecular functions occupied high percentage of total CRP-DEGs. For example, 64% (107/166) and 79% (121/153) of CRP-DEGs were enriched in these molecular functions in the cells treated for 4 h and 48 h, respectively. Additionally, the molecular function of catalytic activity was enriched by 85 and 74 CRP-DEGs in the cells treated for 4 h and 48 h, respectively (Table 3). To find the molecular functions of those commonly regulated CRP-DEGs at various treating times and their involved biological processes, we identified 87 CRP-DEGs with fold change ≥ 2 under at least two treating times (Figure 2A). Most of these genes were consistently upregulated or downregulated under various treatments (Figure 2A). The functional annotation of these genes revealed that the biological processes enriched by the consistently upregulated CRP-DEGs (cuCRP-DEGs) (Cluster a) are mainly related to the immune, taxis, locomotion, and responses; however, those enriched by the consistently downregulated CRP-DEGs (cdCRP-DEGs)(Cluster d) are mainly related to the metabolic and catabolic processes (Figure 2B). The molecular function analysis revealed that the binding, ion binding, and cation binding were significantly enriched by many of cuCRP-DEGs and cdCRP-DEGs (Figure 2B). In addition, the molecular functions of metal and transition metal ion binding were highly enriched by many of cuCRP-DEGs (Figure 2B). Anyway, there was significant difference between the molecular functions of cuCRP-DEGs and cdCRP-DEGs. For example, the chemokine activity was highly enriched by cuCRP-DEGs but catalytic activity was highly enriched by the cdCRP-DEGs (Figure 2B). In addition, two receptor binding GO terms, receptor and chemokine receptor bindings, were significantly enriched by cuCRP-DEGs, but protein, chromatin and nucleotide bindings were enriched by the cdCRP-DEGs (Figure 2B). Summarily, the cuCRP-DEGs are mainly related to immune and response, but the cdCRP-DEGs are mainly related to metabolic processes and catalytic activity. To further clarify the biological functions of CRP-DEGs, we performed a KEGG pathway analysis to the all 365 CRP-DEGs. The results revealed that 24 pathways are significantly (p

DMSA-Coated Iron Oxide Nanoparticles Greatly Affect the Expression

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