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Introduction

Nanoparticles (NPs) have been widely applied in the field of healthcare owing to their unique physicochemical characteristics including large surface-to-volume ratio and small size (1,2). Superparamagnetic iron oxide nanoparticles (SPIONs) have gained attention in medicine as a result of their excellent magnetic properties and low cytotoxicity (3). SPIONs are clinically approved as magnetic resonance imaging contrast agents and have also been used in hyperthermia treatment, chemotherapy and drug delivery (4–7). The biomedical applications of NPs require a complete understanding of their interaction with biological systems. The cytotoxicity of SPIONs remains unclear. For instance, carboxydextran-coated SPIONs did not affect the proliferation and viability of murine macrophages, but dextran-stabilized SPIONs could alter endothelial integrity and function (8,9). Therefore, it is necessary to further assess the biological toxicity of these NPs. The accumulation of iron oxide NPs and release of free iron from the magnetite core may damage iron homeostasis in cells, resulting in inflammation, DNA damage or other toxin responses (10,11). Furthermore, mitogen-activated protein kinases (MAPK) are induced in cells following exposure to NPs (12).

It is well accepted that NPs can deliberately access the vascular system via injection in the form of nano-medicine (13). Monocytes are the portal of NPs into the human body; they develop into macrophages or dendritic cells and are also involved in various diseases and tissue homeostasis (14). Although certain studies have evaluated the toxic effects of iron oxide NPs (15–17), the limited immune toxicity data and its underlying signaling mechanism require further investigation. It is preferable to select human primary cells for studying the bio-safety of NPs, as they are closer to the in vivo condition than non-human cells or immortalized cell lines (18). In the present study, therefore, the effects of Dex-SPIONs on certain key activities of human primary monocyte cells, including cell uptake, viability, pro-inflammatory cytokines (IL-1β and TNF-α) and the potential signaling pathways were focused on.

Materials and methods

Characterization of Dex-SPIONs

Dex-SPIONs were synthesized following a previously described method (19). Briefly, a mixture of 2.508 g dextran T70 (Mw: 70 kDa, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and 3.044 g iron (III) chloride hexahydrate (Shanghai Aladdin Bio-Chem Technology Co., Ltd., Shanghai, China) was dissolved in 20 ml deionized water and placed in a three-neck flask equipped with a mechanical stirrer. A freshly prepared aqueous solution containing 1.27 g of ferrous chloride tetrahydrate (Shanghai Aladdin Bio-Chem Technology Co., Ltd.) dissolved in 2 ml deionized water was added to the mixture above. While being rapidly stirred, 20 ml 7.5% ammonium hydroxide solution was added into the mixture under argon protection. Subsequently, the suspension was heated to 75°C and maintained at this temperature for 30 min while stirring constantly. The black suspension was cooled and centrifuged at a speed of 400 × g for 15 min at 4°C to separate large particles. The ammonium chloride along with excess ammonia and dextran was removed by dialysis using a citrate buffer (0.01 mol/l; Shanghai Aladdin Bio-Chem Technology Co., Ltd.). Characterization of Dex-SPIONs was performed using TEM (Jeol2100f; JEOL, Ltd., Tokyo, Japan). Dynamic light scattering was used to analyze the ζ-potential and hydrodynamic size (Zeta Sizer, Malven Nano ZS90; Malvern Panalytical Ltd., Malvern, UK). The initial concentration of the NPs was determined by quantitative prussian blue assay.

Isolation of human monocyte cells

The present study was approved by the Institutional Review Board of the West China Second University Hospital of Sichuan University (Chengdu, China). Peripheral blood (20 ml) mononuclear cells were obtained from human buffy coat residues (Chengdu Blood Center, Chengdu, China) using gradient centrifugation (400 × g for 30 min at 16°C). Written informed consent was obtained from all patients (n=15; 7 females and 8 males; age, 38±3 years) and samples were collected between October and December 2016). Human monocyte cells [Cluster of differentiation (CD)14+ cells] were purified using magnetic beads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) according to the manufacturer's protocol. Cells with >98% viability were obtained and the purity was 95–98% (data not shown), verified respectively by cytospin preparations and flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA), as previously described (20).

Cell culture

Monocytes were seeded in 24-well plates in RPMI 1640 culture medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS; Invitrogen; Thermo Fisher Scientific, Inc.) and antibiotics (1% solution of penicillin 100 µg/ml and streptomycin 100 µg/ml; Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and incubated at 37°C, 5% (v/v) CO2, and 95% relative humidity for 1 h (to allow enough time for attachment of monocytes). The medium was discarded following adhesion of the cells to the plate. Dex-SPIONs were mixed with RPMI 1640 medium #at a concentration of 13.2 mg/ml to form the stock concentration from which the working concentration was derived. Subsequently, the cells were incubated with Dex-SPIONs (20 or 100 µg/ml) in fresh pre-warmed medium for 24 h.

Transmission electron microscopy (TEM)

Following 24 h of incubation, cells were collected for TEM analysis as previously described (21). Briefly, monocyte cells were washed thrice with PBS and fixed with 2.5% glutaraldehyde (Sigma-Aldrich; Merck KGaA) in PBS at 4°C overnight. The fixed cells were dehydrated and subsequently embedded with a layer of Spurr epoxy resin at 60°C for 24 h (Polysciences Inc., Warrington, PA, USA). Ultrathin sections (60–90 nm) were collected on 200 mesh copper grids prior to staining with uranyl acetate and lead citrate (Polysciences, Inc.) for 30 min at room temperature. Samples were analyzed by TEM (Hitachi, Ltd., Tokyo, Japan; H600IV). Micrographs were processed using Adobe Photoshop CS5 software (Adobe Systems, Inc., San Jose, CA, USA).

Neutral red assays

Cells were seeded into 96-well plates (1.5×105 cells/well) and incubated with Dex-SPIONs (10–100 µg/ml) for 24 h at 37°C. Subsequently, the cells were treated with Neutral Red Cell Proliferation and Cytotoxicology Assay kit (Beyotime Institute of Biotechnology, Shanghai, China) following the manufacturer's protocol and the absorbance was measured at a wavelength of 540 nm using microplate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Flow cytometry

Cells were collected and washed with PBS. Subsequently, cell suspensions (5×105 cells) were stained with anti-CD14 (1:100) in 100 µl PBS with 5% FCS (cat no. 301829; Biolegend Inc., San Diego, CA, USA) for 30 min at room temperature. An annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit (Beijing Solarbio Science & Technology, Co., Ltd., Beijing, China) was used according to the manufacturer's protocol. Cells were subsequently analyzed within 1 h by flow cytometry (BD Biosciences). Flow cytometric data were processed using FlowJo v.8.5.2 (Flowjo, LLC, Ashland, OR).

Quantification of cytokines

Cell supernatants were recovered following 24 h of incubation. The levels of interleukin (IL)-1β (cat. no. P01584) and tumor necrosis factor (TNF)-α (cat. no. P01375) were evaluated using commercially available human ELISA kits (RayBiotech, Inc., Norcross, GA, USA) according to the manufacturer's protocol and the absorbance was measured at 450 nm using a microplate reader (Bio-Rad Laboratories, Inc.). Lipopolysaccharide (LPS; 1 µg/ml; Merck KGaA) was used as a positive control.

To further assess the effects of Dex-SPIONs on the MAPK pathway, cells were incubated with the MAPK inhibitors SB 203580 (for p38; Tocris Bioscience, Bristol, UK), SP 600125 (for JNK; Absin Bioscience Inc., Shanghai, China) and PD98059 [for extracellular regulated kinase (ERK)1/2; Cell Signaling Technology, Inc., Danvers, MA, USA; 10 µM in all cases] for 1 h at 37°C prior to the addition of Dex-SPIONs. The cells were cultured for 24 h at 37°C and cell supernatants were collected to analyze cytokine concentrations.

Western blot analysis

Cells were seeded in 24-well plates (1.2×106 cells/well) in the presence of Dex-SPIONs (100 µg/ml) for 24 h. Cells were harvested, pelleted (centrifugation at 300 × g for 5 min at room temperature) and lysed by adding Western-IP lysis buffer (Beyotime Institute of Biotechnology). Protein concentrations were determined with a bicinchoninic acid protein assay. An equal amount of protein (35 µg) for each sample was subjected to SDS-PAGE (10% and 12% separation gels) and transferred onto polyvinylidene fluoride (PVDF) membranes. Membranes were subsequently blocked with 5% non-fat dry milk for 1 h at room temperature and incubated with anti-GAPDH (1:1,000; cat. no. 97166; CST Biological Reagents Co., Ltd., Shanghai, China), anti-p38 (1:1,000; cat. no. 9212; CST Biological Reagents Co., Ltd.), anti-phosphorylated (p)-p38 (1:1,000; cat. no. 9211; CST Biological Reagents Co., Ltd.), anti-JNK (1:1,000; cat. no. 9252; CST Biological Reagents Co., Ltd.), anti-p-c-Jun N-terminal kinase 1 (JNK; 1:1,000; cat. no. 9251; CST Biological Reagents Co., Ltd.), anti-ERK (1:1,000; cat. no. 4695S; CST Biological Reagents Co., Ltd.) and anti-p-ERK (1:1,000; cat. no. 9101S; CST Biological Reagents Co., Ltd.) overnight at 4°C. The PVDF membranes were incubated with an appropriate horseradish peroxidase-conjugated secondary antibody (1:5,000; cat. no. AP132P; Merck KGaA) for 1 h at room temperature, then protein bands were visualized with a super signal chemiluminescence (ECL) kit (Beyotime Institute of Biotechnology). The densitometric analysis of western blot was performed using Chemi-Doc (Quantity One 4.6.8; Bio-Rad Laboratories, Inc.).

Statistical analysis

Results are presented as the mean ± standard error of mean and the data conformed to Gaussian distribution. Statistical analyses were performed using SPSS 11.0 (SPSS Inc., Chicago, IL, USA). Significant differences were evaluated by one-way analysis of variance followed by Dunnett's post-hoc test (at least three experiments). P<0.05 was considered to indicate a statistically significant difference.

Results

Characterization of Dex-SPIONs

The size of the Dex-SPIONs were first characterized prior to using them in the subsequent experiments. The mean sizes of the NPs were calculated using Adobe Photoshop CS5 following appropriate calibration. Dynamic light scattering was also used to analyze the ζ-potential and hydrodynamic size. As demonstrated in Fig. 1, Dex-SPIONs were observed to be an average of 7 nm in diameter and demonstrated to be spherical in shape. Furthermore, Dex-SPIONs exhibited a negative potential (−11 mV) in cell culture medium and the hydrodynamic size was ~63 nm (Table I). SPIONs are divided into three main categories based on their hydrodynamic diameter; this NP belongs to the standard size SPIONs, which have an overall diameter of 50–150 nm (22).

Figure 1. Transmission electron microscopy of dextran coated-superparamagnetic iron oxide nanoparticle (scale bar, 20 nm).

Table I. Characterization of the particle parameters of Dex-SPION.

Table I.

Characterization of the particle parameters of Dex-SPION.

Nanoparticle	Hydrodynamic size (nm)	ζ-potential (mV)	Initial concentration (mg/ml)
Dex-SPION	62.8	−11	13.2

[i] Dex-SPION, dextran coated-superparamagnetic iron oxide nanoparticle.

Cellular uptake and localization of Dex-SPIONs

To follow the intracellular localization and internalization of Dex-SPIONs, TEM was used to observe the cellular response (Fig. 2). Monocytes treated with Dex-SPIONs (100 µg/ml) exhibited vacuoles with highly electron-dense Dex-SPIONs (Fig. 2B and D) compared with the control group (Fig. 2A and C). Following careful observation of the TEM images, Dex-SPIONs were demonstrated to either be engulfed by monocytes in phagosomes or freed in the cytoplasm (Fig. 2B). There were bulky vacuoles present and a number of pseudopodia from the cell membrane (Fig. 2D). Furthermore, side-scatter light of flow cytometry representing cell granularity was increased following Dex-SPIONs exposure and it also indicated that Dex-SPIONs were phagocytosed by monocytes (Fig. 2E). These results definitively demonstrated an interaction between these NPs and cells.

Figure 2. Cellular uptake of Dex-SPION. Cells were incubated with Dex-SPION (100 µg/ml) for 24 h. The cells were collected and examined by transmission electron microscopy. (A) Human monocyte cells without any treatment. (B) Human monocyte cells treated with Dex-SPION (scale bar: 0.5 µm). Dex-SPIONs were either in phagosomes (black arrows) or free in the cytoplasm (transparent arrows). (C) Higher magnification of cells in black boxed areas (scale bar: 0.2 µm) in A and (D) in B. Large vacuoles (black arrows) and pseudopodia (transparent arrows) of the cell membrane. (E) Cells were collected and detected by flow cytometry. Mean values represent mean fluorescence intensities of side-scatter light. Dex-SPION, dextran-coated superparamagnetic iron oxide nanoparticle.

Effect of Dex-SPIONs on the cell viability

In order to determine whether the Dex-SPIONs that enter the cells evoke a cell viability response, the number of Annexin V+/PI+ (apoptotic or dead) or Annexin V−/PI− (live) cells following Dex-SPIONs exposure at concentrations ranging from 10 to 100 µg/ml was determined. It was demonstrated that Dex-SPIONs exhibited no significant effects on cellular apoptosis and viability (Fig. 3A and B). In addition, cell viability was further analyzed by neutral red assays. As illustrated in Fig. 3C, Dex-SPIONs did not influence the viability of cell even at 100 µg/ml compared with the control group following 24 h of incubation.

Figure 3. Effect of Dex-SPION on cell viability. (A) Cells were treated with Dex-SPION (10–100 µg/ml) for 24 h, stained with Annexin V-FITC/PI and detected by flow cytometry. (B) Graphical representation of these results. (C) Cell viability was analyzed using a neutral red assay. Data were presented as the mean ± standard deviation, n=3. Dex-SPION, dextran coated-superparamagnetic iron oxide nanoparticle; FITC, fluorescein isothiocyanate; PI, propidium iodide.

Effect of Dex-SPIONs on pro-inflammatory cytokines production in human monocyte cells

To examine the activation of human monocyte cells, an ELISA was used to analyze relevant parameters. The expression level of IL-1β was significantly improved following Dex-SPIONs treatment in a concentration-dependent manner (P<0.05 and P<0.01; Fig. 4A). Dex-SPIONs treatment significantly increased the production of TNF-α in the group at 100 µg/ml (P<0.01; Fig. 4B). In addition, dextran did not affect the secretion of cytokines in cells (data not shown). LPS (1 µg/ml) was used as a positive control.

Figure 4. Proinflammatory cytokines production induced by Dex-SPION. Cells were incubated with Dex-SPION for 24 h. (A) The level of IL-1β. (B) The level of TNF-a. Data were presented as the mean ± standard deviation, n=3. *P<0.05, **P<0.01 vs. the control; LPS (1 µg/ml), used as positive control. IL, interleukin; TNF, tumor necrosis factor; LPS, lipopolysaccharide.

Effect of Dex-SPIONs on the MAPK signaling pathway in human monocyte cells

To further investigate the underlying signaling pathway of the activation of monocytes induced by Dex-SPIONs, MAPK p38, JNK, ERK proteins and their respective inhibitors were used. Dex-SPIONs treatment significantly increased the phosphorylation levels of MAPK p38, JNK and ERK compared with the control group (P<0.05; Fig. 5A-C). IL-1β production only decreased significantly in the presence of SP 600125 (JNK inhibitor) or PD 98059 (ERK inhibitor; both P<0.01; Fig. 5D). However, co-treatment of Dex-SPIONs with inhibitors significantly reduced the expression level of TNF-α (P<0.05; Fig. 5E). Additionally, the inhibitors themselves did not modify cytokine expression in cells (data not shown).

Figure 5. Dex-SPION induced the activation of MAPK p-p38, p-JNK and p-ERK in human monocyte cells. Monocytes were incubated with Dex-SPION (100 µg/ml) for 24 h or cells were treated with the MAPK inhibitors SB 203580 (for p38), SP 600125 (for JNK) and PD98059 (for ERK1/2; 10 µM in all cases) for 1 h prior to the addition of Dex-SPION. Cells were subsequently prepared for western blotting and ELISA analyses. (A) p-p38/p38 expression. (B) p-JNK/JNK expression. (C) p-ERK/ERK expression. (D) The expression level of IL-1 β. (E) The expression level of TNF-α. Data were presented as mean ± standard deviation, n=3. #P<0.05 vs. control; *P<0.05, **P<0.01 vs SPION. IL, interleukin; TNF, tumor necrosis factor; ERK, extracellular signal regulated kinase; p-JNK, phosphorylated c-Jun N-terminal kinase 1; MAPK, mitogen-associated protein kinase; Dex-SPION, dextran coated-superparamagnetic iron oxide nanoparticle.

Discussion

SPIONs have enormous potential for biomedical applications, but there remains a gap in the knowledge about their reactions with the human immune system. In the present study, Dex-SPIONs were demonstrated to be uptaken by human monocyte cells and the cells were subsequently activated through the MAPK signaling pathway. To a certain extent, the results of the present study offered a better understanding and more convincing experimental evidence for the issue of SPIONs safety.

Monocytes are crucial innate immune cells, professional phagocytes whose main function is to recognize, ingest and degrade foreign invaders including bacteria and NPs (23). The results of the present study demonstrated that Dex-SPIONs were internalized by human monocyte cells and located in phagosomes or freed in the cytoplasm, which is similar to other studies (24,25). Certain spherical vesicles resembling lysosomes fully packed with Dex-SPIONs in the cytoplasm were observed. It has been demonstrated that most endocytic routes of NPs are concentrated in the lysosomes, which leads to lysosomes being the common site for accumulation of NPs (26).

It is well documented that the cellular uptake of NPs have a crucial role in influencing certain cellular functions, including cell viability and inflammation (27). Whether SPIONs are cytotoxic or not is controversial. Certain studies have demonstrated SPIONs to be safe, while others have demonstrated SPIONs toxicity (18,28,29). It may depend on the size of the NPs, any modifications or cell types studied. Coating is vital to stabilize SPIONs as it can decrease the toxicity of SPIONs by preventing aggregation and leakage of free iron ions. Dextran has excellent biocompatibility and has been used in the clinic for a long time, but it has been reported to modify cell-cell interaction and cell viability (30). However, the results of the present study demonstrated that dextran had no impact on the production of cytokines. Furthermore, Dex-SPIONs showed no cytotoxicity and can't increase in apoptosis in human monocyte cells. These results indicated that these NPs have a good biocompatibility.

Bulky vacuoles and a number of pseudopodia of the cell membrane were observed, indicating the potential activation of human monocyte cells following Dex-SPIONs exposure. When undergoing activation, monocytes can secrete various pro-inflammatory cytokines, which have an important role in immune and inflammatory responses. Of these cytokines, IL-1β and TNF-α are the commonly used markers of monocytes activation (31). To further evaluate the activation of cells, the expression levels of IL-1β and TNF-α were analyzed. The production of IL-1β and TNF-α were enhanced following Dex-SPIONs treatment. In one case, bare-iron oxide NPs increased the secretion of TNF-α as well as the generation of reactive oxygen species in RAW 264.7 cells (32). It is noteworthy that cytokines, in particular pro-inflammatory cytokines can be useful tools in evaluating immunotoxicity of NPs (33). According to the literature and the results of the present study, it was inferred that Dex-SPIONs may induce immunotoxicity of monocytes by enhancing the production of pro-inflammatory cytokines, but this conclusion requires further testing in vivo.

p38, JNK and ERK, which are all the members of the MAPK family, are involved in a number of functions, including inflammation, cytokine production, transcriptional and apoptosis (34). In order to prove the potential signaling mechanism of cell activation, the phosphorylation levels of p38, JNK and ERK were measured. It is well documented that the MAPK signaling pathway can be induced in response to a diverse range of extracellular stresses including chemicals and cytokines (35,36). Among these stresses, certain studies have reported that NPs can induce MAPK pathway activation, such as ZnO and CuO (37,38). Recently, Couto et al (39) demonstrated that iron oxide NPs could cause cytokine secretion and that the activation of MAPK p38 and JNK are involved in this effect. In the present study, Dex-SPIONs were verified to increase the expression levels of p-p38, p-JNK and p-ERK. Furthermore, MAPK inhibitors also inhibited the production of pro-inflammatory cytokines (IL-1β and TNF-α). Based on these results, it was hypothesized that iron oxide NPs-caused inflammatory responses that were associated with the MAPK signaling pathway. The data from the present study demonstrated that there is an interaction between human monocyte cells and Dex-SPIONs, and this should be considered when deciding their various applications in the biomedical field. Future studies in animal models should enhance understanding of the health effects of these nanoparticles and may guide the risk assessment of their applications.

In conclusion, it demonstrated that Dex-SPIONs were engulfed by human monocyte cells and displayed the ability to activate monocytes via the induction of the formation of pseudopodia and the production of pro-inflammatory cytokines. In addition, these inflammatory responses possibly depended on the activation of MAPK signaling pathway.

Acknowledgements

Not applicable.

Funding

The present study was financially supported by the National Natural Science Foundation of China (grant no. 81471848) and the National Key Basic Research Program of Chain (grant no. 2013CB933903).

Availability of data and materials

The analyzed data sets generated during the study are available from the corresponding author on reasonable request.

Authors' contributions

QW designed the study and performed experiments. TM and TF performed the western blot analysis. CY performed the flow cytometry analysis. YG provided the nanoparticles, directed the project and interpreted the data. HL supervised the project and contributed to the study design. QW and YG wrote the paper. All authors discussed the results and contributed to the manuscript.

Ethics approval and consent to participate

The present study was approved by the Institutional Review Board of the West China Second University Hospital of Sichuan University (Chengdu, China). Written informed consent was obtained.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References