Magnetic nanoparticles were synthesized, and stored in the dark at 4 °C. Dulbecco’s modified Eagle’s medium (DMEM) was purchased from GIBCO Company. Penicillin and streptomycin were purchased SIGMA Company. TNF-α ELISA Kit was purchased from BOSTER Company. All of other chemicals were of reagent grade and were used as received without further purification. Double distilled water was used for all the experiments.

Fe₃O₄ nanoparticles were synthesized by chemical coprecipitation of Molday 22 . Typically, a solution of mixture of FeCl₃ and FeSO₄ (molar ratio 2:1) was prepared under N₂ protecting, followed by the slow addition of enough ammonia aqueous solution with vigorous stirring for 30 min. The black Fe₃O₄ precipitates were obtained and washed immediately with distilled water for five times by magnetic separation. The final precipitates were dispersed in distilled water with concentration of 0.128 M and pH 3.0, and oxidized into more stable MNPs (γ-Fe₂O₃) by air at the temperature of 90 °C.

According to the process described in the literature 23 24, MNPs were coated with DMSA and GLU. Finally stable aqueous sol DMSA-MNPs (DMSA-Fe₂O₃), and GLU-MNPs (GLU-Fe₂O₃) were obtained. Similarly, APTS-MNPs (APTS-Fe₂O₃) were prepared according to literature 25 . The part of above samples was dried into powder at room temperature under vacuum.

The particle size and morphology of the coated MNPs was determined by transmission electronic microscopy (TEM, JEOL, JEM-200EX). Powder X-ray diffraction (XRD, Rigaku, D/Max-RA, λ = 1.5405 × 10⁻¹⁰ m, CuK) was used to determine the crystal structure of MNPs. Surface charge measurements were performed with a zeta potential analyzer (BECKMAN, Delsa 440SX). The magnetic measurements were carried out with a vibrating sample magnetometer (VSM, Lakeshore 7407). Fourier transform infrared (FTIR) spectroscopy measurements were performed on a Bruker Fourier transform spectrometer model VECTOR22 using KBr pressed discs.

Sprague-Dawley (SD) rat aortic SMCs were grown from explants of normal SD rat aorta fragments. Cells were further cultured in Dulbecco’s modified Eagle medium (DMEM) containing 15% fetal bovine serum (FBS), penicillin (100 μg/mL), and streptomycin (100 μg/mL), in 5% CO₂chamber.

All the coated MNPs was sterilized with filter-film (pore size, 0.22 μm) and sonicated before dilution into DMEM culture medium to ensure even particle suspension. Then, the MNPs were diluted with DMEM at different concentration and added into the plates in triplicate for a further specified time after the normal medium was removed. All control cells were cultured in the absence of any particles. Every experiment was repeated at least three times independently.

Cellular uptake of MNPs was evaluated according to the method of Petri-Fink 26 . The supernate of cells on the 6-well plates was removed and cells were thoroughly washed with PBS and resuspended in 2 M HCl (1 mL/well of a 6-well plate) at 37 °C for 2 h. The protein concentration of mixture was determined at 280 nm by Ultraviolet Visible Spectrophotometer (UV–vis). 1 mL of 5% solution of K₄[Fe(CN)₆ in H₂O was added, and the absorbance of samples was read after 10 min at 690 nm. A standard curve of an aqueous FeCl₃ · 6H₂O solution was treated in the same conditions to quantify the amount of cellular uptake of MNPs. The cellular uptake of MNPs was expressed at the amount of Fe₂O₃ (weight, μg) per milligram of protein.

The SMC incubated with DMSA-MNPs for 24 h were washed with PBS and fixed in 4% glutaraldehyde solution for 1 h at 4 °C. The cells were postfixed in 1% osmium tetroxide for 1 h at room temperature and washed. Then, cells were scraped and concentrated in 2.5% agar in 0.05 M cacodylate buffer. The obtained samples were then treated with 2% uranyl acetate solution for 1 h and subsequently dehydrated by means of ethanol/water solutions, with increasing ethanol content and embedded in epoxy resin 27 . The samples were cut at 70 nm (ultrathin sections) with an ultramicrotome. Ultrathin sections were transferred to the 300 mesh copper grid and stained with 5% uranyl acetate. The copper grid was observed on a transmission electron microscope (TEM, HITACHIH-600) at 80 kV.

After incubation for a period of time, cell culture supernate was collected and centrifuged at 8,000×gfor 30 min to remove cell debris and nanoparticles. Tumor necrosis factor α (TNF-α) protein concentrations in the supernate were measured using an ELISA kit (RAT TNF-α, ELISA KIT, BOSTER) according to the manufacturer’s instructions.

Viability of SMC was determined by using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide) assay. After incubation the supernate was removed and 200 μL MTT solution (0.5 mg/mL in DMEM medium) was added at 37 °C for 2 h. Then, cells were rinsed two times with PBS and 150 μL extracting solution (0.04 M HCl in isopropanol) was added to each well of 96-well plates. The plates were placed for 15 min at ambient temperature to dissolve the dyes and the dye extract was transferred to 96 well Elisa plates. Absorbance was assayed at 570 nm by Ultra Microplate Reader ELX808 IU (Bio-Tek) and the cell viability was expressed in percent based on the control.

Magnetic nanoparticles were synthesized by chemical coprecipitation and stable magnetic fluid was obtained via coating by DMSA, APTS, and GLU. The morphology and structure of particles were observed by TEM, and the images indicate that most of the particles are quasi-spherical and with an average diameter of 10 nm. The TEM image and size distribution of particles are shown in Fig. 1 The same morphology and size distribution can avoid the difference of size effect. The phase identification of the uncoated MNPs was performed by XRD. As shown in Fig. 2, the result shows that the MNPs are inverse cubic spinel structure 28 . Compared with the theoretical values, the reduction of d-spacing obtained experimentally is due to the lattice constrictions for nanosized particles 25 .

Fourier transform infrared spectroscopy was used to confirm that the DMSA, APTS, and GLU were successfully coated on the nanoparticles. The FTIR spectra of MNPs, DMSA-MNPs, APTS-MNPs, and GLU-MNPs are shown in Fig. 3All samples show broad band at 3400 cm⁻¹indicative of the presence of –OH groups on nanoparticles surface. Compared with the uncoated MNPs, all the coated MNPs possess absorption bands in 2956 and 2922 cm⁻¹due to stretching vibration of C–H bond. In addition, for DMSA-MNPs, the band in 1715 cm⁻¹appears due to stretching vibration of C=O bond, which reveals the existence of DMSA. For APTS-MNPs, bands in 1091 and 1051 cm⁻¹are attributed to stretching vibration of C–N bond and Si–O bond, band in 932 cm⁻¹is attributed to bending vibration of –NH₂. In the spectrum of GLU-MNPs, the bands in 1091 and in 932 cm⁻¹are also observed because the existence of C–O and –NH₂group, which is the strong evidence that GLU is bonded to the surface of the nanoparticles.

Zeta (ξ) potential measurement as a function of pH has been performed to confirm the surface charge properties and the presence of DMSA, APTS, and GLU on the surface of MNPs. Figure 4shows that the DMSA-MNPs have high negative potential at the pH range, while the APTS and GLU-MNPs are positively charged at lower pH and negatively at higher pH and their isoelectric points (IEP) are 7.2 and 7.9, respectively. The difference of charge properties of them attribute to the ionization of the functional groups, such as –COOH, –SH and –NH₂at different pH. According to results, we can make sure that the surface charge property is negative for DMSA-MNPs, positive for APTS-MNPs, and nearly neutral for GLU-MNPs in the physicochemical states (pH 7.4).

The magnetic property of the MNPs was measured by VSM. Figure 5shows the magnetization of the coated MNPs as a function of an external field at ambient temperature. The three of MNPs possess similar magnetization loops and saturation magnetization (50 emu/g) because of the same structure and size distribution. The synthesized MNPs also show a superparamagnetic behavior, as evidenced by zero coercivity and remanance on the magnetization loops.

The cellular uptake amount is counted based on the Fe₂O₃per milligram protein that released from the cells. For the DMSA-MNPs, the cellular uptake of MNPs by SMC increases with increasing concentration from 0.001 to 0.5 mg/mL in DMEM, as shown in Fig. 6a. The uptake also shows the same behavior with increasing of incubation time from 24 to 72 h when the concentration of DMSA-MNPs is 0.1 mg/mL (Fig. 6b). Briefly, the cellular uptake shows a clear dose and incubation time dependent patterns under the experiment conditions.

The cellular uptake indicates a function of surface properties of MNPs. From Fig. 6c, there is obvious difference among the three types of MNPs at the same concentration (0.1 mg/mL) for the same incubation time (24 h). The uptake amount of APTS-MNPs, GLU-MNPs, and DMSA-MNPs are 3.72, 4.60, and 8.98 μg per milligram protein, respectively. We infer that the different surface properties, such as charges of surface molecules, result in the different affinity with SMC 2729 30 31 32. According to the results, it is promising to facilitate or alter uptake of SMC by altering the surface properties of MNPs.

The thin section TEM images indicate that the DMSA-MNPs are incorporated into SMC after 24 h of incubation at 0.1 mg/mL of concentration. As shown in Fig. 7, the MNPs are clearly visible and distinct from the cellular matter because of their high electron density. The particles or their aggregates are distributed outside, on the surface of the membrane and inside of cell. We infer that the nanoparticles or their aggregates are absorbed on the membrane due to their small size effect, and incorporated by endocytosis vesicles through the deformation of the membrane, and then dispersed in cytoplasm of SMC. We can observe that the particles are swallowed by lysosome in cytoplasm from Fig. 7b and c. However, the fate of MNPs during cellular degradation is a key and unknown question.

Tumor necrosis factor α is a polypeptide cytokine that promotes antitumor and immune responses 33 , also called pro-inflammatory cytokine. For SMC, TNF-α is released from cells when the cells are damaged, and then the free TNF-α will promote the inflammatory damage of cells inversely. The supernate of SMC incubated with DMSA-MNPs under the same experiment conditions was collected and mixed. Then, the TNF-α was assayed by ELISA. The results were shown in Fig. 8 The level of TNF-α is normal compared with control, which means that DMSA-MNPs did not elicit the production of the pro-inflammatory cytokine TNF-α. Therefore, DMSA-MNPs cannot lead to SMC inflammatory damage in vitro. The other two types of MNPs show the same results (the data not shown). In conclusion, the three types of MNPs display the biocompatibility with SMC. Using our chosen endpoints, SMC do not damaged by the MNPs.

The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide) assay is a simple nonradioactive colorimetric assay to measure cell cytotoxicity, proliferation, or viability 34 . The active SMC is able to convert this dye into a water-insoluble dark-blue formazan by reductive cleavage of the tetrazolium ring 35 . Formazan crystals, then, was dissolved in acidification isopropanol by measuring the absorbance of the solution at 570 nm, and the resultant value is related to the number of living cells.

The viability of SMC is apparently decreased with the increasing of the concentration and incubation time of DMSA-MNPs compared with the control, as shown in Fig. 9a and b. The data were analyzed by statistical method and the result indicates there is statistical difference when the concentration of magnetic nanoparticles is more than 0.1 mg/mL (p < 0.05). Here, the viability means the activity, the cytotoxicity and the number of SMC. The decrease of viability may be resulted from the damage, proliferation inhibition of SMC by DMSA-MNPs. Considering the TNF-α level, we can infer that the decrease of viability is caused from proliferation inhibition of SMC by DMSA-MNPs.

However, the viability of SMC has not shown obviously statistical difference when incubated with three types of MNPs at the same concentration (0.1 mg/mL) and incubation time (24 h), which demonstrates that the properties of MNPs (used in the experiments) hardly have effect on the viability of SMC. The data are shown in Fig. 9c.