Characterization of Gel-FeS NPs
The gelatin stabilized FeS NPs were simply synthesized by co-precipitation method .The precursor containing ferrous ion of FeSO4 and Na2S are co-precipitate in nitrogen protected aqueous solutions under continuous stirring. Considering that FeS is unstable and easily oxidized in aqueous solution, we introduced biocompatible molecules gelatin into the reaction solution . The introduced gelatin not only improves the dispersion and stability of FeS NPs, but also improves the biocompatibility. Fig. 1A presents UV-Vis spectra of FeSO4, Na2S, gelatin and Gel-FeS NPs, there are negligible absorption of the individual solutions of FeSO4, Na2S and gelatin at longer wavelength region over 250 nm. After mixing these three reagents and reaction, the black and turbid product was observed, and it had a strong absorbance in the wavelength range of 200-600 nm. Satisfactorily, although the Gel-FeS NPs storage at 4 °С for a week, there was no notable change in the UV-Vis spectra of Gel-FeS NPs, implying the good physically and chemically stabilization of Gel-FeS NPs in aqueous solution. The morphology and size distribution of Gel-FeS NPs were measured with TEM and dynamic light scattering (DLS) analyzer. The TEM image shows that Gel-FeS NPs were well-dispersed with nonuniform size in aqueous solution (Fig. 1B), which was consistent with the size distribution of the Gel-FeS NPs in the range of 77.7 ± 16.4 nm (inset map).
In order to further analyze the molecular structure and chemical bond information of Gel-FeS NPs, FTIR spectra of Gel-FeS NPs and gelatin were compared in the Fig. 1C to study the functional groups differences. The peaks appeared at 3434 cm−1 assigned to the O−H stretching vibrations from adsorbed water and the stabilizer gelation. The abundant O−H bonds in gelatin were contributed to the formation of strong intermolecular hydrogen bonds between gelatin and FeS NPs, which improved the stability of Gel-FeS NPs effectively. Moreover, the absorption peaks around at 1645 cm−1 and 1107 cm−1 attributed to the −COO− and C−O−C stretching vibrations, respectively. The FTIR results indicated that the gelation with functional groups such as O−H and −COO− were tightly attached to the surface of FeS NPs. The modification of gelatin provided electrostatic repulsion and steric hindrance for FeS to avoid oxidation and aggregation . Subsequently, the XRD diffractogram of Gel-FeS NPs were analyzed (Fig. 1D), the two typical peaks at 2θ = 23.0° and 47.0° were indicative of FeS, and the diffraction peak at 2θ = 49.8° corresponding to (2 0 0) reflection of FeS, indicating the crystal form of FeS in the presence of gelatin . In addition, the peak appeared at 2θ = 36.4° corresponded to iron oxides, probably attributing to partial oxidation of Gel-FeS NPs. These results show that Gel-FeS NPs is synthesis successful.
Subsequently, the components and surface functional groups of the Gel-FeS NPs were characterized by XPS analysis. The XPS full scans spectrum of Gel-FeS NPs is shown in the Fig. 2A, demonstrating five obvious peaks at binding energy of 163.30, 399.14, 530.98, 582.20 and 710.57 eV, which were corresponded to S 2p, C 1s, N 1s, O 1s and Fe 2p orbital, respectively. The high resolution XPS spectra of C 1s could be resolved into three peaks at 284.81, 286.12, 287.87, 288.52 eV, indicating the presence of C−C, C−O−C, C=O and O−C=O bonds in Gel-FeS NPs, respectively (Fig. 2B). Besides, Fig. 2C shows the high resolution XPS spectra of Fe 2p, the peak centered at 710.92 eV assigned to Fe(Ⅱ)−S species, which were the main forms of elements Fe in Gel-FeS NPs. And the peaks at 719.04 and 724.33 eV were attributed to Fe(Ⅱ)−O and Fe(Ⅲ)−O, respectively, implying the Gel-FeS NPs partly oxidized during the preparation and storage process as article reported . Moreover, the peak at 161.50 eV was ascribed to FeS in high resolution XPS spectra of S 2p (Fig. 2D), corresponding to the analysis result of Fe 2p. Additionally, there were two peaks at 167.41 and 168.30 eV of SO42−, which indicated that some sulphate impurities could not be removed thoroughly.
Inhibitory Effect of Ferrous Ion on PRRSV Proliferation
First, we verified the antiviral activity of ferrous ion, and investigated whether the synthetic raw materials FeSO4, Na2S and gelatin themselves can inhibit the proliferation of PRRSV. MARC-145 cells were selected, cell viability was monitored by the MTT assay to estimate the cytotoxicity of such raw material and to define experimental conditions. Since the raw material ferrous sulfate is easily oxidized, we choose ferrous ammonium sulfate solution to explore the biocompatibility of ferrous ions. In this way, we cultured MARC-145 cells in the presence of raw materials at different concentrations. Cells cultured in the absence of raw material were used as the control experiment. as the data result in Fig. S1A, the cell survival rate was about 75% at the concentration of 60.0 µg/mL, in addition, the cell viability is greatly reduced after incubation with 80.0 µg/mL of ferrous ions. However, there is little cytotoxicity at the concentration of 40.0 µg/mL, the concentration at 40.0 µg/mL was selected to explore the antiviral activity of three synthetic raw materials by indirect immunofluorescence assay.
As depicted in Fig. S1B, the red fluorescence signal representing PRRSV N protein reduced significantly after treatment with FeSO4, whereas no visible differences were seen after treatment with Na2S and gelatin, indicating that the FeSO4 in synthetic raw materials of Gel-FeS NPs had antiviral activity on PRRSV. This result confirmed our hypothesis that ferrous ion has antiviral activity. And implied it is the Fe (II) in Gel-FeS NPs that plays a dominate role in inhibiting PRRSV proliferation.
Gel-FeS NPs Indicating Enhanced Biocompatibility
Cell viability was monitored by the MTT assay to estimate the cytotoxicity of the prepared particles and to define experimental conditions. After incubation with Gel-FeS NPs for 12, 24, 36, 48 h, the potential cytotoxicity of Gel-FeS NPs on MARC-145 cells was detected by MTT assay. As depicted in Fig. 3, negligible cytotoxicity of the Gel-FeS NPs treated MARC-145 cells can be observed at the concentration below 430.0 µg/mL for 12, 24, 36 and 48 h. Compared with the cytotoxicity of the raw materials for Gel-FeS NPs synthesis, the biocompatibility of Gel-FeS NPs was significantly improved. According to the results of inductively coupled plasma-mass spectrometry (the experimental results are not shown in figures), the mass fraction of Fe in Gel-FeS NPs is 23.5%. We converted the content of Fe in Gel-FeS NPs, 430.0 µg/mL of Gel-FeS NPs contains 101.0 µg/mL iron element. In contrast, the modification of gelatin greatly improves the biocompatibility of ferrous ions.
Gel-FeS NPs Exhibit Inhibitory Effect on PRRSV Proliferation
Based on the results of the above cytotoxicity experiments and the concentration control of the prepared samples, we finally chose four concentrations (0, 85.0, 170.0, 255.0, 340.0 µg/mL) to study the antiviral activity of Gel-FeS NPs against PRRSV further. The inhibitory effect of Gel-FeS NPs on PRRSV was evaluated based on indirect immunofluorescence assay. In Fig. 4, it could be found that the red fluorescence signal representing PRRSV N protein in cytoplasm of MARC-145 cells declined significantly, showing the PRRSV content in cells reduced. There was almost no difference in the nuclei stained blue, which once again showed the good biocompatibility of Gel-FeS NPs.
Furthermore, the viral infectivity of PRRSV treated with Gel-FeS NPs was determined for quantitative analysis by plaque assay. Plaque reduction assay was carried out to evaluate the viral content in cells treated with Gel-FeS NPs or not as shown in Fig. 5, the titers of PRRSV decreased significantly in a dose-dependent manner by Gel-FeS NPs compared to the control groups, with the most predominant reduction of ~103-fold, that is an encouraging data. In a word, the above experimental results demonstrated the excellent antiviral effect of Gel-FeS NPs on PRRSV proliferation.
Gel-FeS NPs Inhibit PRRSV Proliferation by Multi-Stage
In order to explore the effect of Gel-FeS NPs on virus life cycle, the ability of direct inactivation PRRSV by Gel-FeS NPs was investigated. As described in the experimental method section of the supporting information, the results of plaque assay showed that PRRSV content decreased dramatically (Fig. 6A), indicating that Gel-FeS NPs possess virucidal activity in vitro.
The virus infection involves a series of stages, each of which may be a potential target of antiviral drugs. According to the reported life cycle characteristics of PRRSV and classical experimental methods, the effect of Gel-FeS NPs on each process of PRRSV proliferation was explored. The number of plaques can directly reflect the effect of Gel-FeS NPs on the proliferation of the virus. As shown in Fig. 6B and 6C, PRRSV content was decreased ~ 10-fold by Gel-FeS NPs during the two stages respectively, indicating that Gel-FeS NPs inhibit the adsorption and invasion processes of PRRSV.
Following invasion by receptor-mediated endocytosis and disassembly, replicase polyproteins were produced under the guide of PRRSV genome positive strand RNA. This process is isolated as a genome replication process for PRRSV, so the level of PRRSV negative-sense RNA open reading frame 7 (ORF7) gene was quantified by RT-qPCR assay to assess the influence of Gel-FeS NPs on PRRSV replication. As described in Fig. 6D, the level of PRRSV negative-sense RNA ORF7 gene slightly decreased after treatment with Gel-FeS NPs at 340.0 mg/mL, suggesting that Gel-FeS NPs had a moderate inhibition effect on PRRSV replication. Finally, the release experiment of PRRSV progeny virus was carried out, the Gel-FeS NPs had no influence on PRRSV release because there was no significant change observed in the virus content in either intracellular or supernatant (Fig. 6E, F). In general, Gel-FeS NPs inhibited on PRRSV proliferation in MARC-145 cells by inhibiting the adsorption, invasion, replication but not the release stages of PRRSV.