Introduction

Severe Acute Respiratory syndrome coronavirus 2 (SARS-CoV-2) belongs to the Coronaviridae family, and it is an enveloped virus with a positive-sense, single-stranded RNA (ssRNA) genome of around 30 kb that can infect humans and other mammals. SARS-CoV-2 infection has spread globally, ultimately provoking the coronavirus pandemic that caused over 776 million confirmed cases up to October 2024 and over 7 million deaths globally, although these numbers are most likely underestimated.1 Influenza viruses (IVs) belong to the Orthomyxoviridae family and they are also enveloped viruses but with a segmented, negative-sense, ssRNA genome. IVs are divided into types A, B, C and D, with the type A (IAV) and B (IBV) producing global epidemics in humans each year.2 Vaccination programs are implemented to combat IV infections but nevertheless, the World Health Organization (WHO) considers there are 1 billion infections every year, 3–5 million severe cases and around 500,000 deaths due to these viral infections. IAVs can be further subdivided into subtypes based on their viral surface glycoproteins hemagglutinin (HA, H1-H18) and neuraminidase (NA, N1-N11), H1N1 and H3N2 being the subtypes seasonally circulating in humans nowadays. However, sporadic human infection by different subtypes of avian viruses have become increasingly more common since 1997, raising the threat of a possibly devastating pandemic if any of these viruses evolve and become efficiently transmitted among humans.3–5

Current options available to treat COVID-19 include a range of antiviral agents or immune modifiers. However, novel antiviral agents or treatment strategies will be necessary to better reduce the ongoing morbidity and mortality caused by SARS-CoV-2. Remdesivir and Molnupiravir are among the best studied antiviral agents, which act by disrupting RNA transcription and viral replication, respectively.6,7 Similarly PaxlovidTM, which combines Nirmatrelvir (a SARS-CoV-2 3C-like main protease (Mpro) inhibitor that inhibits the growth of the virus) and Ritonavir (a boosting agent, that enhances Nirmatrelvir’s performance), represents a promising treatment to significantly reduce hospitalization and the death of non-hospitalized patients who are at high risk of progressing to a severe illness.8 Other current COVID-19 treatments include monoclonal antibodies (mAbs) targeting SARS-CoV-2.9 However, the large-scale production of mAbs is extremely time consuming, labor-intensive and costly,10 and the emergence of antigenically distinct viral strains diminishes their efficacy.6

Several antiviral agents have been approved to combat IV infections, although most target viral proteins and therefore, their use may lead to the selection of influenza strains resistant to these drugs. For example, amantadine11 and rimantadine12 are effective antiviral agents that target viral M2 proton channel activity but as IAV strains resistant to these drugs have been reported,11,12 these drugs are no longer recommended for IAV treatment. NA inhibitors are currently available, such as zanamivir, oseltamivir, peramivir and laninamivir,13 and while it is generally more difficult for IV strains to become resistant to NA inhibitors than to amantadine, it is still possible.13,14 Thus, there remains a need to improve and synthesize new drugs to control IAV infections.

Due to the constant need to produce new strategies to combat SARS-CoV-2 and IAV infection, the possible antiviral activity of nanoparticles (NPs) is now being explored. The antiviral activity of metal and metal oxide NPs has been studied in relation to multiple viruses.15–18 The selection of viral strains resistant to these agents appears to be negligible, and they could have also a broad spectrum against different viruses and opportunist pathogens that become virulent in unhealthy individuals. In this sense, we previously showed that iron oxide NPs (IONPs) and iron oxyhydroxide NPs (IOHNPs) impair the replication and transcription of SARS-CoV-2, as well as the production of infectious viruses by cells in culture. These effects were evident when these cells were treated either before or after infection, and it is an effect that is probably due to changes in oxidative stress and iron metabolism induced by the treatment with the NPs.19

Plaque inhibition assays and quantitative real-time PCR (RT-qPCR) of viral transcripts in the presence of glycine-coated IONPs suggested these have antiviral activity against IAV. Moreover, this activity appeared to involve the reaction of iron oxide with -SH groups of proteins in the cell, inactivating these proteins.20 In addition, IONPs can catalyze lipid peroxidation of the viral lipid envelope due to their ability to induce the production of reactive oxygen species (ROS), thereby dampening the infectivity of the H1N1, H5N1 and H7N9 IAV subtypes.21

Silver NPs (Ag-NPs) can interact with IAVs, efficiently preventing their infection of cultured cells22 and of mice,23 diminishing any lung pathology and enhancing the survival of mice after infection. Furthermore, Ag-NP/chitosan composites with different NP sizes are active against H1N1 IAV, as witnessed by the reduced viral titers in viral suspensions after treatment with Ag-NP/Ch, and with smaller Ag-NPs displaying stronger antiviral activity against IAV.24 In addition, the antiviral activity of Ag-NPs coated with influenza NA inhibitors like oseltamivir (Ag-NP-OTV)25 and zanamivir (Ag-NP-ZNV)26 has been analyzed, showing that the treatment of cells with the Ag-NPs, Ag-NP-OTV and Ag-NP-ZNV after the IAV infection reduces viral titers, being this effect higher for the Ag-NP-OTV and Ag-NP-ZNV than for Ag-NPs, and that, as expected, the Ag-NP-OTV and Ag-NP-ZNV affect the IAV NA activity, likely affecting the attachment of the virus to the host cells.25,26

Selenium NPs (Se-NPs) also display antiviral activity against IAVs, with data indicating that their antiviral activity can be further potentiated by coating them with the NA inhibitors zanamivir27 or oseltamivir,28 with the M2 inhibitor amantadine,29 or with the influenza polymerase inhibitor ribavirin.30 Moreover, the activity of Se-NPs against IAVs can be enhanced by coating the Se-NPs with b-thujaplicin (TP), an antimicrobial pentatrieneone (Se-NPs-TP).31 Interestingly, the treatment of mice with Se-NPs-TP after IV infection prevents the lung pathology induced by infection and viral replication, and consequently enhances the survival of these mice.31

Among the different IONPs we tested for antiviral activity against SARS-CoV-2 in tissue culture cells in our study mentioned above, we found that magnetic IONPs produced by thermal decomposition in organic medium, with a core diameter of 10 nm and coated with DMSA (DMSA-IONP-10), were the IONPs with the strongest antiviral activity.19

Several years ago, our group studied the toxicity and biotransformation of DMSA-IONP-10 in mice over three months. The results of these studies showed that after five retro-orbital intravenous (i.v.) injections, DMSA-IONP-10 nanoparticles accumulated in spleen, liver and lung tissues where they undergo a process of biodegradation, and although some signs of very mild toxicity were observed post-administration, these were transient and did not compromise mouse survival and health status 3 months after administration, supporting the use of DMSA-IONP-10 in biomedical applications.32 Therefore, because the excellent antiviral activity in vitro of DMSA-IONP-10 and their biocompatibility in mice, we decided to test in vivo the antiviral activity of DMSA-IONP-10.

Here, DMSA-IONP-10 were shown to efficiently impair the production of infectious SARS-CoV-2 in mice, as well as IAV in cultured cells or mice, suggesting that these NPs may serve to combat SARS-CoV-2 and IAV infections. Furthermore, our results show that treating infectious viruses with DMSA-IONP-10 reduces viral infectivity, and that treating cells with these IONPs affects their oxidative stress and iron metabolism, which probably accounts for their antiviral activity.

Materials and Methods Materials

Iron(III) acetylacetonate 99% (Fe(acac)3) and benzyl ether 99% (BE) were purchased from Acros Organics (Geel, Belgium); oleic acid 80% GPR Rectapur® (OA) and acetone (anhydrous) were purchased from VWR (Leicestershire); 1,2-dodecanediol 90% (ODA), ethanol (99.5% - ACS reagent), dimercaptosuccinic acid (DMSA, ~98%), dimethyl sulfoxide (DMSO, ≥99.7%) and potassium ferricyanide 0.8% were obtained from Sigma Aldrich (San Luis, MO, USA); oleylamine (OAM) 70% technical grade, was obtained from Merck; toluene 99.5% EssentQ® was purchased from Scharlau (Madrid); Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Gibco (Invitrogen, CA); Fetal Bovine Serum (FBS) was obtained from Fisher; Mouse Serum (MS) was obtained from Sigma-Aldrich; PrestoBlue reagent was obtained from ThermoFisher Scientific; the anti-Nucleoprotein antibody was purchased from ATCC (HB-65: H16-L10-4R5); the total RNA extraction kit was purchased form Omega Bio-tek (GA, USA); the High-Capacity cDNA Reverse-Transcription kit, SYBR Green PCR Master and TaqMan gene expression assays were obtained from Applied Biosystems; the High Pure RNA Isolation Kit was purchased from Roche; C57BL/6 male mice were purchased from Envigo; agarose was obtained from Pronadisa; RNAlater was purchased from Ambion; glutaraldehyde, osmium tetroxide and epoxy resin TAAB 812 were obtained from TAAB Laboratories; DHR probe was purchased from Molecular probes, (Carlsbad, CA, USA).

Synthesis and Physicochemical Characterization of IONPs

Magnetic iron oxide cores of 10 nm in diameter were prepared by thermal decomposition of Fe(acac)3 in BE, in the presence of OA, OAM and ODA, according to reference.33 In brief, the mixture containing the iron precursor (0.1 M), OA, OAM and ODA (molar ratio of Fe(acac)3:OA:OAM:ODA= 1:3:3:2) was stirred mechanically at 100 rpm with nitrogen for 1.5 h. The reactor was then heated up to 200°C at 3°C/min, left for two hours and then heated again to 286°C at 9°C/min. The product was then washed three times with a mixture of toluene (99.5%) and ethanol (1:2 v/v), sonicated for 15 min and separated magnetically.

Particles were transferred to water by ligand exchange using DMSA to displace OA and OAM. The IONPs were first precipitated from the hydrophobic suspension (50 mg) by adding ethanol, centrifuging and removing the solution, and the precipitate was then dispersed in 20 mL of toluene and mixed with 5 mL of DMSO containing DMSA (100 mg). The mixture was kept in a rotary shaker for 24 h and after discarding the supernatant, the IONPs coated with DMSA were washed several times with ethanol. Finally, the IONPs were redispersed in alkaline water, dialyzed and filtered through a 0.2 μm membrane, thereafter referred to as DMSA-IONP-10.

The IONP particle size was determined by Transmission Electron Microscopy (TEM), with the iron oxide phase analyzed by X-ray diffraction (Bruker D8 Advance with Cu Kα radiation: data not shown) and the colloidal stability (ie hydrodynamic size and surface charge) studied by dynamic light scattering (DLS: Zetasizer, Malvern Instrument). Elemental analyses were performed by inductively coupled plasma optical emission spectroscopy after aqua regia digestion (ICP-OES: Perkin Elmer-2400) and the magnetic properties of the NPs using a SQUID magnetometer at room temperature and 5 K (Quantum Design MPMS-5), with a maximum magnetic field ±5 T. The NPs were lyophilized and then, packed in a gelatin capsule to measure the hysteresis loop, normalizing the magnetization values as a function of the Fe concentration calculated by ICP-OES. The amount and composition of the coating was determined by Fourier Transform Infrared Spectroscopy (FTIR) in a Bruker IFS 66 V-S spectrometer and a Nicolet FT-IR 20SXC. Finally, a thermogravimetric (TG) analysis was performed on a Seiko TG/DTA 320U device.

Cell Cultures

The African Green monkey kidney-derived epithelial Vero E6 cells were obtained from ATCC (CRL-1586), Vero cells expressing the TMPRSS2 protease (Vero-TMPRSS2) were obtained from Japanese Collection of Research Bioresources (JCRB) Cell Bank in Japan (JCRBno. JCRB1819), Madin-Darby canine kidney (MDCK) cells were obtained from ATCC (CCL-34), and human lung adenocarcinoma A549 cells were obtained from ATCC (CCL-185). All cell lines were all grown in DMEM supplemented with 25 mm HEPES, 10% FBS and 100 U/mL streptomycin.

Analysis of IONP Cytotoxicity

PrestoBlue assay. A549 cells were seeded in 96-well plates at a density of 3 × 104 cells per well in a final volume of 100 μL. After a 24 h incubation at 37°C, the A549 cells were treated with different concentrations of DMSA-IONP-10 (0–500 µg Fe/mL) for an additional 24 h. The PrestoBlue reagent was then added to each well for 2 h in the same culture conditions and the fluorescence was measured (560 nm excitation; 590 nm emission). The PrestoBlue reagent contains a blue, cell-permeable, non-fluorescent solution of resazurin that can be metabolized inside cells to a red and fluorescent compound called resorufin, a shift that serves as an indicator of cell viability. Cell viability is indicated by the number of fluorescent treated cells relative to the fluorescent untreated cells.

Quantification of Intracellular Iron After IONP Treatment

Vero E6 and A549 cells were seeded in a 6-well plate (1 x 105 cells per well) and cultured for 24 h at 37°C. The cells were then incubated for 3, 6 or 24 h with DMSA-IONP-10 (50 or 250 μg Fe/mL). Alternatively, the Vero E6 cells in 6-well plates were infected with IAV (multiplicity of infection – MOI 1) and 1 h post-infection (hpi), the cells were treated for 24 h with the highest concentration of DMSA-IONP-10. Subsequently, the cells were washed three times with phosphate buffer saline (PBS) to remove the non-internalized NPs, they were harvested and then counted in a Neubauer chamber. The samples were digested with 1 mL of HNO3 for 1 h at 90°C and the amount of iron per cell was measured by ICP-OES.

Antiviral Activity of IONPs in Tissue Cultured Cells

The prophylactic effect of DMSA-IONP-10 was assessed by treating confluent monolayers of Vero E6 and A549 cells (in 24-well plates) with these particles at 50 or 250 µg Fe/mL for 24 h. The cells were then infected with IAV (A/PuertoRico/8/1934 strain or PR8 strain) for 24 and 48 h at a MOI 1, collecting the cell culture media at 24 and 48 hpi for titration. Besides, to assess the prophylactic effect of IONPs on virus replication and transcription, the same experiment was performed but the cells were collected at 6 and 16 hpi to purify their total RNA for gene expression analysis.

To analyze the therapeutic effect of DMSA-IONP-10, confluent monolayers of Vero E6 and A549 cells (24-well plates) were infected with IAV (MOI 1), and the IONPs were added to the media at 1 hpi at the same concentrations as those used to study the prophylactic effect. The medium was collected at 24 and 48 hpi, and titrated, and total RNA to assess therapeutic effect on virus replication and transcription was purified from cells collected at 6 and 16 hpi. IAV virus titrations were performed in MDCK cells grown in 96-well plates and infected over 14 h with 10-fold serial dilutions of the virus at 33°C. An immunofocus assay (focus-forming units [FFU]/mL) was then performed using the HB-65 anti-Nucleoprotein antibody.

The Effect of IONPs on Virus Replication and Transcription

To analyze whether the therapeutic and prophylactic treatment of cells with DMSA-IONP-10 influences viral replication and/or transcription directly, total RNA from untreated and DMSA-IONP-10-treated Vero E6 cells, either mock-infected or IAV-infected was collected at 6 and 16 hpi, as explained above. Total RNA was extracted using a total RNA extraction kit, according to the manufacturer’s protocol. RT-qPCR was performed following an adapted protocol34 in which purified RNA (1 µg) was reverse-transcribed to cDNA using a High-Capacity cDNA Reverse-Transcription kit and the NPvRNA-5’-GGCCGTCATGGTGGCGAATGAATGGACGAAAAACAAGAATTGC-3´ or NPmRNA-5´-CCAGATCGTTCGAGTCGTTTTTTTTTTTTTTTTTCTTTAATTGTC-3´ primers, complementary to the viral RNA (vRNA) NP segment and the viral NP gene mRNA, respectively. RT-qPCR was then performed using the SYBR Green PCR Master mix and the Fw-NPvRNA 5´-CTCAATATGAGTGCAGACCGTGCT-3´ and Rv-NPvRNA-5´-GGCCGTCATGGTGGCGAAT-3´ primers, complementary to the NP vRNA segment and the Fw-NPmRNA-5´- CGATCGTGCCTTCCTTTG-3´and Rv-NPmRNA-5´-CCAGATCGTTCGAGTCGT3´ primers, complementary to the NP gene mRNA. The transcripts amplified were quantified using the threshold cycle (2− ΔΔCT) method.35

Analysis of DMSA-IONP-10 Treatment on Viral Infectivity

Cell culture supernatants containing 105 FFU IAV, were treated for 2 h at 37°C with medium containing DMSA-IONP-10 at 250 or 50 µg Fe/mL, or with medium alone. The infectious virus titers were then evaluated through an immunofocus assay in MDCK cells, as indicated above.

The Antiviral Effect of DMSA-IONP-10 in SARS-CoV-2 and IAV-Infected Mice

C57BL/6 male mice, 12-weeks-old or 6-8-weeks-old, were purchased one week before inoculation with SARS-CoV-2 or IAV, respectively, and they were housed under pathogen-free conditions at the Animal Health Research Center (INIA-CISA, Madrid, Spain), or at the animal facilities of the National Center for Biotechnology (Centro Nacional de Biotecnología – Consejo Superior de Investigaciones Científicas – CNB-CSIC, Madrid, Spain). All the protocols involving mice were approved by the CSIC ethics committee for animal experimentation and by the Division of Animal Protection of the regional government of Madrid, and they followed National and European Union legislation on animal experimentation (PROEX125.7/21 for IAV infections and PROEX49.6/23 for SARS-CoV-2 infections). All mice involved in the in vivo experiments were given ad libitum access to the same diet throughout the study. Mice were mildly anesthetized with ketamine/xylazine and then, inoculated intranasally with 2000 FFU/mice of IAV (strain A/PuertoRico/8/1934) or with 5000 FFU/mice of a SARS-CoV-2 mouse-adapted strain described previously.36 Subsequently, at 4 hours post-infection (hpi), and every 24 hours over the next three days, under mild isoflurane anesthesia, mice were retro-orbitally injected with DMSA-IONP-10 at a concentration of 0.16 mg Fe/mouse per injection, representing a total dose of 0.64 mg Fe/mouse after 4 injections, or with PBS as control (SARS-CoV-2 + IONPs, IAV +IONPs, SARS-CoV-2+ PBS, and IAV +PBS groups). Besides, for IAV and SARS-CoV-2 in vivo experiment, a group of mice was left uninfected and inoculated with PBS as control (N=4). A total of 6, 8, 10, and 9 mice per SARS-CoV-2 + IONPs, SARS-CoV-2+ PBS, IAV + IONPs, and IAV + PBS groups, respectively, were used for the in vivo experiments. The body weight of mice was evaluated throughout the in vivo experiments and the body weight loss was determined relative to the starting weight. Mice losing more than 25% of their initial body weight were considered to have reached the experimental end-point and were sacrificed humanely. At 4 days post-infection (dpi), all the mice were sacrificed and their lungs were extracted for analysis.

IAV and SARS-CoV-2 replication was evaluated by assessing the viral titers in the lungs at 4 dpi. As such, the right superior and middle lung lobules were extracted and homogenized, and IAV titers were determined in immunofocus assays on MDCK cells as indicated above. By contrast, the SARS-CoV-2 titers were determined in plaque titration assays on Vero-TMPRSS2 cells. To this end, Vero-TMPRSS2 cells were grown in 24-well plates and infected with 10-fold serial dilutions of the virus. After 1 h absorption, the cells were overlaid with low electroendosmosis agarose and incubated for 3 days at 37°C. The cells were then fixed with 10% formaldehyde in PBS and permeabilized with 20% methanol, and the viral plaques were visualized and counted using crystal violet, and expressed as plaque forming units (PFU) per mL of lung homogenate. In addition, total RNA was extracted from the right inferior and post-caval lobules, and the Cxcl10, Il-1b, Sod2 and Duox2 mRNA in the lungs were analyzed at 4 dpi. Hence, the left lung lobules were extracted and incubated in RNA later at 4°C for 24 h prior to adding the lungs to RNA lysis buffer and homogenizing them manually in a dounce homogenizer. Total RNA was extracted from the homogenized lungs using the total RNA kit. RT was then performed for 2 h at 37°C to generate cDNAs using the High-Capacity cDNA transcription kit and random hexamers. qPCR analysis using TaqMan gene expression assays specific for the murine Cxcl10 (Mm00445235_m1) and Il-1b (Mm00434228_m1), and specific for the mouse GAPDH gene (Mm99999915_g1) were then carried out. Alternatively, the cDNAs were then analyzed by qPCR with a Power SYBR Green PCR mix and primers specifically designed to amplify Mus musculus transcripts for the genes Sod2 and Duox2 (Table S1), synthesized by Sigma-Aldrich. The data were analyzed using the threshold cycle (2− ΔΔCT) method35 and normalized to the GAPDH expression.

Analyses of the Presence of DMSA-IONP-10 in Lung Tissues

The presence of DMSA-IONP-10 in lung tissues was evaluated in TEM images. As such, approximately 1 mm3 of lung tissue was fixed for 30 min at room temperature in 4% paraformaldehyde (PFA) and 2.5% glutaraldehyde diluted in PBS. The tissue was then transferred to fresh buffered fixative solution for 1h at room temperature and then left overnight at 4°C. The tissue was then washed with PBS, post-fixed for 1 h at 4°C with 1% osmium tetroxide in potassium ferricyanide 0.8% and then maintained for 1h in 2% aqueous uranyl acetate at 4°C. After washing with distilled water, the tissues were dehydrated with increasing concentrations of acetone, embedded in epoxy resin TAAB 812 and polymerized in epoxy resin (100%) over 2 days at 60°C. The resin blocks were then trimmed and ultrathin, 70 nm thick sections were obtained on an UC6 ultramicrotome (Leica Microsystems). The sections were transferred to 200 mesh nickel grids (Gilder) and stained at room temperature for 20 min with saturated aqueous uranyl acetate and for 1 min in lead citrate 0.2% (Electron Microscopy Sciences). The sections were visualized on a JEOL JEM 1400 Flash electron microscope (operating at 100 kV) and micrographs were obtained with a Gatan OneView digital camera at various magnifications.

In addition, the left lung lobule was extracted and used for ICP-OES analysis after nitric acid and aqua regia digestion, and for magnetic measurements with a SQUID magnetometer after lyophilization and packing in a gelatin capsule without cotton to avoid contamination. Hysteresis loops were recorded at room temperature and at 5 K, and the magnetic behavior of the NPs was used as a reference to quantify them in the lungs.

Analysis of Oxidative Stress in Cells Treated with DMSA-IONP-10 Dihydrorhodamine 123 (DHR) Staining

The production of ROS was quantified by dark-field confocal staining with the DHR probe, a non-fluorescent ROS indicator that can be oxidized inside cells to the fluorescent rhodamine 123. A549 cells were cultured on coverslips in 24-well plates for 24 h and the cells were then treated for 24 h with a DMSA-IONP-10 suspension (250 μg Fe/mL). As a positive control of oxidative stress, the A549 cells were incubated for 1 h with H2O2 (1 mm). The coverslips were then washed with PBS and the cells incubated for 30 min with DHR (diluted 1:500 in medium) under cell culture conditions. After washing with PBS, the cells were fixed for 15 min with PFA (4%), stained for 10 min with DAPI (diluted 1:500 in PBS), washed and mounted with Fluoromount-G. Images were obtained under a dark-field Leica TCS SP5 confocal microscope with the 63X oil objective, using a 488 nm laser light for dark-field acquisition of the IONPs and quantifying the DHR signal intensity with Image J software.

RT-qPCR Analysis of the Effect of the IONPs on the Expression of Genes Involved in the Antioxidant Response

A549 cells were treated for 24 h with a DMSA-IONP-10 suspension (250 μg Fe/mL), collected, and the total RNA from untreated and treated cells was extracted using the High Pure RNA Isolation Kit. The RNA was quantified with NanoDrop, and 2 μg of RNA was reverse-transcribed to cDNA using the High-Capacity cDNA Reverse Transcriptase kit and random primers. The cDNA was then analyzed by qPCR with a Power SYBR Green PCR mix and primers specifically designed to amplify Homo sapiens transcripts of the following genes (see Table S1): catalase (CAT); dual oxidases 1 and 2 (DUOX1, DUOX2); superoxide dismutase 1, 2 and 3 (SOD1, SOD2, SOD3); and thioredoxin reductase 2 (TXNRD2). The primers were synthesized by Sigma-Aldrich, and the RT-qPCR data was analyzed by the threshold cycle (2− ΔΔCT) method35 and normalized to GAPDH expression.

The Effect of Oxidative Stress Induction on IAV Replication

Confluent monolayers of Vero E6 and A549 cells (24-well plates) were treated for 24 h with N-Acetyl-L-Cysteine (NAC, 200 µM), a recognized ROS scavenger,37 or left untreated as a control. The cells were then infected with IAV (MOI 1) and the extracellular medium containing the virus was replaced at 1 hpi with a suspension of the DMSA-IONP-10 and NAC (200 µM), or with a suspension of the DMSA-IONP-10 without NAC as a control. After 48 h the medium was collected and the virus was titrated in an immunofocus assay, as described above.

The Effect of DMSA-IONP-10 Treatment on Iron Metabolism Genes

To analyze the effect of IONPs on genes involved in iron metabolism, Vero E6 cells were treated for 24 h with a DMSA-IONP-10 suspension (250 μg Fe/mL), or these cells were infected with IAV and 1 hpi, they were treated with the same concentration of DMSA-IONP-10 for 24 h. The total RNA from both untreated and treated cells was extracted with the High Pure RNA Isolation Kit (Roche), quantified using NanoDrop, and 2 μg of RNA was reverse-transcribed to cDNA using the High-Capacity cDNA Reverse Transcriptase kit and random primers. The cDNA was used to perform RT-qPCR with a Power SYBR Green PCR mix and primers designed specifically to amplify transcripts for the: Transferrin Receptor (TFRC), SLC11A2 (encoding divalent metal transporter 1, DMT1), SLC48A1 (encoding heme transporter 1, HRG1), SLC40A1 (encoding ferroportin, FPN1), iron responsive element binding protein 2 (IREB2) and lipocalin 2 (LCN2, encoding neutrophil gelatinase-associated lipocalin-NGAL) according to the Chlorocebus sabaeus predicted mRNA sequences (NCBI taxonomy number 60711, see Table S2).19 The primers were synthesized by Sigma-Aldrich, and the RT-qPCR data was analyzed by the threshold cycle (2− ΔΔCT) method35 and normalized to GAPDH expression.

Statistical Analysis

All the data are presented as the mean ± standard deviation (SD) and analyzed with a Student’s t-test, one-way or two-way analysis of variance (ANOVA), applying a Tukey’s and Dunnett's test to calculate the differences between the distinct values. Values of p < 0.05 were considered statistically significant: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. GraphPad Prism version 9.5.1 was used for all the statistical analyses.

Results DMSA-IONP-10 Production and Physicochemical Characterization

As mentioned in the introduction, in a previous study, our group evaluated the antiviral potential of various coated IONPs against SARS-CoV-2 infections in vitro. Among the different IONPs we tested, we found that magnetic IONPs produced by thermal decomposition in organic medium, with a core diameter of 10 nm and coated with DMSA (DMSA-IONP-10), were the IONPs with the strongest antiviral activity against SARS-CoV-2 in tissue culture cells.19 Therefore, we wanted to evaluated the antiviral activity of DMSA-IONP-10 against SARS-CoV-2 in vivo in a mouse model of infection.

For this study a new batch of DMSA-IONP-10 was produced and the main physicochemical characteristic of this new batch was ascertained (Figure 1), confirming that this batch was indistinguishable from previous batches of DMSA-IONP-10 used in previous studies of the group19,32,38–41 DMSA-IONP-10 have spherical shape (Figure 1A) with a size distribution of 10 ± 2 nm (Figure 1B). The magnetization measurements confirmed the superparamagnetic behavior of the DMSA-IONP-10 at room temperature (RT, see Figure 1C). The characteristic infrared bands detected through Fourier-transform infrared spectroscopy (FTIR) demonstrated that the IONPs were coated with the corresponding organic compounds (Figure 1D), showing peaks at 1625 and 1383 cm−1 for DMSA and Fe−O bond-specific IR bands between 400 and 850 cm−1. To quantify the amount of polymer bound to the iron oxide core of the DMSA-IONP-10, thermogravimetric (TG) analysis was performed. The mass percentage of the coatings was approximately 15% for the DMSA-IONP-10 (Figure 1E). Finally, the stability of DMSA-IONP-10 was assessed using dynamic light scattering (DLS) measurements after incubating the IONPs in water, PBS, or DMEM supplemented with 10% mouse serum (MS, DMEM-MS) for 0, 24, 48, 72 or 96 hours (Figure 1F). The results obtained show that the DMSA-IONP-10 have a similar hydrodynamic size in water (~86.11 ± 5.27 nm) and PBS (~124.78 ± 16.62 nm), and that this size remains constant over time, indicating that they are stable, even in PBS, which is the vehicle used for administration in the animal model. Regarding the hydrodynamic size of DMSA-IONP-10 incubated in DMEM-MS, a condition that simulates the environment in the bloodstream following intravenous administration in the in vivo model, we observed that the NPs increase in size (283.0 ± 48.65 nm) due to interactions with serum proteins and the formation of a protein corona, a process previously described by the group with similar NPs.32,38,42 Nevertheless, it is important to note that their stability is not compromised, as only a single peak is observed in the size distribution, and their polydispersity indices indicate they remain monodisperse. These results are concomitant with those previously shown by our group,32 and confirm that DMSA-IONP-10 are feasible for in vivo applications.

Figure 1 Physicochemical characterization of DMSA-IONP-10.

Notes: (A) TEM images of spherical DMSA-IONP-10. Scale bar: 40 nm. (B) Distribution of the iron core diameter (nm) of at least 200 IONPs. (C) Magnetic properties of DMSA-IONP-10, showing superparamagnetic behavior at room temperature and ferromagnetic behavior at 5 K. (D) FTIR spectra of DMSA-IONP-10. Characteristic IR 1625, 1383 and 1140 cm−1 bands were detected corresponding to COO−, OH and CO groups. Iron-oxygen bands were detected at 850–400 cm−1. (E) Density of the DMSA coating determined by thermogravimetric (TG) analysis showing 17.54% DMSA coverage of the iron core surface. (F) Characterization of the stability of DMSA-IONP-10 over time (0, 24, 48, 72 and 96 h) determined by DLS. DMSA-IONP-10 were incubated during the mentioned hours in water, in PBS, and in DMEM with 10% MS. Polydispersity indices (PDI) are shown in all cases.

DMSA-IONP-10 Accumulated in the Lung After I.v. Injection

In a previous study by our group, we had observed that after retro-orbital i.v. injections in mice of biocompatible DMSA-IONP-10, these nanoparticles were detected in the lungs and to a lesser extend in spleen and liver.38 Following these observations, we conducted a study of the biodistribution, toxicity and degradation of DMSA-IONP-10 in vivo, which confirmed our previous observations and allowed us to characterize more precisely when the presence of nanoparticles begins to be detected in the different organs and the time it takes them to be degraded. In this study DMSA-IONP-10 NPs were detected in the lungs as early as 30 min after i.v. injection and they persisted for up to 90 days post-inoculation, although gradual degradation was evident over time, indicating that the IONPs are cleared from the body.32 To confirm that the new DMSA-IONP-10 batch synthesized for this study reached the lungs in mice and to quantify the DMSA-IONP-10 that accumulated there, three different doses of DMSA-IONP-10 were i.v. injected retro-orbitally into C57BL/6 mice on consecutive days (4 days) to reach total doses of 0.48, 0.64 and 0.80 mg Fe per mouse after the four injections, with 0.80 mg Fe per mouse after the four injections being the maximum dose that can be administered in retro-orbital i.v. injections for four consecutive days. One day after the last dose was administered, the mice were sacrificed, their lungs were extracted and lyophilized, and the presence of IONPs was confirmed by magnetic measurements in a SQUID magnetometer (Supplemental Figure S1A). This technique detects the presence of IONPs in the tissue, quantifying them through the magnetic behavior of the IONPs as a function of the field. There were considerable differences in the magnetization curves between the lung tissue samples from control mice injected with PBS and lung tissue samples from mice injected with DMSA-IONP-10, when administered at doses of 0.48 mg Fe/mouse and 0.64 mg Fe/mouse, reflecting the accumulation of DMSA-IONP-10 in the lungs (Supplemental Figure S1B). A non-linear dependence of magnetization with the field (“S” shape) was evident, characteristic of ferromagnetic materials like magnetite with zero coercivity at room temperature (ie superparamagnetic behavior) and non-zero coercivity (350–400 Oe) at 5 K, similar to the IONP curves (Figure 1C). By contrast, magnetization has a linear dependence with the field at both temperatures for the PBS control samples, diamagnetic in most cases (negative) or paramagnetic (positive) when there is some residual blood in the lungs. Lung tissue samples from mice treated with the highest dose of 0.80 mg Fe/mouse were not measured in a SQUID magnetometer because at this dose, although all the mice remain alive and no obvious changes in the body weight of mice were observed during the treatments at any of the doses (Supplemental Figure S1C), some signs of toxicity (eye necrosis, piloerection) were observed, and therefore, we decided not to use this dose for the antiviral activity studies.

SARS-CoV-2 Antiviral Effect of DMSA-IONP-10 in Mice

Having confirmed that the batch of DMSA-IONP-10 synthetized for this study could reach the lungs (Supplemental Figure S1B), we evaluated the antiviral activity of these nanoparticles against SARS-CoV-2 in vivo in a mouse model of infection. For these experiments, the dose of DMSA-IONP-10 that allows the highest nanoparticle concentration detected in the lungs without induction of toxicity in mice was used: a total dose of 0.64 mg Fe/mouse administered in 4 i.v. injections of 0.16 mg Fe/mouse for each individual injection. C57BL/6 mice were inoculated intranasally with 5000 PFU of a mouse-adapted strain of SARS-CoV-2, which reproduces many aspects of the disease seen in humans.36 At 4 hpi and every 24 hours over the next three days, mice were intravenously inoculated with 0.16 mg Fe/injection of DMSA-IONP-10, reaching a total of 0.64 mg Fe/mouse after 4 injections (SARS-CoV-2 + IONPs group), or intravenously inoculated with PBS (SARS-CoV-2 + PBS group). The mice were then sacrificed at 4 dpi (Figure 2A) and the presence of IONPs in the lungs were evaluated by SQUID magnetometer to confirm the superparamagnetic behavior in DMSA-IONP-10 treated mice compared to PBS treated control mice (Figure 2B). By measuring the maximum magnetization in the lungs at 5K (0.004 emu/g – 0.0013 emu/g of organ) and comparing this with that for the IONPs (110 emu/g Fe), the NPs that reached the lungs was estimated to be between 0.036 mg Fe and 0.012 mg Fe per gram of organ. Taking into account an average weight of 0.2 grams per lung, this represents between the 1.13 and 0.34% of the injected dose. The weight loss evaluated over the 4 days of infection showed that infected mice lost weight as a consequence of infection (Figure 2C), as expected,36 although all the mice remained alive during the 4-day-experiment. Interestingly, SARS-CoV-2-infected mice treated with the DMSA-IONP-10 lost slightly less weight than the untreated SARS-CoV-2-infected control mice (Figure 2C), further suggesting, as observed in the non-infected mice (Supplemental Figure S1C), that the treatment of mice with DMSA-IONP-10 is not toxic to the animals. Finally, the viral titers in the lungs at 4 dpi were determined through a plaque lysis analysis in Vero-TMPRSS2 cells. In the infected mice, a 3-fold reduction was observed in the DMSA-IONP-10 inoculated mice relative to the control mice treated with the vehicle alone (Figure 2D), reflecting the antiviral activity of DMSA-IONP-10 in vivo.

Figure 2 SARS-CoV-2 antiviral effect of DMSA-IONP-10 in mice.

Notes: Groups of 12-week-old mice were inoculated intranasally with 5000 FFU/mouse. At 4 hpi, and over the next 3 days, mice were inoculated intravenously with DMSA-IONPs-10 (N = 6) or with PBS as a control (N = 8), and they were then sacrificed at 4 dpi. (A) Experimental timeline. (B) Presence of DMSA-IONP-10 in the lungs detected by SQUID magnetometer, showing ferromagnetic behavior at 5 K in the DMSA-IONP-10-treated mice compared to the paramagnetic behavior in the PBS control mice. The graph represents an example of one lung of the DMSA-IONP-10-treated mice versus one lung of the PBS treated mice after SARS-CoV-2 infection. (C) Body weights of SARS-CoV-2 infected mice were monitored over 4 days after receiving PBS (grey) or DMSA-IONP-10 (blue). (D) Viral titer evaluated by plaque analysis. (E and F) Inflammatory responses were evaluated by the expression of the proinflammatory cytokines Cxcl10 (E) and Il-1b (F) by RT-qPCR at 4 dpi. Increased mRNA levels in SARS-CoV-2-infected DMSA-IONP-10-treated or untreated mice were expressed as the fold change relative to the mock-infected, non-treated, control mice. Each dot corresponds to one IAV-infected mouse and the data is analyzed with a t-test: *p < 0.05 and ns, no significant differences.

A very strong inflammatory response is correlated with disease severity after SARS-CoV-2 infections in patients.43,44 To further assess whether treating mice with the NPs could reduce the inflammation provoked by SARS-CoV-2 infection, dampening the SARS-CoV-2-induced pathology, the expression of the pro-inflammatory cytokines Cxcl10 and Il-1b in the mouse lungs was assessed by RT-qPCR. Weaker Cxcl10 expression was observed in infected mice treated with DMSA-IONP-10 relative to those infected mice administered with the vehicle alone (Figure 2E). Furthermore, while Il-1b expression was enhanced in the infected mice that received the vehicle, it did not increase when these mice were treated with the IONPs (Figure 2F), suggesting that the NPs might be beneficial to the infected hosts by reducing lung viral titers, and by leading to a decreased inflammatory response induced after SARS-CoV-2 infection.

Analysis of DMSA-IONP-10 Uptake and Toxicity in A549 Cells

Given the antiviral activity of DMSA-IONPs-10 against SARS-CoV-2 in both infected cultured cells19 and mice (this article), we investigate the potential antiviral activity of these NPs against IAV, another relevant respiratory virus. The potential antiviral effect of DMSA-IONP-10 against IAV was first assessed in infected cell lines. For these experiments, we first determined the working concentrations for DMSA-IONP-10 in the two model cell lines commonly used to study IAV infection, Vero E6 and A549 cells. The PrestoBlue assay was used to determine cell viability, assessing mitochondrial metabolism as an indirect measure of cell viability. The working concentration of DMSA-IONP-10 for Vero E6 cells was determined previously to be 250 µg Fe/mL, with cell viability remaining above 95% at this concentration.19 The working dose of DMSA-IONP-10 in human lung epithelial A549 cells was determined using a PrestoBlue assay after a 24 h treatment with different concentrations of DMSA-IONP-10. DMSA-IONP-10 did not induce strong cytotoxicity, as the viability was >80% at concentrations as high as 250 μg Fe/mL (Figure 3A). Therefore, we chose to use a working concentration of 250 µg Fe/mL for DMSA-IONP-10 for both cell lines,19 as well as a second lower working concentration (50 μg Fe/mL) at which Vero E6 and A549 cell viability was nearly 100%, to assess whether these treatments have any dose-dependent effects.

Figure 3 Evaluation of DMSA-IONP-10 toxicity and iron uptake in A549 cells.

Notes: (A) Viability of A549 cells after a 24h treatment with different doses of DMSA-IONP-10, as measured with the PrestoBlue fluorometric test. (B) Iron uptake over time measured by ICP-OES after treating the cells with 50 and 250 μg Fe/mL DMSA-IONP-10. The data represents the mean and SD from three independent experiments.

To investigate DMSA-IONP-10 uptake by A549 cells, cells were incubated for 3, 6 or 24 h with the two concentrations of DMSA-IONP-10 (50 or 250 μg Fe/mL) and the IONPs internalized by the cells were assessed by ICP-OES, measuring the intracellular iron concentration of the cultured cells. The dose and time-dependent iron uptake of DMSA-IONP-10 by Vero E6 cells has been published previously19 and the maximal iron uptake by A549 cells was observed with the highest dose of DMSA-IONP-10 (250 μg Fe/mL) at 6 h (5.55 pg Fe/cell: Figure 3B). Thus, both cell lines internalized the DMSA-IONP-10.

Effect of DMSA-IONPs-10 on Infectious Viral Production

To analyze the effect of DMSA-IONP-10 on IAV production, Vero E6 cells were treated with these particles for 24 h before infection (prophylactic effect) or for 1 h after infection (therapeutic effect), measuring the viral titers in the cell culture supernatants at 24 and 48 hpi (Figure 4A). The maximum DMSA-IONP-10 concentration had a prophylactic effect and reduced viral titers at 48 hpi by 98.8% (Figure 4A). In terms of the therapeutic effect, the highest, non-cytotoxic concentration of DMSA-IONP-10 reduced the viral titers at 48 hpi by 97% (Figure 4A). Therefore, therapeutic and prophylactic treatment of cells with DMSA-IONP-10 significantly diminished the production of infectious IAV viruses.

Figure 4 Prophylactic and therapeutic antiviral effect of DMSA-IONP-10 in cells.

Notes: Vero E6 (A) or A549 (B) cells were treated with DMSA-IONP-10 at the two different concentrations indicated, or left untreated (control cells), and 24 h after treatment the cells were infected with IAV (prophylactic treatment). Alternatively, Vero E6 (A) or A549 (B) cells were infected with IAV and 1 hpi they were treated with DMSA-IONP-10 (therapeutic treatment). (A and B) Cell culture supernatants were collected at 24 and 48 hpi, and the viral titer was determined by an immunofocus assay in MDCK cells. Viral titers were assessed and represented relative to the titers in control, untreated cells (%). The data (mean ± SD) are from three independent experiments and analyzed by two-way ANOVA followed by a Dunnett’s multiple comparison test: **p < 0.01; ***p < 0.001; and ****p < 0.0001; and ns, no significant differences.

To confirm that the IAV antiviral effect of DMSA-IONP-10 also occurs in infected human cells, similar experiments were performed on A549 human lung adenocarcinoma cells (Figure 4B). The highest concentration of DMSA-IONP-10 had a prophylactic effect, reducing the viral titers in cell culture supernatants by 99.97% and 99.95% at 24 and 48 hpi, respectively (Figure 4B). In therapeutic terms, the highest DMSA-IONP-10 concentration reduced viral titers by 91% at 24 hpi (Figure 4B).

The Effect of IONPs on Viral Replication and Transcription

To analyze whether the therapeutic and prophylactic treatment of cells with DMSA-IONP-10 influences viral replication and/or transcription directly, Vero E6 cells were exposed to DMSA-IONP-10 before or after IAV infection, and genomic RNA (vRNA) and NP mRNA expression was determined by RT-qPCR at 6 and 16 hpi to evaluate viral replication and transcription, respectively. After prophylactic exposure, the expression of vRNA and NP mRNA did not decrease in cells treated with DMSA-IONP-10 relative to the untreated control cells, except for the 2-fold reduction in NP mRNA at 16 hpi when the highest dose of DMSA-IONP-10 was administered (Figure 5A). The therapeutic treatment of cells with both doses of DMSA-IONP-10 after IAV infection only reduced the amounts of NP mRNA at 6 hpi, (Figure 5B), a reduction that did not persist at 16 hpi. These results strongly suggest that treating cells with DMSA-IONP-10 before or after the infection did not significantly affect viral replication or transcription.

Figure 5 Effect of DMSA-IONP-10 on viral replication, transcription and infectivity.

Notes: (A and B) Effect of treatment of cells with DMSA-IONP-10 before or after the infection on IAV replication and transcription. (A) Vero E6 cells were treated with DMSA-IONP-10 at two different concentrations, or left untreated (control cells), and 24 h after treatment the cells were infected with IAV (MOI 1) for an additional 6 or 16 h. (B) Vero E6 cells were infected with IAV and 1 hpi they cells were treated with DMSA-IONP-10 for 6 or 16 h as indicated in A. (A and B) Total RNA was extracted at 6 and 16 hpi, and the vRNA, NP mRNA and GAPDH RNA expressed was quantified by RT-qPCR. The vRNA and mRNA expression was normalized to that of GAPDH, and represented relative to the expression in control, untreated cells (%). (C) The effect of DMSA-IONP-10 treatment on virus infectivity. Infectious viruses were incubated with medium containing DMSA-IONP-10 at 50 and 250 μg Fe/mL for 2 h at 37°C, and virus infectivity was then evaluated in an immunofocus assay, determining the viral titers relative to those in the controls treated with medium alone (%). The data (mean ± SD) were representative of three independent experiments and they were evaluated by two-way ANOVA followed by a Dunnett’s multiple comparison test: *p < 0.05 and ns, no significant differences.

The Effect of DMSA-IONP-10 Treatment on Virus Infectivity

Treatment of IAV with iron oxide nanozymes reduces viral infectivity by catalyzing lipid peroxidation of the viral envelope.21 To analyze whether treating the viruses with DMSA-IONP-10 reduces their infectivity, cell culture supernatants containing viruses were exposed to medium containing DMSA-IONP-10 at 250 and 50 μg Fe/mL for 2 h, or with medium alone, and the infectious virus titers were then evaluated. The treatment of viruses with the highest concentration of DMSA-IONP-10 reduced the viral titers 3-fold (Figure 5C), suggesting that DMSA-IONP-10 can directly dampen viral infectivity.

In vivo Antiviral Effect of DMSA-IONP-10 Against IAV

Since promising results were observed with both the prophylactic and therapeutic treatment of cells in vitro, reducing the production of IAV infectious viruses, the antiviral activity of DMSA-IONP-10 was evaluated in vivo. C57BL/6 mice were inoculated intranasally with IAV (A/PuertoRico/8/1934 strain) at 2000 FFU/mouse and at 4 hpi, and on the following 3 days, mice were injected retro-orbitally with DMSA-IONP-10 (0.16 mg Fe/mice per injection, resulting in a total dose of 0.64 mg Fe/mouse after the 4 injections) (+IAV PR8 + IONPs group) or injected with PBS (+IAV PR8 + PBS group) (Figure 6A). These mice were sacrificed at 4 dpi, and the IONPs that reached the lungs in this setting was quantified by measuring the magnetic moment of the lungs at 5 K (between 0.0084 emu/g and 0.0022 emu/g) and compared to the magnetic behavior of the IONPs (110 emu/g Fe: Figure 6B). Between 0.075 mg Fe and 0.02 mg Fe was detected in the lungs per gram of organ (Figure 6B), representing between 2.2% and 0.6% of the injected dose (0.64 mg Fe), assuming an average weight of 0.2 grams per lung. In addition, the presence of DMSA-IONP-10 in the lungs was corroborated in TEM images, showing these IONPs to accumulate in the lung after infection and DMSA-IONP-10 treatment (Supplemental Figure S2). Significantly, the infected mice, either treated or not with DMSA-IONP-10, lost no significant weight during the 4 days of the experiment (Figure 6C), suggesting that in this animal model, neither IAV, nor the DMSA-IONP-10 treatment cause significant disease in the mice. Interestingly, the viral titers in the lungs at 4 dpi were 4-fold lower in the DMSA-IONP-10 inoculated mice than in the control mice treated with the vehicle alone (Figure 6D), reflecting the antiviral effects of the DMSA-IONP-10 in vivo.

Figure 6 Influenza antiviral effect of DMSA-IONP-10 in mice.

Notes: Groups of 6 to 8-week-old mice were inoculated intranasally with 2000 FFU/mice and at 4 hpi, and every day over the next 3 days, the mice were inoculated intravenously with DMSA-IONP-10 (N = 10) or with PBS as a control (N = 9), sacrificing them at 4 dpi. (A) Experiment timeline. (B) The levels of iron in mice were evaluated at 4 dpi by SQUID magnetometer at 5 K. The graph represents an example of one lung of the DMSA-IONP-10 treated mice versus one lung of the PBS treated mice after IAV infection. (C) The weight of the was assessed just before infection and daily from day 0 to day 4 after infection. (D) Viral titers in the DMSA-IONP-10 treated and control mouse lungs were measured with an immunofocus assay in MDCK cells at 4 dpi. (E and F) Cxcl10 (E) and Il-1b (F) expression in the mouse lungs was evaluated by RT-qPCR at 4 dpi. Increases in mRNA in IAV-infected, DMSA-IONP-10 treated mice or untreated mice were expressed as the fold change relative to mock infected mice as controls. Each dot corresponds to one IAV-infected mouse and the data was analyzed with a t-test: *p < 0.05 and ***p < 0.001.

An exacerbated inflammatory response has been correlated with disease severity after IAV infection in vivo.45,46 Thus, to further assess whether treating mice with the NPs could reduce the inflammation induced after IAV infection, pro-inflammatory Cxcl10 and Il-1b cytokine expression in the mouse lungs from infected animals treated with DMSA-IONP-10 or with vehicle alone, was measured by RT-qPCR, and compared to the expression in non-treated, non-infected mice. A mild decrease in Cxcl10 and Il-1b expression was observed after infection in the mice treated with the NPs relative to those treated with PBS (Figure 6E and 6F). These data correlate with the lower virus titers observed in the mouse lungs from DMSA-IONP-10-treated mice, compared to the viral titers in vehicle-treated mice and suggest that NP treatment of mice might benefit the host by leading to a milder inflammation after IAV infection.

Induction of Oxidative Stress as a Consequence of DMSA-IONP-10 Cell Uptake and Its Effect on the IONP’s Antiviral Activity

It is well known that one of the cytotoxic effects of IONPs is the production of ROS, which in turn triggers oxidative stress in cells. We previously showed that one of the possible antiviral mechanisms of IONPs against SARS-CoV-2 infection in Vero E6 cells was the induction of oxidative stress.19 To determine whether oxidative stress induced by DMSA-IONP-10 could be partially responsible for their antiviral effect against IAV in infected cells, it is important to determine the capacity of these IONPs to induce ROS in these cells. ROS induction was studied in A549 cells, since we previously described that DMSA-IONP-10 induce ROS in Vero E6 cells (18). A549 cells were treated for 24 h with DMSA-IONP-10 (250 μg Fe/mL) and then, the cells were stained with the DHR probe that only fluoresces inside the cells when oxidized by ROS. DMSA-IONP-10-treated cells induced a 3.5-fold change in DHR fluorescence relative to the control A549 cells (1 a.u. of fluorescence) (Figure 7A), whereas in Vero E6 cells DMSA-IONP-10 produced a 10-fold change in DHR fluorescence relative to the control.19

Figure 7 Effect of DMSA-IONP-10 on the induction of oxidative stress and how oxidative stress influences the antiviral activity of DMSA-IONP-10.

Notes: (A) ROS generation observed by DHR fluorescence and quantitative image analysis of DHR fluorescence intensity using Image J software: Control (-) untreated A549 cells; and control (+) A549 cells incubated with 1mM H2O2. Images were taken with a 63X oil objective under a 3X zoom and the DHR fluorescence of at least 200 cells/condition is shown. (B) Quantification of gene expression by RT-qPCR (mRNA levels) in A549 cells after treatment with DMSA-IONP-10. The expression following oxidative stress was compared to that in untreated cells and the data were normalized to the expression of GAPDH. (C) Effect of ROS on the antiviral activity of DMSA-IONP-10. Confluent Vero E6 cells were treated for 24 h with N-acetylcysteine (NAC) or left untreated as a control. The cells were then infected with IAV (MOI 3) and 1 hpi, the extracellular medium containing the virus was replaced with a suspension of DMSA-IONPs-10, with or without NAC (200 µM). The medium was collected from the cells at 48 hpi and titrated. The data are the mean ± SD (N = 3). Differences on ROS production were analyzed with one-way analysis of variance (ANOVA) with a Dunnett's multiple comparisons test, differences on antioxidant gene expression were analyzed with a t-test and differences on the effect of ROS on virus titer were analyzed with a one-way ANOVA with Tukey’s multiple comparison test: *p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001.

The oxidative stress induced was further studied by assessing the antioxidant responses induced by DMSA-IONP-10 in A549 cells, quantifying the expression of antioxidant enzymes like catalase (Cat), superoxide dismutases (SOD 1, 2 and 3), dual oxidases (DUOX 1 and 2), and thioredoxin reductase 2 (TXNRD2), by RT-qPCR.47 We previously showed that DMSA-IONP-10 induced the expression of DUOX1, DUOX2, SOD1, SOD2, SOD3 and thioredoxin domain containing 2 (TXNDC2) antioxidant genes in Vero E6 cells19 and here, we found that DMSA-IONP-10 induced the expression of DUOX1, DUOX2, SOD2 and SOD3 in A549 cells (Figure 7B). Hence, DMSA-IONP-10 appear to induce significant oxidative stress in Vero E6 and A549 cells, activating different antioxidant machineries (