Silver-Zinc oxide nanocomposites: a green approach to mitigate herpes simplex virus type 1 challenges

Abstract

Herpes simplex virus type 1 (HSV-1) remains a significant global health challenge due to its high prevalence, recurrent infections, emergence of drug-resistant viral strains, and adverse effects associated with conventional antiviral therapies. In this study, silver–zinc oxide nanocomposites (Ag–ZnONPs) were synthesized using Nigella sativa extract and evaluated for antiviral activity against HSV-1 alongside ZnONPs, AgNPs, and calcined Ag–ZnONPs (Ag–ZnONPs-K). Characterization confirmed nanoparticle formation with sizes of ~ 35–50 nm and zeta potentials ranging from − 15.5 to − 25.5 mV. Cytotoxicity assays showed CC₅₀ values of 700 µg/mL (ZnONPs), 35 µg/mL (AgNPs), and 80 µg/mL (Ag–ZnONPs-K), while Ag–ZnONPs showed no cytotoxicity within the tested range. In post-treatment assays, Ag–ZnONPs achieved 95% viral inhibition at 120 µg/mL (SI = 12), comparable to ZnONPs (97% at 300 µg/mL, SI = 13) and higher than AgNPs (83% at 25 µg/mL). These results demonstrate strong in vitro antiviral activity of Ag–ZnONPs against HSV-1, particularly in post-infection conditions.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-51618-4.

Introduction

Herpes simplex virus type 1 (HSV-1) is one of the most common human pathogens with a global prevalence. HSV-1 mainly causes oral and ocular herpes, herpes encephalitis, neonatal infections, and skin infections, and is transmitted through direct contact with infected wounds, viral droplets, or secretions¹. Currently, the first-line treatment for HSV-1 is the nucleoside analogs acyclovir (ACV), famciclovir (FCV), and valacyclovir (VCV), which inhibit viral replication by inhibiting viral DNA polymerase². Due to frequent relapses of the disease and non-completion of the treatment regimen, HSV-1 can develop drug resistance, which is caused by mutations in the thymidine kinase and DNA polymerase genes of the virus³. However, in addition to the challenges caused by drug resistance, the use of these drugs can lead to side effects such as headache, nausea, and even serious complications such as nephrotoxicity and electrolyte imbalance^4,5. These challenges highlight the need to develop new and potent therapeutic approaches to combat HSV-1.

In recent decades, nanotechnology has become a promising area in the synthesis of diverse nanoparticles, especially metal and metal oxide nanoparticles, to interact with biological agents and combat a wide range of human pathogens^6–8. Among them, zinc oxide nanoparticles (ZnONPs) have been recognized as safe by the Food and Drug Administration (FDA) and can be widely used in the cosmetic and medical industries. Research has demonstrated that ZnONPs possess broad-spectrum antiviral activity, particularly against viruses such as rhinovirus, human immunodeficiency virus, influenza, and HSV^9–11. These nanoparticles prevent viruses from binding to host cell receptors, ultimately preventing viruses from entering the host cell. In addition, by producing reactive oxygen species (ROS) under certain conditions, they can damage the genetic material of viruses and effectively inactivate them¹².

Silver nanoparticles (AgNPs) are another highly valuable type of metal nanoparticles due to their exceptional antimicrobial and antiviral properties. Similar to ZnONPs, AgNPs can also produce ROS. AgNPs interact with the lipid membrane or surface proteins of viruses, changing the shape of viral particles and preventing them from binding to and entering cells. They can also prevent viral replication, proliferation, and spread by binding to the viral genome^13,14. Given the distinct antiviral properties of ZnONPs and AgNPs, a combination of these materials may exhibit a synergistic effect. Together, they can enhance each other’s antioxidant properties and provide a larger contact surface with the virus, preventing virus entry and replication or disrupting viral structures. This synergistic potential requires further investigation and research¹⁵.

The synthesis of nanoparticles is carried out by various methods, including physical and chemical processes, which unfortunately cause serious environmental concerns with high energy consumption and the use of hazardous chemical materials. In response to these problems, green synthesis methods that utilize natural resources, minimize the use of harmful chemicals, and improve energy consumption have recently attracted much attention^16,17.

Despite the promising antiviral properties of ZnONPs and AgNPs individually, limited research has explored their synergistic effects in nanocomposite form against HSV-1, particularly using eco-friendly green synthesis methods. Previous studies have primarily focused on chemically synthesized nanoparticles or their activity against other viruses, such as SARS-CoV-2 or influenza, leaving a gap in understanding their potential for HSV-1-specific applications. HSV-1 specifically matters due to its high global prevalence (affecting over 3.7 billion people under age 50), lifelong latency leading to recurrent infections, increasing drug resistance to conventional treatments like acyclovir, and associated complications including encephalitis and neonatal herpes, which pose significant public health burdens. This study fills this gap by synthesizing Ag-ZnONPs via a green method using Nigella sativa aqueous extract, evaluating their antiviral efficacy against HSV-1 in both virucidal and post-treatment assays, and comparing them to individual components. By doing so, we provide a novel, environmentally sustainable alternative that addresses drug resistance and side effects while enhancing antiviral potency through synergy. Therefore, we hypothesize that the Ag–ZnONPs nanocomposite synthesized via a green method will exhibit enhanced antiviral activity against HSV-1 compared to its individual components due to potential synergistic effects.

Materials and methods

Synthesis of ZnONPs, AgNPs, and Ag-ZnONPs

Nigella sativa seeds were purchased from the regional market and were certified by the Herbarium of Islamic Azad University of Tehran. To obtain the water-based extract, 60 g of seed powder were mixed with 600 mL of double-distilled water and heated at 80 °C for 30 min. The resulting solution was then centrifuged at 2000 rpm, and the supernatant was passed through Whatman No. 1 filter paper to eliminate impurities.

For the synthesis of ZnONPs, based on the reference article with slight modifications to the conditions,100 ml of a 90 mM zinc acetate dihydrate (Zn(CH₃CO₂)₂·2H2O, Merck, Germany) solution was placed at 60 °C in a heater stirrer, and 40 ml of black seed aqueous extract was added dropwise while stirring vigorously. The pH of the solution was brought to 12 by adding a 5 M NaOH solution. After 30 min, first, a color change and then the precipitation of ZnONPs were observed. After washing twice by means of distilled water, the precipitate was oven-dried at 80 °C and collected¹⁸.

The method for synthesizing AgNPs was modified slightly in the incubation process based on the referenced article. Briefly, 100 mL of 1 mM silver nitrate (AgNO3, Merck, Germany) solution was heated to 80 °C on a heater stirrer, and 10 mL of black seed extract was added dropwise. The mixture was incubated at room temperature without pH adjustment. After 4 h, first, the color change and then the precipitation of AgNPs were observed. After washing twice, the sediment was dried at 80˚C and collected¹⁹.

To disperse Ag on zinc oxide, this procedure was modified based on the reference article with slight adjustments. A total of 0.1 g of synthesized ZnONPs was suspended in 3 mL of distilled water via sonication and added to 100 mL of 1 mM AgNO3 solution at 80 °C while being strongly stirred. The solution’s pH was modified to 9 using 5 M NaOH. A shift in color from cream to brown followed by precipitation was observed. The residue was washed twice and dried at 80 °C to obtain the Ag-ZnONPs. Additionally, to remove any possible impurities from the plant extract, a portion of the precipitate was subsequently calcined at 400 °C for 2 h in a furnace, resulting in the Ag-ZnONPs-K. Both nanoparticle types were collected and used for further analysis²⁰.

Characterization of nanoparticles

Characterization of the nanoparticles was performed using several techniques. X-ray diffraction (XRD, Philips PW1730) confirmed the crystal structure, while UV-visible spectrophotometry (Thermo Scientific Biomate 5) verified the synthesis. Fourier transform infrared (FTIR) spectroscopy (Thermo Scientific Nicolet Avatar 380) identified surface functional groups. Zeta potential analysis (Horiba SZ-100) was used to determine the surface charge, while morphological and elemental characterization was performed using FESEM-EDX (TESCAN MIRA4). Transmission electron microscopy (TEM, Philips Electron Microscopes 208 S) confirmed Ag dispersed on ZnONPs.

Cell line and virus

Vero cells, sourced from African green monkey kidney tissue, were obtained from the Virology Group, Iran University of Medical Sciences (Tehran, Iran). Were grown in high-glucose Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Invitrogen, USA). The growth medium was supplemented in the presence of 10% heat-inactivated fetal bovine serum (FBS; Gibco, Invitrogen, USA), 2 mM L-glutamine (Merck, Germany), and 100 U/mL penicillin plus 100 µg/mL streptomycin (Sigma-Aldrich, USA). The cells were incubated at 37 °C in a 5% CO₂ atmosphere. To assess antiviral properties, the study used the HSV-1 KOS strain, and its replication in the Vero cells was monitored. The Reed and Muench method was used to determine viral titer²¹, measuring the TCID₅₀/mL. The viral samples were then aliquoted and stored in sterile microtubes at −70 °C for subsequent experiments.

Cytotoxicity assay

The cytotoxicity of each synthesized nanoparticle was assessed in Vero cells. The cells were plated in a 96-well microtiter plate (Sorfa Life Science, China) at a density of 1 × 10⁴ cells per well and incubated for 24 h at 37 °C with 5% CO₂. Afterward, the cells were treated with various concentrations of nanoparticles and incubated at 37 °C with 5% CO₂ for an additional 48 h. Following incubation, the medium was removed, and an aliquot of 10-µL MTT solution (5 mg/mL; Bio-Idea, Iran) was dispensed into each well. 3-hour incubation at 37 °C was performed for the plate. The formazan crystals were subsequently dissolved in dimethyl sulfoxide (DMSO; Bio-Idea, Iran), and the absorbance was measured at 550 nm using a microplate reader (Hiperion MPR 4+, Germany). Cell viability in each well was expressed as a percentage relative to the untreated control group.

Antiviral activity assessment

Virucidal assay

To evaluate virucidal activity, 100 µL of each nanoparticle at concentrations ≤ CC₂₀ (20% cytotoxic concentration) was mixed with 100 µL of HSV-1 suspension (100 TCID₅₀/mL) and placed in a humidified incubator at 37 °C with 5% CO₂ for 3 h. At the same time, 100 µL of 100 TCID₅₀/mL virus suspension was incubated with nanoparticle-free culture medium serving as the virus control. Vero cell monolayers were subsequently exposed to the mixtures and incubated for an additional hour at 37 °C. After that, the supernatant was discarded, and the cells were washed three times with PBS to remove unbound viruses. Fresh DMEM with 2% FBS was added, and the cells were incubated at 37 °C for 48 h. Each experiment was performed in three independent biological replicates, and within each experiment, all conditions were tested in triplicate.

Cell post-treatment assay

A total of 1 × 10⁴ Vero cells were added to each well of a 96-well plate and incubated for 24 h at 37 °C under 5% CO₂. After removing the medium, HSV-1 (100 TCID₅₀/mL) was added to the cells, and they were incubated at 37 °C for 1 h. After removing the virus by aspiration, Cells were gently washed with phosphate-buffered saline (PBS) to eliminate any unbound virus. Next, different non-cytotoxic concentrations (≤ CC₂₀) of nanoparticles were added to the infected cells. Acyclovir was used as a positive antiviral control in the post-treatment assay under the same experimental conditions as the tested nanoparticles. The drug was evaluated at concentrations of 25, 75, and 100 µg/mL, and viral load reduction was quantified using real-time PCR. At the same time, infected cells in other wells were treated with only cell culture media as virus controls. The plate was placed in an incubator at 37 °C in a 5% CO₂ humidified atmosphere for 48 h until the virus controls exhibited typical cytopathic effects (CPEs). All experiments were conducted as three independent biological replicates, with each condition tested in triplicate within each experiment.

Quantitative real-time PCR (qPCR) analysis

The effect of ZnONPs, AgNPs, Ag-ZnONPs, and Ag-ZnONPs-K on HSV-1 infection in Vero cells was assessed using qPCR. The supernatants of virus-infected Vero cells, obtained from virucidal and post-treatment assays, were used to extract HSV-1 genomic DNA. The extraction process was carried out using the Nucleic acid extraction kit for viruses (BehGene Biotechnology, Iran) in accordance with the manufacturer’s guidelines. The UL30 gene in HSV-1 was targeted using specific sequences. The forward primer sequence is 5’-ATCGGCGAGTACTGCATACA-3’, the reverse primer sequence is 5’-GAGCTCCAGATGGGCAA-3’, and the probe sequence is 5’-HEX-ATTCCCTGCTGTGTGGGCCA- BHQ1-3’. The primer length is 75 bp²².

Real-time PCR was carried out in a total volume of 25 µL, comprising 12.5 µL of RealQ Plus 2x Master Mix for Probe, without ROX™ (Ampliqon, Denmark (, 2 µL of the forward primer, 1 µL of the reverse primer, 1 µL of the probe, 5 µL of template DNA, and 3.5 µL of nuclease-free water. The reactions were performed on the Rotor-Gene Q system (Qiagen, Germany) under the following thermal cycling conditions: an initial denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s.

To establish the reference standard, the pUC57 vector carrying the desired viral sequence was used. We generated a series of ten-fold dilutions (from 10⁻¹ to 10⁻¹⁰) of the synthesized plasmid to create a standard curve in conjunction with the experimental samples. Plasmid copy number was calculated with the aid of a web-based DNA copy number calculator from Technology Networks. The qPCR assay reached a detection limit (LOD) of 3 copies per microliter (µL). All assays were conducted in triplicate, with both positive and negative controls included in each experimental run.

Selectivity Index (SI) calculation

The selectivity index (SI) was calculated to evaluate the safety and antiviral efficacy of the synthesized nanoparticles against HSV-1 in Vero cells. SI values were obtained by dividing the 50% cytotoxic concentration (CC₅₀) by the 50% inhibitory concentration (IC₅₀) derived from the cytotoxicity and antiviral assays, respectively, using the following formula:

Statistical analysis

All experiments were performed as three independent biological replicates. Within each independent experiment, all conditions were tested in triplicate (technical replicates). Therefore, each data point represents the mean ± standard deviation (SD) of nine measurements (n = 9). The distribution of the data were analyzed using the Kolmogorov-Smirnov test. Based on the results of this test, Analysis of Variance (ANOVA) was conducted to examine the effect of different concentrations of nanoparticles on viral inhibition. Subsequently, a Tukey post-hoc test was employed to identify inter-group differences. A p-value < 0.05 was considered statistically significant. All statistical analyses were carried out using SPSS 27.0 software (SPSS Inc., Chicago, Illinois, USA; https://www.ibm.com/products/spss-statics), and graphical representations were produced using GraphPad Prism (version 9, graphpad software, San Diego, CA, USA; http//.graphpad.com) software. Error bars in all graphs represent mean ± standard deviation (SD).

Results

Characterization of nanoparticles

After observing the color change and precipitation of nanoparticles, the detection of the optical absorption peak in the expected range through UV-Vis analysis confirmed the successful synthesis of nanoparticles. For ZnONPs, the expected range is 350–380 nm according to the references. In this study, the peak was observed at 361 nm, indicating efficient synthesis^18,23,24.

AgNPs exhibit localized surface plasmon resonance (LSPR), and the peak of surface plasmon resonance (SPR) in these nanoparticles depends on their size and shape. Typically, the SPR peak in AgNPs occurs in the range of 400–450 nm. In our synthesized nanoparticles, it was observed at 414 nm; verify the synthesis of these nanoparticles^19,25,26. For Ag-ZnONPs, the distinct peak at 416 nm confirms the effective dispersing of Ag in the ZnO matrix. This peak is related to the Ag surface plasmon resonance (LSPR). The strong LSPR peak associated with Ag effectively overshadows any absorption peaks that may arise from ZnO, rendering them undetectable in this spectral region^27–29. (Fig. 1)

Fig. 1.

UV–Vis spectra corresponding to the synthesized nanoparticles: A: ZnONPs exhibit a peak at 361 nm. B: AgNPs display a characteristic SPR peak at 414 nm, indicating nanoscale silver formation. C: Ag-ZnONPs show a distinct peak at 416 nm, indicating successful Ag dispersion in the ZnO matrix via surface plasmon resonance (LSPR), which dominates the spectrum.

Zeta potential analysis was performed to evaluate the surface charge and colloidal stability of the nanoparticle suspensions. In general, when the zeta potential values for positive or negative nanoparticles are greater than 30 mV, it can be said that the electrostatic repulsion is strong and that we have a stable system³⁰. As shown in the Fig. 2, in this study, the measured zeta potential values for AgNPs are − 25.5 mV, which indicates a strong negative charge, and due to the strong repulsive forces between the particles, this nanoparticle shows relatively good stability. For ZnONPs and Ag-ZnONPs, the measured values were − 15.5 mV and − 17.9 mV, respectively. These values indicate moderate stability because the negative value indicates electrostatic repulsion that helps prevent aggregation^31,32.

Fig. 2.

Zeta potential characterization of the nanoparticle samples: A: ZnONPs exhibit − 15.5 mV, indicating moderate colloidal stability. B: AgNPs show − 25.5 mV, reflecting strong negative charge and relatively high stability. C: Ag-ZnONPs have − 17.9 mV, suggesting moderate stability with sufficient electrostatic repulsion.

In the FTIR spectrum of ZnONPs, the peak at 3429.78 cm⁻¹ corresponds to hydroxyl (-OH) or phenol groups, while the peak at 2924.52 cm⁻¹ represents C-H stretching vibrations of alkanes, indicating the presence of organic molecules from the plant extract. These functional groups act as reducing agents to convert Zn²⁺ ions into ZnO and serve as capping agents, preventing aggregation and stabilizing the nanoparticles^33–36. The band observed at 2343.09 cm⁻¹, assigned to O = C=O stretching, likely corresponds to adsorbed CO₂ or carbonyl-containing molecules, which contribute to surface passivation of the nanoparticles³⁷.

Additional peaks at 1519.63 cm⁻¹ and 1071.26 cm⁻¹ are attributed to C = C stretching, and C-O stretching, respectively, play a role in surface stabilization and control over particle morphology^34,38. The distinct peak at 410.76 cm⁻¹ corresponds to Zn-O stretching, confirming the favorable synthesis of ZnONPs^34,38.

For AgNPs, the peaks at 3285.14 cm⁻¹ and 2923.56 cm⁻¹ correspond to O-H groups from phenolic compounds and C-H stretching in alkanes, respectively. These organic functional groups play a vital role in reducing Ag ions (Ag⁺ to Ag⁰) and stabilizing the nanoparticles^25,36,39,40. Peaks at 1743.33 cm⁻¹, 1649.8 cm⁻¹, and 1387.53 cm⁻¹ are assigned to C = O stretching, C = C or N-H bending, and C-H bending or nitro groups, respectively, indicating the presence of bioactive compounds that aid in nanoparticle stabilization^36,41,42. The peaks at 1238.08 cm⁻¹ and 1038.48 cm⁻¹ correspond to C-O bonds from plant-derived organic compounds, further enhancing nanoparticle stability³⁶. The absorption bands observed at 872.6 cm⁻¹ and 545.76 cm⁻¹ are characteristic of Ag–O stretching and providing strong evidence for the successful formation of silver-oxygen bonds. This finding indicates a minor surface oxidation of the AgNPs, which is expected during green synthesis because oxygen-containing functional groups (such as hydroxyl and carbonyl groups) in the plant extract can interact with the nanoparticle surface. Nevertheless, the particle cores remain metallic Ag⁰, as the phytochemicals in the extract mainly function as reducing and stabilizing agents, facilitating the conversion of Ag⁺ ions to metallic silver nanoparticles^43–46. (Fig. 3).

Fig. 3.

Fourier Transform Infrared (FTIR) Spectral Characterization of Synthesized Nanoparticles. (A) ZnO nanoparticles (ZnONPs), (B) Silver nanoparticles (AgNPs), (C) Silver-doped ZnO nanoparticles (Ag-ZnONPs), and (D) Overlaid FTIR spectra for comparative analysis of functional groups and chemical bonding in the synthesized nanoparticle formulations.

For Ag-ZnONPs, the peaks at 3382.53 cm⁻¹ and 2925.48 cm⁻¹ indicate the presence of hydroxyl (-OH) or amino (-NH) groups and C-H stretching from alkanes or fatty acids, respectively. These functional groups, originating from plant biomolecules, contribute to nanoparticle reduction and stabilization^{34,38,40,47,48}. The peak at 2088.53 cm⁻¹ suggests possible chemical interactions or bond formation between Ag and ZnO components^45,49,50. Additionally, the peak at 1654.62 cm⁻¹ corresponds to C = C bonds or N-H bending in aromatic or amide compounds, which likely enhance nanoparticle stability⁵¹. Finally, the incorporation of silver into the ZnO matrix is confirmed by the peak at 705.82 cm⁻¹, associated with Zn-O and Ag-O bonds^45,52(Fig. 3).

X-ray diffraction (XRD) analysis of all three synthesized nanoparticles was carried out using Cu-Kα radiation (λ = 1.5406 Å) at varying 2θ values, and the data were analyzed using the High Score Plus (version 3.0, Malvern Panalytical; http://www.malvernpanalytical.com.) software. The analysis confirmed their crystalline structure, while the absence of additional peaks indicated their purity.

Based on JCPDS card no. 36-1451, ZnONPs exhibited a hexagonal wurtzite crystal structural. typical diffraction peaks at 31.81°, 34.63°, and 36.35°, corresponding to the (100), (002), and (101) planes, respectively. Additional peaks were observed at 47.6°, 56.84°, 62.94°, 68.1°, 69.2°, 72.6°, and 77.1°, which are associated with the (102), (110), (103), (112), (201), (004), and (202) planes, demonstrate the crystalline nature of the synthesized nanoparticles^53–55(Fig. 4).

Fig. 4.

Crystallographic patterns of the three nanoparticle systems: A: ZnONPs show hexagonal wurtzite peaks. B: AgNPs exhibit FCC peaks with minor surface oxidation. C: Ag-ZnONPs display ZnO wurtzite peaks along with reflections of Ag, confirming successful Ag dispersion.

The XRD pattern of the AgNPs, as indexed to JCPDS card No. 01-075-1532, confirms that the synthesized nanoparticles exhibit a cubic crystal structure. Distinct and characteristic peaks were observed at 32.44°, 46.4°, and 54.9° that are related to (111), (200), and (220), respectively. The remaining peaks, as shown in the Fig. 4, further validate the face-centered cubic (FCC) structure of the silver nanoparticles, while the presence of minor reflections suggests slight surface oxidation of the particles, which is also consistent with the FTIR analysis^56–58.

For Ag-ZnONPs, the XRD pattern reveals characteristic peaks corresponding to the hexagonal wurtzite structure of ZnO. The prominent peaks at 31.8°, 34.4°, and 36.3° are related to the (100), (002), and (101) crystallographic planes of ZnO, which are typical and significant for verifying the ZnO phase. These peaks align with the JCPDS card No. 00-036-1451. Additionally, peaks at 36.6°, 47.5°, 56.6°, and 62.8° correspond to the (111), (200), (102), and (110) planes of ZnO, further supporting the presence of ZnO in the sample. Furthermore, peaks at 36.3° and 36.6° can be attributed to silver oxide, specifically corresponding to the (111) and (200) planes of silver oxide, as indicated by JCPDS card No. 00-001-1041. The presence of silver (Ag) is further confirmed by TEM analysis, which shows silver nanoparticles dispersed within the ZnO matrix. This combination of XRD and TEM results clearly supports the effective incorporation of silver into the ZnO structure^59–61 (Fig. 4).

In this study, the morphological structures of the synthesized nanoparticles were examined using scanning electron microscopy (SEM), as shown in Fig. 5, and their elemental compositions were evaluated using energy dispersive X-ray (EDX) analysis, as shown in Fig. 6. Transmission electron microscopy (TEM) analysis was also performed only for Ag-ZnONPs. SEM images of ZnONPs show that they have a spherical morphology with an average size of approximately42 nm. This indicates a uniform crystallization process. However, slight aggregation is noticeable in certain regions, likely resulting from the nanoparticles high surface energy⁶².

Fig. 5.

Morphological characterization of nanoparticles via FESEM and TEM: A, B: ZnONPs exhibit spherical shapes with slight aggregation, consistent with uniform crystallization. C, D: AgNPs display well-distributed particles (~ 50 nm) with minor aggregation. E, F: Ag-ZnONPs show successful dispersion of Ag on the ZnO matrix (FESEM: E, TEM: F).

Fig. 6.

Energy-dispersive X-ray (EDX) spectra of the nanoparticles Shows the elemental composition of the synthesized nanoparticles. A: ZnONPs consist of Zn (76.38%) and O (23.62%). B: AgNPs contain Ag (62.05%) and O (37.95%). C: Ag-ZnONPs display Zn (53.46%), O (32.67%), and Ag (13.87%), confirming dispersion of silver into the ZnO matrix.

EDX analysis of this nanoparticle shows 76.38% Zn at ~ 8.6 keV and 23.62% O at ~ 0.5 keV, showed the expected stoichiometry of ZnO. These results are in line with previous studies for the synthesis of ZnONPs^55,63. SEM images of AgNPs show a uniform distribution of quasi-spherical particles with an average size of 50 nm validate the efficient synthesis. However, minor aggregation is also visible in the images, likely due to the high surface energy of the nanoparticles⁶⁴. EDX analysis reveals that Ag constitutes approximately 62.05% of the total composition, while oxygen (O) accounts for approximately 37.95%. The silver (Ag) signal appears at approximately 3 keV, and the oxygen (O) signal at approximately 0.5 keV, which is consistent with the results of our study^65,66. TEM and SEM analysis of Ag-ZnONPs provides clear evidence of the favorable dispersion of silver on the ZnO matrix. The approximate size of the nanoparticles is reported to be 35 nm, which is desirable and expected. In the EDX analysis of Ag-ZnO, the oxygen signal was detected at approximately 0.5 keV, with a weight% of 32.67%, and zinc at approximately 8.6 keV with a weight% of 53.46%, confirming ZnO as the main matrix of the nanoparticles. Ag, detected at 3 keV, showed a weight% of 13.87% and an atomic percentage of 4.30%, verified its presence in the ZnO matrix. The lower atomic percentage of Ag compared to its weight% can be attributed to its higher atomic weight compared to zinc and oxygen. These results are in line with previous studies^60,67. A summary of the physicochemical properties of the synthesized nanoparticles is presented in Table 1.

Table 1.

Summary of physicochemical characteristics of synthesized nanoparticles.

Nanoparticle (NP)	Size (nm)	Zeta Potential (mV)	Structure	Morphology
ZnO NPs	~ 42 nm	−15.5	Hexagonal Wurtzite	Spherical
Ag NPs	~ 50 nm	−25.5	Cubic	Quasi-spherical
Ag-ZnO NPs	~ 35 nm	−17.9	ZnO Hexagonal Wurtzite with Ag oxide presence	Quasi-spherical

Cytotoxicity effects of nanomaterials

The MTT assay results demonstrated that cell viability decreased to 20% (CC₂₀) and 50% (CC₅₀) in comparison to control cells when ZnONPs were applied at concentrations of 500 and 700 µg/mL, respectively. AgNPs exhibited CC₂₀ and CC₅₀ values at 30 and 35 µg/mL, respectively. For Ag-ZnONPs, cell viability decreased to 20% (CC₂₀) at a concentration of 140 µg/mL; however, a 50% reduction in cell viability (CC₅₀) was not observed even at the highest tested concentration. Ag-ZnONPs-K demonstrated CC₂₀ and CC₅₀ values at 70 and 80 µg/mL, respectively (Fig. 7). Therefore, at concentrations ≤ CC₂₀, the virucidal potency of ZnONPs, AgNPs, Ag-ZnONPs, and Ag-ZnONPs-K against HSV-1 was investigated. Additionally, in the post-treatment assay, the ability of these nanoparticles to inhibit HSV-1 replication in Vero cells was assessed at concentrations ≤ CC₂₀.

Fig. 7.

Cytotoxicity of Nanoparticles on Vero cells. ZnONPs, AgNPs, Ag-ZnONPs, and Ag-ZnONPs-K reduced cell viability at different concentrations.

Virucidal assay

The results of the virucidal assay demonstrated that incubation of HSV-1 with AgNPs, ZnONPs, Ag-ZnONPs, and Ag-ZnONPs-K for 3 h was associated with a concentration-dependent reduction in virus-induced cytopathic effects (CPE) (Fig. 8).

Fig. 8.

Inhibition rate of HSV-1 by nanoparticles in virucidal and post-infection assays at different concentrations. All nanoparticles showed concentration-dependent antiviral effects in virucidal and post-infection assays, with ZnONPs and Ag-ZnONPs achieving the highest inhibition rates. Acyclovir served as a positive control in post-infection assays.

Furthermore, the Real-Time PCR analysis showed that increasing the nanoparticles concentration resulted in a corresponding decrease in viral load during the 3-hour incubation period. Based on the viral load data obtained by the Real-Time PCR technique and comparisons with the virus control group, the levels of viral inhibition were calculated. In the case of AgNPs, incubation at concentrations of 5, 15, and 25 µg/mL resulted in viral inhibition rates of > 1%, 35%, and 59%, respectively (P = 0.006). ZnONPs exhibited higher antiviral activity, with inhibition rates of 46%, 54%, and 57% at concentrations of 100, 200, and 300 µg/mL, respectively (P < 0.001). For Ag-ZnONPs, incubation at concentrations of 40, 80, and 120 µg/mL led to inhibition rates of 36%, 49%, and 64%, respectively (P < 0.001). Similarly, Ag-ZnONPs-K demonstrated concentration-dependent inhibition of viral replication, with inhibition rates of 48%, 57%, and 69% at concentrations of 20, 40, and 60 µg/mL, respectively (P = 0.005) (Fig. 8).

Post-treatment assay

The addition of non-toxic concentrations of the synthesized nanoparticles significantly reduced HSV-1-induced CPEs in infected cells (Fig. 8) (Fig. 9 ). Real-time PCR analysis further revealed that AgNPs at concentrations of 5, 15, and 25 µg/mL significantly reduced HSV-1 genomic DNA copies, with viral inhibition rates of 73%, 81%, and 83%, respectively (P < 0.001).

Fig. 9.

Inhibition of HSV-1-induced cytopathic effects (CPEs) in Vero cells following treatment with nanoparticles at different concentrations in the post-treatment assay. A: Ag-ZnONPs at 120 µg/mL, B: Virus control (VC), C: Ag-ZnO.K at 60 µg/mL, D: VC.

ZnONPs showed viral inhibition rates of 88%, 95%, and 97% at concentrations of 100, 200, and 300 µg/mL, respectively (P < 0.001). In parallel, Ag-ZnONPs achieved inhibition rates of 87%, 90%, and 95% at concentrations of 40, 80, and 120 µg/mL, respectively (P < 0.001).

In the case of Ag-ZnONPs-K, the lowest tested concentration (20 µg/mL) exhibited minimal inhibition (1%), while increasing the concentration to 40 µg/mL and 60 µg/mL significantly enhanced the viral inhibition rates to 69% and 80%, respectively (P < 0.001) (Table 2). In parallel Compared to the synthesized nanoparticles, acyclovir demonstrated a higher antiviral efficacy at all tested concentrations, achieving near-complete viral inhibition at 100 µg/mL. However, certain nanoparticle formulations showed considerable inhibitory effects, suggesting their potential as alternative or complementary antiviral agents. Although acyclovir was included as a positive control, IC₅₀ and selectivity index (SI) values were not calculated for this compound, which may limit direct quantitative comparison with the nanoparticles. (Fig. 8) (Table 3) (Figs. 9 and 10).

Table 2.

Comparison of the antiviral activity between different concentrations of Nanoparticles against HSV-1 in the virucidal and post-treatment assay Shows concentration-dependent inhibition of HSV-1 in virucidal and post-treatment assays. AgNPs inhibited the virus up to ~ 59% (virucidal) and ~ 83% (post-treatment). ZnONPs reached ~ 57% and ~ 97%, Ag-ZnONPs up to ~ 64% and ~ 95%, and Ag-ZnONPs-K showed up to ~ 69% and ~ 80% inhibition, respectively, indicating enhanced antiviral activity at higher concentrations.

Setting	Virucidal				Post infection
Ag	5 (µg/ml)	15 (µg/ml)	25 (µg/ml)	p-value	5 (µg/ml)	15 (µg/ml)	25 (µg/ml)	p-value
Inhibition %	0.60 ± 0.57	34.70 ± 18.38	59.25 ± 16.19*	0.006	73.10 ± 9.06*	80.90 ± 5.14*	83.30 ± 3.48*	< 0.001
ZnO	100 (µg/ml)	200 (µg/ml)	300 (µg/ml)	p-value (µg/ml)	100 (µg/ml)	200 (µg/ml)	300 (µg/ml)	p-value
Inhibition %	45.60 ± 5.09*	54.10 ± 1.13*	56.50 ± 1.84*	< 0.001	88.17 ± 4.23*	94.60 ± 0.87*	96.73 ± 0.71*	< 0.001
ZnO-Ag	40 (µg/ml)	80 (µg/ml)	120 (µg/ml)	p-value	40 (µg/ml)	80 (µg/ml)	120 (µg/ml)	p-value
Inhibition %	35.95 ± 15.77*	49.05 ± 3.32*	63.75 ± 3.61*ᵟ	< 0.001	87.03 ± 2.12*	90.00 ± 1.48*	94.87 ± 2.20*ᵟ	< 0.001
ZnO-Ag (K)	20 (µg/ml)	40 (µg/ml)	60 (µg/ml)	p-value	20 (µg/ml)	40 (µg/ml)	60 (µg/ml)	p-value
Inhibition %	47.90 ± 9.33*	56.90 ± 16.55*	69.20 ± 19.80*	0.005	1.07 ± 0.38	69.03 ± 17.69*ᶲ	80.20 ± 3.21*ᶲ	< 0.001

Table 3.

Acyclovir’s dose-dependent effect on HSV-1 viral load reduction in post treatment assay.

Material	Compared groups	Average difference (%)	CI (Confidence interval) (%)	P-value
Acyclovir	25 µg/mL vs. VC	93.4	92-94.7	< 0.001*
	25 µg/mL vs.75 µg/mL	5.1	3.2–6.9	0.002*
	25 µg/mL vs.100 µg/mL	6.6	4.5–8.6	0.005*
	75 µg/mL vs.100 µg/mL	1.5	0.2–2.8	< 0.001*
	75 µg/mL vs. VC	98.5	97.1–99.7	0.03*
	100 µg/mL vs. VC	100	96.7-103.2	< 0.001*

Fig. 10.

Selectivity Index (SI) values of ZnO NPs, Ag NPs, ZnO–Ag NPs and ZnO–Ag (K) NPs in the virucidal and post-treatment assay against HSV-1.

Selectivity Index (SI) calculation

Among the tested nanoparticles, ZnO NPs and ZnO-Ag NPs (non-calcined) exhibit the most favorable selectivity profiles in the post-treatment assay (SI = 13 and 12, respectively) (Table 4), suggesting a relatively wide therapeutic window for inhibiting HSV-1 replication with limited cytotoxicity to Vero cells. Ag NPs show particularly strong selectivity in the post-treatment approach (SI = 11), consistent with its high inhibition even at low concentrations. Calcination (ZnO-Ag (K) NPs) appears to reduce overall biocompatibility (lower CC₅₀), resulting in lower SI values (Fig. 10).

Table 4.

Selectivity Index of the synthesized nanoparticles against HSV-1 in Vero cells.

Nanomaterial	Assay type	CC₅₀ (µg/mL)	IC₅₀ (µg/mL)	Selectivity Index (SI)
Ag NPs	Virucidal	33	21	1.6
	Post treatment	33	3	11
ZnO NPs	Virucidal	650	152	4.3
	Post treatment	650	50	13
ZnO-Ag NPs	Virucidal	240	83	2.9
	Post treatment	240	20	12
ZnO-Ag (K) NPs	Virucidal	88	35	2.5
	Post treatment	88	35	2.5

Discussion

According to the findings of the present study, green-synthesized ZnONPs, AgNPs, Ag-ZnONPs, and Ag-ZnONPs-K demonstrated measurable antiviral activity against HSV-1, with notable differences observed between nanoparticle types and treatment conditions. Among the tested materials, ZnO nanoparticles and Ag–ZnO nanocomposites showed relatively higher selectivity indices compared to Ag nanoparticles, particularly in post-treatment assays, suggesting improved antiviral performance under intracellular conditions. These findings are generally consistent with previous studies on metal and metal oxide nanoparticles. For instance, Pan et al. reported that AgNPs inhibit HSV-1 primarily through interactions with viral surface glycoproteins, thereby blocking viral attachment and entry. However, in the present study, AgNPs exhibited comparatively lower SI values than ZnO-based formulations, suggesting a narrower therapeutic window. This discrepancy may be attributed to differences in particle size, green synthesis approach, and surface chemistry, all of which critically influence nanoparticle–virus interactions and cellular uptake⁶⁸. Similarly, Melk et al. demonstrated that green-synthesized ZnONPs exert enhanced antiviral effects when applied after viral entry. In agreement with these findings, ZnONPs in the present study showed greater efficacy in post-treatment conditions compared to virucidal assays, suggesting that their antiviral activity may extend beyond viral attachment and involve intracellular stages of the viral life cycle. A plausible explanation is that ZnONPs may interfere with viral replication processes, potentially through oxidative stress-mediated damage and disruption of viral protein synthesis⁶⁹. The superior performance of ZnONPs compared to AgNPs may be attributed to their ability to generate reactive oxygen species (ROS) and release Zn²⁺ ions. These ions have been reported to inhibit viral polymerase activity and interfere with replication. In addition, the relatively smaller particle size and favorable surface charge of ZnONPs synthesized in this study may enhance cellular internalization, thereby improving antiviral efficiency⁷⁰. The Ag–ZnO nanocomposites evaluated in this study exhibited intermediate to high antiviral activity, supporting the hypothesis that combining Ag and ZnO may result in synergistic or additive effects. Previous studies, including those by Dolatyari and Rostami, have suggested that such nanocomposites can enhance antiviral performance due to combined mechanisms of action. However, in the present study, while improved activity was observed compared to AgNPs alone, the enhancement over ZnO nanoparticles was modest, indicating that the degree of synergy may depend on synthesis conditions and compositional ratios⁷¹.

The observed superiority of post-treatment over virucidal activity suggests that the nanoparticles may act more effectively during intracellular stages of the viral life cycle rather than solely through direct viral inactivation. This observation is consistent with recent studies reporting significant antiviral effects of nanoparticles in post-infection conditions. For example, Hamidzade et al. (2024) demonstrated that a copper oxide–graphite nanocomposite significantly reduced HSV-1 viral load when applied after viral entry, indicating strong intracellular antiviral activity. Similarly, Jwaziri et al. (2025) showed that magnesium oxide nanoparticles retained potent antiviral efficacy in post-treatment assays, further supporting the involvement of intracellular mechanisms in nanoparticle-mediated viral inhibition. These findings collectively reinforce the hypothesis that the antiviral effect of nanoparticles extends beyond direct virucidal action. However, it is important to note that intracellular uptake, ROS generation, and viral protein expression were not directly assessed in the present study; therefore, these mechanistic interpretations should be considered plausible hypotheses rather than definitive conclusions^72,73. Despite the observed antiviral activity, the efficacy of the nanoparticles did not reach that of Acyclovir, which remains the gold standard antiviral agent for HSV-1 due to its specific inhibition of viral DNA polymerase. The nanoparticles evaluated in this study likely exert their effects through more indirect and multifactorial mechanisms, which may account for their comparatively lower potency⁷⁴. Overall, the findings of this study contribute to the growing body of evidence supporting the antiviral potential of metal oxide nanoparticles, particularly ZnO-based systems. We observed a concentration-dependent reduction in viral genomic copies; however, the precise mechanisms underlying the antiviral effects of these nanoparticles remain unclear. In summary, green-synthesized ZnONPs, AgNPs, Ag-ZnONPs, and Ag-ZnONPs-K exert potent, dose-dependent antiviral activity against HSV-1, with Ag incorporation significantly enhancing both virucidal and post-infection efficacy. Compared to chemical synthesis approaches such as Dolatyari et al., our green synthesis offers environmental sustainability and potentially improved safety. Despite the promising antiviral activity observed, several limitations should be acknowledged. Intracellular ROS generation and viral protein expression (e.g., HSV-1 proteins such as UL30 and gD) were not directly evaluated, and time-of-addition experiments were not performed, limiting the precise identification of the stage of viral inhibition. In addition, although extract-only and vehicle control groups were included, further studies are needed to fully separate the independent effects of Nigella sativa extract and the nanoparticles. These limitations have been clearly presented in the Discussion and framed as important considerations for future studies.Future research will focus on ROS quantification, protein-level analysis (e.g., Western blot), time-course experiments, and in vivo studies to further elucidate the antiviral mechanisms and support the translational potential of these nanomaterials.

Conclusion

This study is among the first to evaluate the antiviral activity of green-synthesized ZnONPs, AgNPs, and Ag/ZnO nanocomposites against HSV-1. Our results suggested that these nanomaterials demonstrated the ability to inhibit viral replication in a dose-dependent manner. their antiviral effects may involve interactions with viral particles and potentially blocking attachment and entry into host cells or interfering with intracellular replication. Notably, Ag/ZnO nanocomposites exhibit higher efficacy compared to individual nanoparticles. These findings support the potential of these nanomaterials as antiviral agents. However, further mechanistic studies and in vivo investigations are necessary to confirm these observations and assess their safety and therapeutic applicability.

Electronic Supplementary Material

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Author contributions

R. Babaei: Methodology, initial draft writing, data analysis. S. Daneshjoo: Methodology, nanoparticle synthesis, preparation, supervision. S. J. Kiani: Methodology, data analysis, manuscript review. A. Tavakoli: Supervision, project conceptualization, corresponding author.

Funding

This research received no external funding.

Data and code availability

All data generated or analyzed during this study are included in this published article. Raw data supporting the findings of this study are available in the Supplementary Information.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval

This study was approved by the Ethics Committee of Islamic Azad University, Tehran Medical Sciences, Tehran, Iran (Ethics code: IR.IAU.PS.REC.1403.188).