Mesoporous silica nanoparticle-mediated delivery of a synergistic 5-miRNA combination inhibits HSV-2 infection

Abstract

Herpes simplex virus type 2 (HSV-2) is a widespread sexually transmitted pathogen responsible for genital herpes and associated with serious complications, including neonatal infections and increased HIV susceptibility. Although antiviral agents such as acyclovir are available, the emergence of drug resistance and viral latency necessitate the development of novel therapeutic approaches. RNA interference has emerged as a promising strategy against HSV-2. In our previous work, we identified a combination of five host-targeting microRNAs - miR-374a, miR-181a, miR-195, miR-29b, and miR-211, that significantly inhibited HSV-2 replication through modulation of the PI3K/AKT (phosphoinositide 3-kinase/protein kinase B) signaling pathway. In the current study, we developed a mesoporous silica nanoparticle (MSN)-based delivery system to enhance the stability of this miR-Combo. The MSN formulation was characterized for particle size, polydispersity index, zeta potential and surface morphology of developed particles by transmission electron microscopy analysis. In vitro studies in THP-1 macrophages confirmed the formulation’s biocompatibility and potent antiviral activity. Moreover, in a murine model of genital HSV-2 infection, intravaginal administration of the miRNA-loaded nanoparticles (N-miR-Combo) significantly reduced viral gene expression at both transcriptional and translational levels. Furthermore, N-miR-Combo significantly suppressed HSV-2-induced pro-inflammatory mediators, while concurrently upregulating the anti-inflammatory cytokine, demonstrating dual antiviral and immunomodulatory activity relevant to HSV-2-associated immunopathology. These findings highlight the promise of MSN-mediated combinatorial miRNA delivery as an innovative and promising preclinical candidate for further therapeutic development against HSV-2 infection and antiviral resistance.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-55793-2.

Introduction

Herpes simplex virus type 2 (HSV-2) is a highly prevalent sexually transmitted pathogen and the leading cause of genital herpes¹. The World Health Organization estimates that over 520 million people aged 15-49 years are infected with HSV-2 globally, underscoring its significant public health burden². Beyond the hallmark recurrent painful genital lesions, HSV-2 infection is associated with severe complications including neonatal herpes, meningitis, and a substantially increased risk of acquiring and transmitting HIV-1³. The virus establishes latency in sensory neurons, leading to lifelong persistence and periodic reactivation, which contributes to continued viral transmission and recurrent disease episodes⁴. Current treatment options for HSV-2 rely primarily on nucleoside analogues such as acyclovir and valacyclovir, which inhibit viral DNA polymerase. Despite advances in understanding HSV-2 biology, therapeutic strategies remain largely limited to these nucleoside analogues, which neither eliminate latent reservoirs nor mitigate the excessive inflammatory responses that exacerbate genital infection^5,6. Moreover, long-term use can lead to drug resistance, especially in immunocompromised individuals, and systemic administration may result in side effects, including nephrotoxicity and gastrointestinal disturbances⁵. The lack of a prophylactic or therapeutic vaccine further exacerbates the need for novel and more effective antiviral strategies that can overcome these limitations.

In recent years, host-directed therapies have emerged as promising alternatives to conventional antiviral agents. Among them, RNA interference (RNAi) has gained considerable attention for its ability to silence specific viral or host genes involved in viral replication and pathogenesis^7,8. MicroRNAs (miRNAs), a class of small non-coding RNAs, play a pivotal role in post-transcriptional regulation of gene expression and are increasingly recognized as important modulators of viral infections. Several studies have shown that specific host miRNAs can either promote or restrict viral replication by modulating cellular signaling pathways^8,9. Recent reviews have shown the significant role of miRNAs in regulating viral infections such as SARS-CoV-2, dengue, HSV-1, EBV, HIV, and HPV. These non-coding small RNAs may enhance or suppress the course of the viruses by targeting the RNAs of the cells or viruses^10,11. miR-142 was found as the regulator of TIM-1 that contributes to SARS-CoV-2 internalization and has the possible application in the treating COVID-19 and the other viruses¹². HSV-2 infection has been often associated with other STIs like HIV and HPV¹³. In case of HPV, miR-29 was identified to target YY1 and CDK6, which are positive regulators of HPV E6/E7 expression and represent its tumor-suppressive function¹⁴. The genomes of HPV might encode viral miRNAs such as HPV-16 miR-1, miR-2, and miR-3 to regulate host genes responsible for oncogenesis¹⁵. In case of HIV infections, miR-196b and miR-1290 were found to target the HIV-1 3’ UTR, suppressing viral production and infectivity¹⁶. Utilizing endogenous or synthetic miRNAs to target host pathways hijacked by HSV-2 may offer a novel strategy to limit viral replication while potentially minimizing the risk of resistance^7,17. However, most studies focus on single miRNAs, leaving unexplored the possibility that rationally designed combinations of miRNAs could exert synergistic antiviral and immunomodulatory effects. For example, individual host miRNAs such as miR-36 and miR-138 have demonstrated efficacy in restricting HSV-2 replication in specific cell types^18,19. To date, no study has explored a rationally designed combination of miRNAs capable of simultaneously suppressing viral replication and modulating host inflammation in macrophages, key players in HSV-2 pathogenesis. In our previous work, we identified a rationally selected combination of five host-targeting miRNAs - miR-374a, miR-181a, miR-195, miR-29b, and miR-211, which exhibited significant antiviral activity against HSV-2 in vitro by modulating the PI3K/AKT signaling pathway²⁰. This combinatorial strategy enables synergistic regulation of host cellular pathways to disrupt multiple stages of the viral life cycle. Nevertheless, the clinical translation of miRNA-based therapeutics remains challenged by enzymatic degradation in biological fluids, limited cellular uptake, off-target effects, and rapid clearance from mucosal sites.

To address these limitations, we developed and characterized an MSN-based nanocarrier system for delivering the miR-Combo targeting HSV-2 infection. The formulation was extensively evaluated for its physicochemical properties and in vitro cytocompatibility. Using THP-1 cell-based infection model, we assessed the antiviral efficacy of the nanoformulation and its ability to suppress HSV-2 replication. To validate its potential in vivo efficacy, we employed a murine model of genital HSV-2 infection, where intravaginal administration of the miRNA-loaded nanoparticles resulted in significant reductions in viral gene expression at both transcriptional and translational levels.

Overall, we designed a combinatorial miRNA approach targeting host pathways important for HSV-2 replication and inflammation, and delivered it using mesoporous silica nanoparticles to overcome the barriers of vaginal administration, including nuclease degradation, mucus entrapment, and poor cellular uptake. Through this approach, we demonstrate that rationally selected host miRNAs, when stabilized and efficiently delivered, can exert dual antiviral and immunomodulatory effects in both in vitro and in vivo settings. Our findings underscore the promise of MSN-mediated combinatorial miRNA delivery as a potent and innovative preclinical strategy with therapeutic potential to combat HSV-2 infection. This host-directed, nanomedicine-based approach holds potential not only to improve clinical outcomes but also to address key challenges in current antiviral therapies, including resistance and latency.

Materials and methods

Cells and virus

Vero cells, an African green monkey kidney epithelial cell line (ATCC: CCL-81™, Manassas, VA, USA), were used for the propagation of HSV-2. The cells were cultured and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Waltham, MA, USA), 20 mM HEPES buffer, and 1% penicillin-streptomycin (Pen-Strep) antibiotic solution. The human monocyte/macrophage cell line THP-1 (ATCC: TIB-202™, ATCC, Manassas, VA, USA) was utilized for cytotoxicity assays and in vitro validation studies. THP-1 macrophages were selected as the in vitro cellular model because macrophages are among the first immune cells recruited to the site of HSV-2 infection in the genital mucosa and play a critical role in early innate antiviral defense, inflammatory cytokine production, and immunopathology²¹. Also, our prior work identified and validated the five miRNAs used in this study was conducted in THP-1 macrophages, making this the most appropriate model for direct translational follow-up²⁰. THP-1 cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 20 mM HEPES, 1 mM sodium pyruvate, and 1% Pen-Strep. For differentiation into macrophages, THP-1 cells were treated with 10 nM phorbol 12-myristate 13-acetate (PMA) for 24 hours, followed by resting in complete RPMI medium (PMA-free) for an additional 24 hours.

Herpes simplex virus type-2 (HSV-2; ATCC: VR-734D™, Manassas, VA, USA) was propagated in Vero cells maintained in 2% FBS-containing DMEM for 2-3 days. Viral titers were determined by standard plaque assay following established protocols.

Preparation of the miR-combo

The five selected miRNAs (miR-374a, miR-181a, miR-195, miR-29b, and miR-211) were obtained as miRNA mimics or inhibitors. The miRNA mimics and inhibitors were procured from Thermo Fisher Scientific (Waltham, MA, USA) with the following catalogue numbers: hsa-miR-195-5p mimic (MC10827), hsa-miR-181a-5p mimic (MC10421), hsa-miR-374a-5p mimic (MC10112), hsa-miR-29b-3p mimic (MC10103), and hsa-miR-211-5p inhibitor (MH10168). The mimics are chemically modified, double-stranded RNA molecules engineered to functionally replicate endogenous miRNAs, while the inhibitors are single-stranded oligonucleotides designed to suppress endogenous miRNA activity. Each incorporated stability-enhancing chemical modifications, including partial 2′-O-methylation and phosphorothioate backbone linkages, as provided by the manufacturer. These modifications are widely reported to improve nuclease resistance, enhance in vivo stability, and reduce off-target immune activation.

Lyophilized miRNAs were reconstituted in nuclease-free water to prepare 10 μM stock solutions and stored at –20 °C . For nanoparticle formulation, the five miRNAs were combined to generate the final miRNA mixture at the specified concentrations (miR-374a: 36.46 nM, miR-181a: 41.37 nM, miR-195: 32.44 nM, miR-29b: 15.99 nM, and miR-211: 28.05 nM), as described previously²⁰. This mixture was subsequently used for loading into mesoporous silica nanoparticles as described in Section "miRNA loading onto MSN-PEI".

A scrambled miRNA (sc-miR) mimic with no significant homology to known mammalian genes (validated using BLAST and TargetScan) was purchased from Thermo Fisher Scientific (Waltham, MA, USA) and used as a negative control. Placebo nanoparticles (without miRNA) and virus-only groups were also included as additional negative controls.

Preparation and characterization of mesoporous silica nanoparticles-based miRNA formulation

Synthesis of mesoporous silica nanoparticles (MSNs)

MSNs were synthesized using a modified sol-gel method as previously described²². Briefly, 24 mL of 25% (w/v) aqueous cetyltrimethylammonium chloride (CTAC; Sigma-Aldrich, MO, USA) was dissolved in 36 mL of deionized water, followed by the addition of 0.2 g triethanolamine (TEOA; Sigma-Aldrich, MO, USA) to prepare the surfactant solution. Simultaneously, a silica precursor solution was prepared by mixing 17.5 mL chlorobenzene with 2.5 mL tetraethyl orthosilicate (TEOS; Sigma-Aldrich, MO, USA). The surfactant and silica precursor solutions were combined, stirred at 500 rpm, and incubated at 60°C for 12 hours. The resulting MSNs were collected by centrifugation, washed sequentially with ethanol and deionized water to remove residual reagents, dried at room temperature, and finally calcined at 550°C for 5 hours to eliminate the surfactant template.

Surface functionalization and polyethylenimine (PEI) coating

To enhance colloidal stability and biocompatibility, calcined MSNs were surface-modified using 3-(trihydroxysilyl) propyl-methyl phosphonate (THPMP; Sigma-Aldrich). Sixty milligrams of MSNs were dispersed in 20 mL deionized water, followed by the addition of 10 mL of 56 mM THPMP solution. The mixture was stirred at 40°C for 2 hours. The THPMP-modified MSNs were collected by centrifugation and washed with deionized water.

For polyethylenimine coating, 150 mg of 10 kDa PEI (Sigma-Aldrich) was dissolved in 15 mL of 100 mM carbonate buffer (pH 9.6). The THPMP-modified MSNs were suspended in the PEI solution and stirred at room temperature for 4 hours to facilitate electrostatic interaction. The resulting PEI-coated nanoparticles (MSN-PEI) were collected by centrifugation, washed thoroughly with water, and air-dried.

miRNA loading onto MSN-PEI

The miR-Combo comprising miR-374a, miR-181a, miR-195, miR-29b, and miR-211 was added at a final concentration of ~ 150 nM to an MSN-PEI suspension (5 mg/mL in phosphate buffer, pH 7.4). The mixture was incubated at room temperature for 30 minutes to allow electrostatic interaction between the miRNAs and the PEI-coated MSNs. The miRNA-loaded nanoparticles were collected by centrifugation, washed with 2 mL sterile deionized water to remove unbound miRNAs, and finally resuspended in 2 mL phosphate buffer (pH 7.4) or appropriate culture medium for further experiments.

Physicochemical characterization

The hydrodynamic particle diameter, polydispersity index (PDI), and zeta potential of the nanoparticles were measured using dynamic light scattering (DLS) with a Zetasizer Nano-ZS (Malvern Instruments, Worcestershire, UK). Morphological features were assessed using transmission electron microscopy (TEM). For TEM analysis, a drop of nanoformulation was placed on wax paper, transferred to a 300-mesh copper grid, stained with 2% (w/v) phosphotungstic acid, and air-dried. TEM imaging was conducted using a Thermo Fisher Scientific Talos L120C microscope operating at 70 kV.

In vitro haemolysis assay

The haemolysis assay was performed to evaluate the haemocompatibility of the miRNA-loaded MSNs in comparison with naked miR-Combo. Fresh blood was collected from healthy animals into EDTA-coated tubes to prevent coagulation. The blood samples were centrifuged at 5000 rpm for 15 minutes, and the plasma supernatant was carefully discarded. The remaining erythrocyte pellet was washed three times with phosphate-buffered saline (PBS; pH 7.4) to remove any residual plasma and cellular debris. After each wash, the samples were centrifuged again under identical conditions, and the erythrocytes were resuspended in PBS to obtain a homogeneous RBC suspension.

For the haemolysis test, 96-well plates were prepared by adding 20 µL of each test formulation, miRNA-loaded MSNs, MSN placebo, naked miRNA, 1% Triton X-100 (positive control), and PBS (negative control), to separate wells containing 180 µL of the diluted RBC suspension. The samples were incubated at room temperature for one hour to allow interaction between the RBCs and test formulations. Following incubation, the plates were centrifuged, and the absorbance of the supernatant was measured at 570 nm using a microplate reader (ELISA reader) to determine the extent of haemolysis. The percentage of haemolysis was calculated using the following equation, where OD represents the optical density of each sample^23,24:

Plasma pharmacokinetics study

Animal treatment and blood collection

Following HSV-2 infection, mice were randomly divided into groups. One group received naked miRNA, while the other was administered miRNA encapsulated in MSNs. Both formulations were suspended in sterile saline and administered intravaginally as a single dose of 5 mg/kg (10 µL of 10 µg/µL concentration for each mouse of ~25 g). At specified time intervals post-dosing (0, 0.5, 1, 2, 4, 6, and 12 hours), blood samples were collected from three animals per group at each time point via retro-orbital bleeding into EDTA-coated tubes to prevent coagulation. The collected samples were immediately mixed with an equal volume of lysis-loading buffer (Clarity OTX kit; Phenomenex, CA) and stored at –80 °C until further analysis.

miRNA extraction and HPLC analysis

For quantification, circulating miRNA was extracted from plasma using the Clarity OTX kit (Phenomenex, CA), following the manufacturer’s protocol, with minor modifications using a Thermo Scientific HyperSep Vacuum Manifold (Thermo Fisher Scientific, Rockwood, TN). Equal volumes of lysis-loading buffer were combined with the plasma prior to loading onto the solid-phase extraction columns (Clarity OTX, 100 mg/3 mL; Phenomenex). Columns were pre-conditioned with methanol and equilibrated with equilibration buffer (10 mM phosphate, pH 5.5). After sample loading, columns were rinsed twice with equilibration buffer, followed by washing with wash buffer (10 mM phosphate, pH 5.5/50% acetonitrile). Bound miRNA was eluted using elution buffer containing 100 mM ammonium bicarbonate (pH 8.0), 40% acetonitrile, and 10% tetrahydrofuran. The eluates were concentrated via vacuum concentrator to a final volume of approximately 100 μL for chromatographic analysis²⁵.

miRNA quantification was performed using a validated ion-pair high-performance liquid chromatography (HPLC) method. The analysis was carried out on an Alliance HPLC system (Waters 2695 Separation Module) coupled with a Waters 2998 photodiode array detector (Waters, Milford, MA). Separation was achieved using a Clarity 3 μm Oligo-RP column (50 × 2.0 mm; Phenomenex) with an injection volume of 1 μL. The mobile phase consisted of 20 mM triethylamine-acetic acid buffer (pH 7) and a gradient of 5-12% acetonitrile, delivered at a flow rate of 0.2 mL/min. Detection was conducted at 269 nm, and chromatograms were processed using Empower Pro software (Waters Corporation, Milford, MA).

Pharmacokinetic data analysis

Pharmacokinetic (PK) parameters were calculated using non-compartmental analysis with WinNonlin software version 6.0 (Pharsight, Mountain View, CA). The plasma concentration-time profiles following vaginal administration were further analyzed using PK Functions for Microsoft Excel (Pharsight Corporation, CA). Key PK parameters, including maximum plasma concentration (Cmax), area under the concentration-time curve from 0 to 12 hours (AUC_0–12), and elimination half-life (t_1/2), were determined for both naked miRNA and N-miR-Combo formulations using PK Solution 2.0 software.

Cell viability assay

The cytotoxicity of the NP- miR-Combo was assessed in THP-1 cells using the MTT assay. Cells were seeded in 96-well plates at a density of 2 × 10⁵ cells/well and incubated overnight. Placebo and scrambled miRNA-loaded MSNs (N-Sc miR-Combo) served as controls. Cells were then treated with varying concentrations of miRNA-loaded MSNs for 24 hours. Following treatment, 20 µL of MTT reagent (5 mg/mL) was added to each well, and optical density (OD) was measured at 550 nm and 630 nm using a multimode plate reader. The 50% cytotoxic concentration (CC₅₀) was calculated from dose-response curves.

Quantitative PCR (qPCR)

For in vitro antiviral and immunomodulatory studies, THP-1 cells were seeded at a density of 2 × 10⁶ cells/well in 6-well plates; differentiation conditions (10 nM PMA for 24 hours followed by 24-hour rest in PMA-free medium); treatment regimen for antiviral studies (pre-treatment with 100 nM N-miR-Combo or appropriate controls for 24-48 hours prior to HSV-2 infection at MOI = 1); time points for sample collection (4, 8, and 24 hours post-infection). Total RNA was extracted from THP-1 cells and vaginal tissues using the TRIzol reagent via organic extraction. cDNA synthesis was performed using the SuperScript™ III First-Strand Synthesis System (18080-051; Invitrogen, Waltham, MA, USA). The synthesized cDNA served as the template for real-time PCR amplification of the HSV-2 viral gene UL30, inflammatory markers, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase).

qPCR was carried out using 2× GoTaq® qPCR SYBR Green Master Mix (A6001; Promega, Madison, WI, USA) and 200 nM of gene-specific primers. Primer sequences for each target gene are provided in Supplementary Table 1. The reaction mix included the master mix, forward and reverse primers, template cDNA, and nuclease-free water to a final volume of 20 µL. Reactions were run under optimized cycling conditions appropriate to each primer set. Gene expression was quantified using the 2^–ΔΔCt method. GAPDH served as the internal control to normalize expression levels. All qPCR assays adhered to the MIQE guidelines.

Immunoblot analysis

For protein analysis, THP-1 cells were seeded and differentiated as described in Section "Quantitative PCR (qPCR)". Treatment and infection conditions mirrored those described for qPCR analysis. Protein samples were collected at 8 and 24 hours post-infection. At designated endpoints, THP-1 cells and vaginal tissue samples were washed with ice-cold PBS and lysed in RIPA buffer supplemented with protease inhibitors. Proteins were separated by SDS-PAGE and transferred onto PVDF membranes. After blocking with 5% blocking solution or non-fat dry milk (NFDM) for 1 hour at room temperature, membranes were incubated overnight at 4°C with primary antibodies specific to NF-κB p65 (Cell Signaling Technology, 8242), IL-1β (Cell Signaling Technology, 12242), HSV-2 proteins (ICP8: Santa Cruz Biotechnology, SC-56992; ICP27: Santa Cruz Biotechnology, SC-69806; gB: Abcam, ab18687), and GAPDH (G-9) (Santa Cruz Biotechnology, SC-365062-HRP).

Subsequently, membranes were probed with appropriate HRP-conjugated secondary antibodies. Detection was performed using the Pierce™ ECL Western Blotting Substrate (Thermo Scientific, Waltham, MA, USA: 32209) and visualized on a Chemidoc imaging system (BioRad, Hercules, CA, USA). Membranes were stripped using 1× ReBlot Plus Strong Antibody Stripping Solution and reprobed with GAPDH antibody. Band intensities were quantified using ImageJ software v1.53a (NIH) and normalized to GAPDH.

In vivo infection model

Female BALB/c mice (7 weeks old, 18-25 g) were used for in vivo evaluation. To synchronize the estrous cycle, 50 mice (5 per group for 10 groups) were pre-treated subcutaneously with 2 mg medroxyprogesterone acetate (MPA) five days prior to infection. Animals were allocated to treatment groups by simple randomization using a random number table, ensuring that body weights were comparable across groups at baseline. The group size was chosen based on previously published HSV-2 murine studies that successfully demonstrated antiviral efficacy with similar numbers, while also adhering to the principle of minimizing animal use in line with ethical guidelines^26–29. For prophylactic assessment, nanoparticles were administered intravaginally (10 µL; 10 µg/µL concentration) 24 hours before infection, followed by intravaginal infection with 10 µL HSV-2 stock (1 × 10⁷ PFU/mL). The 24-hour prophylactic window was selected based on standard protocols used in prophylactic murine genital herpes models. This timeframe has been consistently employed in comparable published murine HSV-2 prophylactic studies using nanoparticle-based intravaginal delivery systems^26,30. Also, our previously published in vitro optimization data²⁰, demonstrated that the individual miRNAs and the miR-Combo showed maximal antiviral activity and PI3K/AKT pathway dysregulation at 8–24 hours post-infection.

Post-infection, animals were monitored daily for 7-10 days for clinical signs, including body weight changes, lesion development (scored 0-5 performed by an observer blinded to treatment group allocation; 5 indicating severe ulceration), vaginal swelling, redness, and behavioural alterations. On day 10, animals were euthanized using CO₂ asphyxiation or cervical dislocation in accordance with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020). Vaginal tissues were harvested, snap-frozen in liquid nitrogen for RNA/protein analysis or preserved in RNAlater.

All animal experiments were reviewed and approved by the Institutional Animal Ethics Committee (IAEC), RPMC-University of Calcutta, under CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals, Government of India) registration number 1148/PO/Re/S/07/CPCSEA, with study protocol number IAEC/RPMC/2022/5. All animal experimental methods were performed in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and regulations.

Statistical analysis

All in vitro experiments were performed with n=3 independent biological replicates unless otherwise stated, and all in vivo groups comprised n=5 mice per group. Data are presented as mean ± standard deviation (SD). Normality of data distribution was assessed using the Shapiro-Wilk test prior to parametric analysis; all in vitro datasets passed normality testing (p>0.05). Statistical analysis was performed using GraphPad Prism 5. Differences between groups were assessed using one-way ANOVA followed by Tukey’s post hoc test. Body weight and lesion score data were analyzed by two-way ANOVA with Tukey’s post hoc correction. Survival data were analyzed using the log-rank (Mantel-Cox) test. A p-value <0.05 was considered statistically significant. Statistical significance is indicated in all figures as *p<0.05; **p<0.01; ***p<0.001; ns = not significant.

Results

Our earlier investigation illuminated several key findings²⁰; we first mapped the expression landscape of five specific miRs (miRNAs) within macrophages during HSV-2 infection. This revealed a dynamic interplay, with some miRs showing increased expression, while others were suppressed in response to the virus. Armed with this knowledge, we tested the impact of manipulating these miRs individually. Ectopic expression of miR mimics for the downregulated miRs or inhibitors for the upregulated miRs led to a significant reduction in HSV-2 infection within infected cells, suggesting their direct involvement in the viral life cycle. Furthermore, we sought to harness the synergistic power of multiple miRs. Through in vitro experimentation and systems biology tools, we identified an optimal combination of these five miRs that exhibited the most potent anti-HSV-2 activity. This miR-Combo was then rigorously validated in vitro which confirmed its ability to effectively attenuate HSV-2 infection in macrophages, paving the way for further exploration.

Nano-encapsulation: delivering miR-combo with mesoporous core-cone silica nanoparticles

Nano-formulation preparation

To translate the in vitro observations into a viable antiviral delivery approach, we needed a robust delivery system. We selected mesoporous core-cone silica nanoparticles (MSN) for their biocompatibility, high loading capacity, and controlled release properties³¹. The miR-Combo was loaded into MSN following a modified co-precipitation method. Briefly, MSN was synthesized using tetraethyl orthosilicate (TEOS) as a silica source and cetyltrimethylammonium chloride (CTAC) as a template. The miR-Combo was then adsorbed onto the MSN by incubation in an aqueous solution under optimized conditions to ensure maximum encapsulation efficiency without compromising the integrity of the miR-Combo.

Characterization of Mesoporous Silica Nanoparticles (MSNs)

The synthesized MSNs were characterized for their hydrodynamic diameter, polydispersity index, and surface charge to assess the integrity and suitability of the nanoformulation for miRNA delivery. Dynamic light scattering (DLS) analysis revealed an average hydrodynamic diameter of 83.21 ± 1.03 nm for blank MSNs (Supplementary Fig 1) and 90.19 ± 1.53 nm for miRNA-loaded MSNs (Fig. 1).

Fig. 1.

Particle size and polydispersity index (PDI) of miRNA-loaded MSNs. Dynamic light scattering (DLS) analysis of miRNA-loaded mesoporous silica nanoparticles (N-miR-Combo). Blank MSNs used as a control are presented in Supplementary Figure 1.

The PDI of the miRNA-loaded MSN formulation or N-miR-Combo was 0.3419, indicating a relatively narrow size distribution, while the PDI of blank MSNs (N-Blank) was 0.3104 (Supplementary Fig 1). These observations indicate that miRNA loading did not substantially alter the particle size distribution. The minimal change in hydrodynamic diameter likely reflects the incorporation of miRNAs within the mesoporous structure or their adsorption onto the nanoparticle surface without inducing aggregation. A PDI value below 0.5 indicates acceptable uniformity in particle size and supports the suitability of the formulation for systemic administration and enhanced biodistribution.

Zeta potential analysis

Zeta potential measurements were conducted to further confirm surface modifications and electrostatic interactions throughout the formulation process. The blank MSNs exhibited a zeta potential of 25.81 ± 1.26 mV (Supplementary Figure 2), whereas the miRNA-loaded formulation (N-Combo-miR) showed a zeta potential of 19.43 ± 3.58 mV following PEI coating and miRNA loading (Fig. 2). The observed positive surface charge is expected to facilitate cellular uptake through favourable electrostatic interactions with negatively charged cell membranes. The slight reduction in zeta potential after miRNA loading likely reflects partial charge neutralization resulting from electrostatic interactions between the negatively charged miRNAs and the positively charged PEI-coated MSN surface. Importantly, the retained positive surface charge suggests that the nanoparticles maintain sufficient electrostatic stability for effective cellular interaction and delivery.

Fig. 2.

Surface charge analysis of mesoporous silica nanoparticles: Zeta potential distribution of miRNA-loaded mesoporous silica nanoparticles (N-miR-Combo) following PEI coating and miRNA loading. The N-miR-Combo formulation exhibited a positive zeta potential, indicating successful surface modification and electrostatic interactions between the PEI-coated MSN surface and the loaded miRNAs. Zeta potential analysis of blank MSNs (N-Blank) is presented in Supplementary Figure 2.

Transmission Electron Microscopy (TEM) analysis

Transmission electron microscopy was utilized to investigate the morphological characteristics of the synthesized MSNs. As shown in Figure 3, the MSNs exhibited spherical morphology with well-distributed particle sizes. The particles demonstrated a distinct mesoporous architecture, evidenced by the presence of visible pores within the silica matrix, confirming the successful formation of the mesostructure during the synthesis process. The particle size observed by TEM ranged from approximately 59.2 to 100 nm, which corroborated the hydrodynamic diameter measured via DLS analysis. The size and shape uniformity of the MSNs are essential features that contribute to their reproducible behaviour in biological systems, including cellular uptake and biodistribution. TEM images of blank nanoparticles (N-Blank) showing comparable morphology and mesoporous structure are provided in Supplementary Figure 3. Some degree of clustering was observed in TEM analysis, likely due to drying during sample preparation. However, DLS and zeta potential analysis indicated colloidal stability in aqueous suspension. The structural integrity and nanoscale porosity of the MSNs are critical for facilitating high surface area and efficient miRNA loading, supporting their potential as a robust delivery platform for nucleic acids^32–34.

Fig. 3.

Morphological characterization of MSNs by Transmission electron microscopy (TEM). TEM images of the N-miR-Combo formulation. The observed morphology corroborates particle size measurements obtained by dynamic light scattering. TEM images of N-Blank are provided in Supplementary Figure 3. Scale bars represent nanometer dimensions.

In vitro haemolysis evaluation of miRNA-loaded MSN formulation

Haemolysis, or red blood cell (RBC) rupture, is a critical parameter for assessing the hemocompatibility of nanoparticle formulations. In this study, the negative control (PBS) exhibited negligible haemolytic activity, similar to that observed with MSNs without miRNA (placebo formulation). As expected, treatment with Triton X-100, a potent membrane-disrupting agent commonly used as a positive control in hemocompatibility assays, induced substantial haemolysis, thereby confirming assay sensitivity through pronounced RBC lysis^35,36. Encouragingly, RBCs exposed to miRNA-loaded MSNs exhibited minimal membrane damage, with only 0.79% haemolysis, closely resembling the placebo-treated control. Additionally, naked miR-Combo treatment resulted in 0.398% haemolysis, further confirming the biocompatibility of both formulations. These results demonstrated that the miRNA-loaded MSN formulation exhibits excellent hemocompatibility, supporting its potential safety for in vivo applications. These results are in agreement with previous work by Sartaj et al., who similarly assessed erythrocyte compatibility while evaluating the anticancer efficacy of Ribociclib-loaded nanoparticles, thereby validating the relevance of haemolysis studies in nanomedicine safety assessment³⁷.

Plasma pharmacokinetics of miRNA-loaded MSN nanoparticles

The pharmacokinetic profile of miRNA delivered via mesoporous silica nanoparticles was evaluated in comparison to naked miRNA combination following vaginal administration. Ion-pair high-performance liquid chromatography (HPLC) analysis demonstrated optimal assay linearity across the range of 50-10,000 ng/mL (r = 0.9984), with a lower limit of detection (LLOD) of 17.29 ng/mL and a limit of quantification (LLOQ) of 52.41 ng/mL.

The plasma concentration-time profiles for N-miR-Combo and naked miR-Combo are presented in Fig. 4. To confirm assay specificity, blank MSNs (N-Blank) were administered intravaginally as a negative control, and MSN formulation of Scrambled-miR (N-Sc-miR) as experimental control. No detectable miRNA signal was observed in plasma of N-Blank-treated animals at any time point, whereas the N-Sc-miR exhibited plasma miRNA levels comparable to those of N-miR-Combo, thereby validating that the pharmacokinetic profiles observed for the miRNA formulations are solely attributable to the miRNA cargo, independent of miRNA sequence. Following administration of miRNA, the MSN formulations achieved a significantly higher maximum plasma concentration (C_max) of 19187.61 ± 152.76 ng/mL for N-miR-Combo and 19796.11 ± 256.14 ng/mL for N-Sc-miR, compared to 7612.20 ± 90.32 ng/mL observed for the naked miRNA group.

Fig. 4.

Plasma concentration-time profiles of naked miRNA and miRNA-loaded MSNs following intravaginal administration in mice. Plasma levels of miRNA were measured at designated time points after intravaginal delivery of either N-Blank, miR-Combo, N-miR-Combo, or N-Sc-miR. Data are presented as mean ± standard deviation from three independent replicates.

The area under the concentration-time curve from 0 to 12 hours (AUC_0-12) was markedly higher for N-miR-Combo (148322.16 ± 128.69 ng·h/mL) compared to free miR-Combo (19592.53 ± 54.86 ng·h/mL), while N-Sc-miR exhibited comparable exposure (137187.92 ± 164.21 ng·h/mL), indicating that nanoparticle-mediated delivery governs pharmacokinetics independent of miRNA sequence. In contrast, N-Blank (blank MSN without miRNA loading) showed negligible systemic exposure, remaining close to baseline levels as expected. These pharmacokinetic differences are further summarized in Table 1, which highlights the superior performance of MSN-based miRNA delivery compared to the conventional naked miR-Combo. The improved pharmacokinetic behavior of the MSN formulation can be attributed to several factors. Poor aqueous solubility of naked miRNA often results in incomplete dissolution, limiting its absorption and leading to reduced C_max and AUC values. Nanoparticle encapsulation offers protection against enzymatic degradation and reduces clearance by the reticuloendothelial system, further improving bioavailability³⁸. Collectively, these findings support the potential of mesoporous silica nanoparticles as an effective delivery platform for improving the pharmacokinetics and preclinical antiviral activity of miRNA-based treatments in the context of HSV-2 infection.

Table 1.

Pharmacokinetic parameters of naked miRNA and MSN NPs in mice following vaginal administration.

Parameters	N-Blank	miR-Combo	N-Sc-miR	N-miR-Combo
Cmax (ng/mL)	UD	7612.20 ± 90.32	19796.11 ± 256.14	19187.61 ± 152.76
Tmax (hour)	UD	1	2	1
AUC0-12 (ng.h/mL)	UD	19592.53 ± 54.86	137187.92 ± 164.21	148322.16 ± 128.69
t1/2 (h)	UD	1.73 ± 0.06	18.15 ± 0.09	17.97 ± 0.12

*n = 3; AUC_0-12, Area under the plasma concentration-time curve from time zero to time of last measurable concentration; C_max, Peak plasma concentration; T_max, Time to peak plasma concentration; t_1/2, Plasma terminal half-life. UD, Undetermined.

Anti-HSV-2 effects of MSN with miR-combo in vitro

Cytotoxicity assessment

The biocompatibility of the nanoformulations was evaluated using an MTT assay on THP-1 cells to determine their potential cytotoxic effects. Three formulations were tested: MSNs alone (N-Placebo), MSNs loaded with scrambled miRNA (N-Sc-miR), and MSNs loaded with the miR-Combo (N-miR-Combo). Dose-response curves were generated by exposing cells to nanoparticle concentrations ranging from 0 to 2800 nM, and cell viability was assessed accordingly (Fig. 5).

Fig. 5.

Cytotoxicity of MSN formulations on THP-1 cells. MTT assay showing dose-dependent viability of THP-1 cells treated with (A) N-Placebo, (B) N-Sc-miR, and (C) N-miR-Combo (0-2800 nM) as mean ± standard deviation from three independent replicates. All formulations exceeded the ISO 10993-5 biocompatibility threshold of 70% cell viability at this concentration.

All formulations exhibited minimal cytotoxicity at concentrations up to 200 nM. The calculated 50% cytotoxic concentration (CC₅₀) values were 480 nM for N-Placebo, 456 nM for N-Sc-miR, and 460 nM for N-miR-Combo, indicating good biocompatibility across all controls. At 200 nM, N-Placebo, N-Sc-miR, and N-miR-Combo demonstrated cell viability of 89.2 ± 3.1%, 91.4 ± 2.6%, and 92.8 ± 2.1%, respectively. All formulations exceeded the 70% viability threshold defined as biocompatible by ISO 10993-5, confirming their safety at the selected working concentration. Based on these results, a concentration of 100-200 nM, well below the CC₅₀ threshold, was selected for subsequent antiviral studies to ensure safety.

In vitro antiviral efficacy of miRNA-loaded nanoparticles against HSV-2

The antiviral efficacy of the miRNA-loaded mesoporous silica nanoparticles was assessed by quantifying HSV-2 gene expression at both mRNA and protein levels using quantitative PCR and Western blotting, respectively. THP-1 macrophages infected with HSV-2 at an MOI of 1 and harvested at 4-, 8-, and 24-hours post-infection (hpi) were pre-treated with 100 nM of N-miR-Combo. Control conditions included cells treated with N-Placebo, N-Sc-miR, or left untreated.

For mRNA analysis, total RNA was extracted at each time point, and qPCR was performed using primers specific for the HSV-2 UL30 gene, with GAPDH as the internal control. The N-miR-Combo treatment resulted in a significant, time-dependent reduction in UL30 expression compared to the virus control. A marginal inhibition (7.9%) was observed at 4 hpi, followed by a substantial 64.5% reduction at 8 hpi and a striking 98% suppression at 24 hpi (Fig. 6A). Neither N-Placebo nor N-Sc-miR treatments produced any significant changes in UL30 expression, confirming the specificity of the antiviral response to the miR-Combo.

Fig. 6.

In vitro antiviral activity of N-miR-Combo against HSV-2. Cells were pre-treated with MSN formulations for 24-48 h prior to HSV-2 infection. RNA and protein were collected at 4 hpi, 8 hpi, and 24 hpi. (A) qPCR analysis of HSV-2 UL30 gene expression in infected THP-1 cells at 4, 8, and 24 hpi with or without N-miR-Combo treatment. (B) Western blot analysis of HSV-2 gB and ICP8 protein expression at 8 and 24 hpi following treatment with N-Placebo, N-Sc-miR, or N-miR-Combo. GAPDH was used as loading control. Results are presented as the means and standard deviations from three independent experiments. ns, nonsignificant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

To corroborate these findings at the protein level, Western blot analysis was conducted to evaluate the expression of key HSV-2 proteins ICP8 and glycoprotein B (gB), both essential for viral replication and entry. Consistent with the qPCR results, N-miR-Combo-treated cells showed a marked decrease in ICP8 and gB protein levels at both 8 and 24 hpi compared to only virus infected control (Fig. 6B). No significant changes were observed in cells treated with N-Placebo or N-Sc-miR, further confirming the targeted antiviral action of the N-miR-Combo formulation.

These findings collectively establish the potent in vitro anti-HSV-2 activity of the N-miR-Combo formulation, demonstrating its capacity to inhibit viral gene expression and protein synthesis in a time-dependent and miRNA-specific manner.

N-miR-combo attenuates HSV-2-induced inflammation and viral replication in macrophages

HSV-2 infection is known to trigger a robust inflammatory response, contributing to tissue damage and disease progression. To evaluate the immunomodulatory effects of N-miR-Combo, we analyzed the mRNA expression levels of key inflammatory mediators: NF-κB, IL-1β, TNF-α, IL-6, and IL-10 in HSV-2-infected THP-1 macrophages treated with or without N-miR-Combo. As expected, infection with HSV-2 led to a strong upregulation of pro-inflammatory genes. NF-κB expression increased 86.4-fold at 8 hpi and 33.8-fold at 24 hpi compared to the mock control (Fig. 7A). IL-1β was upregulated 62.9-fold and 29.4-fold at 8 and 24 hpi, respectively (Fig. 7B). Similarly, TNF-α showed 73.1-fold and 66.1-fold elevation, while IL-6 was induced 49.8-fold and 108.8-fold at 8 and 24 hpi, respectively (Fig. 7C-D).

Fig. 7.

N-miR-Combo suppresses HSV-2-induced inflammation and viral replication in THP-1 macrophages. qPCR data showing mRNA expression of inflammatory markers (A) NF-κB, (B) IL-1β, (C) TNF-α, (D) IL-6, and (E) IL-10 at 8 and 24 hpi. (F) Expression of HSV-2 UL30 gene in the same sample set. (G) Western blot analysis of NF-κB, IL-1β, and HSV-2 ICP8 protein expression at 8 and 24 hpi. GAPDH was used as the internal control. Results are presented as the means and standard deviations from three independent experiments. ns, nonsignificant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Remarkably, N-miR-Combo treatment significantly suppressed this inflammatory cascade. NF-κB expression was reduced by 86.6% at 8 hpi and 75.4% at 24 hpi compared to HSV-2-only infected cells (Fig. 7A). IL-1β levels dropped by 99.9% at 8 hpi and 47.9% at 24 hpi, while TNF-α levels were reduced by 74.6% and 82.1% at the respective time points (Fig. 7B-C). IL-6 expression was decreased by 93.8% at 8 hpi and 98.6% at 24 hpi (Fig. 7D), reflecting potent anti-inflammatory activity.

Consistent to these suppressive effects, N-miR-Combo treatment significantly enhanced the expression of the anti-inflammatory cytokine IL-10 in HSV-2-infected cells. At 8 hpi, IL-10 expression was elevated by 2.98-fold, and 1.6-fold at 24 hpi compared to HSV-2-only (Fig. 7E). This indicates that N-miR-Combo not only dampens pro-inflammatory signaling but also promotes immune regulation and resolution of inflammation.

These findings confirm that N-miR-Combo significantly mitigates the HSV-2-induced inflammatory response by targeting both upstream (NF-κB) and downstream effectors (IL-1β, TNF-α, IL-6) while simultaneously enhancing IL-10 expression. In the same sample set, the expression of the HSV-2 UL30 gene, a key marker of viral replication, was markedly reduced following N-miR-Combo treatment (Fig. 7F), reinforcing the dual action of the formulation in suppressing both viral replication and the associated inflammatory response. Collectively, these results underscore the preclinical antiviral activity of N-miR-Combo in controlling HSV-2 infection and reducing immunopathology.

To further validate these effects at the protein level, Western blot analysis was conducted to assess the expression of ICP8, a viral protein essential for HSV-2 replication, along with NF-κB and IL-1β. PMA-differentiated THP-1 cells were infected with HSV-2 (MOI = 1) and treated with N-Placebo, N-Sc-miR, or N-miR-Combo. Comparisons were made at both 8 and 24 hpi under infected and uninfected conditions (Fig. 7G).

Likewise, transcript-level data, N-miR-Combo-treated cells showed a pronounced reduction in ICP8 protein levels at both time points compared to HSV-2 infected control, confirming its antiviral effect. Moreover, protein expression of NF-κB and IL-1β was also strongly upregulated in HSV-2-infected cells, reflecting activation of canonical inflammatory pathways. However, treatment with N-miR-Combo led to a substantial reduction in both proteins, mirroring the suppression observed at the mRNA level. These findings confirm that N-miR-Combo modulates both the host inflammatory response and viral replication at transcriptomic and proteomic levels, reinforcing its promise as a dual-function agent against HSV-2.

N-miR-combo effectively suppresses HSV-2 infection in murine model of HSV-2 infection

To validate the in vivo antiviral efficacy of the miRNA-loaded mesoporous silica nanoparticles, we employed a well-established murine model of genital HSV-2 infection. Female BALB/c mice (7 weeks old, 18-25 g - 5 mice per group) were pre-treated subcutaneously with medroxyprogesterone acetate (MPA) to synchronize the estrous cycle and enhance susceptibility to intravaginal HSV-2 challenge. Mice were intravaginally administered PBS (Sham), MSNs alone (N-Placebo), MSNs with scrambled miRNA (N-Sc-miR), or the miR-Combo (N-miR-Combo), followed by intravaginal inoculation with HSV-2 (10 µL, 1 × 10⁷ PFU/mL).

Ten days post-infection, vaginal tissues were collected for quantitative PCR and western blot analysis to evaluate viral burden at both transcript and protein levels. Quantitative PCR revealed a 67.7-fold reduction in the expression of the UL30 gene in the N-miR-Combo-treated mice compared to virus-only controls (Fig. 8A), indicating strong suppression of viral replication in vivo and aligning with the antiviral efficacy observed in vitro. To further investigate the breadth of antiviral activity, we examined the expression of key HSV-2 protein representing distinct phases of viral replication: ICP27 (immediate early), ICP8 (early), and gB (late). Western blot analysis of vaginal tissue lysates demonstrated robust expression of all three proteins in HSV-2-infected mice, while N-miR-Combo-treated mice showed marked reductions in their levels (Fig. 8B), suggesting effective inhibition of viral replication at multiple stages. In contrast, mice treated with N-Placebo or N-Sc-miR showed no appreciable changes, confirming the specificity of the miR-Combo. GAPDH served as the internal loading control for all samples.

Fig. 8.

In vivo efficacy of N-miR-Combo in a murine model of genital HSV-2 infection. (A) Quantitative PCR analysis of HSV-2 UL30 gene expression in vaginal tissues collected 10 days post-infection. (B) Western blot analysis of HSV-2 proteins ICP27 (immediate early), ICP8 (early), and gB (late) in vaginal tissue of N-miR-Combo-treated mice. GAPDH served as loading control. Results are presented as the means and standard deviations from three independent experiments. ns, non-significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Beyond molecular outcomes, we monitored clinical parameters including body weight loss, lesion formation, and survival over a 10 days post-infection period. HSV-2-infected control mice exhibited progressive weight loss beginning at day 3 post-infection, peaking at 17.3 ± 2.5% by day 10 (Fig. 9A), while N-miR-Combo-treated animals experienced significantly less weight loss (4.0 ± 1.0%). Vaginal lesion scores followed a similar trend, with untreated infected mice reaching maximum scores of 5.0, whereas the N-miR-Combo group peaked at only 1.0 by day 5 and declined thereafter (Fig. 9B).

Fig. 9.

Clinical Outcome and antiviral efficacy of N-miR-Combo against HSV-2 infection in vivo. (A) Percentage of mean body weight loss after HSV-2 challenge. Mice (n = 5 per group) were weighed daily following intravaginal HSV-2 infection. Y-axis represents body weight loss in percentage and X-axis represents days’ post-infection (Day 1-10). (B) Mean lesion score after HSV-2 challenge. Vaginal lesion severity was scored on a 0-5 scale each day post-infection (Y-axis), with 0 = no lesion and 5 = very severe lesions; X-axis = days post-infection (1-10). (C) Percent survival after HSV-2 challenge. Y-axis represents percent survival over 10 days post-infection; X-axis = days 1-10. All data are presented as mean ± standard deviation (SD); n = 5 mice per group. Differences between groups were considered statistically significant where p < 0.05.

Survival analysis further underscored the preclinical antiviral activity of the formulation. While the virus-only, N-Placebo + HSV-2, and N-Sc-miR + HSV-2 groups showed survival rates of 20-40%, the N-miR-Combo-treated group exhibited 80% survival, nearly equivalent to the uninfected or mock-treated controls (Fig. 9C). Mice from each treatment group taken between days 6 and 10 post-infection, along with quantitative symptom scoring, are presented in Fig. 10A-C. These visuals clearly demonstrate the clinical benefit conferred by N-miR-Combo treatment, showing reduced lesion severity, improved physical condition. HSV-2-infected mice without treatment displayed marked weight loss, pronounced vaginal swelling and severe lesion scores, with only 20% survival observed between days 6 and 10 post-infection (Fig. 10A-C). In contrast, mice infected with HSV-2 and treated with N-miR-Combo exhibited significantly reduced disease burden, including minimal weight loss (Fig. 10A), markedly reduced vaginal swelling and lesion scores (Fig. 10B), and a substantial survival benefit of 80% by day 10 (Fig. 10C). The Sham control, N-Placebo, miRNA Combo alone, and N-miR-Combo (uninfected) groups exhibited no or only minimal disease symptoms, confirming the safety and tolerability of the formulation. These clinical and visual assessments further support the potent in vivo antiviral effect of N-miR-Combo in mitigating HSV-2-induced morbidity and mortality.

Fig. 10.

N-miR-Combo reduces clinical symptoms and improves survival in HSV-2-infected mice. (A) Body weight changes were monitored daily in HSV-2-infected mice with or without treatment. (B) Disease severity was scored based on vaginal swelling, lesion formation, and overall clinical symptoms on a scale from 0 to 5. (C) Survival graphs demonstrate the protective effect of N-miR-Combo treated mice compared to virus-only controls. All data are presented as mean ± standard deviation (SD); n = 5 mice per group. Differences between groups were considered statistically significant where p < 0.05. Statistical analysis was performed using two-way ANOVA and log-rank test for survival.

Overall, these in vivo findings strongly support the preclinical antiviral activity of N-miR-Combo as a novel, host-targeted nanomedicine for HSV-2 infection. By delivering a synergistic combination of antiviral miRNAs directly to the site of infection, this approach effectively suppresses viral replication, attenuates inflammation, improves clinical outcomes, and enhances survival, demonstrating its promise as a dual-action strategy against genital herpes.

Discussion

Herpes simplex virus type 2 remains a global health burden, particularly due to its lifelong latency, recurrent reactivation, and lack of a definitive cure³⁹. Conventional antiviral agents like acyclovir offer limited benefits in reducing viral infection and recurrence, while concerns over drug resistance further emphasize the need for innovative therapeutic strategies. miRNA-based therapeutics have emerged as a promising platform to regulate both viral and host cellular pathways⁷. Building upon our earlier findings on HSV-2-associated miRNA dysregulation in macrophages, this study presents a robust, nanotechnology-enabled approach to deliver a synergistic miR-Combo (N-miR-Combo) for dual antiviral and immunomodulatory action.

Our study presents a novel miR-Combo delivered through a nanoparticle delivery system targeting HSV-2 via combinatorial gene silencing. A key distinction of our strategy lies in the use of a rationally selected 5-miRNA combination (miR-374a, miR-181a, miR-195, miR-29b, and miR-211), which offers significant advantages over single-miRNA approaches. Each of these miRNAs has previously been involved in regulating cellular pathways relevant to viral replication and immune responses, but their integration into a combinatorial therapy provides a unique opportunity to simultaneously target multiple facets of HSV-2 pathogenesis²⁰. Compared to single miRNA approaches, which frequently exhibit limited antiviral efficacy due to viral escape and pathway redundancy, often requiring high doses that increase the risk of toxicity⁴⁰, our use of a synergistic miR-Combo and N-miR-Combo aligns with a growing body of literature advocating for multi-targeted strategies⁴¹. Similar miRNA combination approaches have shown promise against other viruses, such as HIV and HCV, where multiple miRNAs or siRNAs targeting different viral or host genes yielded more robust suppression and reduced resistance development^16,42–44. Our earlier study established that a panel of five cellular miRs (miR-374a, miR-181a, miR-195, miR-29b, and miR-211) exhibited altered expression profiles with 5 or more-fold change in THP-1 macrophages following HSV-2 infection. miR-374a, miR-181a, miR-195, miR-29b were upregulated upon HSV-2 infection whereas miR-211 was downregulated during HSV-2 infection. Importantly, although each miRNA regulates different cellular pathways, all of them converge on the PI3K/AKT signaling cascade, a pathway consistently hijacked by HSV-2 to promote replication²⁰. miR-374a can indirectly inactivate PI3K/AKT signaling by directly targeting CCND1 (Cyclin D1), which plays a role in sustaining AKT phosphorylation and downstream cascade⁴⁵. miR-181a is known to modulate T-cell receptor signaling and innate immune responses. miR-195 targets cyclin D1, whereas both miR-195 and miR-181a target PI3K expression, leading to the suppression of AKT1 in the signaling cascade. By downregulating this pathway, it interferes with viral replication and survival. miR-29b targets CASP3, thereby influencing apoptosis through the Bax/Bcl2 axis and ultimately regulating cell survival during infection. miR-211 impacts the PI3K/AKT pathway through its targets CAV1 and SOS1²⁰. By selecting these five miRNAs, we ensured that multiple upstream regulators and downstream effectors of the PI3K/AKT axis were simultaneously targeted. Also, the ectopic expression of individual miR-mimics or -inhibitors demonstrated the capacity to suppress HSV-2 replication in vitro. Through computational modelling using the Box-Behnken design and experimental validation, from our previous study, further confirmed that this specific combination provides synergistic inhibition of HSV-2 replication compared to individual miRNAs. The synergy arises because each miRNA targets distinct but converging host pathways, particularly the PI3K/AKT signaling axis and inflammatory mediators, which HSV-2 exploits for efficient replication. The combinatorial design minimized redundancy while maximizing complementary targeting of viral replication and host-pathway modulation. The use of a combination of miRNAs targeting multiple genes offers several advantages over single-target approaches^41,46. It may enhance the overall antiviral efficacy by simultaneously targeting multiple steps in the viral replication cycle. The miR-Combo effectively restricted HSV-2 infection in THP-1 macrophages in vitro, warranting further investigation.

However, the naked miRNAs are inherently unstable, susceptible to nuclease degradation, and inefficient in cellular uptake. To overcome these hurdles and facilitate targeted delivery, we employed mesoporous silica nanoparticles as a delivery platform. The nanocarrier systems have been explored to improve the stability, delivery efficiency, and therapeutic index of nucleic acid-based drugs. Mesoporous silica nanoparticles provide an attractive solution to these challenges. MSNs offer distinct advantages compared to other nanoparticle systems as their high surface area and tunable mesopores allow for efficient miRNA loading and controlled release²², while the rigid silica framework protects miRNAs against enzymatic degradation²³. MSNs were selected as the delivery platform owing to several properties that uniquely address the challenges of intravaginal miRNA administration. The vaginal mucosal environment poses significant barriers, including nuclease-rich secretions, protective mucus layers, and rapid clearance, all of which compromise the stability and uptake of naked oligonucleotides²⁴. Unlike liposomal or polymeric nanoparticles, which often suffer from aggregation or premature release in mucosal fluids, MSNs provide enhanced colloidal stability and mechanical robustness, ensuring prolonged retention at mucosal surfaces^25,26. Moreover, when functionalized with polyethyleneimine (PEI) for nucleic acid binding and hyaluronic acid (HA) for targeted delivery, MSNs can facilitate efficient condensation, protection, and intracellular delivery of miRNAs, an advantage that has been demonstrated in mucosal delivery of siRNAs^27,28. Importantly, prior studies have shown that MSN-based carriers achieve superior biocompatibility and reduced cytotoxicity compared to high-charge-density cationic polymers such as lipoplexes or PEI-only formulations^29,30. Furthermore, intravaginal administration of such nanoparticles allows for localizeddrug delivery at the site of HSV-2 infection, reducing systemic exposure and potential side effects^31,32.

The use of MSN enhances miRNA stability and delivery efficiency⁴⁷. Wang et al. demonstrated that MSNs can effectively deliver miRNA-26a to macrophages, suppressing pro-inflammatory cytokine production and promoting osteogenic differentiation, highlighting their potential for osteoimmune modulation²². Similarly, MSNs have been successfully used to deliver microRNAs like miR-200c-3p to breast cancer cells, resulting in tumor suppression and decreased metastasis, showcasing their potential as a safe and efficient delivery system⁴⁸. Additionally, MSNs have been effectively used to deliver siRNAs targeting HCV, achieving significant viral suppression, while prior studies have shown that Janus-type rod-shaped magnetic MSNs loaded with ganciclovir (GCV) and functionalized with PEG-g-PLL demonstrate improved drug-loading efficiency and faster release, highlighting their potential in HSV-TK/GCV gene therapy systems^49,50. Collectively, these findings highlight the remarkable versatility of MSNs as a platform for nucleic acid and drug delivery in a wide range of therapeutic applications. Other nanoparticles, such as liposomes and polymeric nanoparticles, have been explored for nucleic acid delivery; however, MSN offers high biocompatibility, controlled drug release, and efficient cellular uptake⁵¹. The choice of MSNs as the delivery vehicle was based on their well-established high-loading capacity and ability to protect the encapsulated miRNAs from degradation^22,52. While gold or silver nanoparticles have also been explored for drug delivery, MSNs offer several advantages, including lower cost, ease of synthesis, and tunable pore size^53–56. Our findings further support the potential of MSNs as a versatile platform for delivering miRNA-based therapies that simultaneously target viral replication and host inflammation in HSV-2 infection. Therefore, N-miR-Combo was formulated using mesoporous core-cone silica nanoparticles. We hypothesised that the synergistic action of multiple miRNAs delivered by MSNs would result in enhanced antiviral activity and reduced off-target effects.

MSN was synthesized via a modified co-precipitation method using TEOS and CTAC⁵⁷. The miR was subsequently adsorbed onto the MSN surface under optimized conditions. Our MSN synthesis resulted in a uniform particle size distribution. A stable colloidal suspension was indicated by a positive zeta potential after polyethylenimine coating and miRNA loading. TEM confirmed the core-cone morphology of the MSN and the successful encapsulation of the miR-Combo within the mesoporous matrix. The successful synthesis and characterization of the MSNs confirmed their suitability for drug delivery. This efficient delivery system significantly enhanced the stability and bioavailability of the miRNAs, as demonstrated by the improved plasma pharmacokinetics (higher C_max and AUC) compared to naked miRNAs. The pharmacokinetic profile demonstrated T_max of 60 minutes for both naked miR-Combo and MSN-encapsulated formulation N-miR-Combo. This rapid appearance in circulation is consistent with the high vascularity of the vaginal mucosa and the well-established ability of nanoparticles to traverse mucosal epithelial barriers via transcytosis and paracellular transport pathways^58,59. The short plasma half-life of naked miR-Combo (t_1/2 = 1.73 h) reflects rapid renal clearance and nuclease-mediated degradation, which are well-documented limitations of unmodified oligonucleotides in systemic circulation⁶⁰. In contrast, the markedly prolonged half-life of the MSN formulation (t_1/2 = 17.97 h) suggests sustained miRNA release from the nanoparticle matrix and possible tissue retention, contributing to the higher AUC observed for N-miR-Combo. Such enhanced delivery is consistent with findings from other studies demonstrating the efficacy of MSNs in delivering miRNAs to various cell types, including macrophages for immunomodulation and cancer cells for tumor suppression^22,61.

Importantly, the cytotoxicity assays highlighted the biocompatibility of our formulation, with N-miR-Combo demonstrating tolerability at doses that retained antiviral efficacy. This contrasts with challenges often observed with other cationic carriers, such as liposomes or high-molecular-weight PEI polymers, which can provoke dose-dependent toxicity^62,63. Our formulation offers an encouraging balance between stability, delivery efficiency, and safety.

The in vitro antiviral activity of N-miR-Combo was assessed in HSV-2-infected THP-1 macrophages, where qPCR analysis demonstrated a marked reduction in viral load. Importantly, UL30 gene expression was substantially suppressed at both early and later stages post-infection in N-miR-Combo-treated cells compared to controls, indicating strong and sustained inhibition of viral replication. Immunoblotting confirmed a marked reduction in HSV-2 protein expression (ICP8 and gB) in the N-Combo-miR group. The qPCR and Western blot results demonstrated that the MSNs loaded with the miR-Combo significantly reduced the expression of both the UL30 gene and the viral proteins in HSV-2-infected cells. Our data indicate how N-miR-Combo shapes both viral replication and host responses. The suppression of UL30, ICP8, and gB expression following N-miR-Combo treatment is consistent with significant inhibition of viral replication. However, since these proteins represent sequential stages of the viral lifecycle (early: UL30/ICP8; late: gB), their coordinate downregulation may reflect inhibition at an upstream stage, such as early transcription or viral DNA replication, with consequent reduction in downstream viral protein accumulation, rather than necessarily implying independent interference at each individual stage. Further mechanistic dissection will be required to precisely define the step(s) of the viral lifecycle targeted by the N-miR-Combo. Unlike earlier reports where single host miRNAs such as miR-36 or miR-138 restricted HSV-2 replication only in specific cell types^18,19, our rationally designed combination with the help of MSN exerts a more comprehensive antiviral effect, particularly within macrophages, a critical immune cell reservoir. In addition to its antiviral activity, our study reveals that N-miR-Combo significantly modulates the host inflammatory response to HSV-2 infection. HSV-2 is known to trigger robust innate immune activation, largely mediated through the NF-κB pathway, resulting in elevated levels of pro-inflammatory cytokines such as IL-6, IL-1β, and TNF-α^64–67. These cytokines are not only markers of inflammation but also contributors to tissue damage, lesion severity, and disease progression in HSV-2 infections^68,69. Previous research has demonstrated that excessive inflammatory responses are linked to worse disease outcomes, and immunomodulatory therapies targeting cytokine pathways can ameliorate tissue damage^70,71. Our results demonstrate that HSV-2 infection led to a significant upregulation of IL-6, IL-1β, and TNF-α, corroborating previous reports of intense inflammatory signaling during active infection^72,73.

Strikingly, N-miR-Combo treatment significantly suppressed the expression of these cytokines in THP-1 macrophages. IL-1β levels were substantially reduced at early time points, and NF-κB expression declined markedly by 24 hours post-infection, pointing to effective upstream regulatory control. Collectively, our findings demonstrate that the miR-Combo delivered by MSNs not only targets viral replication pathways but also attenuates the inflammatory milieu by suppressing NF-κB activation and its downstream cytokines. This dual effect, viral suppression and immunomodulation, is particularly advantageous, as it may limit the immunopathological consequences of HSV-2 infection, reduce lesion formation, and improve healing outcomes.

In parallel, we observed a significant upregulation of IL-10 expression following N-miR-Combo treatment, indicating enhanced activation of endogenous anti-inflammatory responses. IL-10 is a key cytokine involved in maintaining immune homeostasis and preventing tissue damage during viral infections⁷⁴. Its increased expression suggests that N-miR-Combo not only dampens excessive inflammation but actively promotes immune regulation. Our observation is consistent with previous reports linking IL-10 induction to protection against HSV-related immunopathology⁷⁵. The simultaneous downregulation of pro-inflammatory cytokines and NF-κB, along with IL-10 upregulation, supports the therapeutic potential of cytokine modulation in improving lesion resolution and minimizing immunopathological damage during HSV infection^75,76.

Western blot results further validated this immunomodulatory effect by showing significant downregulation of IL-1β and NF-κB proteins post N-miR-Combo treatment, whereas IL-10 was upregulated. This is consistent with studies showing that elevated IL-1β and NF-κB drive immune-mediated damage during viral infections^77–79. Reduction in these proteins implies decreased activation of the inflammasome and cytokine storm, a mechanism that has been explored for therapeutic targeting in HSV and other viral diseases^80,81. Furthermore, the N-miR-Combo was evaluated in a murine model of vaginal HSV-2 infection. Mice were pre-treated intravaginally with the N-miR-Combo and then infected with HSV-2, followed by molecular analysis of viral gene and protein expression in vaginal tissues. Our results demonstrated N-miR-Combo significantly reduced the expression of UL30 (viral gene) in vaginal tissue from HSV-2-infected mice. This indicates a substantial reduction in viral mRNA in the N-miR-Combo treated group compared to untreated infected controls, indicating that MSNs effectively delivered the miRNAs to the target tissue, leading to a reduction in viral load and suppression of HSV-2 replication in vivo. The choice of the UL30 gene as a target for miRNA-mediated silencing was based on its essential roles in HSV-2 replication. UL30 encodes the viral DNA polymerase, which is critical for viral DNA synthesis⁸². By targeting this essential viral gene, the N-miR-Combo effectively inhibited viral replication and reduced the burden of HSV-2 infection.

Additionally, in vivo Western blot analysis of viral proteins (ICP27, ICP8, and gB) provided deeper insights into the suppression of viral replication by N-miR-Combo. Each of these proteins represents a critical stage in the HSV-2 life cycle: ICP27, an immediate early protein, is involved in host transcriptional shutoff; ICP8, a single-stranded DNA-binding protein, is essential for viral DNA replication and recombination⁸³; and gB, a late-stage structural glycoprotein required for virion assembly^84–88. Their downregulation indicates that N-miR-Combo interferes with multiple stages of the viral replication cycle. Previous studies have shown that knockdown of ICP27 or gD significantly reduces viral titers, supporting the relevance of these targets^26,88–91. The reduced expression of these viral markers in the N-miR-Combo group, as opposed to the N-Placebo and N-Sc-miR groups, suggests that the miR-Combo may disrupt early transcriptional events as well as virion formation and egress, thereby contributing to a holistic antiviral effect. Additionally, the intravaginal route of administration, closely mimicking the natural infection pathway, adds translational relevance to our findings. Together, our observations highlight the potential of N-miR-Combo not only as a direct antiviral but also as an anti-inflammatory agent, capable of mitigating both viral burden and host-mediated tissue damage during HSV-2 infection.

Furthermore, in vivo evaluation of the N-miR-Combo revealed significant mitigation of clinical symptoms associated with HSV-2 infection. Disease progression in HSV-2-infected mice, as evident by mean body weight loss, lesion score, and survival percentage, was effectively curtailed following N-miR-Combo treatment . N-miR-Combo treated mice exhibited significantly reduced disease severity scores compared to HSV-2-infected controls. The onset of symptoms was delayed, and their severity was significantly lower throughout the 10-day infection period. Mice infected with HSV-2 exhibited marked weight loss starting from day 3 post-infection, peaking between days 6-8, which is consistent with acute systemic illness resulting from active viral replication and inflammation. In contrast, mice treated with N-miR-Combo showed significantly reduced body weight loss compared to both untreated virus-infected groups and those treated with N-Placebo or N-Sc-miR, highlighting the protective systemic effects of the miR-Combo. Vaginal lesion scores were substantially decreased in the N-miR-Combo group, with most animal exhibiting only mild symptoms, in contrast to the severe lesions observed in virus-only and control-treated groups. The maximum lesion severity in untreated or control nanoparticle-treated HSV-2-infected groups reached score 4-5, whereas N-miR-Combo-treated animals predominantly exhibited only mild symptoms (score 1-2), suggesting a pronounced local antiviral and anti-inflammatory effect of the miRNA cargo at the site of infection. Importantly, survival rates were markedly improved in the N-miR-Combo group, with 100% survival observed compared to 40% in the HSV-2 control group highlighting the strong protective potential of the N-miR-Combo formulation against HSV-2-induced morbidity and mortality as revealed by clinical monitoring. To complement these findings, the critical late phase of infection (days 6-10), a period during which disease severity typically peaks and survival divergence becomes most evident, was observed. Our data indicated that N-miR-Combo not only mitigated early disease progression but also provided sustained protection at later stages, preventing the sharp decline in survival and escalation in lesion severity observed in untreated and control groups. Thus, N-miR-Combo not only reduced the expression of essential viral genes and proteins (UL30, ICP27, ICP8, gB) across distinct stages of the viral life cycle but also ameliorated clinical outcomes, including body weight loss, lesion severity, and mortality. Mice receiving either miRNA alone or non-targeting controls (N-Sc-miR or miR-Combo without nanoparticles) did not show significant clinical improvements or survival benefits. Similarly, the N-Placebo group failed to exhibit antiviral activity, highlighting that the delivery system alone, in the absence of active miRNA cargo, does not confer protection. The reductions in viral gene/protein expression, lesion severity, and mortality observed in N-miR-Combo-treated mice should not be seen merely as endpoints but as evidence that coordinated targeting of viral replication and inflammatory signaling can reshape the trajectory of HSV-2 infection. Unlike conventional antivirals such as acyclovir, which primarily limit DNA polymerase activity but fail to address inflammatory damage, N-miR-Combo demonstrates that addressing both sides of the virus-host interaction can confer superior clinical protection. The marked improvement in survival in our model, therefore, reflects not only viral control but also the mitigation of tissue injury. Our findings underscore the critical role of the mesoporous silica nanoparticle platform in miRNA stabilization, cellular uptake, and controlled release at the site of infection. The combination of five miRNAs effectively reduced viral replication in vitro, and nanoparticle formulation improved stability and bioavailability, exhibiting significant in vitro and in vivo efficacy. While our study demonstrates promising antiviral efficacy of the miRNA-loaded MSNs, we acknowledge the importance of addressing long-term safety and potential off-target effects. miRNAs, by nature, can regulate multiple transcripts, raising the possibility of unintended gene modulation. To minimize this, we selected miRNAs with validated host targets converging on the PI3K/AKT pathway and employed a scrambled-miRNA control to confirm specificity in our assays. However, comprehensive transcriptomic and proteomic studies would be necessary to fully rule out off-target regulation. Also, our findings demonstrate short-term protection against acute HSV-2 disease within a 10-day infection window. Further longer-term studies, including lethal-dose challenge models and latency monitoring, are necessary to confirm whether the observed effects translate into durable protection.

In the present study, the antiviral efficacy of the MSN-miRNA formulation was evaluated using a prophylactic murine model, in which the miRNA formulation was administered prior to HSV-2 challenge. While this design allowed us to assess the capacity of the formulation to prevent or limit early viral replication, it does not directly address its effectiveness when administered after infection. Therefore, future studies will be required to evaluate the therapeutic potential of this platform in post-infection treatment models, which will be important for establishing the clinical relevance and translational applicability of this MSN–miRNA delivery strategy. A limitation of the present study is that biodistribution analyses were not performed. The tissue-level fate of intravaginally administered N-miR-Combo, including its potential distribution to draining lymph nodes, systemic organs, and sensory neurons, remains to be fully characterized. Such biodistribution profiling, together with evaluation of renal and hepatic clearance kinetics, will be an important component of future preclinical safety studies. These investigations will provide a clearer understanding of the tissue-specific pharmacokinetics and pharmacological behavior of our nanoformulation. Additionally, formal histological evaluation of vaginal tissue was not performed but the preliminary safety evidence is supported by multiple independent observations. The hemolysis assay confirmed excellent hemocompatibility of the N-miR-Combo formulation with less than 0.79% hemolysis, well within the accepted safety threshold. The MTT cytotoxicity assay demonstrated greater than 89% cell viability at the working concentration of 200 nM in THP-1 macrophages, confirming biocompatibility at the cellular level. Furthermore, nanoparticle-treated uninfected animals did not exhibit any overt clinical signs of local mucosal irritation, abnormal vaginal swelling, bleeding, or behavioural distress throughout the 10-day observation period, providing indirect in vivo evidence of mucosal tolerability. Collectively, these multi-level safety indicators suggest that intravaginal administration of N-miR-Combo is well-tolerated without causing gross local tissue damage. Nevertheless, we fully recognize that formal histopathological assessment including hematoxylin and eosin staining, inflammatory infiltrate scoring, and epithelial integrity evaluation represents an essential next step in the preclinical safety characterization of our formulation. Future studies will therefore include detailed histological evaluation of vaginal tissue following both single-dose and repeat-dose intravaginal administration of N-miR-Combo, together with biodistribution analysis and long-term safety profiling. These investigations will provide a more complete understanding of the local tissue response and systemic safety profile of this nanoformulation and will form an important component of the preclinical development pipeline prior to potential translational advancement of the MSN-miRNA platform.

In addition, although polyethyleneimine coating of MSNs improved miRNA loading and endosomal escape, cationic polymers are known to trigger cytotoxicity and inflammatory responses at higher doses. Previous reports have shown that PEI can induce dose-dependent toxicity and immune activation⁹². But in our in vivo HSV-2 model, no overt signs of mucosal irritation, systemic toxicity, or weight loss unrelated to infection were observed. Nevertheless, the possibility of local mucosal inflammation or systemic distribution of nanoparticles cannot be completely ruled out. Also, our model focused on macrophage responses and vaginal tissue infection; given the neuronal latency of HSV-2, future studies should evaluate whether such miR-Combos can penetrate neuronal reservoirs or prevent reactivation, possibly by integrating additional miRNAs like miR-138 that are active in neurons. We also acknowledge that all experiments were conducted with a single laboratory-adapted HSV-2 strain (ATCC VR-734D). Clinical HSV-2 isolates exhibit considerable genetic heterogeneity, and the antiviral efficacy of N-miR-Combo against clinical strains and drug-resistant variants (e.g., acyclovir-resistant TK-deficient strains) remains to be evaluated. Future studies should include multiple HSV-2 clinical isolates. Also, our in vitro studies were performed exclusively in THP-1 macrophages, which, while a well-established HSV-2 infection model and relevant to macrophage-mediated pathogenesis, does not recapitulate the full spectrum of HSV-2 target cells including epithelial cells, dendritic cells, and sensory neurons. The MSN delivery system was not designed to selectively target macrophages, and therefore its antiviral activity in other relevant cell types remains to be characterized. Studies in primary cells, vaginal epithelial cell lines, neuronal cell and co-culture systems will be required to evaluate cell-type-specific antiviral and immunomodulatory effects, in parallel with expanded in vivo studies building upon the proof-of-concept data presented in our study. It is important to clarify that the in vivo antiviral outcomes reported here were assessed by quantifying viral gene expression (UL30 mRNA) and protein levels (ICP27, ICP8, and gB) in vaginal tissue lysates at a single endpoint (Day 10 post-infection). These measurements reflect intracellular viral replication activity within tissue and should not be interpreted as direct quantification of viral shedding, which conventionally requires detection of infectious virus or viral nucleic acids in genital secretions obtained from vaginal swabs. Assessment of viral shedding will be an important component of future studies evaluating the translational potential of the MSN-miRNA platform.

Our findings highlight the promise of combinatorial miRNA therapy as a next-generation antiviral strategy. By integrating synergistic host-targeting miRNAs with a robust nanocarrier system, we demonstrated effective suppression of viral replication, mitigation of inflammatory damage, and improved survival in vivo. Our work contributes to a growing recognition that antiviral therapies must address both viral replication and host-pathogen interactions to achieve durable benefits. Future work will therefore require dedicated biodistribution, histopathological, and chronic toxicity studies to comprehensively assess tissue accumulation and long-term inflammatory responses. The translational pathway will require further optimization of dosing, long-term safety profiling, and eventually, evaluation in clinical settings. However, the principle established through our study is that rationally designed miR-Combos can be stabilized and delivered using nanotechnology to combat HSV-2 efficiently.

Conclusions

Our journey to combat HSV-2 infection began with a foundation laid in our previous work, where we explored the potential of microRNAs as preclinical antiviral agents²⁰. Our research highlights the potential of MSN-based miRNA delivery for HSV-2 treatment. The cytotoxicity assays confirmed N-Combo-miR biocompatibility, while in vitro studies demonstrated significant suppression of HSV-2 viral genes and proteins. The in vivo findings further validated these effects, showing reduced inflammation and viral loads in N-Combo-miR-treated mice. In vitro and in vivo studies demonstrated the potential of miRNA-loaded MSNs as a promising antiviral delivery approach for HSV-2 infections. The miRNA-loaded MSNs effectively inhibited viral gene expression and protein synthesis, reduced the HSV-2 induced inflammation in vitro and reduced viral load in vivo. Compared to free miR-Combos, the MSN delivery system enhanced stability and bioavailability, improving therapeutic outcomes.

Our findings suggest that the MSN-miRNA platform represents a promising preclinical antiviral strategy for HSV-2 infection, providing proof-of-concept for miRNA-based nano-delivery systems. Future studies will be required to evaluate long-term immune responses, biodistribution, and optimized dosing strategies in order to further define the translational potential of this approach. Compared with conventional antiviral drugs, N-Combo-miR offers a targeted antiviral approach that interferes with viral replication while potentially modulating host immune responses. Overall, our findings provide a strong preclinical foundation for the development of miRNA-based nanomedicine approaches against HSV-2 infection.

Author contributions

Debashree Dass: Investigation, Data curation, Methodology, Validation, Formal analysis, Writing - original draft; Anwesha Banerjee: Investigation, Methodology, Formal analysis, Writing - original draft; NS Suneesh: Investigation, Data curation, Methodology; Artha Mondal: Investigation, Methodology; Santanu Rana: Visualization, Resources, Supervision, Writing - review and editing; Abdul Arif Khan: Software, Resources, Writing - review and editing; Shobhit Kumar: Investigation, Methodology, Validation, Formal analysis, Writing - original draft, Writing - review and editing; Anupam Mukherjee: Conceptualization, Visualization, Validation, Resources, Supervision, Writing - review and editing, Fund acquisition, Project administration.

Funding

This research was supported by Anusandhan National Research Foundation (ANRF), Department of Science and Technology (DST), Government of India (Grant No. CRG/2021/000190). D Dass was supported by the CSIR - Senior Research Fellowship, A Banerjee supported by the DHR Young Scientist Fellowship and NS Suneesh supported by the ICMR Junior Research Fellowship. The overall support provided by the ICMR - National Institute of Translational Virology and AIDS Research, Pune and the ICMR - National Institute of Virology, Pune.

Data availability

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.

Declarations

Competing interest

The authors declare no competing interests.

Ethics

All animal experiments were reviewed and approved by the Institutional Animal Ethics Committee (IAEC), RPMC-University of Calcutta, under CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals, Govt. of India) registration No. 1148/PO/Re/S/07/CPCSEA, with Study Protocol No. IAEC/RPMC/2022/5. The animal experimental procedures were performed in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and applicable regulations. Animals were humanely euthanized after completion of the experiments in compliance with the American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals (2020).