Evaluation of the antiviral effect of casein nanoparticles (CNP) loaded with Plantago major extract (PME-CNP) on herpes simplex virus

Abstract

Background

Herpes simplex virus (HSV) infections remain a global health challenge due to their persistence, limited treatment options and drug resistance. In this study, the antiviral potential of Plantago major extract (PME) encapsulated in casein nanoparticles (CNPs) against HSV-1 was investigated.

Methods

PME was extracted with ethanol by Soxhlet extraction, concentrated and analyzed by gas chromatography-mass spectrometry. PME-loaded CNPs (PME-CNPs) were prepared by cross-linking casein with calcium chloride and subsequent centrifugation. The nanoparticles were characterized in terms of size, morphology (dynamic light scattering, scanning electron microscopy), chemical interactions (Fourier transform infrared spectroscopy), crystallinity (X-ray diffraction), thermal stability (differential scanning calorimetry) and encapsulation efficiency (UV-visible spectroscopy). The in vitro release kinetics were investigated using dialysis. Antiviral activity was tested in Vero cells using cytotoxicity assays, viral titration (50% Tissue Culture Infectious Dose, TCID₅₀) and three treatment modes: virucidal (pre-treatment of the virus), prophylactic (pre-treatment of the cells) and therapeutic (post-infection). Statistical significance (p < 0.05) was determined using ANOVA.

Results

The PME-CNPs exhibited uniform size (118.6 nm), stability (zeta potential: −19.4 mV) and spherical morphology. The encapsulation efficiency was 82.1%, with sustained release (76.8% over 2 h, first-order kinetics). PME-CNPs showed lower cytotoxicity than free PME. Antiviral assays showed significant inhibition of HSV-1: virucidal (2.7-log reduction), prophylactic (1.2-log) and therapeutic (1.9-log at 1 h post-infection), outperforming free PME (p < 0.05). Early post-infection treatment indicates inhibition of viral entry/replication.

Conclusions

PME-CNPs improve bioavailability, sustain release, and enhance antiviral efficacy against HSV-1, supporting their potential as a plant-based nanotherapeutic.

Introduction

Herpes simplex virus (HSV) infections are a major health problem worldwide. An estimated 3.7 billion people under the age of 50 are affected worldwide. HSV-1 and HSV-2, the two primary serotypes, cause a spectrum of clinical manifestations ranging from mild mucosal lesions to severe infections such as herpes encephalitis and keratitis [1, 2]. The persistent nature of HSV infections, characterized by a latency period in the neural ganglia and periodic reactivation, makes their treatment particularly difficult [3]. Despite decades of research, HSV infections are still incurable. Current therapeutic approaches focus primarily on treating symptoms and suppressing viral replication during active episodes [4, 5].

Conventional antiviral agents, particularly acyclovir and its derivatives, have been the cornerstone of HSV treatment for decades [6]. However, these synthetic drugs have notable limitations, including the emergence of drug-resistant viral strains, potential toxicity with prolonged use, and insufficient efficacy in immunocompromised patients [7]. These limitations underscore the urgent need for novel antiviral strategies with increased efficacy, improved safety profiles and alternative mechanisms of action to combat drug-resistant viral variants [8].

Medicinal plants have long served as valuable resources for the development of therapeutics against various diseases, including viral infections [9, 10]. Plantago major, commonly known as broadleaf plantain, has been used for centuries in the traditional medicine of many cultures. This perennial herb contains a variety of bioactive compounds, including flavonoids, iridoid glycosides, terpenoids and phenolic compounds, which together contribute to its proven anti-inflammatory, antimicrobial and wound-healing properties [11, 12]. Recent scientific research has shown promising antiviral activity of Plantago major extracts (PME) against various viral pathogens, although comprehensive studies specifically targeting HSV are still limited [13].

The therapeutic potential of bioactive compounds from plants is often limited by factors such as poor bioavailability, limited stability and insufficient cell penetration [14]. Nanotechnology-based drug delivery systems offer a promising approach to overcome these limitations by improving the pharmacokinetic and pharmacodynamic profiles of bioactive compounds [15–17]. Such nanocarrier systems have demonstrated efficacy across diverse therapeutic applications including cancer treatment, wound healing, and infectious diseases, with benefits including improved drug stability, reduced systemic toxicity, and enhanced therapeutic indices compared to conventional formulations [18–21]. Among the various nanocarrier systems, casein nanoparticles (CNPs) have proven to be particularly attractive candidates for drug delivery. Casein, the predominant protein in milk, has inherent biocompatibility, biodegradability and exceptional binding capacity for various bioactive substances. In addition, CNPs have controlled release properties and can protect encapsulated substances from degradation in physiological environments [22, 23].

The integration of PME with CNPs represents an innovative approach that may combine the intrinsic antiviral properties of the plant extract with the enhanced delivery capabilities of nanoparticle systems. This synergistic combination could facilitate targeted delivery of bioactive compounds to viral infection sites, improve their stability and bioavailability, and potentially enhance their therapeutic efficacy against HSV infections. Despite the theoretical promise of this approach, empirical studies evaluating the antiviral efficacy of CNPs loaded with Plantago major extract against HSV remain absent from the scientific literature.

We hypothesize that encapsulation of Plantago major extract in casein nanoparticles will (1) improve the physicochemical stability and bioavailability of PME bioactive compounds, (2) provide sustained release of antiviral components, (3) reduce cytotoxicity compared to free extract, and (4) enhance antiviral efficacy against HSV-1 through improved cellular delivery and multi-stage viral inhibition.

This study aims to develop and comprehensively characterize PME-loaded casein nanoparticles (PME-CNPs) and evaluate their antiviral potential against HSV-1. The specific objectives are:

1. Formulation and physicochemical characterization: To synthesize PME-CNPs using calcium-crosslinked casein and characterize their particle size, surface charge, morphology, chemical interactions (FTIR), crystallinity (XRD), and thermal properties (DSC).

2. Encapsulation efficiency and release kinetics: To determine the entrapment efficiency and drug loading capacity of PME in CNPs, and to evaluate the in vitro release profile and kinetic modeling to elucidate the release mechanism.

3. Cytotoxicity assessment: To determine the safe, non-toxic concentrations of PME, CNPs, and PME-CNPs in Vero cells using neutral red uptake assay.

4. Antiviral efficacy evaluation: To investigate the antiviral activity of PME-CNPs against HSV-1 through three complementary approaches: Virucidal assay, Prophylactic assay, Therapeutic assay.

5. Mechanism elucidation: To determine whether PME-CNPs primarily affect viral attachment, entry, or intracellular replication stages by analyzing time-dependent therapeutic efficacy.

6. Comparative analysis: To statistically compare the antiviral efficacy of PME-CNPs versus free PME and unloaded CNPs to demonstrate the added value of nanoencapsulation.

The outcomes of this study are expected to contribute to the development of novel plant-derived nanotherapeutics for HSV infections, offering potential advantages over conventional antiviral agents in terms of efficacy, safety, and ability to overcome viral resistance mechanisms.

Materials and methods

Extraction of Plantago major

Plantago major extract (PME) was obtained by Soxhlet extraction using ethanol (99.9%, Merck, Germany) as the extraction solvent. Dried and pulverized Plantago major leaves (50 g) were placed in a cellulose thimble and subjected to continuous extraction in a Soxhlet apparatus containing 500 mL of ethanol. The extraction was performed for 6 h at 78 ± 2 °C (controlled by a heating mantle with thermostat), which corresponds to approximately 95% of ethanol’s boiling point (78.37 °C at 1 atm). This temperature was selected to ensure efficient extraction of bioactive compounds while preventing excessive solvent evaporation and thermal degradation of heat-sensitive phytochemicals such as flavonoids and iridoid glycosides. Following extraction, the resulting dark green extract was filtered through Whatman No. 1 filter paper to remove particulate matter. The filtrate was concentrated under reduced pressure using a rotary evaporator (Heidolph Laborota 4000, Germany) at 50 °C and 100 mbar until complete solvent removal. The final extract was analyzed by gas chromatography-mass spectrometry (GC-MS) to identify and characterize the chemical constituents present in the extract.

Preparation of casein nanoparticles loaded with Plantago major extract (PME-CNPs)

Bovine milk casein (purity ≥ 90%) was used as the nanocarrier material. Casein nanoparticles were prepared by a calcium-induced crosslinking method. Bovine casein powder (2.0 g) was dissolved in distilled water (100 mL) under magnetic stirring (500 rpm, 25 ± 2 °C) for 30 min until a clear, slightly opalescent solution was obtained. The pH of the casein solution was 6.8 ± 0.2 (natural pH, not adjusted). PME (30 mg) was dissolved in 1.2 mL ethanol and added to 60 mL of the prepared casein solution. The solution was stirred for 2 h to achieve effective loading of the extract in the casein. In addition, 0.8 mL of CaCl2 solution (0.5 M) was added to this solution. The resulting slightly turbid solution was stirred for 45 min. The solutions were centrifuged at 3000 rpm for 15 min to remove the larger microparticles. The ethanol was removed from the supernatant using a vacuum flash evaporator. The supernatant with the nanoparticles was used for further studies. The final concentration of the PME was 0.5 mg/mL. The addition of the CaCl2 solution triggers the rearrangement of the protein chains and the soluble casein present in the solution transforms into a micellar scaffold, which contributes to the formation of dense nanoparticles. The calcium ions facilitate the cross-linking of the casein molecules, leading to the formation of stable nanoparticles with a uniform size distribution.

Characterization of PME-CNPS

The characterization of PME-CNPS was performed using different analytical techniques. The mean particle size and zeta potential of PME-CNPS were measured by dynamic light scattering (DLS), which provided information on the colloidal stability and dispersion of the particles. The surface morphology of CNP and PME-CNPS was investigated by scanning electron microscopy (SEM), which allowed detailed observation of the structural features. In addition, Fourier transform infrared (FTIR) spectra of the PME, CNP and PME-CNPS were recorded in the range of 4000–600 cm-¹ to identify the major functional peaks and evaluate possible chemical interactions. X-ray diffraction (XRD) analysis was also performed using a Smart Lab high performance powder X-ray diffractometer to determine the crystalline properties of the extract and formulations. Differential scanning calorimetry (DSC) was performed by placing samples of the PME, CNP, PME-CNPS and their physical mixture in sealed aluminum dishes. The samples were heated at a rate of 10 °C/min in a nitrogen atmosphere to study their thermal behavior. To evaluate entrapment efficiency and drug loading, 10 ml of each prepared formulation was mixed with an equal volume of distilled water (10 ml) in a 20 ml test tube. The resulting aqueous suspension was sonicated for 10 min using a probe ultrasonicator. For formulations containing the PME, the unincorporated extract was removed by centrifuging the suspension at 10,000 rpm for 30 min. The supernatant was then collected and analyzed using a UV-visible spectrophotometer at 420 nm (with distilled water as blank) to determine the concentration of unincorporated extract. The entrapment efficiency was calculated using the following equation:

To determine the loading capacity, 20 mL of the formulation was centrifuged at 10,000 rpm for 30 min. The amount of unloaded PME in the supernatant was then quantified using the UV-visible spectrophotometric method at 420 nm. The drug loading for each batch was calculated according to the following equation:

The in vitro release study was conducted by filling 20 mL of the formulation into pre-swollen dialysis bags and placing these into 400 mL of distilled water, which served as the dissolution medium in a USP type II apparatus. The paddle rotation was set at 50 rpm, and the temperature was maintained at 37 ± 0.5 °C. At predetermined time intervals (10, 20, 30, 40, 50, 60, 90, and 120 min), 2 mL aliquots were withdrawn, filtered, and replaced with an equal volume of fresh medium kept at the same temperature. The absorbance of each filtrate was measured at 420 nm using a UV-visible spectrophotometer to determine the concentration of the released PME. Finally, the drug release data of PME-CNPS were analyzed by fitting them into both zero-order and first-order kinetic models to elucidate the release mechanism of the encapsulated extract.

Cell culture and virus Preparation

Vero cells were obtained from the Department of Virology, School of Public Health, Tehran University of Medical Sciences. Vero cells (ATCC CCL‑81) were maintained in DMEM supplemented with 10% heat‑inactivated FBS, 100 U mL⁻¹ penicillin and 100 µg mL⁻¹ streptomycin at 37 °C, 5% CO₂; cells were used between passages 5 and 20, seeded at 1 × 10⁴ cells well⁻¹ for 96‑well plates and 1 × 10⁵ cells well⁻¹ for 24‑well plates, sub‑cultured every 3–4 days when 80–90% confluent by washing with PBS, incubating with 0.05% trypsin‑EDTA for 3 min at 37 °C, neutralising with complete medium, centrifuging at 300 × g for 5 min, resuspending and reseeding; viability was confirmed (> 95% by trypan‑blue) before each experiment and cultures were screened monthly for mycoplasma. This detailed protocol now ensures full reproducibility of the cell‑culture conditions used throughout the study. Herpes simplex virus type 1 (HSV-1) was obtained from the Pasteur Institute of Iran. The infectious virus dose was determined using the TCID₅₀ (Tissue Culture Infectious Dose) assay.

Cytotoxicity assay

To determine the non-toxic concentrations of the test materials (PME, CNP and PME-CNPS) for Vero cells, a neutral red assay was performed. The neutral‑red uptake assay was selected instead of the more common MTT assay because casein nanoparticles and the polyphenolic components of Plantago major can reduce MTT tetrazolium salts non‑specifically, whereas neutral‑red measures lysosomal uptake and is less prone to interference from protein‑based nanocarriers, providing a more accurate assessment of cell viability for Vero cells. In brief, Vero cells (1 × 10⁵ cells per well) were seeded in 96-well microplates. The plates were incubated at 37 °C and 5% CO₂ until approximately 80% confluent monolayers were formed. Cells were washed with pre-warmed phosphate buffered saline (PBS) and then serial dilutions of PME, CNP and PME-CNPS prepared in DMEM were added to the Vero cells. After 48 h of incubation at 37 °C and 5% CO₂, the cells were washed with PBS and filtered neutral red dye was added to all wells. The plates were incubated for 3 h at 37 °C. After incubation, the cells were washed with PBS and the neutral red destaining solution was added. The plates were placed on a shaker in the dark for 10 min. The optical density (OD) in each well was measured at 540 nm (reference wavelength 650 nm). The highest concentration of each test material showed low cytotoxicity in the Vero cells. Thus, it considered an appropriate non-toxic concentration for the subsequent antiviral assays.

TCID₅₀ (50% tissue culture infectious Dose) assay

To determine the antiviral efficacy of the test materials, a TCID₅₀ (50% Tissue Culture Infectious Dose) assay was conducted using Vero cells and Herpes simplex virus type 1 (HSV-1). The assay involved infecting Vero cells with serial dilutions of HSV-1 and monitoring the viral cytopathic effect (CPE) to calculate the TCID₅₀ value. Vero cells were cultured in 24-well plates and infected with serial dilutions of HSV-1. The infected plates were incubated in a CO₂ incubator at 37 °C for one hour. After the incubation period, cells were washed with phosphate-buffered saline (PBS), and DMEM was added to all wells. The 24-well plates were incubated at 37 °C with 5% CO₂ and monitored daily for the presence of viral cytopathic effect (CPE). The CPE of HSV-1 appears as rounding and enlargement of infected cells. The final results were recorded after 7 days, and the TCID₅₀ value was calculated using the Reed & Muench method.

Antiviral activity assays

The potential antiviral activity of PME, CNP, and PME-CNPS against HSV-1 was evaluated in three different modes of treatment:

1. Pre-infection Treatment of Virus (Virucidal Assay).

   To determine the direct virucidal effect of test materials on viral particles, appropriate non-toxic concentrations of PME, CNP, and PME-CNPS (determined from the neutral red assay) were used to prepare HSV-1 dilutions. The mixtures were incubated for one hour at 4 °C. After the incubation period, the virus dilutions were used to infect Vero cells, and the TCID₅₀ assay was performed as described previously. The 24-well plates were examined daily for viral CPE, and the final results were reported after 7 days.

2. Pre-infection Treatment of Cells (Prophylactic Assay).

   Vero cells cultured in 24-well plates were treated with non-toxic concentrations of PME, CNP, and PME-CNPS and incubated for 2 h at 37 °C with 5% CO₂. After the incubation period, the test materials were removed from the cells, and the cells were washed with PBS. Subsequently, the cells were infected with serial dilutions of HSV-1. The TCID₅₀ assay was performed as previously described, and the final results were read at the end of day 7.

3. Post-infection Treatment of Cells (Therapeutic Assay).

   After incubating the cell monolayer with HSV-1 for one hour, Vero cells were washed with PBS, and appropriate concentrations of PME, CNP, and PME-CNPS prepared in DMEM were added to the cell monolayer at different time points: 1, 4, 8, 12, and 24 h post-infection. The test plates were incubated for 7 days in a CO₂ incubator at 37 °C and monitored daily for the presence of CPE.

Statistical analysis

All experiments were performed in triplicate, and results were expressed as mean ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test for multiple comparisons. P values less than 0.05 were considered statistically significant. GraphPad Prism software (version 8.0) was used for statistical analysis and graphical representation of data.

Results

Characterization of CNPs

Particle Size, polydispersity Index, and zeta potential measurements

Dynamic light scattering (DLS) analysis revealed that the unloaded casein nanoparticles (CNP) had a mean particle size of 87.3 ± 4.2 nm with a polydispersity index (PDI) of 0.182 ± 0.023, indicating a homogeneous size distribution. After loading with PME, the PME-CNPS showed a moderate increase in size to 118.6 ± 5.9 nm with a PDI of 0.245 ± 0.031, confirming the successful incorporation of the extract, while the nanoparticle dimensions remained below 120 nm. The zeta potential measurements showed that the unloaded CNP had a surface charge of −23.8 ± 2.1 mV, while the PME-CNPS had a slightly reduced negative charge of −19.4 ± 1.8 mV (Fig. 1). This slight reduction in the negative surface charge of the nanoparticles after loading with the extract can be attributed to the partial neutralization of the surface charges by the bioactive components contained in the PME. Despite this reduction, the zeta potential values remained in the range that provides sufficient electrostatic repulsion for good colloidal stability in aqueous suspension. The narrow PDI values observed for both formulations also confirm the uniformity of the particle size distribution, which is critical for consistent biological activity and stability of the nanoparticulate systems.

Fig. 1.

Particle size, polydispersity index (PDI), and zeta potential of unloaded casein nanoparticles (CNP) and Plantago major extract-loaded casein nanoparticles (PME-CNPS)

Surface morphology analysis

Scanning electron microscopy (SEM) revealed distinct morphological features of both the unloaded casein nanoparticles (CNP) and the PME-loaded casein nanoparticles (PME-CNPS) (Fig. 2). The unloaded CNP showed predominantly spherical shapes with smooth surfaces and a uniform size distribution. These nanoparticles appeared as discrete units with minimal aggregation and had a diameter of 75–95 nm, which confirmed the DLS measurements. The PME-CNPS formulation retained the spherical morphology of the unloaded particles, but showed a slight increase in size with diameters of 105–125 nm. The surface of the loaded nanoparticles appeared less smooth compared to the unloaded CNP, indicating successful encapsulation of the Plantago main extract components within and on the surface of the casein matrix. Some minimal bonding between the particles was observed in the PME-CNPS samples, although the nanoparticles remained predominantly well dispersed. Both formulations exhibited good structural integrity with no visible fractures or deformations. SEM analysis confirmed the nanoscale dimensions determined by DLS measurements and provided visual evidence of the successful formation of nanoparticles with relatively homogeneous size distribution.

Fig. 2.

Scanning electron microscopy (SEM) images of unloaded casein nanoparticles (CNP) and plantago major extract-loaded casein nanoparticles (PME-CNPS). Scale bar = 200 nm

FTIR spectroscopic analysis

The FTIR spectroscopic analysis provided valuable insights into the chemical composition and possible interactions between the casein nanoparticles and the PME (Fig. 3). The spectrum of the pure PME showed characteristic absorption bands at 3412 cm-¹ (O-H stretching vibrations), 2925 cm-¹ (C-H stretching vibrations), 1734 cm-¹ (C = O stretching vibrations of carboxyl groups), 1622 cm-¹ (C = C stretching vibrations of aromatic rings) and 1072 cm-¹ (C-O stretching vibrations), which indicate the presence of polyphenols, flavonoids and other phytochemical constituents. The unloaded casein nanoparticles (CNP) showed distinct protein-related bands at 3295 cm-¹ (N-H stretching), 1655 cm-¹ (amide I, C = O stretching), 1536 cm-¹ (amide II, N-H bending and C-N stretching) and 1245 cm-¹ (amide III), which are the typical structural features of casein protein. The FTIR spectrum of PME-CNPS showed a combination of peaks of both the extract and the casein nanoparticles, with notable changes. The O-H stretching band of the extract shifted from 3412 cm-¹ to 3385 cm-¹ in PME-CNPS, while the amide I band of casein shifted from 1655 cm-¹ to 1648 cm-¹. In addition, the intensity of the C = O stretching band at 1734 cm-¹ was reduced in the PME-CNPS compared to the pure extract. These spectral changes indicate hydrogen bonding and electrostatic interactions between the functional groups of casein and the bioactive components of PME. The slight shifts in the characteristic peaks without the appearance of completely new absorption bands confirm that the extract was successfully loaded into the casein nanoparticles primarily through non-covalent interactions, thereby preserving the chemical integrity of the bioactive compounds. These observations indicate that the encapsulation process did not result in significant chemical changes in the extract components, possibly preserving their antiviral activity against the herpes simplex virus.

Fig. 3.

FTIR spectra of pure Plantago major extract, unloaded casein nanoparticles (CNP), and plantago major extract-loaded casein nanoparticles (PME-CNPS)

X-Ray diffraction analysis

X-ray diffraction (XRD) analysis revealed significant differences in the crystallographic properties between the native casein protein and the extract-loaded casein nanoparticles (PME-CNPS) (Fig. 4). The diffractogram of the native casein protein showed semi-crystalline properties with peaks of medium intensity at 2θ values of approximately 9.8°, 19.2° and 23.5°, reflecting the partially ordered secondary and tertiary protein structure. These peaks correspond to the α-helical and β-sheet structural elements typically found in casein micelles and aggregates. In contrast, the PME-CNPS formulation showed a predominantly amorphous pattern characterized by a broad diffuse halo around 20.1° with significantly reduced peak intensities. The sharp crystalline peaks observed in the pure PME (at 15.3°, 22.7°, 28.4° and 33.6°) were either strongly attenuated or completely absent in the PME-CNPS diffractogram. Instead, the pattern showed subtle shoulders at around 16.2° and 23.1°, indicating the partial retention of some ordered structures within the nanoparticle matrix. This significant reduction in crystallinity of the PME-CNPS compared to native casein and pure extract indicates successful molecular integration of the bioactive components of the extract into the casein nanoparticle structure. The transformation to a predominantly amorphous state confirms that the process of nanoparticle formation has effectively disrupted the original crystalline arrangements of both the casein protein and the extract components, resulting in a more homogeneous nanoparticulate system. The dispersion at the molecular level achieved in the PME-CNPS formulation indicates a potential improvement in the dissolution properties and bioavailability of the encapsulated bioactive compounds, which may contribute to enhanced antiviral activity against herpes simplex virus through improved cellular delivery of the therapeutic components.

Fig. 4.

X-ray diffraction (XRD) patterns of native casein protein, pure Plantago major extract, and plantago major extract-loaded casein nanoparticles (PME-CNPS)

The predominately amorphous diffraction pattern of PME‑CNPs indicates that the crystalline lattice of both the native casein protein and the phytochemicals in PME has been disrupted. Amorphous solids possess higher free‑energy and lack a long‑range ordered structure, which translates into (i) enhanced dissolution and solubility of the encapsulated phytochemicals, (ii) more rapid and uniform diffusion of the active compounds from the polymer matrix, and (iii) greater molecular dispersion of the extract within the nanoparticle. These physicochemical advantages are directly linked to the observed sustained‑release profile (first‑order kinetics) and to the superior antiviral activity of PME‑CNPs compared with free PME, because a larger fraction of the bioactive molecules remains bioavailable and can interact with the virus or the host cell during the early stages of infection.

Differential scanning calorimetry analysis

Differential scanning calorimetry (DSC) thermograms provided valuable insights into the thermal behavior and physical state of the components in the different formulations (Fig. 5). The DSC curve of the pure PME showed a broad endothermic peak at 89.3 °C due to loss of residual moisture, followed by several characteristic endothermic peaks at 158.7 °C, 223.5 °C and 276.2 °C due to melting and decomposition of the various phytochemical constituents present in the extract. The unloaded casein nanoparticles (CNP) showed a distinct thermal profile with a broad endothermic dehydration peak around 78.6 °C and a protein denaturation endotherm at 205.4 °C, representing the thermal transitions typical of the casein protein matrix. The physical mixture of PME and casein showed an additive thermal pattern with slightly shifted but still recognizable endothermic peaks of both components, including the characteristic peaks of the extract at 155.9 °C, 220.8 °C and 274.1 °C, albeit with reduced intensity. In contrast, the DSC thermogram of PME-CNPS showed significant changes in thermal behavior compared to both the individual components and their physical mixture. The characteristic endothermic peaks of the extract were either strongly attenuated or completely absent in PME-CNPS. The formulation exhibited a broader dehydration endotherm around 81.2 °C and an altered peak of protein denaturation at 210.8 °C. Notably, the disappearance of the sharp endothermic peaks associated with the crystalline components of the extract indicates their transformation to an amorphous or molecularly dispersed state within the casein nanoparticle matrix. These changes in thermal profile are strong evidence of effective encapsulation of the PME within the casein nanoparticles rather than mere physical mixing.

Fig. 5.

Differential scanning calorimetry (DSC) Thermograms of pure Plantago major extract, unloaded casein nanoparticles (CNP), Physical mixture of Plantago major extract and casein, and Plantago major extract-loaded casein nanoparticles (PME-CNPS)

Entrapment efficiency and drug loading capacity of PME-CNPS

The entrapment efficiency (EE) and drug loading capacity (LC) of casein nanoparticles loaded with Plantago major extract (PME-CNPS) were evaluated to determine the ability of the formulation to encapsulate and retain the bioactive components of the extract. The results summarized in Table 1 show that the PME-CNPS formulations exhibited high entrapment efficiency, ranging from 78.3% to 85.6%, with a mean EE of 82.1 ± 3.2%. This indicates that most of the Plantago major extract was successfully entrapped into the casein nanoparticle matrix during the preparation process.

Table 1.

Entrapment efficiency and loading capacity of PME-CNPS formulations

Formulation batch	Total extract added (mg)	Unentrapped extract (mg)	Total excipients (mg)	Entrapment efficiency (%)	Loading capacity (%)
F1	50	10.8	300	78.3 ± 2.1	12.4 ± 0.8
F2	50	9.2	300	81.6 ± 1.8	13.1 ± 0.6
F3	50	7.2	300	85.6 ± 2.4	14.7 ± 1.1
Mean ± SD	-	-	-	81.8  ±  2.1	13.4 ±  0.8

Data are presented as mean ± standard deviation (SD) for triplicate measurements. The bold text "Mean ± SD" indicates the mean and standard deviation of the corresponding values (Entrapment efficiency (%) and Loading capacity (%)) calculated from the three formulation batches (F1, F2, and F3)

The loading capacity (LC) of the formulations was also evaluated and showed values between 12.4% and 14.7%, with an average LC of 13.5 ± 1.1%. These results indicate that the nanoparticles were able to absorb a significant proportion of the extract relative to the total mass of the formulation (extract + excipients). The slight variations in EE and LC across batches may be attributed to differences in the initial extract-to-excipient ratios or minor inconsistencies during the nanoprecipitation and sonication steps.

The UV-visible spectrophotometric analysis at 420 nm confirmed minimal residual unentrapped extract in the supernatant after centrifugation, further validating the efficiency of the encapsulation process.

In vitro release kinetics of PME-CNPS

The release profile of PME from casein nanoparticles (PME-CNPS) was evaluated under simulated physiological conditions (37 ± 0.5 °C, 50 rpm paddle rotation) using a dialysis bag method in distilled water. The cumulative drug release over 120 min is presented in Fig. 6; Table 2. The results revealed a biphasic release pattern characterized by an initial burst release phase (0–30 min), followed by a sustained release phase (30–120 min).

Fig. 6.

In vitro release profile of casein nanoparticles loaded with Plantago major extract (PME-CNPS)

Table 2.

Cumulative in vitro release of Plantago major extract from PME-CNPS

Time (minutes)	Cumulative release (%)
10	12.5 ± 1.6
20	24.3 ± 1.9
30	32.4 ± 2.1
40	41.8 ± 2.7
50	50.6 ± 3.2
60	58.7 ± 3.5
90	68.9 ± 2.4
120	76.8 ± 2.8

Data are expressed as mean ± SD (n = 3)

Within the first 30 min, 32.4 ± 2.1% of the encapsulated extract was released, likely due to the diffusion of surface-associated or loosely bound Plantago major components. By 60 min, the cumulative release reached 58.7 ± 3.5%, and by the end of the 120-minute study period, 76.8 ± 2.8% of the total entrapped extract had been released. The sustained release phase suggests that the casein matrix effectively controlled the diffusion of the extract over time, which is critical for maintaining prolonged antiviral activity.

To elucidate the release mechanism, the data were fitted to zero-order and first-order kinetic models (Table 3). The first‑order model provided the highest correlation coefficient (R² = 0.982) and therefore best described the release behavior of PME from the casein matrix. In a first‑order process the release rate is proportional to the amount of drug remaining in the carrier; as the concentration of encapsulated PME decreases, the diffusion driving force diminishes, leading to a gradual slowdown of release. This is consistent with the biphasic pattern observed experimentally (an initial burst followed by a sustained phase). The Korsmeyer‑Peppas analysis yielded a release exponent n = 0.62, which lies between 0.45 (Fickian diffusion) and 0.89 (non‑Fickian, anomalous transport) for spherical systems. This indicates that the release of PME from PME‑CNPs is governed by a combination of Fickian diffusion through the hydrated casein network and polymer relaxation/swelling of the casein matrix. The Higuchi model (R² = 0.931) also supports a diffusion‑controlled mechanism, but its fit is inferior to the first‑order model because Higuchi assumes a constant diffusion path, which is not fully applicable to the swelling casein matrix. Consequently, the release of PME from casein nanoparticles can be described as a concentration‑dependent diffusion process that is modulated by matrix relaxation. The predominance of the first‑order kinetics reflects the gradual depletion of the drug reservoir within the nanoparticles, while the n‑value from the Korsmeyer‑Peppas model confirms the contribution of polymer relaxation to the sustained release phase. The initial burst (≈ 32% released within 30 min) ensures rapid availability of PME to inactivate extracellular HSV‑1 particles (virucidal effect), whereas the subsequent first‑order release maintains therapeutic concentrations intracellularly, supporting the observed prophylactic and therapeutic efficacy at early post‑infection time points.

Table 3.

Kinetic modeling of PME-CNPS release data

Model	Equation	R²	Rate constant (k)
Zero-order	Qt=Q0+k0t	0.874	0.642% min⁻¹
First-order	lnQt=lnQ0 − k1t	0.982	0.015 min⁻¹
Higuchi	Qt=kH √t​	0.931	2.84% min⁻¹⁄²
Korsmeyer-Peppas	Qt/Q∞=kKtn	0.957	0.112 min⁻ⁿ

Qt = amount of drug released at time t; Q0 = initial amount of drug; k = release rate constant

Cytotoxicity assay

The cytotoxicity of Plantago major extract (PME), unloaded casein nanoparticles (CNP), and PME-loaded casein nanoparticles (PME-CNPS) was evaluated on Vero cells using the neutral red uptake assay. Serial dilutions of each test material (1:5, 1:10, 1:25, 1:50, 1:100, 1:200, and 1:400) were prepared in DMEM and incubated with Vero cells for 48 h. Cell viability was quantified by measuring optical density at 540 nm (reference wavelength 650 nm). The highest non-toxic concentrations, defined as those causing < 50% reduction in cell viability, were determined for each formulation (Table 4). PME exhibited the lowest non-toxic concentration at a 1:25 dilution (equivalent to 175 mg/L), while CNP demonstrated minimal toxicity up to a 1:10 dilution (corresponding to 0.08% w/v). Notably, the PME-CNPS formulation showed a non-toxic concentration at a 1:20 dilution (equivalent to 150 mg/L of PME), indicating that encapsulation of the extract into casein nanoparticles slightly reduced its cytotoxicity compared to the free extract. These non-toxic concentrations were selected for subsequent antiviral assays to ensure that any observed antiviral effects were not confounded by cellular toxicity.

Table 4.

Non-Toxic concentrations of PME, CNP, and PME-CNPS on Vero cells

Test material	Highest non-toxic dilution	Corresponding concentration	Cell viability (%)
PME	1:25	175 mg/L	92.3 ± 3.1
CNP	1:10	0.08% w/v	89.7 ± 2.8
PME-CNPS	1:20	150 mg/L (PME equivalent)	94.5 ± 2.4

Data are expressed as mean ± SD (n = 3). Non-toxic concentrations were defined as those causing < 50% reduction in cell viability compared to untreated controls.

Determination of virus infectious titer

The infectious titer of the HSV-1 stock was quantified using the 50% tissue culture infectious dose (TCID₅₀) assay in Vero cells. Serial 10-fold dilutions of the viral stock were prepared, and Vero cell monolayers in 24-well plates were infected in triplicate. After a 1-hour adsorption period at 37 °C in a 5% CO₂ incubator, the inoculum was removed, and cells were overlaid with fresh DMEM. Plates were monitored daily for viral cytopathic effects (CPE), characterized by cell rounding, enlargement, and detachment from the culture surface (Fig. 7). The final CPE observations were recorded on day 7 post-infection. The Reed & Muench method was used to calculate the TCID₅₀ value, which represents the viral dilution required to induce CPE in 50% of infected wells. The initial infectious titer of the HSV-1 stock was determined to be log₁₀ TCID₅₀ = 6.2, corresponding to 1.58 × 10⁶ TCID₅₀ units/mL. This high titer confirmed the robustness of the viral stock for subsequent antiviral efficacy experiments.

Fig. 7.

Cytopathic effects (CPE) of HSV-1 infection in Vero cells

Antiviral activity assays

Direct virucidal effect

The direct effect of PME, CNP, and PME-CNPS on HSV-1 infectivity was evaluated by incubating the virus with each test material for 1 and 2 h before infection of Vero cells. The results are presented in Fig. 8A. As shown in Fig. 8A, PME reduced the HSV-1 titer by 1.9 and 2.2 log units after 1 and 2 h of incubation, respectively. CNP showed minimal virucidal activity with reductions of 0.4 and 0.5 log units. Notably, PME-CNPS demonstrated the strongest virucidal effect with reductions of 2.4 and 2.7 log units after 1 and 2 h, respectively. All reductions were statistically significant compared to the control (p < 0.05).

Fig. 8.

Direct virucidal and prophylactic antiviral effects of Plantago major extract and casein nanoparticle formulations against HSV-1. (A) Virucidal effect: Direct inactivation of HSV-1 after co-incubation with non-toxic concentrations of PME, CNP, or PME-CNPs for 1 h (light bars) or 2 h (dark bars) at 4 °C prior to infection of Vero cells. PME-CNPs demonstrated superior virucidal activity with 2.4-log₁₀ and 2.7-log₁₀ reductions in viral titer after 1 h and 2 h incubation, respectively, significantly exceeding free PME (1.9-log₁₀ and 2.2-log₁₀) and unloaded CNP (0.4-log₁₀ and 0.5-log₁₀). (B) Prophylactic effect: Inhibition of viral infection following 2-hour pre-treatment of Vero cells with test materials before HSV-1 challenge. PME-CNPs reduced viral titer by 1.2-log₁₀, outperforming free PME (0.8-log₁₀) and CNP (0.3-log₁₀, not significant). Data are expressed as log₁₀ TCID₅₀/mL (mean ± SD, n = 3). Statistical significance: ***p < 0.001, **p < 0.01, *p < 0.05 vs. control (untreated virus); ###p < 0.001, ##p < 0.01 PME-CNPs vs. free PME (one-way ANOVA with Tukey’s post-hoc test). The enhanced virucidal and prophylactic activities of PME-CNPs suggest multiple mechanisms including direct viral inactivation through interaction with envelope glycoproteins and prevention of viral attachment to host cell receptors

Prophylactic effect

The protective effect of test materials was evaluated by pre-treating Vero cells with non-toxic concentrations of PME, CNP, and PME-CNPS for 2 h prior to HSV-1 infection. The results are presented in Fig. 8B. As shown in Fig. 8B, pre-treatment of cells with PME and PME-CNPS resulted in significant reductions in HSV-1 titers (0.8 and 1.2 log units, respectively) compared to control (p < 0.05). In contrast, CNP showed only a minor reduction (0.3 log units) that was not statistically significant (p = 0.068).

Therapeutic effect

To determine whether test materials could inhibit HSV-1 replication after establishment of infection, PME, CNP, and PME-CNPs were added to infected Vero cells at different time points (1, 4, 8, 12, and 24 h) post-infection. This time-course experiment was designed to identify the stages of the viral replication cycle most susceptible to inhibition by the test materials. The results, presented in Fig. 9A, demonstrated significant time-dependent antiviral activity. All test materials exhibited maximum therapeutic efficacy when administered 1 h post-infection, with activity declining progressively at later time points (Fig. 9A). PME-CNPs demonstrated superior antiviral performance compared to free PME and unloaded CNP across all time points examined (p < 0.001). At 1 h post-infection, PME-CNPs reduced viral titer by 1.9-log₁₀ (from 6.2 to 4.3 log₁₀ TCID₅₀/mL), significantly exceeding the efficacy of free PME (1.4-log₁₀ reduction, p < 0.001) and CNP (0.6-log₁₀ reduction, p < 0.001). This enhanced early-stage activity suggests that nanoencapsulation facilitates more efficient delivery of PME bioactive compounds to sites of active viral replication.The therapeutic efficacy of PME-CNPs remained substantial even when administered at intermediate time points, achieving 1.6-log₁₀, 1.5-log₁₀, and 1.0-log₁₀ reductions at 8, 12, and 4 h post-infection, respectively. In contrast, free PME showed more modest activity at these time points (1.2-log₁₀, 1.1-log₁₀, and 0.6-log₁₀ reductions, respectively). By 24 h post-infection, when multiple rounds of viral replication have occurred, PME-CNPs still maintained significant antiviral activity (0.7-log₁₀ reduction, p = 0.002), while free PME exhibited only marginal efficacy (0.4-log₁₀ reduction, p = 0.042). Unloaded CNP demonstrated minimal therapeutic activity, with modest effects observed only at early time points (0.6-log₁₀, 0.5-log₁₀, and 0.4-log₁₀ reductions at 1, 8, and 4 h, respectively) and no significant inhibition at 12–24 h post-infection (p > 0.05). This confirms that the antiviral activity of PME-CNPs is primarily attributable to the encapsulated PME components rather than the casein nanocarrier itself. Statistical analysis of time-dependent efficacy variations revealed critical insights into the mechanism of action. For PME-CNPs, the difference in antiviral activity between 1 h and 24 h post-infection was highly significant (mean difference: 1.2-log₁₀, p < 0.001), indicating that the formulation is most effective during the initial stages of infection. Similarly, significant differences were observed between 1 h versus 4 h (0.9-log₁₀, p < 0.001) and 8 h versus 24 h (0.9-log₁₀, p < 0.001). However, no significant difference was detected between 8 and 12 h post-infection (0.1-log₁₀, p = 0.748), suggesting relatively stable activity during this intermediate window of the replication cycle. Free PME exhibited a similar temporal pattern but with less pronounced overall efficacy. The difference between 1 h and 24 h post-infection was 1.0-log₁₀ (p < 0.001), while the difference between 1 h and 4 h was 0.8-log₁₀ (p < 0.001). As with PME-CNPs, no significant difference was observed between 8 and 12 h (p = 0.748), indicating that both formulations target similar stages of the viral life cycle, albeit with different potencies. To visualize the comparative antiviral performance across all conditions, a heatmap was constructed showing log₁₀ reduction relative to untreated control at each time point (Fig. 9B). The heatmap clearly illustrates that PME-CNPs consistently exhibit the highest antiviral activity (darkest red coloration) across all time points, with particularly intense activity concentrated at early infection stages (1–8 h). Free PME displays intermediate activity (moderate red), while CNP shows minimal effect (pale yellow to white), particularly at late time points.The time-dependent decline in therapeutic efficacy for both PME and PME-CNPs suggests that these formulations primarily interfere with early events in the HSV-1 replication cycle. HSV-1 entry into host cells occurs within the first 1–2 h post-infection, followed by immediate-early gene expression (2–4 h), early gene expression and viral DNA replication (4–8 h), late gene expression (8–12 h), and virion assembly and egress (> 12 h). The peak activity of PME-CNPs at 1 h post-infection indicates potential inhibition of viral entry, membrane fusion, or nuclear transport of viral genomes. The sustained but declining activity at 4–12 h suggests additional effects on viral DNA replication or early protein synthesis, while the reduced efficacy at 24 h-when progeny virions have already been assembled and released-confirms that late-stage events are less susceptible to inhibition.The superior performance of PME-CNPs compared to free PME at all time points supports the hypothesis that casein nanoencapsulation enhances the therapeutic potential of Plantago major extract through multiple mechanisms. First, nanoparticles may facilitate more efficient cellular uptake via endocytosis, delivering higher intracellular concentrations of bioactive compounds to sites where viral replication occurs. Second, the sustained release properties of PME-CNPs may maintain therapeutic concentrations of antiviral components throughout the infection period, whereas free PME may be more rapidly degraded or cleared. Third, the nanoformulation may protect labile phytochemicals from enzymatic or chemical degradation in the cellular environment, preserving their antiviral activity. Collectively, these findings demonstrate that PME-CNPs possess robust time-dependent therapeutic activity against HSV-1, with maximum efficacy during the early stages of infection when intervention can most effectively limit viral spread. The consistent outperformance of PME-CNPs relative to free PME across all experimental conditions provides strong evidence that nanotechnology-based delivery enhances the antiviral potential of plant-derived bioactive compounds.

Fig. 9.

Time-dependent therapeutic antiviral activity of test materials against HSV-1 when administered at different time points post-infection. (A) Line graph showing viral titers (log₁₀ TCID₅₀/mL) when non-toxic concentrations of PME, CNP, or PME-CNPs were added to HSV-1 infected Vero cells at 1, 4, 8, 12, or 24 h post-infection. PME-CNPs exhibited the strongest therapeutic effect across all time points, with maximum activity observed at 1 h post-infection (1.9-log₁₀ reduction) and declining efficacy at later time points (0.7-log₁₀ at 24 h). Free PME showed moderate activity (1.4-log₁₀ to 0.4-log₁₀), while unloaded CNP demonstrated minimal effect. The time-dependent decline in efficacy indicates that PME-CNPs primarily target early stages of the viral replication cycle, particularly viral entry and early gene expression. (B) Heatmap visualization of log₁₀ reduction relative to untreated control at each time point. Color intensity represents magnitude of antiviral activity (white: no reduction; dark red: maximum reduction ≥ 2.0-log₁₀). PME-CNPs display consistently higher activity (darker red) than PME or CNP across all time points, with peak activity concentrated at early infection stages (1–8 h). Data are mean ± SD (n = 3). ***p < 0.001, **p < 0.01, *p < 0.05 vs. control at the same time point (one-way ANOVA). The superior performance of PME-CNPs compared to free PME supports the hypothesis that nanoencapsulation enhances cellular uptake and delivery of bioactive compounds to sites of viral replication

Discussion

The development of antiviral therapies using bioactive phytochemicals has attracted considerable attention due to their potentially low toxicity, broad-spectrum activity and lower likelihood of resistance development [24, 25]. In this study, we investigated the antiviral efficacy of PME (PME) encapsulated in casein nanoparticles (PME-CNPS) against herpes simplex virus type 1 (HSV-1). The physicochemical characterization, in vitro release kinetics and antiviral assays show that PME-CNPS is a promising nanoformulation to enhance the therapeutic potential of PME. The particle size, polydispersity index (PDI) and zeta potential of PME-CNPS were characterized in detail to ensure their suitability for biological applications. The unloaded casein nanoparticles (CNP) exhibited a mean size of 87.3 ± 4.2 nm (PDI = 0.182), which increased to 118.6 ± 5.9 nm (PDI = 0.245) after loading with PME. This moderate increase in size (~ 35 nm) confirms successful encapsulation while maintaining a size well below 150 nm, a critical threshold for efficient cellular uptake and tissue penetration [26]. Nanoparticles in the 100–200 nm range are ideal to avoid rapid renal clearance and RES uptake, thus prolonging circulation time [27, 28].

The low PDI values (< 0.3) for both CNP and PME-CNPS indicate a monodisperse size distribution, which is essential for reproducible pharmacokinetics and antiviral efficacy. Polydisperse systems (PDI > 0.3) often suffer from inconsistent drug release and variable cellular interactions [29, 30]. Our results are consistent with studies on casein-based nanoparticles encapsulating curcumin (PDI ~ 0.2) and EGCG (PDI ~ 0.18) showing uniform dispersion and sustained release [31, 32].

The zeta potential of PME-CNPS (− 19.4 ± 1.8 mV) remained within the range required for colloidal stability (≥ ± 20 mV), despite a slight reduction compared to uncharged CNP (− 23.8 mV). This reduction is attributed to partial charge neutralization by cationic or neutral phytochemicals in PME (e.g. flavonoids, polysaccharides), as reported in similar systems [33–35]. Importantly, the negative surface charge may enhance interactions with HSV-1 virions that possess positively charged glycoproteins (e.g. gB, gC), which are critical for attachment to the host cell [36, 37]. Anionic nanoparticles, such as sulfated polysaccharide-coated liposomes, have shown strong affinity for HSV glycoproteins and block viral entry [38–40].

Morphological analysis by SEM confirmed the DLS data and revealed spherical, well-dispersed nanoparticles with smooth surfaces in unloaded CNP and slightly roughened surfaces in PME-CNPS. The surface roughness in PME-CNPS likely reflects the incorporation of PME components into the casein matrix, a phenomenon observed in zein nanoparticles loaded with Artemisia annua extract [41].

Structural findings from FTIR, XRD and DSC confirmed the successful encapsulation of PME. The FTIR spectra showed shifts of characteristic peaks (e.g. the O-H stretching from 3412 cm-¹ to 3385 cm-¹ in PME-CNPS), indicating hydrogen bonding between the phenolic groups of PME and the amide groups of casein. Such non-covalent interactions maintain the chemical integrity of bioactive compounds, as seen in PLGA nanoparticles loaded with Glycyrrhiza glabra extract [42].

The XRD patterns showed a transition from the semi-crystalline structure of native casein to an amorphous state in PME-CNPS, indicating dispersion of PME at the molecular level in the nanoparticles. Amorphous formulations often exhibit better dissolution rate and bioavailability than crystalline counterparts [43]. Similarly, DSC thermograms showed the disappearance of the endothermic peaks of PME (e.g. 158.7 °C) in PME-CNPS, confirming the transformation of PME components from a crystalline to an amorphous or molecularly dispersed state. This is consistent with studies on curcumin-loaded casein nanoparticles, where encapsulation suppressed the melting peak of curcumin at 176 °C [44]. The conversion of the extract and casein from a semi‑crystalline to an amorphous state, as demonstrated by the XRD data, is a hallmark of successful molecular encapsulation. Amorphous formulations are known to exhibit markedly higher apparent solubility and faster dissolution rates than their crystalline counterparts [45, 46]. In the context of PME‑CNPs, this amorphous matrix ensures that the flavonoids, iridoid glycosides and phenolic acids are released in a controlled yet readily accessible manner, thereby maximizing their interaction with HSV‑1 particles and with intracellular viral targets. Consequently, the amorphous nature of the nano‑carrier is a central contributor to the enhanced antiviral efficacy reported in this study. The PME‑CNPs exhibited a zeta potential of ‑19.4 ± 1.8 mV, i.e., a moderately negative surface charge. In the field of nanomedicine a near‑neutral or mildly negative charge is generally regarded as optimal for systemic delivery because it reduces nonspecific adsorption of plasma proteins (opsonins) and therefore limits rapid recognition by the mononuclear phagocyte system (MPS) in the liver and spleen [47, 48]. Highly positive particles are quickly cleared by the MPS due to electrostatic attraction to negatively charged cell membranes, whereas strongly negative particles can also be opsonised through complement activation. The modestly negative potential of our casein‑based nanocarrier strikes a balance: it is low enough to avoid immediate MPS uptake, yet sufficiently negative to maintain colloidal stability and prevent aggregation during storage and in biological media. Although the present work is limited to in‑vitro antiviral assays, the surface‑charge profile suggests that PME‑CNPs would have a prolonged circulation time in vivo, thereby increasing the probability of reaching HSV‑infected tissues.

The entrapment efficiency (EE) and the loading capacity (LC) are decisive factors for the therapeutic potential of a nanoformulation. PME-CNPS achieved an EE of 82.1 ± 3.2% and an LC of 13.5 ± 1.1%, outperforming many systems loaded with plant extracts. For example, chitosan nanoparticles loaded with Azadirachta indica extract exhibited an EE of 74% [49], while zein nanoparticles loaded with Artemisia annua achieved an EE of 78% [50]. The high EE in PME-CNPS can be attributed to the amphiphilic nature of casein, which enables strong hydrophobic and hydrogen-bonding interactions with the polyphenols of PME (e.g. aucubin, ursolic acid) [51].

The biphasic release profile of PME-CNPS — characterised by an initial release burst (32.4% within 30 min) followed by sustained release (76.8% over 120 min) — is ideal for antiviral therapy. The burst phase provides rapid availability of PME to neutralize free virions, while the sustained phase maintains therapeutic concentrations to inhibit viral replication.

Kinetic modeling revealed that PME-CNPS follows first-order release kinetics (R² = 0.982), meaning that the release rate is concentration-dependent. This is in contrast to zero-order kinetics (R² = 0.874), where the release is independent of the concentration. First-order release is common in matrix-based systems such as casein nanoparticles, where diffusion through the polymer network determines the release of the drug [52]. The observed kinetics are consistent with EGCG-loaded casein nanoparticles, which showed first-order release over 48 h [53].

The sustained release phase is particularly advantageous for combating HSV-1 as the virus undergoes multiple replication cycles (6–8 h per cycle) [54, 55]. A prolonged supply of the bioactive components of PME (e.g. flavonoids, iridoid glycosides) could interfere with viral entry, genome replication and capsid assembly over several cycles [56].

While HPLC is the gold standard for quantifying individual phytochemicals, we used UV-Vis spectroscopy (420 nm) to determine entrapment efficiency (EE) and loading capacity (LC) due to its simplicity and suitability for preliminary screening of total Plantago major extract (PME) content. This approach leverages the broad absorption of PME’s phenolic compounds but lacks specificity for individual bioactives (e.g., quercetin, aucubin) and may be affected by matrix interference.

The ethanolic Plantago major extract used in this study contains a complex mixture of flavonoids (quercetin, luteolin, apigenin), iridoid glycosides (aucubin, catalpol), phenolic acids (caffeic, ferulic acid) and terpenoids [13, 57]. Several of these compounds have been examined individually for HSV‑1 inhibition. For example, quercetin reduced HSV‑1 plaque formation and was shown to block viral attachment and DNA‑polymerase activity [58]. Caffeic acid and luteolin have also been reported to inhibit HSV‑1 replication, albeit with lower potency [59, 60].

Surface‑charge considerations in contemporary nanomedicine. Early drug‑delivery research often advocated the use of highly charged nanoparticles (|ζ| > 30 mV) because strong electrostatic repulsion prevented particle aggregation in vitro. Subsequent in‑vivo investigations, however, revealed that excessive surface charge- positive or negative-promotes rapid opsonisation and clearance by MPS, especially in the liver and spleen, and can increase systemic toxicity [47, 48]. A near‑neutral surface charge (|ζ| ≈ 10–20 mV) that still provides sufficient colloidal stability is now considered ideal for achieving long circulation times and evading immune surveillance. The PME‑CNPs prepared in this study exhibit a zeta potential of − 19.4 ± 1.8 mV, which is modestly negative enough to maintain dispersion (|ζ| > 15 mV) yet close enough to neutrality to minimise MPS recognition. Consequently, the charge profile of our casein‑based nanocarrier is expected to support prolonged systemic exposure and reduce off‑target toxicity, thereby enhancing the likelihood that the encapsulated Plantago major phytochemicals reach HSV‑infected tissues.

The antiviral activity of PME-CNPS was comprehensively evaluated by direct virucidal, prophylactic and therapeutic assays, with statistical comparisons with unloaded CNP and free PME. Direct virucidal effect: PME-CNPS showed the strongest virucidal activity, reducing HSV-1 titers by 2.7 log units after 2 h of incubation. This exceeded the effect of free PME (2.2 log units) and uncontaminated CNP (0.5 log units). The increased efficacy of PME-CNPS is likely due to the following factors:

1. Improved solubility and stability of the hydrophobic components of PME (e.g. ursolic acid) in the casein matrix.

2. Multivalent interactions between the anionic nanoparticles and cationic viral glycoproteins leading to virion aggregation or structural disruption [61].

Similar results were reported for sulfated polysaccharide nanoparticles that inactivated HSV-1 by glycoprotein binding [62, 63].

Prophylactic effect: Pretreatment of Vero cells with PME-CNPS reduced HSV-1 infection by 1.2 log units, compared to 0.8 log for free PME. This indicates that PME-CNPS effectively blocks viral attachment:

1. Occupying heparan sulfate receptors on host cells, preventing HSV-1 glycoprotein C/D binding.

2. Formation of a protective barrier on the cell surface through electrostatic interactions.

Casein nanoparticles alone (CNP) showed a minimal prophylactic effect (0.3 log reduction), confirming that antiviral activity is primarily driven by PME.

Therapeutic effect: PME-CNPS showed superior inhibition of HSV-1 replication when administered at different time points post-infection. Specifically, PME-CNPS reduced viral titer by 1.9 log units even when added 24 h post-infection, while free PME only achieved a 0.4 log reduction. This sustained activity underlines the benefits of nanoencapsulation:

1. Protection of PME from degradation in the cellular environment.

2. Enhanced cellular uptake by endocytosis, allowing PME to target intracellular stages of the viral life cycle (e.g. genome replication, protein synthesis).

The stronger therapeutic effect at early time points (1–8 h post-infection) suggests that PME-CNPS primarily affects viral entry and early gene expression, which is consistent with the mechanisms of other herbal antivirals such as EGCG [64, 65].

Statistical analysis (Tables 3 and 4) confirmed that PME-CNPS significantly outperformed free PME at all time points (p < 0.001), with the largest difference observed 1 h post-infection (1.9 log reduction vs. 1.4 log for PME). This underscores the importance of early intervention in HSV-1 infections, where nanoformulations can maximize therapeutic outcomes.

The antiviral mechanisms of PME-CNPS can be traced back to the synergistic effects of:

1. Direct inactivation of virions: The polyphenols of PME (e.g. aucubin, quercetin) disrupt viral envelope glycoproteins and capsid proteins through hydrogen bonding and hydrophobic interactions [66].

2. Blocking viral entry: The anionic surface of PME-CNPS competes with HSV-1 for binding to heparan sulfate proteoglycans on host cells.

3. Inhibition of intracellular replication: Sustained release of PME components can suppress viral DNA polymerase and proteases involved in capsid assembly [67, 68].

The neutral red uptake test showed that PME-CNPS was less cytotoxic than free PME, with a non-toxic concentration of 150 mg/L (PME equivalent) compared to 175 mg/L for PME. This improvement is likely due to the controlled release of PME from the casein matrix, which reduces the immediate exposure of cells to high concentrations of bioactive compounds. Casein nanoparticles alone (CNP) showed minimal toxicity up to 0.08% w/v, which is consistent with their demonstrated biocompatibility [69].

An important consideration is whether residual CaCl₂ from nanoparticle crosslinking could enhance uptake via transfection-like mechanisms. However, this is unlikely because: (1) calcium is consumed during casein crosslinking and removed by centrifugation (residual < 0.5 mM, ~ 5,000× lower than transfection protocols); (2) our system lacks phosphate buffer required for CaPO₄ formation; (3) PME-CNPs have negative surface charge (ζ = −19.4 mV), creating electrostatic repulsion unlike near-neutral transfection particles; and (4) unloaded CNP showed minimal activity (0.3–0.6 log₁₀) while PME-CNPs achieved 1.2–2.7 log₁₀ reductions (p < 0.001), confirming efficacy derives from encapsulated PME, not residual calcium.

While the 1.9–2.7 log₁₀ reduction in HSV-1 TCID₅₀ observed with PME-CNPs is statistically significant (p < 0.001), it falls below the typical efficacy thresholds required for standalone antiviral therapies in pharmaceutical development. This level of viral suppression, though promising for a proof-of-concept study, primarily demonstrates the formulation’s potential to inhibit early stages of infection (e.g., viral entry/replication) rather than achieve complete viral clearance. Given that HSV-1 titers in optimized cultures can reach 10⁸–10⁹ TCID₅₀/mL, these results suggest PME-CNPs may be better suited as an adjunct or prophylactic therapy-particularly in combination with conventional antivirals like acyclovir- enhance overall efficacy. Future studies will focus on in vivo validation, mechanistic characterization of viral inhibition, and optimization of the nanoformulation to improve potency.

Study limitations and future directions

The present study provides solid in vitro evidence that encapsulation of Plantago major extract in casein nanoparticles significantly enhances its antiviral activity against HSV-1 in all tested modes (virucidal, prophylactic, and therapeutic), while simultaneously reducing cytotoxicity compared to the free extract. The formulation exhibited favourable physicochemical properties, high encapsulation efficiency, excellent colloidal stability under the tested conditions, and a desirable sustained-release profile, all of which contributed to superior performance over the non-encapsulated extract. These findings represent a meaningful advance in plant-based nanotherapeutics for herpes simplex virus and justify continued development of this platform.

Nevertheless, as an early-stage in vitro investigation, several limitations must be acknowledged and addressed in subsequent studies. First, viral titers were determined solely by TCID₅₀ assay; although reliable and widely accepted, complementary quantification by plaque assay and/or qPCR would strengthen the reported log-reduction values. Second, the Plantago major extract was characterized only by GC-MS and UV-visible absorbance without quantitative profiling of key antiviral markers (aucubin, catalpol, verbascoside, quercetin glycosides, etc.), which prevents full standardization and identification of the most active constituents. Third, entrapment efficiency and loading capacity were measured by a centrifugation-based method immediately after preparation in water; while the values obtained (EE ≈ 82%, LC ≈ 13.5%) are encouraging, this technique can overestimate true encapsulation in protein-based systems, and no data are yet available on payload retention in serum-containing media or under physiological ionic strength. Fourth, long-term storage stability, serum stability, and behaviour in biological fluids remain uninvestigated; casein nanoparticles are known to be sensitive to divalent ions and pH changes, so premature aggregation or burst release in vivo cannot be excluded at this stage. Fifth, cellular uptake mechanism, intracellular trafficking, and endosomal escape efficiency were not examined; the superior therapeutic effect observed may therefore arise partly from extracellular release rather than true cytosolic delivery. Finally, and most importantly, no in vivo studies have been performed to date. Without animal efficacy, pharmacokinetic, biodistribution, and safety data, the clinical relevance of the promising in vitro results remains to be established.

To move this nanotherapeutic candidate toward preclinical and eventual clinical evaluation, the following studies are recommended: (1) full phytochemical standardization of PME by UPLC-MS and establishment of marker-based quality control; (2) re-determination of EE/LC and release kinetics in 10–50% serum and PBS using dialysis or ultrafiltration methods; (3) comprehensive stability testing (storage at 4 °C and 25 °C, freeze-drying, serum incubation up to 72 h); (4) cellular uptake and trafficking studies in Vero and human keratinocyte/neuron models using fluorescently labelled nanoparticles and confocal/live-cell imaging; (5) detailed mechanism-of-action studies including viral attachment/entry inhibition assays, time-of-addition with qRT-PCR of immediate-early/early/late genes, and glycoprotein-binding experiments; (6) in vivo proof-of-concept in established HSV-1 models (mouse flank, guinea pig genital, or rabbit ocular keratitis) with measurement of lesion score, viral load in tissues, and latency; and (7) preliminary pharmacokinetic and acute toxicity evaluation after topical, intravaginal, or systemic administration.

Addressing these points systematically will clarify the true potential of PME-loaded casein nanoparticles and determine whether this green, biocompatible, and cost-effective nanoformulation merits advancement into preclinical development as a novel therapeutic option for HSV-1 infections.

Conclusion

This study provides robust and comprehensive evidence that encapsulation of Plantago major ethanolic extract within casein nanoparticles (PME-CNPs) markedly enhances its antiviral efficacy against HSV-1 while simultaneously improving its safety profile. The nanoformulation exhibited excellent physicochemical characteristics—uniform nanoscale size (118.6 nm), favourable zeta potential, high encapsulation efficiency (82.1%), and a desirable biphasic release profile governed by first-order kinetics—resulting in significantly greater virucidal (2.7-log reduction), prophylactic (1.2-log), and therapeutic (up to 1.9-log at 1 h post-infection) activity compared to free extract and unloaded nanoparticles (p < 0.001). The superior performance observed across all stages of the viral life cycle, combined with reduced cytotoxicity and sustained intracellular availability of bioactive phytochemicals, strongly supports the hypothesis that casein-based nanoencapsulation overcomes the inherent limitations of crude plant extracts, namely poor solubility, low stability, and limited cellular penetration. By synergistically integrating the well-established antiviral properties of Plantago major with the proven advantages of a biocompatible, biodegradable, and GRAS-status protein nanocarrier, this work presents a promising, green, and cost-effective nanotherapeutic platform for herpes simplex virus infections. The ability of PME-CNPs to outperform free extract even when administered post-infection highlights their potential clinical relevance, particularly in recurrent or drug-resistant HSV episodes where early and sustained intervention is critical. These findings not only validate casein nanoparticles as a highly effective delivery vehicle for complex herbal extracts but also reinforce the immense therapeutic potential of combining ethnopharmacology with modern nanotechnology. PME-CNPs merit immediate progression to in vivo efficacy, pharmacokinetic, and safety studies in relevant animal models, with a clear path toward topical or systemic formulations that could offer a safer, more accessible, and resistance-breaking alternative or adjunct to current acyclovir-based therapies for one of the world’s most prevalent viral infections.

Author contributions

A.A. designed the study, performed the experiments, and drafted the manuscript. H.Z.-Z. contributed to the nanoparticle synthesis, characterization, and data analysis. He also conceptualized the study, supervised the research, and revised the manuscript critically. S.T.H. conducted the antiviral assays and data interpretation. M.A.H. contributed to data analysis, and revised the manuscript. All authors read and approved the final manuscript.

Funding

This research received no specific grant from any funding agency.

Data availability

All data generated or analyzed during this study are included in this published article. Raw datasets are available from the corresponding author upon reasonable request.

Declarations

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