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

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.