Repurposing CNS drugs Zonisamide and Perampanel conjugated with silver nanoparticles as an anti-amoebic agent in combating Granulomatous Amebic Encephalitis (GAE) and Primary Amebic Meningoencephalitis (PAM) infection caused by Acanthamoeba castellanii and Naegleria Fowleri.

Acanthamoeba castellanii (A. castellanii) and Naegleria fowleri (N. fowleri) are free-living amoebae that cause Granulomatous Amoebic Encephalitis (GAE) and Primary Amoebic Meningoencephalitis (PAM), respectively rare but often fatal central nervous system infections. Current treatment options are limited by poor blood-brain barrier (BBB) penetration and inconsistent therapeutic efficacy. In this study, we explored the repurposing of the central nervous system drugs zonisamide and perampanel, conjugated with silver nanoparticles (AgNPs), as a novel anti-amoebic strategy. Drug-nanoparticle conjugates were synthesized and physicochemically characterized, demonstrating stable colloidal properties. Both zonisamide-AgNPs and perampanel-AgNPs exhibited significant, dose-dependent amoebicidal activity. At 100 μ g /mL, zonisamide-AgNPs and perampanel-AgNPs reduced the viability of A. castellanii and N. fowleri trophozoites by up to 77.8% and 80.7%, respectively. In addition, the conjugates inhibited encystation and excystation in A. castellanii and markedly reduced N. fowleri-mediated host cell cytopathogenicity by up to 95%. Cytotoxicity assays using human HaCaT and SH-SY5Y cell lines revealed moderate toxicity, with perampanel-AgNPs displaying higher cytotoxicity (80% at 100 μ g /mL) compared to zonisamide-AgNPs (60%). Mechanistic investigations indicated that zonisamide-AgNPs induced elevated reactive oxygen species (ROS), suggesting oxidative stress-mediated amoebicidal activity. In contrast, perampanel-AgNPs exerted their effects through ROS-independent mechanisms. Overall, these findings demonstrate the potential of AgNP-conjugated CNS drugs as dual-function therapeutic agents against neuroinvasive amoebic infections and support further in vivo evaluation of this nanotherapeutic approach.