Development and characterization of an Eugenol–Shilajit–Chitosan nano-gel composite with anticancer activity against oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) accounts for over 90% of oral malignancies and remains associated with high morbidity, recurrence, and poor long-term survival rates. To address these clinical challenges, the development of multifunctional nanotherapeutic systems capable of improving active stability and localized anticancer activity has become a vital focus in oral oncology and dental nanomedicine. The present study aimed to fabricate, structurally characterize, and biologically evaluate a novel Shilajit/Eugenol/Chitosan (SEC) biohybrid nanocomposite against human OSCC cell lines. The SEC nanocomposite was synthesized via homogenization and amalgamator-assisted mixing. Comprehensive physicochemical characterization (UV–Visible spectroscopy, FTIR, SEM, XRD, and zeta potential analysis) confirmed the successful incorporation and stabilization of shilajit and eugenol within the chitosan polymer matrix through intermolecular hydrogen bonding and electrostatic interactions. Dynamic light scattering revealed a monodisperse nanoscale distribution with an average particle size of 366.1 nm, accompanied by strong colloidal stability indicated by a highly positive zeta potential of + 47.3 mV. Biological profiling established that the SEC nanocomposite possesses robust radical-scavenging activity (ABTS IC 50 = 34.089 ± 0.93 µg/mL; ORAC = 39.784 ± 1.33 µM TE/L) alongside potent 5-lipoxygenase inhibitory action (IC 50 = 1.12 ± 0.03 µg/mL), confirming its dual antioxidant and anti-inflammatory capacity. MTT cytotoxicity assays demonstrated significant dose-dependent antiproliferative efficacy against human OSCC cell lines, displaying IC 50 values of 7.645 µg/mL (SCC-9), 4.687 µg/mL (SCC-15), and 3.702 µg/mL (SCC-25). Mechanistic flow cytometric analysis confirmed that this anticancer action was driven by marked G0/G1 phase cell cycle arrest and significant induction of apoptotic programmed cell death in SCC-25 cells. While these results provide strong in vitro proof-of-concept, the findings reflect preliminary cell line evaluations and warrant further validation in complex 3D culture platforms and in vivo models. Collectively, these findings demonstrate that the developed SEC nanocomposite represents a highly effective, multifunctional nanoplatform with therapeutic potential for oral cancer treatment and localized dental applications.