Biogenic Ag/AgCl nanocomposites induce apoptosis via mitochondrial pathway and reduce size of 3D HT29 colorectal cancer spheroids

This study reports the successful green synthesis of silver/silver chloride nanocomposites using Opopanax hispidus extract. Characterization analyses confirmed the formation of Ag/AgCl nanostructures with good stability. A distinct surface plasmon resonance peak at 433 nm and absorbance over 30 days indicated strong colloidal stability. X-ray diffraction analysis revealed the coexistence of metallic silver and silver chloride phases, while transmission electron microscopy images showed polydisperse, spherical to quasi-spherical nanoparticles ranging from 8.02 to 51.68 nm. Energy dispersive spectroscopy further confirmed the elemental composition, including a notable chlorine signal supporting AgCl formation. The cytotoxic effects of the synthesized nanocomposites were assessed using a three-dimensional spheroid model of HT29 colorectal cancer cells. A dose-dependent reduction in both cell viability and spheroid integrity was observed, with significant effects at concentrations of 100 and 200 μg/mL. Western blot analysis revealed apoptosis induction via the mitochondrial pathway, with downregulation of anti-apoptotic Bcl-X_S/L and upregulation of pro-apoptotic Bax and cleaved caspase-3. This study is the first to investigate the anticancer effects of green-synthesized silver/silver chloride nanocomposites in a three-dimensional colorectal cancer spheroid model. The findings demonstrate the therapeutic potential of these nanocomposites and emphasize their molecular specificity within a physiologically relevant tumor microenvironment.