Bioorthogonal click chemistry-mediated conjugation of an anti-DR4 antibody to gold nanorods enhances its pro-apoptotic activity under near infra-red-light stimulation

• A novel surface conjugation strategy enables precise attachment of therapeutic antibodies to gold nanorods without loss of biological activity. • This targeted design enhances cancer cell death while allowing simultaneous imaging through the unique optical features of the nanomaterials. • The combined therapeutic and diagnostic functionality offers a new pathway for selective and personalized cancer treatment. • The work demonstrates how advanced materials engineering can overcome major limitations in conventional cancer drug delivery systems. Pro-apoptotic receptor activators (PARAs), including second-generation agonist antibodies and ligands targeting the TNF receptor superfamily such as TRAIL (TNF-Related Apoptosis-Inducing Ligand), represent a promising class of cancer therapeutics. Enhancing their apoptotic efficacy through multivalency or controlled thermal stimulation has been previously demonstrated. In this study, we report the design and optimization of gold nanorod (GNR)-based nanoconjugates for targeted PARA delivery under near-infrared (NIR)-induced mild hyperthermia. Three conjugation strategies—EDC/NHS coupling, Schiff base formation, and strain-promoted azide–alkyne cycloaddition (SPAAC)—were systematically evaluated. Surface functionalization of GNRs with –COOH, –NH 2 , and −DBCO groups was confirmed via UV–vis spectroscopy, surface-enhanced Raman spectroscopy (SERS), X-ray photoelectron spectroscopy (XPS), zeta potential analysis, and transmission electron microscopy (TEM). Bioactivity assays indicated that SPAAC-mediated click chemistry enabled superior retention of PARA functionality compared to conventional methods. Notably, GNRs conjugated with an anti-DR4 antibody selectively triggered apoptosis in cancer cells upon NIR exposure. These results demonstrate that SPAAC-mediated functionalization enables precise, bioactive nanocarriers for enhanced cancer cell apoptosis under photothermal control