A ratiometric NIR-II photoacoustic nanoprobe for non-invasive tracking of natural killer cell activation in vivo

Adoptive natural killer (NK) cell-based immunotherapy holds immense promise for cancer treatment, yet the lack of non-invasive tools for real-time in vivo activation monitoring hinders therapeutic evaluation. Herein, we report AuNR@P18, a smart ratiometric photoacoustic (PA) nanoprobe designed to track NK cell activation. Constructed by conjugating a granzyme B (GrB)-responsive dye (P18) onto gold nanorods (AuNRs), the probe utilizes the AuNR core as a stable NIR-II reference (PA 970 nm ), and P18 as an activatable signal (PA 740 nm ) upon GrB-mediated cleavage. Comprehensive assays confirmed that AuNR@P18 labeling (50 µg/mL) preserves NK cell viability, phenotype, cytokine secretion, and cytotoxicity. In vitro , co-culture with tumor cells triggered a significant increase in the PA 740 nm /PA 970 nm ratio, accurately reporting activation. Critically, in vivo studies in tumor-bearing mice demonstrated that intravenous infusion of labeled NK cells enabled non-invasive visualization of both tumor homing and subsequent activation within the microenvironment. This biocompatible nanoplatform offers a promising strategy for longitudinal monitoring of adoptive NK cell therapy, providing crucial insights for optimizing treatment protocols and elucidating anti-tumor mechanisms.