A macrophage-targeted ratiometric upconversion nanoprobe for high-fidelity imaging of nitric oxide dynamics during immune polarization and plasticity.

Visualizing nitric oxide (NO) dynamics during macrophage polarization is critical for understanding immune regulation yet remains challenging due to the poor photostability and specificity of existing probes. Herein, we engineered a macrophage-targeted ratiometric nanoprobe (ManNAc-UCNPs) to monitor rapid NO fluctuations during immune plasticity. The probe integrates a photostable core-shell-shell upconversion nanoparticle (UCNP) donor with an NO-responsive dye acceptor via a self-calibrating Luminescence Resonance Energy Transfer (LRET) mechanism. Surface modification with N-Acetyl-D-mannosamine (ManNAc) enables active targeting to macrophage mannose receptors. The nanoprobe exhibits superior photostability compared to organic dyes, high selectivity against interfering species, and a low detection limit of 3.38 nM. In vitro experiments, including co-culture models, demonstrated precise macrophage-specific uptake and excellent long-term colloidal stability. Crucially, the probe successfully differentiated M0, M1 (high NO), and M2 (low NO) phenotypes and enabled the real-time, high-fidelity visualization of dynamic macrophage re-polarization processes. This study provides a robust tool for dissecting immune signaling pathways and evaluating immunomodulatory therapies.