Natural killer cells labeled with radionuclide-embedded double-layer gold nanoparticles conjugated to a mitochondrial localization sequence for PET/CT imaging and NK cell-based immunotherapy.

BACKGROUND

Noninvasive, sensitive monitoring of natural killer (NK) cells in vivo remains challenging due to limited nanoparticle uptake. We developed a radionuclide-embedded double-layer gold nanoparticle (RI-DLGNP) platform labeled with iodine-124 or iodine-125 and conjugated with mitochondrial localization sequence (MLS) peptides to enhance uptake and enable PET/CT tracking.

METHODS

RI-DLGNPs were synthesized, radiolabeled, and conjugated with MLS peptides. NK cells were labeled with MLS-RI-DLGNPs, and labeling efficiency and stability were evaluated. Cell viability and function were assessed post-labeling. In vivo PET/CT imaging was performed after intravenous injection to monitor NK cell distribution for up to 48 h. Cytotoxicity was evaluated via caspase-3 activation in D54-CR cells and in a luciferase-expressing RKO model.

RESULTS

MLS conjugation improved NK cell uptake and labeling efficiency, with stable radiolabel retention. NK cell viability and function were preserved. PET/CT imaging enabled longitudinal tracking up to 48 h, showing predominant accumulation in lungs, liver, and spleen. Labeled NK cells demonstrated enhanced cytotoxicity, evidenced by increased caspase-3 activation and target-dependent effects.

CONCLUSIONS

MLS-conjugated RI-DLGNPs enable stable labeling and high-sensitivity PET/CT imaging for real-time monitoring of NK cells, supporting further preclinical validation for immunotherapy applications.