Smart Gold Nanoprobe for CT Imaging of Granzyme-B Activity: towards Early Immunotherapy Response Monitoring

Abstract Immunotherapy effectiveness varies widely among patients, strongly impacting outcomes and long-term survival. Early detection of therapeutic response is essential, as timely treatment adjustments can improve prognosis. However, conventional diagnostic tools often lack sensitivity for early response detection, and current imaging techniques are poorly suited for real-time monitoring of immunotherapy efficacy. To overcome this limitation, we developed a hybrid nanoprobe based on gold nanospheres functionalized with a Granzyme B-cleavable peptide−polyethylene glycol conjugate (GNS@pep-PEG). This nanoprobe detects Granzyme B (GzmB) activity using computed tomography (CT). The probe remains colloidally stable in circulation and becomes selectively activated in the tumor microenvironment when GzmB is released by immune effector cells during effective immunotherapy. Peptide cleavage removes the PEG shell, exposing hydrophobic cholesterol groups that trigger nanoparticle aggregation and enhance CT contrast. In vitro, the nanoprobe showed selective activation in a co-culture model of B16F10 melanoma and CTLL-2 cytotoxic T cells, while monocultures confirmed GzmB specificity. In vivo, GzmB-treated mice exhibited increased nanoprobe accumulation and enhanced CT contrast at injection sites. This proof-of-concept study validates the gold-based nanoprobe platform’s performance, ensuring enzyme activity and opening the possibility of following immunotherapy treatment outcomes.