Distinguishing Breast Cancer Subtypes via Ratiometric Photoacoustic Sensing of pH and Cu ²⁺ in the Tumor Microenvironment

Triple-negative breast cancer (TNBC) is characterized by a lack of effective therapeutic targets, high invasiveness, and a propensity for metastasis, making the development of highly specific and sensitive early diagnostic and personalized treatment strategies particularly urgent. Leveraging the distinct features of the TNBC tumor microenvironment─specifically, acidic pH and abnormal Cu2+ enrichment─this study designed and constructed a pH/Cu2+ dual-responsive ratiometric photoacoustic (PA) nanoprobe, termed LPCR NP, for precise in vivo PA imaging and the differential diagnosis of TNBC. The nanoprobe consists of a pH/Cu2+ dual-responsive cyanine dye (LET-PCu) and a reference dye (IR140), exhibiting excellent biocompatibility and stability. In vitro experiments confirmed that under acidic conditions and in the presence of an elevated Cu2+ concentration, the probe exhibited a significant increase in optical absorption, which led to a robust enhancement of PA signal at 720 nm, demonstrating excellent dual-activation behavior. Meanwhile, IR140 maintained a stable reference PA signal at 820 nm, allowing the LPCR NP to generate highly distinguishable ratiometric PA signals. In a subsequent in vivo double-blind study, the nanoprobe successfully differentiated between MDA-MB-231 (TNBC) and MCF-7 (non-TNBC) tumor models through PA imaging. This nanoprobe not only provides a novel tool for noninvasive and accurate diagnosis of TNBC but also holds significant translational potential in theranostic applications such as monitoring the efficacy of copper depletion therapy and screening eligible patients.