Prostate cancer is the second most prevalent malignancy in men globally, with delayed clinical management due to the lack of reliable technologies for early metastasis monitoring and precise imaging. To address this challenge, we developed a self-assembled DNA tetrahedral nanostructure (TDN)-based nanoprobe targeting prostate-specific membrane antigen (PSMA). This platform aims to enable targeted in vivo imaging of prostate cancer, offering a transformative strategy for non-invasive diagnosis and real-time metastasis surveillance.

The tetrahedral DNA nanostructure (TDN) was synthesized via stepwise annealing of four phosphorothioate-modified DNA single strands, forming a stable 3D framework with 7 nm edge lengths. PSMA-specific aptamers (binding affinity Kd = 4.3 nM) were conjugated at the vertices, while near-infrared fluorophore Cy5.5 was linked to the side chains. Structural integrity and assembly efficiency were validated through native polyacrylamide gel electrophoresis (PAGE, Lanes 1-6) and atomic force microscopy (AFM). Targeting specificity was assessed using flow cytometry and confocal imaging in PSMA-positive (LNCaP) and PSMA-negative (PC3) cell lines. In vivo biodistribution and tumor-targeting efficacy were evaluated via fluorescence imaging in orthotopic prostate cancer xenograft mouse models.

The TDN demonstrated exceptional serum stability (>24 hours vs. <2 hours for linear DNA) and achieved 4.1-fold higher fluorescence intensity in PSMA-positive cells compared to PSMA-negative controls (p<0.001). In vivo imaging revealed 3.2-fold greater accumulation of Cy5.5-TDN at tumor sites than non-targeted counterparts within 24 hours, with fluorescence signal intensity quantitatively confirming its high-sensitivity detection capability.

We successfully engineered a PSMA-targeted DNA tetrahedral nanoprobe with superior stability, precise tumor specificity, and high imaging contrast, establishing a reliable platform for non-invasive prostate cancer diagnosis and metastasis monitoring. This modular design paves the way for future integration of therapeutic agents to realize theranostic integration.