Enhanced NIR-II Nanoparticle Probe for PSMA-Targeted Molecular Imaging and Prostate Cancer Diagnosis.

INTRODUCTION: Prostate-specific membrane antigen (PSMA) is a well-established biomarker overexpressed in prostate cancer (PCa). However, existing PSMA-targeted imaging probes suffer from radiation exposure, limited tissue penetration, and inadequate intraoperative performance. To overcome these challenges, we developed a novel near-infrared-II (NIR-II) fluorescent nanoprobe for both in vivo and ex vivo imaging of PCa. METHODS: An organic semiconducting polymer (OSP(12)) with strong NIR-II fluorescence and excellent photostability was self-assembled into nanoparticles (NPs) using DSPE-PEG-Mal. These OSP(12) NPs were then conjugated with ACUPA-SH, (S)-2-[3-((S)-5-amino-1-carboxypentyl)ureido]pentanedioic acid, a thiol-modified glutamate-urea-lysine derivative that specifically targets PSMA, via a maleimide-thiol click reaction to form PSMA-OSP(12) NPs. The probe's targeting specificity was assessed using PSMA positive and negative cell lines under NIR-II imaging. For in vivo evaluation, subcutaneous xenograft tumors were established in BALB/c nude mice. Animals were randomly assigned to PSMA-OSP(12) NP, blocking (ACUPA pre-injection), and control (OSP(12) NPs) groups (n = 3 per group). Ex vivo tumor slice imaging was performed on fresh tissue sections. Biosafety was evaluated in healthy mice (n=5) through hematological, biochemical, and histopathological analyses. RESULTS: PSMA-OSP(12) NPs exhibited excellent optical properties in the NIR-II window, including high photostability, negligible autofluorescence, and deep tissue penetration. In vitro assays confirmed selective binding to PSMA-positive cells, while in vivo imaging demonstrated sustained tumor accumulation with a peak TBR of 7.40 ± 1.28 at 48 h post-injection. This performance significantly surpassed OTL78 and Cy-KUE-OA, enabling flexible surgical planning and real-time intraoperative guidance. In ex vivo tissue imaging, PSMA-OSP(12) NPs provided high-contrast tumor delineation without systemic administration. Biosafety evaluations revealed no significant systemic toxicity, and biodistribution analysis indicated hepatic metabolism and biliary clearance. CONCLUSION: PSMA-OSP(12) NPs are a promising NIR-II fluorescent probe with excellent tumor specificity, deep tissue imaging capability, and good biocompatibility, supporting their application in fluorescence-guided surgery and ex vivo tumor evaluation.