Introduction

According to American Cancer Society’s estimates, there will be 299,010 new cases of prostate cancer in 2024, and about 35,250 deaths from prostate cancer. Surgical removal of the tumor has been the main treatment for localized prostate cancer, but it shows a more than 60% recurrence rate after radical prostatectomy, mainly due to the difficulty in visualizing areas of prostate cancer invasion. The imprecise surgery commonly results in positive surgical margins and damage or removal of muscles and nerves surrounding the prostate. Recently, fluorescent imaging guided surgery (FIGS) emerges and attracts extensive attention helping surgeons identify anatomic landmarks and surgical planes.  In the meanwhile, a new noninvasive therapeutic method, photothermal therapy (PTT), has gained momentum for cancer treatment. PTT is a technique that convert light energy to heat energy to induce protein denaturation and cell membrane damage, leading to rapid cell death[1]. Thus, searching for dual-functional fluorescent reagent that can improve the visualization of tumor margins and simultaneously eliminate residual tumor tissue has become a new research hotspot.

Goal

ICG is an FDA approved near-infrared (NIR) reagent used for fluorescence-based intraoperative imaging with minimal toxicity [2]. ICG also has a potential application for photothermal therapy. This study aims to find a dual-functional fluorescent reagent which can selectively bind to prostate cancer biomarker PSMA and can be effectively used in FIGS and PTT.

Methods

PSMA-1-ICG was synthesized using our highly specific, negatively charged, urea-based PSMA ligand PSMA-1 [3] and its structure was confirmed by Mass Spectrometry (MS). To verify if PSMA-1-ICG binds selectively to PSMA, binding assay of PSMA-1-ICG was performed in PSMA positive PC3pip cells and PSMA negative PC3flu cells. To detect the in vivo uptake of PSMA-1-ICG, Nu/Nu mice were inoculated with both PC3pip and PC3flu cells. PSMA-1-ICG (100 nmol/kg body weight) was administered via tail vein injection and fluorescent images were taken by IVIS spectrum at different time points. To verify whether PSMA-1-ICG generate heat under the light stimulation, PSMA-1-ICG solutions in PBS were illuminated with irradiance of 400 mW/cm2 and radiant exposure of 240 J/cm2 (Using Modulight) for10 min and the temperature of the solution was measured every minute.

Results

In in vitro binding assay, PSMA-1-ICG only showed specific binding to PC3pip cells with equilibrium dissociation constant (KD) at 15.3 nM (determined by GraphPad Prism 10); no specific binding was observed in PC3flu cells. In vivo fluorescence imaging studies demonstrated that PSMA-1-ICG selectively accumulated in PC3-pip tumor tissue and the uptake started slowly at around 2h, reached peak at 72h, then slowly decreased. At peak time, the signal in PC3pip tumors was about 5-fold higher compared to signal in PC3flu tumors. From PSMA-1-ICG heat generation assay it was found that the temperature of 20uM PSMA-1-ICG solution reached to about 54oC under the light stimulation, when the distance was 4mm between the tip of the fiber and the surface of solution. In contrast, no temperature change was observed in PBS at the same conditions. These results indicate that PSMA-1-ICG has good photo-thermal effect.

Conclusion

By targeting PSMA, PSMA-1-ICG showed selective binding to PSMA positive PC3pip cells and tumor tissue, which makes it a good probe for fluorescence image guided surgery for localized prostate cancer. Also, PSMA-1-ICG can generated heat under light stimulation. The highest temperature can reach 54oC in the 20uM solution, which indicated its cell killing capability by PTT. The approach here will provide a new theranostic approach for the treatment of prostate cancer. Further in vitro and in vivo studies to verify PSMA-1-ICG application in FIGS and PTT are ongoing. 

Acknowledgement

This work is supported by the National Science Foundation under Grant No. 2320090.

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