Purpose

Fibroblast activation protein inhibitor (FAPI) -based probes have been widely studied in the diagnosis of various malignant tumors with positron emission tomography/computed tomography (PET/CT). However, current imaging studies of FAPI-based probes face challenges in rapid clearance rate and potential false-negative results. Furthermore, FAPI has been rarely explored in optical imaging. Considering this, further modifications are imperative to improve the properties of FAPI-based probes to address existing limitations and broaden their application scenarios. In this study, we rationally introduced methylene blue (MB) to FAPIs, thereby imparting nuclei-targeting and fluorescence imaging capabilities to the probes. Furthermore, we evaluated the added value of FAPI-based fluorescence imaging to traditional PET/CT, exploring the potential application of FAPI-based probes in intraoperative fluorescence imaging.

Methods

A new FAPI-based probe, namely NOTA-FAPI-MB, was designed for both PET/CT and fluorescence imaging by conjugation of MB. The targeting efficacy of the probe was evaluated on fibroblast activation protein (FAP)-transfected cell line and human primary cancer-associated fibroblasts (CAFs). Subsequently, PET/CT and fluorescence imaging were conducted on tumor-bearing mice. The tumor detection and boundary delineation were assessed by fluorescence imaging of tissues from hepatocellular carcinoma (HCC) patients.

Results

NOTA-FAPI-MB demonstrated exceptional targeting ability towards FAP-transfected cells and CAFs in comparison to NOTA-FAPI. This benefit arises from the cationic methylene blue (MB) affinity for anionic nucleic acids. PET/CT imaging of tumor-bearing mice revealed significantly higher tumor uptake of [¹⁸F]F-NOTA-FAPI-MB (standard uptake value of 2.20 ± 0.31) compared to [¹⁸F]F-FDG (standard uptake value of 1.66 ± 0.14). In vivo fluorescence imaging indicated prolonged retention at the tumor site, with retention lasting up to 24 h. In addition, the fluorescent probes enabled more precise lesion detection and tumor margin delineation than clinically used indocyanine green (ICG), achieving a 100.0% (6/6) tumor-positive rate for NOTA-FAPI-MB while 33.3% (2/6) for ICG. These findings highlighted the potential of NOTA-FAPI-MB in guiding intraoperative surgical procedures.

Conclusions

The NOTA-FAPI-MB was successfully synthesized, in which FAPI and MB simultaneously contributed to the targeting effect. Notably, the nuclear delivery mechanism of the probes improved intracellular retention time and targeting efficacy, broadening the imaging time window for fluorescence imaging. In vivo PET/CT demonstrated favorable performance of NOTA-FAPI-MB compared to [¹⁸F]F-FDG. This study highlights the significance of fluorescence imaging as an adjunct technique to PET/CT. Furthermore, the encouraging results obtained from the imaging of human HCC tissues hold promise for the potential application of NOTA-FAPI-MB in intraoperative fluorescent surgery guidance within clinical settings.

中文翻译：

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合理设计的基于 FAPI 04 的核靶向分子探针，可增强 PET/CT 和荧光成像的肿瘤摄取

目的

基于成纤维细胞激活蛋白抑制剂（FAPI）的探针已在正电子发射断层扫描/计算机断层扫描（PET/CT）诊断各种恶性肿瘤中得到广泛研究。然而，目前基于 FAPI 的探针的成像研究面临快速清除率和潜在假阴性结果的挑战。此外，FAPI 在光学成像领域很少得到探索。考虑到这一点，有必要进一步修改以提高基于 FAPI 的探针的性能，以解决现有的局限性并拓宽其应用场景。在本研究中，我们合理地将亚甲蓝（MB）引入到FAPI中，从而赋予探针核靶向和荧光成像能力。此外，我们评估了基于 FAPI 的荧光成像相对于传统 PET/CT 的附加值，探索基于 FAPI 的探针在术中荧光成像中的潜在应用。

方法

一种新的基于 FAPI 的探针，即 NOTA-FAPI-MB，设计用于通过 MB 结合进行 PET/CT 和荧光成像。在成纤维细胞激活蛋白（FAP）转染的细胞系和人原代癌症相关成纤维细胞（CAF）上评估了探针的靶向功效。随后，对荷瘤小鼠进行 PET/CT 和荧光成像。通过肝细胞癌（HCC）患者组织的荧光成像评估肿瘤检测和边界描绘。

结果

与 NOTA-FAPI 相比，NOTA-FAPI-MB 对 FAP 转染细胞和 CAF 表现出卓越的靶向能力。这一优势源自阳离子亚甲蓝 (MB) 对阴离子核酸的亲和力。荷瘤小鼠的 PET/CT 成像显示，与 [ 18 F]F-FDG（标准摄取值为 1.66）相比，[ 18 F]F-NOTA-FAPI-MB 的肿瘤摄取显着更高^（标准摄取值为^2.20 ± 0.31）±0.14）。体内荧光成像显示在肿瘤部位的滞留时间较长，滞留时间长达 24 小时。此外，与临床使用的吲哚菁绿（ICG）相比，荧光探针能够实现更精确的病灶检测和肿瘤边缘勾画，NOTA-FAPI-MB的肿瘤阳性率达到100.0%（6/6），而NOTA-FAPI-MB的肿瘤阳性率为33.3%（2/6）。 ）对于 ICG。这些发现凸显了 NOTA-FAPI-MB 在指导术中手术过程中的潜力。

结论

成功合成了NOTA-FAPI-MB，其中FAPI和MB同时发挥了靶向作用。值得注意的是，探针的核递送机制改善了细胞内保留时间和靶向功效，拓宽了荧光成像的成像时间窗口。体内 PET/CT 显示 NOTA-FAPI-MB 与 [ ¹⁸ F]F-FDG 相比具有良好的性能。这项研究强调了荧光成像作为 PET/CT 辅助技术的重要性。此外，从人类 HCC 组织成像中获得的令人鼓舞的结果为 NOTA-FAPI-MB 在临床环境中术中荧光手术指导中的潜在应用带来了希望。