Guangdong Huixuan Pharmaceutical Technology Co. Ltd.

Myocardial fibrosis, a key pathological feature of hypertensive heart disease (HHD), remains diagnostically challenging due to limited clinical tools. In this study, a FAPI-targeted uptake mechanism previously reported by our group, originally developed for tumor imaging, is extended to the detection of myocardial fibrosis in HHD using [¹⁸F]F-NOTA-FAPI-MB. The diagnostic performance of this tracer is compared with those of [¹⁸F]F-FDG, [¹⁸F]F-FAPI-42, and [¹⁸F]F-NOTA-FAP2286, and its potential for fluorescence imaging is also evaluated. Myocardial fibrosis associated with HHD was induced in Sprague-Dawley rats using isoproterenol. Evaluation of cardiac structure and function was conducted through echocardiography. Following each administration of a radioactive tracer via tail vein injection, static PET/CT imaging was performed at 1 and 2 h after injection. Additionally, cardiac excision allowed for ex vivo fluorescence imaging using NOTA-FAPI-MB. All three FAP-targeting probes enabled the precise localization and quantification of fibrotic activity within affected myocardial regions. Among them, [¹⁸F]F-NOTA-FAPI-MB demonstrated the most pronounced increase in TBR from 1 to 2 h in the ISO group and exhibited the highest 2 h/1 h ratio. Blocking experiments revealed limited competitive inhibition of the [¹⁸F]F-NOTA-FAPI-MB uptake by unlabeled FAPI-42. Ex Vivo cardiac fluorescence imaging further confirmed the significant accumulation of NOTA-FAPI-MB. [¹⁸F]F-FAPI-42 exhibited an inferior retention ability at the target site compared to [¹⁸F]F-NOTA-FAPI-MB. In contrast, [¹⁸F]F-NOTA-FAP2286 showed a lower uptake and the weakest retention in fibrotic regions among the three tracers. However, [¹⁸F]F-FDG imaging showed no significant changes in myocardial metabolic activity in HHD, suggesting that [¹⁸F]F-FDG imaging is not effective in detecting cardiac abnormalities associated with HHD. The successful translation of the MB-driven uptake mechanism from tumors to HHD is evidenced by the enhanced myocardial accumulation of [¹⁸F]F-NOTA-FAPI-MB via MB synergy, confirming its mechanism-based targeting. Although further clinical validation is required, this radiopharmaceutical holds strong potential as a dual-modality imaging agent for HHD with a reduced radiation burden.

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.

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.

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.

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.

Paraquat (PQ) -induced pulmonary fibrosis poses a significant medical challenge due to limited treatment options and high mortality rates. Consequently, there is an urgent need for early diagnosis and accurate staging to facilitate appropriate treatment strategies. In this study, we assessed the diagnostic potential of [18F]F-FAPI-42 PET/CT imaging for early detection and disease staging in a rat model of PQ-induced lung fibrosis.

After administering 80 mg/kg of PQ orally to Sprague-Dawley rats, we intravenously injected 3-3.5 MBq of [18F]F-FAPI-42 on day 7, 14, and 21 post-dosing. Dynamic PET/CT imaging was carried out for one hour immediately after the administration of [18F]F-FAPI-42. Subsequently, the lung tissues were collected for Hematoxylin and Eosin (HE) staining, Masson’s trichrome staining, and NOTA-FAPI-04-MB fluorescent probe staining. Data analysis was performed using the Imalytics preclinical software, and the mean standardized uptake value (SUVmean) was calculated.

PET signals revealed that in areas with evident lesions on CT, the SUVmean on day 14 was significantly higher than on day 7 and 21, indicating that changes in fibrosis activity levels contribute to the staging of pulmonary fibrosis. Additionally, the NOTA-FAPI-04-MB fluorescent probe staining also demonstrated the most pronounced probe uptake on day 14. In regions without apparent lesions on CT, the SUVmean gradually increased from day 7 to day 21, reflecting ongoing fibrotic activity. Moreover, HE staining and Masson’s trichrome staining did not reveal pulmonary fibrosis, while PET imaging was able to detect it, serving the purpose of early diagnosis. At 30 min and 60 min, the target-to-background ratio (TBR) of the PQ groups on day 7, 14, and 21 was significantly higher than the control group, suggesting a high specificity of [18F]F-FAPI-42 binding to activated fibroblasts.

[18F]F-FAPI-42 PET/CT imaging enables early diagnosis and staging of PQ-induced pulmonary fibrosis, demonstrating its feasibility and potential for characterizing early disease stages.