English

• As a metabolic disorder, diabetes readily leads to severe complications affecting multiple organs and systems of the body, posing significant challenges to individuals, the healthcare system, and the socio-economy, particularly in low- and middle-income countries [1,2]. Studies have demonstrated that lifestyle modifications and risk factor reduction can delay the onset of type 2 diabetes in individuals with early pancreatic β-cell impairment and significantly improve their quality of life post-diagnosis [3]. Therefore, screening high-risk individuals is crucial for the early prevention and management of diabetes [4,5]. However, effective early screening methods for diabetes are currently lacking [6]. Blood glucose, the gold standard for diabetes diagnosis, has limited utility for early detection due to the remarkable compensatory capacity of pancreatic β-cell, which masks blood glucose dysregulation until a large part of β-cell function is impaired [2,7-9]. While the functional loss of β-cell mass (BCM) is directly related to islet damage and has the potential for early diabetes screening [10-12], there are currently few effective strategies for monitoring the loss of functional BCM [13,14]. Consequently, developing an effective early screening strategy for high-risk individuals holds significant implications for diabetes prevention and management.

  Insulinoma is a rare neuroendocrine tumor arising from pancreatic β-cell, typically presenting as a solitary nodule [15]. While predominantly benign, it often leads to excessive insulin secretion, causing endocrine disorders that significantly impair quality of life and can even be fatal [16,17]. Conventional imaging modalities often face challenges in localizing insulinoma due to their small size, with limited specificity and inability to precisely delineate lesions for diagnosis and treatment [18,19]. While endoscopic ultrasound (EUS) and calcium stimulation (ASVS) offer higher sensitivity, they require complex procedures performed by experienced specialists, posing significant implementation hurdles [14,20]. Therefore, there is a need to develop a highly sensitive, highly specific, non-invasive, and user-friendly detection strategy to enable the detection of insulinoma.

  Positron emission tomography/computed tomography (PET/CT) is an advanced imaging modality that synergistically integrates PET and CT technologies [21-23]. Initially, a CT scan is performed to acquire high-resolution anatomical images [24]. Subsequently, a PET scan is conducted to detect early functional changes, which may precede detectable structural alterations [25-27]. The resulting images amalgamate the detailed anatomical information provided by CT with the metabolic and functional data obtained from PET [28]. This comprehensive approach enhances diagnostic accuracy, enabling clinicians to more precisely localize and evaluate lesions [25,29]. However, the PET/CT detection methods currently used in clinical applications generally have low specificity and are susceptible to interference from non-specific uptake [30,31]. PET/CT detection of BCM and insulinoma is still under research [14]. Although some progress has been made, challenges remain [24,30]. Therefore, further development of efficient PET/CT molecular probes is needed to address these issues.

  In this study, we employed a previously reported molecular recognition panel consisting of two RNA aptamers as a specific recognition molecule for pancreatic β-cell [32]. Gallium-68 (⁶⁸Ga) was used as the reporting molecule. 1,4,7-Triazacyclononane-1,4,7-triacetic acid (NOTA)-modified aptamers were conjugated with 68Ga through chelation, thereby constructing a PET molecular imaging probe (⁶⁸Ga-NOTA-Ap) with specific recognition capability (Fig. 1). Studies have found that the two RNA aptamers, named m12–3773 and 1–717, achieved highly specific imaging of pancreatic islets by recognizing clusterin and transmembrane p24 trafficking protein 6 (TMED6) on the surface of pancreatic β-cell, respectively [32]. Studies showed that combining these two RNA aptamers, which have the ability to recognize different subpopulations of β-cell, can maximize the signal-to-noise ratio for β-cell identification by targeting different β cell-specific epitopes [32]. Intravenous administration presents a significant challenge for molecular probes constructed from RNA aptamers [33,34]. RNA aptamers are highly susceptible to degradation by RNases in the blood, leading to a loss of recognition capability and hindering specific targeted imaging [35,36]. To address this issue, we introduced fluorination at the 2′ position of the ribose of cytosine (C) and uracil (U), and methoxylation at the 2′ position of the ribose of adenine (A) and guanine (G) [34,37]. The modified RNA aptamers were renamed M-m12–3773 and M-1–717, respectively. These modifications enhanced the RNases resistance of the RNA aptamers, thereby significantly improving the stability of ⁶⁸Ga-NOTA-Ap in the blood. Through a series of studies, we successfully achieved imaging of rat islets using ⁶⁸Ga-NOTA-Ap, observed changes in BCM in Sprague-Dawley rats with pancreatic β-cell damage, and demonstrated that ⁶⁸Ga-NOTA-Ap can not only monitor islets but also effectively image insulinoma in NOD/SCID mice through recognition of abnormal β-cell proliferation, significantly broadening its application range. The successful construction of this new PET molecular imaging probe holds significant potential clinical value for diabetes monitoring and insulinoma diagnosis.

  Figure 1

  

  Figure 1.  Schematic of nucleoside modifications stabilizing ⁶⁸Ga-labeled RNA aptamers for pancreatic β-cell and insulinoma imaging. This study primarily consists of two parts: (Ⅰ) The 2′-O-methyl modifications on C and U, and 2′-fluoro modifications on A and G respectively, improved the stability and RNases resistance of RNA aptamers while maintaining their K_d values. (Ⅱ) The ⁶⁸Ga-labeled modified RNA aptamers not only characterized the damaged islets but also imaged the insulinoma.

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  As a major class of aptamers, RNA aptamers exhibit a remarkable ability to recognize target molecules and have been extensively studied across various fields, leading to significant advancements [34,38,39]. However, due to the inherent chemical structure of RNA aptamers, they are highly susceptible to degradation by ubiquitous RNases in the environment, leading to a loss of activity and limiting their application in biomedical detection [40,41]. In this study, we adopted general nucleoside modification techniques to enhance the stability of RNA aptamers (Fig. 2a). We applied 2′-fluoro modifications to the ribose of C and U, and 2′-O-methyl modifications to the ribose of A and G, on the m12–3773 and 1–717 RNA aptamers, which were reported in the literature to specifically recognize clusterin and TMED6 proteins on the surface of pancreatic β-cells [32,42]. Through this modification strategy, we aim to enhance the stability of RNA aptamers.

  Figure 2

  

  Figure 2.  Stability and recognition assessment of modified RNA aptamers. (a) Schematic representation of RNA aptamers with modified nucleosides. (b, c) Stability assessment of 1–717 and M-1–717 in serum using PAGE analysis. (d, e) Stability assessment of m12–3773 and M-m12–3773 in serum using PAGE analysis. (f) The stability of unmodified and modified RNA aptamers against RNases was tested using PAGE. Lanes 2 and 4 show 1–717 and m12–3773 after RNases digestion, while lanes 1 and 3 are their controls without RNases. Similarly, lanes 6 and 8 show M-1–717 and M-m12–3773 after RNases digestion, with lanes 5 and 7 as their controls without RNases. (g, j) Recognition capabilities of M-1–717 and M-m12–3773 were validated using FCM with RIN-M5f and INS-1 cells. APC, allophycocyanin. (h, k) FCM data was used to calculate K_d values of M-1–717 and M-m12–3773 for recognizing target cells using RIN-M5f and INS-1 cells. Data are presented as mean ± standard deviation (SD) (n = 3). (i, l) LSCM images of M-1–717 and M-m12–3773 recognizing RIN-M5f and INS-1 cells. DAPI, 4′, 6-diamidino-2-phenylindole. The scale bar in (i, l) represents 50 µm.

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  The experimental results indicated a significant improvement in the stability of modified-nucleoside RNA aptamers. As shown in Figs. 2b and d, when the unmodified m12–3773 and 1–717 were incubated with 10% fetal bovine serum (FBS), polyacrylamide gel electrophoresis (PAGE) revealed that all RNA aptamers were degraded by RNases in the FBS within 0.5 h. In contrast, the modified aptamers, M-m12–3773 and M-1–717, remained stable in FBS for up to 12 h (Figs. 2c and e). This demonstrates that the nucleoside modification strategy substantially enhances the stability of RNA aptamers in serum. To further confirm the nuclease resistance of the modified RNA aptamers, we directly incubated both unmodified and modified RNA aptamers with RNases. The PAGE results showed that the modified RNA aptamers remained stable for at least 15 min at 37 ℃ when in direct contact with RNases, whereas the unmodified RNA aptamers were completely degraded under the same conditions (Fig. 2f). The results directly indicate that nucleoside modification significantly enhances the stability of RNA aptamers in serum, providing substantial resistance to nuclease degradation. This improvement ensures the successful implementation of subsequent imaging studies.

  Although modifying nucleotides in RNA aptamers can significantly enhance their stability and resistance to RNases degradation [43,44], such modifications may affect the spatial conformation and thermodynamic stability of the RNA aptamers [34,45]. These changes can increase the steric hindrance between the RNA aptamers and their target molecules, leading to decreased recognition capability or even loss of function [46,47]. Therefore, we utilized RIN-M5f and INS-1 cells, which are commonly used to study damage of islet and insulinoma, to investigate the recognition capabilities of M-1–717 and M-m12–3773.

  As illustrated in Figs. 2g and j, flow cytometry (FCM) data revealed that M-1–717 and M-m12–3773 exhibit good recognition capabilities for both RIN-M5f and INS-1 cells. Moreover, according to previous studies, the combination of two RNA aptamers demonstrates superior recognition capabilities for target molecules compared to the individual RNA aptamer [32]. Therefore, the recognition capabilities of the combination of the modified two RNA aptamers were also investigated, and the results demonstrated that this combination exhibits excellent recognition capabilities for both RIN-M5f and INS-1 cells (Figs. 2g and j). Additionally, we evaluated the K_d values of the two modified RNA aptamers using FCM. The data showed that the K_d values of M-1–717 for RIN-M5f and INS-1 cells were 45.29 and 145.1 nmol/L, respectively, while the K_d values of M-m12–3773 for these two cell types were 67.42 and 72.87 nmol/L, respectively (Figs. 2h and k). These results are consistent with those reported in the literature, indicating that the modified RNA aptamers retain good recognition capabilities [32]. Furthermore, laser scanning confocal microscopy (LSCM) images confirmed that both M-1–717 and M-m12–3773, when used individually, exhibit good recognition capabilities for RIN-M5f and INS-1 cells, and that their mixture also demonstrates excellent recognition capabilities for both cell types (Figs. 2i and l). Consistent with literature [32], combining these two RNA aptamers, which can recognize different subpopulations of β-cell, maximizes the signal-to-noise ratio for β-cell identification by targeting different β cell-specific epitopes. Therefore, the cell recognition ability demonstrated by the mixture of M-1–717 and M-m12–3773 is superior to their recognition ability when used individually. These results indicate that introducing 2′-fluoro modifications to cytidine (C) and uridine (U), along with 2′-O-methyl modifications to adenosine (A) and guanosine (G), improves the stability and nuclease resistance of RNA aptamers while maintaining their K_d values.

  Before using radioactive isotopes for PET/CT imaging, M-1–717 and M-m12–3773 were labeled with the fluorescent molecule indocyanine green (ICG) for imaging. This was done to study the in vivo metabolism and islet recognition capabilities of the two modified RNA aptamers while minimizing radiation exposure risks. The animal experiments conducted in this study were approved by the Animal Ethics Committee of Renji Hospital, in compliance with relevant ethical guidelines and regulations. Following previous studies, M-1–717 and M-m12–3773 were combined into an RNA aptamer panel to recognize the target tissue [32]. ICG is a hydrophilic fluorescent dye approved by the Food and Drug Administration (FDA) for clinical use [48]. Compared to anthocyanin dyes with hydrophobic properties, ICG causes less interference in imaging for this study [49]. Therefore, it was covalently linked to aptamers and used for imaging studies in nude mice in this research.

  After injecting the ICG-labeled RNA aptamer panel into mice via the tail vein, imaging was performed for main organs of nude mice at various time points using a small animal imaging system (Fig. S1a in Supporting information). The results showed that the fluorescence signal in the pancreas was strongest 1 h after administration, and it disappeared by 3 h (Figs. S1b and c in Supporting information). This provides a reference for subsequent PET/CT imaging. Additionally, images from the small animal imaging system in Fig. S1b and the fluorescence intensity quantification in Fig. S1c indicate that the liver and kidneys are the primary metabolic organs for the RNA aptamer panel, consistent with previously reported RNA in vivo metabolic pathways. To further determine the binding sites of the RNA aptamer panel on pancreatic tissue, the dissected pancreas was subjected to fluorescence histopathological analysis. Figs. S1d and e (Supporting information) clearly show the fluorescence of M-m12–3773 and M-1–717 in the islets, indicating that these modified RNA aptamers have the capability to bind to the islets. Through the aforementioned results, the metabolism of M-m12–3773 and M-1–717 in nude mice and their recognition of islets were preliminarily determined using fluorescent molecules, providing a reference for subsequent PET/CT imaging.

  ⁶⁸Ga is a significant radioactive isotope with important applications in nuclear medicine [50]. Its half-life of approximately 68 min means that it remains in the body for a relatively short duration, which reduces radiation damage to tissues [51]. The production of ⁶⁸Ga via a ⁶⁸Ge/⁶⁸Ga generator is simple, rapid, and convenient for clinical use [51]. Moreover, ⁶⁸Ga can bind with various ligands, enabling specific imaging of targeted molecules, making it an ideal radiotracer [17]. This study utilizes the ability of NOTA to chelate ⁶⁸Ga, attaching ⁶⁸Ga to the terminal of RNA aptamers modified with NOTA, thereby imparting specific recognition capabilities to ⁶⁸Ga. Building upon previous methods for chelating ⁶⁸Ga with NOTA-modified DNA, this study optimized the experimental procedure for chelating ⁶⁸Ga with NOTA-modified RNA aptamers (Fig. S2 in Supporting information) [52]. The final conditions were established as a pH of 4 and a temperature of 95 ℃ for the chelation reaction, and the integrity of the RNA aptamer's post-reaction was assessed using PAGE (Fig. S3 in Supporting information), resulting in the construction of the ⁶⁸Ga-NOTA-Ap RNA aptamer radiotracer.

  To validate the detection capability of ⁶⁸Ga-NOTA-Ap for islet damage, an islet damage model in rats was established using STZ, and after monitoring blood glucose levels until fasting blood glucose reached 23.4 mmol/L, ⁶⁸Ga-NOTA-Ap was administered via the tail vein, with subsequent PET/CT imaging of the rats performed at various time points (Fig. 3a). As shown in Figs. 3b and c, before STZ modeling, the tissue-specific recognition of ⁶⁸Ga-NOTA-Ap allowed clear visualization of pancreas in the rats. After STZ modeling, the pancreatic images became blurred, indicating that ⁶⁸Ga-NOTA-Ap can effectively monitor islet damage in rats. Further analysis of the PET/CT images and real-time radioactive signal quantification of various tissues and organs revealed that following islet damage, pancreatic uptake of ⁶⁸Ga-NOTA-Ap significantly decreased, indicating that ⁶⁸Ga-NOTA-Ap can effectively indicate islet damage (Fig. 3d and Fig. S4 in Supporting information). Additionally, a reduction in the hepatic uptake of ⁶⁸Ga-NOTA-Ap was also observed post-modeling. In-depth investigation of this phenomenon revealed that STZ modeling led to metabolic abnormalities in the liver and the formation of numerous cavities in liver tissue sections (Figs. S5 and S6 in Supporting information), indicating liver damage and impaired metabolic capacity. Consequently, the reduced uptake of ⁶⁸Ga-NOTA-Ap by the liver is associated with decreased liver function, resulting in slower hepatic clearance of ⁶⁸Ga-NOTA-Ap and subsequent accumulation in the blood (Fig. S4 in Supporting information). This explains why the hepatic signal in Fig. 3c is weaker than that in Fig. 3b, due to the reduced uptake following damage.

  Figure 3

  

  Figure 3.  ⁶⁸Ga-NOTA-Ap monitoring of islet damage in Sprague-Dawley rats. (a) Schematic of the STZ-induced islet damage model construction and detection using ⁶⁸Ga-NOTA-Ap via PET/CT. (b, c) Comparison of PET/CT images showing the pancreas in normal (b) and model (c) rats at different time points. (d) Comparison of pancreas-to-muscle radionuclide signal ratio between normal and mode rats at different time points. (e) Comparison of ⁶⁸Ga-NOTA-Ap signal intensity in various tissues and organs between the model and normal rats when fasting blood glucose levels in the model group have not reached diabetic standards (n = 5). (f) Comparison of the pancreatic-to-muscle ratio of ⁶⁸Ga-NOTA-Ap signal intensity between normal and model rats (n = 5). Data are presented as mean ± SD. *P < 0.05.

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  To further assess the detection capability of ⁶⁸Ga-NOTA-Ap for islet damage, an attempt was made to use ⁶⁸Ga-NOTA-Ap for islet imaging in rats before fasting blood glucose levels reached diabetic thresholds after modeling (Table S2 and Fig. S7 in Supporting information). The study found that, although fasting blood glucose levels in rats increased following STZ modeling, they did not meet the criteria for diabetes. At this stage, islet damage could be clearly identified by analyzing the pancreatic uptake of ⁶⁸Ga-NOTA-Ap (Fig. 3e). Additional statistical analysis, comparing the pancreatic-to-muscle ratio, demonstrated that ⁶⁸Ga-NOTA-Ap significantly differentiated between normal and damaged islets, with data in normal rats being notably higher than in model group (Fig. 3f).

  Insulinoma originates from pancreatic β-cells and is formed by the proliferation of these cells [15]. Although it is usually benign, the uncontrolled excessive secretion of insulin leads to symptoms of hypoglycemia in patients, which can be severe enough to cause coma, thereby significantly impacting the patient's quality of life [16]. The hypoglycemic symptoms caused by insulinoma are typically intermittent, making it difficult to diagnose insulinoma through blood glucose and insulin level testing [28]. Additionally, insulinoma is usually small in size, resulting in weak signals on conventional imaging, which makes localization challenging [14]. Although EUS and ASVS have relatively high diagnostic sensitivity, they are technically complex and require specially trained and experienced operators, which are not available in all medical institutions [14,20].

  Considering the correlation between insulinoma and pancreatic islet tissue, combined with the recognition abilities of M-1–717 and M-m12–3773 for RIN-M5f and INS-1 cells (Fig. 2), this study attempts to use ⁶⁸Ga-NOTA-Ap for imaging insulinoma (Fig. 4a). After injecting ⁶⁸Ga-NOTA-Ap via the tail vein into mice with subcutaneous insulinoma models, PET/CT scans were performed at 1 and 2 h post-injection. As shown in Figs. 4b and c, the tumor regions exhibited radioactive signals, indicating that ⁶⁸Ga-NOTA-Ap has good recognition capability for insulinoma. The biodistribution of ⁶⁸Ga-NOTA-Ap at 1 h post-injection also demonstrated strong signals in the tumor (Fig. 4d). To further validate the tumor recognition capability of ⁶⁸Ga-NOTA-Ap, tumor tissues were dissected from the tumor-bearing mice and analyzed using fluorescence immunohistochemistry, which showed that the tumor tissue regions exhibited a strong binding capacity for ⁶⁸Ga-NOTA-Ap (Fig. 4e). These results demonstrate that ⁶⁸Ga-NOTA-Ap effectively recognizes insulinoma, highlighting its potential for use in insulinoma detection.

  Figure 4

  

  Figure 4.  ⁶⁸Ga-NOTA-Ap imaging of insulinoma in NOD/SCID mice. (a) Schematic representation of insulinoma detection using ⁶⁸Ga-NOTA-Ap via PET/CT. (b, c) PET/CT images of ⁶⁸Ga-NOTA-Ap for detecting subcutaneous insulinoma at 1 h (b) and 2 h (c) post-administration. (d) Biodistribution of ⁶⁸Ga-NOTA-Ap in various tissues and organs of mice. (e) Fluorescence immunohistochemistry images of the tumor tissue, with pink indicating the binding sites of RNA Aptamer Panel.

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  In summary, based on the modification of RNA aptamers, we developed a novel radionuclide probe, ⁶⁸Ga-NOTA-Ap, which incorporates RNA aptamers specific to pancreatic β-cell surface proteins clusterin and TMED6. By modifying the 2′ ribose with fluorine groups for C and U, and methoxy for A and G, the RNA aptamers' original recognition capabilities were preserved while significantly enhancing their stability and resistance to RNases hydrolysis [32]. The modification preserves the aptamers' efficacy in physiological conditions without significantly altering their affinity (Fig. S8 in Supporting information), making them more suitable for intravenous administration. Utilizing rat models with pancreatic islet damage and subcutaneous insulinoma models in mice demonstrates that ⁶⁸Ga-NOTA-Ap not only monitors pancreatic islet damage, showing potential for early detection of high-risk individuals for diabetes and control of diabetes onset, but also reflects its application value in detecting insulinoma. These findings provide valuable insights into the future application of radionuclide probes in the diagnosis and management of metabolic diseases, offering a promising avenue for advancing medical imaging and treatment strategies.

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  Acknowledgments

  This work was supported by National Natural Science Foundation of China (No. 82002241), National Key Research and Development Program of China (No. 2020YFA0909000), and "Clinic Plus" Outstanding Project (No. 2024ZY012) from Shanghai Key Laboratory for Nucleic Acid Chemistry and Nanomedicine.

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2024.110804.

  
  
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  Figure 1  Schematic of nucleoside modifications stabilizing ⁶⁸Ga-labeled RNA aptamers for pancreatic β-cell and insulinoma imaging. This study primarily consists of two parts: (Ⅰ) The 2′-O-methyl modifications on C and U, and 2′-fluoro modifications on A and G respectively, improved the stability and RNases resistance of RNA aptamers while maintaining their K_d values. (Ⅱ) The ⁶⁸Ga-labeled modified RNA aptamers not only characterized the damaged islets but also imaged the insulinoma.

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  Figure 2  Stability and recognition assessment of modified RNA aptamers. (a) Schematic representation of RNA aptamers with modified nucleosides. (b, c) Stability assessment of 1–717 and M-1–717 in serum using PAGE analysis. (d, e) Stability assessment of m12–3773 and M-m12–3773 in serum using PAGE analysis. (f) The stability of unmodified and modified RNA aptamers against RNases was tested using PAGE. Lanes 2 and 4 show 1–717 and m12–3773 after RNases digestion, while lanes 1 and 3 are their controls without RNases. Similarly, lanes 6 and 8 show M-1–717 and M-m12–3773 after RNases digestion, with lanes 5 and 7 as their controls without RNases. (g, j) Recognition capabilities of M-1–717 and M-m12–3773 were validated using FCM with RIN-M5f and INS-1 cells. APC, allophycocyanin. (h, k) FCM data was used to calculate K_d values of M-1–717 and M-m12–3773 for recognizing target cells using RIN-M5f and INS-1 cells. Data are presented as mean ± standard deviation (SD) (n = 3). (i, l) LSCM images of M-1–717 and M-m12–3773 recognizing RIN-M5f and INS-1 cells. DAPI, 4′, 6-diamidino-2-phenylindole. The scale bar in (i, l) represents 50 µm.

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  Figure 3  ⁶⁸Ga-NOTA-Ap monitoring of islet damage in Sprague-Dawley rats. (a) Schematic of the STZ-induced islet damage model construction and detection using ⁶⁸Ga-NOTA-Ap via PET/CT. (b, c) Comparison of PET/CT images showing the pancreas in normal (b) and model (c) rats at different time points. (d) Comparison of pancreas-to-muscle radionuclide signal ratio between normal and mode rats at different time points. (e) Comparison of ⁶⁸Ga-NOTA-Ap signal intensity in various tissues and organs between the model and normal rats when fasting blood glucose levels in the model group have not reached diabetic standards (n = 5). (f) Comparison of the pancreatic-to-muscle ratio of ⁶⁸Ga-NOTA-Ap signal intensity between normal and model rats (n = 5). Data are presented as mean ± SD. *P < 0.05.

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  Figure 4  ⁶⁸Ga-NOTA-Ap imaging of insulinoma in NOD/SCID mice. (a) Schematic representation of insulinoma detection using ⁶⁸Ga-NOTA-Ap via PET/CT. (b, c) PET/CT images of ⁶⁸Ga-NOTA-Ap for detecting subcutaneous insulinoma at 1 h (b) and 2 h (c) post-administration. (d) Biodistribution of ⁶⁸Ga-NOTA-Ap in various tissues and organs of mice. (e) Fluorescence immunohistochemistry images of the tumor tissue, with pink indicating the binding sites of RNA Aptamer Panel.

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