博文
Near-infrared II fluorescent probe for lung metastatic tumor
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Volume 426, 1 March 2025, 137005
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a
Key Laboratory of Haikou Trauma, Key Laboratory of Hainan Trauma and Disaster Rescue, Key Laboratory of Emergency and Trauma, Ministry of Education, The First Affiliated Hospital of Hainan Medical University, Hainan Medical University, Haikou 571199, China
b
Engineering Research Center for Hainan Bio-Smart Materials and Bio-Medical Devices, College of Emergency and Trauma, Hainan Medical University, Haikou 571199, China
c
Department of Breast Surgery, The First Affiliated Hospital of Hainan Medical University, Haikou 570102, China
Received 10 September 2024, Revised 5 November 2024, Accepted 24 November 2024, Available online 26 November 2024, Version of Record 29 November 2024.
https://doi.org/10.1016/j.snb.2024.137005
Highlights•Glutathione-Activated Near-infrared II Fluorescent Probe (LJ-GSH) was developed.
•The probe LJ-GSH showed high specificity and suitable response sensitivity for the detection of GSH in vitro.
•LJ-GSH enables dynamic visualization of the biological role of GSH within the physiological environment.
•Precise lung metastasis imaging and fluorescence-guided tumor resection have been achieved using LJ-GSH.
Abstract
Accurate identification of intraoperative tumor lesions and effective treatment are crucial for improving surgical outcomes. Near-infrared (NIR) fluorescence imaging demonstrates advantages over traditional medical approaches in tumor interventions, garnering significant attention. However, clinically available imaging agents are generally limited by their "always on" characteristics, which can lead to non-specific imaging interference and "false-positive" results. In this context, we present a glutathione-activated NIR-II probe, LJ-GSH, designed for metastatic tumor imaging and specific imaging-guided tumor resection. LJ-GSH initially exhibits quenched fluorescence due to the weak electron-donating effect of the thiophenol moiety, which is recovered at 815/910 nm upon activation by the overexpressed levels of glutathione (GSH) in tumor cells and tissues, significantly enhancing the specificity of tumor imaging. This unique characteristic positions LJ-GSH as a reliable fluorescent sensor for monitoring GSH dynamics during physiological events. Notably, the probe's NIR-II emission feature markedly improves imaging contrast and resolution, facilitating real-time identification and imaging of lung metastatic lesions. With the aid of high-specific NIR-II imaging guidance, tumor tissues can be precisely resected, with the residual negative margin diameter reduced to approximately 0.2 mm. We envision that our tailored probe may offer an attractive option for clinical applications.
Near-infrared II
Glutathione-Activated
Fluorescent probe
Intraoperative tumor imaging
Lung Metastatic Diagnosis
IntroductionMalignancies, characterized by the uncontrolled growth of cancer cells and a high risk of metastasis, present a significant threat to human life and health [1], [2], [3]. Precisely monitoring the dynamic changes in tumors allows for early diagnosis and on-site evaluation of tumor development and progression, thereby enabling personalized therapy and enhancing patient survival rates [4], [5], [6]. In recent years, clinical imaging modalities such as CT, PET, and MRI have witnessed significant advances in tumor detection and localization. However, highly sensitive preoperative and intraoperative diagnosis of tumor remains a challenge for the above imaging modalities, primarily due to their limited resolution, potential radiation exposure risks, and inability to offer timely feedback on treatment outcomes during surgery [7], [8]. As an alternative tracer technology, fluorescence imaging poses significant superiority over the above medical imaging approaches in tumor interventions, benefiting from its high sensitivity, non-invasive, and fast feedback, thus, having attracted widely interesting [9], [10], [11], [12].
Recently, efforts have focused on designing and developing fluorescent-based agents for disease diagnosis and treatment. Compared to conventional imaging probes in the visible region, fluorescence imaging in the NIR-II region (1000–1700 nm) exhibits deeper tissue penetration and higher spatiotemporal resolution due to reduced tissue autofluorescence and photon scatter, enhancing accuracy and reliability in disease detection [13], [14], [15], [16], [17], [18]. Clinically approved NIR fluorescent contrast agents, such as indocyanine green (ICG) and methylene blue (MB), have been utilized for tumor detection and guiding tumor treatment [19], [20]. However, these imaging probes typically face the challenge of short tumor retention and quick clearance from the body. Additionally, many of the currently available clinical agents display the “always on” characteristic, where they illuminate disease sites through self-accumulation rather than being specifically activated by target molecules of interest, which leads to poor imaging contrast and compromised detection accuracy. To address the issue mentioned above, stimulus-responsive fluorescence imaging strategies that can activate signal light-up only in the presence of biomarkers or pathological environments can afford higher tumor-to-normal tissue ratios (T/N ratio) and real-time biological information, making the activatable modality a preferred choice for disease diagnosis and therapy evaluation [21], [22], [23], [24], [25].
To develop the activation-responsive system for tumor imaging, it is crucial to carefully select a biomarker that is associated with the tumor. Studies have shown that the rapid angiogenesis, proliferation, and metastasis of tumors are linked to the overexpression of reactive oxygen species (ROS) [26], [27]. To circumvent the tumor-related oxidative stress, Glutathione (GPx4) and NADPH act synergistically to maintain a cellular redox homeostasis environment via the generation of endogenous antioxidants, represented by the reduced state of glutathione (GSH). Elevated levels of GSH are commonly found in many types of tumors, and have been considered as the key indicator for discrimination of tumor region from the normal tissue [28], [29], [30], [31], [32]. Moreover, mitochondria are gaining increasing attention due to their critical role as the hub of metabolic activity and their potential as targets for cancer treatment. Consequently, the development of mitochondria-targeted fluorescent probes has emerged as a significant focus of research. To date, several GSH-activatable fluorescent molecular probes have been developed for detecting endogenous GSH and for further exploration of its biological role [33], [34], [35], [36], [37], [38]. Despite significant advancements in GSH sensors designed for in vivo tumor imaging and therapy intervention, challenges remain. These challenges are partly due to the limited emission wavelength (less than 700 nm), undesirable mitochondrial targeting capabilities and the inadequate sensitivity of these sensors in detecting endogenous GSH levels, which typically range within the millimolar concentration.
To address earlier issues mentioned, herein, we report the GSH-activatable NIR-I/II fluorescent probe for specific tumor detection and image-guided tumor resection (Scheme 1). In this study, a carboxy-modified heptamethine cyanine (Cy-7) derivative was chosen as the fluorescence reporter due to its excellent biocompatibility and intrinsic ability to target mitochondria [39], [40], [41]. Additionally, p-Methoxy thiophenol functionality was integrated into the Cy-7 fluorophore to serve as a GSH-specific response site and fluorescence quencher, resulting in the fabrication of the NIR fluorescent sensor LJ-GSH. Initially, LJ-GSH exhibits a weak fluorescence signal due to the fluorescence quencher effect of the thiophenol site. However, upon exposure to a solution containing GSH, the GSH reacts with the probe through aromatic nucleophilic substitution, leading to the formation of a thiol skeleton with enhanced electron transfer and the subsequent emission of the bright optical signal. In vitro studies have shown that LJ-GSH demonstrates a suitable response sensitivity (0−10 mM GSH) and high specificity for GSH, with an emission maximum at 815/910 nm. This unique characteristic makes LJ-GSH a reliable fluorescent tool for discriminating tumor cells from normal ones based on GSH content discrepancy. Additionally, distinct fluctuations in GSH levels during the oxygen-glucose deprivation model process were monitored with real-time fluorescence imaging. More importantly, in combination with its NIR-II emission and GSH-specific activation characteristics, the probe LJ-GSH demonstrated promising capabilities for the precise localization of subcutaneous tumors and lung metastatic lesions, achieving the accurate removal of tumors with negative margins as small as 0.2 mm in diameter.
Scheme 1. (a) Schematic illustration of probe LJ-GSH design and response strategy. (b) Schematic describing the mechanism of utilizing LJ-GSH for NIR-II imaging-guided lung metastatic lesions diagnosis.
Design and synthesis of GSH-responsive NIR-II probe LJ-GSH
Developing a fluorescent diagnostic probe for tumor applications, the ideal imaging agent should meet the following prerequisites: (1): NIR fluorescence emission for deep-tissue penetration and minimize auto-fluorescence; (2) tumor-related biomarker activation to enhance tumor imaging contrast (3): a highly specific response to the biomarker for increasing the accuracy in tumor diagnostics. To this end, we selected Cy derivative as the fluorescent scaffold considering its NIR emits characteristic, easy modification and favorable biocompatibility. Then, p-Methoxy-modified thiophenol, as the GSH reaction site and a fluorescence quencher, was incorporated into Cy skeleton to prepare probe LJ-GSH. The synthesis of the probe followed the route shown in Scheme S1, and the compound was characterized using nuclear magnetic resonance (1H/13C NMR) and high-resolution mass spectrometry (ESI-HRMS)
Fig. 1. (a) Absorption spectra of LJ-GSH (10 μM) upon the addition of 0–10 mM GSH; (b) NIR-I, and (c) NIR-II FL spectrum of LJ-GSH (10 μM) after treatment with different concentrations of GSH (0–10 mM). (d) Titration curve of LJ-GSH (10 μM) to 0–10 mM GSH; (e) Linear relationship between fluorescence intensity at 815 nm and [GSH] in the range of 0.25–2.0 mM; (f) NIR-I FL response of 10 μM LJ-GSH to various interfering species and GSH. λex = 808 nm for NIR-II, and λex = 720 nm for NIR-I.
Fig. 2. (a-i) Cells were only incubated with probe (10 μM) for 30 min. (a-ii) Cells were pre-treated with 1 mM NEM for 20 min, and then cells were incubated with LJ-GSH for 30 min. (a-iii) Cells were treated with GSH-ethyl-ester (1 mM) and probe (10 μM) for 30 min. Cells were pre-incubated with H2O2 (0.2 mM, a-vi), 5 μg/mL Lps (a-v), and 2.5 μg/mL Lps (a-vi) for 2 h, and then incubated with LJ-GSH (10 μM) for 30 min. (b) Fluorescence intensity of Aa−f group cells. Data denote mean±s.d. (n=3, **P<0.01, ***P<0.001, ****P<0.0001). The images were recorded with 633 nm excitation and 725−800 nm collection. Scale bars: 25 μm.
Fig. 3. (ai-iv) Time-dependent (0–90 min) real-time GSH imaging with LJ-GSH (10 μM) in 4T1 cells under oxygen-glucose deprivation (glucose-free (0 mM: 0 G), 1 % O2). (a-v-a-viii) After 90 min oxygen-glucose deprivation, cells were incubated with normal medium containing (25 mM glucose) and 21 % O2 for different reperfusion time, 30 min (a-v), 60 min (a-vi), 90 min (a-vii), 120 min (a-viii), subsequent, LJ-GSH (10 μM) were added to cells for another 30 min incubation. (b) Fluorescence intensity of ai−aviii group cells. Data denote mean±s.d. The images were recorded with 633 nm excitation and 725−800 nm collection. Scale bars: 25 μm.
Fig. 4. (a-i) Time-dependent in vivo fluorescence imaging of GSH in tumor bearing mice at NIR-I window and (a-ii) at NIR-II window. (b) NIR-I and (d) NIR-II Fluorescence intensity in tumor region at various time points (0–24 h) after i.v. injection of LJ-GSH (100 μL, 1 mM). (c) T/N ratios after 24 h i.v. injection of probes at NIR-I window, and (e) at NIR-II window. (f) Time-dependent T/N ratios after i.v. injection of probes. (g)-(h) representative ex vivo fluorescence images of tumor and major organs at 24 h post-injection of LJ-GSH. (i) Normalized NIR-II fluorescence intensity of the tumor and other organs in panels g. (j) NIR-II image-guided subcutaneous tumor resection. (k) SBR of the resected tumor (ROI-A) and post-surgery position (ROI-B). (l) Line profile of photon counts from j. (m)-(n) H&E staining results of excised tumor margin, Scar bar: 200 μm. (o) Kaplan–Meier survival rate curve of five mice with or without tumor resection. NIR-I window: Ex = 745 nm, Em: 780−825 nm. NIR-II images were collected under 808 nm excitation, 900 nm long-pass filter, 40 mW/cm2, and exposure time: 20 ms. n=3, Data denote mean±s.d. (n=3, **P<0.01, ***P<0.001, ****P<0.0001).
Fig. 5. (a) Schematic illustration the timeline of lung metastasis model establishment and real-time tumor imaging. (b) Representative bioluminescence image. (c)-(d) NIR-II imaging of lung tissue dissected from lung metastatic mice after 24 h post-injection of (100 nM) LJ-GSH. (e) SBR of lung metastatic tumors according to c and d (marked as a-f and control as g). (f) H&E, ki 67, and F4/80 immunofluorescence staining of the resected lung metastatic nodules (ROI-a) Scar bar: 1 mm.
4. ConclusionIn summary, based on cyanine fluorophore, we reported the GSH-activated NIR-I/II fluorescent probe, LJ-GSH, for specific lung metastasis imaging and fluorescence-guided surgical navigations. The probe initially exhibits fluorescence quenching due to the inhibited electron transfer process from p-Methoxy thiophenol functionality to cyanine moiety. Upon interaction with GSH, LJ-GSH converts to its thiol form through GSH-mediate nucleophilic substitution, releasing NIR-I/II emission (815/910 nm) with recovery of electron transfer. In vitro analysis tests have shown probe displays high specificity and suitable sensitivity to GSH, making LJ-GSH a promising tool for imaging GSH dynamics in cells. Benefiting from the high resolution and high-specific NIR-II fluorescence imaging from LJ-GSH, subcutaneous and metastasis breast lesions are clearly distinguished from surrounding normal tissue, with negative margins as small as 0.25 mm in diameter. We expected that LJ-GSH could offer a valuable approach for exploring the biological functions of GSH and potentially serve as a therapeutic technology for GSH-related tumours.
https://blog.sciencenet.cn/blog-2438823-1462349.html
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