A dual-enzyme-activated ruthenium complex fluorescent probe, and a preparation method and application thereof
By using a dual-enzyme activated ruthenium complex fluorescent probe, employing a triple pyridine ruthenium complex and a spray formulation, the false-positive problem of existing fluorescent probes has been solved, enabling precise identification and resection of tumors and normal tissues.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- QINGDAO UNIV
- Filing Date
- 2026-02-11
- Publication Date
- 2026-06-02
Smart Images
Figure CN122127370A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of molecular probe technology, specifically to a dual-enzyme activated ruthenium complex fluorescent probe, its preparation method, and its application. Background Technology
[0002] Gliomas are the most common primary malignant tumors of the central nervous system. Their invasive growth characteristics lead to blurred boundaries between the tumor and normal brain tissue, posing a significant challenge to complete surgical resection. Surgical resection is currently the primary treatment method, but incomplete resection easily leads to recurrence, while excessive resection may damage key brain functional areas, causing permanent neurological deficits. Therefore, achieving precise, real-time visualization of tumor boundaries during surgery is crucial to improving the surgical outcomes of gliomas.
[0003] Currently, commonly used intraoperative navigation technologies such as magnetic resonance imaging (MRI) and computed tomography (CT) have significant limitations: the equipment is bulky, cannot provide real-time imaging, has limited resolution, and brain drift after craniotomy can cause the images to mismatch with the real-time anatomical position, affecting the accuracy of localization.
[0004] Fluorescence imaging technology offers new insights for intraoperative navigation due to its advantages such as high sensitivity, high resolution, and real-time observation. Among them, responsive (on-off) fluorescent probes only emit fluorescence in response to specific tumor microenvironment stimuli, such as abnormally expressed enzymes, low pH values, or high levels of reactive oxygen species. Compared to normally lit probes, they can significantly reduce background signal and improve the signal-to-noise ratio.
[0005] However, most reported responsive probes are single-factor activated, such as responding to only one enzyme or one active substance. This design has an inherent flaw: transient or localized increases in this single factor may occur in inflammatory areas surrounding the tumor or in some normal tissues, leading to false positives in fluorescence signals, misleading surgical decisions, and causing incorrect removal of normal tissue or tumor residue.
[0006] Therefore, developing a fluorescent probe that can simultaneously respond to two highly expressed biomarkers in gliomas, achieving low false positive rates and high tumor recognition specificity, is a pressing technical problem to be solved in this field.
[0007] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention
[0008] To address the aforementioned technical problems, this invention provides a dual-enzyme activated ruthenium complex fluorescent probe, its preparation method, and its application, thereby resolving the issues raised in the background section.
[0009] I. The design concept of this invention patent is explained in detail below:
[0010] The first step, target selection and logical design: Research has found that glutathione S-transferase and azoreductase are specifically highly expressed in glioma tissue, at levels five to ten times and eight to fifteen times higher than in normal brain tissue, respectively, making them ideal molecular targets. The fluorescence output of the probe Ru-GA in this invention is designed to require simultaneous biocatalysis by both glutathione S-transferase and azoreductase for activation. This dual-target recognition mechanism fundamentally avoids false positives caused by increased activity of a single enzyme under inflammatory or other pathological conditions, significantly improving the specificity and signal-to-noise ratio for tumor tissue recognition.
[0011] The second step, optimization of the fluorescent group and its performance: To meet the stringent requirements of intraoperative real-time navigation for optical performance and application, this design abandons traditional organic fluorescent dyes, such as rhodamine and fluorescein, which have drawbacks such as small stokes. Innovatively, a terpyridine ruthenium complex (II) is selected as the fluorescent reporter group. This group has the following key advantages: First, its luminescence mechanism based on metal-to-ligand charge transfer can generate stokes larger than 160 nm, effectively separating excitation and emission light and avoiding interference from the intraoperative excitation source on visual judgment; second, the excitation light is located in the visible light region, exhibiting low phototoxicity and good photostability; third, as an ionic complex, it possesses excellent water solubility and can be directly formulated into a pure water spray system, enabling immediate intraoperative spraying and imaging.
[0012] The third step is probe structure design and activation mechanism: The probe Ru-GA consists of three parts: a bipyridine ruthenium complex, a glutathione S-transferase specific recognition group, and an azo reductase specific recognition group. Due to the strong electron-withdrawing effect of the nitro group, there is a strong photoinduced electron transfer effect within the probe structure, which quenches the probe fluorescence. When glutathione S-transferase and azo reductase are present simultaneously, they react with the probe, causing the nitro group structure to leave, and the red fluorescence of the ruthenium complex to be strongly restored and enhanced (the reaction principle is as follows). Figure 4 This avoids false positives caused by inflammation, significantly increases the ratio of tumors to normal tissue, and assists clinicians in accurately identifying and removing tumor tissue.
[0013] II. The specific solution of this invention patent is as follows:
[0014] A dual-enzyme activated ruthenium complex fluorescent probe, the structural formula of which is as follows: Figure 1 As shown in Chinese formula I:
[0015] The preferred synthetic reaction route for the dual-enzyme activated ruthenium complex fluorescent probe is as follows: Figure 2 As shown; among them, the compound of formula I is named Ru-GA.
[0016] The preferred method for synthesizing the dual-enzyme activated ruthenium complex fluorescent probe specifically includes the following steps:
[0017] (1) Compound 1 and Compound 2 were heated under nitrogen protection in ethanol and refluxed overnight. After the reaction was completed, the solvent was evaporated to dryness, purified by silica gel column chromatography, and potassium nitrate was replaced with potassium hexafluorophosphate to obtain Compound 3.
[0018] (2) The compound 3 obtained in step (1) was dissolved in dichloromethane. Under the protection of ice bath and nitrogen, a dichloromethane solution of boron tribromide was slowly added dropwise. The reaction was then carried out at room temperature for 7 hours. After the reaction was completed, water was added to quench the reaction. The organic phase was separated, the solvent was evaporated to dryness, and the compound was purified by silica gel column chromatography. Potassium nitrate was replaced with potassium hexafluorophosphate to obtain compound 4.
[0019] (3) The compounds 4, 2,4-dinitrofluorobenzene and potassium carbonate obtained in step (2) were mixed in acetonitrile and heated under nitrogen protection and refluxed overnight. After the reaction was completed, the fluorescent probe Ru-GA was obtained by solvent evaporation, silica gel column chromatography purification and potassium hexafluorophosphate was used to replace potassium nitrate.
[0020] Preferably, the molar ratio of compound 1 to compound 2 is 1:1.
[0021] Preferably, the molar ratio of compound 4 to 2,4-dinitrofluorobenzene is 1:2.
[0022] Preferably, in steps (1), (2) and (3), the eluent used in the silica gel column chromatography purification is a mixed solvent system of acetonitrile-potassium nitrate-water.
[0023] Application of a dual-enzyme activated ruthenium complex fluorescent probe as described above in the preparation of glioma detection products.
[0024] Preferably, the glioma detection product is an in vitro diagnostic reagent used for the identification and fluorescence imaging of glioma cells.
[0025] Preferably, the glioma detection product is a surgical navigation reagent.
[0026] Preferably, the product formulation of the glioma detection product is a spray.
[0027] The present invention provides a dual-enzyme activated ruthenium complex fluorescent probe, its preparation method, and its application, which have the following beneficial effects:
[0028] (1) The fluorescent probe of this invention adopts a dual-enzyme synergistic activation strategy of glutathione S-transferase and azoreductase. Its fluorescence signal is specifically triggered only in the tumor microenvironment where both enzymes are highly expressed. Experiments have shown that this design can clearly distinguish between tumor cells and normal cells, accurately illuminate the tumor area in the in vivo model, thereby effectively reducing false positive interference and significantly improving the imaging signal-to-noise ratio and diagnostic accuracy.
[0029] (2) The fluorescent probe needle of this invention uses a terpyridine ruthenium(II) complex as the fluorophore, which has the optical properties of a large stokes, effectively separating excitation light and emission light, minimizing the interference of excitation light on the surgical field during surgery, and facilitating the doctor's observation of tumor boundaries. This complex has both good photostability and low phototoxicity, making it suitable for long-term intraoperative illumination and observation. Its excellent hydrophilic properties allow the probe to be directly formulated into a water-based spray, making it easy to use.
[0030] (3) The fluorescent probe of the present invention is made into a spray form, which can be sprayed directly. Under blue light excitation, the tumor area can quickly produce visible red fluorescence, thereby guiding the surgeon to perform precise resection in real time and dynamically. Postoperative pathological verification shows that the fluorescence signal is highly consistent with the tumor tissue, confirming that the probe can effectively assist in achieving precise and safe tumor resection. Attached Figure Description
[0031] Figure 1 The structural formula of the dual-enzyme activated ruthenium complex fluorescent probe in this invention is shown below.
[0032] Figure 2 This is a schematic diagram of the synthetic reaction route for the dual-enzyme activated ruthenium complex fluorescent probe in this invention.
[0033] Figure 3 The above is the proton NMR spectrum of the probe Ru-GA in this invention;
[0034] Figure 4 The reaction mechanism diagram of probe Ru-GA with AZoR and GSTs;
[0035] Figure 5 This is the activity result of GL216 cells after being cultured for 24 h with different concentrations of the probe Ru-GA in this invention;
[0036] Figure 6 The images show fluorescence imaging of the probe Ru-GA after co-incubation with GL261 glioma cells and BV2 normal glial cells for different times in this invention.
[0037] Figure 7 Fluorescence imaging of GL261 glioma cells pretreated with different inhibitors and co-incubated with the probe Ru-GA;
[0038] Figure 8This is a graph showing the changes over time in in vivo fluorescence imaging of the probe Ru-GA in the brains of tumor-bearing and normal mice.
[0039] Figure 9 In vivo fluorescence imaging of tumor-bearing mouse brain tissue after pretreatment with different inhibitors and spraying with the probe Ru-GA;
[0040] Figure 10 This is a schematic diagram of a mouse glioma resection surgery guided by traditional in vivo imaging and Ru-GA probe fluorescence imaging.
[0041] Figure 11 HE-stained pathological sections of normal brain tissue and tumor tissue resected in stages under the guidance of the Ru-GA probe. Detailed Implementation
[0042] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0043] To address the aforementioned technical problems, this invention provides a dual-enzyme activated ruthenium complex fluorescent probe, its preparation method, and its application, thereby resolving the issues raised in the background section.
[0044] Example 1: Synthesis of the probe Ru-GA
[0045] The synthetic route of probe Ru-GA is as follows: Figure 1 As shown.
[0046] Synthesis of compound 3: Compound 1 (200 mg, 0.29 mmol) and compound 2 (88 mg, 0.29 mmol) were dissolved in 10 mL of ethanol and stirred under nitrogen protection at 80 °C for 10 h. The product was evaporated to dryness and purified by silica gel chromatography using CH3CN-KNO3-H2O. Potassium nitrate was replaced with potassium hexafluorophosphate to obtain compound 3.
[0047] Synthesis of compound 4: Compound 3 (82.7 mg, 0.1 mmol) was dissolved in CH2Cl2 (5 mL) under ice bath. Under nitrogen protection, the CH2Cl2 solution of BBr3 (5 mL) was added dropwise to the compound 3 solution using a constant pressure dropping funnel. The mixture was stirred at room temperature for 5 h, then deionized water (5 mL) was added and stirred for 30 min. The organic phase was separated and evaporated to dryness. The mixture was purified by silica gel chromatography using CH3CN-KNO3-H2O. After replacing potassium nitrate with potassium hexafluorophosphate, compound 4 was obtained.
[0048] Synthesis of probe Ru-GA: Compound 4 (50 mg, 0.063 mmol), 2,4-dinitrofluorobenzene (23.4 mg, 0.126 mmol, compound 5), and K2CO3 (34.5 mg, 0.25 mmol) were dissolved in CH3CN (15 mL) under nitrogen protection and refluxed overnight at 82 °C with stirring. The crude product was evaporated to dryness and purified by silica gel chromatography using CH3CN-KNO3-H2O. Potassium nitrate was replaced with potassium hexafluorophosphate to obtain Ru-GA. Figure 3 ).
[0049] Characterization data of the proton NMR spectrum: 1 H NMR (600 MHz, CD3CN): 8.86-8.81 (m, 2H), 8.79-8.74 (m, 3H), 8.73-8.66 (m, 4H), 8.45-8.40 (m, 2H), 8.12-8.06 (m, 4H), 8.04-7.99 (m, 4H), 7.94-7.91 (m, 2H), 7.83-7.79 (m, 5H), 7.69-7.6 (m, 2H), 7.47-7.42 (m, 4H), 7.4-7.35 (m, 3H), 7.26-7.23 (m, 1H), 7.03-6.9 (m, 1H), 6.87 (d, J=6Hz, 2H), 3.16 (s, 6H).
[0050] Example 2: Cytotoxicity Evaluation Experiment of Probe Ru-GA
[0051] The cytotoxicity of this probe was evaluated using the MTT assay. Figure 5 As shown, in the range of 0-20 μM, cells (GL261) incubated with different concentrations (0, 1, 3, 5, 10, 15, 20 μM) of probe Ru-GA for 24 h maintained a cell viability of over 80%, indicating that probe Ru-GA has low cytotoxicity.
[0052] Example 3: Specific fluorescence imaging and mechanism verification of probe Ru-GA on glioma cells
[0053] The specificity of the probe Ru-GA in recognizing glioma cells was validated by fluorescence imaging. Glioma cells GL261 and normal glioma cells BV2 were co-incubated with the probe Ru-GA (10 M) for different time periods, and the fluorescence imaging results are shown below. Figure 6 As shown.
[0054] Experimental results showed that the red fluorescence signal generated by the probe Ru-GA in GL261 cells gradually increased with prolonged incubation time, reaching a peak at 30 minutes. In contrast, the fluorescence signal of normal glial cells BV2 remained weak, demonstrating that Ru-GA can specifically distinguish tumor cells that highly express the target enzyme.
[0055] It should be noted that, in order to clearly identify cell locations and perform colocalization analysis, the nuclear dye DAPI was used to label the nuclei of all cells with blue fluorescence in the experiment. Figure 6 The overlay image shown in the middle Merge is formed by fusing the red fluorescence channel of Ru-GA with the blue fluorescence channel of DAPI, thereby simultaneously displaying the activated probe signal (red) and the nuclear location of all cells (blue) in the same field of view, visually confirming that the red fluorescence signal originates from inside the cell.
[0056] To verify that the intracellular red fluorescence signal is induced by the reaction of the probe Ru-GA with GSTs and AzoR in glioma cells, GL261 cells were pretreated with the GST inhibitor ethacrylic acid (R1), the AzoR inhibitor sulfasalic acid (R2), and both inhibitors (R1+R2) simultaneously. After incubation with Ru-GA (10 M) for 30 min, imaging was performed. The imaging results are shown below. Figure 7 As shown.
[0057] Figure 7 Imaging results showed that GL261 cells treated with two inhibitors and then incubated with the probe Ru-GA exhibited significantly reduced intracellular red fluorescence signals. These imaging results demonstrate that the probe Ru-GA can achieve specific fluorescence imaging of glioma cells through its specific fluorescence response to GSTs and AzoR.
[0058] It should be noted that complete inhibition of enzyme activity within cells is almost impossible. Furthermore, since glutathione S-transferase and azoreductase are highly expressed in tumor cells, the uptake and efflux of inhibitors by cells will result in a small number of enzyme molecules remaining active, inevitably leading to weak fluorescence in normal glial cells BV2.
[0059] Example 4: In vivo imaging experiment
[0060] Next, the feasibility of using the probe Ru-GA for fluorescence imaging and intraoperative tumor resection of in situ gliomas was investigated. First, in situ gliomas were established in the brains of 6-8 week old C57BL / 6 mice using GL261 cells and a stereotaxic instrument. Two weeks later, the mice were anesthetized and underwent craniotomy. The probe Ru-GA (10 mM) was sprayed onto the surface of the brain tissue after craniotomy using PBS. Simultaneously, normal mice were anesthetized, underwent craniotomy, and treated with Ru-GA. Fluorescence imaging was performed using a small animal in vivo imaging system. The imaging results are shown below. Figure 8 As shown. Figure 8 Imaging results showed that fluorescence in the brain tissue of glioma-bearing mice significantly increased over time, and the fluorescence intensity was significantly stronger than that of normal mice. Semi-quantitative analysis of fluorescence intensity indicated that, after ten minutes of treatment with the Ru-GA probe, Ru-GA spraying could rapidly achieve differential imaging between glioma tissue and normal brain tissue.
[0061] To verify that the aforementioned fluorescence signal was generated by the reaction of the probe Ru-GA with GSTs and AzoR in glioma tissue, mouse glioma tissue was pretreated with the GSTs inhibitor ethacrylic acid (R1), the AzoR inhibitor sulfasalic acid (R2), and both inhibitors (R1+R2) simultaneously, followed by spraying with Ru-GA (10 M) in PBS solution. The imaging results are shown below. Figure 9 As shown. Figure 9 Imaging results showed that the fluorescence signal of glioma-bearing mouse brain tissue was significantly weakened after treatment with two inhibitors followed by Ru-GA probe spraying. These imaging results indicate that Ru-GA probe spraying can achieve specific fluorescence imaging of glioma tissue through its fluorescence response to GSTs and AzoR.
[0062] Given the excellent imaging capabilities of Ru-GA spray for glioma tissue, we subsequently applied it to intraoperative guided resection of mouse gliomas. Figure 10 In mice with gliomas, bright red fluorescence was observed in the glioma tissue under 450 nm light after Ru-GA spray treatment. The glioma tissue with red fluorescence was surgically removed under fluorescence guidance. Subsequently, the removed brain tissue was treated with Ru-GA spray again, and the process was repeated three times until no red fluorescence signal was observed, indicating that the glioma tissue had been completely removed.
[0063] In addition, histological analysis of the excised glioma tissue was performed using HE staining. HE staining results showed that ( Figure 11The tissues removed using fluorescence guidance were all glioma tissues, with significantly deepened nuclear staining and a significantly increased nuclear-to-cytoplasmic ratio. These experimental results demonstrate that the Ru-GA probe spray can achieve specific fluorescence imaging of glioma tissues, and its application in intraoperative guided resection of mouse gliomas shows good accuracy.
[0064] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims
1. A dual-enzyme activated ruthenium complex fluorescent probe, characterized in that, Its structural formula is shown in Formula I: 。 2. A method for synthesizing a dual-enzyme activated ruthenium complex fluorescent probe according to claim 1, characterized in that, The synthetic reaction route is as follows: ； Among them, the compound of formula I is named Ru-GA.
3. The method for synthesizing the dual-enzyme activated ruthenium complex fluorescent probe according to claim 2, characterized in that, Includes the following steps: (1) Compound 1 and Compound 2 were heated under nitrogen protection in ethanol and refluxed overnight. After the reaction was completed, the solvent was evaporated to dryness, purified by silica gel column chromatography, and potassium nitrate was replaced with potassium hexafluorophosphate to obtain Compound 3. (2) The compound 3 obtained in step (1) was dissolved in dichloromethane. Under the protection of ice bath and nitrogen, a dichloromethane solution of boron tribromide was slowly added dropwise. The reaction was then carried out at room temperature for 7 hours. After the reaction was completed, water was added to quench the reaction. The organic phase was separated, the solvent was evaporated to dryness, and the compound was purified by silica gel column chromatography. Potassium nitrate was replaced with potassium hexafluorophosphate to obtain compound 4. (3) The compounds 4, 2,4-dinitrofluorobenzene and potassium carbonate obtained in step (2) were mixed in acetonitrile and heated under nitrogen protection and refluxed overnight. After the reaction was completed, the fluorescent probe Ru-GA was obtained by solvent evaporation, silica gel column chromatography purification and potassium hexafluorophosphate was used to replace potassium nitrate.
4. The method for synthesizing the dual-enzyme activated ruthenium complex fluorescent probe according to claim 3, characterized in that, In step (1), the molar ratio of compound 1 to compound 2 is 1:
1.
5. The method for synthesizing the dual-enzyme activated ruthenium complex fluorescent probe according to claim 3, characterized in that, In step (3), the molar ratio of compound 4 to 2,4-dinitrofluorobenzene is 1:
2.
6. The method for synthesizing the dual-enzyme activated ruthenium complex fluorescent probe according to claim 3, characterized in that, In steps (1), (2), and (3), the eluent used in the silica gel column chromatography purification is a mixed solvent system of acetonitrile-potassium nitrate-water.
7. The application of the dual-enzyme activated ruthenium complex fluorescent probe as described in claim 1 in the preparation of glioma detection products.
8. The application of the dual-enzyme activated ruthenium complex fluorescent probe according to claim 7 in the preparation of glioma detection products, characterized in that, The glioma detection product is an in vitro diagnostic reagent used for the identification and fluorescence imaging of glioma cells.
9. The application of the dual-enzyme activated ruthenium complex fluorescent probe according to claim 7 in the preparation of glioma detection products, characterized in that, The glioma detection product is a surgical navigation reagent.
10. The application of the dual-enzyme activated ruthenium complex fluorescent probe according to claim 7 in the preparation of glioma detection products, characterized in that, The product for detecting gliomas is in the form of a spray.