Tumor-targeting near-infrared fluorescent probes for monitoring hypoxia, methods of making and uses thereof

Abstract

The application discloses a tumor-targeting near-infrared fluorescence probe capable of monitoring hypoxia, a preparation method and application, and relates to the technical field of fluorescence detection; the fluorescence probe comprises a core fluorophore composed of a boron dipyrromethene derivative, a PSMA targeting group composed of two KUEs and a probe composed of a nitro group, wherein the boron dipyrromethene derivative serves as a fluorophore core; the nitro aromatic group serves as a sensing unit and inhibits fluorescence emission before activation through a photoinduced electron transfer mechanism; meanwhile, the two KUE motifs are functionalized to have high-affinity PSMA binding capacity; the fluorescence probe XN-NTR of the application can specifically target prostate cancer, can specifically react to nitroreductase highly expressed by tumor tissues, can realize precise definition of tumor and normal tissues in clinical surgical resection of prostate cancer, and has the characteristics of rapid response, high sensitivity, good selectivity, good biological safety, in-vivo tissue imaging and the like.

Description

Technical Field

[0001] This invention relates to the field of fluorescence detection technology, specifically to a near-infrared fluorescent probe for tumor targeting and monitoring hypoxia, its preparation method, and its application. Background Technology

[0002] Prostate cancer is an increasingly common type of cancer among men, and surgical resection remains the primary treatment, effectively reducing tumor burden and alleviating the psychological burden on patients. While wide resection offers therapeutic advantages, the surgical risks cannot be ignored. Traditional methods for determining intraoperative margins rely mainly on visual examination and tactile feedback; both methods are inherently subjective and lack objective, real-time, and precise definition.

[0003] To address this issue, fluorescence image-guided surgery (FIGS) has emerged as a promising adjunctive technique, enabling real-time visualization of tumors through fluorophore-based imaging. This navigational technology can accurately identify occult malignant tissues and positive surgical margins, thereby protecting surrounding healthy tissues while completely removing the tumor. Furthermore, combining fluorescent probes with tumor-specific biomarkers significantly enhances the specificity of this technique. These advantages help surgeons achieve negative surgical margins, thereby reducing recurrence rates and complications.

[0004] The effectiveness of fluorescence-guided surgery is highly dependent on the selection of appropriate molecular targets. Prostate-specific membrane antigen (PSMA), a type II transmembrane glycoprotein overexpressed in prostate cancer and expressed at very low levels in normal tissues, is an ideal molecular imaging target. Therefore, fluorescent probes targeting PSMA are being actively developed, as they can selectively accumulate in cancerous tissues, potentially enabling precise surgical guidance. However, traditional optical probes often employ a continuous activation mode, requiring a long waiting time to obtain sufficient tumor-background contrast; while activatable ("off-on") probes emit light only upon interaction with the target tissue, minimizing background signal. Among various tumor markers, nitroreductase (NTR) is a clinically significant tumor-associated enzyme, widely studied due to its upregulation in the typical hypoxic environment of solid tumors such as prostate cancer. Although several NTR-responsive fluorescent probes have been reported, none have been successfully applied to intraoperative navigation in prostate cancer due to insufficient tumor targeting ability. Recently, Zhang et al. reported a PSMA-targeted fluorescent molecular rotor that triggers an on-off response through enzyme residue interactions. However, this molecular rotor is highly sensitive to microenvironmental factors (such as polarity and viscosity), which may produce false positive signals.

[0005] It is evident that the boundary between tumor lesions and normal tissue remains difficult to define precisely during prostate cancer surgical resection. Therefore, developing a specific, activatable fluorescent probe that can target prostate cancer and accurately identify the boundary between cancer lesions and normal tissue has great application value. Summary of the Invention

[0006] This invention addresses the technical problem that traditional optical probes, which mostly employ continuous activation modes, require long waiting times to obtain sufficient contrast, resulting in significant time consumption. Furthermore, activatable probes are highly sensitive to microenvironmental factors, easily generating false positive signals and affecting guidance reliability. This leads to the inability to accurately delineate tumors and normal tissues during clinical surgical resection of prostate cancer. The aim is to provide a tumor-targeting near-infrared fluorescent probe capable of monitoring hypoxia, its preparation method, and its application. This fluorescent probe, XN-NTR, can specifically target prostate cancer and specifically reacts to nitroreductase (NTR), which is highly expressed in tumor tissue. This enables precise delineation of tumors and normal tissues during clinical surgical resection of prostate cancer, featuring rapid response, high sensitivity, good selectivity, good biosafety, and the ability to perform in vivo tissue imaging.

[0007] The present invention is achieved through the following technical solution.

[0008] The first objective of this invention is to provide a near-infrared fluorescent probe for tumor targeting and monitoring hypoxia, the fluorescent probe comprising a core fluorophore composed of a boron dipyrrolemethylene derivative, a PSMA targeting group composed of two KUEs, and a probe composed of nitro groups.

[0009] The fluorescent probe of this invention uses a borodipyrrole methylene (BODIPY) derivative as the fluorophore core. This structure is easy to modify, has high quantum yield, NIR emission characteristics, and deep tissue penetration ability. The nitro aromatic group serves as the sensing unit, which inhibits fluorescence emission before activation through photoinduced electron transfer (PET) mechanism. At the same time, to ensure prostate cancer specificity, the fluorescent probe of this invention is functionalized with two KUE (lysine-ureido-glutamic acid) motifs, which have high affinity PSMA binding ability.

[0010] This invention achieves dual selectivity (enzyme activation and receptor targeting) by coupling a specific NTR-responsive core (nitroaromatic group) with a PSMA ligand (KUE), significantly enhancing imaging specificity in hypoxic prostate tumors. Under hypoxic conditions, intracellular NTRs, in the presence of nicotinamide adenine dinucleotide phosphate (NAD(P)H) as an electron donor, reduce the nitroaromatic group, releasing the fluorophore XN-NH2 and generating a strong NIR signal. The specificity of XN-NTR was confirmed by comparing its imaging performance in PSMA-positive (22Rv1(PSMA++)) cells with other cell lines, indicating that the fluorophore specifically accumulates and activates in 22Rv1 cells. Importantly, XN-NTR can also precisely visualize the boundary between tumor lesions and normal tissue in clinical human prostate cancer samples without the need for additional adjuvants.

[0011] Therefore, the fluorescent probe of this invention can specifically target prostate cancer and specifically react to nitroreductase (NTR) highly expressed in tumor tissue, thereby achieving precise delineation of tumor and normal tissue during clinical surgical resection of prostate cancer. Experiments have demonstrated that the fluorescent probe of this invention has the characteristics of rapid response, high sensitivity, good selectivity, good biosafety, and the ability to perform in vivo tissue imaging.

[0012] Furthermore, the structural formula of the fluorescent probe is shown in Formula I:

[0013] .

[0014] Furthermore, the response time of the fluorescent probe is ≤20 min, and the detection limit of the fluorescent probe is 0.037 μg / mL.

[0015] The second objective of this invention is to provide a method for preparing a near-infrared fluorescent probe for tumor targeting and monitoring hypoxia, comprising the following steps:

[0016] (1) 2,2'-((((1E,1'E)-(5,5-difluoro-1,3,7,9-tetramethyl-10-(2-nitrophenyl)-5H-4λ 4 ,5λ 4 -Dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborhexacyclohexanetriene, 4-formylphenoxyacetic acid, and piperidine were added to acetonitrile, stirred and refluxed overnight, and the reaction solution was concentrated under reduced pressure and purified to obtain NO2-Bo 636 -2COOH;

[0017] (2) NO2-Bo 636-2COOH, N-hydroxysuccinimide, 1-hydroxybenzotriazole monohydrate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and pyridine were dissolved in DMF and stirred overnight. The reaction solution was purified to obtain NO2-Bo 636 -2NHS;

[0018] (3) NO2-Bo 636 -2NHS, PSMAI, 1-hydroxybenzotriazole monohydrate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and pyridine were dissolved in dichloromethane, magnetically stirred overnight, the reaction solution was concentrated under reduced pressure, purified and lyophilized to obtain the fluorescent probe XN-NTR.

[0019] Further, in step (1), 2,2'-((((1E,1'E)-(5,5-difluoro-1,3,7,9-tetramethyl-10-(2-nitrophenyl)-5H-4λ 4 ,5λ 4 The molar ratio of dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborhexacyclohextriene, 4-formylphenoxyacetic acid, and piperidine is (0.24-0.3):(0.7-0.8):(0.024-0.03).

[0020] Furthermore, in step (2), NO2-Bo 636 The molar ratio of -2COOH, N-hydroxysuccinimide, 1-hydroxybenzotriazole monohydrate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and pyridine is (0.1-0.15):(0.2-0.25):0.2:0.5:(0.6-0.65).

[0021] Furthermore, in step (3), NO2-Bo 636 The molar ratio of -2NHS, PSMAI, 1-hydroxybenzotriazole monohydrate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and pyridine is (0.05-0.06):(0.07-0.08):0.10:(0.05-0.1):0.6.

[0022] Furthermore, the reaction solution in step (1) is purified using a rapid liquid chromatograph equipped with a rapid silica column, and the reaction solutions in steps (2) and (3) are purified using preparative chromatography.

[0023] Furthermore, the reaction temperature in step (1) is 75-80℃, and the reaction temperatures in steps (2) and (3) are room temperature.

[0024] The third objective of this invention is to provide an application of a tumor-targeting near-infrared fluorescent probe capable of monitoring hypoxia in the preparation of tumor detection reagents.

[0025] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0026] 1. Strong tumor targeting: The fluorescent probe of this invention can specifically target tumor cells and tissues that highly express the PSMA receptor and be taken up by cells through endocytosis;

[0027] 2. High specificity: The fluorescent probe of this invention is based on photoinduced emission electron transfer (PET) mechanism. Under normal circumstances, the fluorescence of the probe is quenched due to the nitro structure. Only when NTR and coenzyme NADH are present at the same time is the probe activated by the electron provided by coenzyme NADH, which reduces the nitro group to amino group, thereby restoring the fluorescence and generating a strong near-infrared fluorescence signal, thus achieving high specificity detection.

[0028] 3. Rapid response: The fluorescent probe of this invention can react completely with NTR within 20 min, which can greatly shorten the reaction time compared with the reported probe response time (>100 min);

[0029] 4. High sensitivity: The detection limit of the fluorescent probe of this invention reaches 0.037 μg / mL, which greatly improves the detection sensitivity;

[0030] 5. High selectivity: The fluorescent probe of this invention will only show significant fluorescence enhancement when NTR and coenzyme NADH are added simultaneously;

[0031] 6. High biocompatibility: The fluorescent probe molecules of this invention have high biocompatibility with cells and organisms;

[0032] 7. Specific targeting of prostate cancer cells: The fluorescent probe molecules of this invention specifically target cancer cells with PSMA receptors via the KUE group, and it has been demonstrated that they enter cells via endocytosis and specifically respond to the NTR of tumor cells;

[0033] 8. Vivo tissue imaging: The fluorescent probe of this invention can successfully distinguish the NTR and hypoxia level inside tumor tissue and achieve precise localization of the boundary between tumor and normal tissue;

[0034] 9. High time stability: The fluorescent probe of this invention has good stability and can image NTR activity for a long time. Attached Figure Description

[0035] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be considered as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort. In the drawings:

[0036] Figure 1 This is a synthetic route diagram of XN-NTR in Embodiment 1 of the present invention;

[0037] Figure 2 The characterization results of the XN-NTR prepared in Example 1 are shown, where (i) is the 1H NMR spectrum, (ii) is the 1C NMR spectrum, and (iii) is the high-resolution mass spectrum.

[0038] Figure 3 Functional theory calculations were performed on the XN-NTR prepared in Example 1 and its dye.

[0039] Figure 4 Docking simulation and interaction diagram of XN-NTR and NTR active cavity prepared in Example 1;

[0040] Figure 5 The fluorescence emission spectra of XN-NTR and NTR prepared in Example 1 are shown below;

[0041] Figure 6 The fluorescence emission diagrams of the XN-NTR prepared in Example 1 and its response to NTR at different time points are shown.

[0042] Figure 7 The corresponding performance diagrams of XN-NTR prepared in Example 1 and NTR are shown, where (A) is the fluorescence spectrum of XN-NTR after reaction with NTR of different concentrations, and (B) is the linear relationship between fluorescence intensity and NTR concentration.

[0043] Figure 8 The selectivity performance of the XN-NTR prepared in Example 1 against 25 common interfering substances was tested.

[0044] Figure 9 The results show the cytotoxicity of different concentrations of probe XN-NTR against 22RV1 cells with different PSMA expression levels under normoxic and hypoxic conditions. Among them, (A) and (C) are the test results after culturing 22RV1 and transfected 22RV1 (PSMA++) cells at 20% O2, respectively; (B) and (D) are the test results after culturing 22RV1 and transfected 22RV1 (PSMA++) cells at 1% O2, respectively.

[0045] Figure 10The results of intracellular fluorescence imaging of XN-NTR prepared in Example 1 with different PSMA expression levels are shown; (A) is an imaging image of prostate cancer cells with different PSMA expression levels under hypoxic conditions, and (B) is a fluorescence intensity quantification map.

[0046] Figure 11 The fluorescence imaging results of XN-NTR prepared in Example 1 under different hypoxic and inhibitor conditions are shown in (A) confocal images of cancer cells 22RV1 (PSMA++) after pretreatment with NTR inhibitors dicumarol (Dic, 50 μM) or KUE peptide (100 μM) under conditions of 20% and 1% O2; (B) fluorescence intensity quantification map.

[0047] Figure 12 From top to bottom, the images show two-dimensional, three-dimensional, and Z-axis scanning depth fluorescence images of XN-NTR prepared in Example 1 after incubation for 20 minutes in human prostate cancer tissue and normal tissue sections;

[0048] Figure 13 Fluorescence images of human prostate cancer tissue sections incubated with XN-NTR prepared in Example 1 at different time intervals;

[0049] Figure 14 Fluorescence image, H&E staining image, and combined field image of XN-NTR prepared in Example 1 in human prostate cancer tissue. Detailed Implementation

[0050] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

[0051] Embodiments of the present invention will now be described in detail with reference to the accompanying drawings. However, unnecessary details may be omitted. For example, detailed descriptions of well-known matters and repetitive descriptions may be omitted. This is to avoid making the following description unnecessarily lengthy and to facilitate understanding by those skilled in the art.

[0052] The "scope" disclosed in this invention is defined in the form of a lower limit and an upper limit. A given scope is defined by selecting a lower limit and an upper limit, which define the boundaries of the specific scope. The scope defined in this way can include or exclude end values, and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a scope.

[0053] Unless otherwise specified, all embodiments and optional embodiments of the present invention can be combined with each other to form new technical solutions.

[0054] Unless otherwise specified, all technical features and optional technical features of this invention can be combined to form new technical solutions.

[0055] Unless otherwise specified, the terms "comprising" and "including" as used in this invention can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other substances not listed may also be included, or that only the listed substances may be included.

[0056] Unless otherwise specified, all steps of the present invention may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0057] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0058] It should be noted that, unless otherwise specified, the experimental methods used in the embodiments are conventional methods. The materials, reagents, methods, and instruments used, unless otherwise specified, are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0059] Example 1

[0060] Reference Figure 1 The synthetic route diagram shown illustrates a method for preparing a tumor-targeting near-infrared fluorescent probe (XN-NTR) capable of monitoring hypoxia, comprising the following steps:

[0061] (1) 2,2'-((((1E,1'E)-(5,5-difluoro-1,3,7,9-tetramethyl-10-(2-nitrophenyl)-5H-4λ 4 ,5λ 4-Dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborhexacyclohextriene (86 mg), 4-formylphenoxyacetic acid (126 mg, 0.7 mmol), and piperidine (3 mL) were added to acetonitrile (27 mL) and refluxed overnight with magnetic stirring. The reaction mixture was concentrated under reduced pressure with silica gel. Subsequently, purification was performed using a rapid liquid chromatography system equipped with a rapid silica gel column: elution was performed using a linear gradient of dichloromethane and methanol (wavelength: 650 nm; flow rate: 30 mL / min; time-methanol percentage: 0.0 min, 0%; 1.0 min, 0%; 17.5 min, 75%; 20 min, 75%). NO2-Bo was collected from the corresponding fractions. 636 -2COOH, concentrated under reduced pressure, yielded 44 mg of product, yield 27.6%.

[0062] (2) NO2-Bo 636 -2COOH (69.3 mg, 0.1 mmol), N-hydroxysuccinimide (NHS, 23 mg, 0.2 mmol), 1-hydroxybenzotriazole monohydrate (27.1 mg, 0.2 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (96 mg, 0.5 mmol), and 60 μL of pyridine were dissolved in 3 mL of DMF (HPLC grade, Sigma) and magnetically stirred overnight at room temperature. The products were separated by preparative chromatography: the gradient conditions were water / acetonitrile (using ultrapure water and HPLC grade acetonitrile) at a flow rate of 12 mL / min (linear gradient from 9:1 to 1:9 over 20 min, followed by a hold for 25 min). The products were collected, stored at -80°C overnight, and then lyophilized to give a dark green powder, namely NO2-Bo 636 -2NHS. Yield: 73.7 mg (83.1%).

[0063] (3) The above NO2-Bo 636 -2NHS (44.4 mg, 0.05 mmol), PSMAI (34.2 mg, 0.07 mmol), 1-hydroxybenzotriazole monohydrate (13.5 mg, 0.1 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (96 mg, 0.5 mmol), and 60 μL of pyridine were dissolved in dichloromethane (DCM, 3 mL). The reaction mixture was magnetically stirred overnight at room temperature.

[0064] (4) Trifluoroacetic acid (TFA, 0.75 mL) was then added, and stirring was continued for one hour. The solution was then concentrated under reduced pressure. The crude product was redissolved in DMF (2 mL) and purified by preparative chromatography: the gradient conditions were water / acetonitrile (using ultrapure water and HPLC-grade acetonitrile), the flow rate was 12 mL / min (the linear gradient was from 90:10 to 10:90 over 20 minutes, followed by a hold for 25 minutes). The product was collected, stored at -80°C overnight, and then lyophilized to obtain a dark green powder, which was the target fluorescent probe XN-NTR.

[0065] Example 2

[0066] XN-NTR Characterization and Mechanism

[0067] The XN-NTR obtained in Example 1 was characterized, as follows: Figure 2 As shown, the positions of the probe in the proton spectrum and the number of protons are all corresponding one-to-one, as can be seen from the proton NMR spectrum, carbon NMR spectrum and high-resolution mass spectrometry. The calculated value of the mass spectrometry data is [M+Na]=1318.45339, and the measured value is 1318.45321. Therefore, it can be fully confirmed that the XN-NTR fluorescent probe of this invention has been successfully synthesized.

[0068] The working mechanism of the XN-NTR of this invention is as follows: Because the KUE group specifically targets PSMA overexpressed in prostate cancer, this probe can be easily taken up by prostate cancer cells, enter the cell via endocytosis, and specifically respond to the overexpressed NTR within the cell. During this process, due to the PET luminescence mechanism, the fluorescence of the probe is initially in a "off" state; upon response to the NTR, the nitro group of XN-NTR is converted to an amino group, PET disappears, leading to the recovery of the fluorescence signal and the formation of a strong fluorescence signal. Figure 3 As shown, the LUMO orbital of the XN-NTR fluorescent probe molecule of the present invention lies between the HOMO-LUMO orbital of XN-NH2 formed after its NTR response, which is consistent with the luminescence mechanism of PET; and its band gap is 1.0949 eV (less than XN-NH2), which indicates that the XN-NTR fluorescent probe molecule is more reactive and readily reacts with the target analyte.

[0069] Combination Figure 4 As shown, the XN-NTR fluorescent probe molecules of this invention can easily bind to the surface of NTR. (From...) Figure 4 Computer simulation (docking) results show that the probe XN-NTR does indeed form a stable covalent bond with the active site of the NTR, with a binding energy of -7.57 kcal / mol, indicating that the probe can stably act on the active site of the NTR to produce an in situ fluorescence response.

[0070] Example 3

[0071] XN-NTR optical testing

[0072] The reaction system for optical testing was as follows: Each well of a 384-well plate had a total volume of 100 μL, including 5 μL of a 100 μM stock solution of the fluorescent probe XN-NTR, 10 μL of a 100 μg / mL NTR stock solution, 10 μL of a 5 mM NADH stock solution, and 85 μL of PBS solution at pH 7.4. The probe stock solution was prepared from DMSO, and the final test system contained 5% DMSO at a final concentration of 5 μM. The NTR concentration was 10 μg / mL, and the NADH concentration was 500 μM. The reaction system was incubated at 37°C and 1000 rpm for 40 min in the dark. The detection results are as follows:

[0073] (1) such as Figure 5 As shown, the response performance of XN-NTR to NTR was tested. Specifically, under the condition of NTR concentration of 10 μg / mL and using a 600 nm wavelength laser as the excitation wavelength, a 10.5-fold fluorescence enhancement was observed after the reaction, while the fluorescence decreased significantly after adding the inhibitor dicoumarin (Dic). These changes in photophysical properties confirm that XN-NTR can be activated by NTR and release a fluorophore, causing fluorescence enhancement. These data indicate that XN-NTR can effectively detect changes in NTR activity.

[0074] (2) For example Figure 6 As shown, the time-kinetic response of XN-NTR to NTR was tested. Specifically, in a PBS test system (0.1M, 5% DMSO) at pH = 7.4, with XN-NTR and NTR concentrations of 5 μM and 10 μg / mL, respectively, the reaction time kinetics were tested at 37℃. The kinetic results showed that the reaction of XN-NTR and NTR reached equilibrium in 20 min (λ). ex = 600 nm). And it can remain stable for up to 80 minutes.

[0075] (3) such as Figure 7 As shown in (A), the concentration-response performance of XN-NTR to NTR was tested. Specifically, in a PBS test system (0.1 M, 5% DMSO) at pH = 7.4, with an XN-NTR concentration of 5 μM, different concentrations of NTR (0-10 μg / mL) were added, and the reaction was carried out at 37℃ for 20 min to obtain the final concentration gradient (λ). ex= 600 nm). The results showed that, within the NTR concentration range of 0 to 10 μg / mL, the fluorescence intensity of XN-NTR after reacting with NTR gradually increased with increasing NTR concentration. Its working concentration curve is shown below. Figure 7 As shown in (B), the detection limit for NTR is calculated to be 0.037 μg / mL.

[0076] (4) such as Figure 8 As shown, the selectivity of XN-NTR against 25 other interfering substances was tested. Specifically, in a PBS test system (0.1 M, 5% DMSO) at pH 7.4, with an XN-NTR concentration of 5 μM, 25 common analytes were added for reaction, namely: Cu 2+ Zn 2+ , K + Mg 2+ Fe 3+ Fe 2+ , Cr 3+ Ca 2+ Na + Al 3+ , S 2- HS - SO3 2- SO4 2- CO3 2- The analytes were VC, VE, H2O2, Glu, Cys, GSH, Tyr, BSA, NTR, and Blank. The mixture was then reacted at 37 °C for 20 min. The results showed that the fluorescence changes caused by the other analytes were negligible compared to those caused by NTR, confirming the high selectivity of XN-NTR for NTR.

[0077] Example 4

[0078] Intracellular assay of XN-NTR

[0079] (1) First, the cytotoxicity of probe XN-NTR was tested. Specifically, the standard MTT assay was used for analysis. 22RV1 and transfected 22RV1 (PSMA++) cells were digested with 0.25% trypsin, and then the cells were seeded into a cell suspension in cell culture medium and seeded into 96-well plates. The cells were cultured at 37°C for 24 hours. Probes of different concentrations (0 μM, 0.3125 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM, 60 μM, 80 μM, 100 μM) were added to the well plates and cultured for 24 hours. MTT was added and cultured for 4 hours. Finally, triple lysis buffer was added and incubated for another 2 hours. The absorbance of each well was measured using a microplate reader. Control wells and zeroing wells were also set up. For the MTT assay under hypoxic conditions, 22RV1 cells and transfected 22RV1 (PSMA++) cells are pre-cultured under hypoxic conditions (1% O2) for 2 hours before the above procedures are performed. Figure 9 As shown, the experimental results indicate that XN-NTR has good biocompatibility. At a concentration of 100 μM, XN-NTR did not exhibit significant cytotoxicity to cells, and the cell viability remained above 80% under normoxic conditions.

[0080] (2) Next, intracellular fluorescence imaging of the probe XN-NTR with different PSMA expression levels was tested. Specifically, three types of prostate cancer cells (PC3, 22RV1, and 22RV1 (PSMA++)) were cultured for 2 hours under hypoxic conditions (1% O2), and XN-NTR was added for 1 hour of culture. Finally, confocal fluorescence microscopy was used for imaging. The experimental results are as follows: Figure 10 As shown in (A). According to Figure 10 (B) The quantitative graph shows that the fluorescence signal of 22RV1 (PSMA++) cells transfected with a large amount of PSMA receptor is the highest, which is consistent with the uptake mode of probe XN-NTR in cells.

[0081] (3) Finally, the fluorescence imaging of the probe XN-NTR under different hypoxic and inhibitory conditions was tested. Specifically, 22RV1 (PSMA++) cells were selected and cultured under different oxygen conditions (20% O2 and 1% O2), and XN-NTR (5 μM) was added and cultured for 1 h. Finally, confocal fluorescence microscopy was used for imaging. For the inhibition group, Dic (50 μM) or KUE peptide (100 μM) were used for pre-culture for 2 h before imaging. The experimental results are as follows: Figure 11 As shown in (A). Based on the corresponding fluorescence quantization Figure 11Data in (B) showed that under hypoxic conditions, XN-NTR exhibited the strongest fluorescence signal in 22RV1 (PSMA++) cells. The addition of the inhibitor Dic resulted in a corresponding decrease in fluorescence signal, indicating that the enhanced fluorescence signal was indeed due to NTR. Similarly, the addition of KUE resulted in a corresponding decrease in fluorescence signal, consistent with the XN-NTR uptake mechanism. High concentrations of KUE inhibited the uptake of XN-NTR by the PSMA receptor.

[0082] These results all indicate that XN-NTR can specifically target prostate cancer cells that overexpress PSMA and can perform specific fluorescence imaging of NTR expression and hypoxia levels within cancer cells.

[0083] Example 5

[0084] Clinical tissue imaging testing of XN-NTR

[0085] (1) First, the imaging of human prostate cancer tissue samples by the probe XN-NTR was tested. Specifically, human prostate cancer tissue and adjacent (normal) tissue were obtained from clinically diagnosed prostate cancer patients by biopsy. Tumor samples were sliced ​​into 3×3 mm thin slices and co-incubated with XN-NTR (60 μM) for 20 minutes. Adjacent tissue served as a negative control, while fresh tumor slices incubated only with PBS buffer constituted the PBS control group; in addition, tumor slices pretreated with an NTR inhibitor (Dic, 1.0 mM) for 1 hour constituted the inhibitor control group. The experimental results are as follows: Figure 12 As shown, no significant fluorescence signal was observed in normal tissue; however, a strong fluorescence signal was detected in tumor sections co-incubated with XN-NTR, with a tissue penetration depth of up to 200 micrometers, sufficient to cover the entire section. As expected, the fluorescence signal in the inhibitor group samples was significantly reduced, confirming that the fluorescence intensity is specifically dependent on NTR enzyme activity, with a signal-to-background ratio of up to 7.9.

[0086] (2) Next, the time stability imaging of the probe XN-NTR on human prostate cancer tissue samples was tested. Specifically, the slides were incubated with XN-NTR solution (60 μM) for 0 to 180 minutes. Fluorescence imaging was performed using confocal fluorescence microscopy at different incubation times. The experimental results are as follows: Figure 13 As shown, the fluorescence intensity gradually increased within the first 20 minutes and remained stable between 20 and 180 minutes, demonstrating that the signal is suitable for long-term imaging.

[0087] (3) Finally, the ability of the XN-NTR probe to identify the boundary between tumor and normal tissue in human prostate cancer tissue was tested. Specifically, after incubating the slide with XN-NTR solution for 20 min, fluorescence imaging was performed using a confocal fluorescence microscope, and the tissue was stained with H&E. Imaging was then performed under the same field of view using a microscope. Figure 14 As shown, the experimental results indicate that the red fluorescent region highly coincides with the region of moderately differentiated squamous cell carcinoma with necrosis, confirming the co-localization of the probe signal and tumor morphological features.

[0088] These results demonstrate that the fluorescent probe XN-NTR prepared in this invention has extremely high diagnostic reliability due to its ability to achieve direct in-situ imaging of tumor biomarkers, and can clearly delineate tumor boundaries, showing significant application potential in intraoperative navigation and rapid clinical tissue biopsy assessment.

[0089] Finally, it should be noted that the above specific embodiments are only used to describe the purpose, technical solution, and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation of the present invention and is not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the foregoing specific embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions or improvements can be made to some or all of the technical features. These modifications, equivalent substitutions, and improvements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A near-infrared fluorescent probe for tumor targeting and monitoring hypoxia, characterized in that, The fluorescent probe comprises a core fluorophore composed of a boron dipyrrolemethylene derivative, a PSMA targeting group composed of two KUEs, and a probe composed of nitro groups.

2. The near-infrared fluorescent probe for tumor targeting and monitoring hypoxia according to claim 1, characterized in that, The structural formula of the fluorescent probe is shown in Formula I: 。 3. The near-infrared fluorescent probe for tumor targeting and monitoring hypoxia according to claim 1, characterized in that, The response time of the fluorescent probe is ≤20 min, and the detection limit of the fluorescent probe is 0.037 μg / mL.

4. A method for preparing a tumor-targeting near-infrared fluorescent probe for monitoring hypoxia as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) 2,2'-((((1E,1'E)-(5,5-difluoro-1,3,7,9-tetramethyl-10-(2-nitrophenyl)-5H-4λ 4 ,5λ 4 -Dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborhexacyclohexanetriene, 4-formylphenoxyacetic acid, and piperidine were added to acetonitrile, stirred and refluxed overnight, and the reaction solution was concentrated under reduced pressure and purified to obtain NO2-Bo 636 -2COOH; (2) NO2-Bo 636 -2COOH, N-hydroxysuccinimide, 1-hydroxybenzotriazole monohydrate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and pyridine were dissolved in DMF and stirred overnight. The reaction solution was purified to obtain NO2-Bo 636 -2NHS; (3) NO2-Bo 636 -2NHS, PSMAI, 1-hydroxybenzotriazole monohydrate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and pyridine were dissolved in dichloromethane, magnetically stirred overnight, the reaction solution was concentrated under reduced pressure, purified and lyophilized to obtain the fluorescent probe XN-NTR.

5. A near-infrared fluorescent probe for tumor targeting and monitoring hypoxia according to claim 4, characterized in that, In step (1), 2,2'-((((1E,1'E)-(5,5-difluoro-1,3,7,9-tetramethyl-10-(2-nitrophenyl)-5H-4λ 4 ,5λ 4 The molar ratio of dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborhexacyclohextriene, 4-formylphenoxyacetic acid, and piperidine is (0.24-0.3):(0.7-0.8):(0.024-0.03).

6. The near-infrared fluorescent probe for tumor targeting and monitoring hypoxia according to claim 4, characterized in that, In step (2), NO2-Bo 636 The molar ratio of -2COOH, N-hydroxysuccinimide, 1-hydroxybenzotriazole monohydrate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and pyridine is (0.1-0.15):(0.2-0.25):0.2:0.5:(0.6-0.65).

7. A near-infrared fluorescent probe for tumor targeting and monitoring hypoxia according to claim 4, characterized in that, In step (3), NO2-Bo 636 The molar ratio of -2NHS, PSMAI, 1-hydroxybenzotriazole monohydrate, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and pyridine is (0.05-0.06):(0.07-0.08):0.10:(0.05-0.1):0.

6.

8. A near-infrared fluorescent probe for tumor targeting and monitoring hypoxia according to claim 4, characterized in that, The reaction solution in step (1) was purified using a rapid liquid chromatograph equipped with a rapid silica column, and the reaction solutions in steps (2) and (3) were purified using preparative chromatography.

9. A near-infrared fluorescent probe for tumor targeting and monitoring hypoxia according to claim 4, characterized in that, The reaction temperature in step (1) is 75-85℃, and the reaction temperature in steps (2) and (3) is room temperature.

10. The application of a tumor-targeting near-infrared fluorescent probe capable of monitoring hypoxia as described in any one of claims 1-3 in the preparation of tumor detection reagents.

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