Preparation and application of granzyme B response near-infrared two-region ratiometric fluorescent probe

Abstract

The invention discloses a preparation method and application of a granzyme B response near-infrared two-region ratiometric fluorescent probe (DCGA). The DCGA combines the NIR-II dual-emission fluorescence characteristic of rare earth ions with a ratio-type signal response mode, the sensitivity and signal stability of in-vivo fluorescence imaging are greatly improved, ultralow or enhanced F1060nm fluorescence signals are respectively shown before and after interaction with granzyme B (GzmB), F1525nm fluorescence signals are almost kept unchanged, and the DCGA can be used for detecting the in-vivo fluorescence imaging of the granzyme B (GzmB). Therefore, the self-calibration ratio fluorescence imaging GzmB activity change is realized. The DCGA is applied to tumor treatment of immunotherapy drugs, and responders and non-responders of immunotherapy can be distinguished in a non-invasive early stage through ratio fluorescence signals of the DCGA. Therefore, the fluorescent probe can be used as an effective GzmB active NIR-II ratio fluorescence imaging tool, and is expected to provide powerful support for immunotherapy research, precision medicine and personalized diagnosis and treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of medical biomaterials, and in particular to a preparation method and application of a granzyme B-responsive near-infrared second-region ratiometric fluorescent probe. Background Art

[0002] T lymphocytes (T cells) in the tumor microenvironment are the primary anti-tumor immune effector cells. Their number, location, and activation level directly influence the efficacy of immunotherapy, making them an important target for tumor immunotherapy. Activated T cells have a direct cytotoxic effect on tumor cells. Accurate, rapid, and dynamic imaging of their activation status helps monitor the body's response to immunotherapy and provides an early indicator of the true immune response, making it a key indicator for evaluating immunotherapy efficacy. Granzyme B (GzmB) is a key serine protease involved in T cell cytotoxicity. Monitoring changes in its activity can identify activated T cells and assess the immune response. Molecular imaging technology provides important technical support for real-time, noninvasive, and in vivo visualization of T cell activation. However, commonly used noninvasive medical imaging techniques in clinical practice, such as computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET), suffer from low sensitivity, high background signal, and the inability to achieve multi-channel imaging, making it difficult to dynamically visualize the T cell activation process in real time. Therefore, there is an urgent need to develop new strategies for dynamic visualization imaging of T cell activation with high specificity and sensitivity to provide technical support for research related to tumor immunotherapy.

[0003] Fluorescence imaging technology offers advantages such as fast imaging speed, high temporal and spatial resolution, high sensitivity, and real-time dynamic monitoring. It is an important means for obtaining and monitoring information on the distribution and function of immune cells in real time. Currently, visible or near-infrared (NIR-I) fluorescent probes are primarily used to image immune cell surface markers or effector molecules to assess T cell status changes within the tumor microenvironment. However, visible / NIR-I fluorescence imaging often suffers from limitations such as shallow tissue penetration, strong autofluorescence, and high background noise. Near-infrared (NIR-II) fluorescence imaging, 1000-1700 nm, can effectively reduce background fluorescence signals, increase tissue penetration, and enhance imaging contrast, making it more suitable for real-time visualization of T cell dynamics within the living tumor microenvironment. However, imaging techniques that rely on single fluorescence intensity changes are often subject to interference from factors such as uneven probe distribution, nonspecific enrichment, and microenvironmental variations (pH, polarity, temperature, etc.), making it difficult to quantitatively measure the concentration of target molecules and accurately obtain quantitative information on the target.

[0004] Therefore, in order to achieve real-time monitoring of T cells and their status in the tumor microenvironment at the in vivo level, it is urgent to develop a new type of fluorescent probe. Summary of the Invention

[0005] The purpose of the present invention is to provide a novel granzyme B-responsive rare earth NIR-II ratiometric fluorescent nanoprobe (DCGA) to dynamically monitor the activation state of T cells by responding to granzyme B in tumor immunotherapy, to predict the immune response in real time and non-invasively, and to provide decision support for personalized immunotherapy.

[0006] To achieve the above object, the present invention adopts the following technical solutions: A granzyme B-responsive near-infrared second-region ratiometric fluorescent probe, comprising the following components: (a) A rare earth doped core-shell near-infrared second-zone luminescent nanoparticle β-NaErF4@NaYF4@NaYF4:x%Nd@NaYF4 nanoparticle, wherein the core of the luminescent nanoparticle is doped with the rare earth element Er 3+ β-NaErF4, the shell layer includes a buffer layer NaYF4, a light-emitting layer NaYF4:x%Nd and a passivation layer NaYF4, wherein 5≤x≤50, x is the doping molar mass ratio of Nd; (b) Amphiphilic lipids that cause the phase transition of luminescent nanoparticles include DSPE-PEG2000-Mal and DSPE-PEG2000, with a mass ratio of 1:2. (c) an active peptide specifically cleaved by granzyme B protease, wherein the active peptide is Tyr(SO3) AIEFDSGC; (d) Near-infrared light absorber A1094.

[0007] Furthermore, the synthesis method of the β-NaErF4@NaYF4@NaYF4:x%Nd@NaYF4 nanoparticles comprises the following steps: (1) ErCl3, oleic acid and octadecene were mixed in a molar ratio of 1:(10~100):(10~100), heated to 100℃~200℃ under argon atmosphere, and after returning to room temperature, NH4F and NaOH were mixed in a molar ratio of 1:(0.2~2) and added to the reaction system. The mixture was stirred for 10~100 minutes, and then the solution was heated to 200℃~400℃ for 20~200 minutes. The mixture was centrifuged to obtain β-NaErF4. (2) β-NaErF4, oleic acid and octadecene were mixed in a molar ratio of 1:(10~100):(10~100), heated to 200℃~400℃ under argon protection, and Y-OA precursor and Na-TFA-OA precursor were alternately added to the reaction system in a molar ratio of 1:(0.2~2). The mixture was stirred for 20~200 minutes and centrifuged to obtain β-NaErF4@NaYF4. (3) β-NaErF4@NaYF4, oleic acid and octadecene were mixed in a molar ratio of 1:(10~100):(10~100), heated to 200℃~400℃ under argon protection, and Y:x%Nd-OA precursor was evenly added to the reaction system and stirred for 20~200 minutes to obtain β-NaErF4@NaYF4@NaYF4:10%Nd; (4) β-NaErF4@NaYF4@NaYF4:x%Nd was mixed with oleic acid and octadecene, and heated to 200℃~400℃ under argon protection. Y-OA precursor and Na-TFA-OA precursor were alternately added to the reaction system at a molar ratio of 1:(0.2~2). The mixture was stirred for 20~200 minutes and centrifuged to obtain β-NaErF4@NaYF4@NaYF4:x%Nd@NaYF4 nanoparticles. The ratio of Y-OA precursor and Na-TFA-OA precursor to β-NaErF4@NaYF4@NaYF4:x%Nd was 1:(0.5~5):(0.2~2).

[0008] Furthermore, the Y-OA precursor in step (2) is prepared by reacting 2.5 mmol of Y(CH3COO)3·4H2O, 10 mL of oleic acid and 15 mL of octadecene at 140 °C under argon protection; the Na-TFA-OA precursor is prepared by reacting 4 mmol of Na-TFA and 10 mL of oleic acid at 70 °C under reduced pressure; the alternation time is 10 min, wherein the amount ratio of the Y-OA precursor and the Na-TFA-OA precursor to β-NaErF4 is 1:(0.5~5):(0.1~1).

[0009] Furthermore, in step (3), the Y:x%Nd-OA precursor is prepared by reacting 0.25 mmol of Nd(CH3COO)3, 2.25 mmol of YCl3, 10 mL of oleic acid and 15 mL of octadecene at 140 °C under argon protection; the dosage ratio of the Y:x%Nd-OA precursor to β-NaErF4@NaYF4 is 1:(0.5~5), where 5≤x≤50.

[0010] The diameter of the above-mentioned β-NaErF4@NaYF4@NaYF4:x%Nd@NaYF4 nanoparticles ranges from 20 to 400 nm, with an excitation wavelength of 808 nm and emission wavelengths of 1060 nm and 1525 nm.

[0011] The method for synthesizing the granzyme B-responsive near-infrared region II ratiometric fluorescent probe comprises the following steps: (1) β-NaErF4@NaYF4@NaYF4:x%Nd@NaYF4 nanoparticles and amphiphilic lipids were dissolved in 4-40 mL of chloroform at a mass ratio of 1:1~10, and DCNPs@PEG were obtained by self-assembly. The lipids are amphiphilic, and lipid-coated NIR-II luminescent nanoparticles DCNPs@PEG were obtained by self-assembly, which enabled them to be efficiently dispersed and stabilized in aqueous solution.

[0012] (2) The active peptide and DCNPs@PEG were dissolved in 4-40 mL of deionized water at a mass ratio of 1:30-300, stirred for 0.5-5 hours, and centrifuged to obtain DCNPs@GzmB; the active peptide had a thiol group at the C-terminus, and was covalently reacted with Mal through Michael addition reaction to obtain DCNPs@GzmB, which could specifically respond to granzyme B.

[0013] (3) A1094 and DCNPs@GzmB were dissolved in deionized water at a mass ratio of 1:100~500, stirred for 0.5-5 hours, and centrifuged to obtain DCNPs@G-A1094 (DCGA). The maximum absorption wavelength of A1094 is 1094 nm, which is similar to that of Nd 3+ The emission signals at 1060 nm overlap well. A1094 binds to the sulfonate group (SO3 - ) to obtain the responsive NIR-II ratiometric fluorescent probe DCGA. 1060 nm The fluorescence signal is suppressed.

[0014] When the excitation wavelength of the probe is 808 nm, the fluorescence signal at 1060 nm is suppressed. The probe exhibits enhanced F 1060 nm Fluorescence signal, while F 1525 nm The fluorescence signal remained almost unchanged, and the activity of GzmB protease was similar to that of F 1060 nm / F 1525 nm The change in ratio is positively correlated.

[0015] The present invention also provides the above-mentioned granzyme B response near infrared second region ratio fluorescence probe for distinguishing CD8 + In the application of T cell activation, the probe is 1060 nm / F 1525 nm Ratio signal in differentiating CD8 + T cell activation status.

[0016] The present invention also provides the use of the aforementioned granzyme B-responsive near-infrared second-region ratiometric fluorescent probe in non-invasive early differentiation of immunotherapy responses and non-responses.

[0017] The present invention also provides the use of the aforementioned granzyme B-responsive near-infrared second-region ratiometric fluorescent probe in non-invasive in vivo imaging monitoring of early-stage tumor immunotherapy.

[0018] The present invention selects Er 3+ and Nd 3+ ions act as activators and are confined in the core and luminescent shell, respectively, to achieve dual emission in the NIR-II window under a single 808 nm excitation. 3+ / Nd 3+ Energy dissipation caused by energy migration between ions, in Er-containing 3+ Doped core and Nd 3+ A buffer NaYF4 interlayer with appropriate thickness (d1, 1-5nm) is introduced between the doped luminescent shell layers to avoid the 3+ and Nd 3+ In addition, a passivation NaYF4 coating with appropriate thickness (d2, 1-5 nm) is added to the outermost layer to minimize the Nd 3+ The emission is quenched by surface defects and solvents.

[0019] Preferably, the Nd 3+ 5≤x≤50, for example, 5%, 10%, 20%, 30%, 40% and 50%. When doped in the above specific ratio, as Nd 3+ With the increase of concentration, the luminescence intensity at 1060 nm increased slightly, which was attributed to the increase in the number of luminescence centers. 3+ The 1525 nm emission of the Er is almost unaffected, indicating that the Er is successfully suppressed by the NaYF4 buffer layer with a thickness of d1. 3+ / Nd 3+ However, when Nd 3+ As the doping concentration continues to increase, the 1060 nm emission intensity decreases significantly, which may be due to the higher concentration of Nd 3+ Doping induces the adjacent Nd 3+ Stronger cross-influences.

[0020] The beneficial effects of the present invention are mainly reflected in: (1) The DCGA probe developed in this paper combines the NIR-II dual-emission fluorescence characteristics of rare earth ions with a ratiometric signal response mode, significantly improving the sensitivity and signal stability of in vivo fluorescence imaging. It has deeper tissue penetration and higher imaging contrast in the NIR-II window, which can significantly improve the visualization of immunotherapy effects in deep tissues.

[0021] (2) The responsive DCGA probe in this invention can non-invasively and early differentiate between responders and non-responders to immunotherapy through its ratiometric fluorescence signal, which is faster and more efficient than traditional methods that rely on tumor volume changes. This feature makes it a powerful tool for evaluating the efficacy of treatment in the early stages and provides a scientific basis for optimizing personalized treatment strategies.

[0022] (3) The DCGA probe developed in this invention has a flexible design. It can integrate functional modules (responsive peptides or recognition molecules) that respond to multiple immune factors and functional proteases through modular design, and can be combined with multimodal imaging technology to support multi-target, dynamic molecular imaging. In addition, it can be further applied to patient-derived tumor models (PDX) to verify its sensitivity and reliability in human-like tumor microenvironments, showing good clinical translation potential and applicable to various disease models and precision medicine scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram of the process for preparing rare earth NIR-II luminescent nanoparticles according to Example 1.

[0024] Figure 2 These are characterization images of the rare earth NIR-II luminescent nanoparticles prepared in Example 1, where (a) is a transmission electron microscope image, (b) is a dynamic light scattering image, (c) is a high-angle annular dark-field scanning transmission electron microscope, and (d) is an X-ray diffraction image.

[0025] Figure 3 This is a schematic diagram of the process for preparing the GzmB-responsive rare earth NIR-II ratiometric fluorescent probe according to Example 2.

[0026] Figure 4 Characterization diagrams of the GzmB-responsive rare earth NIR-II ratiometric fluorescent probe prepared in Example 2, where (a) is a transmission electron microscopy image, (b) is a dynamic light scattering image, (c) is a Zeta potential image, and (d) is a UV-visible-near-infrared absorption spectrum image.

[0027] Figure 5 The optical performance test diagram of the GzmB-responsive rare earth NIR-II ratiometric fluorescent probe prepared in Example 2, where (a) is the fluorescence emission spectrum of DCNPs@GzmB and the absorption spectrum of A1094, (b) is the fluorescence emission spectrum of DCGA after adding different concentrations of A1094 (0-80 μg / mL), and (c) is the fluorescence ratio of DCGA (F 1060 nm / F 1525 nm) versus A1094 concentration (0-80 μg / mL).

[0028] Figure 6These are graphs verifying the GzmB response performance of the GzmB-responsive rare earth NIR-II ratiometric fluorescent probe prepared in Example 2, where (a) is the NIR-II fluorescence and corresponding ratio image after incubation of DCGA and GzmB, (b) is the ratio calculated using ImageJ software for the image in Figure (a), and (c) is the fluorescence emission spectrum when DCGA and GzmB are co-incubated.

[0029] Figure 7 These are graphs verifying the responsiveness specificity of the GzmB-responsive rare earth NIR-II ratiometric fluorescent probe prepared in Example 2, where (a) is the NIR-II fluorescence and corresponding ratio graph after incubation of DCGA with different substrates, and (b) is the ratio graph calculated for the image in (a) using ImageJ software.

[0030] Figure 8 The results of in vitro detection of GzmB at the cellular level by probe DCGA in Application Example 1 are shown in Figure 1, where (a) shows the interaction of DCGA with Hepa1-6 cells and unactivated CD8 + T cells, activated CD8 + T cells, and activated CD8 T cells pre-treated with the GzmB inhibitor Ac-IEPD-CHO + NIR-II fluorescence and corresponding ratio images after T cell incubation. (b) is the ratio image calculated using ImageJ software for the image in (a).

[0031] Figure 9 Application Example 2: Probe DCGA for CD8 + Schematic diagram of NIR-II fluorescence imaging of T cell activation.

[0032] Figure 10 This is the NIR-II fluorescence imaging result of the probe DCGA for CD8+ T cell activation in vivo in Application Example 2, and the NIR-II fluorescence images and ratio diagrams of the tumor site under treatment with different therapeutic drugs.

[0033] Figure 11 Application Example 2: Probe DCGA for CD8 + The results of NIR-II fluorescence imaging of T cell activation, where (a) is the image of the T cell activated by using ImageJ software 24 hours after the injection of the probe. Figure 10 (a) The ratio graph calculated from the middle image, and (b) the correlation between the GzmB concentration of isolated tumors under different treatments and the ratio signal of DCGA in tumors was analyzed using a linear regression model).

[0034] Figure 12Schematic diagram of application example 3 of the probe DCGA for early prediction of in vivo anti-tumor immune response, where (a) is the treatment timeline and (b) is the chemical structure diagram of different immunomodulators.

[0035] Figure 13 This is the NIR-II fluorescence imaging result of the in vivo anti-tumor immune response using the DCGA probe in Application Example 3, and the in vivo NIR-II fluorescence images and ratio diagrams of tumor mice in different treatment groups.

[0036] Figure 14 The results of NIR-II fluorescence imaging of in vivo anti-tumor immune response by probe DCGA in Application Example 3 are shown in Figure 3, where (a) is Figure 13 The corresponding fluorescence ratio signal (F 1060nm / F 1525nm ) values, (b) are the images obtained using ImageJ software. Figure 10 Ratio plot calculated from the image.

[0037] Figure 15 The results of application example 3, DCGA, for early prediction of in vivo anti-tumor immune response, are shown in Figure 1, where (a) is the relative tumor volume of tumor-bearing mice on day 10 under different treatments, (b) is the relative tumor volume of tumor-bearing mice on day 22, and (c) is the correlation between the relative tumor volume on day 22 and the ratio of DCGA in the tumor on day 10.

[0038] Figure 16 This is a summary diagram of the manual, used to illustrate the synthesis and response principle of the probe. DETAILED DESCRIPTION

[0039] The present invention will be further described below with reference to the accompanying drawings and specific examples, which are intended to help those skilled in the art more fully understand and implement the present invention. It should be understood that the examples listed are only some embodiments of the present invention, not all embodiments. These specific examples are intended only to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0040] Example 1 Synthesis of rare earth NIR-II luminescent nanoparticles DCNP (β-NaErF4@NaYF4@NaYF4:10%Nd@NaYF4 S1: Y-OA precursor: Add 2.5 mmol of Y(CH3COO)3·4H2O, 10 mL of oleic acid (OA), and 15 mL of octadecene (ODE) into a flask, stir and mix, then heat to 140 °C and stir under argon for 1 hour to obtain a transparent Y-OA precursor solution (0.1 M).

[0041] S2: Y:10%Nd-OA precursor: Add 0.25 mmol of Nd(CH3COO)3, 2.25 mmol of YCl3, 10 mL of OA and 15 mL of ODE into a flask, stir and mix, then heat to 140 °C and stir under argon protection for 1 hour to obtain a transparent Y:10%Nd-OA precursor solution (0.1 M).

[0042] S3: Na-TFA-OA precursor: Add 4 mmol of sodium trifluoroacetate (Na-TFA) and 10 mL of OA into a flask, stir and mix, then heat to 70 °C and stir under reduced pressure for 30 min to obtain a transparent Na-TFA-OA precursor solution (0.4 M).

[0043] S4: β-NaErF4 core nanoparticles (Core): 1 mmol of ErCl3, 7.5 mL of OA, and 17.5 mL of ODE were added to a flask, heated to 130 °C under an argon atmosphere, and then cooled to room temperature. Next, 10 mL of methanol containing 4 mmol of NH4F and 2.5 mmol of NaOH was added to the reaction solution and stirred for 40 minutes. Subsequently, the mixture was heated to 70 °C and held for 1 hour. Finally, the solution was heated to 280 °C and held under a slow flow of argon for 90 minutes. The nanoparticles were isolated by centrifugation and dispersed in 10 mL of cyclohexane to obtain a β-NaErF4 core nanoparticle dispersion for subsequent use.

[0044] S5: β-NaErF4@NaYF4 Nanoparticles (Core-Shell): 5 mL of 0.5 mmol of β-NaErF4 core nanoparticle dispersion (S4), 8 mL of OA, and 12 mL of ODE were added to a three-necked flask. The flask was heated to 80 °C under reduced pressure for 30 minutes to remove cyclohexane. Subsequently, the flask was heated to 280 °C under an argon atmosphere, and 1 mL of a 0.1 M Y-OA precursor solution and 0.5 mL of a 0.4 M Na-TFA-OA precursor solution were alternately injected into the reaction system at 10-minute intervals. After 16 injections, the reaction solution was held at 280 °C for 90 minutes. The final core-shell nanocrystals were separated by centrifugation and dispersed in 5 mL of cyclohexane to obtain a β-NaErF4@NaYF4 nanoparticle dispersion for subsequent use.

[0045] S6: β-NaErF4@NaYF4@NaYF4:10%Nd nanoparticles (Core-shell-shell): Add 5 mL of 0.5 mmol of β-NaErF4@NaYF4 nanoparticle dispersion (S5), 8 mL of OA, and 12 mL of ODE to a three-necked flask. Repeat the procedure of step S5, replacing the Y-OA precursor solution (0.1 M) with a Y:10%Nd-OA precursor solution (0.1 M), and perform two injection cycles.

[0046] S7: β-NaErF4@NaYF4@NaYF4:10%Nd@NaYF4 nanoparticles (DCNPs): 5 mL of 0.5 mmol of β-NaErF4@NaYF4@NaYF4:10%Nd nanoparticle dispersion (S6), 8 mL of OA and 12 mL of ODE were added to a three-necked flask and the above method was repeated. 1 mL of Y-OA precursor solution (0.1 M) and 0.5 mL of Na-TFA-OA precursor solution (0.4 M) were alternately injected into the reaction system at intervals of 10 minutes, and a total of 8 cycles of injection were performed. The final core-shell-shell-shell nanocrystals were separated by centrifugation and dispersed in 10 mL of cyclohexane for subsequent use. The preparation process of DCNPs is as follows: Figure 1 shown.

[0047] In order to verify the structural characteristics of the GzmB responsive rare earth NIR-II ratiometric fluorescent probe, the DCNPs prepared in Example 1 were characterized by transmission electron microscopy (TEM), dynamic light scattering, X-ray diffractometer, and high-angle annular dark-field scanning transmission electron microscopy. Figure 2 As shown, the successful synthesis of core-shell structured DCNPs was demonstrated.

[0048] Example 2 Synthesis of GzmB-responsive rare earth NIR-II ratiometric fluorescent probe DCGA S1: Maleimide-functionalized DCNPs (DCNPs@PEG): DCNPs (20 mg), DSPE-mPEG2000 (40 mg), and DSPE-mPEG2000-Mal (20 mg) were dissolved in 10 mL of chloroform. Chloroform was then slowly evaporated in a fume hood to yield lipid-modified DCNPs (DCNPs@PEG). The mixture was then centrifuged at 15,000 rpm and washed with deionized water to obtain the product, which was stored at 4°C until further use.

[0049] S2: Conjugation of GzmB substrate peptide to DCNPs@PEG (DCNPs@GzmB): GzmB substrate peptide (Tyr(SO3)AIEFDSGC, 2 mg) and DCNPs@PEG (40 mg) were dispersed in 10 mL of deionized water and stirred at 500 rpm for 2 hours. The mixture was then centrifuged at 15,000 rpm and washed with deionized water to obtain the product, which was stored at 4 °C until further use.

[0050] S3: A1094 molecules are combined with DCNPs@GzmB (DCNPs@G-A1094, i.e., DCGA): 40 μL of dye A1094 (2 mg / mL, dissolved in DMF, CAS: 3031640-11-3) was added to 1 mL of the prepared DCNPs@GzmB (4 mg / mL) aqueous solution and stirred at room temperature for 1 hour. The mixture was then centrifuged at 15,000 rpm and washed with deionized water to obtain the GzmB-responsive rare earth NIR-II ratiometric fluorescent probe DCGA. The preparation process of DCGA is as follows: Figure 3 shown.

[0051] The DCGA probe prepared in Example 2 was characterized by TEM. Figure 4 As shown in a, the DCGA probe is well dispersed, with an obvious PEG hydration layer on the surface. The particle size is about 80×50 nm. The particle size and Zeta potential of the DCGA prepared in Example 2 were characterized using a Zeta potential analyzer. The results are shown in Figure 4 As shown in b and c, the hydrodynamic diameter of the nanoparticles changed from 68 nm to 91 nm after surface modification. DCNPs@PEG, DCNPs@GzmB, and DCGA all exhibited negative surface charges and good dispersion in aqueous solutions, indicating their good potential for in vivo biological applications. The UV-Vis-NIR absorption spectroscopy test results are shown in Figure 4 As shown in Figure d, DCGA has obvious absorption peaks at 216 nm and 1094 nm, indicating that the peptide and A1094 were successfully modified.

[0052] The optical performance of the DCGA probe prepared in Example 2 was tested using a fluorescence spectrometer. Figure 5 As shown in a, since the absorption of A1094 at 1094 nm significantly overlaps with the emission of DCNPs@GzmB at 1060 nm, A1094 can quench the Nd in DCNPs through the FRET effect. 3+ The fluorescence spectrum results are as follows: Figure 5As shown in Figures b and c, the fluorescence emission intensity of the DCGA probe at 1060 nm decreased exponentially with the increase of A1094 concentration. Compared with the original DCNPs@GzmB, the fluorescence intensity of DCGA at 1060 nm was quenched by approximately 85.3% when the A1094 concentration was 80 μg / mL.

[0053] To further verify the GzmB response to rare earth NIR-II ratiometric fluorescence probe to detect GzmB activity, the DCGA prepared in Example 2 was tested for NIR-II fluorescence imaging and spectral changes in the presence / absence of GzmB to explore its responsiveness to GzmB. Figure 6 As shown in a and 6b, after DCGA (1 mg / mL) was incubated with GzmB (0.2 μM) in PBS buffer (pH 7.4) at 37°C for 2 h, DCGA showed a strong affinity for GzmB at F under 808 nm excitation. 1060 nm The photoluminescence signal of the NIR-II fluorescence was significantly enhanced, while the fluorescence at 1525 nm remained stable, as defined by the NIR-II fluorescence ratio (F 1060 nm / F 1525 nm ) increased by 5.1 times. This response phenomenon was further verified by fluorescence spectroscopy. Figure 6 c, where Er 3+ The fluorescence intensity at 1525 nm remained almost unchanged, while Nd 3+ The fluorescence intensity at 1060 nm increased significantly, indicating that A1094-Tyr(SO3)AIEFDSGC (IEFD is the recognition sequence) was hydrolyzed by GzmB, releasing A1094 and reducing the fluorescence energy transfer effect.

[0054] To further investigate the specificity of DCGA response to GzmB, the probe (1 mg / mL) was incubated with GzmB (0.2 μM) or other possible interfering substances (including hydrogen peroxide (H2O2), alkaline phosphatase (ALP), γ-glutamyltransferase (GGT), tyrosinase (TYR), cathepsin C (CTSC), and neutrophil elastase (NE)) in PBS buffer (pH 7.4) at 37°C for 2 h. Figure 7 As shown, only in the presence of GzmB, the F 1060 nm The fluorescence intensity and the corresponding ratiometric signal increased significantly. The results showed that DCGA was highly specific for GzmB under physiological conditions and exhibited excellent specific response ability compared with other biomolecules, demonstrating its potential for in vivo specific detection of GzmB.

[0055] Application Example 1: In vitro detection ability of DCGA for GzmB at the cellular level To investigate the selective response of DCGA to intracellular GzmB, we used GzmB-positive activated CD8 + T cells, GzmB-negative Hepa1-6 cells, and unactivated CD8 + The probe (0.5 mg / mL) was incubated with cells at 37°C for 4 h, and the treated cells were collected by centrifugation. The fluorescence signal changes were monitored using the NIR-II fluorescence imaging system. Figure 8 As shown, compared with unactivated CD8 + Compared with the T cell and Hepa 1-6 cell groups, activated CD8 + F of DCGA in T cell group 1060 nm The fluorescence intensity and ratio signal were significantly enhanced, indicating that the probe restored the fluorescence signal under the specific activation of GzmB. + After T cells were pretreated with GzmB inhibitor (Ac-IEPD-CHO), the fluorescence signal change was completely inhibited, further verifying the key role of GzmB in the restoration of DCGA fluorescence signal. These results indicate that DCGA can activate CD8 + T cells efficiently respond to GzmB, thereby enabling sensitive detection of T cell activation.

[0056] Application Example 2 DCGA for CD8 + NIR-II fluorescence imaging of T cell activation To demonstrate that DCGA can image the activation state of CTL (cytotoxic T cells) developed for the tumor immune microenvironment in vivo, the present invention selected Hepa1-6 as a cell model and tested the ability of DCGA to specifically image GzmB protease released by CTL cells in a Hepa 1-6 tumor-bearing mouse model.

[0057] First, we evaluated the feasibility of DCGA for in vivo imaging of GzmB by directly activating T cells in tumors. The experiment was divided into G1, G2, and G3 groups. Group G1 was treated with PBS (control), group G2 was treated with a GzmB inducer (intratumoral injection of phorbol myristate acetate (PMA, CAS: 16561-29-8) and ionomycin (CAS: 56092-82-0)), and group G3 was treated with a GzmB inhibitor (Ac-IEPD-CHO combined with PMA and ionomycin). The detailed treatment steps are shown in [ 1 ]. Figure 9At the beginning of the experiment, tumor-bearing mice were injected with DCGA (100 μL, 4 mg / mL, in saline) via the tail vein, and the first NIR-II fluorescence imaging was performed 12 hours after injection to observe the initial state of T cells. Subsequently, the mice were treated with a GzmB inducer (50 μL of saline containing 100 ng PMA and 5 μg ionomycin for intratumoral injection), a GzmB inhibitor (10 μg Ac-IEPD-CHO combined with PMA and ionomycin), or PBS (control). NIR-II fluorescence imaging was performed 24 hours after DCGA injection, and the signal at the tumor site was quantitatively analyzed.

[0058] Imaging results such as Figure 10 and Figure 11 As shown in Figure 2, DCGA gradually accumulated in the tumor site 12 hours after injection. 1060 nm and F 1525 nm Signal intensity varies between individual mice, but the ratio signal (F 1060 nm / F 1525 nm ) were almost identical, indicating no significant difference in GzmB protease activity within the tumor. In contrast, in the second imaging 24 hours after injection, the GzmB inducer-treated group showed a significant difference in F 1060 nm The fluorescence recovery in the 447-well channel was stronger, and the ratiometric imaging signal was enhanced, indicating that the GzmB protease activity was higher. The fluorescence ratio (F 1060 nm / F 1525 nm ) were 1.58-fold and 1.69-fold higher than those in the GzmB inhibitor group and PBS-treated group, respectively, indicating that T cells were activated and secreted GzmB. In addition, linear regression analysis showed a positive correlation between the ratio signal of DCGA and the concentration of GzmB in Hepa 1-6 tumors (R 2 = 0.8417, r = 0.9175). Therefore, DCGA can be used for NIR-II ratiometric fluorescence in vivo imaging of T cell activation status.

[0059] Application Example 3: DCGA for early prediction of anti-tumor immune response in vivo To activate the immune response, three immunotherapy drugs were selected to treat tumor-bearing mice: NLG919 (CAS: 1402836-58-1), BMS-1 ​​(CAS: 1675201-83-8), and S-(2-boronoethyl)-L-cysteine ​​hydrochloride (BEC, CAS: 63107-40-4). Each drug was dissolved according to the instructions and then diluted to normal saline. The treatment and imaging diagram is shown below. Figure 12As shown, the G1 group was given PBS; the G2 group was given NLG919 (15 mg / kg); the G3 group was given BMS-1 ​​(15 mg / kg); and the G4 group was given BEC (15 mg / kg). Dosing was performed every three days. After completing three rounds of immunotherapy, each mouse was intravenously injected with DCGA (100 μL, 4 mg / mL, in saline). NIR-II fluorescence imaging and quantitative analysis were performed at the planned time points.

[0060] Imaging results such as Figure 13 and Figure 14 As shown, compared with the control group (G1: PBS), the tumor sites in the immunotherapy groups (G2-G4) were located in the F 1060 nm There is stronger fluorescence recovery in the channel. Due to the difference in probe enrichment and clearance efficiency, the single channel (F 1060 nm or F 1525 nm Fluorescence signals can vary significantly during in vivo imaging, affecting the accuracy of monitoring treatment progress. Therefore, self-calibrated ratiometric signal analysis offers significant advantages. In the immunotherapy groups, the fluorescence ratios in the NLG919, BMS-1, and BEC treatment groups were 1.2-fold, 1.41-fold, and 1.89-fold higher than those in the control group, respectively.

[0061] Subsequently, the therapeutic effects of different immunomodulators on Hepa 1-6 tumor-bearing mice were evaluated by measuring tumor volume growth. Figure 15 As shown in Figure 13, compared with the control group, despite the differences in ratio signals, the relative tumor volumes of the treatment groups did not show significant differences on day 10 after treatment (13a), while significant tumor suppression changes were observed on day 22 (13b). Therefore, DCGA can achieve early non-invasive in vivo monitoring of immune responses, and the time of detecting changes in immunotherapy effects is earlier than the time when tumor volume changes show statistical differences. To verify whether the tumor fluorescence ratio on day 10 can predict the final treatment outcome, the signal ratio value (F 1060 nm / F 1525 nm Correlation analysis was performed between the tumor signal ratio (DFR) and relative tumor volume (day 22). The results showed a negative correlation between the tumor signal ratio and relative tumor volume (R² = 0.8535, r = -0.9239). Therefore, in vivo fluorescence ratio imaging using DCGA provides an early, non-invasive, and accurate method for predicting treatment efficacy.

[0062] It should be noted that although the above examples have been described herein, this does not limit the scope of patent protection of the present invention. Therefore, based on the innovative concept of the present invention, changes and modifications made to the embodiments herein, or equivalent structural or equivalent process transformations made using the contents of the present invention specification, and direct or indirect application of the above technical solutions to other related technical fields are all included in the scope of patent protection of the present invention.

Claims

1. A granzyme B-responsive near-infrared second-region ratiometric fluorescent probe, characterized by: The fluorescent probe consists of the following components: (a) A rare earth doped core-shell near-infrared second-zone luminescent nanoparticle β-NaErF4@NaYF4@NaYF4:x%Nd@NaYF4 nanoparticle, wherein the core of the luminescent nanoparticle is doped with the rare earth element Er 3+ β-NaErF4, the shell layer includes a buffer layer NaYF4, a light-emitting layer NaYF4:x%Nd and a passivation layer NaYF4, wherein 5≤x≤50, x is the molar mass ratio of Nd doping; (b) Amphiphilic lipids that cause the phase transition of luminescent nanoparticles include DSPE-PEG2000-Mal and DSPE-PEG2000, with a mass ratio of 1:2; (c) an active peptide specifically cleaved by granzyme B protease, wherein the active peptide is Tyr(SO3) AIEFDSGC; (d) Near-infrared light absorber A1094.

2. The granzyme B-responsive near-infrared second-region ratiometric fluorescent probe according to claim 1, characterized in that: The synthesis method of the β-NaErF4@NaYF4@NaYF4:x%Nd@NaYF4 nanoparticles comprises the following steps: (1) ErCl3, oleic acid and octadecene were mixed in a molar ratio of 1:(10~100):(10~100), heated to 100℃~200℃ under argon atmosphere, and after returning to room temperature, NH4F and NaOH were mixed in a molar ratio of 1:(0.2~2) and added to the reaction system. The mixture was stirred for 10~100 minutes, and then the solution was heated to 200℃~400℃ for 20~200 minutes. The mixture was centrifuged to obtain β-NaErF4. (2) β-NaErF4, oleic acid and octadecene were mixed in a molar ratio of 1:(10~100):(10~100), heated to 200℃~400℃ under argon protection, and Y-OA precursor and Na-TFA-OA precursor were alternately added to the reaction system in a molar ratio of 1:(0.5~5). The mixture was stirred for 20~200 minutes and centrifuged to obtain β-NaErF4@NaYF4. (3) β-NaErF4@NaYF4, oleic acid and octadecene were mixed in a molar ratio of 1:(10~100):(10~100), heated to 200℃~400℃ under argon protection, and Y:x%Nd-OA precursor was evenly added to the reaction system and stirred for 20~200 minutes to obtain β-NaErF4@NaYF4@NaYF4:10%Nd; (4) β-NaErF4@NaYF4@NaYF4:x%Nd was mixed with oleic acid and octadecene, and heated to 200 ℃~400 ℃ under argon protection. Y-OA precursor and Na-TFA-OA precursor were alternately added to the reaction system at a molar ratio of 1:(0.5~5). The mixture was stirred for 20~200 minutes and centrifuged to obtain β-NaErF4@NaYF4@NaYF4:x%Nd@NaYF4 nanoparticles. The molar ratio of Y-OA precursor and Na-TFA-OA precursor to β-NaErF4@NaYF4@NaYF4:x%Nd was 1:(0.5~5):(0.2~2).

3. The granzyme B-responsive near-infrared second-region ratiometric fluorescent probe according to claim 2, characterized in that: The Y-OA precursor in step (2) is prepared by reacting 2.5 mmol of Y(CH3COO)3·4H2O, 10 mL of oleic acid and 15 mL of octadecene at 140°C under argon protection; the Na-TFA-OA precursor is prepared by reacting 4 mmol of Na-TFA and 10 mL of oleic acid at 70°C under reduced pressure; the alternation time is 10 min, and the molar ratio of the Y-OA precursor and the Na-TFA-OA precursor to β-NaErF4 is 1:(0.5~5):(0.1~1).

4. The granzyme B-responsive near-infrared second-region ratiometric fluorescent probe according to claim 2, characterized in that: In step (3), the Y:x%Nd-OA precursor is prepared by reacting 0.25 mmol of Nd(CH3COO)3, 2.25 mmol of YCl3, 10 mL of oleic acid and 15 mL of octadecene at 140 °C under argon protection; the molar ratio of the Y:x%Nd-OA precursor to β-NaErF4@NaYF4 is 1:(0.5~5), where 5≤x≤50.

5. The granzyme B-responsive near-infrared second-region ratiometric fluorescent probe according to claim 1, characterized in that: The synthetic method of the fluorescent probe comprises the following steps: (1) β-NaErF4@NaYF4@NaYF4:x%Nd@NaYF4 nanoparticles and amphiphilic lipids were dissolved in 4-40 mL of chloroform at a mass ratio of 1:1-10, and DCNPs@PEG were obtained by self-assembly. (2) Dissolve the active peptide and DCNPs@PEG in 4-40 mL of deionized water at a mass ratio of 1:30-300, stir for 0.5-5 hours, and centrifuge to obtain DCNPs@GzmB; (3) A1094 and DCNPs@GzmB were dissolved in deionized water at a mass ratio of 1:100-500, stirred for 0.5-5 hours, and centrifuged to obtain DCNPs@G-A1094.

6. The granzyme B-responsive near-infrared second-region ratiometric fluorescent probe according to claim 1, characterized in that: The diameter of the β-NaErF4@NaYF4@NaYF4:x%Nd@NaYF4 nanoparticles ranges from 20 to 400 nm, the excitation wavelength is 808 nm, and the emission wavelengths are 1060 nm and 1525 nm.

7. The granzyme B-responsive near-infrared second-region ratiometric fluorescent probe according to claim 1, characterized in that: When the excitation wavelength of the probe is 808 nm, the fluorescence signal at 1060 nm is suppressed. After interacting with GzmB, the probe exhibits an enhanced F1060 nm fluorescence signal, while the F1525 nm fluorescence signal remains almost unchanged. The activity of the GzmB protease is positively correlated with the change in the F1060 nm / F1525 nm ratio.

8. A granzyme B-responsive near-infrared second-region ratiometric fluorescent probe according to any one of claims 1 to 7 for differentiating CD8 + Application in T cell activation, characterized by: The probe differentiates CD8 + T cell activation status.

9. Use of the granzyme B-responsive near-infrared second-region ratiometric fluorescent probe according to any one of claims 1 to 7 in non-invasive early differentiation of immunotherapy responders and non-responders.

10. Use of the granzyme B-responsive near-infrared second-region ratiometric fluorescent probe according to any one of claims 1 to 7 in non-invasive in vivo imaging monitoring of early-stage tumor immunotherapy.

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