Granzyme b specific response nanoprobes, preparation method and application thereof

Abstract

The application belongs to the technical field of biological medical materials, and particularly relates to a granzyme B specific response nano probe and a preparation method and application thereof. The granzyme B specific response nano probe comprises a near-infrared down-conversion nano probe, and a pentapeptide IEFDK-fluorescence quenching modification group is modified on the surface of the near-infrared down-conversion nano probe; the pentapeptide IEFDK-fluorescence quenching modification group is derived from a pentapeptide IEFDK-fluorescence quencher with a structure shown in formula (1). The granzyme B specific response nano probe provided by the application can specifically respond to granzyme B, realize immune effect monitoring of a tumor site, and thus realize detection of tumor immune responses after different radiotherapy schemes are processed. Formula (1).

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biomedical materials, and particularly relates to a granzyme B specific response nano probe and a preparation method and application thereof. BACKGROUND

[0002] Radiotherapy (RT) is a method of using high-energy ionizing radiation to kill cancer cells to treat tumors, which is one of the three major means of tumor treatment together with surgery and chemotherapy. In addition to having a direct killing effect on tumor cells, radiotherapy also has potential immunomodulatory properties. Through acute damage to DNA by radiation, it directly induces tumor immunogenic cell death, triggers the body's anti-tumor immune response, produces an effect similar to an "in situ vaccine", and can also act on tumors by inducing the production of various cytokines to regulate the immune microenvironment. However, the effect of radiotherapy on the tumor microenvironment is bidirectional, and depending on the dose, fractionation method and tumor location, it can have different immunomodulatory effects. Therefore, if the activated immune effect of different radiotherapy doses can be monitored, it is expected to guide the selection of clinical radiotherapy regimens.

[0003] Currently, there are few objective and direct methods to monitor the activated immune effect of radiotherapy. At present, the downstream biomarkers of radiotherapy, such as reactive oxygen species ROS, apoptosis protein caspase-3 and DNA damage-related protein gammaH2AX, are detected by imaging methods including computed tomography (CT), magnetic resonance imaging (MRI) and positron emission tomography (PET) to monitor the effect of radiotherapy under different doses and fractionation methods. However, first of all, these markers are non-specific to biological immune response and cannot accurately and effectively evaluate the effect of radiotherapy on tumor immunity. Secondly, imaging methods have a certain radiation, the imaging method is relatively cumbersome, the detection time is long and the cost is high, so dynamic monitoring of the effect of radiotherapy cannot be achieved. SUMMARY

[0004] The first object of the present application is to provide a new granzyme B specific response nano probe which can specifically respond to granzyme B to achieve immune effect monitoring of tumor sites.

[0005] The second object of the present application is to provide a preparation method of the granzyme B specific response nano probe.

[0006] The third object of the present application is to provide the granzyme B specific response nano probe prepared by the above-mentioned method.

[0007] The fourth object of the present application is to provide the application of the above-mentioned granzyme B specific response nano probe in imaging of the activated immune effect of radiotherapy.

[0008] The granulase B specific response nano probe provided by the application comprises a near-infrared down-conversion nano probe, and the surface of the near-infrared down-conversion nano probe is modified with a pentapeptide IEFDK-fluorescence quenching modifier group; the near-infrared down-conversion nano probe is NaErF4@NaYF4 with a core-shell structure, the inner core component of the NaErF4@NaYF4 is NaErF4, and the outer shell component is NaYF4; the pentapeptide IEFDK-fluorescence quenching modifier group is derived from a pentapeptide IEFDK-fluorescence quencher with a structure shown in formula (1):

[0009] Formula (1).

[0010] The preparation method of the granulase B specific response nano probe provided by the application comprises the following steps:

[0011] S1. Coating a connection layer on the surface of the near-infrared down-conversion nano probe, wherein the connection layer has active groups capable of reacting with the pentapeptide IEFDK-fluorescence quencher with a structure shown in formula (1), to obtain a modified nano probe;

[0012] S2. Contacting the modified nano probe with the pentapeptide IEFDK-fluorescence quencher to make the pentapeptide IEFDK-fluorescence quencher react with the active groups on the connection layer of the modified nano probe, so as to be chemically connected to the surface of the connection layer of the modified nano probe;

[0013] Formula (1).

[0014] The key of the application lies in modifying the surface of the near-infrared down-conversion nano probe with a pentapeptide IEFDK-fluorescence quenching modifier group, wherein the near-infrared down-conversion nano probe has good near-infrared two-region light emitting performance, the pentapeptide IEFDK is isoleucine-glutamic acid-phenylalanine-aspartic acid-lysine (Ile-Glu-Phe-Asp-Lys), the fluorescence quencher is IR806 capable of specifically absorbing 808 nm laser, and the introduction of the pentapeptide IEFDK-fluorescence quenching modifier group can endow the probe with good specific granulase B responsiveness, specifically responding to the presence of granulase B at the tumor site, so that the detection of the immune response of the tumor after different radiotherapy schemes can be realized. In addition, the granulase B specific response nano probe provided by the application has high stability, good dispersibility and uniform particle size distribution. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 It is a transmission electron microscope image of the down-conversion nano particle inner core (NaErF4) obtained in Example 1.

[0016] Figure 2 It is a transmission electron microscope image of the down-conversion nano particle (NaErF4@NaYF4) obtained in Example 1.

[0017] Figure 3 The near-infrared emission spectrum of the down-conversion nanoparticles (NaErF4@NaYF4) obtained in Example 1;

[0018] Figure 4 The in vivo imaging diagram of the breast cancer tumor model mouse corresponding to the granzyme B-specific responsive nanoprobe obtained in Example 1;

[0019] Figure 5 The emission spectrum of the granzyme B-specific responsive nanoprobe obtained in Example 1 in the in vitro granzyme B-responsive imaging;

[0020] Figure 6 The near-infrared imaging diagram of the granzyme B-specific responsive nanoprobe obtained in Example 1 in the in vitro granzyme B-responsive imaging. DETAILED DESCRIPTION

[0021] The granzyme B-specific responsive nanoprobe provided by the application comprises a near-infrared down-conversion nanoprobe, and the surface of the near-infrared down-conversion nanoprobe is modified with a pentapeptide IEFDK-fluorescence quenching modification group; the pentapeptide IEFDK-fluorescence quenching modification group is derived from a pentapeptide IEFDK-fluorescence quencher with the structure shown in formula (1):

[0022] Formula (1).

[0023] In the application, the near-infrared down-conversion nanoprobe is a rare earth luminescent material NaErF4@NaYF4 with a core-shell structure, the inner core component of the NaErF4@NaYF4 is NaErF4, and the shell component is NaYF4. Among them, the molar ratio of the yttrium element to the yttrium element in the near-infrared down-conversion nanoprobe is preferably 1:(0.8-1.2), such as 1:0.8, 1:0.82, 1:0.85, 1:0.88, 1:0.9, 1:0.92, 1:0.95, 1:0.98, 1:1, 1:1.12, 1:1.15, 1:1.18, 1:1.2 or any value between them.

[0024] In the application, the near-infrared down-conversion nanoprobe has a spherical structure. In addition, the particle size D50 of the near-infrared down-conversion nanoprobe is preferably 10-50 nm, and can be specifically 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm or any value between them.

[0025] In the present application, the granzyme B-specific response nanoprobes preferably further comprise a connecting layer coated on the surface of the near-infrared down-conversion nanoprobes, and the pentapeptide IEFDK-fluorescence quenching modification group is connected to the active group of the connecting layer. The active group of the connecting layer can be at least one of -NHS, -COOH, -NH2, etc. These active groups can react with the amino or carboxyl group on the pentapeptide IEFDK-fluorescence quencher to enable the pentapeptide IEFDK-fluorescence quencher to be connected to the connecting layer. Particularly preferably, the connecting layer is a DSPE-PEG-NHS layer. In addition, the mass ratio of the connecting layer to the molar amount of erbium in the near-infrared down-conversion nanoprobes is preferably (200-500) mg:1 mol, such as 200 mg:1 mol, 220 mg:1 mol, 240 mg:1 mol, 260 mg:1 mol, 280 mg:1 mol, 300 mg:1 mol, 320 mg:1 mol, 340 mg:1 mol, 360 mg:1 mol, 380 mg:1 mol, 400 mg:1 mol, 420 mg:1 mol, 440 mg:1 mol, 460 mg:1 mol, 480 mg:1 mol, 500 mg:1 mol, or any value therebetween.

[0026] The present application provides a preparation method of the granzyme B-specific response nanoprobes, which comprises the following steps:

[0027] S1. Coating a connecting layer on the surface of the near-infrared down-conversion nanoprobes, wherein the connecting layer has an active group capable of reacting with the pentapeptide IEFDK-fluorescence quencher of formula (1), to obtain a modified nanoprobes;

[0028] S2. Contacting the modified nanoprobes with the pentapeptide IEFDK-fluorescence quencher to enable the pentapeptide IEFDK-fluorescence quencher to react with the active group on the connecting layer of the modified nanoprobes and thereby be chemically connected to the surface of the connecting layer of the modified nanoprobes;

[0029] Formula (1).

[0030] In the above preparation process of the granzyme B-specific response nanoprobes of the present application, the down-conversion nanomaterials are first converted to an aqueous phase by a DSPE ligand exchange method and modified with an NHS group, and then the pentapeptide fragment IEFDK that can be specifically cleaved by granzyme B is further connected by an amide reaction, and the fluorescence quencher IR806 is again combined at the end by an amide reaction, thereby synthesizing a new type of nanoprobes for specific granzyme B imaging and biological safety.

[0031] In the present application, the near-infrared down-conversion nanoprobe is a rare earth luminescent material NaErF4@NaYF4 with a core-shell structure, the inner core component of the NaErF4@NaYF4 is NaErF4 and the outer shell component is NaYF4. The NaErF4@NaYF4 with a core-shell structure can be obtained by commercial purchase or prepared according to various existing methods without special limitation.

[0032] In a preferred embodiment, the near-infrared down-conversion nanoprobe is prepared based on a high-temperature thermal decomposition reaction, specifically comprising the following steps:

[0033] S11. Dissolve erbium acetate, oleic acid and 1-octadecene under stirring under inert gas protection, stir the obtained mixture with an alcohol solution containing sodium source I and fluorine source I at 45-55°C for 10-60 min, then remove alcohol until no bubbles are generated in the reaction system, then perform high-temperature decomposition reaction at 250-350°C for 1-5 h, then perform solid-liquid separation, wash the obtained solid product and then disperse it in organic solvent I to obtain a solution containing NaErF4 inner core;

[0034] S12. Dissolve yttrium acetate, oleic acid and 1-octadecene under stirring under inert gas protection, stir the obtained mixture with an alcohol solution containing sodium source II and fluorine source II and the solution containing NaErF4 inner core at 45-55°C for 10-60 min, then remove alcohol until no bubbles are generated in the reaction system, then perform high-temperature decomposition reaction at 250-350°C for 1-5 h, then perform solid-liquid separation, wash the obtained solid product and then disperse it in organic solvent II to obtain a solution containing NaErF4@NaYF4.

[0035] In the preparation process of the near-infrared down-conversion nanoprobes, in step S11, the amount ratio of the erbium acetate, the oleic acid and the 1-octadecene is preferably 1 mmol:(5-10) mL:(10-20) mL. Specifically, the amount ratio of the erbium acetate to the oleic acid can be 1 mmol:5 mL, 1 mmol:6 mL, 1 mmol:7 mL, 1 mmol:8 mL, 1 mmol:9 mL, 1 mmol:10 mL or any value between them. The amount ratio of the erbium acetate to the 1-octadecene can be 1 mmol:10 mL, 1 mmol:12 mL, 1 mmol:14 mL, 1 mmol:16 mL, 1 mmol:18 mL, 1 mmol:20 mL or any value between them. The amount ratio of the sodium source I to the erbium acetate is preferably (2-4):1, such as 2:1, 2.2:1, 2.4:1, 2.6:1, 2.8:1, 3:1, 3.2:1, 3.4:1, 3.6:1, 3.8:1, 4:1 or any value between them. The amount ratio of the fluorine source I to the erbium acetate is preferably (3-5):1, such as 3:1, 3.2:1, 3.4:1, 3.6:1, 3.8:1, 4:1, 4.2:1, 4.4:1, 4.6:1, 4.8:1, 5:1 or any value between them.

[0036] In the preparation process of the near-infrared down-conversion nanoprobes, in step S12, the amount ratio of the yttrium acetate, the oleic acid and the 1-octadecene is preferably 1 mmol:(10-20) mL:(25-40) mL. Specifically, the amount ratio of the yttrium acetate to the oleic acid can be 1 mmol:10 mL, 1 mmol:12 mL, 1 mmol:14 mL, 1 mmol:16 mL, 1 mmol:18 mL, 1 mmol:20 mL or any value between them. The amount ratio of the yttrium acetate to the 1-octadecene can be 1 mmol:25 mL, 1 mmol:28 mL, 1 mmol:30 mL, 1 mmol:32 mL, 1 mmol:34 mL, 1 mmol:36 mL, 1 mmol:38 mL, 1 mmol:40 mL or any value between them. The amount ratio of the sodium source II to the yttrium acetate is preferably (2-4):1, such as 2:1, 2.2:1, 2.4:1, 2.6:1, 2.8:1, 3:1, 3.2:1, 3.4:1, 3.6:1, 3.8:1, 4:1 or any value between them. The amount ratio of the fluorine source II to the yttrium acetate is preferably (3-5):1, such as 3:1, 3.2:1, 3.4:1, 3.6:1, 3.8:1, 4:1, 4.2:1, 4.4:1, 4.6:1, 4.8:1, 5:1 or any value between them.

[0037] In the preparation process of the near-infrared down-conversion nanoprobes, the temperature of the stirring reaction in step S11 and step S12 is independently 45℃-55℃, such as 45℃, 48℃, 50℃, 52℃, 55℃ or any value between them; the time is independently 10min-60min, such as 10min, 20min, 30min, 40min, 50min, 60min or any value between them. The temperature of the high-temperature decomposition reaction in step S11 and step S12 is independently 250℃-350℃, such as 250℃, 260℃, 270℃, 280℃, 290℃, 300℃, 310℃, 320℃, 330℃, 340℃, 350℃ or any value between them; the time is independently 1h-5h, such as 1h, 2h, 3h, 4h, 5h or any value between them.

[0038] In the preparation process of the near-infrared down-conversion nanoprobes, the sodium source I and the sodium source II are each independently at least one of sodium hydroxide, sodium chloride and sodium carbonate. The fluorine source I and the fluorine source II are each independently at least one of ammonium fluoride, sodium fluoride and sodium hydrogen fluoride. The organic solvent I and the organic solvent II are each independently at least one of cyclohexane, methanol, chloroform and acetone. The terms "I" and "II" are only to distinguish the same kind of substances introduced at different positions for the convenience of description, and have no other special meanings.

[0039] In the present application, the method for coating a connecting layer on the surface of near-infrared down-conversion nanoprobes preferably comprises mixing the near-infrared down-conversion nanoprobes with DSPE-PEG-NHS in a volatile solvent under stirring, and drying after the solvent is substantially volatilized. The ratio of the mass of the DSPE-PEG-NHS to the molar amount of erbium in the near-infrared down-conversion nanoprobes is preferably (200-500) mg: 1 mol, such as 200 mg: 1 mol, 220 mg: 1 mol, 240 mg: 1 mol, 260 mg: 1 mol, 280 mg: 1 mol, 300 mg: 1 mol, 320 mg: 1 mol, 340 mg: 1 mol, 360 mg: 1 mol, 380 mg: 1 mol, 400 mg: 1 mol, 420 mg: 1 mol, 440 mg: 1 mol, 460 mg: 1 mol, 480 mg: 1 mol, 500 mg: 1 mol, or any value therebetween. The volatile solvent is preferably at least one selected from chloroform, cyclohexane, toluene, methanol, and acetone. In addition, the ratio of the use amount of the volatile solvent to the DSPE-PEG-NHS is preferably 1 mL: (4-6) mg, such as 1 mL: 4 mg, 1 mL: 4.2 mg, 1 mL: 4.4 mg, 1 mL: 4.6 mg, 1 mL: 4.8 mg, 1 mL: 5 mg, 1 mL: 5.2 mg, 1 mL: 5.4 mg, 1 mL: 5.6 mg, 1 mL: 5.8 mg, 1 mL: 6 mg, or any value therebetween.

[0040] In the present application, the pentapeptide IEFDK-fluorescence quencher can be obtained by commercial purchase or prepared according to various existing methods, and is not particularly limited. In a preferred embodiment, the pentapeptide IEFDK-fluorescence quencher is prepared according to the following method:

[0041] S21. IR-780 is stirred and reacted with 4-mercaptobenzoic acid under the protection of inert gas and in the presence of an organic solvent, and then the obtained reaction product is purified to obtain IR-806;

[0042] S22. The protecting group Fmoc in the Fmoc-Lys(Dde)-CTC resin is removed, the obtained H-Lys(Dde)-CTC resin is coupled with Fmoc-Asp(OtBu)-OH in the presence of benzotriazole-N,N,N,N-tetramethyluronium hexafluorophosphate / N,N-diisopropylethylamine (HBTU / DIEA), and then the protecting group Fmoc in the obtained Fmoc-Asp(OtBu)-Lys(Dde)-CTC is removed and extended by SPPS to obtain Boc-Ile-Glu(OtBu)-Phe-Asp(OtBu)-Lys(Dde)-CTC. Then Dde in the Boc-Ile-Glu(OtBu)-Phe-Asp(OtBu)-Lys(Dde)-CTC is removed, and then the Boc-Ile-Glu(OtBu)-Phe-Asp(OtBu)-Lys(Dde)-CTC is coupled with IR-806 in the presence of 1-hydroxybenzotriazole / diisopropyl carbodiimide (HOBT / DIC), and then cut by trifluoroacetic acid / triisopropylsilane / H2O (TFA / TIS / H2O) to obtain H-Ile-Glu-Phe-Asp-Lys(IR806)-OH.

[0043] In the preparation of the above five-peptide IEFDK-fluorescence quencher, the reaction process in step S21 is shown in reaction formula (1). The molar ratio of IR-780 to 4-mercaptobenzoic acid is preferably 1:(1.5-2.5), and can be 1:1.5, 1:1.8, 1:2, 1:2.2, 1:2.5 or any value therebetween. The organic solvent can be any existing liquid inert medium, such as at least one of N,N-dimethylformamide (DMF), toluene, methanol, acetone and cyclohexane. The purification method can include, for example, removing the solvent from the obtained reaction product, ultrasonically dissolving the residue in dichloromethane, filtering with a filter having a pore size of 0.2-0.6 μm, precipitating the obtained filtrate in ethyl ether, centrifuging, and then vacuum drying the obtained centrifuged product.

[0044]

[0045] Reaction formula (1)

[0046] In the preparation of the above-mentioned pentapeptide IEFDK-fluorescence quencher, in step S22, the method for removing the protecting group Fmoc can be treating with piperidine / DMF solution for 10 min to 60 min. The molar ratio of Fmoc-Lys(Dde)-CTC to Fmoc-Asp(OtBu)-OH is preferably 1: (2-4), such as 1:2, 1:2.2, 1:2.4, 1:2.6, 1:2.8, 1:3, 1:3.2, 1:3.4, 1:3.6, 1:3.8, 1:4, or any value between them. The molar ratio of IR-806 to Fmoc-Lys(Dde)-CTC is preferably (1.1-1.3):1, such as 1.1:1, 1.15:1, 1.2:1, 1.25:1, 1.3:1, or any value between them.

[0047] In the preparation of the above-mentioned granzyme B-specific response nanoprobes, in step S2, the conditions of the contact reaction preferably include a temperature of room temperature (0°C-45°C) and a time of 5 h-24 h. In addition, before the contact reaction, the modified nanoprobes are preferably activated with an NHS group in an organic solvent. The conditions of the NHS group activation preferably include a temperature of room temperature (0°C-45°C) and a time of 10 min-30 min.

[0048] The present application also provides granzyme B-specific response nanoprobes prepared by the above-mentioned method.

[0049] In addition, the present application also provides the use of the granzyme B-specific response nanoprobes in radiotherapy-activated immune effector imaging.

[0050] The present application will be described in detail below through examples.

[0051] Preparation Example 1 This preparation example is used to illustrate the preparation of the pentapeptide IEFDK-fluorescence quencher

[0052] S21. Preparation of fluorescence quencher IR-806

[0053] In a glass bottle, IR-780 (130 mg, 195 µmol), 4-mercaptobenzoic acid (60 mg, 380 µmol) were dissolved in 5 mL of anhydrous DMF, vacuumed first and then stirred at room temperature under nitrogen protection for 17 h, and then the DMF was removed under vacuum at 40°C. The residue was ultrasonically dissolved in 5 mL of CH2Cl2 and filtered through a 0.45 μm PTFE filter, and 50 mL of ether was slowly added to precipitate the product. The precipitate was collected by centrifugation, washed with ether, and vacuum dried to obtain dark red gold crystals, which were IR-806.

[0054] S22. Preparation of pentapeptide IEFDK-fluorescence quencher

[0055] First, starting with 0.2 mmol of Fmoc-Lys(Dde)-CTC resin, Fmoc protecting group was removed by treatment with 20% piperidine / DMF solution for 30 min to obtain H-Lys(Dde)-CTC resin. Then, the coupling reaction of Asp was carried out by HBTU and DIEA, using 0.6 mmol of Fmoc-Asp(OtBu)-OH, 0.6 mmol of HBTU and 1.0 mmol of DIEA, and the reaction was carried out for 1 h to obtain Fmoc-Asp(OtBu)-Lys(Dde)-CTC resin. Then, Fmoc protecting group was removed again by treatment with 20% piperidine / DMF solution for 30 min to obtain H-Asp(OtBu)-Lys(Dde)-CTC resin. Boc-Ile-Glu(OtBu)-Phe-Asp(OtBu)-Lys(Dde)-CTC resin was synthesized by solid phase peptide synthesis (SPPS) method. Dde protecting group was removed by treatment with 3% hydrazine hydrate / DMF solution for 30 min to obtain Boc-Ile-Glu(OtBu)-Phe-Asp(OtBu)-Lys-CTC resin. Then, the coupling reaction of IR-806 was carried out by DIC and HOBT, using 0.24 mmol of IR-806, 0.24 mmol of HOBT and 0.24 mmol of DIC, and the reaction was carried out overnight to obtain Boc-Ile-Glu(OtBu)-Phe-Asp(OtBu)-Lys(IR-806)-CTC. Finally, Boc-Ile-Glu(OtBu)-Phe-Asp(OtBu)-Lys(IR-806)-CTC was cleaved by treatment with a mixed solution of 95% TFA, 2.5% Tis and 2.5% H2O for 3 h to obtain the pentapeptide IEFDK-fluorescence quencher (H-Ile-Glu-Phe-Asp-Lys(IR-806)-OH).

[0056] The pentapeptide IEFDK-fluorescence quencher has the structure shown in formula (1) by nuclear magnetic and infrared detection.

[0057] Comparative Preparation Example 1

[0058] The pentapeptide IEFDK-fluorescence quencher was prepared according to the method of Preparation Example 1, except that in step S22, Boc-Thr-Glu(OtBu)-Phe-Asp(OtBu)-Lys(Dde)-CTC was synthesized in the SPPS process, and the other conditions were the same as those of Preparation Example 1 to obtain the reference pentapeptide IEFDK-fluorescence quencher (H-Thr-Glu-Phe-Asp-Lys(IR-806)-OH).

[0059] Comparative Preparation Example 2

[0060] The preparation of the pentapeptide IEFDK-fluorescence quencher was prepared according to the method of Preparation Example 1, except that step S21 was not included, and in step S22, IR-806 was replaced with the same molar amount of the fluorescence quencher 4-dimethylaminoazobenzene-4'-carboxylic acid (Dabcyl), and the remaining conditions were the same as in Preparation Example 1, to obtain the reference pentapeptide IEFDK-fluorescence quencher (H-Ile-Glu-Phe-Asp-Lys(Dabcyl)-OH).

[0061] Example 1 This example is used to illustrate the preparation of a granzyme B specific response to a nanoprobe

[0062] (1) Synthesis of down-conversion nanoparticle core

[0063] A clean 100 mL three-necked round-bottom flask was charged with 7 mL of oleic acid, 15 mL of 1-octadecene, and 1 mmol of erbium acetate, which was stirred and mixed, vacuumed, then nitrogen was introduced, and the temperature was raised to 140 °C. After the reaction in the bottle was completely dissolved, the mixed solution was cooled to 50 °C, and a previously prepared methanol solution containing 2.5 mmol of sodium hydroxide and 4 mmol of ammonium fluoride was added. The reaction was maintained at 50 °C for 30 min. After the reaction was completed, the temperature was raised to 100 °C to remove the methanol until no bubbles were generated in the solution. The vacuum was applied, nitrogen was introduced, and the cycle was repeated three times. The temperature was raised to 300 °C and reacted for 1.5 h, and then cooled to room temperature. The reaction solution was collected, ethanol was added, and centrifuged at 8000 rpm. Cyclohexane was used for resuspension and precipitation, and ethanol was added for centrifugation. After being washed three times, it was dispersed in 4 mL of cyclohexane to obtain a cyclohexane solution containing 1 mmol of NaErF4 core.

[0064] (2) Synthesis of down-conversion nanoparticles

[0065] A clean 100 mL three-necked round-bottom flask was charged with 7 mL of oleic acid, 15 mL of 1-octadecene, and 1 mmol of erbium acetate, which was stirred and mixed, vacuumed, then nitrogen was introduced, and the temperature was raised to 140 °C. After the reaction in the bottle was completely dissolved, the mixed solution was cooled to 50 °C, and a previously prepared methanol solution containing 2.5 mmol of sodium hydroxide and 4 mmol of ammonium fluoride was added. The reaction was maintained at 50 °C for 30 min. After the reaction was completed, the temperature was raised to 100 °C to remove the methanol until no bubbles were generated in the solution. The vacuum was applied, nitrogen was introduced, and the cycle was repeated three times. The temperature was raised to 300 °C and reacted for 1.5 h, and then cooled to room temperature. The reaction solution was collected, ethanol was added, and centrifuged at 8000 rpm. Cyclohexane was used for resuspension and precipitation, and ethanol was added for centrifugation. After being washed three times, it was dispersed in 4 mL of cyclohexane to obtain a cyclohexane solution containing 1 mmol of NaErF4 core.

[0066] (3) Synthesis of NaErF4@NaYF4 modified NHS

[0067] Take 1 mL of the synthesized NaErF4@NaYF4 cyclohexane solution, add 10 mL of chloroform solution and 50 mg of DSPE-PEG2000-NHS, stir at room temperature until the solvent is completely volatilized and dry for 2 h, then resuspend the residual solid in distilled water DW and wash 3 times, store in DW to obtain NaErF4@NaYF4-NHS (Er / Y-NHS) aqueous solution.

[0068] (4) Synthesis of Granzyme B specific response nanoprobes (NaErF4@NaYF4-IEFDK-IR806)

[0069] Resuspend Er / Y-NHS in 50 mM 2-(N-morpholino) ethanesulfonic acid MES (pH = 8.0) solution, stir at room temperature for 20 min to activate the NHS group, then add the pentapeptide IEFDK-fluorescence quencher (H-Ile-Glu-Phe-Asp-Lys (IR-806)-OH) obtained from Preparation Example 1 and stir overnight for connection. The mass ratio of Er / Y-NHS to pentapeptide IEFDK-fluorescence quencher is 10:1. Collect the precipitate, wash 3 times with DW to obtain Granzyme B specific response nanoprobes (NaErF4@NaYF4-IEFDK-IR806).

[0070] Example 2 This example is used to illustrate the preparation of Granzyme B specific response nanoprobes

[0071] (1) Synthesis of down-conversion nanoparticle core

[0072] Into a clean 100 mL three-necked round-bottom flask, add 5 mL of oleic acid, 10 mL of 1-octadecene and 1 mmol of erbium acetate, stir to mix, vacuumize, then introduce nitrogen and heat to 140 ℃, wait until the reaction in the bottle is completely dissolved, cool the mixed solution to 55 ℃, and add the previously prepared methanol solution containing 2 mmol of sodium hydroxide and 3 mmol of ammonium fluoride, maintain 55 ℃ for 10 min, heat to 100 ℃ to remove methanol until the mixed solution no longer produces bubbles, vacuumize, introduce nitrogen for 3 cycles, then heat to 250 ℃ and react for 5 h, cool to room temperature. Collect the reaction solution, add ethanol and centrifuge at 8000 rpm, resuspend the precipitate in cyclohexane, add ethanol and centrifuge, wash 3 times, then disperse in 4 mL of cyclohexane to obtain a cyclohexane solution containing 1 mmol of NaErF4 core.

[0073] (2) Synthesis of down-conversion nanoparticles

[0074] Into a clean 100 mL three-necked round-bottom flask, 5 mL oleic acid, 20 mL 1-octadecene and 0.5 mmol yttrium acetate were added, and after stirring and mixing, the mixture was vacuumed, followed by nitrogen introduction and heating to 140 ℃. After stirring and dissolving to obtain a transparent solution, the solution was cooled to 55 ℃, and 2 mL of a cyclohexane solution of a NaErF4 inner core and a methanol solution containing 2 mmol of NaOH and 2.5 mmol of ammonium fluoride were sequentially added. The reaction was maintained at 55 ℃ for 10 min, and after the reaction was completed, the temperature was increased to 100 ℃ to remove methanol until no bubbles were generated in the solution. After vacuuming and nitrogen introduction for 3 times, the temperature was increased to 250 ℃, and the reaction was maintained at 250 ℃ for 4 h, and then the solution was cooled to room temperature. The reaction solution was collected, ethanol was added, and centrifugation was performed at 8000 rpm. The precipitate was resuspended in ethanol, and centrifugation was performed. After washing 3 times, the precipitate was dispersed in 4 mL of cyclohexane to obtain a cyclohexane solution of 0.5 mmol of NaErF4@NaYF4 core-shell down-conversion nanoparticles.

[0075] (3) Synthesis of NaErF4@NaYF4 modified NHS

[0076] 1 mL of the synthesized NaErF4@NaYF4 cyclohexane solution was taken, 10 mL of a chloroform solution was added, and 30 mg of DSPE-PEG2000-NHS was added. After stirring at room temperature until the solvent was completely volatilized and drying for 2 h, the residual solid was resuspended in DW and washed 3 times. The NaErF4@NaYF4-NHS (Er / Y-NHS) aqueous solution was obtained and stored in DW.

[0077] (4) Synthesis of granzyme B specific response nanoprobe (NaErF4@NaYF4-IEFDK-IR806)

[0078] The Er / Y-NHS was resuspended in a 50 mM MES (pH=8.0) solution, and the NHS group was activated by stirring at room temperature for 20 min. Then, the pentapeptide IEFDK-fluorescence quencher (H-Ile-Glu-Phe-Asp-Lys(IR-806)-OH) obtained from Preparation Example 1 was added, and the mixture was stirred overnight. The mass ratio of Er / Y-NHS to the pentapeptide IEFDK-fluorescence quencher was 10:1. After the precipitate was collected and washed 3 times with DW, the granzyme B specific response nanoprobe (NaErF4@NaYF4-IEFDK-IR806) was obtained.

[0079] Example 3 This example is used to illustrate the preparation of a granzyme B specific response nanoprobe

[0080] (1) Synthesis of down-conversion nanoparticle inner core

[0081] A clean 100 mL three-necked round-bottom flask was charged with 10 mL of oleic acid, 20 mL of 1-octadecene, and 1 mmol of erbium acetate, stirred and mixed, vacuumed, then nitrogen was introduced, and the temperature was raised to 140 °C. After the reaction in the flask was completely dissolved, the mixed solution was cooled to 45 °C, and a previously prepared methanol solution containing 2.5 mmol of sodium hydroxide and 4 mmol of ammonium fluoride was added. The reaction was maintained at 45 °C for 60 min. After the reaction was completed, the temperature was raised to 100 °C to remove the methanol until no bubbles were generated in the mixed solution. After vacuuming and introducing nitrogen for 3 cycles, the temperature was raised to 350 °C and reacted for 1 h, and then cooled to room temperature. The reaction solution was collected, added with ethanol, and centrifuged at 8000 rpm. The cyclohexane was resuspended and precipitated, added with ethanol, and centrifuged. After being washed 3 times, it was dispersed in 4 mL of cyclohexane to obtain a cyclohexane solution containing 1 mmol of NaErF4 core.

[0082] (2) Synthesis of down-conversion nanoparticles

[0083] A clean 100 mL three-necked round-bottom flask was charged with 10 mL of oleic acid, 20 mL of 1-octadecene, and 1 mmol of erbium acetate, stirred and mixed, vacuumed, then nitrogen was introduced, and the temperature was raised to 140 °C. After the reaction in the flask was completely dissolved, the mixed solution was cooled to 45 °C, and a previously prepared methanol solution containing 2.5 mmol of sodium hydroxide and 4 mmol of ammonium fluoride was added. The reaction was maintained at 45 °C for 60 min. After the reaction was completed, the temperature was raised to 100 °C to remove the methanol until no bubbles were generated in the mixed solution. After vacuuming and introducing nitrogen for 3 cycles, the temperature was raised to 350 °C and reacted for 1 h, and then cooled to room temperature. The reaction solution was collected, added with ethanol, and centrifuged at 8000 rpm. The cyclohexane was resuspended and precipitated, added with ethanol, and centrifuged. After being washed 3 times, it was dispersed in 4 mL of cyclohexane to obtain a cyclohexane solution containing 1 mmol of NaErF4 core.

[0084] (3) Synthesis of NaErF4@NaYF4 modified NHS

[0085] 1 mL of the synthesized NaErF4@NaYF4 cyclohexane solution was taken, 10 mL of chloroform solution was added, and 40 mg of DSPE-PEG2000-NHS was added. The solution was stirred at room temperature until the solvent was completely volatilized and dried for 2 h. Then the residual solid was resuspended in DW and washed 3 times. The NaErF4@NaYF4-NHS (Er / Y-NHS) aqueous solution was obtained and stored in DW.

[0086] (4) Synthesis of granzyme B specific response nanoprobe (NaErF4@NaYF4-IEFDK-IR806)

[0087] The Er / Y-NHS was resuspended in 50 mM MES (pH=8.0) solution, and the NHS group was activated by stirring at room temperature for 20 min, and then the pentapeptide IEFDK-fluorescence quencher (H-Ile-Glu-Phe-Asp-Lys(IR-806)-OH) obtained from Preparation Example 1 was added for overnight stirring for connection. The mass ratio of Er / Y-NHS to the pentapeptide IEFDK-fluorescence quencher was 10:1, and the precipitate was collected and washed with DW for 3 times to obtain the specific response nanoprobe (NaErF4@NaYF4-IEFDK-IR806) for granulase B.

[0088] Comparative Example 1

[0089] The specific response nanoprobe for granulase B was prepared according to the method of Example 1, except that the pentapeptide IEFDK-fluorescence quencher obtained from Preparation Example 1 was replaced by the reference pentapeptide IEFDK-fluorescence quencher obtained from Comparative Preparation Example 1 in the same weight portion, and the other conditions were the same as those of Example 1, to obtain the specific response nanoprobe for granulase B.

[0090] Comparative Example 2

[0091] The specific response nanoprobe for granulase B was prepared according to the method of Example 1, except that the pentapeptide IEFDK-fluorescence quencher obtained from Preparation Example 1 was replaced by the reference pentapeptide IEFDK-fluorescence quencher obtained from Comparative Preparation Example 2 in the same weight portion, and the other conditions were the same as those of Example 1, to obtain the specific response nanoprobe for granulase B.

[0092] Test Example 1

[0093] (1) Morphology and particle size: The down-conversion nanoparticle cores and near-infrared down-conversion nanoprobe obtained in each of the above examples and comparative examples were diluted with cyclohexane, and were uniformly dispersed by ultrasonic treatment. Then, 1 drop of the obtained dispersion liquid was dropped on a transmission electron microscope copper mesh, and after the cyclohexane was completely volatilized, the obtained sample was observed under a transmission electron microscope and photographed. The particle size results are shown in Table 1. The results obtained in Example 1 are shown in Figure 1 and Figure 2 It can be seen from Figure 1 and Figure 2 that the particle size distribution of the synthesized down-conversion nanoparticle cores and near-infrared down-conversion nanoprobe is uniform and has good dispersibility.

[0094] (2) Luminescence performance: The near-infrared down-conversion nanoprobe obtained in each of the above examples and comparative examples was subjected to excitation and emission spectrum measurement. Specifically, an appropriate amount of NaErF4@NaYF4 nanoparticle sample was placed on the sample loading table of a spectrometer, and the near-infrared emission spectrum of the NaErF4@NaYF4 nanoparticle was measured by 808 nm laser excitation. The obtained results are shown in Table 1. The results obtained in Example 1 are shown in Figure 3.from Figure 3 It can be seen that the near-infrared downconversion nanoparticles have good luminescence properties, with the maximum emission peak located at 1525 nm.

[0095] (3) To explore the application of nanoprobes in monitoring radiotherapy-induced tumor immune activation.

[0096] ① Establish a mouse breast cancer tumor model:

[0097] Six-week-old female BALB / c mice were acclimatized for 7 days, and then 4T1 cells (2×10⁻⁶) were introduced. 6 A breast cancer tumor model was established by subcutaneously injecting 4T1 cells into the right leg of mice and continuing to feed them for 7 days. The specific steps were as follows: the mice were fixed, and 1 mL of 4T1 cells were injected subcutaneously into the right leg of the mice using a syringe. The mice were then fed for 7 days. The appearance of a mass under the skin of the right leg of the mice proved that the breast cancer tumor model was successfully established.

[0098] ② Radiotherapy and near-infrared imaging in mice: The granzyme B-specific responsive nanoprobes obtained in the above examples and the reference granzyme B-specific responsive nanoprobes obtained in the comparative examples were used to determine the radiotherapy and near-infrared imaging effects in mice in the following manner. The specific steps are as follows:

[0099] Mice with breast cancer tumors were randomly divided into two groups: (1) control group; and (2) 12 Gy radiation for 2 days. After treatment, synthetically synthesized specific responsive nanoprobes were injected into the tumor, followed by imaging. The results are shown in Table 1. The results obtained in Example 1 are shown in Table 1. Figure 4 . Figure 4 In the diagram, A represents the control group, and B represents the treatment group (12 Gy radiation for 2 days). From Figure 4 It can be seen that the tumor sites in the treatment group showed significant fluorescence recovery, proving that the constructed specific response nanoprobe has good granzyme B response imaging effect.

[0100] (4) Specific response to granzyme B: The specific responsiveness of the nanoprobes obtained in the above examples and comparative examples to granzyme B was tested according to the following method. The specific method and conditions are as follows: The downconversion nanoparticles synthesized in the examples and comparative examples were dissolved in ultrapure water, and then the near-infrared imaging effect of the resulting dispersions was tested in two cases: with granzyme B (granzyme B(+) / granzyme(+)) and without granzyme B (granzyme B(-) / granzyme(-)). If fluorescence imaging was possible after adding granzyme B but not after not adding granzyme B, it indicates that the probe can specifically respond to granzyme B; otherwise, it indicates that it cannot specifically respond to granzyme B. The results are shown in Table 1. Figure 5 and Figure 6 The images show the emission spectra and near-infrared imaging results of the nanoprobe constructed in Example 1 in vitro in response to granzyme B.Figure 5 and Figure 6 As can be seen from the results of Example 1, the nano-probe constructed in Example 1 can specifically correspond to the particle granzyme B.

[0101] Table 1

[0102]

[0103] Although the embodiments of the present application have been shown and described above, it should be understood that the above-described embodiments are exemplary, and should not be construed as limiting the present application, and those ordinarily skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments without departing from the principles and spirit of the present application within the scope of the present application.

Claims

1. A granzyme B-specific responsive nanoprobe, characterized in that, The granzyme B-specific responsive nano probe comprises a near-infrared down-conversion nano probe, and the surface of the near-infrared down-conversion nano probe is modified with a pentapeptide IEFDK-fluorescence quenching modifier; the granzyme B-specific responsive nano probe further comprises a connecting layer, the connecting layer is coated on the surface of the near-infrared down-conversion nano probe, and the pentapeptide IEFDK-fluorescence quenching modifier is bonded to active groups of the connecting layer; the connecting layer is a DSPE-PEG-NHS layer; the near-infrared down-conversion nano probe is NaErF4@NaYF4 with a core-shell structure, the inner core component of the NaErF4@NaYF4 is NaErF4 and the outer shell component is NaYF4; and the pentapeptide IEFDK-fluorescence quenching modifier is a pentapeptide IEFDK-fluorescence quencher with a structure shown in formula (1) and is bonded to the connecting layer through an amide reaction to obtain: Formula (1).

2. The Granzyme B-specific responsive nanoprobes according to claim 1, wherein, The molar ratio of erbium to yttrium in the near-infrared down-conversion nano probe is 1:(0.8-1.2).

3. The Granzyme B-specific responsive nanoprobes according to claim 1, wherein, The near-infrared down-conversion nano probe has a spherical structure, and the particle size D50 is 10-50 nm.

4. The Granzyme B-specific responsive nanoprobe according to any one of claims 1 to 3, wherein, The mass ratio of the connecting layer to the molar amount of erbium in the near-infrared down-conversion nano probe is (200-500) mg:1 mol.

5. A method for preparing the granzyme B-specific responsive nanoprobes according to any one of claims 1 to 4, characterized in that, The method comprises the following steps: S1. Coating a connecting layer on the surface of the near-infrared down-conversion nano probe, the connecting layer having active groups capable of reacting with the pentapeptide IEFDK-fluorescence quencher with a structure shown in formula (1), to obtain a modified nano probe; S2. Contacting the modified nano probe with the pentapeptide IEFDK-fluorescence quencher to react, so that the pentapeptide IEFDK-fluorescence quencher reacts with the active groups on the connecting layer of the modified nano probe and is chemically bonded to the surface of the connecting layer of the modified nano probe; Formula (1).

6. The method of claim 5, wherein the preparation of the Granzyme B-specific responsive nanoprobes is characterized by, The near-infrared down-conversion nano probe is prepared by the following method: S11. Dissolving erbium acetate, oleic acid and 1-octadecene under the protection of inert gas, stirring the obtained mixture with an alcohol solution containing a sodium source I and a fluorine source I at 45-55 DEG C for 10-60 min, removing alcohol until no bubbles are generated in the reaction system, then performing high-temperature decomposition reaction at 250-350 DEG C for 1-5 h, and then performing solid-liquid separation, washing the obtained solid product and then dispersing it in an organic solvent I to obtain a solution containing a NaErF4 inner core; S12. Dissolving yttrium acetate, oleic acid and 1-octadecene under the protection of inert gas, stirring the obtained mixture with an alcohol solution containing a sodium source II and a fluorine source II and the solution containing the NaErF4 inner core at 45-55 DEG C for 10-60 min, removing alcohol until no bubbles are generated in the reaction system, then performing high-temperature decomposition reaction at 250-350 DEG C for 1-5 h, and then performing solid-liquid separation, washing the obtained solid product and then dispersing it in an organic solvent II to obtain a solution containing NaErF4@NaYF4.

7. The method of claim 6, wherein the preparation of the Granzyme B-specific responsive nanoprobes is characterized by, In the preparation process of the near-infrared down-conversion nano probe, in step S11, the amount ratio of the erbium acetate, the oleic acid and the 1-octadecene is 1 mmol:(5-10) mL:(10-20) mL. The dosage ratio of the sodium source I to the erbium acetate is (2-4) mmol: 1 mmol; The dosage ratio of the fluorine source I to the erbium acetate is (3-5) mmol: 1 mmol.

8. The method of claim 6, wherein the preparation of the Granzyme B-specific responsive nanoprobes is characterized by, In the preparation process of the near-infrared down-conversion nano probe, in step S12, the dosage ratio of the yttrium acetate, the oleic acid and the 1-octadecene is 1 mmol: (10-20) mL: (25-40) mL; The dosage ratio of the sodium source II to the yttrium acetate is (2-4) mmol: 1 mmol; The dosage ratio of the fluorine source II to the yttrium acetate is (3-5) mmol: 1 mmol.

9. The method of claim 5-8, wherein the preparation of the Granzyme B-specific responsive nanoprobes is characterized by, The method for coating a connection layer on the surface of the near-infrared down-conversion nano probe comprises stirring and mixing the near-infrared down-conversion nano probe and the DSPE-PEG-NHS in a volatile solvent, drying after the solvent is basically volatilized, and the like. The ratio of the mass of the DSPE-PEG-NHS to the molar amount of erbium in the near-infrared down-conversion nano probe is (200-500) mg: 1 mol; The volatile solvent is at least one selected from chloroform, cyclohexane, toluene, methanol and acetone; The dosage ratio of the volatile solvent to the DSPE-PEG-NHS is 1 mL: (4-6) mg.

10. The granzyme B-specific responsive nano probe prepared by the method of any one of claims 5-9.

11. The use of the granzyme B-specific responsive nano probe according to any one of claims 1-4 and 10 in the preparation of a drug for radiotherapy-activated immune effector imaging.

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