Granzyme B specific response nanoprobe as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to a granzyme B specific response nanoprobe as well as a preparation method and application thereof. The granzyme B specific response nanoprobe 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 quenching agent with a structure as shown in a formula (1). The granzyme B specific response nanoprobe provided by the invention can specifically respond to granzyme B, and realizes monitoring of immune effect of a tumor site, so that detection of tumor immune response after treatment of different radiotherapy schemes is realized. Formula (1)

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a granzyme B-specific responsive nanoprobe, its preparation method, and its application. Background Technology

[0002] Radiation therapy (RT) is a method of treating tumors by using high-energy ionizing radiation to kill cancer cells. It is considered one of the three major methods of cancer treatment, alongside surgery and chemotherapy. Besides its direct killing effect on tumor cells, RT also possesses potential immunomodulatory properties. Through acute DNA damage caused by radiation, it directly induces the death of tumor immunogenic cells, triggering the body's anti-tumor immune response and producing an "in situ vaccine" effect. It can also regulate the immune microenvironment by inducing the production of various cytokines. However, the impact of radiotherapy on the tumor microenvironment is bidirectional; depending on the dose, fractionation method, and tumor location, it may have different immunomodulatory effects. Therefore, monitoring the immune-activating effects of different radiotherapy doses could potentially guide the selection of clinical radiotherapy regimens.

[0003] Currently, there are few objective and direct methods to monitor the immune-activating effects of radiotherapy. The main methods used are imaging techniques, including computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET), to detect various downstream biomarkers of radiotherapy, such as reactive oxygen species (ROS), apoptosis protein caspase-3, and DNA damage-associated protein γH2AX, to monitor the effects of radiotherapy at different doses and fractionation methods. However, firstly, these biomarkers are non-specific to biological immune responses and cannot accurately and effectively assess the effect of radiotherapy on tumor immune activation. Secondly, imaging methods involve radiation exposure, are cumbersome, time-consuming, and costly, thus failing to achieve dynamic monitoring of radiotherapy effects. Summary of the Invention

[0004] The primary objective of this invention is to provide a novel granzyme B-specific responsive nanoprobe that can specifically respond to granzyme B, thereby enabling the monitoring of immune effects at tumor sites.

[0005] The second objective of this invention is to provide a method for preparing a granzyme B-specific responsive nanoprobe.

[0006] A third objective of this invention is to provide a granzyme B-specific responsive nanoprobe prepared by the above method.

[0007] A fourth objective of this invention is to provide the application of the above-mentioned granzyme B-specific responsive nanoprobe in radiotherapy-activated immunotherapy imaging.

[0008] The granzyme B-specific responsive nanoprobe provided by this invention includes a near-infrared downconversion nanoprobe, the surface of which is modified with a pentapeptide IEDK-fluorescence quenching modifier; the near-infrared downconversion nanoprobe is a NaErF4@NaYF4 with a core-shell structure, wherein the core component of NaErF4@NaYF4 is NaErF4 and the shell component is NaYF4; the pentapeptide IEDK-fluorescence quenching modifier is derived from a pentapeptide IEDK-fluorescence quencher with the structure shown in formula (1): Equation (1).

[0009] The method for preparing granzyme B-specific responsive nanoprobes provided by this invention includes: S1. A connecting layer is coated on the surface of the near-infrared downconversion nanoprobe, the connecting layer having an active group that can react with the pentapeptide IEDK-fluorescence quencher with the structure shown in formula (1) to obtain a modified nanoprobe; S2. The modified nanoprobe is contacted and reacted with the pentapeptide IEDK-fluorescence quencher so that the pentapeptide IEDK-fluorescence quencher reacts with the active groups on the modified nanoprobe linking layer and is chemically bonded to the surface of the modified nanoprobe linking layer. Equation (1).

[0010] The key to this invention lies in the surface modification of a near-infrared downconversion nanoprobe with a pentapeptide IEDK-fluorescence quenching group. The near-infrared downconversion nanoprobe exhibits excellent near-infrared II luminescence properties. The pentapeptide IEDK is isoleucine-glutamic acid-phenylalanine-aspartic acid-lysine (Ile-Glu-Phe-Asp-Lys), and the fluorescence quencher is IR806, which specifically absorbs 808nm laser light. The introduction of the pentapeptide IEDK-fluorescence quenching group endows the probe with excellent specific granzyme B responsiveness, specifically responding to the presence of granzyme B at the tumor site. This enables the detection of tumor immune responses after different radiotherapy regimens. Furthermore, the granzyme B-specific responsive nanoprobe provided by this invention exhibits high stability, good dispersibility, and uniform particle size distribution. Attached Figure Description

[0011] Figure 1 Transmission electron microscopy image of the downconversion nanoparticle core (NaErF4) obtained in Example 1; Figure 2 This is a transmission electron microscope (TEM) image of the downconversion nanoparticles (NaErF4@NaYF4) obtained in Example 1; Figure 3 The near-infrared emission spectrum of the downconversion nanoparticles (NaErF4@NaYF4) obtained in Example 1 is shown below. Figure 4 This is an in vivo imaging image of a mouse breast cancer tumor model corresponding to the granzyme B-specific responsive nanoprobe obtained in Example 1. Figure 5 The emission spectrum of the granzyme B-specific responsive nanoprobe obtained in Example 1 is shown in the in vitro granzyme B-responsive imaging. Figure 6 This is a near-infrared image of the granzyme B-specific responsive nanoprobe obtained in Example 1, showing its granzyme B responsiveness in vitro. Detailed Implementation

[0012] The granzyme B-specific responsive nanoprobe provided by this invention comprises a near-infrared downconversion nanoprobe, the surface of which is modified with a pentapeptide IEDK-fluorescence quenching modifying group; the pentapeptide IEDK-fluorescence quenching modifying group is derived from the pentapeptide IEDK-fluorescence quencher with the structure shown in formula (1): Equation (1).

[0013] In this invention, the near-infrared downconversion nanoprobe is a rare-earth luminescent material NaErF4@NaYF4 with a core-shell structure, wherein the core component of NaErF4@NaYF4 is NaErF4 and the shell component is NaYF4. The molar ratio of erbium to yttrium in the near-infrared downconversion 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.

[0014] In this invention, the near-infrared downconversion nanoprobe has a spherical structure. Furthermore, the particle size D50 of the near-infrared downconversion nanoprobe is preferably 10 nm to 50 nm, specifically such as 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, or any value between them.

[0015] In this invention, the granzyme B-specific responsive nanoprobe preferably further includes a linker layer, which coats the surface of the near-infrared downconversion nanoprobe, and the pentapeptide IEDK-fluorescence quenching modification group is bonded to the active group of the linker layer. The active group of the linker layer can be at least one of -NHS, -COOH, -NH2, etc. These active groups can react with the amino or carboxyl groups on the pentapeptide IEDK-fluorescence quencher to bond the pentapeptide IEDK-fluorescence quencher to the linker layer. Particularly preferably, the linker layer is a DSPE-PEG-NHS layer. Furthermore, the ratio of the mass of the connecting layer to the molar amount of erbium in the near-infrared downconversion nanoprobe 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 between them.

[0016] The method for preparing granzyme B-specific responsive nanoprobes provided by this invention includes: S1. A connecting layer is coated on the surface of the near-infrared downconversion nanoprobe, the connecting layer having an active group that can react with the pentapeptide IEDK-fluorescence quencher with the structure shown in formula (1) to obtain a modified nanoprobe; S2. The modified nanoprobe is contacted and reacted with the pentapeptide IEDK-fluorescence quencher so that the pentapeptide IEDK-fluorescence quencher reacts with the active groups on the modified nanoprobe linking layer and is chemically bonded to the surface of the modified nanoprobe linking layer. Equation (1).

[0017] In the preparation process of the above-mentioned granzyme B-specific responsive nanoprobe of the present invention, the downconversion nanomaterial is first converted into an aqueous phase and modified with NHS groups by the DSPE ligand exchange method. Then, the pentapeptide fragment IEFK, which can be specifically cleaved by granzyme B, is further linked by the amide reaction. The end is then bound with the fluorescence quencher IR806 by the amide reaction again, thereby synthesizing a novel nanoprobe for specific granzyme B imaging that is biosafe.

[0018] In this invention, the near-infrared downconversion nanoprobe is a rare-earth luminescent material NaErF4@NaYF4 with a core-shell structure, wherein the core component of NaErF4@NaYF4 is NaErF4 and the shell component is NaYF4. The core-shell structured NaErF4@NaYF4 can be commercially available or prepared using various existing methods, without particular limitation.

[0019] In a preferred embodiment, the near-infrared downconversion nanoprobe is prepared based on a high-temperature thermal decomposition reaction, specifically including the following steps: S11. Erbium acetate, oleic acid and 1-octadecene are dissolved by stirring under inert gas protection. The resulting mixture is reacted with an alcohol solution containing sodium source I and fluorine source I at 45℃~55℃ for 10min~60min. The alcohol is then removed until no more bubbles are generated in the reaction system. After that, the mixture is decomposed at 250℃~350℃ for 1h~5h and then the solid and liquid are separated. The resulting solid product is washed and redispersed in organic solvent I to obtain a solution containing NaErF4 core. S12. Yttrium acetate, oleic acid and 1-octadecene are dissolved by stirring under inert gas protection. The resulting mixture is reacted with an alcohol solution containing sodium source II and fluorine source II and a solution containing NaErF4 core at 45℃~55℃ for 10min~60min. The alcohol is then removed until no more bubbles are generated in the reaction system. After that, the mixture is decomposed at 250℃~350℃ for 1h~5h and then the solid and liquid are separated. The resulting solid product is washed and redispersed in organic solvent II to obtain a solution containing NaErF4@NaYF4.

[0020] In the preparation of the near-infrared downconversion nanoprobe, in step S11, the preferred ratio of erbium acetate, oleic acid, and 1-octadecene is 1 mmol:(5~10) mL:(10~20) mL. Specifically, the ratio of erbium acetate to 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 ratio of erbium acetate to 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 preferred ratio of sodium source I to erbium acetate is (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 preferred ratio of fluorine source I to erbium acetate is (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.

[0021] In the preparation of the near-infrared downconversion nanoprobe, in step S12, the preferred ratio of yttrium acetate, oleic acid, and 1-octadecene is 1 mmol:(10~20) mL:(25~40) mL. Specifically, the ratio of yttrium acetate to 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 ratio of yttrium acetate to 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 preferred ratio of sodium source II to yttrium acetate is (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 preferred ratio of fluorine source II to yttrium acetate is (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.

[0022] In the preparation of the near-infrared downconversion nanoprobe, the stirring reaction temperatures in steps S11 and S12 are each independently 45℃~55℃, such as 45℃, 48℃, 50℃, 52℃, 55℃ or any value between them; the time is each independently 10min~60min, such as 10min, 20min, 30min, 40min, 50min, 60min or any value between them. The high-temperature decomposition reaction temperatures in steps S11 and S12 are each independently 250℃~350℃, such as 250℃, 260℃, 270℃, 280℃, 290℃, 300℃, 310℃, 320℃, 330℃, 340℃, 350℃ or any value between them; the time is each independently 1h~5h, such as 1h, 2h, 3h, 4h, 5h or any value between them.

[0023] In the preparation of the near-infrared downconversion nanoprobe, sodium source I and sodium source II are each independently at least one of sodium hydroxide, sodium chloride, and sodium carbonate. Fluorine source I and fluorine source II are each independently at least one of ammonium fluoride, sodium fluoride, and sodium bifluoride. Organic solvent I and organic solvent II are each independently at least one of cyclohexane, methanol, chloroform, and acetone. The terms "I" and "II" are merely used to distinguish the same type of substance introduced at different locations for ease of description and have no other special meaning.

[0024] In this invention, the method for coating a linker layer onto the surface of a near-infrared downconversion nanoprobe preferably includes stirring and mixing the near-infrared downconversion nanoprobe and DSPE-PEG-NHS in a volatile solvent until the solvent is substantially evaporated, followed by drying. The preferred ratio of the mass of the DSPE-PEG-NHS to the molar amount of erbium in the near-infrared downconversion nanoprobe is (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 between them. The volatile solvent is preferably at least one selected from chloroform, cyclohexane, toluene, methanol, and acetone. Furthermore, the preferred ratio of the volatile solvent to DSPE-PEG-NHS is 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 between them.

[0025] In this invention, the pentapeptide IEDK-fluorescence quencher can be commercially available or prepared using various existing methods, without particular limitation. In a preferred embodiment, the pentapeptide IEDK-fluorescence quencher is prepared according to the following method: S21. IR-780 and 4-mercaptobenzoic acid were reacted under inert gas protection and in the presence of an organic solvent by stirring. The resulting reaction product was then purified to obtain IR-806. S22. The protecting group Fmoc in Fmoc-Lys(Dde)-CTC resin is removed. The resulting H-Lys(Dde)-CTC resin is coupled with Fmoc-Asp(OtBu)-OH in the presence of benzotriazole-N,N,N,N-tetramethylurea hexafluorophosphate / N,N-diisopropylethylamine (HBTU / DIEA). The protecting group Fmoc in the resulting Fmoc-Asp(OtBu)-Lys(Dde)-CTC is then removed and extended by SPPS to obtain Boc-Ile-Glu(OtBu)-Phe-Asp(OtBu)-Lys(Dde). -CTC, then remove Dde from Boc-Ile-Glu(OtBu)-Phe-Asp(OtBu)-Lys(Dde)-CTC, then couple Boc-Ile-Glu(OtBu)-Phe-Asp(OtBu)-Lys(Dde)-CTC with IR-806 in the presence of 1-hydroxybenzotriazole / diisopropylcarbodiimide (HOBT / DIC) and then cleave it with trifluoroacetic acid / triisopropylsilane / H2O (TFA / TIS / H2O) to obtain H-Ile-Glu-Phe-Asp-Lys(IR806)-OH.

[0026] In the preparation of the above-mentioned pentapeptide IEDK-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), specifically 1:1.5, 1:1.8, 1:2, 1:2.2, 1:2.5, or any value between them. The organic solvent can be at least one of various existing liquid inert media, such as N,N-dimethylformamide (DMF), toluene, methanol, acetone, and cyclohexane. The purification method may include, for example, removing the solvent from the obtained reaction product, ultrasonically dissolving the residue in dichloromethane, filtering it through a filter with a pore size of 0.2μm~0.6μm, precipitating the filtrate in diethyl ether, centrifuging it, washing the centrifuged product with diethyl ether, and then vacuum drying it.

[0027]

[0028] Reaction formula (1) In the preparation of the above-mentioned pentapeptide IEDK-fluorescence quencher, in step S22, the protecting group Fmoc can be removed by 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.

[0029] In the preparation of the granzyme B-specific responsive nanoprobes described above, in step S2, the contact reaction conditions preferably include a temperature of room temperature (0℃~45℃) and a time of 5h~24h. Furthermore, before the contact reaction, it is preferable to activate the modified nanoprobes with NHS groups in an organic solvent. The NHS group activation conditions preferably include a temperature of room temperature (0℃~45℃) and a time of 10min~30min.

[0030] The present invention also provides a granzyme B-specific responsive nanoprobe prepared by the above method.

[0031] Furthermore, the present invention also provides the application of the granzyme B-specific responsive nanoprobe in radiotherapy-activated immunotherapy imaging.

[0032] The present invention will be described in detail below through embodiments.

[0033] Preparation Example 1: This preparation example illustrates the preparation of the pentapeptide IEDK-fluorescence quencher. S21. Preparation of fluorescence quencher IR-806 In a glass bottle, IR-780 (130 mg, 195 µmol) and 4-mercaptobenzoic acid (60 mg, 380 µmol) were dissolved in 5 mL of anhydrous DMF. The mixture was first evacuated and then stirred at room temperature for 17 h under nitrogen protection. The DMF was then removed under vacuum at 40 °C. The residue was sonicated and dissolved in 5 mL of CH2Cl2, filtered through a 0.45 μm PTFE filter, and 50 mL of diethyl ether was slowly added to precipitate the product. The precipitate was collected by centrifugation, washed with diethyl ether, and dried under vacuum to obtain deep reddish-gold crystals, which was IR-806.

[0034] S22. Preparation of the pentapeptide IEDK-fluorescence quencher First, starting with 0.2 mmol of Fmoc-Lys(Dde)-CTC resin, the resin was treated with 20% piperidine / DMF solution for 30 min to remove the Fmoc protecting group, yielding H-Lys(Dde)-CTC resin. Next, an Asp coupling reaction was carried out using HBTU and DIEA, with 0.6 mmol of Fmoc-Asp(OtBu)-OH, 0.6 mmol of HBTU, and 1.0 mmol of DIEA, reacting for 1 h to obtain Fmoc-Asp(OtBu)-Lys(Dde)-CTC resin. Then, the resin was again treated with 20% piperidine / DMF solution for 30 min to remove the Fmoc protecting group, yielding H-Asp(OtBu)-Lys(Dde)-CTC resin. Finally, Boc-Ile-Glu(OtBu)-Phe-Asp(OtBu)-Lys(Dde)-CTC resin was synthesized via solid-phase peptide synthesis (SPPS). The 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, IR-806 coupling reaction was carried out by DIC and HOBT, and the reaction was carried out overnight with 0.24 mmol of IR-806, 0.24 mmol of HOBT and 0.24 mmol of DIC to obtain Boc-Ile-Glu(OtBu)-Phe-Asp(OtBu)-Lys(IR-806)-CTC. Finally, the Boc-Ile-Glu(OtBu)-Phe-Asp(OtBu)-Lys(IR-806)-CTC was cleaved for 3 hours using a mixed solution of 95% TFA, 2.5% Tis and 2.5% H2O to obtain the pentapeptide IEDK-fluorescence quencher (H-Ile-Glu-Phe-Asp-Lys(IR-806)-OH).

[0035] According to NMR and infrared detection, the pentapeptide IEDK-fluorescence quencher has the structure shown in formula (1).

[0036] Comparative Preparation Example 1 The pentapeptide IEDK-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 during the SPPS process. The other conditions were the same as in Preparation Example 1, and the reference pentapeptide IEDK-fluorescence quencher (H-Thr-Glu-Phe-Asp-Lys(IR-806)-OH) was obtained.

[0037] Comparative Preparation Example 2 The pentapeptide IEDK-fluorescence quencher was prepared according to the method of Preparation Example 1, except that step S21 was omitted and step S22 was performed by replacing IR-806 with the same molar amount of fluorescence quencher 4-dimethylamineazobenzene-4'-carboxylic acid (Dabcyl). The remaining conditions were the same as in Preparation Example 1, and the reference pentapeptide IEDK-fluorescence quencher (H-Ile-Glu-Phe-Asp-Lys(Dabcyl)-OH) was obtained.

[0038] Example 1 This example illustrates the preparation of granzyme B-specific responsive nanoprobes. (1) Synthesis of downconversion nanoparticle cores Add 7 mL of oleic acid, 15 mL of 1-octadecene, and 1 mmol of erbium acetate to a clean 100 mL three-necked round-bottom flask. After stirring and mixing, evacuate the flask, then purge with nitrogen and heat to 140 °C. Once all the reactants in the flask have dissolved, cool the mixture to 50 °C and add a pre-prepared methanol solution containing 2.5 mmol of sodium hydroxide and 4 mmol of ammonium fluoride. Maintain the reaction at 50 °C for 30 min. After the reaction is complete, heat to 100 °C to remove methanol until no more bubbles are produced in the mixture. Evacuate the flask, purge with nitrogen three times, then heat to 300 °C and react for 1.5 h. Cool to room temperature. Collect the reaction solution, add ethanol, and centrifuge at 8000 rpm. Resuspend the precipitate in cyclohexane, centrifuge with ethanol, wash three times, and disperse in 4 mL of cyclohexane to obtain a cyclohexane solution containing 1 mmol of NaErF4 core.

[0039] (2) Synthesis of downconversion nanoparticles To a clean 100 mL three-necked round-bottom flask, add 7 mL of oleic acid, 15 mL of 1-octadecene, and 0.5 mmol of yttrium acetate. After stirring and mixing, evacuate the flask, then purge with nitrogen and heat to 140 °C. Stir to dissolve and obtain a clear solution, then cool to 50 °C. Add 2 mL of a NaErF4 core-shell cyclohexane solution and a methanol solution containing 1.25 mmol of NaOH and 2 mmol of ammonium fluoride sequentially. Maintain the reaction at 50 °C for 30 min. After the reaction is complete, heat to 100 °C to remove methanol until the solution stops bubbling. Evacuate the flask, purge with nitrogen three times, then heat to 300 °C and react for 1.5 h. Cool to room temperature. Collect the reaction solution, add ethanol, and centrifuge at 8000 rpm. Resuspend the precipitate in cyclohexane, centrifuge with ethanol, wash three times, and disperse in 4 mL of cyclohexane to obtain a cyclohexane solution of 0.5 mmol NaErF4@NaYF4 core-shell underconversion nanoparticles.

[0040] (3) Synthesis of NaErF4@NaYF4 modified NHS 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 evaporated and dry for 2 h, then resuspend the residual solid in distilled water (DW) and wash 3 times, and store in DW to obtain NaErF4@NaYF4-NHS (Er / Y-NHS) aqueous solution.

[0041] (4) Synthesize granzyme B specific responsive nanoprobe (NaErF4@NaYF4-IEFDK-IR806) Er / Y-NHS was resuspended in 50 mM 2-(N-morpholine) ethanesulfonic acid MES (pH=8.0) solution and stirred at room temperature for 20 min to activate the NHS group. Then, the pentapeptide IEDK-fluorescence quencher (H-Ile-Glu-Phe-Asp-Lys(IR-806)-OH) obtained from Preparation Example 1 was added and stirred overnight for linkage. The mass ratio of Er / Y-NHS to pentapeptide IEDK-fluorescence quencher was 10:1. The precipitate was collected and washed three times by DW to obtain the granzyme B specific responsive nanoprobe (NaErF4@NaYF4-IEFDK-IR806).

[0042] Example 2 This example illustrates the preparation of granzyme B-specific responsive nanoprobes. (1) Synthesis of downconversion nanoparticle cores Add 5 mL of oleic acid, 10 mL of 1-octadecene, and 1 mmol of erbium acetate to a clean 100 mL three-necked round-bottom flask. After stirring and mixing, evacuate the flask, then purge with nitrogen and heat to 140 °C. Once all the reactants in the flask have dissolved, cool the mixture to 55 °C and add a pre-prepared methanol solution containing 2 mmol of sodium hydroxide and 3 mmol of ammonium fluoride. Maintain the reaction at 55 °C for 10 min. After the reaction is complete, heat to 100 °C to remove methanol until no more bubbles are produced in the mixture. Evacuate the flask, purge with nitrogen three times, then heat to 250 °C 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, centrifuge with ethanol, wash three times, and disperse in 4 mL of cyclohexane to obtain a cyclohexane solution containing 1 mmol of NaErF4 core.

[0043] (2) Synthesis of downconversion nanoparticles Add 5 mL of oleic acid, 20 mL of 1-octadecene, and 0.5 mmol of yttrium acetate to a clean 100 mL three-necked round-bottom flask. After stirring and mixing, evacuate the flask, then purge with nitrogen and heat to 140 °C. Stir to dissolve and obtain a clear solution, then cool to 55 °C. Add 2 mL of cyclohexane solution containing NaErF4 core and methanol solution containing 2 mmol of NaOH and 2.5 mmol of ammonium fluoride sequentially. Maintain the reaction at 55 °C for 10 min. After the reaction is complete, heat to 100 °C to remove methanol until the solution stops bubbling. Evacuate the flask, purge with nitrogen three times, then heat to 250 °C and react for 4 h. Cool to room temperature. Collect the reaction solution, add ethanol, and centrifuge at 8000 rpm. Resuspend the precipitate in cyclohexane, centrifuge with ethanol, wash three times, and disperse in 4 mL of cyclohexane to obtain a cyclohexane solution of 0.5 mmol NaErF4@NaYF4 core-shell underconversion nanoparticles.

[0044] (3) Synthesis of NaErF4@NaYF4 modified NHS Take 1 mL of the synthesized NaErF4@NaYF4 cyclohexane solution, add 10 mL of chloroform solution and 30 mg of DSPE-PEG2000-NHS, stir at room temperature until the solvent is completely evaporated and dry for 2 h, then resuspend the residual solid with DW and wash 3 times, and store in DW to obtain NaErF4@NaYF4-NHS (Er / Y-NHS) aqueous solution.

[0045] (4) Synthesize granzyme B specific responsive nanoprobe (NaErF4@NaYF4-IEFDK-IR806) Er / Y-NHS was resuspended in 50 mM MES (pH=8.0) solution and stirred at room temperature for 20 min to activate the NHS group. Then, the pentapeptide IEDK-fluorescence quencher (H-Ile-Glu-Phe-Asp-Lys(IR-806)-OH) obtained from Preparation Example 1 was added and stirred overnight for connection. The mass ratio of Er / Y-NHS to pentapeptide IEDK-fluorescence quencher was 10:1. The precipitate was collected and washed three times by DW to obtain the granzyme B specific responsive nanoprobe (NaErF4@NaYF4-IEFDK-IR806).

[0046] Example 3 This example illustrates the preparation of granzyme B-specific responsive nanoprobes. (1) Synthesis of downconversion nanoparticle cores Add 10 mL of oleic acid, 20 mL of 1-octadecene, and 1 mmol of erbium acetate to a clean 100 mL three-necked round-bottom flask. After stirring and mixing, evacuate the flask, then purge with nitrogen and heat to 140 °C. Once all the reactants in the flask have dissolved, cool the mixture to 45 °C and add a pre-prepared methanol solution containing 2.5 mmol of sodium hydroxide and 4 mmol of ammonium fluoride. Maintain the reaction at 45 °C for 60 min. After the reaction is complete, heat to 100 °C to remove methanol until no more bubbles are produced in the mixture. Evacuate the flask, purge with nitrogen three times, then heat to 350 °C and react for 1 h. Cool to room temperature. Collect the reaction solution, add ethanol, and centrifuge at 8000 rpm. Resuspend the precipitate in cyclohexane, centrifuge with ethanol, wash three times, and disperse in 4 mL of cyclohexane to obtain a cyclohexane solution containing 1 mmol of NaErF4 core.

[0047] (2) Synthesis of downconversion nanoparticles To a clean 100 mL three-necked round-bottom flask, add 10 mL of oleic acid, 16 mL of 1-octadecene, and 0.5 mmol of yttrium acetate. After stirring and mixing, evacuate the flask, then purge with nitrogen and heat to 140 °C. Stir to dissolve and obtain a clear solution, then cool to 45 °C. Add 2 mL of a NaErF4 core-shell cyclohexane solution and a methanol solution containing 1.25 mmol of NaOH and 2 mmol of ammonium fluoride sequentially. Maintain the reaction at 45 °C for 40 min. After the reaction is complete, heat to 100 °C to remove methanol until the solution stops bubbling. Evacuate the flask, purge with nitrogen three times, then heat to 320 °C and react for 3.8 h. Cool to room temperature. Collect the reaction solution, add ethanol, and centrifuge at 8000 rpm. Resuspend the precipitate in cyclohexane, centrifuge with ethanol, wash three times, and disperse in 4 mL of cyclohexane to obtain a cyclohexane solution of 0.5 mmol NaErF4@NaYF4 core-shell underconversion nanoparticles.

[0048] (3) Synthesis of NaErF4@NaYF4 modified NHS Take 1 mL of the synthesized NaErF4@NaYF4 cyclohexane solution, add 10 mL of chloroform solution and 40 mg of DSPE-PEG2000-NHS, stir at room temperature until the solvent is completely evaporated and dry for 2 h, then resuspend the residual solid with DW and wash 3 times, and store in DW to obtain NaErF4@NaYF4-NHS (Er / Y-NHS) aqueous solution.

[0049] (4) Synthesize granzyme B specific responsive nanoprobe (NaErF4@NaYF4-IEFDK-IR806) Er / Y-NHS was resuspended in 50 mM MES (pH=8.0) solution and stirred at room temperature for 20 min to activate the NHS group. Then, the pentapeptide IEDK-fluorescence quencher (H-Ile-Glu-Phe-Asp-Lys(IR-806)-OH) obtained from Preparation Example 1 was added and stirred overnight for connection. The mass ratio of Er / Y-NHS to pentapeptide IEDK-fluorescence quencher was 10:1. The precipitate was collected and washed three times by DW to obtain the granzyme B specific responsive nanoprobe (NaErF4@NaYF4-IEFDK-IR806).

[0050] Comparative Example 1 The reference granzyme B-specific responsive nanoprobe was prepared according to the method of Example 1, except that the pentapeptide IEDK-fluorescence quencher obtained from Preparation Example 1 was replaced by the same weight of the reference pentapeptide IEDK-fluorescence quencher obtained from Comparative Preparation Example 1. The other conditions were the same as in Example 1, and the reference granzyme B-specific responsive nanoprobe was obtained.

[0051] Comparative Example 2 The reference granzyme B-specific responsive nanoprobe was prepared according to the method of Example 1, except that the pentapeptide IEDK-fluorescence quencher obtained in Preparation Example 1 was replaced by the same weight of the reference pentapeptide IEDK-fluorescence quencher obtained in Comparative Preparation Example 2. The other conditions were the same as in Example 1, and the reference granzyme B-specific responsive nanoprobe was obtained.

[0052] Test Example 1 (1) Morphology and Particle Size: The downconversion nanoparticle cores and near-infrared downconversion nanoprobes obtained in the above examples and comparative examples were diluted with cyclohexane and ultrasonically dispersed to ensure uniformity. One drop of each dispersion was then placed on a copper grid of a transmission electron microscope. After the cyclohexane had completely evaporated, the particles were observed and photographed under a transmission electron microscope. The particle size results are shown in Table 1. The results obtained in Example 1 are shown in Table 1. Figure 1 and Figure 2 .from Figure 1 and Figure 2 It can be seen that the synthesized downconversion nanoparticle core and near-infrared downconversion nanoprobe have uniform particle size distribution and good dispersibility.

[0053] (2) Luminescence properties: The near-infrared downconversion nanoprobes obtained in the above examples and comparative examples were subjected to excitation and emission spectroscopy measurements. Specifically, an appropriate amount of NaErF4@NaYF4 nanoparticle sample was placed on the spectrometer sample stage and excited with an 808nm laser. The near-infrared emission spectrum of the NaErF4@NaYF4 nanoparticles was measured, and the results are shown in Table 1. Among them, the results obtained in Example 1 are shown in Table 1. Figure 3 .from Figure 3It can be seen that the near-infrared downconversion nanoparticles have good luminescence properties, with the maximum emission peak located at 1525 nm.

[0054] (3) To explore the application of nanoprobes in monitoring radiotherapy-induced tumor immune activation. ① Establish a mouse breast cancer tumor model: 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.

[0055] 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: 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). 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.

[0056] (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 6The results show that the nanoprobe constructed in Example 1 can specifically respond to granzyme B.

[0057] Table 1

[0058] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A granzyme B-specific responsive nanoprobe, characterized in that, The granzyme B-specific responsive nanoprobe includes a near-infrared downconversion nanoprobe, the surface of which is modified with a pentapeptide IEDK-fluorescence quenching modifier; the near-infrared downconversion nanoprobe is a core-shell structure NaErF4@NaYF4, the core component of which is NaErF4 and the shell component is NaYF4; the pentapeptide IEDK-fluorescence quenching modifier is derived from a pentapeptide IEDK-fluorescence quencher with the structure shown in formula (1): Equation (1).

2. The granzyme B-specific responsive nanoprobe according to claim 1, characterized in that, The molar ratio of erbium to yttrium in the near-infrared downconversion nanoprobe is 1:(0.8~1.2).

3. The granzyme B-specific responsive nanoprobe according to claim 1, characterized in that, The near-infrared downconversion nanoprobe has a spherical structure and a particle size D50 of 10 nm to 50 nm.

4. The granzyme B-specific responsive nanoprobe according to any one of claims 1 to 3, characterized in that, The granzyme B-specific responsive nanoprobe also includes a linker layer, which is coated on the surface of the near-infrared downconversion nanoprobe, and the pentapeptide IEDK-fluorescence quenching modification group is linked to the active group of the linker layer. The connecting layer is a DSPE-PEG-NHS layer; The ratio of the mass of the connecting layer to the molar amount of erbium in the near-infrared downconversion nanoprobe is (200~500) mg:1 mol.

5. A method for preparing a granzyme B-specific responsive nanoprobe, characterized in that, The method includes the following steps: S1. A connecting layer is coated on the surface of the near-infrared downconversion nanoprobe, the connecting layer having an active group that can react with the pentapeptide IEDK-fluorescence quencher with the structure shown in formula (1) to obtain a modified nanoprobe; S2. The modified nanoprobe is contacted and reacted with the pentapeptide IEDK-fluorescence quencher so that the pentapeptide IEDK-fluorescence quencher reacts with the active groups on the modified nanoprobe linking layer and is chemically bonded to the surface of the modified nanoprobe linking layer. Equation (1).

6. The method for preparing the granzyme B-specific responsive nanoprobe according to claim 5, characterized in that, The near-infrared downconversion nanoprobe was prepared according to the following method: S11. Erbium acetate, oleic acid and 1-octadecene are dissolved by stirring under inert gas protection. The resulting mixture is reacted with an alcohol solution containing sodium source I and fluorine source I at 45℃~55℃ for 10min~60min. The alcohol is then removed until no more bubbles are generated in the reaction system. After that, the mixture is decomposed at 250℃~350℃ for 1h~5h and then the solid and liquid are separated. The resulting solid product is washed and redispersed in organic solvent I to obtain a solution containing NaErF4 core. S12. Yttrium acetate, oleic acid and 1-octadecene are dissolved by stirring under inert gas protection. The resulting mixture is reacted with an alcohol solution containing sodium source II and fluorine source II and a solution containing NaErF4 core at 45℃~55℃ for 10min~60min. The alcohol is then removed until no more bubbles are generated in the reaction system. After that, the mixture is decomposed at 250℃~350℃ for 1h~5h and then the solid and liquid are separated. The resulting solid product is washed and redispersed in organic solvent II to obtain a solution containing NaErF4@NaYF4.

7. The method for preparing the granzyme B-specific responsive nanoprobe according to claim 6, characterized in that, In the preparation of near-infrared downconversion nanoprobes, in step S11, the ratio of erbium acetate, oleic acid and 1-octadecene is 1 mmol:(5~10) mL:(10~20) mL. The ratio of sodium source I to erbium acetate is (2~4):1; The ratio of fluorine source I to erbium acetate is (3~5):

1.

8. The method for preparing the granzyme B-specific responsive nanoprobe according to claim 6, characterized in that, In the preparation of near-infrared downconversion nanoprobes, in step S12, the ratio of yttrium acetate, oleic acid and 1-octadecene is 1 mmol:(10~20) mL:(25~40) mL. The ratio of sodium source II to yttrium acetate is (2~4):1; The ratio of fluorine source II to yttrium acetate is (3~5):

1.

9. The method for preparing granzyme B-specific responsive nanoprobes according to any one of claims 5 to 8, characterized in that, The method of coating a linker layer onto the surface of a near-infrared downconversion nanoprobe includes mixing the near-infrared downconversion nanoprobe with DSPE-PEG-NHS in a volatile solvent until the solvent is basically evaporated, and then drying it. The ratio of the mass of the DSPE-PEG-NHS to the molar amount of erbium in the near-infrared downconversion nanoprobe is (200~500) mg:1 mol; The volatile solvent is selected from at least one of chloroform, cyclohexane, toluene, methanol, and acetone; The ratio of the volatile solvent to DSPE-PEG-NHS is 1 mL:(4~6) mg.

10. A granzyme B-specific responsive nanoprobe prepared by the method according to any one of claims 5 to 9.

11. The application of the granzyme B-specific responsive nanoprobe according to any one of claims 1 to 4 and 10 in radiotherapy-activated immunotherapy imaging.

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