Ratio fluorescent probe for adoptive NK cell marking and living body tracing and application thereof

Abstract

The invention provides a ratiometric fluorescent probe for adoptive NK cell marking and living body tracing and application of the ratiometric fluorescent probe. The ratiometric fluorescent probe comprises rare earth down-conversion nanoparticles of a core-shell structure, granzyme B response type polypeptide and a sensitizer, the surface of the rare earth down-conversion nano particle with the core-shell structure is coated with stearoyl phosphatidyl ethanolamine-polyethylene glycol. The ratiometric fluorescent probe disclosed by the invention has the characteristics of variable fluorescence signals under 808nm and 980nm laser excitation and self-carrying reference signals, and errors and instrument errors caused by different attenuation coefficients in tissues can be eliminated. The fluorescent probe can be used for adoptive NK marking, does not influence the biological characteristics of adoptive NK cells, and can be used for tracing the biological distribution and survival conditions of the adoptive NK cells in a host living body in a fluorescence imaging living body and evaluating the curative effect of adoptive NK cell treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of immune cell labeling, and relates to a ratio fluorescent probe used for adoptive NK cell labeling and in vivo tracing and an application thereof. Background Art

[0002] Natural Killer Cell (NKC) is a type of innate immune lymphocyte that does not require pre-sensitization, is not restricted by the major histocompatibility complex, can directly and quickly kill tumor cells, and has the advantages of not causing graft-versus-host disease and good safety. Therefore, adoptive immune cell therapy based on NK cells has good clinical application prospects in tumor immunotherapy. However, the transplantation efficiency and therapeutic effect of adoptive NK cell therapy cannot be fully and accurately grasped. Using cell live tracing technology to study the fate of adoptive NK cells in vivo, it is possible to obtain functional information of adoptive NK cells in tumor tissues, including infiltration (homing, migration and distribution) and activation status, which is helpful to evaluate the transplantation efficiency and efficacy of adoptive NK cells and develop new therapies based on adoptive NK cell transplantation.

[0003] Commonly used cell live tracing technologies mainly include optical imaging, magnetic resonance imaging (MRI), ultrasound imaging, and nuclear medicine imaging (PET). Among them, MRI has the disadvantages of low resolution, long detection time, high cost, and poor imaging effect on hard tissues such as bones; ultrasound imaging has poor resolution, poor imaging effect on lungs and bones, and difficult image interpretation; nuclear medicine imaging technology requires radioactive nuclide labeling of cell-specific markers, which poses a risk of radioactive radiation. The current near-infrared II (NIR-II) optical imaging technology has the advantages of high signal-to-background ratio (SBR), non-invasiveness, real-time feedback capability, high sensitivity, convenient operation, and low cost, and can be used to dynamically observe the in vivo functional information of cells.

[0004] At present, the fluorescent materials commonly used for fluorescence in vivo imaging in the NIR-II region mainly include single-walled carbon nanotubes (SWNTs), organic dyes (Organic Dyes), quantum dots (Quantum Dots, QDs) and rare earth doped nanoparticles (Rare Earth Doped Nanoparticles, RENPs). SWNTs have defects such as poor biocompatibility and low quantum yield (Quantum Yield, QY), which limit their development in biological imaging; most organic dyes have photobleaching effects, making it difficult to conduct long-term cell tracking studies; QDs are more likely to accumulate in cells and cannot be used for functional imaging of adoptive cells; RENPs have the advantages of a wide fluorescence lifetime range, high fluorescence stability, and low cytotoxicity, which is conducive to the application in adoptive NI cell tracking.

[0005] Traditional fluorescence imaging mostly uses a single-channel, single-signal source mode. The measurement of fluorescence is easily affected by factors such as spontaneous fluorescence, changes in the body environment (pH, polarity, temperature, etc.) and changes in excitation intensity, making it difficult to achieve quantitative detection of visual information. Ratio probes with two or more signals and built-in correction can effectively eliminate interference from factors such as photobleaching, excitation light intensity, and background signals, significantly improving the sensitivity and specificity of fluorescence imaging and achieving in vivo visualization and quantitative analysis of adoptive NK cell transplantation.

[0006] Granzyme B (GzmB), which is mainly secreted by immune cells, is an important killer factor in human immunotherapy. It can induce target cell death by activating the caspase-mediated apoptosis pathway, and can also promote cell death by cleaving substances such as α-tubulin in the cell. Through enzyme cleavage design, GzmB-specific response polypeptides are selected to connect RENPs and sensitizers. After NK cells are activated, GzmB will be released to cleave the polypeptide, so that RENPs and sensitizers are separated, achieving the effect of fluorescence recovery. However, the prior art does not disclose how to select materials that can be used for NK cell labeling.

[0007] Therefore, in this field, constructing a GzmB-responsive ratio probe with high sensitivity and high fluorescence stability has great application prospects for the labeling of NK cells and visualization of functional information. Summary of the invention

[0008] In view of the deficiencies of the prior art, the object of the present invention is to provide a ratio fluorescent probe for adoptive NK cell labeling and in vivo tracing and its application. The probe of the present invention is a ratio fluorescent probe based on rare earth down-conversion nanomaterials. The probe can have two signals of reference and response with the same wavelength at two excitation wavelengths of 808nm and 980nm. After GzmB response, the fluorescence intensity under 808nm laser excitation is weakened, and the fluorescence intensity under 980nm laser excitation remains unchanged. The ratio of the two signals is linearly related to the GzmB concentration within a certain range. And because it comes with a reference signal, the probe can eliminate the error caused by different attenuation coefficients in the tissue and the instrument error.

[0009] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0010] On the one hand, the present invention provides a ratio fluorescent probe, which includes rare earth down-conversion nanoparticles (DCNPs) with a core-shell structure, a granzyme B (GzmB) responsive polypeptide and a sensitizer, wherein the surface of the rare earth down-conversion nanoparticles with a core-shell structure is coated with stearoyl phosphatidylethanolamine-polyethylene glycol (DSPE-PEG), and the granzyme B responsive polypeptide and the polyethylene glycol on the surface of the rare earth down-conversion nanoparticles with a core-shell structure, as well as the sensitizer and the granzyme B responsive polypeptide are connected through amide bonds formed by dehydration condensation of amino groups and carboxyl groups.

[0011] The ratio fluorescent probe of the present invention has the characteristics of variable fluorescence signal under 808nm and 980nm laser excitation and self-contained reference signal, which can eliminate the error caused by different attenuation coefficients in tissues and instrument errors. It can be used for adoptive NK labeling without affecting the biological characteristics of adoptive NK cells, and can also be used for fluorescent imaging to trace the biological distribution and survival of adoptive NK cells in the host body, and evaluate the efficacy of adoptive NK cell therapy.

[0012] Preferably, the rare earth down-conversion nanoparticles of the core-shell structure are NaYF4:20% Yb, 2% Er@NaYF4:30% Nd, wherein NaYF4:20% Yb, 2% Er represents the core, and NaYF4:30% Nd represents the shell. The raw materials of the rare earth can be acetate, nitrate, chloride, oxide, etc. The selection of rare earth elements can be changed as needed. According to different fluorescence signals, Er, Tm, Ho and other ions can be selected as luminescent ions. According to the absorption cross-section size of the excitation signal, Yb, Nd and other ions can be selected as sensitizing ions to improve the excitation light absorption efficiency of the luminescent ions. Optically inert and low phonon energy materials are selected as matrix materials, such as oxides, fluorides, fluoride oxides, sulfides, etc. In this paper, fluoride NaYF4 is selected as the matrix material, which has low lattice phonon energy, can effectively inhibit non-radiative transitions, and improve fluorescence quantum yield.

[0013] In the present invention, a layer of DSPE-PEG is coated on the surface of the rare earth down-conversion nanoparticles, thereby improving the biological stability of the material and facilitating surface modification.

[0014] Preferably, the granzyme B-responsive polypeptide is selected from a polypeptide containing the granzyme B-responsive sequence IEFDSG.

[0015] Preferably, the granzyme B-responsive polypeptide is selected from GIEFDSGGC or KIEFDSGKC, etc., and can be designed according to requirements (net charge, polypeptide molecular weight, water solubility, special groups, etc.).

[0016] Preferably, the sensitizer can be selected from organic dyes having a large absorption cross section at the excitation signal, such as IR808, IR806, etc., as required.

[0017] In another aspect, the present invention provides a method for preparing the ratiometric fluorescent probe as described above, the preparation method comprising the following steps:

[0018] (1) Using rare earth acetate as raw material, oleic acid as surface ligand, and 1-octadecene as solvent, rare earth down-conversion nanoparticles (DCNPs) coated with oleic acid on the surface were obtained by coprecipitation and seed-mediated core-shell growth;

[0019] (2) sonicating the rare earth down-conversion nanoparticles coated with oleic acid and the amphoteric material DSPE-PEG-NH2 in a solvent, removing the solvent, and centrifuging and washing to obtain the rare earth down-conversion nanoparticles coated with stearoyl phosphatidylethanolamine-polyethylene glycol that are stable in an aqueous phase;

[0020] (3) dispersing the rare earth down-conversion nanoparticles coated with stearoyl phosphatidylethanolamine-polyethylene glycol described in step (2) in a buffer solution, adjusting the pH to 5-6, adding an activator for activation, and then adding a granzyme B responsive polypeptide, stirring, to obtain rare earth down-conversion nanoparticles modified with a granzyme B responsive polypeptide (DCNPs@GzmB);

[0021] (4) The sensitizer is dispersed in a buffer solution, the pH is adjusted to 5-6, an activator is added for activation, and the rare earth down-conversion nanoparticles (DCNPs@GzmB) modified with the granzyme B responsive polypeptide described in step (3) are added, and stirred in the dark to obtain the ratiometric fluorescent probe (DCNPs@GzmB-IR808).

[0022] Preferably, the rare earth acetate in step (1) comprises any one of yttrium acetate, ytterbium acetate, erbium acetate or neodymium acetate, or a combination of at least two thereof.

[0023] Preferably, the total molar amount of the rare earth element in the rare earth acetate in step (1) is 1, mainly to standardize and simplify the experimental conditions to ensure the repeatability and comparability of the experiment. If necessary, it can also be adjusted according to actual conditions.

[0024] Preferably, the rare earth down-conversion nanoparticles in step (1) are NaYF4,:20% Yb,2% Er@NaYF4,:30% Nd.

[0025] Preferably, the preparation method of NaYF4,:20%Yb,2%Er@NaYF4,:30%Nd comprises the following steps:

[0026] A. Dissolve yttrium acetate, ytterbium acetate and erbium acetate in a mixed solution of oleic acid and 1-octadecene to fully dissolve the rare earth acetate. Mix a methanol solution of ammonium fluoride and a methanol solution of sodium oxychloride at room temperature and add them to the rare earth acetate solution. Heat the obtained mixed solution, cool it to room temperature, wash it, and disperse it in cyclohexane to obtain the product NaYF4: 20% Yb, 2% Er.

[0027] B. Dissolve yttrium acetate and neodymium acetate in a mixed solution of oleic acid and 1-octadecene to fully dissolve the rare earth acetate. At room temperature, mix a methanol solution of ammonium fluoride with a methanol solution of sodium oxychloride and an ethanol solution of NaYF4:20% Yb, 2% Er obtained in step A, and add the mixture to the rare earth acetate solution. Heat the mixture, cool it to room temperature, wash it, and disperse it in cyclohexane to obtain NaYF4:20% Yb, 2% Er@NaYF4:30% Nd.

[0028] Preferably, the method for fully dissolving the rare earth acetate in step A and step B is to heat the mixed solution to 50°C-80°C (for example, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C or 80°C), maintain vacuum for 20-35min (for example, 20min, 25min, 30min, 33min or 35min), heat to 90°C-110°C (for example, 90°C, 95°C, 100°C, 105°C or 110°C), keep warm for 10-20min (for example, 10min, 13min, 15min, 18min or 20min), continue to heat to 150°C-160°C (for example, 150°C, 155°C, 158°C or 160°C), and keep warm for 30-50min (for example, 30min, 35min, 38min, 40min, 45min, 48min or 50min).

[0029] Preferably, the mixed solution obtained in step A and step B is heated in a manner as follows: slowly heated to 47°C-50°C (e.g., 47°C, 48°C, 49°C, or 50°C) at a rate of 0.5°C / min-2°C / min (e.g., 0.5°C / min, 1°C / min, 1.5°C / min, or 2°C / min), maintained for 30-50min (e.g., 30min, 35min, 38min, 40min, 45min, 48min, or 50min), and then heated to 65°C-80°C (e.g., The mixture is stirred at a temperature of 65°C, 70°C, 75°C or 80°C, maintained under vacuum for 20 min-30 min (for example, 20 min, 25 min, 28 min or 30 min), heated to 100°C-120°C (for example, 100°C, 105°C, 110°C, 115°C or 120°C), maintained for 3-5 min, and then heated to 298°C-300°C and stirred for 30-50 min (for example, 30 min, 35 min, 38 min, 40 min, 45 min, 48 min or 50 min).

[0030] Preferably, the molar ratio of yttrium acetate, ytterbium acetate, erbium acetate and ammonium fluoride in step A is 0.78:0.2:0.02:4.

[0031] Preferably, the molar ratio of yttrium acetate, ytterbium acetate, erbium acetate and sodium hydroxide in step A is 0.78:0.2:0.02:2.5.

[0032] Preferably, the molar ratio of yttrium acetate, neodymium acetate and ammonium fluoride in step B is 0.78:0.2:0.02:4.

[0033] Preferably, the molar ratio of yttrium acetate, neodymium acetate and sodium hydroxide in step B is 0.78:0.2:0.02:2.5.

[0034] Preferably, the washing in step A and step B is performed by alternating washing with cyclohexane and ethanol.

[0035] Preferably, the mass ratio between the rare earth down-conversion nanoparticles coated with oleic acid on the surface of step (2) and the amphoteric material DSPE-PEG-NH2 is between 1:2-1:6, for example, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5 or 1:6.

[0036] In the present invention, the amphoteric material can also be used by mixing DSPE-PEG and DSPE-PEG-NH2. When mixed, the mass percentage of DSPE-PEG-NH2 in the mixture is 5-10%.

[0037] Preferably, the solvent in step (2) is chloroform.

[0038] Preferably, the ultrasonication time in step (2) is 10 to 30 min, for example, 10 min, 15 min, 20 min, 25 min, 28 min or 30 min.

[0039] Preferably, the buffer solution in step (3) is a MES buffer solution.

[0040] In the present invention, the pH is adjusted to 5-6 in step (3), for example, 5, 5.3, 5.5, 5.8 or 6.

[0041] Preferably, the activating agent in step (3) is EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide).

[0042] Preferably, the molar ratio of the rare earth down-conversion nanoparticles coated with stearoyl phosphatidylethanolamine-polyethylene glycol to EDC in step (3) is 1:5-1:15, for example, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:13, 1:15, etc.

[0043] Preferably, in step (3), the ratio of the amount of rare earth down-conversion nanoparticles coated with stearoyl phosphatidylethanolamine-polyethylene glycol, EDC and NHS is 1:10:10-1:10:20, for example, 1:10:10, 1:10:12, 1:10:14, 1:10:15, 1:10:16, 1:10:18 or 1:10:20.

[0044] Preferably, the activation time in step (3) is 20 to 60 min, for example, 10 min, 15 min, 20 min, 25 min, 28 min, 30 min, 35 min, 38 min, 40 min, 45 min, 50 min, 55 min or 60 min.

[0045] Preferably, in step (3), the ratio of the amount of the granzyme B responsive polypeptide to the amount of DSPE-PEG-NH2 is between 1:1 and 2:1, for example, 1:1, 1.3:1, 1.5:1, 1.8:1 or 2:1.

[0046] Preferably, the stirring time in step (3) is 6-24 hours, for example 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours or 24 hours.

[0047] Preferably, the stirring in step (3) is followed by centrifugation and washing 2 to 3 times.

[0048] Preferably, the buffer solution in step (4) is a MES buffer solution.

[0049] In the present invention, the pH is adjusted to 5-6 in step (4), for example, 5, 5.3, 5.5, 5.8 or 6.

[0050] Preferably, in step (4), the sensitizer is dissolved in an organic solvent and then dispersed in a buffer solution, and the organic solvent is selected from any one of DMF, DMSO or methanol or a combination of at least two thereof.

[0051] Preferably, the activating agent in step (4) is EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide).

[0052] Preferably, the molar ratio of the rare earth down-conversion nanoparticles modified with the granzyme B-responsive polypeptide to the sensitizer in step (4) is 1:1-1:4, for example, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5 or 1:4.

[0053] Preferably, the activation time in step (4) is 20 to 60 min, for example, 10 min, 15 min, 20 min, 25 min, 28 min, 30 min, 35 min, 38 min, 40 min, 45 min, 50 min, 55 min or 60 min.

[0054] Preferably, the dark-protected stirring time in step (4) is 6-24 hours, for example 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours or 24 hours.

[0055] Preferably, the stirring in the dark in step (4) is followed by centrifugation and washing 2 to 3 times.

[0056] In another aspect, the present invention provides the use of the ratiometric fluorescent probe as described above in adoptive NK cell labeling or in vivo tracing.

[0057] In another aspect, the present invention provides use of the ratiometric fluorescent probe as described above in detecting the activation of adoptive NK cells.

[0058] Compared with the prior art, the present invention has the following beneficial effects:

[0059] The present invention uses rare earth down-conversion nanoparticles DCNPs with the same near-infrared second-zone fluorescence wavelength under 980nm and 808nm excitation as a substrate, and uses a unique design scheme to couple organic small molecule dyes as sensitizers on the surface of DCNPs through GzmB specific response peptides as a bridge to construct a highly sensitive, ratiometric fluorescent probe that can be used for cell labeling and activation information detection.

[0060] The DCNPs@GzmB-IR808 ratiometric fluorescent nanoprobe constructed in the present invention is used for labeling adoptive NK cells without affecting the biological characteristics of adoptive NK cells, and further uses optical imaging to track the labeled adoptive NK cells in vivo.

[0061] The DCNPs@GzmB-IR808 ratiometric fluorescent nanoprobe constructed in the present invention can be used for optical imaging to trace the biodistribution and survival of adoptive NK cells in the host's living body, and to evaluate the efficacy of adoptive NK cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 The transmission electron microscope images of the rare earth down-conversion nanoparticles (DCNPs) prepared in Example 1, A is the Core structure, and B is the Core-Shell structure.

[0063] Figure 2 This is the absorption spectrum of DCNPs prepared in Example 1.

[0064] Figure 3 This is the emission fluorescence spectrum of the DCNPs prepared in Example 1 under 808nm and 980nm laser excitation at 1525nm.

[0065] Figure 4This is the emission fluorescence spectrum of the DCNPs prepared in Example 1 before and after coupling with the sensitizer, under 808nm laser excitation at 1525nm.

[0066] Figure 5 This is the emission fluorescence spectra of the ratiometric fluorescent probe prepared in Example 1 before and after GzmB response, under 808nm and 980nm laser excitation at 1525nm, wherein Figure A is 808nm laser excitation, and Figure B is 980nm laser excitation.

[0067] Figure 6 This is the fluorescence spectrum of the ratiometric fluorescent probe prepared in Example 1 under 808 nm laser excitation in different time periods after GzmB response.

[0068] Figure 7 This is the fluorescence spectrum of the ratiometric fluorescent probe prepared in Example 1 under 980nm laser excitation in different time periods after GzmB response.

[0069] Figure 8 This is the cytotoxicity of the ratiometric fluorescent probe prepared in Example 1 on adoptive NK cells.

[0070] Fig. 9 This is a scanning transmission electron microscope image of the product prepared in Comparative Example 1.

[0071] Fig.10 The fluorescence spectra of the product prepared in Comparative Example 1 under 808nm and 980nm laser excitation, wherein Figure A is 808nm laser excitation and Figure B is 980nm laser excitation.

[0072] Fig.11 This is the XRD pattern of DCNPs prepared in Example 1. DETAILED DESCRIPTION

[0073] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0074] Example 1

[0075] In this embodiment, a ratiometric fluorescent probe is provided, and the preparation method thereof is as follows:

[0076] (1) Weigh 0.2075 g of yttrium acetate, 0.0736 g of ytterbium acetate, and 0.0072 g of erbium acetate, add them to a mixed solution of 6 mL of oleic acid and 15 mL of 1-octadecene, heat to 80°C, maintain vacuum for 35 min, continue to heat to 100°C, maintain heat for 25 min, heat to 150°C, maintain heat for 40 min, allow the rare earth acetate to completely dissolve, and cool to room temperature.

[0077] Weigh 148.16 mg of ammonium fluoride (NH4F) and 100 mg of sodium hydroxide (NaOH), dissolve them in 10 mL and 2.5 mL of methanol respectively, mix them, add them to the above solution, heat to 47.5 ° C, keep for 35 minutes, continue to heat to 65 ° C, remove methanol for 10 minutes, continue to heat to 80 ° C, keep vacuum for 20 minutes, continue to heat to 100 ° C, keep for 3 minutes, continue to heat to 298 ° C, keep warm for 50 minutes, and cool to room temperature. Add ethanol and cyclohexane to wash alternately, centrifuge 3 times at 6000g for 15 minutes, disperse in 10 mL of cyclohexane to obtain NaYF4: 20% Yb, 2% Er, and store at 4 ° C.

[0078] (2) Weigh 0.1862 g of yttrium acetate and 0.1018 g of neodymium acetate and add them to a mixed solution of 6 mL of oleic acid and 15 mL of 1-octadecene, heat to 80°C, maintain vacuum for 35 min, continue to heat to 100°C, maintain heat for 25 min, heat to 150°C, maintain heat for 40 min, allow the rare earth acetate to completely dissolve, and cool to room temperature.

[0079] Weigh 148.16 mg of ammonium fluoride (NH4F) and 100 mg of sodium hydroxide (NaOH), dissolve them in 10 mL and 2.5 mL of methanol respectively, add ethanol to the DCNPs in Example 1, centrifuge at 6000 g for 10 min, mix the three, add them to the above solution, heat to 47.5 ° C, keep for 35 min, continue to heat to 65 ° C, remove methanol for 10 min, continue to heat to 80 ° C, keep vacuum for 20 min, continue to heat to 100 ° C, keep for 3 min, continue to heat to 298 ° C, keep warm for 50 min, and cool to room temperature. Add ethanol and cyclohexane to wash alternately, centrifuge 3 times at 6000 g for 15 min, disperse in 10 mL of cyclohexane to obtain NaYF4,: 20% Yb, 2% Er @ NaYF4,: 30% Nd, and store at 4 ° C.

[0080] (3) Weigh 75 mg DSPE-PEG-NH2, take 0.1 mmol DCNPs (1 mL), add 5 mL chloroform, mix and ultrasonicate for 10 min, use a rotary evaporator to remove the chloroform, add 2 mL ultrapure water to disperse the material, centrifuge and wash three times at 14000 rpm for 10 min, and then disperse in 1 mL ultrapure water.

[0081] (4) Take 100ul DCNPs-PEG (about 2mg), weigh 17.5mg EDC and 11.5mg NHS, disperse in MES buffer with a pH of 5-6 and activate for 30min, then add 4mg GzmB responsive peptide (manufacturer: Nanjing GenScript Biotechnology Co., Ltd., model (production number): C765T511G0-1 / PE7896), use 0.1M sodium hydroxide solution to adjust the pH to 7-7.5, stir in the dark at 700rpm, overnight, then at 14000rpm for 10min, centrifuge and wash 3 times, disperse in 100ul ultrapure water, and store at 4°C.

[0082] (5) Weigh 1 mg of sensitizer IR808 and dissolve it in DMF to a concentration of 1 mg / mL. Weigh 2 mg of EDC and 2 mg of NHS and take 20 ug of IR808. Mix the three in MES buffer and activate for 30 min. Then add 90 ug of the product in step (4) and use MES buffer to replenish the system to 1 mL. Oscillate in the dark at 600 rpm overnight, then at 14,000 rpm for 10 min. Wash by centrifugation three times, disperse in 90 ul of ultrapure water and store at 4°C.

[0083] Transmission electron microscopy (TEM), XRD, UV-Vis spectrophotometer (UV-Vis), fluorescence spectrometer and other methods were used to characterize the DCNPs@GzmB-IR808 ratiometric fluorescent nanoprobe prepared above. The specific test results are as follows:

[0084] (1) Transmission electron microscopy (TEM)

[0085] Transmission electron microscope (FEI Talos F200X G2, USA) images show the fine structure of the Core and Core-Shell structured nanoparticles of DCNPs. Figure 1 Figure A shows the distribution and morphology of the Core of DCNPs at a scale of 50 nm, which shows that the DCNPs synthesized by the co-precipitation method have uniform size, consistent morphology, and are distributed in a hexagonal phase. Figure B shows the transmission electron microscopy image of the Core-Shell structure of DCNPs at a scale of 50 nm, indicating that the synthesized DCNPs have the characteristics of uniform size and consistent morphology.

[0086] (2) XRD characterization

[0087] XRD (Bruker D8 ADVANC, Germany) can be used to characterize the crystal structure of the material. By comparing with JCPDS standard card NO.28-1192, the test results are as follows: Fig.11As shown, the measured XRD diffraction peaks match the standard spectrum, confirming that the crystal structure of the material is NaYF4 (hexagonal system). The diffraction peaks of the two materials almost overlap, indicating that their crystal structures are roughly the same, but the slight differences may represent changes in the crystal after doping with different rare earth elements, such as slight changes in the lattice constant, thus proving the successful synthesis of the material.

[0088] (3) Visible-Near Infrared Absorption (Vis-NIR)

[0089] Visible-near infrared absorption spectrum, indicating the characteristic spectrum of the core and core-shell structure synthesis of DCNPs nanoparticles. Figure 2 As can be seen from the figure, in the wavelength range of 200-800nm, the Core-Shell structure has more absorption peaks than the Core structure.

[0090] (4) Fluorescence spectrum

[0091] The fluorescence spectra under 808nm and 980nm laser excitation show the fluorescence emission properties of the DCNPs structure. Figure 3 , shown are the emission spectra of DCNPs materials in the same organic phase under different laser excitations. The emission intensity of DCNPs under 980nm laser excitation is significantly higher than that under 808nm laser excitation, indicating that the neodymium ions in the Shell structure also play a sensitizing role in DCNPs.

[0092] (5) Fluorescence spectroscopy of fluorescent probe sensitized

[0093] Fluorescence spectrum changes of ratiometric fluorescent nanoprobes constructed with DCNPs nanomaterials excited by 808nm laser. Figure 4 , as shown in the 808nm laser excitation emission spectra of DCNPs coupled with GzmB responsive peptide and DCNPs coupled with responsive peptide and then coupled with IR808 at the same concentration. It shows that after coupling with sensitizer IR808, the fluorescence has a significant change, proving the existence of sensitization effect.

[0094] (6) Ratio fluorescence spectroscopy

[0095] GzmB was co-incubated with DCNPs@GzmB-IR808 ratiometric fluorescent nanoprobe for 1 h, and the fluorescence signal of the nanoprobe was detected using a fluorescence spectrometer. Figure 5 , 1 h after GzmB digestion, the fluorescence signal of DCNPs@GzmB-IR808 nanoprobe under 808nm laser excitation was significantly weakened, and the fluorescence signal under 980nm laser excitation remained basically unchanged, indicating that the sensitizer IR808 fell off from the surface of DCNPs.

[0096] (7) Changes of ratio fluorescence spectra over time

[0097] GzmB was co-incubated with DCNPs@GzmB-IR808 ratiometric fluorescent nanoprobe for 1 to 3 h, and the fluorescence signal of the nanoprobe was detected using a fluorescence spectrometer. Figure 6 and Figure 7 , Figure 6 is the emission spectrum of the nanoprobe under 808nm laser excitation, Figure 7 This is the emission spectrum of the nanoprobe under 980nm laser excitation.

[0098] (8) Cytotoxicity

[0099] Different concentrations of DCNPs@GzmB-IR808 ratiometric fluorescent nanoprobes such as 20, 40, 60, 80, 100, 200, 300, 400, 500 ug / mL were incubated with NK cells for 24 h, and the cell proliferation and activity detection kit (CCK8) (Japan Tongren Chemical Research Institute (Shanghai)) was used for detection. Figure 8 The DCNPs@GzmB-IR808 ratio fluorescent nanoprobe was incubated with adoptive NK cells for 24 hours and did not cause cell damage or apoptosis, indicating that the DCNPs@GzmB-IR808 ratio fluorescent nanoprobe is not cytotoxic.

[0100] Example 2

[0101] In this embodiment, a ratiometric fluorescent probe is provided, and the preparation method thereof is as follows:

[0102] (1) Weigh 0.3624 g of erbium acetate and add it to a mixed solution of 6 mL of oleic acid and 15 mL of 1-octadecene. Heat to 80°C and keep under vacuum for 35 min. Continue to heat to 100°C and keep warm for 25 min. Heat to 150°C and keep warm for 40 min to completely dissolve the rare earth acetate. Cool to room temperature.

[0103] Weigh 148.16 mg of ammonium fluoride (NH4F) and 100 mg of sodium hydroxide (NaOH), dissolve them in 10 mL and 2.5 mL of methanol respectively, mix them, add them to the above solution, heat to 47.5°C, keep for 35 minutes, continue to heat to 65°C, remove methanol for 10 minutes, continue to heat to 80°C, keep vacuum for 20 minutes, continue to heat to 100°C, keep for 3 minutes, continue to heat to 298°C, keep warm for 50 minutes, and cool to room temperature. Add ethanol and cyclohexane to wash alternately, centrifuge at 6000g for 15 minutes 3 times, disperse in 10 mL of cyclohexane to obtain NaErF4, and store at 4°C.

[0104] (2) Weigh 0.2661 g of yttrium acetate and add it to a mixed solution of 6 mL of oleic acid and 15 mL of 1-octadecene. Heat to 80°C and keep under vacuum for 35 min. Continue to heat to 100°C and keep warm for 25 min. Heat to 150°C and keep warm for 40 min to completely dissolve the rare earth acetate. Cool to room temperature.

[0105] Weigh 148.16 mg of ammonium fluoride (NH4F) and 100 mg of sodium hydroxide (NaOH), dissolve them in 10 mL and 2.5 mL of methanol respectively, add ethanol to the DCNP-Core in (1), centrifuge at 6000 g for 10 min, mix the three, add them to the above solution, heat to 47.5 ° C, keep for 35 min, continue to heat to 65 ° C, remove methanol for 10 min, continue to heat to 80 ° C, keep vacuum for 20 min, continue to heat to 100 ° C, keep for 3 min, continue to heat to 298 ° C, keep warm for 50 min, and cool to room temperature. Add ethanol and cyclohexane to wash alternately, centrifuge 3 times at 6000 g for 15 min, disperse in 10 mL of cyclohexane to obtain NaErF4@NaYF4,: 30% Nd, and store at 4 ° C.

[0106] (3) Weigh 75 mg DSPE-PEG-NH2, take 0.1 mmol DCNPs (1 mL), add 5 mL chloroform, mix and ultrasonicate for 10 min, use a rotary evaporator to remove the chloroform, add 2 mL ultrapure water to disperse the material, centrifuge and wash three times at 14000 rpm for 10 min, and then disperse in 1 mL ultrapure water.

[0107] (4) Take 100ul DCNPs-PEG (about 2mg), weigh 17.5mg EDC and 11.5mg NHS, disperse in MES buffer with a pH of 5-6 and activate for 30min, then add 4mg GzmB responsive peptide (manufacturer: Nanjing GenScript Biotechnology Co., Ltd., model (production number): C765T511G0-1 / PE7896), use 0.1M sodium hydroxide solution to adjust the pH to 7-7.5, stir in the dark at 700rpm, overnight, then at 14000rpm for 10min, centrifuge and wash 3 times, disperse in 100ul ultrapure water, and store at 4°C.

[0108] (5) Weigh 1 mg of sensitizer IR808 and dissolve it in DMF to a concentration of 1 mg / mL. Weigh 2 mg of EDC and 2 mg of NHS and take 20 ug of IR808. Mix the three in MES buffer and activate for 30 min. Then add 90 ug of the product in step (4) and use MES buffer to replenish the system to 1 mL. Oscillate in the dark at 600 rpm overnight, then at 14,000 rpm for 10 min. Wash by centrifugation three times, disperse in 90 ul of ultrapure water and store at 4°C.

[0109] Embodiment 3:

[0110] In this embodiment, a ratiometric fluorescent probe is provided, and the preparation method thereof is as follows:

[0111] (1) Weigh 0.2075 g of yttrium acetate, 0.0736 g of ytterbium acetate, and 0.0072 g of erbium acetate, add them to a mixed solution of 6 mL of oleic acid and 15 mL of 1-octadecene, heat to 80°C, maintain vacuum for 35 min, continue to heat to 100°C, maintain heat for 25 min, heat to 150°C, maintain heat for 40 min, allow the rare earth acetate to completely dissolve, and cool to room temperature.

[0112] Weigh 148.16 mg of ammonium fluoride (NH4F) and 100 mg of sodium hydroxide (NaOH), dissolve them in 10 mL and 2.5 mL of methanol respectively, mix them, add them to the above solution, heat to 47.5 ° C, keep for 35 minutes, continue to heat to 65 ° C, remove methanol for 10 minutes, continue to heat to 80 ° C, keep vacuum for 20 minutes, continue to heat to 100 ° C, keep for 3 minutes, continue to heat to 298 ° C, keep warm for 50 minutes, and cool to room temperature. Add ethanol and cyclohexane to wash alternately, centrifuge 3 times at 6000g for 15 minutes, disperse in 10 mL of cyclohexane to obtain NaYF4: 20% Yb, 2% Er, and store at 4 ° C.

[0113] (2) Weigh 0.1862 g of yttrium acetate and 0.1018 g of neodymium acetate and add them to a mixed solution of 6 mL of oleic acid and 15 mL of 1-octadecene, heat to 80°C, maintain vacuum for 35 min, continue to heat to 100°C, maintain heat for 25 min, heat to 150°C, maintain heat for 40 min, allow the rare earth acetate to completely dissolve, and cool to room temperature.

[0114] Weigh 148.16 mg of ammonium fluoride (NH4F) and 100 mg of sodium hydroxide (NaOH), dissolve them in 10 mL and 2.5 mL of methanol respectively, add ethanol to the DCNPs in (1) and centrifuge at 6000 g for 10 min to precipitate, mix the three, add them to the above solution, heat to 47.5 ° C, keep for 35 min, continue to heat to 65 ° C, remove methanol for 10 min, continue to heat to 80 ° C, keep vacuum for 20 min, continue to heat to 100 ° C, keep for 3 min, continue to heat to 298 ° C, keep warm for 50 min, and cool to room temperature. Add ethanol and cyclohexane to wash alternately, centrifuge at 6000 g for 15 min 3 times, disperse in 10 mL of cyclohexane to obtain NaYF4,: 20% Yb, 2% Er @ NaYF4,: 30% Nd, and store at 4 ° C.

[0115] (3) Weigh 75 mg DSPE-PEG-NH2 and dissolve it in 200 ul tetrahydrofuran. Take 0.1 mmol DCNPs (1 mL), centrifuge to remove cyclohexane, dissolve it in 200 ul tetrahydrofuran, sonicate for 10 min, mix during sonication, sonicate for another 10 min, add it to 6 mL (5-10 mL) of ultrapure water, stir (100-500 rpm), evaporate tetrahydrofuran overnight (6-12 h), use an ultrafiltration tube (50-100 KDa), ultrafilter and concentrate, wash DCNPs-PEG, and disperse it in 1 mL of ultrapure water.

[0116] (4) Take 100ul DCNPs-PEG (about 2mg), weigh 17.5mg EDC and 11.5mg NHS, disperse in MES buffer with a pH of 5-6 and activate for 30min, then add 4mg GzmB responsive peptide (manufacturer: Nanjing GenScript Biotechnology Co., Ltd., model (production number): C9621HQYG0-1 / PE6503), use 0.1M sodium hydroxide solution to adjust the pH to 7-7.5, stir in the dark at 700rpm, overnight, then at 14000rpm for 10min, centrifuge and wash 3 times, disperse in 100ul ultrapure water, and store at 4°C.

[0117] (5) Weigh 1 mg of sensitizer IR806 and dissolve it in DMF to a concentration of 1 mg / mL. Weigh 2 mg of EDC and 2 mg of NHS. Take 20 μg of IR806 and mix the three in MES buffer. After activation for 30 min, add 90 μg of the product in step (4) and use MES buffer to replenish the system to 1 mL. Oscillate in the dark at 600 rpm for overnight, then at 14,000 rpm for 10 min. Wash by centrifugation three times, disperse in 90 μl of ultrapure water and store at 4°C.

[0118] The ratiometric fluorescent probes prepared in Examples 2 and 3 were also subjected to the same performance tests as in Example 1, and similar results were obtained.

[0119] Comparative Example 1

[0120] In this comparative example, a situation where different synthetic fluorescent probes of rare earth elements fail is provided, and the preparation method thereof is as follows:

[0121] (1) Weigh 0.4514 g of gadolinium nitrate and add it to a mixed solution of 6 mL of oleic acid and 15 mL of 1-octadecene. Heat to 80°C and keep under vacuum for 35 min. Continue to heat to 100°C and keep the temperature for 25 min. Heat to 150°C and keep the temperature for 40 min to completely dissolve the rare earth nitrate. Cool to room temperature.

[0122] Weigh 148.16 mg of ammonium fluoride (NH4F) and 100 mg of sodium hydroxide (NaOH), dissolve them in 10 mL and 2.5 mL of methanol respectively, mix them, add them to the above solution, heat to 47.5°C, keep for 35 minutes, continue to heat to 65°C, remove methanol for 10 minutes, continue to heat to 80°C, keep vacuum for 20 minutes, continue to heat to 100°C, keep for 3 minutes, continue to heat to 298°C, keep warm for 50 minutes, and cool to room temperature. Add ethanol and cyclohexane to wash alternately, centrifuge at 6000g for 15 minutes 3 times, disperse in 10 mL of cyclohexane to obtain NaGdF4, and store at 4°C.

[0123] (2) Weigh 0.1862 g of yttrium acetate and 0.1018 g of neodymium acetate and add them to a mixed solution of 6 mL of oleic acid and 15 mL of 1-octadecene, heat to 80°C, maintain vacuum for 35 min, continue to heat to 100°C, maintain heat for 25 min, heat to 150°C, maintain heat for 40 min, allow the rare earth acetate to completely dissolve, and cool to room temperature.

[0124] Weigh 148.16 mg of ammonium fluoride (NH4F) and 100 mg of sodium hydroxide (NaOH), dissolve them in 10 mL and 2.5 mL of methanol respectively, add ethanol to the DCNPs in (1) and centrifuge at 6000 g for 10 min to precipitate, mix the three, add them to the above solution, heat to 47.5 ° C, keep for 35 min, continue to heat to 65 ° C, remove methanol for 10 min, continue to heat to 80 ° C, keep vacuum for 20 min, continue to heat to 100 ° C, keep for 3 min, continue to heat to 298 ° C, keep warm for 50 min, and cool to room temperature. Add ethanol and cyclohexane to wash alternately, centrifuge 3 times at 6000 g for 15 min, disperse in 10 mL of cyclohexane to obtain NaGdF4@NaYF4,: 30% Nd, and store at 4 ° C.

[0125] Scanning transmission electron microscopy (STEM) and fluorescence spectroscopy showed that the probe synthesis failed, as shown below:

[0126] Scanning transmission electron microscopy: STEM shows the fine structure of Gd DCNPs Core-Shell structure nanoparticles. Fig. 9 , indicating that the distribution and morphology of DCNPs were detected at a scale of 50 nm.

[0127] Fluorescence spectrum: The fluorescence spectrum under 808nm and 980nm laser excitation shows the fluorescence emission performance of DCNPs structure. Fig.10, are the emission spectra of Gd-doped DCNPs and Er-doped DCNPs with the same concentration in the same organic phase under different laser excitations. The images show that the fluorescence signal of Er-doped DCNPs is significantly higher than that of Gd-doped DCNPs under 980nm laser excitation or 808nm laser excitation, and the 808nm signal of Gd-doped DCNPs is almost zero, proving the failure of the experiment.

[0128] The applicant declares that the present invention uses the above-mentioned embodiments to illustrate the ratio fluorescent probe for adoptive NK cell labeling and in vivo tracing and its application, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A ratiometric fluorescent probe, characterized in that: The ratio fluorescent probe comprises rare earth down-conversion nanoparticles with a core-shell structure, a granzyme B responsive polypeptide and a sensitizer. The surface of the rare earth down-conversion nanoparticles with a core-shell structure is coated with stearoyl phosphatidylethanolamine-polyethylene glycol. The granzyme B responsive polypeptide and the polyethylene glycol on the surface of the rare earth down-conversion nanoparticles with a core-shell structure, and the sensitizer and the granzyme B responsive polypeptide are connected through amide bonds formed by dehydration condensation of amino groups and carboxyl groups.

2. The ratiometric fluorescent probe according to claim 1, characterized in that The rare earth down-conversion nanoparticles with core-shell structure are NaYF4,:20%Yb,2%Er@NaYF4,:30%Nd, wherein NaYF4,:20%Yb,2%Er represents the core and NaYF4,:30%Nd represents the shell; Preferably, the granzyme B-responsive polypeptide is selected from a polypeptide containing the granzyme B-responsive sequence IEFDSG; Preferably, the granzyme B-responsive polypeptide is selected from GIEFDSGGC or KIEFDSGKC; Preferably, the sensitizer is selected from organic dyes having a large absorption cross section at the excitation signal; Preferably, the sensitizer is selected from IR808 or IR806.

3. The method for preparing a ratiometric fluorescent probe according to claim 1 or 2, characterized in that: The preparation method comprises the following steps: (1) Using rare earth acetate as a raw material, in the presence of a surface ligand, in a solvent, a coprecipitation method and a seed-mediated core-shell growth method are used to obtain rare earth down-conversion nanoparticles coated with surface ligands; (2) sonicating the rare earth down-conversion nanoparticles coated with surface ligands and the amphoteric material DSPE-PEG-NH2 in a solvent, removing the solvent, and centrifuging and washing to obtain the rare earth down-conversion nanoparticles coated with stearoyl phosphatidylethanolamine-polyethylene glycol that are stable in an aqueous phase; (3) dispersing the rare earth down-conversion nanoparticles coated with stearoyl phosphatidylethanolamine-polyethylene glycol described in step (2) in a buffer solution, adjusting the pH to 5-6, adding an activator for activation, and then adding a granzyme B responsive polypeptide, stirring, to obtain rare earth down-conversion nanoparticles modified with a granzyme B responsive polypeptide; (4) dispersing the sensitizer in a buffer solution, adjusting the pH to 5-6, adding an activator for activation, adding the rare earth down-conversion nanoparticles modified with the granzyme B-responsive polypeptide described in step (3), stirring in the dark, and obtaining the ratiometric fluorescent probe.

4. The preparation method according to claim 3, characterized in that: The rare earth acetate in step (1) comprises any one of yttrium acetate, ytterbium acetate, erbium acetate, gadolinium acetate, holmium acetate, thulium acetate or neodymium acetate, or a combination of at least two thereof; Preferably, the total molar amount of the rare earth element in the rare earth acetate in step (1) is 1; Preferably, the surface ligand in step (1) is oleic acid or oleylamine; Preferably, the solvent in step (1) is selected from 1-octadecene.

5. The preparation method according to claim 3 or 4, characterized in that: The rare earth down-conversion nanoparticles in step (1) are NaYF4,:20% Yb,2% Er@NaYF4,:30% Nd.

6. The preparation method according to claim 5, characterized in that: The preparation method of NaYF4,:20%Yb,2%Er@NaYF4,:30%Nd comprises the following steps: A. Dissolve yttrium acetate, ytterbium acetate and erbium acetate in a mixed solution of oleic acid and 1-octadecene to fully dissolve the rare earth acetate. Mix a methanol solution of ammonium fluoride and a methanol solution of sodium oxychloride at room temperature and add them to the rare earth acetate solution. Heat the obtained mixed solution, cool it to room temperature, wash it, and disperse it in cyclohexane to obtain the product NaYF4: 20% Yb, 2% Er. B. Dissolve yttrium acetate and neodymium acetate in a mixed solution of oleic acid and 1-octadecene to fully dissolve the rare earth acetate. At room temperature, mix a methanol solution of ammonium fluoride with a methanol solution of sodium oxychloride and an ethanol solution of NaYF4:20% Yb, 2% Er obtained in step A, and add the mixture to the rare earth acetate solution. Heat the mixture, cool it to room temperature, wash it, and disperse it in cyclohexane to obtain NaYF4:20% Yb, 2% Er@NaYF4:30% Nd.

7. The preparation method according to claim 6, characterized in that: The method for fully dissolving the rare earth acetate in step A and step B is to heat the mixed solution to 50°C-80°C, maintain vacuum for 20-35 minutes, raise the temperature to 90°C-110°C, keep warm for 10-20 minutes, continue to raise the temperature to 150°C-160°C, and keep warm for 30-50 minutes; Preferably, the mixed solution obtained in step A and step B is heated in the following manner: slowly heated to 47°C-50°C at a rate of 0.5°C-2°C / min, maintained for 30-50min, maintained in vacuum for 20min-30min when reaching 65°C-80°C, slowly heated to 100°C-120°C, maintained for 3-5min, and then heated to 298°C-300°C and stirred for 30-50min; Preferably, the molar ratio of yttrium acetate, ytterbium acetate, erbium acetate and ammonium fluoride in step A is 0.78:0.2:0.02:4; Preferably, the molar ratio of yttrium acetate, ytterbium acetate, erbium acetate and sodium hydroxide in step A is 0.78:0.2:0.02:2.5; Preferably, the molar ratio of yttrium acetate, neodymium acetate and ammonium fluoride in step B is 0.7:0.3:4; Preferably, the molar ratio of yttrium acetate, neodymium acetate and sodium hydroxide in step B is 0.7:0.3:2.5; Preferably, the washing in step A and step B is performed by alternating washing with cyclohexane and ethanol.

8. The preparation method according to any one of claims 3 to 7, characterized in that: In step (2), the mass ratio between the rare earth down-conversion nanoparticles coated with oleic acid and the amphoteric material DSPE-PEG-NH2 is 1:2-1:6; Preferably, the solvent in step (2) is chloroform or tetrahydrofuran; Preferably, the ultrasonication time in step (2) is 10 to 30 minutes; Preferably, the buffer solution in step (3) is a MES buffer solution; Preferably, the activating agent in step (3) is EDC and NHS; Preferably, in step (3), the molar ratio of the rare earth down-conversion nanoparticles coated with stearoyl phosphatidylethanolamine-polyethylene glycol to EDC is 1:5-1:15; Preferably, in step (3), the ratio of the amount of rare earth down-conversion nanoparticles coated with stearoyl phosphatidylethanolamine-polyethylene glycol, EDC and NHS is 1:10:10-1:10:20; Preferably, the activation time in step (3) is 20 to 60 minutes; Preferably, in step (3), the ratio of the amount of the granzyme B responsive polypeptide to the amount of DSPE-PEG-NH2 is 1:1-2:1; Preferably, the stirring time in step (3) is 6-24 hours; Preferably, the stirring in step (3) is followed by centrifugation and washing 2 to 3 times; Preferably, the buffer solution in step (4) is a MES buffer solution; Preferably, in step (4), the sensitizer is dissolved in an organic solvent and then dispersed in a buffer solution, and the organic solvent is selected from any one or a combination of at least two of DMF, DMSO or methanol; Preferably, the activating agent in step (4) is EDC and NHS; Preferably, the molar ratio of the rare earth down-conversion nanoparticles modified with the granzyme B-responsive polypeptide to the sensitizer in step (4) is 1:1-1:4; Preferably, the activation time in step (4) is 20 to 60 minutes; Preferably, the dark-proof stirring time in step (4) is 6-24 hours; Preferably, the stirring in the dark in step (4) is followed by centrifugation and washing 2 to 3 times.

9. Use of the ratiometric fluorescent probe according to claim 1 or 2 in adoptive NK cell labeling or in vivo tracing.

10. Use of the ratiometric fluorescent probe according to claim 1 or 2 in detecting the activation of adoptive NK cells.

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