A cisplatin diagnosis and treatment probe targeting carbonic anhydrase and a preparation method and application thereof

Abstract

The application belongs to the technical field of biological probes, and relates to a cisplatin diagnosis and treatment probe targeting carbonic anhydrase as well as a preparation method and application thereof. A phospholipid-polyethylene glycol compound and a NIR-II region fluorescent probe IR1040 are made into IRNPs-NH2 nanoparticles through a nano precipitation method, amino groups on the surface of the IRNPs-NH2 nanoparticles are covalently connected to p-sulfonamidobenzoic acid and a tetravalent platinum prodrug, and a cisplatin diagnosis and treatment probe IRNPs-SBA / Pt targeting carbonic anhydrase is formed IV . The cisplatin diagnosis and treatment probe can enhance the uptake of the cisplatin diagnosis and treatment probe in tumor cells by utilizing the strong affinity between the p-sulfonamidobenzoic acid group targeting carbonic anhydrase and inhibiting carbonic anhydrase activity and the carbonic anhydrase overexpressed on the surface of cells under a hypoxic condition, further improve the micro-acidity and hypoxic microenvironment of tumor cells, and is beneficial to inhibiting the migration of tumor cells and overcoming the drug resistance problem in cisplatin chemotherapy, and can effectively kill tumor cells.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological probes, and relates to a cisplatin diagnosis and treatment probe targeting carbonic anhydrase as well as a preparation method and application thereof. BACKGROUND

[0002] Carbonic anhydrase (CA) is a kind of metal enzyme in cells and is overexpressed in many tumor cells, such as glioblastoma, colorectal cancer, breast cancer, kidney cancer and pancreatic cancer. CA is involved in the regulation of hypoxic and acidic tumor microenvironment and has become an important target for cancer diagnosis and treatment. Therefore, it is of great significance to construct CA-targeted drugs for tumor imaging and treatment. However, the absorption and fluorescence of the existing CAs-targeted diagnosis and treatment drugs are not in the second near-infrared window (NIR-II) region, and mainly rely on photodynamic therapy (PDT), which may reduce the sensitivity and effectiveness of deep and highly hypoxic pancreatic tumor imaging and treatment.

[0003] Optically active nanoparticles with second near-infrared window (NIR-II) absorption and fluorescence emission provide prospects for cancer diagnosis and treatment. Compared with near-infrared first window (NIR-I) light, NIR-II region light has the advantages of low tissue scattering, deep tissue penetration depth and higher maximum permissible exposure dose (MPE) of laser, which is crucial for generating heat to improve photothermal therapy (PTT) of tumors. In addition, compared with NIR-I fluorescence, NIR-II fluorescence can enhance the penetration depth and reduce the background signal, thereby enhancing the signal-to-noise ratio (SBR) and sensitivity of in vivo imaging. Due to the existence of hypoxia and dense stromal tumor microenvironment (TME) in tumor tissues, the application of existing NIR-II region optically active nanodrugs in tumor treatment still has challenges. In addition, the delivery of NIR-II photons and NIR-II light-excited nanoparticles into deep tumors is not efficient, which greatly reduces the efficacy of PTT on tumors. The commonly used high-power density NIR-II light triggers tumor thermotherapy, which may cause damage and inflammation to the surrounding normal tissues, thereby causing unnecessary side effects. So far, there is still a lack of high-performance NIR-II optically active nanoparticles that can effectively enter tumors and achieve strong PTT efficacy with low-power NIR-II light excitation. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a cisplatin diagnosis and treatment probe targeting carbonic anhydrase as well as a preparation method and application thereof in view of the deficiencies of the prior art.

[0005] Inventive idea: In the present application, phospholipids (DSPE-PEG 2000 -OCH3 and DSPE-PEG 2000-NH2), NIR-II region fluorescent dye IR1040, sulfonamide benzoic acid (SBA) and cisplatin prodrug (Pt IV -COOH) was combined to design and synthesize a cisplatin-targeted carbonic anhydrase-based diagnostic probe IRNPs-SBA / Pt with the following characteristics. IV :First, phospholipids (DSPE-PEG 2000 -OCH3 and DSPE-PEG 2000 -NH2) loaded with NIR-II region fluorescent dye IR1040, phospholipids can improve the water solubility of IR1040 fluorescent molecules; then the amino groups on the phospholipid surface can be covalently linked to sulfonamide benzoic acid (SBA) that specifically recognizes CA and the chemotherapy cisplatin prodrug Pt IV -COOH. Among them, (1) when SBA combines with extracellular CA, it increases the IV SBA can be taken up in the tumor site, and at the same time, it can inhibit the activity of CA, further improve the hypoxic and slightly acidic environment of tumor cells and inhibit the migration of tumor cells; (2) Under low-power NIR-II light excitation, the high photothermal conversion efficiency generated can be used for photothermal treatment of tumors, and the IR1040 fluorescent molecule can generate NIR-II fluorescence for NIR-II fluorescence imaging, and can also generate high photothermal conversion efficiency for photothermal treatment; (3) IRNPs-SBA / Pt IV Under the reduction of glutathione (GSH) highly expressed in tumor cells, the divalent cisplatin original drug is released for chemotherapy of tumors in vivo. The cisplatin diagnostic and therapeutic probe IRNPs-SBA / Pt targeting carbonic anhydrase prepared by the present invention IV It has good applications in NIR-II fluorescence imaging, preparation of anti-tumor drugs, and preparation of tumor diagnostic imaging contrast agents.

[0006] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0007] The present invention discloses a cisplatin diagnostic and therapeutic probe targeting carbonic anhydrase. A phospholipid-polyethylene glycol compound and a NIR-II region fluorescent probe IR1040 are prepared into IRNPs-NH2 nanoparticles by a nanoprecipitation method. The amino groups on the surface of the IRNPs-NH2 nanoparticles are covalently linked to p-sulfonamide benzoic acid and a tetravalent platinum prodrug to form a cisplatin diagnostic and therapeutic probe targeting carbonic anhydrase.

[0008] in,

[0009] The phospholipid-polyethylene glycol compound is DSPE-PEG 2000 -OCH3 and DSPE-PEG 2000 -NH2;

[0010] The structural formula of the NIR-II region fluorescent probe IR1040 is:

[0011]

[0012] The tetravalent platinum prodrug is Pt IV -COOH.

[0013] The phospholipid-polyethylene glycol compound and the NIR-II region fluorescent probe IR1040 are prepared into IRNPs-NH2 nanoparticles by a nanometer precipitation method, the fluorescent probe IR1040 is loaded in the phospholipid-polyethylene glycol compound, and the NIR-II region fluorescent probe IR1040 in the cisplatin diagnosis and treatment probe realizes fluorescence imaging through laser irradiation or realizes photothermal treatment of tumor cells by converting light energy into heat energy through laser irradiation.

[0014] The carbonic anhydrase is a carbonic anhydrase (CA) on the surface of Pan02 cell membranes and / or Hela cell membranes under hypoxic conditions.

[0015] The tetravalent platinum prodrug, i.e., Pt IV The preparation method of -COOH can refer to the prior art Chem.Sci., 2016, 7, 2864.

[0016] In some embodiments, the NIR-II region fluorescent probe IR1040 is prepared by the following steps:

[0017] 1,8-naphthalimide is subjected to a substitution reaction with 5-chloropent-1-yne in the presence of potassium iodide to obtain an intermediate 1; the intermediate 1 is subjected to a Grignard reaction with methyl Grignard reagent under inert gas protection and in anhydrous conditions, and after the Grignard reaction is completed, potassium iodide is added for precipitation to obtain an intermediate 2; the intermediate 2 is subjected to a coupling reaction with 2-chloro-3-(hydroxymethylene)-1-cyclohexen-1-carbaldehyde in the presence of a base and an acid anhydride to obtain the NIR-II region fluorescent probe IR1040.

[0018]

[0019] In some embodiments, the molar ratio of the 1,8-naphthalimide to the 5-chloropent-1-yne and the potassium iodide is 1:1.0-2.0:3.0-4.5; the substitution reaction is performed at a reaction temperature of 120-150°C for 24-48 h.

[0020] In some embodiments, preferably, the molar ratio of the 1,8-naphthalene dicarboxylic acid, 5-chloropent-1-yn-3-ol, potassium iodide is 1:1.5:3.7; the reaction temperature of the substitution reaction is 140°C, and the reaction time is 36h; wherein, the solvent used in the substitution reaction is preferably N,N-dimethylformamide, acetonitrile or acetone, more preferably N,N-dimethylformamide, and the amount of solvent is not limited, as long as the solid raw materials in the system are dissolved and the viscosity is moderate.

[0021] In some embodiments, the methyl Grignard reagent is methyl magnesium chloride; the molar ratio of the intermediate 1, the methyl Grignard reagent, potassium iodide is 1:3.5-5.5:1.5-3.5; the reaction temperature of the Grignard reaction is 55-80°C, and the reaction time is 1-2h; and the inert gas is nitrogen.

[0022] In some embodiments, preferably, the methyl Grignard reagent is methyl magnesium chloride; the molar ratio of the intermediate 1, the methyl Grignard reagent, potassium iodide is 1:4.5:2; the reaction temperature of the Grignard reaction is 60°C, and the reaction time is 1.5h; the inert gas is nitrogen; wherein, the methyl Grignard reagent exists in the form of a solution, the solvent is anhydrous tetrahydrofuran, and the concentration of the methyl Grignard reagent is preferably 3M; the solvent used in the Grignard reaction is preferably anhydrous tetrahydrofuran, and the amount of solvent is not limited, as long as the solid raw materials in the system are dissolved and the viscosity is moderate; and after the Grignard reaction is completed, hydrochloric acid needs to be added to neutralize the unreacted methyl Grignard reagent, the molar ratio of the intermediate 1, the hydrochloric acid is 1:8-12, preferably 1:10, and the hydrochloric acid exists in the form of a 1 mol / L hydrochloric acid aqueous solution.

[0023] In some embodiments, the base is triethylamine or N,N-diisopropyl ethyl amine; the acid anhydride is acetic anhydride; the molar ratio of the intermediate 2, 2-chloro-3-(hydroxy methylene)-1-cyclohexene-1-formaldehyde is 1-2.5:1; the molar volume ratio of the intermediate 2, the base, the acid anhydride is 0.65mmol:0.4-0.6mL:0.4-0.6mL; and the reaction temperature of the coupling reaction is 50-70°C, and the reaction time is 20-60min.

[0024] In some embodiments, preferably, the base is triethylamine; the acid anhydride is acetic anhydride; the molar ratio of the intermediate 2, 2-chloro-3-(hydroxy methylene)-1-cyclohexene-1-formaldehyde is 1.3:1; the molar volume ratio of the intermediate 2, the base, the acid anhydride is 0.65mmol:0.5mL:0.5mL; the reaction temperature of the coupling reaction is 60°C, and the reaction time is 30min; wherein, the solvent used in the coupling reaction is preferably acetic acid, and the amount of solvent is not limited, as long as the solid raw materials in the system are dissolved and the viscosity is moderate.

[0025] In some embodiments, the p-sulfonamide benzoic acid group on the surface of the cisplatin diagnostic and therapeutic probe can target carbonic anhydrase highly expressed on the surface of tumor cells, inhibit the activity of carbonic anhydrase, thereby alleviating the hypoxic environment of tumor cells, lowering extracellular pH and inhibiting tumor cell migration; the cisplatin diagnostic and therapeutic probe releases divalent platinum raw drug under the reduction of glutathione highly expressed in tumor cells, thereby achieving chemotherapy of tumor cells; wherein, the divalent platinum raw drug is cisplatin.

[0026] Among them, the relief of the hypoxic environment of tumor cells promotes the production of reactive oxygen species.

[0027] In some embodiments, the NIR-II region fluorescent probe IR1040 in the cisplatin diagnostic and therapeutic probe achieves fluorescence imaging through a first laser irradiation; wherein the wavelength of the first laser irradiation is 980 nm or 1064 nm.

[0028] In some embodiments, the NIR-II region fluorescent probe IR1040 in the cisplatin diagnostic and therapeutic probe converts light energy into heat energy through a second laser irradiation to achieve photothermal therapy of tumor cells; wherein, the second laser irradiation has a wavelength of 1064 nm and a power density of 0.5 W·cm -2 , the irradiation time is 3 to 10 minutes.

[0029] Furthermore, the present invention discloses a method for preparing the above-mentioned cisplatin diagnostic and therapeutic probe targeting carbonic anhydrase, comprising the following steps:

[0030] (1) Mix IR1040 stock solution with DSPE-PEG 2000 -OCH3、DSPE-PEG 2000 -NH2 and the first solvent are mixed and added to deionized water, and ultrasonication is performed during the addition process. After the ultrasonication, the reaction solution is washed and concentrated by centrifugation, and PBS buffer is added to obtain an IRNPs-NH2 nanoparticle stock solution containing IR1040;

[0031] (2) mixing a 4-aminosulfonylbenzoic acid stock solution with O-benzotriazole-tetramethyluronium hexafluorophosphate, reacting the mixture at -10 to 0°C for 10 to 20 minutes to obtain a first mixed solution; mixing the first mixed solution with the IRNPs-NH2 nanoparticle stock solution containing IR1040 obtained in step (1), adding N,N-diisopropylethylamine, and stirring at room temperature for 4 to 8 hours. After the reaction is completed, the reaction solution is centrifuged and concentrated, and PBS buffer is added to obtain an IRNPs-SBA nanoparticle stock solution containing IR1040;

[0032] (3) mixing the cisplatin prodrug stock solution with O-benzotriazole-tetramethyluronium hexafluorophosphate, and reacting at -10-0℃ for 10-20 minutes to obtain a second mixed solution; mixing the second mixed solution with the IRNPs-SBA nanoparticle stock solution containing IR1040 obtained in step (2), adding N,N-diisopropylethylamine, stirring at room temperature for 4-8 hours, and then centrifuging and concentrating the reaction solution to obtain IRNPs-SBA / Pt containing IR1040 IV nanoparticle stock solution, i.e., a cisplatin diagnosis and treatment probe targeting carbonic anhydrase.

[0033] In some embodiments, the first solvent is tetrahydrofuran or ethanol; the solvent in the IR1040 stock solution is dimethyl sulfoxide; the molar mass ratio of IR1040 to DSPE-PEG 2000 -OCH3, DSPE-PEG 2000 -NH2 is 0.27 μmol:8 mg:2 mg; the mass-volume ratio of DSPE-PEG 2000 -NH2 to the first solvent is 2 mg:0.5-1.5 mL; the volume ratio of the first solvent to deionized water is 0.5-1.5:8-10; the ultrasonic temperature is room temperature, the ultrasonic power is 40 KHz, and the ultrasonic time is 10-15 min; the PBS buffer is 1×, pH 7.4; the concentration of IR1040 in the IRNPs-NH2 nanoparticle stock solution containing IR1040 is 540 μmol / L; the solvent in the 4-aminosulfonylbenzoic acid stock solution is N,N-dimethylformamide, and the solubility of 4-aminosulfonylbenzoic acid in the solution is 1 mg·mL -1 ; the mass ratio of 4-aminosulfonylbenzoic acid to O-benzotriazole-tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine in the 4-aminosulfonylbenzoic acid stock solution is 0.05:0.8-1.0:9.0-10.0; the mass ratio of 4-aminosulfonylbenzoic acid to DSPE-PEG 2000 -NH2 in the 4-aminosulfonylbenzoic acid stock solution is 0.05:2; the concentration of IR1040 in the IRNPs-SBA nanoparticle stock solution containing IR1040 is 540 μmol / L; the solvent in the cisplatin prodrug stock solution is N,N-dimethylformamide, and the concentration of cisplatin prodrug in the solution is 1 mg·mL -1 ; the mass ratio of cisplatin prodrug to O-benzotriazole-tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine in the cisplatin prodrug stock solution is 0.05:0.8-1.0:9.0-10.0; the mass ratio of cisplatin prodrug to DSPE-PEG 2000-NH2 mass ratio is 0.05:2; the IRNPs-SBA / Pt containing IR1040 IV The concentration of IR1040 in the nanoparticle stock solution was 540 μmol / L.

[0034] In some embodiments, preferably, in some embodiments, the first solvent is tetrahydrofuran; the solvent in the IR1040 stock solution is dimethyl sulfoxide, further preferably, the concentration of IR1040 in the IR1040 stock solution is 50 mmol / L; the IR1040 in the IR1040 stock solution is DSPE-PEG 2000 -OCH3、DSPE-PEG 2000 The molar mass ratio of -NH2 is 0.27 μmol: 8 mg: 2 mg; the DSPE-PEG 2000 The mass volume ratio of -NH2 to the first solvent was 2 mg:1 mL; the volume ratio of the first solvent to deionized water was 1:9; the ultrasound temperature was room temperature, the ultrasound power was 40 kHz, and the ultrasound time was 10 min; the PBS buffer was 1×, pH 7.4; the concentration of IR1040 in the IRNPs-NH2 nanoparticle stock solution containing IR1040 was 540 μmol / L; the solvent in the 4-aminosulfonylbenzoic acid stock solution was N,N-dimethylformamide, and the solubility of 4-aminosulfonylbenzoic acid in the solution was 1 mg·mL -1 The mass ratio of 4-aminosulfonylbenzoic acid to O-benzotriazole-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine in the 4-aminosulfonylbenzoic acid stock solution is 0.05:0.9:9.6; the mass ratio of 4-aminosulfonylbenzoic acid to DSPE-PEG in the 4-aminosulfonylbenzoic acid stock solution is 0.05:0.9:9.6. 2000 The mass ratio of IR1040 to IRNPs-SBA nanoparticle stock solution containing IR1040 is 0.05:2; the concentration of IR1040 in the IRNPs-SBA nanoparticle stock solution containing IR1040 is 540 μmol / L; the solvent in the cisplatin prodrug stock solution is N,N-dimethylformamide, and the concentration of cisplatin prodrug in the solution is 1 mg·mL -1 The mass ratio of cisplatin prodrug to O-benzotriazole-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine in the cisplatin prodrug stock solution is 0.05:0.9:9.6; the mass ratio of cisplatin prodrug to DSPE-PEG in the cisplatin prodrug stock solution is 0.05:0.9:9.6. 2000 -NH2 mass ratio is 0.05:2; the IRNPs-SBA / Pt containing IR1040 IV The concentration of IR1040 in the nanoparticle stock solution was 540 μmol / L.

[0035] The application of the above-mentioned carbonic anhydrase-targeted cisplatin diagnosis and treatment probe in preparing an anti-tumor drug is also within the protection scope of the present application.

[0036] The tumor is preferably a cervical cancer tumor or a pancreatic cancer tumor, and more preferably a pancreatic cancer tumor.

[0037] The application of the above-mentioned carbonic anhydrase-targeted cisplatin diagnosis and treatment probe in preparing a tumor photothermal treatment and / or chemotherapy drug is also within the protection scope of the present application.

[0038] The tumor is preferably a cervical cancer tumor or a pancreatic cancer tumor, and more preferably a pancreatic cancer tumor.

[0039] The application of the above-mentioned carbonic anhydrase-targeted cisplatin diagnosis and treatment probe in preparing a tumor diagnosis reagent is also within the protection scope of the present application.

[0040] The tumor is preferably a cervical cancer tumor or a pancreatic cancer tumor, and more preferably a pancreatic cancer tumor.

[0041] The application of the above-mentioned carbonic anhydrase-targeted cisplatin diagnosis and treatment probe in preparing a tumor NIR-II region imaging contrast agent is also within the protection scope of the present application.

[0042] The tumor is preferably a cervical cancer tumor or a pancreatic cancer tumor, and more preferably a pancreatic cancer tumor.

[0043] The application of the above-mentioned carbonic anhydrase-targeted cisplatin diagnosis and treatment probe in preparing a tumor NIR-II region fluorescence imaging / photothermal imaging contrast agent is also within the protection scope of the present application.

[0044] The tumor is preferably a cervical cancer tumor or a pancreatic cancer tumor, and more preferably a pancreatic cancer tumor.

[0045] In the present application, the "hypoxic" and "normoxic" conditions, unless otherwise specified, contain 1% v / v O2 under the hypoxic condition and 20% v / v O2 under the normoxic condition.

[0046] Beneficial effects:

[0047] (1) In the present application, based on the NIR-II region light-excitable IR1040, CA-targeted and clinical first-line chemotherapy anticancer drug cisplatin prodrug, CA-targeted NIR-II region cisplatin diagnosis and treatment nanoparticles IRNPs-SBA / Pt are designed and prepared. IV For PTT and chemotherapy combined treatment under the guidance of NIR-II region fluorescence imaging of pancreatic cancer.

[0048] (2) In the present application, by utilizing the strong affinity of the p-sulfonamidobenzoic acid group (SBA) to CA and the overexpression of carbonic anhydrase (CA) on the cell surface under hypoxic conditions, the uptake of IRNPs-SBA / Pt in tumor cells can be enhanced IV Uptake in tumor cells and IRNPs-SBA / Pt IV Release of Pt (II) under GSH reduction. IRNPs-SBA / Pt IV The SBA on the surface of the tumor cell can significantly inhibit the activity of CA while targeting the overexpression of CA on the surface of hypoxic tumor cells, further improving the micro-acidic and hypoxic microenvironment of tumor cells, which is conducive to inhibiting the migration of tumor cells and overcoming the drug resistance problem faced in subsequent cisplatin chemotherapy.

[0049] (3) Compared with the small molecule cisplatin or the pre-prepared Pt (IV) nanoparticles in the prior art, the cisplatin prodrug is covalently connected to IRNPs-SBA in the present application, which improves the significant chemotherapeutic effect in the treatment of subcutaneous pancreatic cancer tumor model due to the high uptake in tumor sites. At the same time, the cisplatin prodrug needs to be reduced by GSH which is highly expressed in tumor sites to release Pt II with chemotherapeutic effect, reducing the toxic side effects on normal tissues.

[0050] (4) Compared with the nanoparticles targeting the high expression of biomarkers in the tumor in the prior art, the diagnosis and treatment probe in the present application has SBA targeting the overexpression of CA on the surface of hypoxic tumor, which can significantly inhibit the activity of CA while specifically targeting the tumor, thereby causing the improvement of the hypoxic tumor microenvironment, which is conducive to the efficient treatment of tumor.

[0051] (5) Compared with the combination therapy of photothermal therapy and chemotherapy in the prior art, the present application can achieve high-efficiency photothermal conversion efficiency (65.17%) under low-power laser irradiation (0.5 W / cm 2 ), and the dosage of the chemotherapy prodrug (2.0 mg / kg Pt) is also lower than that of the chemotherapy drugs in the prior art.

[0052] (6) In the present application, by utilizing NIR-II region fluorescence imaging, imaging of deep pancreatic sites can be achieved, overcoming the problems faced by other nanoparticles in the prior art when imaging deep tissue penetration depth of diseased tissues. The enzyme-targeted diagnosis and treatment probe IRNPs-SBA / Pt IVNIR-II fluorescence imaging and photothermal imaging can be realized, so that the hypoxic tumor cells can be detected and imaged with high sensitivity and high penetration depth. And by using the PTT produced by inhibiting the CA activity and low-power irradiation and the cisplatin chemotherapy drug reduced by GSH, the "visual" tumor treatment guided by the NIR-II region fluorescence imaging signal of the tumor region is realized, and the tumor cells are further rapidly and effectively killed. BRIEF DESCRIPTION OF DRAWINGS

[0053] The above and / or other aspects of the present application will become apparent and more readily appreciated from the following description, taken in conjunction with the accompanying drawings, in which:

[0054] Figure 1 Synthesis route chart of NIR-II region dye IR1040.

[0055] Figure 2 NMR hydrogen spectrum chart of intermediate 1 prepared in Example 1.

[0056] Figure 3 NMR carbon spectrum chart of intermediate 1 prepared in Example 1.

[0057] Figure 4 High resolution mass spectrum chart of intermediate 1 prepared in Example 1.

[0058] Figure 5 High resolution mass spectrum of intermediate 2 prepared in Example 1.

[0059] Figure 6 NMR hydrogen spectrum chart of NIR-II region fluorescence probe IR1040 prepared in Example 1.

[0060] Figure 7 NMR carbon spectrum chart of NIR-II region fluorescence probe IR1040 prepared in Example 1.

[0061] Figure 8 Maldi-TOF spectrum of NIR-II region fluorescence probe IR1040 prepared in Example 1.

[0062] Figure 9 Design of carbonic anhydrase targeted cisplatin diagnosis and treatment probe IRNPs-SBA / Pt IV and its mechanism chart; wherein, Figure 9 a is a preparation chart, Figure 9 b is a mechanism chart, Figure 9 c is the chemical structure of IR1040, Pt IV -COOH, SBA, cisplatin and the cartoon of CA.

[0063] Figure 10TEM images, DLS analysis and absorption / fluorescence spectra of carbonic anhydrase-targeted cisplatin theranostic probe IRNPs-SBA / Pt IV TEM images, DLS analysis and absorption / fluorescence spectra of carbonic anhydrase-targeted cisplatin theranostic probe IRNPs-SBA / Pt Figure 10 a is TEM images, Figure 10 b is DLS analysis images, Figure 10 c is absorption / fluorescence spectra.

[0064] Figure 11 TEM images, DLS analysis and absorption / fluorescence spectra of carbonic anhydrase-targeted cisplatin theranostic probe IRNPs-SBA / Pt IV TEM images, DLS analysis and absorption / fluorescence spectra of carbonic anhydrase-targeted cisplatin theranostic probe IRNPs-SBA / Pt

[0065] Figure 12 TEM images, DLS analysis and absorption / fluorescence spectra of carbonic anhydrase-targeted cisplatin theranostic probe IRNPs-SBA / Pt IV TEM images, DLS analysis and absorption / fluorescence spectra of carbonic anhydrase-targeted cisplatin theranostic probe IRNPs-SBA / Pt Figure 12 a is binding constant, Figure 12 b is Pt release ability evaluation.

[0066] Figure 13 NIR-II region fluorescence microscopy imaging and cellular uptake ability data of enzyme-targeted cisplatin theranostic probe IRNPs-SBA / Pt IV NIR-II region fluorescence microscopy imaging and cellular uptake ability data of enzyme-targeted cisplatin theranostic probe IRNPs-SBA / Pt Figure 13 a is NIR-II FLI of the specified probe after incubating Pan02 cells under hypoxia or normoxia; Figure 13 b is NIR-II FLI of cell clusters after incubation of control group, IRNPs-Pt IV and IRNPs-SBA / Pt IV ; Figure 13 c is the corresponding quantified fluorescence signal of NIR-II FLI of cell clusters after incubation of control group, IRNPs-Pt IV and IRNPs-SBA / Pt IV ; Figure 13 d is the cellular uptake of cisplatin, IRNPs-Pt IV and IRNPs-SBA / Pt IV (20 μM) after treating Pan02 cells under hypoxia or normoxia, respectively.

[0067] Figure 14 Influence of nanoprobes on extracellular pH value of Pan02 cells and influence on HIF-1α expression in Pan02 cells under different conditions; wherein, Figure 14 a is influence on pH value, Figure 14 b is influence on HIF-1α expression.

[0068] Figure 15ROS assessment and flow cytometry analysis of Pan02 cells treated with the designated probes under hypoxia; wherein, Figure 15 a ROS assessment, Figure 15 b flow cytometry analysis.

[0069] Figure 16 Scratch test of Pan02 cells under hypoxia.

[0070] Figure 17 Cell survival rate data graph of Pan02 cells incubated with the designated probes; wherein, I: IRNPs-SBA (normoxia); II: IRNPs-SBA + laser (normoxia); III: IRNPs-SBA + laser (hypoxia); IV: CisPt (hypoxia); V: IRNPs-SBA / Pt + laser (normoxia); VI: IRNPs-SBA / Pt + laser (hypoxia). IV IV

[0071] Figure 18 Flow cytometry analysis of apoptosis of Pan02 cells treated with the designated probes under hypoxia; wherein, I: PBS; II: laser; III: IRNPs-SBA / Pt + laser. IV IV

[0072] Figure 19 NIR-II FLI of a mouse subcutaneous pancreatic tumor model at different time points after tail vein injection of the designated probes, corresponding fluorescence signal and signal-to-background ratio, and NIR-II fluorescence microscopy imaging of tumor sections.

[0073] Figure 20 NIR-II FLI of different organs ex vivo and uptake of different organs after tail vein injection of IRNPs-SBA / Pt IV ; wherein, Figure 20 a NIR-II FLI of different organs ex vivo, Figure 20 b uptake of different organs; wherein, the organs include heart (H), liver (Li), spleen (Sp), lung (Lu), kidney (Ki), and tumor (T).

[0074] Figure 21 IR thermal imaging of 1064 nm laser irradiation and temperature change curve at the tumor site of mice 24 h after tail vein injection of the designated probes; wherein, Figure 21 a IR thermal imaging, Figure 21 b temperature change curve at the tumor site.​​​​

[0075] Figure 22 Figure 6 is a graph showing the immunofluorescence staining of CA IX expression of tumor tissue sections with pimonidazole (HP6) antibody and HIF-1a antibody; wherein, I: saline; II: IRNPs-OCH3; III: IRNPs-SBA / Pt IV Scale bar: 50 pm.

[0076] Figure 23 Figure 8 is a graph showing the combined PTT and chemotherapy treatment data of tumor by enzyme-targeted cisplatin diagnostic and therapeutic probe IRNPs-SBA / Pt IV; wherein, Figure 23 a is a schematic diagram of Pan02 tumor treatment in mice, Figure 23 b is a photograph of mice before treatment (day 0) and after treatment (day 21), Figure 23 c is the change in tumor volume of mice during treatment, Figure 23 d is the change in body weight of mice, Figure 23 e is a graph showing H&E and TUNEL staining of Pan02 tumor sections; wherein, I: saline; II: saline + laser; III: IRNPs-SBA / Pt IV ; IV: IRNPs-OCH3 + laser; V: IRNPs-SBA + laser; VI: IRNPs-SBA / Pt IV + laser; VII: IRNPs-SBA / Pt IV + laser + IRNPs-SBA / Pt IV (2 doses) at 24 h. Laser: 1064 nm, 0.5 W cm -2 , 10 min. Scale bar: 50 pm. DETAILED DESCRIPTION

[0077] The application will be further described below in conjunction with the drawings and specific examples, but the scope of protection of the application is not limited thereto. In the following examples, the experimental methods described are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0078] The mouse pancreatic cancer Pan02 cells involved in the following examples were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences, and were cultured in DMEM (Dulbecco's Modified Eagle Medium) medium. The medium was supplemented with 10% (v / v) fetal bovine serum (FBS), and 100 units of penicillin and 100 units of streptomycin were added per milliliter of medium. All cells were cultured in a humidified environment (5% CO2) at 37°C.

[0079] Unless otherwise specified, the "hypoxia and normoxia" conditions described in the following examples refer to hypoxia (1% v / v O2) and normoxia (20% v / v O2) (Normoxia).

[0080] Carbonic anhydrase-targeted cisplatin therapeutic probe IRNPs-SBA / Pt designed in the present invention IV The design and mechanism of action of Figure 9 As shown. Carbonic anhydrase-targeted cisplatin therapeutic probe IRNPs-SBA / Pt IV It is prepared by nanoprecipitation and two-step condensation reaction. Specifically, phospholipid-polyethylene glycol compounds and the prepared NIR-II region fluorescent probe IR1040 are first prepared into IRNPs-NH2 by nanoprecipitation, and then p-sulfonamide benzoic acid and tetravalent platinum prodrug are covalently linked to NH2 on the surface of IRNPs-NH2 through condensation reaction to prepare IRNPs-SBA and IRNPs-SBA / Pt. IV When IRNPs-SBA / Pt IV After entering the living body, IRNPs-SBA / Pt IV It can selectively target hypoxic tumor cells and increase its enrichment on tumor cells. IV After the SBA on Pt reacts with CA, it can significantly inhibit the activity of CA and further improve the micro-acidic environment and hypoxic microenvironment of hypoxic tumor cells. Among them, the improvement of the hypoxic microenvironment of tumor cells is conducive to overcoming the drug resistance of chemotherapy drugs during chemotherapy. IV After internalization into tumor cells, under 1064nm laser irradiation, NIR-II fluorescence imaging can be achieved while simultaneously implementing NIR-II photothermal therapy. Furthermore, GSH, which is highly expressed within tumor cells, can reduce the Pt(IV) prodrug to the chemotherapeutic Pt(II). Therefore, the combined treatment of NIR-II photothermal therapy and Pt(II) chemotherapy can achieve significant therapeutic effects on tumor cells.

[0081] Example 1

[0082] 1. Synthesis of NIR-II fluorescent probe IR1040

[0083] The synthetic route of IR1040 is as follows Figure 1 The specific synthesis steps are as follows:

[0084]

[0085] (a) Preparation of Intermediate 1:

[0086] The intermediate 1 (118 mg, 0.5 mmol, prepared in step (a)) was dissolved in 2 mL of anhydrous THF solution, and 0.75 mL of methyl magnesium chloride tetrahydrofuran solution (concentration of 3 M, containing methyl magnesium chloride 2.25 mmol) was added dropwise under N2protection. The Grignard reaction was carried out at 60 °C for 1.5 hours under N2protection. After the reaction was completed, the reaction solution was cooled to room temperature, and 5 mL of hydrochloric acid aqueous solution (concentration of 1 M, containing hydrochloric acid 5 mmol) was added dropwise. After stirring, the reaction solution was removed of THF by rotary evaporation, and then 1 mL of KI aqueous solution (concentration of 1 M, containing KI 1 mmol) was added for precipitation. Filtration gave a red precipitate, which was intermediate 2. The high resolution mass spectrum of intermediate 2 is shown in Figure 2 , the carbon nuclear magnetic resonance spectrum is shown in Figure 3 , the high resolution mass spectrum is shown in Figure 4 , [M+H] + Found 236.1062.

[0087] (b) Preparation of intermediate 2:

[0088] The intermediate 1 (118 mg, 0.5 mmol, prepared in step (a)) was dissolved in 2 mL of anhydrous THF solution, and 0.75 mL of methyl magnesium chloride tetrahydrofuran solution (concentration of 3 M, containing methyl magnesium chloride 2.25 mmol) was added dropwise under N2protection. The Grignard reaction was carried out at 60 °C for 1.5 hours under N2protection. After the reaction was completed, the reaction solution was cooled to room temperature, and 5 mL of hydrochloric acid aqueous solution (concentration of 1 M, containing hydrochloric acid 5 mmol) was added dropwise. After stirring, the reaction solution was removed of THF by rotary evaporation, and then 1 mL of KI aqueous solution (concentration of 1 M, containing KI 1 mmol) was added for precipitation. Filtration gave a red precipitate, which was intermediate 2. The high resolution mass spectrum of intermediate 2 is shown in Figure 5 , [M-I] + Found 234.1271.

[0089] (c) Preparation of fluorescent probe IR1040:

[0090] The intermediate 2 (234.32 mg, 0.65 mmol) and 2-chloro-3-(hydroxy methylene)-1- cyclohexen-1-carboxaldehyde (86.0 mg, 0.5 mmol) were dissolved in 1 mL of acetic acid, and 0.5 mL of triethylamine and 0.5 mL of acetic anhydride solution were added. The coupling reaction was carried out at 60 °C for 30 minutes. After the reaction was completed, the reaction solution was cooled to room temperature, 10 mL of ethyl acetate was added, and recrystallization and filtration were carried out with EtOH. The solid crude product was purified by HPLC to obtain fluorescent probe IR1040 (259.21 mg, 71% yield), a dark green solid. The hydrogen nuclear magnetic resonance spectrum of NIR-II region fluorescent probe IR1040 is shown inFigure 6 As shown, the NMR carbon spectrum is as follows Figure 7 As shown, the Maldi-TOF spectrum is as follows Figure 8 shown.

[0091] 2. Tetravalent platinum prodrug Pt IV Preparation of -COOH

[0092] tetravalent platinum prodrug Pt IV For the specific synthesis steps of -COOH, please refer to the prior art Chem. Sci., 2016, 7, 2864.

[0093] 3. Preparation of diagnostic probes

[0094] 3.1 Preparation of IRNPs-OCH3 Nanoparticles

[0095] 5.4 μL of IR1040 DMSO stock solution (50 mM) was mixed with DSPE-PEG 2000 -OCH3 (10 mg) was dissolved in 1 mL of tetrahydrofuran (THF) to form a clear solution, which was then rapidly injected into 9 mL of deionized water under ultrasonication (ultrasonication temperature at room temperature and ultrasonic power at 40 kHz) for 10 minutes. The resulting aqueous solution was washed three times with deionized water and further concentrated using a 10 kDa ultrafiltration tube at 4,000 rpm for 15 minutes. After concentration, 1 mL of PBS buffer (1×, pH 7.4) was added to obtain an IRNPs-OCH3 stock solution containing IR1040. The concentration of the stock solution IR1040 was approximately 540 μM, which was recorded as IRNPs-OCH3 nanoparticles.

[0096] 3.2. Cisplatin-targeted theranostic probe IRNPs-SBA / Pt IV Preparation

[0097] IR1040 and tetravalent platinum prodrug Pt prepared under items "1" and "2" of this embodiment respectively IV -COOH was used in this step.

[0098] (1) Preparation of IRNPs-NH2 nanoparticles:

[0099] 5.4 μL of IR1040 DMSO stock solution (50 mM) was mixed with DSPE-PEG 2000 -OCH3 (8 mg) and DSPE-PEG 2000-NH2(2 mg) was dissolved in 1 mL of tetrahydrofuran (THF) to form a clear solution, which was then quickly injected into 9 mL of deionized water under ultrasonication (ultrasonic temperature was room temperature, ultrasonic power was 40 KHz) for 10 min. The obtained aqueous solution was washed with deionized water three times, and further concentrated for 15 min under centrifugation at 4,000 r.p.m. using a 10 KDa ultrafiltration tube. After the end of concentration, 1 mL of PBS buffer (1x, pH 7.4) was added to obtain an IRNPs-NH2 nanoparticle stock solution containing IR1040, and the concentration of IR1040 was about 540 mM, which was recorded as IRNPs-NH2 nanoparticles.

[0100] (2) Preparation of IRNPs-SBA nanoparticles:

[0101] To 50 mL of a DMF solution of 4-aminosulfonylbenzoic acid (SBA) with a concentration of 1 mg mL -1 , 0.9 mg of O-benzotriazol- tetramethyluronium hexafluorophosphate (HBTU) was added, and the first mixture was obtained by reacting at 0°C for 10 min. Then, the first mixture was added to the IRNPs-NH2 nanoparticle stock solution prepared in step (1) (the concentration of IR1040 in the IRNPs-NH2 nanoparticle stock solution was 540 mM), and 13 mL of N,N-diisopropylethylamine (DIPEA) was added. The NH2 groups in the IRNPs-NH2 nanoparticles were allowed to react with SBA by stirring at room temperature for 4 hours. After the reaction was completed, the unreacted SBA, DIPEA and HBTU were removed by centrifugation using a 10 KDa ultrafiltration tube, and PBS buffer (1x, pH 7.4) was added after the end of concentration to obtain an IRNPs-SBA nanoparticle stock solution, and the concentration of IR1040 in the stock solution was 540 mM, which was recorded as IRNPs-SBA nanoparticles.

[0102] (3) Preparation of cisplatin diagnosis and treatment probe IRNPs-SBA / Pt nanoparticles: IV

[0103] To 50 mL of a DMF solution of cisplatin prodrug (Pt IV -COOH) with a concentration of 1 mg mL -1 , 0.9 mg of HBTU was added, and the second mixture was obtained by reacting at 0°C for 10 min. Then, the second mixture was added to the IRNPs-SBA nanoparticles prepared in step (2) (the concentration of IR1040 was 540 mM), and 13 mL of DIPEA was added. The free NH2 groups in the IRNPs-SBA were allowed to react with Pt IV -COOH by stirring at room temperature for 4 hours. After the reaction was completed, the unreacted Pt IV ​-COOH, DIPEA and HBTU, and then PBS buffer (1×, pH 7.4) was added after concentration to obtain IRNPs-SBA / Pt IV Nanoparticle stock solution, the concentration of IR1040 in the stock solution is 540 μM, denoted as IRNPs-SBA / Pt IV Nanoparticles.

[0104] 3.3 IRNPs-Pt IV Preparation of nanoparticles

[0105] Add 50 μL of 1 mg mL -1 Cisplatin prodrug (Pt IV 0.9 mg of HBTU was added to a DMF solution of IRNPs-NH2 (prepared in step (2) of item "3.2" of this embodiment) and the mixture was reacted at 0°C for 10 minutes to obtain a mixed solution. The mixed solution was then added to the prepared IRNPs-NH2 nanoparticles (IR1040 concentration was 540 μM) (IRNPs-NH2 nanoparticles prepared in step (2) of item "3.2" of this embodiment), and 13 μL of DIPEA was added. The mixture was stirred at room temperature for 4 hours to allow the NH2 groups in the IRNPs-NH2 to react with the Pt IV -COOH condensation reaction. After the reaction is completed, use 10KDa ultrafiltration tube to remove unreacted Pt IV -COOH, DIPEA and HBTU, and then PBS buffer (1×, pH 7.4) was added after concentration to obtain IRNPs-Pt IV Nanoparticle stock solution, the concentration of IR1040 in the stock solution is 540 μM, denoted as IRNPs-Pt IV Nanoparticles.

[0106] 3.4 IR1048 NPs-SBA / Pt IV Preparation of nanoparticles

[0107] The preparation method is the same as that of "3.2, Cisplatin diagnostic probe IRNPs-SBA / Pt targeting carbonic anhydrase" in this embodiment. IV The difference is that IR1040 is replaced by IR1048, and IR1048 NPs-SBA / Pt is finally prepared. IV Nanoparticles; among them, IR1048 is commercially available with CAS number 155613-98-2.

[0108] Example 2

[0109] In this example, the carbonic anhydrase-targeted cisplatin diagnostic probe IRNPs-SBA / Pt IV (Example 1 "3.2, cisplatin diagnostic probe IRNPs-SBA / Pt targeting carbonic anhydraseIV The optical properties, photothermal performance evaluation, binding constant determination with CA and Pt release ability of the prepared Pt nanostructured ...

[0110] The IRNPs-SBA / Pt used in this example IV From "3.2, Cisplatin diagnostic probe IRNPs-SBA / Pt targeting carbonic anhydrase in Example 1 IV The IR1048 NPs-SBA / Pt used in this example was prepared under the preparation of IV From "3.4, IR1048 NPs-SBA / Pt in Example 1 IV The IRNPs-SBA used was prepared under the preparation of nanoparticles; the IRNPs-SBA used was prepared under the preparation of nanoparticles ...; the IRNPs-SBA used was prepared under the preparation of nanoparticles; the IRNPs-SBA used was prepared IV The IRNPs-Pt IV From "3.3, IRNPs-Pt IV The nanoparticles were prepared under the heading of “Preparation of nanoparticles” and diluted to different concentrations (based on the concentration of IR1040) using PBS buffer (1×, pH 7.4) according to experimental requirements.

[0111] 1. Carbonic anhydrase-targeted cisplatin diagnostic probe IRNPs-SBA / Pt IV Characterization and optical property evaluation of

[0112] (1) Figure 10 As shown in a, the transmission electron microscopy (TEM) image shows that IRNPs-SBA / Pt IV Exists in spherical shape.

[0113] (2) Figure 10 As shown in b, dynamic light scattering (DLS) analysis showed that IRNPs-SBA / Pt IV The average hydrated particle size in PBS buffer (pH=7.4) was 62.1±1.2 nm.

[0114] (3) This example is to evaluate the IRNPs-SBA / Pt IV The optical properties of IRNPs-SBA / Pt IV PBS buffer (1×, pH 7.4) was added to dilute the sample to 20 μM and its spectral properties were tested. Figure 10As shown in c, due to the presence of IR1040, IRNPs-SBA / Pt IV It displays blue in PBS buffer, with maximum absorption and emission wavelengths at 1036 and 1060 nm, respectively, and emits obvious NIR-II fluorescence under 980 nm light excitation.

[0115] 2. Carbonic anhydrase-targeted cisplatin therapeutic probe IRNPs-SBA / Pt IV Photothermal performance evaluation

[0116] In this example, IRNPs-SBA / Pt IV The photothermal performance was tested under 1064nm excitation.

[0117] (1) 540 μM (based on the concentration of IR1040) of IRNPs-SBA / Pt IV PBS buffer (1×, pH 7.4) was added to dilute the mixture to 5 μM, 10 μM, 20 μM, and 30 μM, and then the mixture was washed with water at a power density of 1.0 W·cm -2 IRNPs-SBA / Pt with different concentrations were irradiated by 1064 nm laser. IV The results are as follows Figure 11 As shown in a, IRNPs-SBA / Pt IV The PBS solution temperature increases with the IV In the IRNPs-SBA / Pt IV When the concentration was 30 μM, the temperature gradually increased from 25.2°C to 83.8°C after 10 min of irradiation.

[0118] (2) Further maintain IRNPs-SBA / Pt IV The concentration of 30 μM was irradiated with 1064 nm laser of different powers (0.2, 0.3, 0.4, 0.5 W·cm -2 ) irradiated IRNPs-SBA / Pt IV PBS solution, such as Figure 11 As shown in b, IRNPs-SBA / Pt IV The temperature of PBS solution increases with the increase of laser power density. 2 After continuous irradiation with a 1064 nm laser with a high power density for 10 minutes, the temperature gradually increased from 25.2 °C to 54.3 °C.

[0119] (3) Figure 11 As shown in c, at a power density of 0.5 W cm -2 , 1064nm laser irradiation, IRNPs-SBA / Pt IVThe photothermal conversion efficiency (PCE) of IR1048 NPs-SBA / Pt (30 μM, based on the concentration of IR1040) is 65.17%, which is better than that of IR1048 NPs-SBA / Pt IV (IR1048NPs-SBA / Pt IV The PCE of IR1040 was 21.47% at a concentration of 30 μM (based on the concentration of IR1048), which may be due to the higher molar extinction coefficient of IR1040 than that of commercial IR1048.

[0120] (4) Figure 11 As shown in d, at a power density of 0.5 W cm -2 , 1064nm laser irradiation, IRNPs-SBA / Pt IV (30 μM, based on the concentration of IR1040) showed good photothermal stability, and the light heat generation cycle could be 6 times within 70 minutes, and the maximum heat generation temperature remained basically unchanged.

[0121] The above experimental results confirmed that IRNPs-SBA / Pt IV It exhibits excellent photothermal performance and has certain potential in the PTT process in vitro and in vivo.

[0122] 3. Carbonic anhydrase-targeted cisplatin therapeutic probe IRNPs-SBA / Pt IV Evaluation of hCA I binding ability and Pt release ability

[0123] (1) This example further explored the IRNPs-SBA / Pt IV The affinity between DNSA and CA is determined. DNSA, a sulfonamide-based CA inhibitor, is the target of human carbonic anhydrase (hCA I). It first binds to the active site of hCA I and exhibits enhanced fluorescence at 458 nm after binding. The corresponding K d The value was 1.32±0.35μM. Then IRNPs-SBA / Pt IV , IRNPs-SBA, SBA and IRNPs-Pt IV Titrate hCA I·DNSA solution for competition experiment and calculate the corresponding apparent K d Value. K d The value is calculated using the following formula:

[0124]

[0125] F total : total fluorescence intensity; F obs : observed fluorescence intensity; F ini : Fluorescence intensity in the absence of DNSA; F end : Fluorescence intensity when saturation is reached;

[0126] as well as,

[0127]

[0128] F total : total fluorescence intensity; F obs : observed fluorescence intensity; F ini : Fluorescence intensity in the absence of DNSA; F end : fluorescence intensity when reaching saturation; K probe : Dissociation constant of the specified probe; K DNSA : Dissociation constant of DNSA.

[0129] The analysis results are as follows Figure 12 As shown in a, IRNPs-Pt without SBA IV Compared with IRNPs-SBA / Pt IV The binding of IRNPs-SBA and SBA to hCA I produced similar K d ' values, which were 14.40±5.49nM, 11.64±1.41nM, and 14.11±1.41nM, respectively. IV , IRNPs-SBA, SBA and CA. This efficient binding ability is conducive to their specific uptake in tumor cells.

[0130] (2) In order to evaluate the release ability of Pt drug in this example, IRNPs-SBA / Pt IV (65 μM, based on the concentration of IR1040) were divided into three groups: (1) no treatment group; (2) GSH (10 mM) treatment group at 37 °C (IRNPs-SBA / Pt IV GSH was added to the final concentration of 10 mM); (3) the power density was 0.5 W·cm -2 After irradiation with a 1064 nm laser for 5 min, the cells were treated with GSH (final concentration 10 mM) at 37°C. The amount of Pt released at 0 h, 0.5 h, 1 h, 2 h, 4 h, and 5 h of treatment was determined by ICP.

[0131] The analysis results are as follows Figure 12 As shown in b, IRNPs-SBA / Pt IVPt drug can be effectively released in the presence of GSH. At 5h, the cumulative release of Pt reached nearly 92.98%. However, in the absence of GSH and without laser irradiation, a small amount of Pt drug was released (6.07%) within 5 hours. In addition, under the combined action of laser irradiation and GSH, the Pt release rate was as high as 98.02%, which may be because the heat generated by laser irradiation promotes the kinetics of the reduction reaction, which is conducive to the release of Pt. Therefore, NIR-II region can excite IRNPs-SBA / Pt IV show good stability, high PCE, high binding force with hCA I, rapid release of Pt under GSH and 1064nm laser irradiation, and can be used for further in vitro and in vivo studies.

[0132] Example 3

[0133] In this example, the enzyme-targeted cisplatin diagnostic and therapeutic probe IRNPs-SBA / Pt IV NIR-II region fluorescence microscopy imaging, uptake ability, improvement of hypoxic and micro-acidic environment, ROS production ability and cell migration ability of tumor cells overexpressing CA under hypoxic conditions.

[0134] The IRNPs-SBA / Pt IV used in this example was prepared under the item “3.2, Preparation of enzyme-targeted cisplatin diagnostic and therapeutic probe IRNPs-SBA / Pt IV ” in Example 1; the IRNPs-Pt IV used in this example was prepared under the item “3.3, Preparation of IRNPs-Pt IV nanoparticles” in Example 1; the IRNPs-OCH3 used in this example was prepared under the item “3.1, Preparation of IRNPs-OCH3 nanoparticles” in Example 1; the IRNPs-SBA used in this example was prepared under step (2) of the item “3.2, Preparation of enzyme-targeted cisplatin diagnostic and therapeutic probe IRNPs-SBA / Pt IV ” in Example 1. According to the experimental needs, PBS buffer (1x, pH 7.4) was diluted to different concentrations (based on the concentration of IR1040).

[0135] 1, NIR-II region fluorescence microscopy imaging and cell uptake ability of enzyme-targeted cisplatin diagnostic and therapeutic probe IRNPs-SBA / Pt IV

[0136] In this example, in order to prove the response sensitivity of the nanoprobe to CA in tumor cells, Pan02 cells (about 5x10 4 ​) were inoculated into glass bottom dishes (In Vitro Scientific, D35-20-1-N) and allowed to grow overnight. No probe was added to the blank control group. IRNPs-SBA / Pt IV IRNPs-Pt IV IRNPs-SBA / Pt IV +SBA, IRNPs-OCH3 and four other probes (the concentration of IR1040 was 20 μM) were incubated at 37°C for 3 hours under hypoxia or normoxia. The results showed that compared with other control experimental groups, IRNPs-SBA / Pt IV The NIR-II fluorescence signal of the incubated cells was the strongest ( Figure 13 a) This is because CA is highly expressed on the surface of tumor cells under hypoxia, and IRNPs-SBA / Pt IV The SBA on the surface of the cells can specifically bind to CA. Moreover, NIR-II FLI of Pan02 cell clusters collected after incubation of cells under hypoxia or normoxia with the designated probe further confirmed this ( Figure 13 b and Figure 13 c). In addition, the uptake of Pt in tumor cells under hypoxia or normoxia was determined by inductively coupled plasma mass spectrometry (ICP-OES). IV and IRNPs-SBA / Pt IV (Pt concentration of 20 μM) were treated. Incubated at 37 ° C for 3 hours under hypoxia or normoxia. Data analysis showed that IRNPs-Pt IV Compared with cisplatin, IRNPs-SBA / Pt IV Cellular uptake under hypoxia increased by approximately 2.6-fold and 12-fold, respectively ( Figure 13 d).

[0137] 2. Enzyme-targeted cisplatin diagnostic probe IRNPs-SBA / Pt IV Evaluation of the improvement effect on the slightly acidic and hypoxic microenvironment of Pan02 cells

[0138] In this example, IRNPs-SBA / Pt IV Whether CA activity can be inhibited to regulate the tumor microenvironment in hypoxic Pan02 cells. In order to avoid cell death caused by Pt drugs, further change the tumor microenvironment and slow down the migration of tumor cells, IRNPs-SBA was used instead of IRNPs-SBA / Pt in this example. IV To incubate hypoxic Pan02 cells.

[0139] First, the extracellular pH (pH ePan02 cells (about 1×10 5 ) were seeded in 24-well plates. And allowed to grow overnight. After removing the DMEM medium and washing with PBS (1×, pH 7.4), culture medium containing PBS, IRNPs-OCH3, IRNPs-SBA (the concentration of IR1040 was 20 μM) and SBA (the concentration of SBA was 20 μM) was added respectively and incubated at 37°C for 24 hours in a normoxic or hypoxic environment. At the beginning and end of each experiment, a pH probe was inserted into the culture medium to measure the extracellular pH value. The analysis results are shown in Figure 2. Figure 14 As shown in a, when Pan02 cells were incubated with IRNPs-SBA, SBA and IRNPs-OCH3 under hypoxia for 24 h, the pH of the cell culture medium of the IRNPs-SBA and SBA treatment groups e Significantly reduced pH in hypoxic Pan02 cells e (ΔpH e ) changes by -0.28, which is similar to the free SBA (ΔpH e =-0.32). e The highest was -0.64, and the ΔpH of the IRNPs-OCH3 treatment group e The above results indicate that IRNPs-SBA can effectively inhibit CA activity in hypoxic Pan02 cells. With the effective inhibition of CA activity by IRNPs-SBA and SBA, HIF-1α expression in hypoxic Pan02 cells was significantly downregulated; in contrast, HIF-1α expression in hypoxic Pan02 cells incubated with IRNPs-OCH3 was not affected ( Figure 14 b), indicating that IRNPs-SBA can effectively alleviate the hypoxic environment by inhibiting CA activity.

[0140] 3. Enzyme-targeted cisplatin diagnostic probe IRNPs-SBA / Pt IV Evaluation of the ability of hypoxic Pan02 cells to produce ROS

[0141] In this example, the intracellular reactive oxygen species (ROS) level of 2',7'-dichlorofluorescein diacetate (DCFH-DA) was measured.

[0142] Pan02 cells (about 5×10 4) were inoculated into glass-bottom dishes (In Vitro Scientific, D35-20-1-N) and allowed to grow overnight. After removing DMEM medium and washing with PBS (1x, pH 7.4), cells were incubated with DMEM medium containing PBS (Control group), IR / Rh6G NPs-OCH3, SBA, IR / Rh6G NPs-SBA, IR / Rh6G NPs-SBA / Pt IV (Rh6G and SBA concentrations were 2 and 20 mM, respectively, and the concentration of IR1040 was 20 mM) for 24 h under hypoxia at 37 °C. After removing DMEM medium and washing with PBS (1x, pH 7.4), cells were incubated with medium containing DCFH-DA (20 mM) for 30 min under hypoxia at 37 °C. After removing the medium and washing with PBS, a certain amount of fresh medium was added for imaging.

[0143] The results of the analysis showed that the DCF fluorescence in hypoxic Pan02 cells treated with IRNPs-SBA and SBA was stronger than that in the IRNPs-OCH3 treatment group Figure 15 a). Further flow cytometry analysis confirmed that the DCF fluorescence in hypoxic Pan02 cells treated with IRNPs-SBA or SBA was significantly higher than that in the untreated control group or the IRNPs-OCH3 treatment group Figure 15 b). These results indicated that SBA and IRNPs-SBA upregulated the intracellular ROS level, and the alleviation of the hypoxic environment caused by the inhibition of CA activity promoted the production of ROS. In addition, when Pan02 cells were cultured under hypoxia with IRNPs-SBA / Pt IV The corresponding DCF fluorescence intensity was further increased, which might be due to the consumption of GSH caused by the reduction of Pt IV by GSH, which further led to the accumulation of ROS.

[0144] 4. Enzyme-targeted cisplatin diagnostic and therapeutic probe IRNPs-SBA / Pt IV Evaluation of the inhibitory ability of Pan02 cell migration under hypoxia

[0145] In this example, Pan02 cells were cultured at a density of 2 x 10 5The cells were seeded at a density of 100 cells / well in a 6-well cell culture plate. After growing overnight, they were slowly scratched with a 200μL pipette tip to form a "well" shape, and the cells were imaged under a white light microscope. Mark the imaging position in each well plate. Then, DMEM culture medium containing PBS, IRNPs-OCH3 (the concentration of IR1040 was 20μM) and IRNPs-SBA (the concentration of SBA was 20μM) was added respectively, and the cells were imaged under a white light microscope after incubation at hypoxia and 37°C for 24 hours. The experimental analysis results showed that compared with the hypoxic Pan02 cells treated with PBS or IRNPs-OCH3, the migration speed of the wound boundary in the hypoxic Pan02 cells incubated with IRNPs-SBA was much slower. Compared with the PBS group and the IRNPs-OCH3-treated group, the migration of hypoxic Pan02 cells incubated with IRNPs-SBA was significantly inhibited by ~92.9% ( Figure 16 ), indicating that IRNPs-SBA can inhibit the metastatic ability of tumor cells by inhibiting CA activity.

[0146] Example 4

[0147] In this example, the enzyme-targeted cisplatin diagnostic probe IRNPs-SBA / Pt IV Cell viability assessment and flow cytometric analysis of apoptosis in Pan02 cells.

[0148] The IRNPs-SBA used in this example is the cisplatin diagnostic and therapeutic probe IRNPs-SBA / Pt targeted to carbonic anhydrase in Example 1. IV The IRNPs-SBA / Pt IV From "3.2, Cisplatin diagnostic probe IRNPs-SBA / Pt targeting carbonic anhydrase in Example 1 IV The IR1040 was prepared under the heading of "Preparation of IR1040". According to experimental requirements, it was diluted to different concentrations (based on the concentration of IR1040) using PBS buffer (1×, pH 7.4).

[0149] 1. Enzyme-targeted cisplatin diagnostic probe IRNPs-SBA / Pt IV Cell viability assessment of Pan02 cells

[0150] (1) In this example, the IC 50 The cytotoxicity of the probe IRNPs-SBA to Pan02 cells was investigated by the following steps:

[0151] Cells were seeded in 96-well flat-bottom plates (1×10 4cells) and incubated overnight at 37 °C. Then different concentrations of IRNPs-SBA (0, 1, 2, 10, 20, 40, 100 mM) were added into DMEM medium (100 pL) respectively. Then incubation was divided into three groups, the first group was incubated with IRNPs-SBA for 3 h at 37 °C under normoxia, washed with PBS, and then incubated for another 48 h with fresh medium (Group I). The second group was incubated with IRNPs-SBA for 3 h at 37 °C under normoxia, washed with PBS, and then incubated for another 48 h with fresh medium after 1064 nm (0.5 W-cm -2 ) laser irradiation for 3 min (Group II). The third group was incubated with IRNPs-SBA for 3 h at 37 °C under hypoxia, washed with PBS, and then incubated for another 48 h with fresh medium after 1064 nm (0.5 W-cm -2 ) laser irradiation for 3 min (Group III). After incubation, 50 pL MTT solution (1 mg / mL in PBS) was added into each well. Cells were kept at 37 °C for 4 h, and then the solution in each well was carefully removed. 150 pL DMSO was added to dissolve the purple formazan crystals in the well. The absorbance (OD) at 490 nm in each well was obtained on a microplate reader (Tcan). The absorbance of blank cells (ODcontrol) was used as a control, and the percentage of cell viability in each treatment was calculated by OD divided by ODcontrol. The corresponding IC 50 values were calculated by Prism 7 software. The experimental results are shown in Figure 17 a. IRNPs-SBA (100 pM) also had little cytotoxicity to Pan02 cells under normoxia. However, dose-dependent cytotoxicity was observed after IRNPs-SBA treated Pan02 cells under normoxia or hypoxia with 1064 nm laser irradiation. The IC 50 values of IRNPs-SBA to Pan02 cells under normoxia or hypoxia after 1064 nm laser irradiation were 25.07 ± 8.13 pM and 16.73 ± 4.95 pM Figure 17 c, respectively, indicating that IRNPs-SBA had good PTT effect under NIR-II light (1064 nm) irradiation.

[0152] (2) To investigate the combined treatment effect of PTT and chemotherapy, the cytotoxicity of IRNPs-SBA / Pt IV to Pan02 tumor cells was further evaluated. In this embodiment, cells were seeded in a flat-bottom 96-well plate (1 x 10 4 cells per well) and incubated overnight at 37 °C. Then different concentrations of CisPt or IRNPs-SBA / Pt IV(0, 0.5, 1, 5, 10, 20 and 50 μM) in DMEM medium (100 μL). Then they were divided into three groups for incubation. The first group was cultured with CisPt at 37°C for 3 hours under hypoxia, washed with PBS, and then cultured with fresh medium for another 48 hours (Group IV). The second group was first cultured with IRNPs-SBA / Pt under normoxia. IV The cells were cultured at 37°C for 3 hours, washed with PBS, and then irradiated with 1064 nm (0.5 W·cm -2 ) laser irradiation for 3 minutes and then incubated for 48 hours (V group). The third group was treated with IRNPs-SBA / Pt IV The cells were cultured at 37°C for 3 hours, washed with PBS, and then irradiated with 1064 nm (0.5 W·cm -2 ) Laser irradiation for 3 minutes and then incubate for 48 hours (VI group). After incubation, 50 μL of MTT solution (1 mg / mL in PBS) was added to each well. The cells were kept at 37°C for 4 hours, and then the solution in each well was carefully removed. 150 μL of DMSO was added to dissolve the purple formazan crystals in the wells. The absorbance (OD) at 490 nm in each well was obtained on a microplate reader (Tcan). The absorbance of blank cells (OD control) was used as a control, and the percentage of cell viability in each treatment was calculated by dividing the OD by the OD control, and the corresponding IC50 value was calculated using Prism 7 software. The experimental results are shown in Figure 2. Figure 17 As shown in b, under 1064 nm laser excitation, IR NPs-SBA / Pt IV IC of hypoxic Pan02 cells 50 The value was 0.66±0.28μM, which was significantly lower than that of the IRNPs-SBA+light group (IC 50 =16.73±4.95μM) or cisplatin group (IC 50 =13.44±2.83μM), indicating that the combined treatment of IRNPs PTT and cisplatin released after GSH reduction can significantly inhibit the growth of tumor cells. IV The IC50 of light exposure to Pan02 cells under hypoxia was significantly higher than that under normoxia (IC 50 =1.57±0.73μM) was more than 2 times lower, indicating that IRNPs-SBA / Pt IV More effective in killing hypoxic Pan02 cells ( Figure 17 c).

[0153] 2. Enzyme-targeted cisplatin diagnostic probe IRNPs-SBA / Pt IV Flow cytometric analysis of apoptosis in Pan02 cells

[0154] In this example, Pan02 cells were cultured at 2×10 5 The density of cells / well was seeded in 6-well cell culture plates. After growing overnight, the DMEM medium was removed and washed with PBS (1×, pH 7.4). IV IRNPs-SBA / Pt IV + Laser treatment of Pan02 cells in hypoxia (1% O2) (IR1040 concentration was 20 μM) at 37°C for 24 h. The laser irradiation conditions were 1064 nm laser (0.5 W·cm -2 ) irradiated the cells for 3 minutes. After the incubation, the cells were trypsinized and the cell pellets were stained with Annexin V-FITC (5.0 μL) and propidium iodide (PI) (5.0 μL). After staining, the cell population was analyzed using the FITC and PI channels using a Coulter FC-500 flow cytometer. At least 10,000 cells were used for all experiments. Annexin V-propidium iodide (PI) stained flow cytometric analysis indicated that the IRNPs-SBA / Pt IV After light treatment of Pan02 cells, severe apoptosis occurred in hypoxic Pan02 cells ( Figure 18 ). The above results further confirmed that the IRNPs-SBA / Pt IV The combined therapeutic effect of PTT and chemotherapy on hypoxic Pan02 cells is better than the PTT of IRNPs-SBA and chemotherapy of cisplatin.

[0155] Example 5

[0156] In this example, the enzyme-targeted cisplatin diagnostic probe IRNPs-SBA / Pt IV NIR-II fluorescence imaging of subcutaneous Pan02 cancer in mice and its uptake in different organs of mice.

[0157] The IRNPs-SBA / Pt used in this example IV From "3.2, Cisplatin diagnostic probe IRNPs-SBA / Pt targeting carbonic anhydrase in Example 1 IV The IRNPs-OCH3 used was prepared under the item "3.1, Preparation of IRNPs-OCH3 Nanoparticles" in Example 1; the IRNPs-SBA used was prepared under the item "3.2, Cisplatin diagnostic and therapeutic probe IRNPs-SBA / Pt targeted to carbonic anhydrase" in Example 1. IVwere prepared according to the procedure described in the item of “Preparation of IRNPs-SBA / Pt” under step (2). And according to the experimental needs, diluted to different concentrations (based on the concentration of IR1040) using PBS buffer (1x, pH 7.4).

[0158] 1. Enzyme-targeted cisplatin theranostic probe IRNPs-SBA / Pt IV NIR-II region fluorescence imaging of subcutaneous Pan02 cancer in mice

[0159] After exploring the IRNPs-SBA / Pt IV In the cell level of the killing effect of different tumor cells, further carried out IRNPs-SBA / Pt IV NIR-II region imaging detection of subcutaneous pancreatic cancer in BALB / C mice. In order to establish a mouse model of subcutaneous pancreatic cancer, 9 female BALB / C nude mice aged 4-5 weeks (purchased from Nanjing University Model Animal Research Center (MARC) (Nanjing, China)) were used according to the regulations of the Institutional Animal Care and Use Committee (IACUC). 2x10 6 Pan02 cells, establish xenotransplantation Pan02 tumor, when the average volume of tumor reaches about 120mm 3 When, the mice were randomly divided into three groups (n=3). In order to carry out in vivo fluorescence imaging (980nm) of Pan02 subcutaneous tumor mice, IRNPs-OCH3, IRNPs-SBA and IRNPs-SBA / Pt IV (probe concentration is 540μM (based on IR1040), and the probe is injected into the mouse body 200μL) were injected into the mouse body. As shown in Figure 19 a, the NIR-II FL intensity at the tumor site gradually increased and reached a maximum value at 24h, and the duration was more than 48h. Due to the strong affinity between SBA in IRNPs-SBA / Pt IV and the overexpressed CA on the tumor, the NIR-II FL intensity of IRNPs-SBA and IRNPs-SBA / Pt IV The experimental group at 24h was 3.37 times and 3.49 times (b) higher than that of the mice injected with IRNPs-OCH3, respectively. Figure 19 Corresponding to the signal-to-noise ratio (tumor-to-background ratio, TBR), IRNPs-SBA / Pt IV and IRNPs-SBA were 3.08 times and 2.97 times (b) of the IRNPs-OCH3 group, respectively. Figure 19c). After one day of injection, the tumor was removed after another day of laser irradiation for 10 min. The NIR-II FL microscopy imaging results of the ex vivo tumor tissue sections matched well with the NIR-II FLI, which showed that IRNPs-SBA and IRNPs-SBA / Pt IV Figure 19 d). These data showed that CA-targeted active delivery could greatly improve the tumor uptake of IRNPs-SBA and IRNPs-SBA / Pt IV .

[0160] 2. Enzyme-targeted cisplatin theranostic probe IRNPs-SBA / Pt IV Uptake in different organs of mice

[0161] To investigate the uptake of probe IRNPs-SBA / Pt IV in different organs of mice, the subcutaneous pancreatic cancer model of mice was established as described in item “1” of this example. 200 μL of IRNPs-SBA / Pt IV (540 μM) was injected into the subcutaneous tumor of Pan02 mice via the tail vein, and the tumor (T) and major organs, including heart (H), liver (Li), spleen (Sp), lung (Lu) and kidney (Ki), were collected after 24 h for NIR-II region fluorescence imaging (980 nm). As shown in Fig. Figure 20 a, IRNPs-SBA / Pt IV was mainly distributed in the tumor (T) and had little uptake in other organs, which matched well with the strong NIR-II FLI observed in the ex vivo excised organs.

[0162] Further, to investigate the Pt distribution in different organs in vivo, 200 μL of IRNPs-SBA / Pt IV (540 μM) was injected into the subcutaneous tumor of Pan02 mice via the tail vein, and the tumor (T) and major organs, including heart (H), liver (Li), spleen (Sp), lung (Lu) and kidney (Ki), were collected after 24 h for weighing. The tissues were cut into small pieces, digested with concentrated HNO3 at 120°C overnight, and then the residue in each organ was diluted with 5 mL of 2 wt% HNO3 aqueous solution, and the concentration of platinum was determined by ICP-OES. The uptake values of the tissues were calculated and expressed as %ID / g of the injected dose per gram of tissue for comparison, and the results are shown in Fig. Figure 20 b, the probe IRNPs-SBA / Pt IV had the highest accumulation in the tumor tissue (~12.6 %ID / g), which was significantly higher than that in other organs.​

[0163] Example 6

[0164] In this example, the enzyme-targeted cisplatin diagnostic probe IRNPs-SBA / Pt IV NIR photothermal imaging of subcutaneous Pan02 cancer in mice, immunofluorescence staining of tumor tissue sections, and combined PTT and chemotherapy treatment of tumors.

[0165] The IRNPs-SBA / Pt used in this example IV From "3.2, Cisplatin diagnostic probe IRNPs-SBA / Pt targeting carbonic anhydrase in Example 1 IV The IRNPs-OCH3 used was prepared under the item "3.1, Preparation of IRNPs-OCH3 Nanoparticles" in Example 1; the IRNPs-SBA used was prepared under the item "3.2, Cisplatin diagnostic and therapeutic probe IRNPs-SBA / Pt targeted to carbonic anhydrase" in Example 1. IV Prepared according to step (2) under "Preparation of". And diluted to different concentrations (based on the concentration of IR1040) using PBS buffer (1×, pH 7.4) according to experimental needs.

[0166] 1. Enzyme-targeted cisplatin diagnostic probe IRNPs-SBA / Pt IV NIR photothermal imaging of subcutaneous Pan02 cancer in mice

[0167] Under the guidance of NIR-II FLI, IRNPs-SBA / Pt IV Based on the efficient enrichment at the tumor site, NIR-II region photothermal imaging was performed on a mouse subcutaneous pancreatic cancer model. The establishment of a mouse subcutaneous pancreatic cancer model was as described under "1" in Example 5. 200 μL of saline, IRNPs-OCH3, and IRNPs-SBA / Pt were injected into the tail vein respectively. IV After 24 hours (the probe concentration was 540 μM (based on IR1040), and the probe was injected in 200 μL), each group was irradiated with 1064 nm laser for 10 minutes at a power density of 0.5 W·cm -2 , which is half of the MPE dose). Photothermal imaging (1064nm) results showed that IRNPs-SBA / Pt IV The tumor temperature of the treated mice increased rapidly from 32.8°C to 42.6°C, which was significantly higher than that of the mice treated with normal saline (33.4°C) or IRNPs-OCH3 (38.7°C) ( Figure 21 a and Figure 21b) It is worth mentioning that the temperature of 42.6°C is mild, which is enough to induce tumor hyperthermia while avoiding overheating and damaging surrounding normal tissues.

[0168] 2. Enzyme-targeted cisplatin diagnostic probe IRNPs-SBA / Pt IV Immunofluorescence staining of tumor tissue sections

[0169] In this example, IRNPs-SBA / Pt IV The ability to inhibit CA activity and alleviate tumor hypoxia in vivo. The establishment of a subcutaneous pancreatic cancer model in mice was as described under "1" in Example 5. 200 μL of normal saline, IRNPs-OCH3 and IRNPs-SBA / Pt were injected into the tail vein respectively. IV After 24 h (the probe concentration was 540 μM (based on IR1040), and the probe was injected in 200 μL), for IRNPs-OCH3 and IRNPs-SBA / Pt IV The experimental group was irradiated with 1064 nm laser for 10 min (power density 0.5 W·cm -2 ) and then the tumor was removed and stained. Immunofluorescence staining of tumor tissue Figure 22 It is shown that the injection of IRNPs-SBA / Pt IV The expression of CA in the tumor tissues of mice injected with IRNPs-SBA / Pt was significantly downregulated, but the expression of CA in the tumor tissues of mice injected with saline and IRNPs-OCH3 was not downregulated. IV The expression of HIF-1α in the tumor tissues of mice after IRNPs-SBA / Pt IV It can effectively inhibit CA activity in mouse tumors, thereby alleviating the tumor hypoxic environment, which is beneficial for overcoming cisplatin chemotherapy resistance.

[0170] 3. Enzyme-targeted cisplatin diagnostic probe IRNPs-SBA / Pt IV Combination therapy of PTT and chemotherapy for tumors

[0171] The establishment of subcutaneous pancreatic cancer model in mice was as described in Example 5 under "1". In this example, the tumor size was 120 mm. 3 Pan02 tumor-bearing BALB / C mice were randomly divided into 7 groups, with 5 mice in each group. Then, each group of mice was injected with normal saline (group I), normal saline + light (group II), IRNPs-SBA / Pt IV (Group III), IRNPs-OCH3+light (Group IV), IRNPs-SBA+light (Group V), IRNPs-SBA / Pt IV+ light (V group) and IRNPs-SBA / Pt + light (VI group) IV + light + IRNPs-SBA / Pt IV (2 doses) (VII group) (200 μL of saline injection, the concentration of the probe was 540 μM (based on IR1040), and 200 μL of the probe was injected). One saline, IRNPs-SBA / Pt IV , IRNPs-OCH3 or IRNPs-SBA was injected intravenously, and 24 hours later, the tumors were irradiated with 1064 nm laser (0.5 W-cm -2 ) for 10 minutes. The detailed injection time is shown in Figure 23 a. The body weight and tumor size of each mouse were measured within 21 days.

[0172] From Figure 23 b (1064 nm laser) and Figure 23 c, it can be seen that the mice treated with saline (I group) or saline + light (II group) showed a rapid tumor growth rate, and the average tumor size increased by 8.8 and 8.1 times, respectively, after 21 days, indicating that the PTT effect of 1064 nm laser irradiation alone on the tumor can be ignored. When the mice were treated with IRNPs-SBA / Pt IV (III group) or IRNPs-OCH3 + light (IV group), the tumor growth was slower than that of the saline injection group. On day 21, the average tumor size of groups III and IV increased by 7.2 and 5.4 times, respectively, indicating that the chemotherapy produced by IRNPs-SBA / Pt IV and the PTT efficiency produced by IRNPs-OCH3 were not high. When the mice were treated with IRNPs-SBA + light (V group) or IRNP-SBA / Pt IV + light (VI group), the tumor volume of the two groups decreased within the first 10 days, but then began to grow slowly. Due to the combined effect of PTT and chemotherapy, the average tumor size of the mice treated with IRNPs-SBA / Pt IV + light (VI group) was significantly smaller than that of IRNPs-SBA + light (V group) on day 21 after treatment, indicating that IRNPs-SBA / Pt IV was more effective than IRNPs-SBA in treating subcutaneous Pan02 tumors. However, since only IRNPs-SBA / Pt IV was injected once, the tumor in group VI grew by 2.1 times on day 21, which may be due to the insufficient dose of cisplatin in the tumor cells. In order to improve the combined treatment effect of PTT and chemotherapy, the mice in group VI were further injected with IRNPs-SBA / Pt IVThe mice in the group treated with Pt drugs (group VII) as a supplement to chemotherapy showed a 12.3-fold decrease in tumor volume in 21 days. During the treatment, the mice in these experimental groups showed no significant change in body weight Figure 23 d) These studies show that IRNPs-SBA / Pt IV The combination of PTT conjugated with IR1040 and the released cisplatin chemotherapy has low toxic side effects on normal tissues, but strong anti-tumor activity. This conclusion is in good agreement with the conclusion of hematoxylin and eosin (H&E) staining of the main organs (heart, liver, spleen, lung and kidney) and tumor tissue sections of the mice Figure 23 e) In addition, when the combination of PTT and chemotherapy was used to treat the tumors of mice, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining of tumor tissue showed significant apoptosis, indicating that IRNPs-SBA / Pt IV It is expected to be used for the treatment of Pan02 tumors in vivo.

[0173] The present application provides a kind of carbonic anhydrase targeted cisplatin diagnosis and treatment probe and its preparation method and application idea and method, the method and approach for specifically realizing this technical scheme are many, above-mentioned only preferred embodiment of the present application, it should be pointed out, for the ordinary skilled in the art, without departing from the principles of the present application, can make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application. The components not explicitly described in the embodiment can be realized by existing technology.

Claims

1. A cisplatin diagnostic and therapeutic probe targeting carbonic anhydrase, characterized in that: IRNPs-NH2 nanoparticles were prepared by nanoprecipitation using a phospholipid-polyethylene glycol compound and the NIR-II region fluorescent probe IR1040. The amino groups on the surface of the IRNPs-NH2 nanoparticles were covalently linked to p-sulfonamide benzoic acid and a tetravalent platinum prodrug to form a cisplatin diagnostic and therapeutic probe targeting carbonic anhydrase. in, The phospholipid-polyethylene glycol compound is DSPE-PEG 2000 -OCH3 and DSPE-PEG 2000 -NH2; The structural formula of the NIR-II region fluorescent probe IR1040 is: ； The tetravalent platinum prodrug is Pt IV -COOH; Wherein, the Pt IV The chemical formula of -COOH is: .

2. The cisplatin diagnostic and therapeutic probe targeting carbonic anhydrase according to claim 1, characterized in that: The NIR-II region fluorescent probe IR1040 is prepared by the following steps: 1,8-Naphtholactimide and 5-chloropentyne under the action of potassium iodide undergo a substitution reaction to prepare intermediate 1; intermediate 1 undergoes a Grignard reaction with a methyl Grignard reagent under inert gas protection and anhydrous conditions. After the Grignard reaction, potassium iodide is added for precipitation to prepare intermediate 2; intermediate 2 undergoes a coupling reaction with 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carboxaldehyde under the action of a base and anhydride to obtain the NIR-II region fluorescent probe IR1040; 。 3. The cisplatin diagnostic and therapeutic probe targeting carbonic anhydrase according to claim 2, characterized in that: The molar ratio of the 1,8-naphtholactimide to 5-chloropentyne and potassium iodide is 1:1.0-2.0:3.0-4.5; the reaction temperature of the substitution reaction is 120-150°C and the reaction time is 24-48 h; the methyl Grignard reagent is methylmagnesium chloride; the molar ratio of the intermediate 1 to the methyl Grignard reagent and potassium iodide is 1:3.5-5.5:1.5-3.5; the reaction temperature of the Grignard reaction is 55-80°C and the reaction time is 1-2 h; the inert gas is nitrogen; the base is triethylamine or N,N-diisopropylethylamine; the acid anhydride is acetic anhydride; the molar ratio of the intermediate 2 to 2-chloro-3-(hydroxymethylene)-1-cyclohexene-1-carbaldehyde is 1-2.5:1; the molar volume ratio of the intermediate 2 to the base and the acid anhydride is 0.65 mmol:0.4-0.6 mL:0.4-0.6 mL; the coupling reaction temperature is 50-70°C and the reaction time is 20-60 min.

4. The method for preparing the carbonic anhydrase-targeting cisplatin diagnostic and therapeutic probe according to any one of claims 1 to 3, characterized in that: The steps include: (1) Mix IR1040 stock solution with DSPE-PEG 2000 -OCH3、DSPE-PEG 2000 -NH2 and the first solvent are mixed and added to deionized water, and ultrasonication is performed during the addition process. After the ultrasonication, the reaction solution is washed and concentrated by centrifugation, and PBS buffer is added to obtain an IRNPs-NH2 nanoparticle stock solution containing IR1040; (2) Mixing the 4-aminosulfonylbenzoic acid stock solution with O-benzotriazole-tetramethyluronium hexafluorophosphate, reacting at -10 to 0°C for 10 to 20 minutes to obtain a first mixed solution; mixing the first mixed solution with the IRNPs-NH2 nanoparticle stock solution containing IR1040 obtained in step (1), adding N,N-diisopropylethylamine, and stirring at room temperature for 4 to 8 hours. After the reaction is completed, the reaction solution is centrifuged and concentrated, and PBS buffer is added to obtain an IRNPs-SBA nanoparticle stock solution containing IR1040; (3) The tetravalent platinum prodrug stock solution was mixed with O-benzotriazole-tetramethyluronium hexafluorophosphate, and the mixture was reacted at -10 to 0°C for 10 to 20 minutes to obtain a second mixed solution; the second mixed solution was mixed with the IRNPs-SBA nanoparticle stock solution containing IR1040 obtained in step (2), N,N-diisopropylethylamine was added, and the mixture was stirred at room temperature for 4 to 8 hours. After the reaction was completed, the reaction solution was centrifuged and concentrated, and PBS buffer was added to obtain IRNPs-SBA / Pt containing IR1040. IV Nanoparticle stock solution, a cisplatin theranostic probe targeting carbonic anhydrase.

5. The preparation method according to claim 4, characterized in that The first solvent is tetrahydrofuran or ethanol; the solvent in the IR1040 stock solution is dimethyl sulfoxide; the IR1040 in the IR1040 stock solution is DSPE-PEG 2000 -OCH3、DSPE-PEG 2000 The molar mass ratio of -NH2 is 0.27 μmol: 8 mg: 2 mg; the DSPE-PEG 2000 -NH2 and the first solvent have a mass volume ratio of 2 mg:0.5~1.5 mL; the first solvent and deionized water have a volume ratio of 0.5~1.5:8~10; the ultrasound temperature is room temperature, the ultrasound power is 40 kHz, and the ultrasound time is 10~15 min; the PBS buffer has a pH of 7.4; the concentration of IR1040 in the IRNPs-NH2 nanoparticle stock solution containing IR1040 is 540 μmol / L; the solvent in the 4-aminosulfonylbenzoic acid stock solution is N,N-dimethylformamide, and the solubility of 4-aminosulfonylbenzoic acid in the solution is 1 mg•mL −1 The mass ratio of 4-aminosulfonylbenzoic acid to O-benzotriazole-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine in the 4-aminosulfonylbenzoic acid stock solution is 0.05:0.8~1.0:9.0~10.0; the mass ratio of 4-aminosulfonylbenzoic acid to DSPE-PEG in the 4-aminosulfonylbenzoic acid stock solution is 0.05:0.8~1.0:9.0~10.0; 2000 The mass ratio of -NH2 is 0.05:2; the concentration of IR1040 in the IRNPs-SBA nanoparticle stock solution containing IR1040 is 540 μmol / L; the solvent in the tetravalent platinum prodrug stock solution is N,N-dimethylformamide, and the concentration of the tetravalent platinum prodrug in the solution is 1 mg•mL −1 The mass ratio of the tetravalent platinum prodrug to O-benzotriazole-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine in the tetravalent platinum prodrug stock solution is 0.05:0.8~1.0:9.0~10.0; the mass ratio of the tetravalent platinum prodrug to DSPE-PEG in the tetravalent platinum prodrug stock solution is 0.05:0.8~1.0:9.0~10.

0. 2000 -NH2 mass ratio is 0.05:2; the IRNPs-SBA / Pt containing IR1040 IV The concentration of IR1040 in the nanoparticle stock solution was 540 μmol / L.

6. Use of the carbonic anhydrase-targeting cisplatin diagnostic and therapeutic probe according to any one of claims 1 to 3 in the preparation of anti-tumor drugs.

7. Use of the carbonic anhydrase-targeting cisplatin diagnostic and therapeutic probe according to any one of claims 1 to 3 in the preparation of tumor photothermal therapy and / or chemotherapy drugs.

8. Use of the carbonic anhydrase-targeting cisplatin therapeutic probe according to any one of claims 1 to 3 in the preparation of a tumor diagnostic reagent.

9. Use of the carbonic anhydrase-targeting cisplatin therapeutic probe according to any one of claims 1 to 3 in the preparation of a tumor NIR-II imaging contrast agent.

10. Use of the carbonic anhydrase-targeting cisplatin diagnostic and therapeutic probe according to any one of claims 1 to 3 in the preparation of a tumor NIR-II region fluorescence imaging / photothermal imaging contrast agent.

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