Radioembolic microspheres with secondary targeting capability and preparation method and application thereof

Abstract

The present invention relates to the technical field of clinical medical embolization therapy, in particular to IPC B01J13, and more specifically to radioembolic microspheres with secondary targeting ability and preparation methods and applications thereof. In the present invention, Tyr-FAP-2286 is connected to dextran microspheres by physical adsorption or esterification reaction to prepare dextran microspheres loaded with Tyr-FAP-2286, and then the above-mentioned dextran microspheres loaded with Tyr-FAP-2286 are connected to nuclides to obtain radioactive element-labeled dextran microspheres loaded with Tyr-FAP-2286, i.e., radioembolic microspheres with secondary targeting ability. The radioembolic microspheres in the present invention can cause the death of VX2 tumor cells, inhibit the growth and intrahepatic metastasis of VX2 tumors, while retaining the activity of distal liver cells, further providing experimental basis for functional research and clinical transformation research of radioembolic microspheres.

Description

Technical Field

[0001] The present invention relates to the technical field of clinical medical embolization therapy, in particular to IPC B01J13, and more specifically to radioactive embolization microspheres with secondary targeting capability, and a preparation method and application thereof. Background Art

[0002] Hepatocellular carcinoma (HCC) is the most common primary liver cancer, with the characteristics of hidden onset, high morbidity and mortality. Since liver cancer is mainly supplied by the hepatic artery, and the surrounding normal liver tissue is mainly supplied by the portal vein system and the hepatic artery, transhepatic artery embolization has become one of the important methods for clinical treatment of liver cancer due to its advantages such as minimally invasive and high drug utilization rate. According to the different drug treatment methods combined with arterial embolization, transhepatic artery embolization treatment methods mainly include transarterial chemoembolization (TACE) and transarterial radioembolization (TARE), the latter of which can produce personalized and precise treatment effects of selective internal radiotherapy.

[0003] Currently, TARE embolic agents such as 90 Y Glass Microspheres 90 Y resin microspheres Combined with the good embolization treatment effect achieved in preclinical experiments, the TheraSphere and SIR-Sphere 90 Y can easily escape from the microspheres and enter the blood circulation, causing serious adverse reactions such as bone marrow suppression and pulmonary fibrosis. In addition, TheraSphere and SIR-Sphere are not degradable, and it is still difficult to solve adverse reactions such as ectopic embolism. Therefore, in-depth research on degradable radioactive microspheres is still needed.

[0004] In the prior art, there are documents that construct 131 I-labeled collagen-chitosan composite microspheres 131 I-CCMs, with a labeling rate of about 95.12%, effectively inhibited tumor progression and tumor metastasis after administration. However, chitosan takes a long time to degrade, about 24-34 weeks (Pang F, Li Y, Zhang W, et al. Biodegradable 131Iodine-Labeled Microspheres: Potential Transarterial Radioembolization Biomaterial for Primary Hepatocellular Carcinoma Treatment[J]. Advanced Healthcare Materials, 2020, 9(13): e2000028). Another paper developed a 131 I-labeled polylactic acid microspheres loaded with paclitaxel and copper sulfide nanoparticles can combine chemotherapy, radiotherapy and phototherapy, and can perform SPECT / CT imaging and photoacoustic imaging to monitor the distribution of microspheres in the body. The therapeutic effect of this multifunctional microsphere is better than that of a single treatment. However, the degradation product of polylactic acid microspheres, lactic acid, is an acidic product, which is prone to cause varying degrees of swelling at the site of vascular embolism and nonspecific aseptic inflammatory response (Liu Q, Qian Y, Li P, et al. 131 I-Labeled Copper Sulfide-Loaded Microspheres to Treat Hepatic Tumors via Hepatic Artery Embolization[J].Theranostics,2018,8(3):785-799). In addition, the detection rate of radionuclide targeted drugs is currently low, and the clarity of tumor contours needs to be improved. Summary of the invention

[0005] In order to solve the problems in the prior art, the present invention provides a method for preparing radioembolic microspheres with secondary targeting capability, comprising the following steps:

[0006] The dextran microspheres loaded with Tyr-FAP-2286 are resuspended in a 0.9% NaCl aqueous solution to form a DMs suspension, which is added to an Iodogen tube. The radioactive solution is continuously added to the Iodogen tube, and the suspension is shaken at room temperature for 20 to 30 minutes to obtain a radioactive solution labeled suspension, which is centrifuged to obtain a supernatant and a precipitate, and the precipitate is washed to obtain radioactive element labeled dextran microspheres loaded with Tyr-FAP-2286; the radioactive element labeled dextran microspheres loaded with Tyr-FAP-2286 are radioembolic microspheres with secondary targeting capability.

[0007] Preferably, the ratio of the Tyr-FAP-2286-loaded dextran microspheres to the 0.9% NaCl aqueous solution is 1 mg: (5-15) μL; more preferably, it is 1 mg: 10 μL.

[0008] Preferably, the ratio of the Tyr-FAP-2286-loaded dextran microspheres to the radioactive solution is 1 mg: (5-15) μL; more preferably, it is 1 mg: 10 μL.

[0009] Preferably, the particle size distribution of the Tyr-FAP-2286 loaded dextran microspheres is 10-200 μm; more preferably, it is 20-70 μm, 70-100 μm, 100-150 μm.

[0010] Preferably, the radioactive solution comprises Na 131 I. Na 125 One or more of I.

[0011] Preferably, the Na 131 The concentration of solution I is 300-800 MBq / μL; more preferably, it is 555 MBq / μL.

[0012] Preferably, the shaking method is a constant temperature shaking metal bath.

[0013] Preferably, the centrifugal speed is 5000-10000 rpm, and the centrifugal time is 3-8 minutes; further preferably, the centrifugal speed is 8000 rpm, and the centrifugal time is 5 minutes.

[0014] Preferably, the Tyr-FAP-2286-loaded dextran microspheres include Tyr-FAP-2286@DMs or Tyr-FAP-2286-DMs.

[0015] Preferably, the preparation method of Tyr-FAP-2286@DMs is a physical adsorption method.

[0016] Preferably, the physical adsorption method comprises the following steps:

[0017] S1: adding purified water to dextran microspheres (DMs) to swell the dextran microspheres, adding the fully swollen dextran microsphere suspension to a packed column, and balancing the packed column with purified water to form a DMs packed column;

[0018] S2: Prepare Tyr-FAP-2286 solution with purified water, slowly add the Tyr-FAP-2286 solution into the DMs column, collect the eluate, add it into the DMs column again, repeat for more than 3 times, and obtain the DMs solution adsorbed with Tyr-FAP-2286;

[0019] S3: taking out the DMs solution adsorbed with Tyr-FAP-2286 from the packed column and centrifuging to obtain the supernatant and microsphere precipitate;

[0020] S4: The microsphere precipitate is washed with pure water, centrifuged, the supernatant and precipitate are collected, and the precipitate is placed in a drying oven to dry, and Tyr-FAP-2286@DMs is obtained after drying.

[0021] Preferably, a PD-10 desalting chromatography column is installed at the outlet of the lower layer of the packed column in S1.

[0022] Preferably, the swelling time in S1 is 30 to 60 min; more preferably, it is 30, 40, 50, or 60 min.

[0023] Preferably, the mass fraction of pure water in S1 is more than 3 times that of the dextran microspheres.

[0024] Preferably, the ratio of Tyr-FAP-2286 to pure water in S2 is (50 μg to 500 μg):1 mL, and more preferably, it is 200 μg:1 mL.

[0025] Preferably, the number of repetitions in S2 is 3 to 10 times; more preferably, it is 3, 4, 5, 6, 7 or 8 times.

[0026] Preferably, the volume of the pure water in S4 is 0.5 to 3 mL; more preferably, it is 0.5, 1, 2, or 3 mL.

[0027] Preferably, the drying oven drying temperature is 20 to 30°C; more preferably, 20 to 25°C.

[0028] Preferably, the schematic diagram of the physical adsorption method is shown in Figure 1 .

[0029] Preferably, the preparation method of Tyr-FAP-2286-DMs is an esterification reaction method.

[0030] Preferably, the esterification reaction method comprises the following steps:

[0031] M1: DMs, Tyr-FAP-2286, dicyclohexylcarbodiimide, and 4-dimethylaminopyridine were added to a vial, sealed, and purified water was added to react at 0°C for 2-4 hours, and then reacted at room temperature for 16-24 hours;

[0032] M2: After the reaction is completed, the supernatant 1 and microsphere precipitate are obtained by centrifugation; the microsphere precipitate is washed by centrifugation with pure water, and the precipitate and the supernatant 2 are collected after centrifugation; the precipitate is placed in a drying oven to dry, and Tyr-FAP-2286-DMs is obtained after drying.

[0033] Preferably, the mass ratio of DMs, Tyr-FAP-2286, dicyclohexylcarbodiimide and 4-dimethylaminopyridine in the M1 is 1 mg: (0.5-5) μg: (0.1-1) μg: (0.1-0.5) μg; further preferably, it is 1 mg: 1 μg: 0.5 μg: 0.25 μg.

[0034] Preferably, the ratio of DMs to pure water in the M1 is (100-500) mg:1 mL; more preferably, it is 200 mg:1 mL.

[0035] Preferably, the ratio of DMs to pure water in the M2 is (100-500) mg:1 mL; more preferably, it is 200 mg:1 mL.

[0036] Preferably, the schematic diagram of the esterification reaction method is shown in Figure 2 .

[0037] Preferably, the types of the dextran microspheres include one or more of Sephadex G15, Sephadex G25, and Sephadex G50.

[0038] The present invention successfully prepared Tyr-FAP-2286-DMs by dicyclohexylcarbodiimide catalysis, and after connecting with nuclides, the aggregation of radioactive embolic microspheres with secondary targeting ability at the tumor site was improved, thereby improving the clarity of tumor part imaging and reducing the entry of nuclides into the blood. The inventors found that Tyr-FAP-2286-DMs was successfully catalyzed by radionuclides through the Iodogen method. 125 I and 131 I mark, through 125 The cumulative release rate experiment of I-labeled DMs showed that Tyr-FAP-2286-DMs effectively reduced 125 The probability of I entering the serum. 131 I nuclide solution can kill tumor cells and vascular cells.

[0039] In the present invention, by selecting dextran microspheres DMs, it can be specifically connected to the Tyr-FAP-2286 targeted drug, can be well developed in the liver cancer animal model, and has high safety performance. The inventor creatively discovered that dextran microspheres have a variety of models, Sephadex G15, Sephadex G25, Sephadex G50, etc., with different particle sizes and swelling properties, which can meet individualized administration. At the same time, dextran microspheres can be proved by animal models to be able to be used as carrier embolic agents for radionuclides with a long half-life. Polylactic acid microspheres, as a kind of acidic product, are prone to swelling of varying degrees at the site of vascular embolism, and are also prone to non-specific sterile inflammatory reactions. Compared with polylactic acid microspheres, DMs are neutral, will not stimulate the site of vascular embolism, and will not cause inflammation. At the same time, the possibility of rejection by the body is also reduced, and DMs has excellent biocompatibility.

[0040] The second aspect of the present invention provides a method for preparing radioembolic microspheres with secondary targeting capability. The prepared radioembolic microspheres with secondary targeting capability are radioactive element labeled dextran microspheres loaded with Tyr-FAP-2286.

[0041] Preferably, the Tyr-FAP-2286-loaded dextran microspheres include Tyr-FAP-2286@DMs or Tyr-FAP-2286-DMs.

[0042] The third aspect of the present invention provides an application of radioembolic microspheres with secondary targeting capability, which is applied to the preparation of radioembolic drugs for cancer.

[0043] Preferably, the cancer includes one or more of liver cancer, pancreatic cancer, breast cancer, colorectal cancer, and ovarian cancer.

[0044] Beneficial Effects

[0045] 1. The present invention successfully prepared Tyr-FAP-2286-DMs by dicyclohexylcarbodiimide catalysis, and when connected with nuclides, the aggregation of radioactive embolic microspheres with secondary targeting ability at the tumor site was improved, thereby improving the clarity of tumor part imaging and also reducing the entry of nuclides into the blood.

[0046] 2. In the present invention, by selecting dextran microspheres DMs, they can be specifically connected to the Tyr-FAP-2286 targeting drug, can be better developed in the liver cancer animal model, and have high safety performance.

[0047] 3. The present invention combines dextran microspheres, targeted drugs and radionuclides for the first time, thereby improving the therapeutic effect and safety of the radionuclides. The inventors found that after injecting radioactive embolic microspheres with secondary targeting capability, the radionuclides in the radioactive embolic microspheres have a long retention time in the blood vessels near the tumor area, and are only concentrated in the tumor area without being concentrated in other organs, indicating that the radioactive embolic microspheres have good stability. In addition, the free radionuclides that are not bound to the microspheres will be excreted in the urine after being metabolized by the blood circulation. Therefore, after a relatively large dose of treatment, while maintaining its radiotherapy effect, it also effectively reduces the occurrence of side effects, thereby improving safety performance.

[0048] 4. The radioembolic microspheres with secondary targeting capability in the present invention, after being used for TARE treatment, have strong killing power on tumor cells at the pathological level, can cause large-scale coagulative necrosis of tumor cells, and some tumor cells wrapped in fibers and at the edge of the tumor also undergo necrosis, and tumor activity is largely inhibited. In addition, the distal normal liver cells are in good condition after embolization with radioembolic microspheres, and the activity of normal liver is maintained to the greatest extent while killing the tumor. In short, the radioembolic microspheres with secondary targeting capability in the present invention cause the death of VX2 tumor cells, inhibit the growth and intrahepatic metastasis of VX2 tumors, and retain the activity of distal liver cells, confirming the feasibility of TARE treatment of liver cancer with radioembolic microspheres with secondary targeting capability, and further providing experimental basis for functional research and clinical transformation research of radioembolic microspheres.

[0049] 5. The radioactive embolic microspheres with secondary targeting capability in the present invention can be used to perform real-time positioning of the radioactive embolic microspheres with secondary targeting capability by means of SPECT / CT imaging, through the position of the highlighted part on the image, to determine the embolic position and some related semi-quantitative monitoring to determine the nuclide 131 I distribution in the body. SPECT / CT imaging and data analysis can effectively identify the degree of radionuclide accumulation in tumor and non-tumor areas to evaluate the embolization of the tumor area and predict the therapeutic effect of TARE. SPECT / CT imaging can detect gastric reflux and lung impact in model rabbits after TARE treatment as early as possible, exclude the group of surgical failure as early as possible, and make timely remedial measures in the clinic to reduce adverse reactions and avoid serious sequelae. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 Schematic diagram of physical adsorption synthesis of Tyr-FAP-2286@DMs in the invention content

[0051] Figure 2 Synthesis of Tyr-FAP-2286-DMs by esterification reaction in the present invention

[0052] Figure 3These are HPLC graphs obtained after HPLC detection of Tyr-FAP-2286@DMs, Tyr-FAP-2286-DMs and standard products in Examples 1 and 2. Figure A is the HPLC graph of the standard product Tyr-FAP-2286, Figure B is the HPLC graph of the supernatant solution in Example 1, and Figure C is the HPLC graph of the supernatant solution in Example 2.

[0053] Figure 4 The figure is a nuclide release curve diagram of the radioactive iodine drug delivery system in Examples 3 and 4.

[0054] Figure 5 This is a graph showing the tumor results of a rabbit VX2 liver carcinoma in situ transplanted tumor 8 days after surgery in the performance test.

[0055] Figure 6 Performance test of VX2 liver carcinoma in situ xenografts in rabbits 2 weeks after surgery 18 F-FDG PET imaging

[0056] Figure 7 These are DSA angiography images during the performance test; A is the DSA angiography before embolization, and B is the DSA angiography after embolization.

[0057] Figure 8 The rabbit used in the performance test was 131 The change curve of the ratio of radioactive counts to muscle (background) of the same volume of tumor, liver, lung, and stomach at 0, 10, and 24 hours after I-Tyr-FAP-2286-DMs interventional embolization;

[0058] Fig. 9 This is a graph showing the change in the ratio of the radioactivity counts in the tumor area to the whole body of the rabbit used in the performance test.

[0059] Fig.10 The performance test was performed on the liver of rabbits before and after embolization. 18 F-FDG PET / CT images and statistical graphs of SUVmax changes in the tumor area before and after embolization; A is the model rabbit before and after DMs interventional embolization treatment 18 F-FDG PET / CT image, B is 131 I@DMs-related TARE model rabbits before and after treatment 18 F-FDG PET / CT image, C is 131 I-Tyr-FAP-2286-DMs-related TARE model rabbits before and after treatment 18 F-FDG PET / CT images, D: Change rate of SUVmax value in the tumor area of ​​the model rabbit before and after treatment.

[0060] Fig.11 For Example 2 131H&E staining images of vascular sections after I-Tyr-FAP-2286-DMs embolization therapy; the optical microscope magnifications were 100 and 400 times. 131 15 days after I-Tyr-FAP-2286-DMs embolization, the embolized vessels were pathologically sectioned and stained with H&E. The left side is at a magnification of 100 times; the right side is at a magnification of 400 times.

[0061] Fig.12 The H&E staining images of tumor tissue sections in the performance test; A is the H&E staining image of normal untreated tumor tissue sections, B is the H&E staining image of tumor tissue sections after DMs intervention embolization, and C is the H&E staining image of tumor tissue sections after DMs intervention embolization. 131 H&E staining of tumor tissue sections after I@DMs interventional embolization. D is the H&E staining of tumor tissue sections after I@DMs interventional embolization. 131 H&E staining of tumor tissue sections after I-Tyr-FAP-2286-DMs interventional embolization. The optical microscope magnification was 40 and 400 times. Pathological sections and H&E staining of tumor tissues of the control group that was not treated during the same period were performed 15 days after microsphere interventional embolization.

[0062] Fig.13 Figure 2 is the Bax staining image of tumor tissue slices in the performance test; A is the Bax staining image of tumor tissue slices after DMs intervention embolization, and B is the Bax staining image of tumor tissue slices after DMs intervention embolization. 131 Bax staining of tumor tissue sections after I@DMs interventional embolization. 131 Bax staining of tumor tissue sections after I-Tyr-FAP-2286-DMs interventional embolization. The optical microscope magnification was 100 and 400 times. 15 days after microsphere interventional embolization, the tumor tissue was pathologically sectioned and stained for Bax.

[0063] Fig.14 is the Sirius red staining image of tumor tissue sections in performance test; A is the 131 Sirius red staining of tumor tissue after I@DMs interventional embolization, B is 131 Sirius red staining of tumor tissue after I-Tyr-FAP-2286-DMs interventional embolization. The optical microscope magnification is 200 times. 131 I@DMs, 131 Fifteen days after I-Tyr-FAP-2286-DMs embolization, the tumor tissues were pathologically sectioned and stained with picrosirius red. DETAILED DESCRIPTION

[0064] Example 1

[0065] The first aspect of this embodiment provides a method for preparing radioembolic microspheres with secondary targeting capability, comprising the following steps:

[0066] Take 10 mg of Tyr-FAP-2286-loaded dextran microspheres (20-70 μm) and resuspend them in 100 μL of 0.9% NaCl aqueous solution to form a 100 mg / mL DMs suspension. Add it to the Iodogen tube. In the Iodogen tube, continue to add 100 μL of Na 131 Solution I (555 MBq / μL) was shaken at 25°C in a constant temperature shaking metal bath for 20 minutes to obtain 131 The I-labeled suspension was centrifuged at 8000 rpm for 5 minutes to obtain the supernatant and precipitate. The precipitate was washed to obtain 131 I-labeled dextran microspheres loaded with Tyr-FAP-2286, i.e., radioembolic microspheres with secondary targeting capability.

[0067] The Tyr-FAP-2286-loaded dextran microspheres are Tyr-FAP-2286@DMs.

[0068] The preparation method of Tyr-FAP-2286@DMs is a physical adsorption method.

[0069] The physical adsorption method comprises the following steps:

[0070] S1: Add purified water to dextran microspheres (DMs) to swell for 30 minutes, add the swollen suspension to the packed column, and balance the packed column with purified water to form a DMs packed column;

[0071] S2: 200 μg of Tyr-FAP-2286 was prepared into a Tyr-FAP-2286 solution with 1 mL of pure water, and the Tyr-FAP-2286 solution was slowly added into a DMs packed column (a PD-10 desalting chromatography column was installed at the outlet of the lower layer of the packed column), and the eluate was collected and added into the DMs packed column again, and the process was repeated 7 times to obtain a DMs solution adsorbed with Tyr-FAP-2286;

[0072] S3: The DMs solution adsorbed with Tyr-FAP-2286 in the packed column was taken out and centrifuged at 8000 rpm for 5 minutes to obtain the supernatant and microsphere precipitate;

[0073] S4: Wash the microsphere precipitate with 1 mL of pure water, centrifuge at 8000 rpm for 5 minutes, collect the supernatant and precipitate, place the precipitate in an electric heated air drying oven, and dry it at a constant temperature of 25°C to obtain Tyr-FAP-2286@DMs.

[0074] The dextran microspheres were purchased from Sephadex G15 of Hefei Bomei Biotechnology Co., Ltd.

[0075] The Tyr-FAP-2286 is prepared according to the preparation method in Example 1 of patent CN202211741941.1.

[0076] The second aspect of this embodiment provides a method for preparing radioembolic microspheres with secondary targeting capability. The radioembolic microspheres with secondary targeting capability are prepared by the method. 131 I-labeled Tyr-FAP-2286-loaded dextran microspheres, i.e. 131 I-Tyr-FAP-2286-DMs.

[0077] The Tyr-FAP-2286-loaded dextran microspheres include Tyr-FAP-2286@DMs. The third aspect of this embodiment provides an application of radioembolic microspheres with secondary targeting capability, which are applied to the preparation of radioembolic drugs for cancer.

[0078] The cancer is liver cancer.

[0079] Example 2

[0080] The specific implementation of Example 2 is the same as that of Example 1, except that the Tyr-FAP-2286-loaded dextran microspheres are Tyr-FAP-2286-DMs.

[0081] The preparation method of Tyr-FAP-2286-DMs is an esterification reaction method.

[0082] The esterification reaction method comprises the following steps:

[0083] M1: Add 200 mg of DMs, 200 μg of Tyr-FAP-2286, 100 μg of dicyclohexylcarbodiimide, and 50 μg of 4-dimethylaminopyridine into a 3 mL vial, seal it, add 1 mL of purified water, react at 0 °C for 3 h, and then react at 25 °C for 16 h.

[0084] M2: After the reaction is completed, centrifuge at 8000 rpm for 5 minutes to obtain supernatant 1 and microsphere precipitate; wash the microsphere precipitate with 1 mL of pure water, collect the precipitate and supernatant 2 after centrifugal washing; place the precipitate in an electric heated air drying oven and dry it at a constant temperature of 25°C to obtain Tyr-FAP-2286-DMs.

[0085] Example 3

[0086] The specific implementation of Example 3 is the same as that of Example 1, except that the radioactive solution is Na 125 I solution; the Na 125 The concentration of I solution is 135MBq / μL.

[0087] Example 4

[0088] The specific implementation of Example 4 is the same as that of Example 3, except that the radioactive solution is Na 125 I solution; the Na 125 The concentration of I solution is 135MBq / μL.

[0089] Comparative Example 1

[0090] The first aspect of this comparative example provides a method for preparing radioembolic microspheres with secondary targeting capability, comprising the following steps:

[0091] Take 10 mg (20-70 μm) of dextran microspheres and resuspend them in 100 μL of 0.9% NaCl aqueous solution to form a 100 mg / mL DMs suspension. Add it to the Iodogen tube. In the Iodogen tube, add 100 μL of Na 131 Solution I (555 MBq / μL) was shaken at 25°C in a constant temperature shaking metal bath for 20 minutes to obtain 131 The I-labeled suspension was centrifuged at 8000 rpm for 5 minutes to obtain the supernatant and precipitate. The precipitate was washed to obtain 131 I-labeled dextran microspheres, i.e. 131 I@DMs. Determined by specific activity reagent 131 The supernatant of I-labeled dextran microspheres and the added Na 131 The dosage of the solution is used to determine the dosage of the microspheres.

[0092] Performance Testing

[0093] 1. High performance liquid chromatography (HPLC) using Waters C18 chromatographic column (3.9 mm × 150 mm, 4 μm), acetonitrile as mobile phase, detection wavelength 220 nm, column temperature 25 ± 2 ° C, by comparing with the peak area of ​​the standard product to calculate the mass of Tyr-FAP-2286 contained in Tyr-FAP-2286@DMs and Tyr-FAP-2286-DMs. Figure 3 , the encapsulation efficiency and drug loading of Tyr-FAP-2286 were calculated.

[0094] The specific activity of the supernatant and the added Na in Examples 1, 2 and 3 was determined by a specific activity reagent. 131 I solution dosage to determine the dosage of microspheres. Wash and centrifuge 131 I@DMs and 131 I-Tyr-FAP-2286-DMs (2.7MBq / mg).

[0095] Table 1

[0096] Tyr-FAP-2286 encapsulation efficiency Drug loading rate of Tyr-FAP-2286 Example 1 65.29±2.72％ 0.0356％ Example 2 55.52±7.60％ 0.0278％

[0097] 2. The zeta potential of DMs, Tyr-FAP-2286@DMs and Tyr-FAP-2286-DMs in Examples 1, 2 and 3 was measured using a potentiometer. The results are shown in Table 2. The potential change indicates that the FAP binding peptide analog Tyr-FAP-2286 binds to the surface of DMs.

[0098] Table 2

[0099] Zeta potential / mV Example 1 3.31±0.73 Example 2 1.63±0.74 Comparative Example 1 6.07±1.2

[0100] 3. 10 mg of the 125 I-labeled DMs and Tyr-FAP-2286-DMs were added to 1 mL of 0.9% NaCl solution and 1 mL of serum (fetal bovine serum, purchased from Shanghai Biyuntian Biotechnology Co., Ltd.), respectively, shaken and placed on a shaker. Centrifuge every 2 hours (2, 4, 6, 8, 10, 12, 14, 16, 18 hours), take 40 μL of the supernatant, and add 40 μL of 0.9% NaCl or serum solution. The collected supernatant was monitored for radiation dose using a radioactivity meter and fitted with an in vitro cumulative release curve. The results are shown in Figure 4 .

[0101] Figure 4 Shows the change of time at room temperature (25℃) 125 I Cumulative release. 125 I@DMs, 125 I-Tyr-FAP-2286-DMs all showed a slow and nearly uniform release rate. 125 I@DMs, 125 I-Tyr-FAP-2286-DMs 125 I releases less and at a slower rate. This indicates that the introduction of Tyr-FAP-2286 into the hydroxyl end of DMs through a dicyclohexylcarbodiimide-catalyzed esterification reaction can effectively slow down the release rate of Tyr-FAP-2286. 125 The release rate of I in serum is faster than that in pure water. The esterification synthesis method can effectively reduce the probability of radionuclides entering the blood and reduce the occurrence of side reactions. 125 I and 131 I mark. 125 The cumulative release rate experiment of I-labeled DMs showed that Tyr-FAP-2286-DMs effectively reduced 125 The probability of I entering the serum. 131I nuclide solution can kill tumor cells and vascular cells.

[0102] 4. Animal experiments

[0103] 4.1 Modeling: Fasting was performed on experimental rabbits to prevent the stomach from interfering with the surgical field of view and to avoid intestinal reflux. Male 6-month-old New Zealand white rabbits weighing 2.5±0.3kg were randomly selected and maintained anesthesia under a small animal gas anesthesia machine (gas flow rate was 700mL / min, and the isoflurane concentration was maintained within the range of 2-2.5%). After anesthesia, surgery was performed to implant a 3mm diameter treated VX2 rabbit anaplastic epidermal squamous cell carcinoma tumor. After the tumor was implanted, the liver was gently placed back in place. Before suturing layer by layer, a small amount of penicillin was injected into the abdominal cavity to prevent infection, and iodine cotton swabs were disinfected layer by layer. After suturing layer by layer, the wound was disinfected with iodine, and 200,000 units of penicillin were injected intramuscularly to prevent postoperative infection.

[0104] 4.2 Model verification: 8 days after the rabbit VX2 liver carcinoma in situ transplantation surgery, the rabbit was sacrificed, liver tissue was taken, and the implanted liver lobe was gently pressed by hand to find a lump. After confirming the location, the liver was cut open to expose the tumor. The tumor tissue was fish-like, with clear edges, no liquefaction necrosis, good tumor activity, and a diameter of 5-6 mm (see Figure 5 ), and the surrounding liver was in good condition. Two weeks after the tumor was transplanted into the experimental rabbit, the experimental rabbit was subjected to 18 F-FDG PET / CT imaging was used to monitor the tumor. The specific operation was that the experimental rabbits were fasted for 8 hours, placed in a prone position under small animal gas anesthesia, and anesthetized. 200 μL of 18 F-FDG solution, about 7.2-10.8MBq / mouse. 18 60 minutes after the injection of F-FDG solution, systemic 18 F-FDG PET / CT imaging. Set the CT tube voltage to 120 kV, tube current to 100 mA, and acquisition time to 18 1 hour after F-FDG administration. Results are shown in Figure 5 . 18 F-FDG PET imaging revealed the presence of 18 F-FDG high uptake, the image shows abnormally high radioactivity concentration ( Figure 6 ).

[0105] 4.3 Using the prepared in Examples 2 and 3 131 I@DMs, 131 I-Tyr-FAP-2286-DMs were used for the experiment: 6 model rabbits were randomly selected for interventional surgery, TARE and DMs embolization were performed. The animals were anesthetized under gas anesthesia. After anesthesia, the model rabbits were fixed on the operating table in a supine position with their limbs straightened. Since the thyroid gland is highly iodine-sucking, in order to prevent free131 I enters the thyroid follicular cells through the sodium / iodine co-transporter to interfere with the experiment. The model rabbit is blocked by intraperitoneal injection of an appropriate amount of sodium iodide solution. The inner thigh of the right hind limb of the model rabbit is depilated and disinfected with iodine, and a medical surgical drape is laid. According to the anatomical direction of the femoral artery of the rabbit, the skin and muscle layer are cut open layer by layer with a scalpel, the femoral artery is bluntly separated, and the lower side of the femoral artery, i.e., the distal end, is ligated. According to the Seldinger technique, the femoral artery is punctured with a puncture needle, and a 4F arterial sheath (an arterial sheath flushed with heparin water is inserted to prevent blood coagulation and thrombosis) is inserted. After a small amount of heparin water is injected, a 4F microcatheter is immediately inserted to prevent the model rabbit from bleeding too much and causing blood loss. The 4F arterial sheath is then fixed to the femoral artery wall with surgical sutures. By operating the end handle of the guidewire, the catheter and guidewire are sent into the common hepatic artery through the abdominal aorta. Under the DSA instrument, Uvitra angiography is performed to confirm the positional relationship between the microcatheter and the common hepatic artery and ensure that the microcatheter enters the common hepatic artery. Angiography is performed again, and the location of the hepatic artery and tumor is found according to the shape of the surrounding blood vessels. After the microguidewire entered the proper hepatic artery, Ulvist angiography was used to observe the appearance of a circular area surrounded by multiple blood vessels, which was identified as a tumor. Figure 7 A. After confirming the location of the tumor, the superselective catheterization technique was used to deliver the catheter to the tumor's blood supply artery. After the microcatheter reached the tumor's blood supply artery, the catheter was fixed and the syringe of radioactive microspheres (located in the syringe lead protective sleeve) was slowly pushed into the blood vessel through the three-way. DMs group 2 (20 mg / each) 131 I@DMs (Comparative Example 1) group 2 (20 mg / each, 2.7 MBq / mg) and 131 Two I-Tyr-FAP-2286-DMs (Example 2) mice (20 mg / each, 2.7 MBq / mg) were given the drug and then the microcatheter was flushed with 0.9% NaCl solution. After the drug was given, the vascular embolism near the tumor was observed by ultraviolet injection. Figure 7 B.

[0106] Figure 7 A and Figure 7 B shows that after interventional surgery for DMs, 131 I-Tyr-FAP-2286-DMs were precisely embolized in the blood supply artery of the tumor, with good embolization effect. The peripheral blood vessel blood flow was blocked, which could effectively cut off the main nutritional support of the rabbit VX2 tumor.

[0107] 4.4 SPECT / CT imaging and localization of radioactive microspheres: The experimental rabbits were placed in a prone position under small animal gas anesthesia to maintain anesthesia. The flow rate of isoflurane needs to be reduced. A low-energy high-resolution collimator was used for image acquisition, and the basic acquisition parameters are as follows: energy peak: 364keV, acquisition matrix: 128×128, window width: 20%. The CT tube voltage was 130kV, the CT tube current was 35mA, the inter-slice spacing was selected as 1mm, and the reconstruction thickness was 1mm. 131 I@DMs and 131 The abdomen and legs of rabbits in the I-Tyr-FAP-2286-DMs group were subjected to SPECT / CT imaging (at 0, 10, and 24 h, respectively), and the internal radioactivity counts of a certain volume of normal liver, lung, stomach, whole body, and muscle (background) were measured at 0, 10, and 24 h. The results are shown in Figure 8 The volume of the region of interest (ROI) of the liver was manually outlined on the SPECT / CT image, and the internal radioactivity counts of a certain volume of liver tumor area (primary lesion), normal liver, thigh muscle (background), and whole body were measured. Fig. 9 , from SPECT / CT images, it was found that from 0 to 24h, 131 The ratio of real-time tumor / tumor volume to whole body count in the I-Tyr-FAP-2286-DMs group gradually increased over time, while 131 The ratio of tumor / tumor volume to whole body counts of I@DMs gradually decreased.

[0108] Figure 8 , Fig. 9 This indicates that there is a clear contrast between the tumor and the normal liver, the tumor site remains bright in the SPECT image, and the radioactivity count remains at a high level. 131 I showed a higher tumor-to-normal tissue ratio in the in vivo radiation distance, which is conducive to clearer imaging of the tumor area and maintaining a higher therapeutic radiation dose in the tumor area. The count ratio of normal liver, stomach, lung and muscle (background) from 0 to 24 hours was much lower than the count ratio of tumor and muscle (background), proving that less radiation was irradiated to normal tissues during treatment.

[0109] 131 I@DMs, 131 After TARE treatment with I-Tyr-FAP-2286-DMs, the radioactivity count in the liver tumor site gradually decreased over time, but from immediately after TARE to 72 hours after TARE, the tumor site maintained a high radioactivity concentration, suggesting that 131 The main site of I deposition was the liver, and no obvious high radioactivity distribution was found in other parts and organs. 131 I@DMs, 131 I-Tyr-FAP-2286-DMs has good stability in vivo and can be used in combination with131 I was used for radiotherapy. On the 5th day after surgery, the radioactivity was mainly concentrated in TARE. Because sodium iodide solution was used to block thyroid uptake before treatment, no high radioactivity was found in the thyroid gland from 0 to 24 hours, indicating that the thyroid gland did not uptake 131 I. Confirmation 131 I can be well labeled on DMs or Tyr-FAP-2286-DMs. 131 I can bind firmly to Tyr-FAP-2286-DMs. 131 I@DMs, 131 The therapeutic effect of I-Tyr-FAP-2286-DMs in vivo is stable.

[0110] 4.5 18 F-FDG PET / CT imaging for monitoring tumor activity: an analysis of 131 I@DMs, 131 The TARE of I-Tyr-FAP-2286-DMs and DMs embolization treatment were performed in rabbits 5 days after the experiment. 18 F-FDG PET / CT imaging, 18 F-FDG PET images Fig.10 , no obvious radioactive concentration was found in the liver. Compared with the images before treatment, there was no radioactive uptake in the area corresponding to the tumor before treatment, and no low-density foci were found on the CT images. It was found that the difference in SUV before and after treatment in the radioactive concentration area of ​​the TARE liver was greater than that in the untreated group. 131 Liver before and after I-Tyr-FAP-2286-DMs treatment 18 The SUVmax change rate in the F-FDG high uptake area was 53.62±10.29%. 131 Liver before and after I@DMs treatment 18 The SUVmax change rate in the F-FDG high uptake area was 25.94±4.64%, which was much lower than 131 I-Tyr-FAP-2286-DMs. Liver before and after DMs treatment 18 The SUVmax change rate of the F-FDG high uptake area was 4.40±3.72%, and the area near the tumor showed high radioactivity concentration before and after treatment. 131 I@DMs, 131 After I-Tyr-FAP-2286-DMs treatment, no obvious large-area necrosis was observed near the tumor site, and the rabbits were in good condition after surgery. 131 I@DMs, 131 I-Tyr-FAP-2286-DMs has a good therapeutic effect and good safety in the surrounding normal tissues under CT.

[0111] 4.6 Pathological examination: 15 days after treatment, the experimental rabbits were killed by overdose of anesthesia. The tumors on the liver of the experimental rabbits were taken for pathological sections and H&E staining. The tissues were fixed with 10% neutral formalin solution, then dehydrated, embedded, and then paraffin sectioned. The paraffin sections were 4-5μm thick and stained with hematoxylin and eosin. The tissue structure and cell status were observed under an optical microscope. The results are shown in Figures 11 to 14 .

[0112] Fig.11 Shown in: Model Rabbit 131 Two weeks after I-Tyr-FAP-2286-DMs embolization, the tumor and surrounding tissues were pathologically sectioned and stained with H&E. Under a light microscope, DMs with complete spherical structures could be observed in the peripheral arterial vessels, and the DMs did not show any agglomeration or clumping. This confirmed that DMs with small particle sizes could enter the peripheral blood vessels supplying the tumor along the direction of injection and deposit in the peripheral blood vessels to achieve the effect of blood flow blockage. 131 I-Tyr-FAP-2286-DMs maintained a constant embolization position and did not deviate into surrounding tissues, confirming that DMs can be used as a carrier embolic agent for radionuclides with a long half-life. 131 I had no effect on the structure of DMs. 131 No obvious inflammation-related cells were found near the I-Tyr-FAP-2286-DMs embolized blood vessels, indicating that DMs have good biocompatibility and can effectively avoid vascular inflammation caused by embolization materials and reduce the possibility of body rejection. 131 I-Tyr-FAP-2286-DMs can enter the peripheral blood vessels around the tumor and effectively play the role of terminal vascular embolism, effectively cutting off the nutrient supply of the tumor. No obvious nuclei were found in the vascular cells around DMs, and some vascular cells died, effectively reducing the probability of new blood vessels and preventing the formation of collateral circulation.

[0113] Fig.12 Shows: The tumor contains dense desmoplastic stroma, separating the tumor cells into clusters ( Fig.12 A). After 15 days of DMs embolization, tumor tissue sections showed that the tumor cell nuclei and chromosomes in the nuclei remained darkly stained, and the ratio of the nucleus to the cytoplasm was large. Some cytoplasmic areas became darker, from pink to dark red, and eosinophilic changes occurred ( Fig.12 B). 131 I@DMs showed that a large number of tumor cell nuclei disappeared, but tumor cells in the fiber-wrapped area were active and the chromosomes in the nuclei remained darkly stained. Tumor cells near the blood vessels were darkly stained and densely arranged ( Fig.12 C). 131After I-Tyr-FAP-2286-DMs treatment, tumor cells showed large areas of coagulative necrosis, tumor cell nuclei became sparse, the nuclei became smaller, and nuclear condensation, nuclear dissolution, and nuclear fragmentation occurred. Tumor cells turned from pink to light red, and tumor cell nuclei disappeared in some areas. Distal hepatocytes were in good condition, still arranged radially, maintaining the hepatic cord structure, with relatively large nuclei and no nuclei ( Fig.12 D). 131 I-Tyr-FAP-2286-DMs have a tumor-killing effect and are friendly to distal normal liver tissue.

[0114] Fig.13 Display: After Bax staining of tumor tissue sections, the results showed that 15 days after surgery 131 I@DMs and 131 Compared with the DMs group, the blue-purple nuclei in the I-Tyr-FAP-2286-DMs group were significantly reduced, and some nuclei and cytoplasm appeared brown and Bax-positive, indicating apoptosis or damage of tumor cells. 131 The I-Tyr-FAP-2286-DMs group had the fewest blue-purple normal cell nuclei, with more cytoplasm in light brown, and some parts of the cells were nucleated, which had the most obvious effect on tumor damage, confirming that 131 I-Tyr-FAP-2286-DMs embolization therapy has excellent effect.

[0115] Fig.14 Display: The results of Sirius red staining of tumor tissue showed that 15 days after surgery 131 I@DMs, 131 In the I-Tyr-FAP-2286-DMs treatment group, fibrosis was observed in some tissues around the tumor, with large red-stained areas, but no obvious inflammatory cell infiltration was observed around the fibrous matrix. 131 After I-Tyr-FAP-2286-DMs treatment, there were more tumor fibrosis areas and a significant decrease in viable tumor cells. After tumor cells become fibrotic, FAP-2286 has a chance to bind to FAP in the fibrotic area.

Claims

1. A method for preparing radioembolic microspheres with secondary targeting capability, characterized in that: The following steps are involved: The dextran microspheres loaded with Tyr-FAP-2286 are resuspended in a 0.9% NaCl aqueous solution to form a DMs suspension, which is added to an Iodogen tube. The radioactive solution is continuously added to the Iodogen tube, and the suspension is shaken at room temperature for 20 to 30 minutes to obtain a radioactive solution labeled suspension, which is centrifuged to obtain a supernatant and a precipitate, and the precipitate is washed to obtain radioactive element labeled dextran microspheres loaded with Tyr-FAP-2286; the radioactive element labeled dextran microspheres loaded with Tyr-FAP-2286 are radioembolic microspheres with secondary targeting capability.

2. The method for preparing radioembolic microspheres with secondary targeting capability according to claim 1, characterized in that: The ratio of the Tyr-FAP-2286 loaded dextran microspheres to the radioactive solution is 1 mg: (5-15) μL.

3. The method for preparing radioembolic microspheres with secondary targeting capability according to claim 1, characterized in that: The radioactive solution includes Na 131 I. Na 125 One or more of I.

4. The method for preparing radioembolic microspheres with secondary targeting capability according to claim 1, characterized in that: The particle size distribution of the Tyr-FAP-2286 loaded dextran microspheres is 10-200 μm.

5. The method for preparing radioembolic microspheres with secondary targeting capability according to claim 1, characterized in that: The centrifugal speed is 5000-10000 rpm, and the centrifugal time is 3-8 minutes.

6. The method for preparing radioembolic microspheres with secondary targeting capability according to claim 1, characterized in that: The Tyr-FAP-2286-loaded dextran microspheres include Tyr-FAP-2286@DMs or Tyr-FAP-2286-DMs. The preparation method of the Tyr-FAP-2286@DMs is a physical adsorption method; the preparation method of the Tyr-FAP-2286-DMs is an esterification reaction method.

7. The method for preparing radioembolic microspheres with secondary targeting capability according to claim 6, characterized in that: The physical adsorption method comprises the following steps: S1: adding purified water to the dextran microspheres to make the dextran microspheres swell, adding the completely swollen dextran microsphere suspension to the packed column, and balancing the packed column with purified water to form a DMs packed column; S2: Prepare Tyr-FAP-2286 solution with purified water, slowly add the Tyr-FAP-2286 solution into the DMs column, collect the eluate, add it into the DMs column again, repeat for more than 3 times, and obtain the DMs solution adsorbed with Tyr-FAP-2286; S3: taking out the DMs solution adsorbed with Tyr-FAP-2286 from the packed column and centrifuging to obtain the supernatant and microsphere precipitate; S4: The microsphere precipitate is washed with pure water, centrifuged, the supernatant and precipitate are collected, and the precipitate is placed in a drying oven to dry, and Tyr-FAP-2286@DMs is obtained after drying.

8. The method for preparing radioembolic microspheres with secondary targeting capability according to claim 6, characterized in that: The esterification reaction method comprises the following steps: M1: DMs, Tyr-FAP-2286, dicyclohexylcarbodiimide, and 4-dimethylaminopyridine were added to a vial, sealed, and purified water was added to react at 0°C for 2-4 hours, and then reacted at room temperature for 16-24 hours; M2: After the reaction is completed, the supernatant 1 and microsphere precipitate are obtained by centrifugation; the microsphere precipitate is washed by centrifugation with pure water, and the precipitate and the supernatant 2 are collected after centrifugation; the precipitate is placed in a drying oven to dry, and Tyr-FAP-2286-DMs is obtained after drying.

9. A radioembolic microsphere with secondary targeting capability prepared by the method for preparing radioembolic microsphere with secondary targeting capability according to any one of claims 1 to 8.

10. A use of the radioembolic microspheres with secondary targeting capability according to claim 9, characterized in that: Preparation of radioembolic drugs for cancer treatment.

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