Preparation method and application of radioactive radium-223 labeled alginate microspheres

Abstract

The invention discloses a preparation method of a radioactive radium-223 labeled alginate microsphere, which comprises the following steps: 1, adding isooctane into a sodium alginate solution, and adding surfactants Span80 and Tween80 to form a water-in-oil (W / O) particle emulsion; and step 2, slowly adding a CaCl2 solution or a BaCl2 solution into the water-in-oil W / O particle emulsion prepared in the step 1, continuously and slowly adding a 223RaCl2 injection, and carrying out radiolabeling on the formed microspheres to obtain the radioradium-223 labeled alginate microspheres. The prepared radioactive radium-223 marked alginate microspheres are good in biocompatibility, have the dual functions of internal radiotherapy and embolism, and are simple in synthesis step and good in marking stability. Theoretical and experimental basis is provided for transformation application of alpha nuclide in the TARE direction, and a new effective treatment choice is expected to be provided for vast liver cancer patients.

Description

Technical Field

[0001] The invention belongs to the technical field of clinical medical embolization therapy, and in particular relates to a preparation method and application of alginate microspheres labeled with radioactive radium-223. Background Art

[0002] Over the years, various treatment methods for advanced liver cancer have been continuously innovated, such as transarterial chemoembolization (TACE), targeted therapy, and immunotherapy which has developed rapidly in recent years. However, the mortality rate of liver cancer patients remains high, and there is an urgent need to develop more effective treatment options.

[0003] Transarterial radioembolization (TARE) is one of the classic local interventional treatments. The most commonly used is yttrium-90 ( 90 Y) microspheres. Whether it is the most classic TACE or the promising 90 Y-TARE and other treatments face a problem: residual active tumor cells and hypoxic tumor microenvironment often remain after treatment, leading to poor mid- and long-term efficacy.

[0004] The initial clinical application of the new α-therapeutic nuclide has attracted everyone's attention. The linear energy transfer (LET) of α particles is higher than that of the commonly used β - The radiation is 400-500 times higher, can directly cause DNA double-strand breaks and make it difficult to repair itself, and can induce tumor cell apoptosis without being affected by the cell cycle and environmental oxygen content. It is resistant to conventional chemotherapy, X-rays, γ-ray external radiotherapy and β-rays. - The radiation also kills heterogeneous tumor cells that are insensitive to radiotherapy, and the radiation action distance is short, without affecting the surrounding normal tissues. Due to the high energy of alpha particles, labeling is more difficult. Currently, alpha particles are mostly used to treat tumor bone metastasis. There are few studies on TARE, which is still in its infancy. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing alginate microspheres labeled with radioactive radium-223.

[0006] Another object of the present invention is to provide a use of alginate microspheres labeled with radioactive radium-223 prepared by the method in preparing drugs for arterial radiotherapy embolization.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] The first aspect of the present invention provides a method for preparing alginate microspheres labeled with radioactive radium-223, comprising the following steps:

[0009] The first step is to add isooctane to a sodium alginate solution with a concentration of 0.01 to 0.1 g / mL (preferably 0.025 g / mL), the volume ratio of isooctane to the sodium alginate solution is 1 to 5:1 (preferably 1:1, 2:1), add surfactants Span80 and Tween80, the volume ratio of Span80 to Tween80 is 1 to 8:1 (preferably 5:1), and stir at room temperature for 0.5 to 2 hours (preferably 1 hour) to form a water-in-oil W / O particle emulsion;

[0010] In the second step, a CaCl2 solution with a concentration of 0.01 to 1 g / mL (preferably 0.2 g / mL) or a BaCl2 solution with a concentration of 0.01 to 1 g / mL (preferably 0.33 g / mL) is slowly added to the water-in-oil W / O particle emulsion prepared in the first step, wherein the volume ratio of the CaCl2 solution to the water-in-oil W / O particle emulsion is 1:1 to 4 (preferably 1:2.1, 1:3.1), and the volume ratio of the BaCl2 solution to the water-in-oil W / O particle emulsion is 1:1 to 4 (preferably 1:2.1, 1:3.1), and the addition is continued slowly. 223 RaCl2 injection, radioactively label the formed microspheres, continue stirring for 0.5 to 2 hours (preferably 1 hour), add anhydrous ethanol to break the emulsion, centrifuge to remove the supernatant, collect the precipitate, and fully dry to obtain the radioactive radium-223 labeled alginate microspheres.

[0011] The method for preparing the sodium alginate solution comprises the following steps: adding sodium alginate into ultrapure water, and after it is completely dissolved, standing for at least 2 hours for degassing to obtain a sodium alginate solution with a concentration of 0.01 to 0.1 g / mL (preferably 0.025 g / mL) as an aqueous phase.

[0012] The second aspect of the present invention provides a use of alginate microspheres labeled with radioactive radium-223 prepared by the method in preparing a drug for arterial radiotherapy embolization.

[0013] The third aspect of the present invention provides a use of alginate microspheres labeled with radioactive radium-223 prepared by the method in preparing a drug for treating liver cancer.

[0014] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0015] The radioactive radium-223 labeled alginate microspheres provided by the present invention for treating liver cancer have cheap and readily available raw materials, mild reaction conditions, few synthesis steps, and simple product processing. 90Y microspheres, the radioactive radium-223 labeled alginate microspheres prepared by the present invention emit higher energy rays, which can directly interrupt the DNA double strands of tumor cells, and have a short action distance and little effect on normal liver tissue. The induction of tumor cell apoptosis is not affected by the cell cycle and whether the environment is hypoxic. 223 RaCl2 has been approved by the US FDA for clinical treatment of prostate cancer bone metastasis. 223 Currently, there is a lack of suitable chelators for Ra, which limits its clinical application in the treatment of other tumors. 223 In summary, the radioactive radium-223 labeled alginate microspheres prepared by the present invention can achieve efficient internal radioembolization of liver tumor blood vessels and improve the traditional 90 The situation of radiotherapy resistance after Y microsphere TARE provides a new and promising treatment direction for the majority of liver cancer patients.

[0016] The present invention utilizes 223 Alkaline earth elements calcium and barium of the same family as Ra are co-precipitated by "in-situ doping" to achieve α nuclides 223 Ra is stably labeled on the surface of alginate microspheres. Alginate microspheres are low in cost and have good biocompatibility. The microspheres obtained by the present invention not only have uniform particle size and good biocompatibility, but also 223 Ra labeling is very stable, has significant tumor treatment effects, and has good clinical application prospects.

[0017] The radioactive radium-223 labeled alginate microspheres prepared by the present invention have good biocompatibility, dual functions of internal radiotherapy and embolization, simple synthesis steps and good labeling stability. It provides theoretical and experimental basis for the transformation and application of α nuclides in the direction of TARE, and is expected to provide new and effective treatment options for liver cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 In Example 1 223 Scanning electron microscopy images of Ra / Ca alginate microspheres and element mapping diagram. A is the scanning electron microscopy image and B is the element mapping diagram.

[0019] Figure 2 In Example 1 223 Schematic diagram of the particle size distribution of Ra / Ca alginate microspheres.

[0020] Figure 3 In Example 2 223 Schematic diagram of the particle size distribution of Ra / Ca alginate microspheres.

[0021] Figure 4 In Example 3 223Scanning electron microscope image of Ra / Ba alginate microspheres and schematic diagram of element mapping.

[0022] Figure 5 In Example 1 223 Schematic diagram of the labeling stability of Ra / Ca alginate microspheres.

[0023] Figure 6 yes 223 Schematic diagram of SPECT / CT imaging of Ra / Ca alginate microspheres after embolization of liver tumor via the hepatic artery of rats.

[0024] Figure 7 yes 223 Ra / Ca alginate microspheres and blank alginate microspheres without radionuclide labeling before and after hepatic artery embolization in the treatment of liver tumors in rats 18 Schematic diagram of F-FDG PET / CT comparison. DETAILED DESCRIPTION

[0025] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0026] Example 1

[0027] A method for preparing alginate microspheres labeled with radioactive radium-223 comprises the following steps:

[0028] In the first step, 1.25 g of sodium alginate was added into 50 mL of ultrapure water. After it was completely dissolved, it was allowed to stand for 2 hours to degas, and a sodium alginate solution with a concentration of 0.025 g / mL was prepared as the aqueous phase.

[0029] 50 mL of isooctane was added as the oil phase to 50 mL of a sodium alginate solution with a concentration of 0.025 g / mL, and the volume ratio of isooctane to the sodium alginate solution was 1:1. Surfactants Span80 (5 mL) and Tween80 (1 mL) were added at the same time, and the volume ratio of Span80 to Tween80 was 5:1. The mixture was stirred at room temperature for 1 hour to form an oil-in-water (W / O) particle emulsion with a total volume of 106 mL.

[0030] In the second step, 50 mL of 0.2 g / mL CaCl2 solution was slowly added to 106 mL of the W / O particle emulsion prepared in the first step, and then the mixture was slowly added. 223 RaCl2 injection (10 μci / 200 μL) (purchased from Bayer) was used to radiolabel the formed microspheres. Stirring was continued for 1 hour to promote the coprecipitation reaction. 50 mL of anhydrous ethanol was added to break the emulsion, the supernatant was removed by centrifugation, the precipitate was collected, and the precipitate was obtained after sufficient drying.223 Ra / Ca alginate microspheres, about 500 mg.

[0031] Scanning electron microscopy (Hitachi SU8010) was used to characterize the morphology of the test microspheres. Figure 1 As shown, Figure 1 In Example 1 223 Scanning electron microscopy images of Ra / Ca alginate microspheres and element mapping diagram. A is a scanning electron microscopy image. It can be seen from the figure that the Ra / Ca alginate microspheres were successfully prepared. 223 Ra / Ca alginate microspheres were observed to be perfectly round in shape under a scanning electron microscope, with a slightly rough surface and slight wrinkles, which were presumed to be co-precipitated metal salts. The composition of the microspheres was further verified by elemental analysis using a scanning electron microscope energy dispersive spectrometer (IXRF, USA). Figure 1 As shown in B, B is the element mapping diagram. It can be seen from the figure that calcium is evenly distributed on the surface of the microspheres. 223 Ra is a radioactive nuclide and cannot be directly monitored using an energy spectrometer. It can be detected by the distribution of calcium elements after co-precipitation. 223 Ra is evenly distributed on the surface of the microspheres.

[0032] The particle size distribution of the microspheres synthesized by the reaction was observed comparatively using a laser particle size analyzer (Malvern MS2000). Figure 2 As shown, Figure 2 In Example 1 223 Schematic diagram of the particle size distribution of Ra / Ca alginate microspheres. It can be seen from the figure that the number of microspheres with a diameter of 69.85 μm is the largest.

[0033] For the preparation 223 Radiolabeling stability study of Ra / Ca alginate microspheres:

[0034] 5mg 223 Ra / Ca alginate microspheres were dispersed in 1 mL phosphate buffered saline (PBS) and 1 mL fetal bovine serum (FBS) solution, and the supernatant was separated from the microspheres by centrifuge (speed: 10000 rpm / min) at multiple consecutive time points after preparation (0, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240, 264, 288, 312, 336, 360, 384 h), and the radioactivity counts of the microspheres and supernatant were measured using a GC-2101γ counter (Beijing Pat Biotechnology Co., Ltd.). The alginate microspheres were radioactive after centrifugation, proving that 223 Ra exists in alginate microspheres, achieving 223 In-situ doping labeling of Ra.

[0035] 223 The results of Ra labeling stability of sodium alginate microspheres are as follows Figure 5 As shown, Figure 5 In Example 1 223 Schematic diagram of the labeling stability of Ra / Ca alginate microspheres. 223 On the 16th day after Ra-labeled alginate microspheres, 223 Ra / Ca alginate microspheres 223 The Ra radionuclide labeling rate was 87.8% ± 0.7%; in the FBS solution environment, 223 Ra / Ca alginate microspheres 223 The Ra nuclide labeling rate is 87.2%±0.2%. The above data show that 223 Ra can be continuously and stably present in alginate microspheres, and the stable labeling of radionuclides makes 223 Ra / Ca alginate microspheres have good directional irradiation function, avoiding unnecessary organ irradiation, giving the microspheres good clinical transformation potential.

[0036] The invention was verified by rat liver cancer 223 The therapeutic effect of transarterial radioembolization (TARE) of Ra / Ca alginate microspheres: The rat liver cancer model was established by serial passage of N1S1 liver cancer cells. When the cell density reached 70%-80%, the cells were implanted. The number of liver cancer cells implanted in each rat was about 3×10 6 N1S1 hepatoma cells were mixed with matrix gel and implanted in situ in rat liver. Scanning was performed 1 week after implantation. 18 F-FDG PET / CT, 18 The injected dose of F-FDG was 3.7×10 7 MBq / kg. When the PET / CT image shows a low-density nodule in the liver with 18 The increased F-FDG uptake confirmed that the rat liver cancer model was successfully established.

[0037] 30mg 223 Ra / Ca alginate microspheres were injected into the tumor supply artery through the hepatic artery cannula, and SPECT / CT (Simens Symbia TruePoint) was used to observe the tumor at 1h, 48h, 7d, and 14d after TARE. 223 The distribution of Ra / Ca alginate microspheres in the tumor area and liver was selected 223 The three characteristic energy peaks of Ra decay process were scanned at 85KeV, 154KeV, and 270KeV. The results are as follows Figure 6 As shown, Figure 6 yes 223 Schematic diagram of SPECT / CT imaging of liver tumors after Ra / Ca alginate microspheres were embolized via the hepatic artery of rats.223 Ra / Ca alginate microspheres successfully entered the liver tissue and 223 Ra / Ca alginate microspheres were mainly distributed in the tumor area and could still be observed two weeks after embolization. 223 The accumulation of Ra in the tumor area indicates that 223 The TARE procedure of rat liver cancer using Ra / Ca alginate microspheres was successfully performed and could produce sustained high-energy internal radiotherapy effects.

[0038] Still using after TARE 18 F-FDG PET / CT was used to observe the glucose metabolism in the liver tumor area to evaluate the therapeutic effect. Figure 7 As shown, Figure 7 yes 223 Ra / Ca alginate microspheres and blank alginate microspheres without radionuclide labeling before and after hepatic artery embolization in the treatment of liver tumors in rats 18 Schematic diagram of F-FDG PET / CT comparison. The glucose metabolism of N1S1 tumor was observed at 2d, 1w, and 2w after TARE. As can be seen from the figure, 223 Ra / Ca alginate microsphere group (the figure shows the α-nuclide microsphere group) 18 The uptake of F-FDG was significantly lower than that before surgery, indicating 223 Ra / Ca alginate microspheres have a good therapeutic effect on rat liver cancer.

[0039] Example 2

[0040] A method for preparing alginate microspheres labeled with radioactive radium-223 comprises the following steps:

[0041] In the first step, 1.25 g of sodium alginate was added into 50 mL of ultrapure water. After it was completely dissolved, it was allowed to stand for 2 hours to degas, and a sodium alginate solution with a concentration of 0.025 g / mL was prepared as the aqueous phase.

[0042] 100 mL of isooctane was added as the oil phase to 50 mL of a sodium alginate solution with a concentration of 0.025 g / mL, and the volume ratio of isooctane to the sodium alginate solution was 2:1. Surfactants Span80 (5 mL) and Tween80 (1 mL) were added at the same time, and the volume ratio of Span80 to Tween80 was 5:1. The mixture was stirred at room temperature for 1 hour to form an oil-in-water (W / O) particle emulsion with a total volume of 156 mL.

[0043] In the second step, 50 mL of 0.2 g / mL CaCl2 solution was slowly added to 156 mL of the W / O particle emulsion prepared in the first step, and then the mixture was slowly added. 223RaCl2 injection (10 μci / 200 μL) was added to radioactively label the formed microspheres. Stirring was continued for 1 hour to promote the coprecipitation reaction. 50 mL of anhydrous ethanol was added to break the emulsion, the supernatant was removed by centrifugation, the precipitate was collected, and the precipitate was obtained after sufficient drying. 223 Ra / Ca alginate microspheres, about 300 mg.

[0044] The hydration kinetic diameter distribution is Figure 3 As shown, Figure 3 In Example 2 223 Schematic diagram of particle size distribution of Ra / Ca alginate microspheres. It can be seen from the figure that the number of microspheres with a diameter of 178.25 μm is the largest. Since the water-oil ratio of the reaction system in Example 1 and Example 2 is different, it can be concluded that microspheres with different particle size ranges can be obtained by adjusting the water-oil ratio.

[0045] Example 3

[0046] A method for preparing alginate microspheres labeled with radioactive radium-223 comprises the following steps:

[0047] In the first step, 1.25 g of sodium alginate was added into 50 mL of ultrapure water. After it was completely dissolved, it was allowed to stand for 2 hours to degas, and a sodium alginate solution with a concentration of 0.025 g / mL was prepared as the aqueous phase.

[0048] 50 mL of isooctane was added as the oil phase to 50 mL of a sodium alginate solution with a concentration of 0.025 g / mL, and the volume ratio of isooctane to the sodium alginate solution was 1:1. Surfactants Span80 (5 mL) and Tween80 (1 mL) were added at the same time, and the volume ratio of Span80 to Tween80 was 5:1. The mixture was stirred at room temperature for 1 hour to form an oil-in-water (W / O) particle emulsion with a total volume of 106 mL.

[0049] In the second step, 50 mL of 0.33 g / mL BaCl2 solution was slowly added to 106 mL of the W / O particle emulsion prepared in the first step, and then the mixture was slowly added. 223 RaCl2 injection (10 μci / 200 μL) was added to radioactively label the formed microspheres. Stirring was continued for 1 hour to promote the coprecipitation reaction. 50 mL of anhydrous ethanol was added to break the emulsion, the supernatant was removed by centrifugation, the precipitate was collected, and the precipitate was obtained after sufficient drying. 223 Ra / Ba alginate microspheres, about 600 mg.

[0050] The SEM diagram of the generated alginate microspheres and the element mapping diagram are shown in Figure 4 shown. Figure 4 In Example 3 223Scanning electron microscope image of Ra / Ba alginate microspheres and element mapping diagram. 223 Schematic diagram of scanning electron microscope (SEM) of Ra / Ba alginate microspheres, B is 223 The element distribution mapping diagram of Ra / Ba alginate microspheres shows that the barium-containing 223 Ra / Ba alginate microspheres, barium is evenly distributed on the surface of the microspheres. Compared with calcium, barium has a higher atomic number and is expected to be transformed into functional microspheres that have both CT angiography performance and internal radioembolization.

[0051] Comparative Example 1

[0052] In the first step, 1.25 g of sodium alginate was added into 50 mL of ultrapure water. After it was completely dissolved, it was allowed to stand for 2 hours to degas, and a sodium alginate solution with a concentration of 0.025 g / mL was prepared as the aqueous phase.

[0053] 50 mL of isooctane was added as the oil phase to 50 mL of a sodium alginate solution with a concentration of 0.025 g / mL, and the volume ratio of isooctane to the sodium alginate solution was 1:1. Surfactants Span80 (5 mL) and Tween80 (1 mL) were added at the same time, and the volume ratio of Span80 to Tween80 was 5:1. The mixture was stirred at room temperature for 1 hour to form an oil-in-water (W / O) particle emulsion with a total volume of 106 mL.

[0054] In the second step, 50 mL of 0.2 g / mL CaCl2 solution was slowly added to 106 mL of oil-in-water W / O particle emulsion prepared in the first step, and stirring was continued for 1 hour. 50 mL of anhydrous ethanol was added to break the emulsion, and the supernatant was removed by centrifugation. The precipitate was collected to obtain calcium alginate microspheres.

[0055] Calcium alginate microspheres are injected into the tumor blood supply artery through the hepatic artery cannula to block the tumor blood supply. 18 F-FDGPET / CT was used to observe the glucose metabolism in the tumor area to evaluate the therapeutic effect. Figure 7 As shown, Figure 7 yes 223 Ra / Ca alginate microspheres and blank alginate microspheres without radionuclide labeling before and after hepatic artery embolization in the treatment of liver tumors in rats 18 Schematic diagram of F-FDG PET / CT comparison. Unlabeled 223 After tumor artery embolization, the blank microsphere group (calcium alginate microsphere) of Ra showed 18 F-FDG uptake decreased significantly, but appeared in the tumor area 2 weeks after surgery. 18 The increased uptake of F-FDG indicates tumor recurrence. 223The glucose metabolism in the liver tumor area of ​​the rats treated with Ra / Ca alginate microspheres was significantly lower at 2d, 1w, and 2w after TARE than before surgery. 223 Ra / Ca alginate microspheres have better therapeutic effect on rat liver cancer.

[0056] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. A method for preparing alginate microspheres labeled with radioactive radium-223, characterized in that: The following steps are involved: The first step is to add isooctane to a sodium alginate solution with a concentration of 0.01 to 0.1 g / mL, wherein the volume ratio of isooctane to the sodium alginate solution is 1 to 5:1, and surfactants Span80 and Tween80 are added, wherein the volume ratio of Span80 to Tween80 is 1 to 8:1, and the mixture is stirred at room temperature for 0.5 to 2 hours to form a water-in-oil (W / O) particle emulsion. In the second step, a CaCl2 solution with a concentration of 0.01 to 1 g / mL or a BaCl2 solution with a concentration of 0.01 to 1 g / mL is slowly added to the water-in-oil W / O particle emulsion prepared in the first step, the volume ratio of the CaCl2 solution to the water-in-oil W / O particle emulsion is 1:1 to 4, and the volume ratio of the BaCl2 solution to the water-in-oil W / O particle emulsion is 1:1 to 4. Continue to slowly add 223 RaCl2 injection, radioactively label the formed microspheres, continue stirring for 0.5 to 2 hours, add anhydrous ethanol to break the emulsion, remove the supernatant by centrifugation, collect the precipitate, and fully dry to obtain the radioactive radium-223 labeled alginate microspheres.

2. The method for preparing radioactive radium-223 labeled alginate microspheres according to claim 1, characterized in that: The method for preparing the sodium alginate solution comprises the following steps: adding sodium alginate into ultrapure water, and after the sodium alginate is completely dissolved, standing for at least 2 hours for degassing, so as to obtain a sodium alginate solution with a concentration of 0.01 to 0.1 g / mL as an aqueous phase.

3. The method for preparing radioactive radium-223 labeled alginate microspheres according to claim 2, characterized in that: The concentration of the sodium alginate solution is 0.025 g / mL.

4. The method for preparing radioactive radium-223 labeled alginate microspheres according to claim 1, characterized in that: The concentration of the CaCl2 solution is 0.2 g / mL.

5. The method for preparing radioactive radium-223 labeled alginate microspheres according to claim 1, characterized in that: The concentration of the BaCl2 solution is 0.33 g / mL.

6. The method for preparing radioactive radium-223 labeled alginate microspheres according to claim 1, characterized in that: The volume ratio of Span80 to Tween80 is 5:

1.

7. The method for preparing radioactive radium-223 labeled alginate microspheres according to claim 1, characterized in that: The volume ratios of the CaCl2 solution to the water-in-oil (W / O) particle emulsion are 1:2.1 and 1:3.

1.

8. The method for preparing radioactive radium-223 labeled alginate microspheres according to claim 1, characterized in that: The volume ratios of the BaCl2 solution to the water-in-oil (W / O) particle emulsion are 1:2.1 and 1:3.

1.

9. Use of alginate microspheres labeled with radioactive radium-223 prepared by the method according to any one of claims 1 to 8 in the preparation of a drug for arterial radiotherapy embolization.

10. Use of alginate microspheres labeled with radioactive radium-223 prepared by the method according to any one of claims 1 to 8 in preparing a drug for treating liver cancer.

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