Preparation method of a radioactive in-situ vaccine

Abstract

A method for preparing a radioactive in-situ vaccine includes the following steps: First, prepare calcium carbonate microsphere powder with calcium chloride and sodium carbonate solutions, then prepare mesoporous hydroxyapatite with a phosphate solution and the calcium carbonate microsphere powder, and then add a manganese salt to obtain manganese-doped hydroxyapatite microspheres; then label with a radionuclide to obtain radionuclide-labeled manganese-doped hydroxyapatite microspheres. The radionuclide therapy adopted in the present invention can well confine the rays to the tumor site, and can avoid systemic reactions while continuously irradiating the tumor; the selected manganese adjuvant, as an essential trace element for the human body, is safer, cheaper and easier to obtain. On the other hand, the manganese doped in the microspheres themselves can activate the immune system after release, and in addition, the special mesoporous structure of the microspheres can achieve efficient capture of tumor antigens, maintain a high concentration of antigen in the tumor site, continuously activate the immune system, produce an anti-tumor effect, and achieve universal tumor treatment.

Description

Technical Field

[0001] The present invention relates to the preparation of vaccines, and in particular to a radioactive in-situ vaccine and a preparation method thereof. Background Art

[0002] In recent years, tumor immunotherapy has been carried out in full swing, and therapeutic tumor vaccines have received extensive attention due to their excellent efficacy. Traditional tumor vaccines usually externally administer tumor antigens and combine with adjuvants to activate the immune system, but they can only act on the given antigens, and it is difficult to obtain new antigens, resulting in corresponding limitations. As a strategy of only injecting adjuvants to make the tumor itself become an antigen factory, in-situ vaccines can highly specifically kill tumors while maintaining universality for the population and different tumor types, providing an opportunity to establish a general in-situ immunotherapy strategy.

[0003] The strategy of using radiotherapy combined with immune adjuvants as in-situ vaccines has been widely carried out in preclinical and clinical trials and achieved excellent therapeutic effects, but there are still some problems that can be improved. For example, in vitro radiotherapy cannot achieve continuous antigen release, and side effects cannot be avoided. At the same time, the existing strategy requires repeated injection of adjuvants, with a long treatment course, and small molecule adjuvants such as CpG are also prone to diffusion and metabolism. In addition, the existing in-situ vaccines have low antigen capture efficiency and limited activation of dendritic cells. Therefore, there is an urgent need to design a general in-situ vaccine strategy that can continuously cause antigen release, load immune adjuvants, and efficiently capture antigens. Summary of the Invention

[0004] To achieve the above object, the present invention provides the following technical solutions:

[0005] A method for preparing a radioactive in-situ vaccine, comprising the following steps:

[0006] S1: Add calcium chloride and sodium carbonate solutions with equal volume and a concentration of 0.1 - 1 mol / L to a reactor equipped with a stirring device, rapidly stir the reactor equipped with the stirring device for 10 - 120 S to obtain a white precipitate, wash the white precipitate and then dry it to obtain calcium carbonate microsphere powder.

[0007] S2: Prepare a phosphate solution with a concentration of 0.5 - 2 mol / L. Then add the calcium carbonate microsphere powder obtained in step S1 to the phosphate solution, and mix evenly to obtain solution A. Next, add a manganese salt to solution A to obtain solution B. React solution B at 100 - 200 °C for 12 - 48 h to obtain powder C. Wash powder C and then dry it to obtain manganese-doped hydroxyapatite microspheres. Prepare mesoporous hydroxyapatite as a carrier for vaccines. Phosphate is beneficial for the labeling of radionuclides. At the same time, the crystal structure of hydroxyapatite makes the doping of manganese ions simpler. The formed mesoporous material has a larger specific surface area and a stronger adsorption capacity for antigens. Select manganese as an immune adjuvant, which can activate the cGAS-STING pathway to activate innate immunity and adaptive immunity.

[0008] S3: Take 0.05 - 2 mg of the manganese-doped hydroxyapatite microspheres obtained in step S2 and suspend them in 4 - 160 μL of physiological saline. Then add 5 - 20 μL of a sodium bicarbonate solution with a concentration of 0.1 - 0.5 mol / L to the physiological saline to obtain solution D. Next, add 0.1 - 1 mCi of a radionuclide to solution D to obtain a mixture E. Then adjust the pH value of mixture E to 5 - 10 and perform labeling at 25 - 90 °C for 1 - 120 min to obtain mixture F. Wash F to obtain radionuclide-labeled manganese-doped hydroxyapatite microspheres. Select a radionuclide as a radiation source to continuously irradiate the tumor. Compared with external irradiation, the rays emitted by the continuous decay of the radionuclide can maximize the release of tumor antigens.

[0009] Preferably, in step S2, the phosphate is one of diammonium hydrogen phosphate and ammonium dihydrogen phosphate; the manganese salt is one of manganese chloride, manganese oxide, and manganese dihydrogen phosphate.

[0010] Preferably, in step S2, the molar ratio of the calcium content in the calcium carbonate microsphere powder to the phosphorus content in the phosphate is 1 - 2.

[0011] Preferably, in step S2, the molar ratio of the calcium content in the calcium carbonate microsphere powder to the manganese content in the manganese salt is 9 - 99.

[0012] Preferably, in step S3, the radionuclide is one of lutetium-177, yttrium-90, and radium-223.

[0013] Preferably, the specific process of step S3 is as follows: Take 0.05 - 2 mg of the manganese-doped hydroxyapatite microspheres obtained in step S2 and suspend them in 4 - 160 μL of physiological saline. Then, add 5 - 20 μL of a sodium bicarbonate solution with a concentration of 0.1 - 0.5 mol / L to the physiological saline, and mix evenly to obtain solution D. Next, add 0.1 - 1 mCi of a radionuclide to solution D to obtain a mixture E. Then, adjust the pH value of mixture E to 5 - 10 with hydrochloric acid or sodium hydroxide, and perform labeling for 1 - 120 min under the condition of 25 - 90 °C to obtain a mixture F. Wash F with physiological saline 1 - 5 times to remove the radionuclide, and obtain the radionuclide-labeled manganese-doped hydroxyapatite microspheres.

[0014] The beneficial effects of the present invention are as follows:

[0015] 1. Most of the existing technologies use external radiotherapy as a means to kill tumors. However, the irradiation of tumor sites is limited, and the resulting systemic side effects cannot be avoided. In contrast, the radionuclide therapy selected in the present invention can well confine the rays to the tumor site, avoiding systemic reactions while continuously irradiating the tumor.

[0016] 2. Most of the existing immunoadjuvants are small molecule adjuvants, which are easily degraded by enzymes in the body. In addition, the diffused immunoadjuvants may cause adverse immune side reactions. The manganese adjuvant selected in the present invention, as an essential trace element for the human body, is safer, cheaper, and easier to obtain.

[0017] 3. The manganese-doped hydroxyapatite microspheres designed in the present invention can not only easily achieve efficient loading of radionuclides, but also the manganese doped in the microspheres can activate immunity after release. In addition, the special mesoporous structure of the microspheres can achieve efficient capture of tumor antigens, maintain a high concentration of antigens at the tumor site, continuously activate immunity, produce an anti-tumor effect, and achieve universal tumor treatment.

[0018] The above description is only an overview of the technical solution of the present invention. In order to understand the technical means of the present invention more clearly and implement it according to the content of the specification, the following describes the preferred embodiments of the present invention in detail in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Scanning electron micrograph of calcium carbonate microspheres in Example 3 (right is the surface structure)

[0020] Figure 2 Scanning electron micrograph of manganese-doped hydroxyapatite microspheres in Example 3 (right is the surface structure) DETAILED DESCRIPTION OF THE INVENTION

[0021] The following will further describe in detail the specific implementation manners of the present invention in conjunction with the accompanying drawings and embodiments. The following embodiments are used to illustrate the present invention, but are not used to limit the scope of the present invention.

[0022] Example 1

[0023] S1: Add 100 μL of calcium chloride solution with a concentration of 0.1 mol / L and 100 μL of sodium carbonate solution with a concentration of 0.1 mol / L into a reactor with a stirring device. The reactor with the stirring device is rapidly stirred for 10 s to obtain a white precipitate. The white precipitate is washed three times with water and anhydrous ethanol respectively, and then dried in vacuum at 200 °C for 2 h to obtain calcium carbonate microsphere powder.

[0024] S2: Prepare 9.9 μL of diammonium hydrogen phosphate solution with a concentration of 0.5 mol / L. Then add 4.95 μmol of the calcium carbonate microsphere powder obtained in step S1 into the diammonium hydrogen phosphate solution, and then uniformly mix to obtain solution A. Then add 0.55 μmol of manganese dihydrogen phosphate into solution A to obtain solution B. Solution B is reacted at 100 °C for 12 h to obtain powder C. Powder C is washed 3 times with water and anhydrous ethanol and then dried at 200 °C for 2 h to obtain manganese-doped hydroxyapatite microspheres.

[0025] S3: Take 0.05 mg of the manganese-doped hydroxyapatite microspheres obtained in step S2 and suspend them in 4 μL of physiological saline. Then add 5 μL of sodium bicarbonate solution with a concentration of 0.1 mol / L into the physiological saline to obtain solution D. Then add 0.1 mCi of radioactive 90 YCl3 into solution D to obtain a mixed solution E. Then adjust the pH value of the mixed solution E to 5, and perform labeling at 25 °C for 1 min to obtain a mixture F. F is washed three times with physiological saline to obtain 90 Y-labeled manganese-doped hydroxyapatite microspheres.

[0026] Example 2

[0027] S1: Add 100 μL of calcium chloride solution with a concentration of 1 mol / L and 100 μL of sodium carbonate solution with a concentration of 1 mol / L into a reactor with a stirring device. The reactor with the stirring device is rapidly stirred for 120 s to obtain a white precipitate. The white precipitate is washed three times with water and anhydrous ethanol respectively, and then dried in vacuum at 200 °C for 2 h to obtain calcium carbonate microsphere powder.

[0028] S2: Prepare 9.9 μL of ammonium dihydrogen phosphate solution with a concentration of 2 mol / L, then add 39.6 μmol of the calcium carbonate microsphere powder obtained in step S1 to the ammonium dihydrogen phosphate solution, and then mix evenly to obtain solution A. Then add 0.4 μmol of manganese oxide to solution A to obtain solution B. React solution B at 200 °C for 48 h to obtain powder C. Wash powder C three times with water and anhydrous ethanol and then dry it at 200 °C for 2 h to obtain manganese-doped hydroxyapatite microspheres;

[0029] S3: Take 2 mg of the manganese-doped hydroxyapatite microspheres obtained in step S2 and suspend them in 160 μL of physiological saline. Then add 20 μL of sodium bicarbonate solution with a concentration of 0.5 mol / L to the physiological saline to obtain solution D. Then add 1 mCi of radioactive 223 RaCl2 to obtain a mixed solution E. Then adjust the pH value of the mixed solution E to 10 and carry out labeling at 90 °C for 120 min to obtain a mixture F. Wash F three times with physiological saline to obtain 223 Ra-labeled manganese-doped hydroxyapatite microspheres.

[0030] Example 3

[0031] S1: Add 100 μL of calcium chloride solution with a concentration of 0.5 mol / L and 100 μL of sodium carbonate solution with a concentration of 0.5 mol / L to a reactor equipped with a stirring device. The reactor equipped with a stirring device is rapidly stirred for 30 s to obtain a white precipitate. Wash the white precipitate three times with water and anhydrous ethanol respectively and then dry it at 200 °C under vacuum for 2 h to obtain calcium carbonate microsphere powder;

[0032] S2: Prepare 9.9 μL of diammonium hydrogen phosphate solution with a concentration of 1.5 mol / L, then add 24.8 μmol of the calcium carbonate microsphere powder obtained in step S1 to the diammonium hydrogen phosphate solution, and then mix evenly to obtain solution A. Then add 0.5 μmol of manganese chloride to solution A to obtain solution B. React solution B at 140 °C for 24 h to obtain powder C. Wash powder C three times with water and anhydrous ethanol and then dry it at 200 °C for 2 h to obtain manganese-doped hydroxyapatite microspheres;

[0033] S3: Take 0.5 mg of the manganese-doped hydroxyapatite microspheres obtained in step S2 and suspend them in 80 μL of physiological saline. Then add 10 μL of sodium bicarbonate solution with a concentration of 0.5 mol / L to the physiological saline to obtain solution D. Then add 0.1 mCi of radioactive 177 LuCl3 to obtain a mixed solution E. Then adjust the pH value of the mixed solution E to 7 and carry out labeling at 25 °C for 60 min to obtain a mixture F. Wash F three times with physiological saline to obtain 177 Lu-labeled manganese-doped hydroxyapatite microspheres.

[0034] Example 4

[0035] S1: Add 100 μL of calcium chloride solution with a concentration of 0.5 mol / L and 100 μL of sodium carbonate solution with a concentration of 0.5 mol / L into a reactor with a stirring device. The reactor with the stirring device is rapidly stirred for 30 s to obtain a white precipitate. The white precipitate is washed three times with water and anhydrous ethanol respectively, and then dried in vacuum at 200 °C for 2 h to obtain calcium carbonate microsphere powder;

[0036] S2: Prepare 9.9 μL of diammonium hydrogen phosphate solution with a concentration of 1.5 mol / L. Then add 24.8 μmol of the calcium carbonate microsphere powder obtained in step S1 into the diammonium hydrogen phosphate solution, and then mix evenly to obtain solution A. Then add 0.5 μmol of manganese chloride into solution A to obtain solution B. Solution B is reacted at 140 °C for 24 h to obtain powder C. Powder C is washed 3 times with water and anhydrous ethanol and then dried at 200 °C for 2 h to obtain manganese-doped hydroxyapatite microspheres;

[0037] S3: Take 0.5 mg of the manganese-doped hydroxyapatite microspheres obtained in step S2 and suspend them in 40 μL of physiological saline. Then add 10 μL of sodium bicarbonate solution with a concentration of 0.5 mol / L into the physiological saline to obtain solution D. Then add 0.1 mCi of radioactive 223 RaCl2 into solution D to obtain a mixed solution E. Then adjust the pH value of the mixed solution E to 7, and perform labeling at 25 °C for 60 min to obtain a mixture F. F is washed three times with physiological saline to obtain 223 Ra-labeled manganese-doped hydroxyapatite microspheres.

[0038] The calcium carbonate microsphere powder and manganese-doped hydroxyapatite microspheres prepared in Example 3 are evenly adhered to the carbon conductive adhesive on the electron microscope stage. After sputtering with gold, the shape and size of the microspheres are observed under a scanning electron microscope and photographed. The obtained results are as Figure 1 — Figure 2 shown. It can be Figures 1 - 2 seen that a porous structure appears on the surface of the manganese-doped hydroxyapatite microspheres, greatly increasing the specific surface area, enabling it to complete the function of antigen adsorption.

[0039] The calcium carbonate microspheres and manganese-doped hydroxyapatite microsphere powders obtained in Examples 1-4 are tested with a fully automatic specific surface area analyzer. The obtained data are shown in Table 1 below.

[0040]

[0041] Table 1

[0042] As can be seen from Table 1, the specific surface area of manganese-doped hydroxyapatite microspheres is significantly improved compared to that of calcium carbonate as a carrier, thus greatly enhancing the antigen-loading capacity. Antigens will be carried away by blood or lymph circulation, and the antigens cannot be fully utilized. However, after the microspheres adsorb antigens, they can maintain a relatively high antigen concentration and continuously stimulate the immune response through slow release.

[0043] Take 0.5 mg of the manganese-doped hydroxyapatite microspheres obtained in Examples 1-4 and place them in 10 mL of PBS buffer with pH = 6.8 (simulating the pH of the tumor microenvironment) and 7.4 (simulating the pH of the normal tissue microenvironment) respectively. Each group has three replicates. After centrifuging at 5000 rpm for 5 min at each time point, take out 1 mL of the supernatant, and then add 1 mL of the corresponding buffer. Take out 200 mg of the supernatant, add 300 mg of digestion solution (200 mg of concentrated nitric acid + 100 mg of hydrogen peroxide), and after digestion, add water to make up to 5 g. Prepare a manganese standard curve and test it by ICP OES. The obtained results are as

[0044] shown in Table 2.

[0045] Example Manganese ion release rate at pH 6.8 (%) Manganese ion release rate at pH 7.4 (%) 1 86.25 65.39 2 93.22 70.56 3&4 92.61 71.44

[0046] Table 2

[0047] As can be seen from Table 2, the release of manganese ions from the manganese-doped hydroxyapatite microspheres finally reaches more than 90% in the simulated tumor microenvironment, and thus can activate the immune system.

[0048] Test the adsorption of the manganese-doped hydroxyapatite microspheres obtained in Examples 1-4 to tumor antigens. Steps: Resuscitate various tumor cells, passage and culture them until the logarithmic growth phase, add 0.25% trypsin to digest them, then add growth medium to terminate the digestion, pipette the cells and collect them, centrifuge at 1000 rpm for 5 min, collect the lower precipitate, and wash it three times with PBS to remove the medium. Count and collect 0.5 M cells each in 1.5 mL Ep tubes, with three replicates in each group. Keep the cells on ice and break them under an ultrasonic crusher for 5 min, with the ultrasound on for 3 s and off for 1 s. After centrifuging at 1000 rpm for 5 min, take the supernatant, and measure the protein concentration by the BCA protein quantification method, which is the original protein concentration. Take 0.5 mL of the supernatant and adsorb it with 0.5 mg of manganese-doped hydroxyapatite microspheres dissolved in 0.5 mL of PBS on a shaker at 37 °C and 200 rpm for 4 h. After the adsorption is completed, centrifuge the Ep tubes at 5000 rpm for 5 min, collect the supernatant, and then measure the protein concentration, which is the unadsorbed protein concentration; the obtained results are shown in Table 3.

[0049]

[0050] Table 3

[0051] As can be seen from Table 3, the microspheres have the ability to adsorb and capture various tumor antigens, showing universality. Compared with the existing radioactive microspheres that are mostly only used for hepatic artery embolization therapy with limited application scenarios, this represents a significant progress.

[0052] The manganese-doped hydroxyapatite microspheres obtained in Examples 1-4 were detected for nuclide labeling. Steps: Weigh the corresponding mass of manganese-doped hydroxyapatite microspheres, suspend them in 80 μL of physiological saline, add 10 μL of 0.5 M sodium bicarbonate buffer solution thereto, and then add 100 μCi of radioactive 177 LuCl3 (the activity was measured with a radioactivity meter before addition), and then adjust the pH to 7 with sodium hydroxide (0.1 M), and shake at room temperature for 60 minutes for labeling. After the labeling was completed, the microspheres were obtained by centrifugation at 3000 rpm for 5 min, and then washed three times with physiological saline to obtain 177 Lu-labeled manganese-doped hydroxyapatite microspheres. The radioactivity of the final microspheres was measured with a radioactivity meter, and the labeling rate was obtained by comparing with the added activity. The obtained data are shown in Table 4.

[0053]

[0054]

[0055] Table 4

[0056] As can be seen from Table 4, the microspheres synthesized by the present invention have a high nuclide labeling rate and simple labeling conditions. The nuclide labeling process of existing microspheres is mostly complex and difficult to popularize. For example, 90 Y glass microspheres need to be nuclide-labeled during the microsphere manufacturing process and cannot be labeled and used immediately.

[0057] Each technical feature can be combined arbitrarily. For the sake of concise description, not all possible combinations of the technical features in the above embodiments are described. However, as long as the combinations of these technical features do not conflict, they should be considered as falling within the scope described in this specification.

[0058] The above-described embodiments merely represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed. However, they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the invention patent should be subject to the appended claims.

Claims

1. A method for preparing a radioactive in-situ vaccine, characterized in that, It includes the following steps: S1: Add calcium chloride and sodium carbonate solutions with a concentration of 0.1 - 1 mol / L in equal volumes to a reactor with a stirring device. The reactor with the stirring device is rapidly stirred for 10 - 120 s to obtain a white precipitate. After washing the white precipitate, it is dried to obtain calcium carbonate microsphere powder; S2: Prepare a phosphate solution with a concentration of 0.5 - 2 mol / L. Then add the calcium carbonate microsphere powder obtained in step S1 to the phosphate solution, and then uniformly mix to obtain solution A. Then add a manganese salt to solution A to obtain solution B. Solution B is reacted at 100 - 200 °C for 12 - 48 h to obtain powder C. After washing powder C, it is dried to obtain manganese-doped hydroxyapatite microspheres; S3: Take 0.05 - 2 mg of the manganese-doped hydroxyapatite microspheres obtained in step S2 and suspend them in 4 - 160 μL of physiological saline. Then add 5 - 20 μL of a sodium bicarbonate solution with a concentration of 0.1 - 0.5 mol / L to the physiological saline to obtain solution D. Then add 0.1 - 1 mCi of a radionuclide to solution D to obtain a mixed solution E. Then adjust the pH value of the mixed solution E to 5 - 10, and perform labeling at 25 °C - 90 °C for 1 - 120 min to obtain a mixture F. After washing F, radionuclide-labeled manganese-doped hydroxyapatite microspheres are obtained.

2. The method for preparing a radioactive in-situ vaccine according to claim 1, wherein In step S2, the phosphate is one of diammonium hydrogen phosphate and ammonium dihydrogen phosphate; the manganese salt is one of manganese chloride, manganese oxide, and manganese dihydrogen phosphate.

3. The method for preparing a radioactive in-situ vaccine according to claim 2, characterized in that, In step S2, the molar ratio of the calcium content in the calcium carbonate microsphere powder to the phosphorus content in the phosphate is 1 - 2.

4. The method for preparing a radioactive in-situ vaccine according to claim 2, wherein In step S2, the molar ratio of the calcium content in the calcium carbonate microsphere powder to the manganese content in the manganese salt is 9 - 99.

5. The method for preparing a radioactive in-situ vaccine according to claim 1, characterized in that, In step S3, the radionuclide is one of lutetium - 177, yttrium - 90, and radium - 223.

6. The method for preparing a radioactive in-situ vaccine according to claim 1, wherein The specific process of step S3 is: Take 0.05 - 2 mg of the manganese-doped hydroxyapatite microspheres obtained in step S2 and suspend them in 4 - 160 μL of physiological saline. Then add 5 - 20 μL of a sodium bicarbonate solution with a concentration of 0.1 - 0.5 mol / L to the physiological saline and uniformly mix to obtain solution D. Then add 0.1 - 1 mCi of a radionuclide to solution D to obtain a mixed solution E. Then use hydrochloric acid or sodium hydroxide to adjust the pH value of the mixed solution E to 5 - 10, and perform labeling at 25 °C - 90 °C for 1 - 120 min to obtain a mixture F. Wash F with physiological saline 1 - 5 times to remove the radionuclide, and radionuclide-labeled manganese-doped hydroxyapatite microspheres are obtained.

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