Mineralization modification method of protein microspheres

Abstract

The invention discloses a mineralization modification method of protein microspheres. The method comprises the following steps: dispersing protein microspheres which are not mineralized and modified in deionized water, adding metal salt, and reacting; adding Na2CO3 into the system, and continuously reacting; and after the reaction is finished, centrifuging, collecting the precipitate and washing to obtain the mineralized protein microspheres. And carrying out nuclide labeling on the mineralized protein microspheres to obtain nuclide-labeled mineralized protein microspheres. The nuclide-labeled mineralized protein microspheres can be used in radiation therapy of cancer patients, and can better predict the distribution condition of organisms. The medicine can also be used for treating middle and advanced liver cancer patients.

Description

Technical Field

[0001] This invention belongs to the field of radiopharmaceutical chemistry and nuclear medicine technology, specifically, it relates to a method for mineralization modification of protein microspheres. Background Technology

[0002] Hepatocellular carcinoma accounts for more than 90% of liver cancer cases and is one of the most common and deadliest cancers worldwide. Selective internal radiation therapy (SIRT) uses radioactive yttrium-90 (Yttrium-90). 90 Y) microspheres, delivered via the hepatic artery to specific liver cancer tissue, release high-energy pure beta rays, radiating and killing tumor tissue from within, thus exerting a local anti-tumor effect. Despite its significant efficacy, radioembolization therapy still faces considerable challenges. Firstly, commercially available radioactive microspheres include resin microspheres and glass microspheres, neither of which undergoes natural degradation in the human body and will remain permanently after embolization. Secondly, because the hemodynamics within the hepatic artery and tumor vascular system affect the distribution of microspheres, the leakage of radioactive microspheres into normal liver or lung tissue can cause liver shunting and lung damage. Therefore, to avoid secondary harm to the body during surgery, a preoperative assessment of the patient's condition is necessary.

[0003] Currently, the material used clinically for predicting biodistribution is albumin microspheres (MAA). Albumin microspheres are composed of biodegradable human serum albumin particles, with most particles ranging from 10-40 μm in size (approximately 90%). 90 Y-microspheres possess similar diameter dimensions and offer advantages such as high biocompatibility, low toxicity, and ease of synthesis. Nevertheless, their distribution in the hepatic vascular system and liver tissue is not entirely satisfactory. Studies have shown that albumin microsphere mimicry often leads to overestimation of pulmonary shunt and underestimation of tumor and liver doses. The main reason for this is the difference between albumin microspheres and... 90 The microspheres differ in their physical properties: glass microspheres are mainly composed of silica and alumina, resulting in a density of 3.3 g / mL, while resin microspheres are mainly composed of styrene-divinylbenzene copolymer, with a density of only 1.6 g / mL. These two different densities... 90 The only mimic for Y microspheres is albumin microspheres, which have a density of 1.1 g / mL.

[0004] Therefore, the key focus of the research is how to increase the density of albumin microspheres, so that they not only meet the density standards of resin microspheres, but can also be adjusted experimentally to meet the density standards of glass microspheres. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing protein microspheres and to modify them with different degrees of mineralization to better simulate the distribution of glass microspheres and resin microspheres in vivo. Examples use near-infrared II imaging experiments to verify that the synthesized albumin microspheres have excellent simulation results. Specifically, silica microspheres were synthesized to simulate commercial glass microspheres, polymer microspheres were synthesized to simulate commercial resin microspheres, and albumin microspheres were synthesized and mineralized to different degrees. The results show that weakly mineralized albumin microspheres have a distribution close to that of polymer microspheres in near-infrared II imaging, while strongly mineralized albumin microspheres have a biological distribution closer to that of silica microspheres. This invention solves the accuracy problem of preoperative assessment in liver perfusion imaging and introduces a new perspective for the development of contrast agents.

[0006] In a first aspect, the present invention provides a method for preparing protein microspheres.

[0007] The method for preparing protein microspheres provided by this invention includes the following steps:

[0008] The protein was mixed with physiological saline solution, deionized water, acetate / sodium acetate buffer and cross-linking agent, and reacted under stirring. The temperature was gradually increased from room temperature to 75-85°C, and then kept at this temperature for 8-15 minutes. After stirring was stopped, the reaction mixture was allowed to cool to room temperature to obtain the protein microspheres.

[0009] Furthermore, in the above method, the protein includes at least one of the following: bovine serum albumin, human serum albumin, horse serum albumin, monkey serum albumin, recombinant human albumin, various antibodies, biological enzyme proteins, hormone proteins, preferably human serum albumin and recombinant human serum albumin, and more preferably human serum albumin.

[0010] Furthermore, in the above method, the albumin concentration in the physiological saline solution of the protein can be 10-100 mg / mL, preferably 40-60 mg / mL, and most preferably 50 mg / mL.

[0011] Furthermore, in the above method, the pH value of the acetate / sodium acetate buffer solution is 4.5-5.5, preferably pH=5.

[0012] Furthermore, in the above method, the crosslinking agent is selected from at least one of the following: glutaraldehyde, acetic anhydride, diglycidyl ether, and octyldiimide methyl ester; preferably, the crosslinking agent is glutaraldehyde.

[0013] Furthermore, in the above method, the crosslinking agent is added in the form of a solution, and its concentration can be 0.05-0.5%, specifically 0.1%.

[0014] Furthermore, in the above method, the volume ratio of the protein's physiological saline solution, deionized water, acetate / sodium acetate buffer, and glutaraldehyde solution can be 1:(0.8-1.2):(0.4-0.6):(0.4-0.6), specifically 1:1:0.5:0.5.

[0015] Furthermore, in the above method, the stirring speed is 700-2000 rpm, preferably 900-1500 rpm. According to an embodiment of the present invention, the stirring speed may specifically be 1100 rpm.

[0016] Furthermore, in the above method, the heating is completed within 30-60 minutes, specifically within 40 minutes.

[0017] Furthermore, the above method also includes the step of washing the obtained protein microspheres with deionized water.

[0018] Furthermore, the particle size range of the protein microspheres is 1-200 μm, preferably 10-100 μm, and more preferably 10-40 μm; the density range is 0.9-3.3 g / mL.

[0019] The protein microspheres prepared by the above method are also within the scope of protection of this invention.

[0020] Secondly, the present invention provides a method for mineralizing protein microspheres.

[0021] By adjusting the density of protein microspheres according to the degree of mineralization, modified microspheres with different densities and strong and weak mineralization were obtained.

[0022] The mineralization method for protein microspheres provided by this invention includes the following steps:

[0023] (1) Disperse unmineralized protein microspheres in deionized water, add metal salt, and carry out the reaction;

[0024] (2) Add soluble carbonate and / or soluble sulfate and / or soluble phosphate to the reaction system of step (1) and continue the reaction;

[0025] (3) After the reaction is complete, centrifuge, collect the precipitate and wash it to obtain mineralized protein microspheres.

[0026] Further, the metal salt mentioned in step (1) may be selected from at least one of the following: calcium salt, zinc salt, barium salt; preferably calcium salt. Even further, the calcium salt may be selected from at least one of the following: calcium carbonate, calcium phosphate, calcium chloride, calcium phosphide; preferably calcium chloride.

[0027] Further, the metal salt in step (1) is added in the form of a metal salt solution, the concentration of which can be 0.1 mmol / L-800 mmol / L, preferably 10 mmol / L-100 mmol / L.

[0028] According to an embodiment of the present invention, the mineralization is strong mineralization, and strong mineralization microspheres H-MAA are prepared by using 100 mmol / L CaCl2 solution; the mineralization is weak mineralization, and weak mineralization microspheres L-MAA are prepared by using 30 mmol / L CaCl2 solution.

[0029] Furthermore, the ratio of the protein microspheres, metal salts, soluble carbonates and / or soluble sulfates and / or soluble phosphates is 10 mg: (0.01-0.1) mmol: (0.01-10) mmol.

[0030] Furthermore, the volume ratio of the deionized water to the metal salt solution can be 1:1-5, preferably 1:1.

[0031] Furthermore, the reaction in step (1) is carried out under stirring conditions, and the stirring speed is 50 rpm to 1500 rpm, preferably 110 rpm.

[0032] Furthermore, the reaction time of the reaction described in step (1) can be 0.5-3 hours, preferably 1 hour.

[0033] Further, the soluble carbonate, soluble sulfate, and soluble phosphate mentioned in step (2) are selected from at least one of the following: Na₂CO₃,

[0034] K₂CO₃, MgCO₃, (NH₄)₂CO₃, NaHSO₄ 4, Na2SO 4, K2SO4, Mg2SO4, Mn2SO4, Zn2SO4, Cr2(SO4)3, FeSO4, Fe2(SO4)3, Cu2SO4, K3PO4, Mg3(PO4)2; preferably Na2CO3;

[0035] Furthermore, the soluble carbonate, soluble sulfate, and soluble phosphate mentioned in step (2) are added in the form of their salt solutions, wherein the concentration of the salt solution is 0.01 mol / L to 10 mol / L, preferably 1 mol / L.

[0036] Furthermore, the reaction time in step (2) can be 0.5-3 hours, preferably 1 hour.

[0037] The mineralized protein microspheres prepared by the above method are also within the scope of protection of this invention.

[0038] Thirdly, the present invention provides a method for radionuclide labeling of mineralized protein microspheres.

[0039] The method for radionuclide labeling of mineralized protein microspheres provided by the present invention includes the following steps: dispersing mineralized albumin microspheres in deionized water, and then reacting them with stannous chloride / hydrochloric acid solution and radionuclide solution to obtain radionuclide-labeled mineralized protein microspheres.

[0040] Furthermore, the ratio of the mineralized protein microspheres to deionized water is 10 mg: 1 mL.

[0041] Furthermore, the volume ratio of the deionized water, stannous chloride / hydrochloric acid solution, and radionuclide solution is 1 mL: 0.01 mL: 0.5 mL, respectively.

[0042] Furthermore, the concentration of the stannous chloride / hydrochloric acid solution is 0.5-100 μg / mL, and the concentration of the hydrochloric acid solution used is 0.01 mmol-100 mmol.

[0043] Furthermore, the activity of the radionuclide solution is 0.2-20 mCi.

[0044] Furthermore, the nuclide selection in the nuclide solution 99m Tc, 188 Re, 64 Cu, 137 Cs, 131 I, 192 Ir, 90 One of Y, preferably 99m Tc.

[0045] The radionuclide-labeled mineralized protein microspheres prepared by the above method are also within the scope of protection of this invention.

[0046] Fourthly, the present invention provides the application of radionuclide-labeled mineralized protein microspheres.

[0047] The application includes at least one of the following aspects:

[0048] 1) Application of radionuclide-labeled mineralized protein microspheres in the preparation of imaging agents;

[0049] 2) Application of radionuclide-labeled mineralized protein microspheres in the preparation of drugs for embolization therapy.

[0050] Furthermore, the imaging area after the imaging agent is injected into the body is the whole body.

[0051] Furthermore, the imaging agent includes, but is not limited to, those used for: liver perfusion imaging, lung imaging, myocardial perfusion imaging, lymphatic imaging, brain imaging, kidney imaging, and thyroid imaging.

[0052] Furthermore, the imaging agent is a SPECT / CT imaging agent.

[0053] Furthermore, the embolization treatment can be tumor embolization treatment.

[0054] Furthermore, the tumor may be a solid tumor, including but not limited to: liver cancer, lung cancer, etc.

[0055] Furthermore, the radionuclide-labeled mineralized protein microspheres are used for transarterial chemoembolization therapy of tumors, by injecting the radionuclide-labeled mineralized protein microspheres into the tumor blood vessels to form an embolization.

[0056] The present invention has the following beneficial effects:

[0057] 1. Adjusting the degree of mineralization can regulate the density of protein microspheres, making them more compatible with the density range of glass microspheres and resin microspheres used in SIRT surgery, thus providing more accurate diagnostic results for surgical treatment.

[0058] 2. Using human serum albumin is an advantage. Human serum albumin has a wide range of clinical applications, and known application cases have proven its safety and reliability, as well as its stable carrier efficacy.

[0059] 3. Selection 99m Tc is a major advantage. 99m Tc combined with SPECT imaging can achieve good radionuclide imaging results. It is economical, readily available, easy to label, has a high and stable labeling rate, and the image acquisition program is simple. 99m Tc is one of the most widely used nuclides in clinical nuclear medicine diagnostics.

[0060] 4. Using CaCl2 as a mineralizing material is an advantage of this invention. Calcium is an important component of the human body and is widely distributed in all tissues and organs of the body. It has no toxic side effects on the human body. Attached Figure Description

[0061] Figure 1 This is a schematic diagram of a scanning electron microscope image of MAA particles.

[0062] Figure 2 These are scanning electron microscope images and energy dispersive spectroscopy (EDS) diagrams of L-MAA particles.

[0063] Figure 3 These are scanning electron microscope images and energy dispersive spectroscopy (EDS) diagrams of H-MAA particles.

[0064] Figure 4 Mice were injected via tail vein.99m Tc MAA, 99m Tc H-MAA, 99m Near-infrared 2-zone imaging (a) and schematic diagram of ROI statistical data analysis of major organs of mice were performed 2 hours, 12 hours and 24 hours after Tc L-MAA (b).

[0065] Figure 5 Mice were injected via tail vein. 99m Tc MAA, 99m Tc H-MAA, 99m SPECT / CT images (a) and statistical data analysis of ROI of major organs in mice were performed 1 hour, 2 hours and 6 hours after Tc L-MAA (b).

[0066] Figure 6 The results are the results of various blood routine indicators obtained from mice 7 days after being injected with MAA, H-MAA and L-MAA via the tail vein.

[0067] Figure 7 This is a magnified image of tissue sections of major organs dissected from mice 7 days after tail vein injection of MAA, H-MAA, and L-MAA, stained with H&E.

[0068] Figure 8 This image shows immunofluorescence staining of neutrophils on lung tissue sections dissected from mice 7 days after tail vein injection of MAA, H-MAA, and L-MAA.

[0069] Figure 9 This is a graph showing the degradation of MAA, L-MAA, and H-MAA particles in buffer solutions at different times and pH values. Detailed Implementation

[0070] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0071] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0072] Example 1: Preparation of albumin microspheres

[0073] Human serum albumin (HSA) (20 mg) was dissolved in physiological saline (400 μL), followed by the addition of deionized water (400 μL), acetate / sodium acetate buffer (pH = 5, 200 μL), and glutaraldehyde solution (0.1% by mass, 200 μL), and the reaction was carried out at room temperature. The mixture was stirred at 1100 rpm using a magnetic stirrer, and the temperature was gradually increased to 80 °C over 40 minutes. After heating at 80 °C for 10 minutes, stirring was stopped, and the mixture was allowed to cool naturally to room temperature. The reaction mixture was then centrifuged at 3000 rpm for 3 minutes, and the supernatant was discarded. 1 mL of deionized water was added, and the mixture was stirred until homogeneous. The washing process was repeated three times to obtain albumin microspheres (MAA).

[0074] The morphology of the obtained albumin microspheres was characterized using scanning electron microscopy (SEM). The results showed that ( Figure 1 Albumin microspheres exhibit irregular morphology, with the vast majority of particles having a diameter between 10 and 40 μm.

[0075] Example 2: Weak mineralization modification treatment of albumin microspheres

[0076] 10 mg of albumin microspheres were suspended in 1 mL of H₂O, and 1 mL of CaCl₂ solution (30 mmol / L) was added to the system. After stirring at 110 rpm for 1 hour, 20 μL of Na₂CO₃ solution (1 mol / L) was added dropwise to the mixture. Stirring was continued at 110 rpm for 1 hour, and then the reaction was stopped. The mixture was then centrifuged at 3000 rpm for 3 minutes, and the supernatant was discarded. 1 mL of deionized water was added, and the mixture was stirred until homogeneous. The washing process was repeated three times to obtain L-MAA.

[0077] The obtained L-MAA was characterized and its elemental distribution was analyzed using scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX). The results showed that the L-MAA contained a significant distribution of calcium carbonate, and its morphological characteristics were consistent with those before modification. Figure 2 )

[0078] Example 3: Strong mineralization modification treatment of albumin microspheres

[0079] 10 mg of albumin microspheres were suspended in 1 mL of H₂O, followed by the addition of 1 mL of CaCl₂ (100 mmol / L). After stirring at 110 rpm for 1 hour, 20 μL of Na₂CO₃ (1 mol / L) was added dropwise to the mixture. Stirring was continued at 110 rpm for another hour, and then the reaction was stopped. The mixture was then washed three times with deionized water to obtain H-MAA.

[0080] The obtained H-MAA was characterized and its elemental distribution was analyzed using scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX). The results showed that the H-MAA contained calcium carbonate, and its morphological characteristics were consistent with those before modification. Figure 3 )

[0081] Example 4: Near-infrared two-zone imaging of three different mineralization levels of MAA (MAA, H-MAA, and L-MAA)

[0082] Based on the above results, we further simulated the in vivo distribution of MAAs at different densities using near-infrared II fluorescence imaging. During the synthesis process, ICG (1%, w / w) was added as a contrast agent via hydrophobic interactions to prepare ICG-loaded materials, namely MAA@ICC, H-MAA@ICC, and L-MAA@ICC. Furthermore, ICG-C16 (1%, w / w) was added during the synthesis process to synthesize materials simulating commercially available MAAs. 90 Y resin microspheres and 90 The scanning electron microscopy results of polymer microspheres (Polymer@ICG-C16) and silica microspheres (SiO2@ICG-C16) are as follows: Figure 4 As shown in figure a. Mice were sacrificed 2 h, 12 h, and 24 h after injection, and major organs were extracted for in vivo near-infrared spectroscopy (NIRS) fluorescence imaging.

[0083] The results are as follows Figure 4 As shown in b, according to Figure 4 The statistical analysis shown in b indicates that 2 hours post-injection, the fluorescence signal of the MAA contrast agent was mainly distributed in the lungs and liver. Over time, the liver concentration gradually decreased at 12 hours post-injection, likely due to the contrast agent entering the mouse bloodstream and ICG being metabolized and excreted through the liver. At 2 hours post-injection, polymer microspheres and simulated resin microspheres showed minimal distribution in the liver, primarily concentrated in the lungs. Over time, the liver distribution slowly increased at 12 hours post-injection, possibly because the less dense polymer microspheres changed less rapidly in the blood compared to MAA. Conversely, silica microspheres simulating glass microspheres were mainly distributed in the liver at 2 hours post-injection, with less pronounced distribution in the lungs. Over time, the liver distribution remained significant, necessitating further investigation into this distribution pattern. These results confirm that MAA microspheres cannot effectively mimic resin and glass microspheres.

[0084] Conversely, when comparing polymer microspheres and weakly mineralized microspheres (L-MAA), the fitting results were excellent. L-MAA was mainly distributed in the lungs 2 hours after injection, with increased distribution in the liver at 12 hours and even more pronounced liver distribution at 24 hours, very similar to the distribution of polymer microspheres. The distribution of H-MAA closely correlated with that of silica microspheres, showing significant liver accumulation over time. This result is consistent with the density results, indicating that increased MAA density affects its distribution in the blood. Furthermore, the distribution of mineralized and non-mineralized microspheres also differed significantly across different organs. This suggests that the biodistribution of different commercial microspheres can be modeled by adjusting the degree of mineralization of MAA, thereby improving its applicability as a contrast agent for targeted imaging and therapy.

[0085] Example 5: Radiolabeling and imaging of MAA with different mineralization levels

[0086] Traditionally, MAA particles are radiolabeled for radioactive tracer imaging, using gamma radioisotopes with a half-life of 6.02 hours. 99m Tc. First, disperse 10 mg of albumin microspheres or mineralized albumin microspheres (albumin microspheres prepared in Example 1, L-MAA prepared in Example 2, or H-MAA prepared in Example 3) in 1 mL of deionized water, add 0.01 mL of stannous chloride / hydrochloric acid solution (50 μg / mL, 0.1 mmol HCl), and then add 0.5 mL of... 99m Tc solution (activity 5 mCi) was stirred for 30 minutes. The mixture was washed three times with deionized water, and 1 mL of PBS was added to form a suspension, yielding... 99m Tc-MAA or 99m Tc-H-MAA or 99m Tc-L-MAA. Radiographic imaging was then performed.

[0087] Nine male Balb / c mice (15-20 g, 3-4 weeks old) were randomly divided into three groups: A, B, and C. Each group was intravenously injected with approximately 0.3-0.6 mCi of [a specific drug / method]. 99m Tc-MAA, 99m Tc-H-MAA and 99m Tc-L-MAA. Mice were anesthetized with isoflurane (mixed with air, concentration 2%, flow rate 1.5 L / min), and SPECT / CT imaging was performed at 0.5 h, 2 h, and 6 h. The results showed that there was obvious radioactive distribution 0.5 h after injection, demonstrating that the three MAA contrast agents can rapidly pass through the heart and embolize in the lungs, with more than 63% of the activity concentrated in the lungs. The distribution in other organs was relatively low. For more detailed analysis... 99m Tc-MAA is mainly concentrated in the lungs within 0.5 hours, with lower absorption in other organs and more uniform distribution. 99mTc-L-MAA and 99m Although Tc-H-MAA is mainly concentrated in the lungs, it is related to... 99m Compared to Tc-MAA, both types of mineralized protein microspheres showed higher uptake in the liver. This preferential distribution in the liver was consistent with results observed in NIR-II fluorescence imaging. Two hours after injection, the distribution in the liver, kidneys, and spleen was slightly increased, and there were clear differences in organoid distribution among the three different mineralization levels of the protein microspheres. Six hours after injection, due to… 99m Tc decay and metabolism in the body lead to an overall decrease in radioactivity and a less concentrated distribution, especially at 6 hours, where L-MAA is most prominently distributed in the intestines, while the MAA group has the highest distribution in the lungs compared to the other two groups. Figure 5 )

[0088] Example 6: Biosafety analysis of MAA, H-MAA and L-MAA

[0089] Twelve BALB / c mice (4-6 weeks old, female, weighing approximately 18-20g) were prepared and divided into four groups (A, B, C, and D), with three mice in each group. Each mouse was injected via tail vein with either 150 μL of PBS or 150 μL of albumin microspheres (approximately 12,000 particles) at different mineralization levels (MAA, H-MAA, or L-MAA). Seven days after injection, venous blood was collected from the mice, and the supernatant was obtained by centrifugation. Complete blood count (CBC) parameters were measured. H&E staining was performed on the heart, liver, spleen, lungs, and kidneys of mice in the PBS and MAA (MAA, H-MAA, and L-MAA) groups after seven days of injection. Immunofluorescence staining of neutrophils in the lung tissue was also performed to observe for any lesions.

[0090] Immunofluorescence staining of mouse neutrophils showed no significant changes, and their indices were comparable to those in PBS. Hematological analysis results showed ( Figure 6 All blood cell markers in the mice were within the normal range, comparable to the PBS group. H&E staining results of tissue sections showed... Figure 7 No obvious lesions were observed in any of the major organs of the mice. Immunofluorescence staining of neutrophils in the lung tissue showed the following results. Figure 8 As shown, there was no significant difference between the experimental group and the control group. This indicates that the material has good biocompatibility.

[0091] Example 7: Degradability Experiments of MAA, H-MAA and L-MAA

[0092] Two different pH PBS buffer solutions (pH=5, pH=6) and a pure aqueous solution (pH=7) were prepared. MAA, H-MAA, and L-MAA were respectively prepared into different albumin microsphere solutions of 10 mg / mL. 2 μL samples were taken at 4 h, 12 h, 3 d, and 5 d to observe the degradation of the microspheres under a scanning electron microscope.

[0093] The results are as follows Figure 9 The results show that MAA, H-MAA, and L-MAA undergo slight degradation in a neutral environment (pH=7), and significant degradation in slightly acidic environments (pH=5 and pH=6), especially complete degradation within 5 days under acidic conditions at pH=5. This indicates that the material has excellent biocompatibility.

[0094] The foregoing description of specific exemplary embodiments of this disclosure is for illustrative and explanatory purposes. These descriptions are not intended to limit this disclosure to the precise form disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of this disclosure and their practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments and various different options of this disclosure.

Claims

1. A method for preparing protein microspheres, comprising the following steps: The protein was mixed with physiological saline solution, deionized water, acetate / sodium acetate buffer and cross-linking agent, and reacted under stirring. The temperature was gradually increased from room temperature to 75-85°C, and then kept at this temperature for 8-15 minutes. After stirring was stopped, the reaction mixture was allowed to cool to room temperature to obtain the protein microspheres.

2. The preparation method according to claim 1, characterized in that: The protein is selected from at least one of the following: bovine serum albumin, human serum albumin, horse serum albumin, monkey serum albumin, recombinant human albumin, various antibodies, biological enzyme proteins, hormone proteins, preferably human serum albumin and recombinant human serum albumin, and more preferably human serum albumin; And / or, the protein concentration in the physiological saline solution is 10-100 mg / mL, preferably 40-60 mg / mL, and most preferably 50 mg / mL; And / or, the pH of the acetate / sodium acetate buffer solution is 4.5-5.5, preferably pH 5; And / or, the crosslinking agent is selected from at least one of the following: glutaraldehyde, acetic anhydride, diglycidyl ether, octyldiimide methyl ester; And / or, the stirring speed of the stirring is 700-2000 rpm, preferably 900-1500 rpm; And / or, the heating is completed within 30-60 minutes.

3. The preparation method according to claim 1 or 2, characterized in that: The crosslinking agent is added in solution form at a concentration of 0.05-0.5%. The volume ratio of the protein in physiological saline solution, deionized water, acetate / sodium acetate buffer, and glutaraldehyde solution is 1:(0.8-1.2):(0.4-0.6):(0.4-0.6).

4. Protein microspheres prepared by the method according to any one of claims 1-3.

5. The protein microspheres according to claim 4, characterized in that: The protein microspheres have a particle size range of 1-200 μm, preferably 10-100 μm, and more preferably 10-40 μm; and a density range of 0.9-3.3 g / mL.

6. The mineralization method for the protein microspheres according to claim 4 or 5, comprising the following steps: (1) Disperse the protein microspheres according to claim 4 or 5 in deionized water, add metal salt, and carry out the reaction; (2) Add soluble carbonate and / or soluble sulfate and / or soluble phosphate to the reaction system of step (1) and continue the reaction; (3) After the reaction is complete, centrifuge, collect the precipitate and wash it to obtain mineralized protein microspheres.

7. The mineralization method according to claim 6, characterized in that: The metal salt in step (1) is selected from at least one of the following: calcium salt, zinc salt, barium salt; preferably calcium salt; more preferably, the calcium salt is selected from at least one of the following: calcium carbonate, calcium phosphate, calcium chloride, calcium phosphide; And / or, the metal salt in step (1) is added in the form of a metal salt solution, the concentration of which is 0.1 mmol / L-800 mmol / L, preferably 10 mmol / L-100 mmol / L; And / or, the ratio of the protein microspheres, metal salts, soluble carbonates and / or soluble sulfates and / or soluble phosphates is 10 mg: (0.01-0.1) mmol: (0.01-10) mmol; And / or, the soluble carbonate, soluble sulfate, or soluble phosphate in step (2) is selected from at least one of the following: Na₂CO₃, K₂CO₃, MgCO₃, (NH₄)₂CO₃, NaHSO₄ 4, Na2SO 4, K2SO4, Mg2SO4, Mn2SO4, Zn2SO4, Cr2(SO4)3, FeSO4, Fe2(SO4)3, Cu2SO4, K3PO4, Mg3(PO4)2; preferably Na2CO3; In step (2), the soluble carbonate, soluble sulfate, and soluble phosphate are added in the form of a salt solution, wherein the concentration of the salt solution is 0.01 mol / L to 10 mol / L, preferably 1 mol / L.

8. The mineralization method according to claim 6 or 7, characterized in that: In step (1), the reaction is carried out under stirring conditions, and the stirring speed is 50 rpm-1500 rpm, preferably 110 rpm; And / or, the reaction time of the reaction in step (1) is 0.5-3 hours, preferably 1 hour; And / or, the reaction time of the reaction in step (2) is 0.5-3 hours, preferably 1 hour.

9. Mineralized protein microspheres prepared by the method according to any one of claims 6-8.

10. A method for radionuclide labeling of the mineralized protein microspheres of claim 9, comprising the following steps: dispersing the mineralized protein microspheres of claim 9 in deionized water, and then reacting them with a stannous chloride / hydrochloric acid solution and a radionuclide solution to obtain radionuclide-labeled mineralized protein microspheres.

11. The preparation method according to claim 10, characterized in that: The ratio of the mineralized protein microspheres to deionized water is 10 mg: 1 mL; And / or, the volume ratio of the deionized water, stannous chloride / hydrochloric acid solution and the radionuclide solution is 1 mL: 0.01 mL: 0.5 mL, respectively; And / or, the concentration of the stannous chloride / hydrochloric acid solution is 0.5-100 μg / mL, and the concentration of the hydrochloric acid solution used is 0.01 mmol / L-100 mmol / L; And / or, the activity of the radionuclide solution is 0.2-2 mCi; And / or, the nuclides in the nuclide solution are selected from... 99m Tc, 188 Re, 64 Cu, 137 Cs, 131 I, 192 Ir, 90 One of Y, preferably 99m Tc.

12. The radionuclide-labeled mineralized protein microspheres prepared by the method of claim 10 or 11.

13. The application of the radionuclide-labeled mineralized protein microspheres of claim 12, wherein the application includes at least one of the following aspects: 1) the application of the radionuclide-labeled mineralized protein microspheres in the preparation of imaging agents; 2) the application of the radionuclide-labeled mineralized protein microspheres in the preparation of medicaments for embolization therapy.

14. The application according to claim 13, characterized in that: The imaging agent, once injected into the body, can be used to image the entire body. And / or, the imaging agents include, but are not limited to, those used for: liver perfusion imaging, lung imaging, myocardial perfusion imaging, lymphatic imaging, brain imaging, kidney imaging, and thyroid imaging; And / or, the imaging agent is a SPECT / CT imaging agent; And / or, the embolization treatment is tumor embolization treatment, and further, the tumor is a solid tumor, including but not limited to: liver cancer, lung cancer.

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