Diagnosis and treatment integrated multifunctional radioactive microsphere as well as preparation method and application thereof

Abstract

The invention discloses a diagnosis and treatment integrated multifunctional radioactive microsphere and a preparation method and application thereof.The preparation method comprises the steps that under the weak alkaline condition, pollen powder and a mixed solution containing polyphenol compounds and functional ligands are subjected to coordination polymerization, and pollen microspheres modified by polyphenol compound-functional ligand composite coatings are formed; further, the multifunctional radioactive microspheres which can be efficiently and stably marked by radionuclides are prepared. The microsphere has the functions of treatment, imaging and immune activation, has high radionuclide labeling efficiency, and can be stably retained in tumors for a long time; a clear ultrasonic high-echo signal is presented, and the specific radioactivity concentration of a tumor part is obvious during nuclide imaging. The diagnosis and treatment integrated platform is constructed on the basis of ultrasonic response and nuclide imaging, primary and metastatic tumors can be systematically inhibited when the diagnosis and treatment integrated platform is combined with immunotherapy, safety is good, and the diagnosis and treatment integrated platform has good clinical transformation potential.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a multifunctional radioactive microsphere for diagnosis and treatment integration and a preparation method and application thereof. BACKGROUND

[0002] In the field of nanocarrier application in tumor diagnosis and treatment, the existing technology faces many challenges. Traditional radioactive carriers such as liposomes and polymer nanoparticles have the problem of complex labeling process. The coordination stability of the surface functional groups and radionuclides is insufficient, resulting in low labeling efficiency and easy label loss, which is difficult to meet the clinical demand for drug stability. At the same time, the existing carriers lack effective visualization monitoring means. For example, the magnetic pollen nanocarrier disclosed in Chinese patent CN114247482A realizes metal ion coordination through a dopamine coating, but only relies on single nuclide imaging, which cannot construct a multi-modal monitoring system, making it difficult to track the distribution and dose deposition of the carrier in the tumor site in real time, limiting the realization of precise treatment.

[0003] In addition, the existing radioactive carriers have insufficient tumor targeting and retention capacity, and are easily cleared by the reticuloendothelial system, resulting in insufficient drug concentration at the tumor site or systemic toxicity. For example, the magnetic pollen carrier mentioned above realizes directional movement through magnetism, but the functional layer modified on its surface lacks responsiveness to the tumor microenvironment, and cannot realize sustained release and action enhancement of the drug. More importantly, traditional carriers generally lack immune activation function, and radionuclide therapy can only directly kill tumor cells, making it difficult to activate the body's anti-tumor immune response. While the immunotherapy lacks precise guidance, the combination of the two has the problem of insufficient synergy, which cannot effectively inhibit tumor metastasis and recurrence.

[0004] It is worth noting that the existing technology involving pollen carriers (such as Chinese patent CN114247482A) only utilizes the natural gas sac structure and surface modification ability of pollen, and has not fully involved the application of pollen wall breaking. In fact, the bioactive components such as polysaccharides and polypeptides in the pollen wall can release immune regulatory factors after breaking the wall, activate dendritic cells to enhance antigen presentation, but the existing technology has not combined the bioactivity of pollen with radiotherapy and immunotherapy, and has not constructed a multi-mechanism synergistic treatment system.

[0005] Therefore, the existing radioactive carriers in the prior art have the defects of complex labeling, insufficient visualization monitoring, lack of immune activation, and not utilizing pollen wall breaking for synergistic treatment, and there is an urgent need for a multifunctional radioactive microsphere for diagnosis and treatment integration to realize efficient loading of radionuclides, dual-modal imaging guidance, immune activation, and synergistic treatment, so as to overcome the above problems. SUMMARY

[0006] To solve the above problems in the prior art, the application provides a multifunctional radioactive microsphere for diagnosis and treatment integration and a preparation method and application thereof, aiming to realize efficient and stable loading of radionuclides and tumor targeting retention; through dual-mode imaging guidance of ultrasound / radionuclide, the treatment process can be monitored in real time; and through ultrasound-responsive release of bioactive components, antitumor immunity can be activated, and immunological checkpoint inhibitors can be combined to inhibit primary and metastatic tumors.

[0007] The application is achieved by the following technical solutions:

[0008] The application provides a preparation method of a multifunctional radioactive microsphere for diagnosis and treatment integration, comprising the following steps:

[0009] (1) dispersing pollen in water to obtain a pollen solution; the pollen has a gas sac structure;

[0010] (2) dissolving polyphenolic compounds and functional ligands in water to obtain a mixed solution; mixing the pollen solution obtained in step (1) and the mixed solution, adjusting the pH to 8.0-9.0, and reacting to obtain modified pollen microspheres; the mass ratio of the polyphenolic compounds in the mixed solution to the pollen in the pollen solution is 1:(1-10); the functional ligands are selected from one or more of bisphosphonate compounds, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and diethylene triamine pentaacetic acid (DTPA);

[0011] (3) mixing the modified pollen microspheres obtained in step (2) with a radionuclide solution, and obtaining the multifunctional radioactive microsphere for diagnosis and treatment integration after labeling is completed;

[0012] the radionuclide in the radionuclide solution is selected from one or more of 90 Y, 125 I, 131 I, 111 In, 67 Ga, 177 Lu and 64 Cu.

[0013] In the weak alkaline reaction system, the pollen powder is surface-modified with the mixed solution containing polyphenolic compounds and functional ligands through coordination polymerization reaction, a multifunctional pollen microsphere with efficient coordination ability of radionuclides is constructed, and stable labeling and functional integration of radionuclides are realized. Based on the inherent porous gas sac microstructure, endogenous bioactive components (polysaccharides / proteins) and unique acoustic response characteristics of natural biological materials (such as pollen), a diagnosis and treatment integration platform integrating radiotherapy, imaging diagnosis and immunoregulation functions is constructed.

[0014] Further, in step (1), the pollen is selected from one or more of pine pollen, fir pollen and spruce pollen, preferably pine pollen. The specific surface area of pine pollen is significantly higher than that of fir pollen, providing more abundant attachment sites for subsequent modification.

[0015] Further, in step (1), the concentration of pollen in the pollen solution is 3-6 g / L. If the concentration is too low, the surface coating of pollen particles is insufficient, the effective binding sites are reduced when the radionuclide is labeled, and the labeling rate is reduced. If the concentration is too high, the pollen particles will agglomerate, resulting in uneven coating.

[0016] Further, in step (2), the polyphenolic compound is selected from one or more of dopamine and its derivatives, tannic acid, gallic acid, epigallocatechin gallate, proanthocyanidins and catechin and its derivatives, preferably dopamine and its derivatives, and more preferably dopamine hydrochloride (DA).

[0017] Preferably, in step (2), the functional ligand is a bisphosphonate compound, more preferably bisphosphonate (BP), which is selected from one or more of alendronate sodium, neridronate sodium, olpadronate sodium, risedronate sodium and ibandronate sodium.

[0018] Further, in step (2), the concentration of polyphenolic compound in the mixed solution is 5-15 g / L. If the concentration is too low, the coating thickness is too thin and the radionuclide coordination sites are insufficient. If the concentration is too high, the polyphenolic compound will self-aggregate too quickly, forming a coating with too high density, which will hinder the coordination of the functional ligand with the radionuclide, resulting in a decrease in labeling stability.

[0019] Further, in step (2), the mass ratio of polyphenolic compound to functional ligand is 1:(0.3-0.8).

[0020] Further, in step (2), the volume ratio of the pollen solution to the mixed solution is (8-10):1.

[0021] Preferably, in step (2), the mass ratio of polyphenolic compound in the mixed solution to pollen in the pollen solution is 1:(3-5).

[0022] Further, in step (3), the concentration of radionuclide ions in the radionuclide solution is 0.5-2 mCi / mL.

[0023] Further, in step (3), the mass ratio of the modified pollen microspheres to the radionuclide in the radionuclide solution is 0.05-0.2 mg: 50-100 μCi. If the amount of radionuclide is too low, the image signal intensity is insufficient, and the tumor cannot be accurately located. If the amount of radionuclide is too high, the radionuclide is oversaturated on the surface of the microspheres, resulting in a radiation shielding effect between adjacent radionuclides, and the actual effective radiation dose decreases.

[0024] In the preparation process of the diagnosis and treatment integrated multifunctional radioactive microspheres described in the application, the labeling rate of the radionuclide reaches 95% within 1 minute and can reach 100% after 5 minutes, as verified by the gamma counting method. The multifunctional radioactive microspheres exhibit excellent stability in phosphate buffer solution (PBS) or 10% fetal bovine serum solution; after intratumoral injection, the radioactive retention rate in the tumor site is still over 90% at 7 days.

[0025] In a specific embodiment, a preparation method of diagnosis and treatment integrated multifunctional radioactive microspheres comprises the following steps:

[0026] (1) dispersing pollen in water to obtain a pollen solution; the pollen has a gas sac structure;

[0027] (2) dissolving DA and BP in water to obtain a mixed solution; mixing the pollen solution obtained in step (1) and the mixed solution, adjusting the pH to 8.0-9.0, and reacting to obtain pollen microspheres modified with a polydopamine-bisphosphonate (PDA-BP) composite coating; the mass ratio of DA in the mixed solution to the pollen in the pollen solution is 1:(1-10);

[0028] (3) mixing the pollen microspheres modified with the PDA-BP composite coating obtained in step (2) with a radionuclide solution, and obtaining the diagnosis and treatment integrated multifunctional radioactive microspheres after labeling is completed.

[0029] In the synthesis process of the pollen microsphere material, in a weak alkaline environment, the phosphonic acid groups of BP preferentially specifically bind to the functional groups such as amino groups and hydroxyl groups on the pollen surface through electrostatic attraction and hydrogen bonding; based on the synergistic effect of intermolecular forces, BP further induces the ordered aggregation of DA on the pollen surface through hydrogen bonding and electrostatic interaction; as the alkaline condition promotes the deprotonation of the phenolic hydroxyl group of DA, it triggers the oxidative self-polymerization reaction, and finally a dense and uniform polydopamine (PDA) coating is formed on the surface of the pollen particles; the o-phenol groups in the PDA coating and the phosphonic acid groups of BP form a stable three-dimensional cross-linked structure through multiple action mechanisms such as hydrogen bonding association and ion exchange, successfully constructing a PDA-BP-pollen ternary composite, and laying a foundation for the multifunctional modification of the microspheres.

[0030] The second aspect of the present application provides the multifunctional radio microsphere for diagnosis and treatment integration prepared by the preparation method of the first aspect.

[0031] The multifunctional radio microsphere for diagnosis and treatment integration provided by the present application has the functions of treatment, imaging and immune activation. The microsphere takes pollen particles as a carrier, is modified with a composite coating on the surface and is loaded with radionuclides, and realizes the integration of multi-modal diagnosis and treatment functions through structural design.

[0032] The third aspect of the present application provides the application of the multifunctional radio microsphere for diagnosis and treatment integration of the second aspect in ultrasonic imaging and / or nuclide imaging.

[0033] The multifunctional radio microsphere for diagnosis and treatment integration provided by the present application has definite application value in the field of ultrasonic imaging and nuclide imaging. Based on the acoustic reflection characteristics of the pollen sac structure, ultrasonic high-echo imaging can be realized. At the same time, through radionuclide labeling, quantitative monitoring of the radioactive distribution of tumor sites can be realized with the help of nuclear medicine equipment. The synergistic effect of the two imaging modes can realize real-time tracking of the spatial distribution and dynamic retention process of the microsphere in the tumor tissue, and provide imaging basis for precise diagnosis and treatment.

[0034] The fourth aspect of the present application provides the application of the multifunctional radio microsphere for diagnosis and treatment integration of the second aspect in preparing brachytherapy drugs.

[0035] The multifunctional radio microsphere for diagnosis and treatment integration provided by the present application can be used for preparing brachytherapy drugs, and realizes the precise release of radionuclides in the tumor through local injection. When applied, ultrasonic treatment (10-30 kHz, 50-150 W) can enhance the retention efficiency of the microsphere in the tumor tissue and promote the controlled release of radionuclides, improve the local deposition effect of the radiotherapy dose, and reduce the whole body radiation toxicity.

[0036] The fifth aspect of the present application provides the application of the multifunctional radio microsphere for diagnosis and treatment integration of the second aspect in preparing immune combination therapy drugs.

[0037] Further, when applied, the multifunctional radio microsphere for diagnosis and treatment integration is subjected to ultrasonic treatment (10-30 kHz, 50-150 W), the pollen wall is broken by ultrasonic treatment, endogenous crude polysaccharides and proteins are released, dendritic cell (DC) maturation is activated, and the production of pro-inflammatory cytokines is promoted. Flow cytometry detection shows that the expression amount of co-stimulatory molecules CD80 / CD86 on the surface of DC cells is up-regulated, the secretion amount of pro-inflammatory cytokines interferon-γ (INF-γ), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) is increased by 2-3 times, and the antigen presentation ability is significantly enhanced.

[0038] Further, the immune combination therapy drug further comprises an anti-cell programmed death-ligand 1 antibody (anti-PD-L1 antibody, referred to as a-PD-L1).

[0039] The diagnosis and treatment integrated multifunctional radioactive microspheres provided by the application can increase the amount of cytotoxic T lymphocytes (CD8 + T) infiltration in the tumor microenvironment by 40% (verified by flow cytometry), and when combined with a-PD-L1, can relieve immune checkpoint inhibition, synergistically enhance anti-tumor immune response, promote T cell killing of tumor cells, and achieve systemic treatment effect of primary tumor inhibition and metastatic tumor clearance.

[0040] The sixth aspect of the application provides the use of the diagnosis and treatment integrated multifunctional radioactive microspheres of the second aspect in the preparation of a tumor treatment drug, and the tumor includes a primary tumor and / or a metastatic tumor.

[0041] Further, the primary tumor includes a 4T1 breast cancer single tumor.

[0042] Further, the metastatic tumor includes a CT26 colon cancer double tumor.

[0043] The application has the following beneficial effects:

[0044] 1. The diagnosis and treatment integrated multifunctional radioactive microspheres prepared by the application realize efficient loading of radionuclides and tumor microenvironment responsive retention through surface engineering modification. Based on the high acoustic impedance characteristics of the pollen air sac structure, it presents a significantly high echo signal in the ultrasonic field, and at the same time, it is labeled with radionuclides to display tumor-specific concentration in nuclear medicine equipment imaging.

[0045] 2. The diagnosis and treatment integrated multifunctional radioactive microspheres provided by the application play an anti-tumor role through a double mechanism. The rays emitted by the radionuclides form a high-dose radiation field in the tumor, directly inducing apoptosis; and the polysaccharide / protein complex released by ultrasound triggers the activation of innate immunity, promoting CD8 + T cells to infiltrate the tumor, and when combined with a-PD-L1, it can relieve immune suppression and enhance distant effects, achieving systemic inhibition of primary and metastatic tumors.

[0046] 3、In the 4T1 breast cancer tumor-bearing mouse model, the tumor growth inhibition rate of the diagnosis and treatment integrated multifunctional radioactive microspheres combined with ultrasound treatment group provided by the application reaches 100%, and 80% of the mice survive for 30 days; in the CT26 colon cancer bilateral tumor model, the combined immunotherapy group not only eliminates the primary tumor, but also completely eliminates the distal untreated tumor, and the survival rate of the mice reaches 100%. Histopathological analysis shows that the heart, liver, spleen, lung, kidney and other organs have no radioactive damage or immune-related toxicity, and the blood biochemical indicators remain within the normal range, fully verifying the safety and clinical transformation potential of the application in solid tumor treatment. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 SEM image of pine pollen particles in Example 1.

[0048] Figure 2 SEM image of PDA-BP composite coating modified pine pollen microspheres in Example 1.

[0049] Figure 3 OM image of pine pollen particles in Example 1.

[0050] Figure 4 OM image of PDA-BP composite coating modified pine pollen microspheres in Example 1.

[0051] Figure 5 Zeta potential data graph of Pine pollen and PDA-BP+Pine pollen in Example 1.

[0052] Figure 6 FTIR graph of PDA-BP+Pine pollen, Pine pollen, BP and PDA in Example 1.

[0053] Figure 7 Ultrasound imaging graph of PDA-BP+Pine pollen.

[0054] Figure 8 Ultrasound imaging graph of PDA-BP+Pine pollen after cell wall breaking.

[0055] Figure 9 Content data graph of Crude Polysaccharide and Protein released by PDA-BP+Pine pollen after cell wall breaking.

[0056] Figure 10 Flow cytometry detection of the expression levels of CD80 and CD86 on DC cells in each treatment group in Test Example 2.

[0057] Figure 11This is a data graph of the secretion of pro-inflammatory cytokines (INF-γ, TNF-α and IL-6) in each treatment group in Test Example 2 detected by ELISA method.

[0058] Figure 12 The graph shows the radiolabeling efficiency data of pine pollen and PDA-BP+pine pollen.

[0059] Figure 13 for 90 The results of the labeling stability test of pine pollen microspheres modified with Y-labeled PDA-BP composite coating.

[0060] Figure 14 Intratumoral injection of mice in Test Example 3 90 Ultrasound imaging after Y-PDA-BP+Pine pollen microspheres.

[0061] Figure 15 For test case 3 90 Y-PDA-BP+Pine pollen group and 90 In vivo imaging of mice in the Y-PDA-BP+Pine pollen-broken group, tumor radioactivity intensity data, and tumor radionuclide percentage data.

[0062] Figure 16 For test case 3 90 %ID / g data of various organs of tumor-bearing mice in the Y-PDA-BP+Pine pollen-broken group.

[0063] Figure 17 For the PBS group, Pine pollen group, 90 Y-PDA-BP+Pine pollen group, 90 The tumor volume data of mice in the Y-PDA-BP+Pine pollen-broken group (left) and the survival rate data of mice (right).

[0064] Figure 18 For the PBS group, Pine pollen group, 90 Y-PDA-BP+Pine pollen group, 90 H&E staining of the Y-PDA-BP+Pine pollen-broken group (upper left), TUNEL section staining (lower left), and the proportion of positive cell apoptosis after TUNEL staining (right).

[0065] Figure 19 For the PBS group, Pine pollen group,90 Y-PDA-BP+Pine pollen group, 90 H&E-stained sections of the main organs (heart, liver, spleen, lung, and kidney) of mice in the Y-PDA-BP+Pine pollen-broken group.

[0066] Figure 20 These are the T cell flow cytometry test results and statistical graphs, mouse survival rate data graphs, and mouse tumor volume data graphs of normal mice and recovered secondary infected mice in test example 4; among them, (a) is the T cell flow cytometry test result graph, (b) is the statistical graph corresponding to the T cell flow cytometry test result, (c) is the mouse survival rate data graph, and (d) is the mouse tumor volume data graph.

[0067] Figure 21 For the PBS group, a-PD-L1 group, 90 Y-PDA-BP+Pine pollen+a-PD-L1 group, 90 Y-PDA-BP+Pine pollen-broken group, 90 Data graph of mouse survival rate in the Y-PDA-BP+Pine pollen+a-PD-L1-broken group.

[0068] Figure 22 For the PBS group, a-PD-L1 group, 90 Y-PDA-BP+Pine pollen+a-PD-L1 group, 90 Y-PDA-BP+Pine pollen-broken group, 90 Data graph of right tumor volume of mice in the Y-PDA-BP+Pine pollen+a-PD-L1-broken group.

[0069] Figure 23 For the PBS group, Anti-PD-L1 group, 90 Y-PDA-BP+Pine pollen+Anti-PD-L1 group, 90 Y-PDA-BP+Pine pollen-broken group, 90 Data graph of left tumor volume of mice in the Y-PDA-BP+Pine pollen+Anti-PD-L1-broken group.

[0070] Figure 24 For the PBS group, a-PD-L1 group, 90 Y-PDA-BP+Pine pollen+a-PD-L1 group,90 Y-PDA-BP+Pine pollen-broken group, 90 CD8 + T and Granzyme B, and their statistical graphs; wherein (a) is CD8 + T, (b) is Granzyme B, (c) is CD8 + T, and (d) is Granzyme B.

[0071] Figure 25 For the PBS group, the a-PD-L1 group, 90 Y-PDA-BP+Pine pollen+a-PD-L1 group, 90 Y-PDA-BP+Pine pollen-broken group, 90 CD8 + T and Granzyme B, and their statistical graphs; wherein (a) is CD8 + T, (b) is Granzyme B, (c) is CD8 + T, and (d) is Granzyme B.

[0072] Figure 26 For the PBS group, the a-PD-L1 group, 90 Y-PDA-BP+Pine pollen+a-PD-L1 group, 90 Y-PDA-BP+Pine pollen-broken group, 90 H&E staining sections of the main organs (heart, liver, spleen, lung, kidney) of mice in the Y-PDA-BP+Pine pollen+a-PD-L1-broken group. DETAILED DESCRIPTION

[0073] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0074] The present application is further described so that others skilled in the art can better understand the present application and can better practice it, by referring to the accompanying drawings and specific embodiments. The embodiments are not intended to limit the present application.

[0075] The following examples were carried out using conventional equipment in the art. The following examples were carried out using conventional equipment in the art. The experimental methods in the following examples, unless otherwise specified, were generally carried out under conventional conditions, or under conditions recommended by the manufacturer. The following examples were carried out using various raw materials, unless otherwise specified, conventional commercially available products were used, and the specifications were conventional specifications in the art.

[0076] In the following examples, pollen particles were treated as follows: an appropriate amount of commercial pollen powder was dissolved in 1x PBS buffer, and after being fully suspended, it was placed in a test tube rack for 15 min. Using the physical property that pollen has a lower density than water, the upper layer of floating pollen particles was collected to complete the preliminary cleaning. Subsequently, the cleaned pollen particles were resuspended in PBS buffer and centrifuged at 1500 rpm for 15 min. The supernatant and the impurities at the bottom of the tube were discarded, and the upper layer of floating pollen particles was collected. The above centrifugation-washing operation was repeated until no visible impurities were deposited at the bottom of the tube. Finally, the purified pollen particles were transferred to a freeze-drying machine, pre-frozen at -80°C for 24 h, and then vacuum freeze-dried (pressure < 10 Pa). The obtained pollen particles were sealed and stored at 4°C.

[0077] Example 1

[0078] A multifunctional radioactive microsphere for diagnosis and treatment integration 90 The preparation method of Y-PDA-BP+Pine pollen includes the following steps:

[0079] (1) 0.4 g of treated pine pollen particles (Pine pollen) were weighed and dispersed in 90 mL of pure water, stirred for 30 min to form a uniform dispersion system, and a pine pollen solution was obtained;

[0080] (2) Dissolve 0.1 g of DA and 0.05 g of alendronate sodium (BP) in 10 mL of pure water to obtain a DA-BP mixed solution; add 10 mL of the DA-BP mixed solution dropwise to 90 mL of the pine pollen solution obtained in step (1) under continuous stirring, adjust the pH of the mixed system to 8.5, and stir thoroughly at room temperature for 6 h. After the reaction, centrifuge the system at 3000 rpm for 15 min, collect the precipitate, and wash it three times with PBS buffer to remove unreacted DA and BP, thereby obtaining pine pollen microspheres modified with a PDA-BP composite coating (PDA-BP+Pine pollen), which are freeze-dried (pre-frozen at -80°C for 24 h, vacuum <10 Pa), and sealed and stored in a refrigerator at 4°C for later use.

[0081] (3) 0.1 mg of the PDA-BP composite coating modified pine pollen microspheres obtained in step (2) was mixed with 100 μCi 90 YCl3 solution (concentration of 1 mCi / mL) was mixed and incubated at room temperature for 5 minutes under shaking. After labeling, the unbound nuclides were removed by centrifugation at 3000 rpm for 5 minutes. After washing, the multifunctional radioactive microspheres ( 90 Y-PDA-BP+Pinepollen).

[0082] Example 2

[0083] A multifunctional radioactive microsphere integrating diagnosis and treatment ( 177 The preparation method of Lu-labeled microspheres comprises the following steps:

[0084] (1) Weigh 0.5 g of treated fir pollen particles, disperse them in 100 mL of pure water, and stir for 10 min until uniformly suspended to obtain a fir pollen solution;

[0085] (2) 0.15 g of tannic acid (TA) and 0.07 g of risedronate sodium (BP) were dissolved in 12 mL of borate buffer (pH = 8.0), mixed with the fir pollen solution at a volume ratio of 8:1, and reacted at 40°C for 4 h. After the reaction, the system was centrifuged at 3000 rpm for 15 min, the precipitate was collected and washed three times with PBS buffer to remove unreacted TA and BP. The modified fir pollen microspheres were freeze-dried (pre-frozen at -80°C for 24 h, vacuum <10 Pa), and sealed and stored in a refrigerator at 4°C until use.

[0086] (3) 0.15 mg of the modified fir pollen microspheres obtained in step (2) were mixed with 75 μCi 177 Lu 3+ The solution (concentration of 0.75 mCi / mL) was mixed, shaken at 37°C for 30 min, centrifuged at 3000 rpm for 3 min to remove unbound radionuclides, and washed to obtain177 Lu-labeled microspheres.

[0087] Example 3

[0088] A preparation method of a multifunctional radioactive microsphere (I-labeled microsphere) for diagnosis and treatment integration, comprising the following steps: 64 Cu-labeled microspheres, comprising the following steps:

[0089] (1) 0.3 g of treated spruce pollen particles were weighed and dispersed in 50 mL of pure water, stirred for 15 min to uniformly suspend, to obtain a spruce pollen solution;

[0090] (2) 0.1 g of procyanidin and 0.04 g of DOTA were dissolved in 5 mL of Tris-HCl buffer (pH = 8.8), mixed with the spruce pollen solution at a volume ratio of 10:1, and reacted at room temperature for 8 h after activation. After the reaction, the system was centrifuged at 3000 rpm for 15 min, the precipitate was collected and washed with PBS buffer for 3 times to remove unreacted procyanidin and DOTA, and modified spruce pollen microspheres were formed. After freeze-drying treatment (-80℃ pre-freezing for 24 h, vacuum degree <10 Pa), it was sealed and stored in a 4℃ refrigerator for standby;

[0091] (3) 0.05 mg of modified spruce pollen microspheres obtained in step (2) were mixed with 50 μCi of 64 Cu 2+ solution (concentration of 1 mCi / mL), incubated at 37℃ for 15 min, centrifuged at 3500 rpm for 3 min to remove unbound nuclides, and washed to obtain 64 Cu-labeled microspheres.

[0092] Example 4

[0093] A preparation method of a multifunctional radioactive microsphere (I-labeled microsphere) for diagnosis and treatment integration, comprising the following steps: 131 I-labeled microspheres, comprising the following steps:

[0094] (1) 0.6 g of treated pine pollen particles were weighed and dispersed in 100 mL of pure water, stirred for 30 min to uniformly suspend, to obtain a pine pollen solution;

[0095] (2) 0.1 g DA and 0.05 g alendronate sodium (BP) were dissolved in 10 mL pure water to obtain a DA-BP mixed solution; 50 mL of the pine pollen solution obtained in step (1) was mixed with 10 mL of the mixed solution, the pH was adjusted to 8.5, and the mixture was reacted at 30°C for 5 h. After the reaction, the system was centrifuged at 3000 rpm for 15 min, the precipitate was collected and washed three times with PBS buffer to remove unreacted DA and BP, and pine pollen microspheres modified with a PDA-BP composite coating were obtained. The microspheres were freeze-dried (pre-frozen at -80°C for 24 h, vacuum <10 Pa), and sealed and stored in a refrigerator at 4°C for later use;

[0096] (3) 0.1 mg of the PDA-BP composite coating modified pine pollen microspheres obtained in step (2) was mixed with 100 μCi Na 131 I solution (concentration of 0.5 mCi / mL) was mixed and incubated at room temperature for 5 min under shaking. After labeling, the unbound nuclides were removed by centrifugation at 2500 rpm for 5 min. 131 I labeled microspheres.

[0097] Example 5

[0098] A multifunctional radioactive microsphere integrating diagnosis and treatment ( 111 The preparation method of the microspheres (labeled with In) comprises the following steps:

[0099] (1) Weigh 0.35 g of treated pine pollen particles, disperse them in 70 mL of pure water, and stir for 30 min until uniformly suspended to obtain a pine pollen solution;

[0100] (2) 0.1 g DA and 0.03 g DTPA were dissolved in 7 mL ammonia solution (pH = 9.0), mixed with pine pollen solution at a volume ratio of 10:1, and reacted at 30°C for 6 h. After the reaction, the system was centrifuged at 3000 rpm for 15 min, the precipitate was collected and washed three times with PBS buffer to remove unreacted DA and DTPA. DTPA was covalently coupled with PDA through the amino group to obtain PDA-DTPA composite coating modified pine pollen microspheres, which were freeze-dried (pre-frozen at -80°C for 24 h, vacuum degree <10 Pa) and sealed and stored in a refrigerator at 4°C for use.

[0101] (3) 0.12 mg of the PDA-DTPA composite coating modified pine pollen microspheres obtained in step (2) were mixed with 60 μCi 111 In 3+ The solution (concentration is 1 mCi / mL) was mixed and incubated at room temperature for 10 min. After labeling, the unbound nuclides were removed by centrifugation at 3000 rpm for 4 min. 111 In labeled microspheres.

[0102] Test Example 1

[0103] The pine pollen microspheres before and after modification in Example 1 were characterized by scanning electron microscopy (SEM) and optical microscopy (OM); the particle size distribution of the pine pollen microspheres before and after modification in Example 1 was statistically analyzed by means of particle size analysis software (Nano measurer); the surface charge characteristics of the pine pollen microspheres before and after modification in Example 1 were detected by a Zeta potential analyzer; the formation of chemical bonds and intermolecular interactions were verified by Fourier transform infrared spectroscopy (FTIR) analysis.

[0104] Figure 1 SEM image of pine pollen particles in Example 1, Figure 2 SEM image of pine pollen microspheres modified by PDA-BP composite coating in Example 1, Figure 3 OM image of pine pollen particles in Example 1, Figure 4 OM image of pine pollen microspheres modified by PDA-BP composite coating in Example 1. The SEM and OM images show that the surface of the pine pollen particles before modification is smooth, oval, symmetrical on both sides, and has a typical near-hemispherical air sac structure. After modification by PDA-BP, the surface of the pine pollen particles is uniformly covered with a dense PDA-BP composite coating, Figure 4 It was observed that the optical contrast of the modified pine pollen microspheres was significantly enhanced. The particle size analysis software measured the diameter of the modified pine pollen microspheres to be 57-84 μm, which is larger than the diameter of the pine pollen particles before modification, indicating that the PDA-BP composite coating was successfully deposited on the surface of the pine pollen particles, and that it meets the size requirements of radioactive embolization microspheres.

[0105] Figure 5 Zeta potential data graph of Pine pollen and PDA-BP+Pine pollen in Example 1. Zeta potential analysis showed that the surface potential of the pine pollen particles before modification was negative, while the potential after modification by PDA-BP composite coating was significantly increased, confirming that the coating material changed the surface charge characteristics of the microspheres.

[0106] Figure 6 FTIR graph of PDA-BP+Pine pollen, Pine pollen, BP and PDA in Example 1, from Figure 6 It can be seen that after modification of the pine pollen particles by PDA-BP composite coating, the intensity and position of the characteristic functional group absorption peaks changed significantly: at 3420 cm -1 and 1250 cm -1 The characteristic peaks of PDA and BP appeared, while the position, intensity and shape of the original peaks changed. These spectroscopic evidences show that the pine pollen particles and PDA, BP form a composite coating structure through chemical bonding and intermolecular interactions.

[0107] SEM and OM observation confirmed the uniformity of the coating, and the particle size increment and FTIR functional group changes jointly evidenced the successful construction of PDA-BP composite coating. The polarity shift of zeta potential provided a charge matching basis for subsequent radionuclide coordination, while the displacement and enhancement of characteristic peaks in FTIR verified the synergistic interaction mechanism between PDA-BP-pollen at the molecular level, providing a solid physicochemical basis for the functional modification of microspheres.

[0108] Test Example 2

[0109] The ultrasonic response immune stimulation function of the pine pollen particles and the PDA-BP composite coating modified pine pollen microspheres in Example 1 was detected:

[0110] (1) The acoustic response characteristics of the PDA-BP composite coating modified pine pollen microspheres were monitored using an ultrasonic detector, and a pulsed ultrasonic device (20 kHz, 100 W) was used for wall breaking. The specific process is as follows: 1 mg of PDA-BP composite coating modified pine pollen microspheres were suspended in 1 mL of PBS, and after 5 min of ultrasonic treatment, the supernatant was collected by centrifugation, and the contents of crude polysaccharide and protein were determined. The PDA-BP composite coating modified pine pollen microspheres before and after wall breaking were monitored by in vivo ultrasonic imaging using an ultrasonic imaging device.

[0111] (2) Take mouse bone marrow-derived dendritic cells (DC), inoculate 1×10 6 cells / mL in a 24-well plate, and add the following treatment group samples:

[0112] Control group: PBS;

[0113] Pine pollen particle group: 30 μg / mL pine pollen particles;

[0114] Pine pollen particle combined with ultrasonic group (Pine pollen-broken): 30 μg / mL pine pollen particles + ultrasonic treatment (20 kHz, 100 W, 5 min);

[0115] PDA-BP composite coating modified pine pollen microsphere group: 30 μg / mL PDA-BP composite coating modified pine pollen microspheres;

[0116] PDA-BP composite coating modified pine pollen microsphere combined with ultrasonic group (PDA-BP+Pine pollen-broken): 30 μg / mL PDA-BP composite coating modified pine pollen microspheres + ultrasonic treatment (20 kHz, 100 W, 5 min);

[0117] After 24h incubation, flow cytometry was used to detect the promoting effect of each treatment group on the maturity of DC cells and statistical analysis was performed.

[0118] (3) The cell culture supernatant of each treatment group was collected, and the secretion amount of pro-inflammatory cytokines (such as INF-γ, TNF-α and IL-6) was detected by enzyme-linked immunosorbent assay (ELISA).

[0119] The detection results are as follows:

[0120] Figure 7 And Figure 8 The ultrasonic imaging analysis of PDA-BP composite coating modified pine pollen microspheres showed that the PDA-BP composite coating modified pine pollen microspheres showed clear dot-like high echo signals before and after the wall was broken.

[0121] After ultrasonic treatment, Figure 9 The content data graph of crude polysaccharide (Crude Polysaccharide) and protein (Protein) released by PDA-BP composite coating modified pine pollen microspheres after the wall was broken is shown in Figure 9 From the data, it can be seen that the PDA-BP composite coating modified pine pollen microspheres released 3.3mg / g of crude polysaccharide and 0.74mg / g of protein after the wall was broken, confirming that ultrasonic triggering can effectively release endogenous bioactive ingredients.

[0122] The flow cytometry detection and statistical results of whether the substances released by ultrasonic PDA-BP composite coating modified pine pollen microspheres can promote the maturation of DC cells are shown in Figure 10 The graph of flow cytometry detection of the expression level of CD80 and CD86 on DC cells in each treatment group in Test Example 2 is shown in Figure 10 Compared with other treatment groups, the promoting effect of the combined ultrasonic group on the maturation of DC cells was the most significant (P<0.001).

[0123] The positive expression rate of the co-stimulatory molecule CD80 on the surface of DC cells in the pine pollen particle combined ultrasonic group was 51.4%, which was significantly higher than that of the control group (24.2%) and the pine pollen particle group (44.7%); the positive expression rate of the co-stimulatory molecule CD86 on the surface of DC cells in the PDA-BP composite coating modified pine pollen microsphere combined ultrasonic group was 58.2%, which was significantly higher than that of the control group (24.2%) and the PDA-BP composite coating modified pine pollen microsphere group (35.3%). The above data show that the bioactive ingredients released by ultrasonic triggering can effectively activate the antigen presentation function of DC cells.

[0124] The ELISA detection results of whether the substances released by ultrasonic PDA-BP composite coating modified pine pollen microspheres can promote the secretion of cytokines are shown in Figure 11 Figure 11 ​Figure 2 shows the data graph of the secretion amount of proinflammatory cytokines (INF-γ, TNF-α and IL-6) in each treatment group in Test Example 2 detected by ELISA method, 90 Y-PDA-BP+Pine pollen microspheres combined with ultrasound can significantly induce the secretion of proinflammatory cytokines, including INF-γ, TNF-α and IL-6, suggesting 90 Y-PDA-BP+Pine pollen microspheres can effectively initiate anti-tumor immune response under the mediation of ultrasound.

[0125] Test Example 3

[0126] The radioactivity labeling efficiency and stability of pine pollen microspheres modified by pine pollen particles and PDA-BP composite coating in Example 1 were detected:

[0127] (1) In vitro study: co-incubate the radionuclide 90 Y with pine pollen particles or pine pollen microspheres modified by PDA-BP composite coating at room temperature for 5 min to obtain 90 Y-labeled pine pollen particles or pine pollen microspheres modified by PDA-BP composite coating; measure the radioactivity counts in the supernatant and precipitate using a gamma counter, and calculate the radioactivity labeling efficiency of 90 Y-labeled pine pollen particles or pine pollen microspheres modified by PDA-BP composite coating;

[0128] Place the 90 Y-labeled pine pollen microspheres modified by PDA-BP composite coating in a 10% fetal bovine serum solution and incubate at 37°C, and detect the radioactivity labeling efficiency by centrifugation-counting method to characterize the labeling stability.

[0129] (2) Establish a 4T1 breast cancer-bearing BALB / c mouse model, and subcutaneously inoculate the mice with 4T1 breast cancer cells (1×10 6 cells per mouse) when the tumor volume reaches 100mm 3 The mice were randomly divided into 2 groups:

[0130] 90 Y-PDA-BP+Pine pollen group: intratumorally inject 90 Y-PDA-BP+Pine pollen microspheres (50μCi per mouse);

[0131] Combined ultrasound group ( 90 Y-PDA-BP+Pine pollen-broken): intratumorally inject 90 Y-PDA-BP+Pine pollen microspheres (50μCi per mouse) + ultrasound treatment (20kHz, 100W, 5min).

[0132] Two mice were selected from each group and tested at 0 h, 12 h, 2 days, 4 days, and 6 days after injection using the following method:

[0133] Ultrasound imaging: Use a high-frequency ultrasound device to monitor the acoustic signals of microspheres in the tumor. In vivo imaging: Use an imaging system to observe the distribution of radioactivity in the tumor and major organs and calculate radioactive decay. Biodistribution measurement: After sacrifice, the tumor, heart, liver, spleen, lungs, kidneys and other organs are collected and the radioactivity counts are measured using a gamma counter. The percentage of injected dose per gram of tissue (%ID / g) and tumor retention rate are calculated.

[0134] The test results are as follows:

[0135] from Figure 12 As can be seen from the radiolabeling efficiency data graph, the radiolabeling efficiency of pine pollen microspheres modified with PDA-BP composite coating reaches 95% within 1 minute, and the labeling rate can reach 100% after 5 minutes, which is significantly higher than that of pine pollen particles. Figure 13 As can be seen from the results of the labeling stability test, 90 After incubation of Y-labeled PDA-BP composite coating modified pine pollen microspheres in 10% fetal bovine serum solution for 72 h, the labeling stability remained at 100%, confirming its excellent nuclide coordination stability.

[0136] Intratumoral injection in mice 90 Ultrasound imaging of Y-PDA-BP+Pine pollen microspheres Figure 14 As shown, the tumor 90 Y-PDA-BP+Pine pollen microspheres showed clear high echo signals.

[0137] Figure 15 for 90 Y-PDA-BP+Pine pollen group and 90 In vivo imaging of mice in the Y-PDA-BP+Pine pollen-broken group, tumor radioactivity intensity data, and tumor radionuclide percentage data. Figure 16 for 90 %ID / g data of various organs of tumor-bearing mice in the Y-PDA-BP+Pinepollen-broken group, from Figure 15 and Figure 16 It can be seen that the radioactivity concentration in the tumor site is significant. Except for the tumor, the radioactivity background values ​​of other organs are all below the detection threshold. 90The retention rate of Y-PDA-BP+Pine pollen microspheres is more than 90%, which is significantly higher than that of normal tissues, proving that the microspheres have long-term tumor targeting retention capacity and provide sustained dose support for local radiotherapy.

[0138] Test Example 4

[0139] The Y-PDA-BP+Pine pollen microspheres prepared in Test Example 1 90 The inhibitory effect of Y-PDA-BP+Pine pollen microspheres on primary tumors was tested by the following method:

[0140] A 4T1 breast cancer single tumor-bearing BALB / c mouse model was established, and the mice were subcutaneously inoculated with 4T1 breast cancer cells (1×10 6 cells / mouse) when the tumor volume reached 100mm 3 The mice were randomly divided into 4 groups (5 mice per group) when the tumor volume reached 100mm

[0141] Control group: intratumoral injection of PBS;

[0142] Pine pollen particle group: intratumoral injection of unlabeled pine pollen particles (50 μL);

[0143] 90 Y-PDA-BP+Pine pollen group: intratumoral injection of 90 Y-PDA-BP+Pine pollen microspheres (50 μCi / mouse);

[0144] Combined ultrasound group ( 90 Y-PDA-BP+Pine pollen-broken): intratumoral injection of 90 Y-PDA-BP+Pine pollen microspheres (50 μCi / mouse) and ultrasound treatment (20 kHz, 100 W, 5 min) was applied on the 3rd day after injection.

[0145] During the treatment, the body weight and tumor volume of the mice were recorded, and the survival status was observed. On the 7th day after injection, 1 mouse was randomly selected from each group and sacrificed, and the tumor and major organs (heart, liver, spleen, lung, kidney) were collected to make histopathological sections for hematoxylin-eosin (H&E) staining and terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) immunohistochemical staining for apoptosis detection.

[0146] The cured mice (tumor completely regressed for more than 20 days) after treatment were re-inoculated with 4T1 breast cancer cells (1×10 6 cells / mouse) after two months, and the tumor growth and immune cell changes were monitored simultaneously. Normal mice were inoculated with 4T1 breast cancer cells (1×10 6weight, tumor volume and survival of the mice after the second infection; the T cell subgroups of the normal mice and the mice after the second infection were analyzed by flow cytometry.

[0147] The test results are as follows:

[0148] Figure 17 The test results show that the mice in the combined ultrasound group have no significant fluctuations in weight, the tumor volume is almost completely eliminated within 20 days, and the survival rate reaches 80% after 30 days; the mice in the non-ultrasound group have a significant increase in tumor volume, and all the mice die, with a survival rate of 0%. 90 The tumor volume of the mice in the Y-PDA-BP+Pine pollen group does not show obvious regression, and the survival rate reaches 60% after 30 days; the tumor volume of the mice in the PBS group and the Pine pollen group shows exponential growth, and all the mice die, with a survival rate of 0%.

[0149] Figure 18 The test results show that the mice in the combined ultrasound group have no significant fluctuations in weight, the tumor volume is almost completely eliminated within 20 days, and the survival rate reaches 80% after 30 days; the mice in the non-ultrasound group have a significant increase in tumor volume, and all the mice die, with a survival rate of 0%. Figure 18 It can be seen from the above that, 90 The tumor tissue of the mice in the Y-PDA-BP+Pine pollen-broken group shows large areas of necrotic foci, and the normal tissue structure is significantly damaged, while the other groups only show slight damage, indicating that 90 The Y-PDA-BP+Pine pollen microspheres combined with ultrasound can efficiently induce programmed death of tumor cells.

[0150] The other organs of the mice in each group were subjected to H&E staining and biological safety evaluation, as shown in the following table: Figure 19 As shown in the table, no radioactive damage or immune-related inflammatory cell infiltration was observed in the H&E stained sections of the main organs (heart, liver, spleen, lung, kidney), and the blood biochemical indicators were within the normal reference range, confirming that the combined treatment regimen has good biological safety.

[0151] Figure 20 The test results of the flow cytometry test of the T cells of the normal mice and the mice after the second infection in Test Example 4 are shown in the following figures and tables: the flow cytometry shows that the number of memory T cells in the mice after the second infection of 4T1 cells is significantly increased compared with the normal mice, confirming that the treatment regimen can induce long-term anti-tumor immune memory; no tumor formation was observed in the mice after the second infection of 4T1 cells within 90 days, and the survival rate reached 100% within 100 days.

[0152] Test Example 5

[0153] Test Example 1 prepared 90 The inhibitory effect of Y-PDA-BP+Pine pollen microspheres synergistic immunotherapy on metastatic tumors was tested using the following method:

[0154] A BALB / c mouse model with bilateral CT26 colon cancer was established. CT26 colon cancer cells were inoculated into the bilateral axilla of the mice (0.5×10 6 cells / head, right axilla 1×10 6 cells / cell), and wait until the right tumor volume reaches 100 mm 3 The mice were randomly divided into 5 groups (5 mice in each group):

[0155] Control group: PBS was injected into the right tumor;

[0156] Immunotherapy group (a-PD-L1): PBS was injected into the right tumor, and a-PD-L1 (35 μg / time) was injected intravenously on days 4, 6, and 8;

[0157] Combined treatment group A ( 90 Y-PDA-BP+Pine pollen+a-PD-L1): Intratumoral injection on the right side 90 Y-PDA-BP + Pine pollen microspheres (50 μCi / mouse) + intravenous injection of a-PD-L1;

[0158] Combined treatment group B ( 90 Y-PDA-BP+Pine pollen-broken): right intratumoral injection 90 Y-PDA-BP + Pinepollen microspheres (50 μCi / mouse) + ultrasonic treatment (20 kHz, 100 W, 5 min) on the 3rd day after injection;

[0159] Combined treatment group C ( 90 Y-PDA-BP+Pine pollen+a-PD-L1-broken): Intratumoral injection on the right side 90 Y-PDA-BP + Pine pollen microspheres (50 μCi / mouse) + ultrasonic treatment (20 kHz, 100 W, 5 min) on the 3rd day after injection + intravenous injection of a-PD-L1 (35 μg / time) on the 4th, 6th and 8th days.

[0160] During the treatment, the weight and bilateral tumor volume of the mice were recorded, and the survival status was observed. On the 10th day after injection, one mouse was randomly selected from each group and sacrificed. The bilateral tumors and major organs (heart, liver, spleen, lung, and kidney) were collected, and histopathological sections were prepared for H&E staining. CD8 +The expression levels of T cells and Granzyme B (Gra B).

[0161] The test results are as follows:

[0162] Figure 21 The survival rate data of mice in each group are shown in the following graphs, Figure 22 The right tumor volume data of mice in each group are shown in the following graphs, Figure 23 The left tumor volume data of mice in each group are shown in the following graphs. As shown in Figure 21 , Figure 22 and Figure 23 , 90 After intratumoral treatment of the right tumor, the left untreated metastatic tumor growth of the mice in the Y-PDA-BP+Pine pollen+a-PD-L1-broken group was also completely inhibited, and both tumors gradually regressed, with a 100% survival rate of the mice within 40 days, compared with the rapid growth of the left tumor in other groups.

[0163] Figure 24 The CD8 + T and Granzyme B immunostained H&E staining images and statistical graphs of the right tumors of mice in each group are shown in the following graphs, Figure 25 The CD8 + T and Granzyme B immunostained H&E staining images and statistical graphs of the left tumors of mice in each group are shown in the following graphs, Figure 24 and Figure 25 From the immunofluorescence double-stained (CD8 + T / Gra B) images, it can be seen that in the combined treatment group C, both tumors had large areas of necrotic lesions, and the tumor vascular structure was significantly damaged; activated cytotoxic T cells diffusely infiltrated the tumor microenvironment, and the number of CD8 + T cells and Gra B cells was much higher than that in other groups, indicating that radiotherapy-immunotherapy combination can effectively induce systemic anti-tumor immune response.

[0164] The other organs of the mice in each group were subjected to H&E staining and biological safety evaluation, as shown in Figure 26 , the H&E staining sections of the main organs (heart, liver, spleen, lung, kidney) showed no radioactive damage or immune-related inflammatory cell infiltration, and the blood biochemical indicators were within the normal reference range, confirming that the combined treatment regimen had good biological safety.

[0165] In summary, the present application constructs a diagnosis and treatment integrated microsphere platform with high radionuclide loading capacity (labeling efficiency up to 100%) by surface engineering modification of natural pine pollen with PDA-BP composite coating. Based on the high acoustic impedance characteristics and radionuclide labeling function of pollen sac structure, the platform realizes the multi-modal diagnosis and treatment integration of ultrasound imaging and radionuclide imaging, and at the same time releases endogenous immune adjuvant through ultrasound triggering, combined with immune checkpoint inhibitors to significantly induce anti-tumor abscopal effect, realizing complete inhibition of primary tumor and systemic clearance of metastatic lesions in 4T1 breast cancer and CT26 colon cancer tumor-bearing mouse models, and no obvious toxic damage to major organs. This multifunctional microsphere system of "radiotherapy-imaging-immunity" trinity provides an innovative technical path for precise diagnosis and treatment and immune combination therapy of malignant tumors, and shows good clinical transformation application potential.

[0166] Obviously, the above embodiments of the present application are only examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. Those skilled in the art should understand that on the basis of the above description, other different forms of changes or variations can also be made. Here, it is not necessary and also impossible to exhaust all the embodiments. Any modification, equivalent replacement and improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the claims of the present application.

Claims

1. A method for preparing multifunctional radioactive microspheres for integrated diagnosis and treatment, characterized in that: The following steps are involved: (1) dispersing pollen in water to obtain a pollen solution; wherein the pollen has an air sac structure; (2) dissolving a polyphenol compound and a functional ligand in water to obtain a mixed solution; mixing the pollen solution obtained in step (1) with the mixed solution, adjusting the pH to 8.0-9.0, and reacting to obtain modified pollen microspheres; the mass ratio of the polyphenol compound in the mixed solution to the pollen in the pollen solution is 1:(1-10); the functional ligand is selected from one or more of bisphosphonate compounds, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, 1,4,7-triazacyclononane-1,4,7-triacetic acid, and diethylenetriaminepentaacetic acid; (3) mixing the modified pollen microspheres obtained in step (2) with a radionuclide solution, and obtaining the multifunctional radioactive microspheres for integrated diagnosis and treatment after labeling; The radionuclide solution contains radionuclides selected from 90 Y. 125 I. 131 I. 111 In, 67 Ga, 177 Lu and 64 One or more of Cu.

2. The preparation method according to claim 1, characterized in that In step (1), the pollen is selected from one or more of pine pollen, fir pollen and spruce pollen; in step (2), the polyphenol compound is selected from one or more of dopamine and its derivatives, tannic acid, gallic acid, epigallocatechin gallate, proanthocyanidins and catechin and its derivatives.

3. A multifunctional radioactive microsphere integrated with diagnosis and treatment prepared by the preparation method according to claim 1 or 2.

4. Use of the multifunctional radioactive microspheres integrated with diagnosis and treatment according to claim 3 in ultrasound imaging and / or radionuclide imaging.

5. Use of the multifunctional radioactive microspheres integrated with diagnosis and treatment according to claim 3 in the preparation of brachytherapy drugs.

6. The use according to claim 5, characterized in that During application, the multifunctional radioactive microspheres integrating diagnosis and treatment are ultrasonically treated.

7. Use of the multifunctional radioactive microspheres integrating diagnosis and treatment according to claim 3 in the preparation of combined immunotherapy drugs.

8. The use according to claim 7, characterized in that During use, the multifunctional radioactive microspheres integrating diagnosis and treatment are ultrasonically treated; the combined immunotherapy drug promotes the production of proinflammatory cytokines, wherein the proinflammatory cytokines are selected from one or more of interferon-γ, tumor necrosis factor-α and interleukin-6.

9. The use according to claim 7 or 8, characterized in that The immunotherapy combination drug also includes anti-programmed cell death-ligand 1 antibody.

10. Use of the multifunctional radioactive microspheres integrated with diagnosis and treatment according to claim 3 in the preparation of tumor therapeutic drugs.

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