Organic metal framework ultrasonic contrast agent for activating tumor immunity as well as preparation method and application of organic metal framework ultrasonic contrast agent

Abstract

The invention relates to an organic metal framework ultrasonic contrast agent for activating tumor immunity as well as a preparation method and application of the organic metal framework ultrasonic contrast agent. The organic metal framework ultrasonic contrast agent comprises a zeolite imidazole framework 8, an azo initiator loaded on the zeolite imidazole framework 8 and targeted mucopolysaccharide modified on the surface of the zeolite imidazole framework 8, and the particle size of the organic metal framework ultrasonic contrast agent is 165-201 nm. The invention also provides a preparation method of the ultrasonic contrast agent. According to the organic metal framework ultrasonic contrast agent for activating tumor immunity, the enrichment of the contrast agent at a tumor part is enhanced through specific binding of targeted mucopolysaccharide and tumor high-expression CD44, the azo initiator is decomposed to generate nitrogen and alkyl radicals after ultrasonic irradiation, the nitrogen and the alkyl radicals are changed into micron-sized bubbles to play a role, and the tumor immunity is activated. The compound has good tumor killing effect and ultrasonic imaging effect, can be used for preparing tumor sonodynamic therapy drugs, and realizes integration of tumor diagnosis and treatment.

Description

Technical Field

[0001] The invention relates to an organic metal framework ultrasound contrast agent for activating tumor immunity and a preparation method and application thereof, belonging to the technical field of ultrasound molecular imaging. Background Art

[0002] Biodegradable metal nanomaterials have the advantages of high permeability and retention effect, functional diversity and stimulus-responsive drug controlled release. In addition, their large specific surface area and high porosity also promote the drug loading capacity of organic metal frameworks, which is expected to become an effective treatment strategy for malignant tumors. However, metal ions have a short half-life in blood components and potential adverse reactions. The surface modification of nanomaterials can improve the internalization and targeting ability of metal ions, which is crucial to improving the delivery accuracy of metal ions. Combining targeting molecules helps nanomedicines bind to specific receptors on tumor cell membranes, thereby enhancing their cellular uptake and reducing systemic adverse reactions.

[0003] Contrast-enhanced ultrasonic (CEUS) is widely used in the diagnosis of tumors and inflammation. Compared with two-dimensional ultrasound, ultrasound contrast agents can increase the resolution of ultrasound diagnosis. Currently, the commonly used contrast agents are mostly nano-microbubbles and nano-droplets. These contrast agents contain gas or droplets, and the outer membrane is a lipid or polysaccharide structure. However, due to their poor stability, they are not easy to store and decay quickly.

[0004] Azo initiators are easily decomposed into nitrogen and alkyl free radicals under ultrasound, but their chemical structure is unstable. Zeolite imidazolate framework material-8 (ZIF-8) is a non-toxic organic metal framework constructed by zinc ions and 2-methylimidazole. It has high porosity and specific surface area and is a good carrier for azo initiators. It is structurally stable under neutral conditions (such as in blood), but can be degraded and released under low pH conditions in tumor cells. The hyaluronic acid modified on its surface can target tumor cells that highly express CD44 (hyaluronic acid-specific receptor). At the same time, nitrogen can be used for ultrasound contrast imaging to achieve real-time visual drug targeted release detection. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides an organic metal framework ultrasound contrast agent for activating tumor immunity and its preparation method and application. The contrast agent has excellent imaging ability, high biological safety, and strong tumor targeting ability, ultrasound contrast ability and tumor killing ability.

[0006] Terminology Note:

[0007] Zeolitic Imidazolate Framework-8 (ZIF-8): It is a metal-organic framework material of metal ions and organic ligands. It has a rich pore structure. Under acidic conditions, the structure of ZIF-8 will collapse and release the drugs encapsulated inside, so it is widely used as a pH-responsive drug release carrier material.

[0008] The technical solution of the present invention is as follows:

[0009] An organic metal framework ultrasound contrast agent for activating tumor immunity comprises zeolite imidazole skeleton-8, an azo initiator loaded on the zeolite imidazole skeleton-8, and a targeted mucopolysaccharide modified on the surface of the zeolite imidazole skeleton-8, and the particle size of the organic metal framework is 165-201 nm.

[0010] Preferably, according to the present invention, the azo initiator is one or more combinations of 2,2-azobis(2methylpropionamidine)dihydrochloride (AIBA), 2,2'-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (AIBI) or 2,2'-azabis(2-imidazolin)dihydrochloride (AIPH).

[0011] More preferably, the azo initiator is 2,2′-azabis(2-imidazoline) dihydrochloride (AIPH).

[0012] Preferably according to the present invention, the loading amount of the azo initiator is 75-85%.

[0013] Preferably according to the present invention, the targeted mucopolysaccharide is one or more combinations of chondroitin sulfate, keratan sulfate or hyaluronic acid.

[0014] Further preferably, the targeting mucopolysaccharide is sodium hyaluronate, and the molecular weight of sodium hyaluronate is 8000 to 12000 Da.

[0015] The method for preparing the organic metal framework ultrasound contrast agent for activating tumor immunity comprises the following steps:

[0016] (1) dissolving a zinc source, an organic ligand and an azo initiator in an organic solvent respectively to obtain a zinc source mother liquor, an organic ligand mother liquor and an azo initiator mother liquor;

[0017] (2) mixing the zinc source mother liquor obtained in step (1) and the azo initiator mother liquor evenly to obtain a mixed solution; then dropping the organic ligand mother liquor into the mixed solution, stirring and reacting for 3 to 5 hours in a dark environment after the dropwise addition is completed, then standing for 3 to 5 hours, centrifuging and drying to obtain a zeolite imidazole framework-8 loaded with an azo initiator;

[0018] (3) dispersing the zeolite imidazole framework-8 loaded with azo initiator obtained in step (2) in the targeted mucopolysaccharide solution, stirring and mixing evenly, and obtaining an organic metal framework ultrasound contrast agent that activates tumor immunity after dialysis purification and freeze drying.

[0019] Preferably according to the present invention, in step (1), the zinc source is zinc nitrate, the organic ligand is dimethylimidazole, and the organic solvent is methanol.

[0020] Preferably according to the present invention, in step (1), the concentration of the zinc source mother solution is 15-25 mg / mL, the concentration of the organic ligand mother solution is 175-185 mg / mL, and the concentration of the azo initiator mother solution is 5.5-6.5 mg / mL.

[0021] Preferably according to the present invention, in step (2), the volume ratio of the zinc source mother liquor, the organic ligand mother liquor and the azo initiator mother liquor is 1:1:1.

[0022] Preferably, according to the present invention, in step (2), the stirring reaction temperature is 4° C., the stirring reaction time is 4 hours, and the standing time is 4 hours.

[0023] Preferably according to the present invention, in step (3), the concentration of the targeted mucopolysaccharide solution is 0.5-2 mg / ml.

[0024] Preferably, according to the present invention, in step (3), the mass volume ratio of the zeolite imidazole framework-8 loaded with azo initiator to the targeted mucopolysaccharide solution is (3-5):1, unit: mg / mL.

[0025] The application of the above-mentioned organic metal framework ultrasound contrast agent that activates tumor immunity in the preparation of tumor sonodynamic therapy drugs.

[0026] The experimental steps not described in detail in the present invention are all carried out according to conventional operations in the art.

[0027] Technical features and beneficial effects of the present invention:

[0028] 1. The organic metal framework ultrasound contrast agent for activating tumor immunity provided by the present invention is to load an azo initiator on a zeolite imidazole framework-8, and to modify the targeted mucopolysaccharide on the surface of the zeolite imidazole framework-8, with a particle size of 165 to 201 nm. Among them, the zeolite imidazole framework-8 has a rich pore structure as a metal organic framework carrier, and has a high encapsulation rate. The metal framework structure will collapse and release the internal encapsulated drug in the tumor microenvironment under acidic conditions. The structure is stable in the blood and has high biosafety. And by modifying hyaluronic acid on the surface of ZIF-8, not only the biocompatibility of the ultrasound contrast agent is increased, but also the ability of the ultrasound contrast agent to target tumors is increased, and ultrasound imaging of the tumor site is easy. At the same time, the ultrasound contrast agent is a gas generator using an azo initiator as an ultrasound contrast agent. After reaching the tumor site under the protection of ZIF-8, it decomposes and produces nitrogen under the action of ultrasound. Compared with the nano droplets and nano microbubbles commonly used in the prior art, the stability of the contrast agent can be significantly improved and the retention time in the tumor can be extended.

[0029] 2. The organic metal framework ultrasound contrast agent for activating tumor immunity provided by the present invention firstly specifically binds to the targeted mucopolysaccharide and the highly expressed CD44 in the tumor, thereby enhancing the enrichment of the contrast agent at the tumor site. After ultrasonic irradiation, the azo initiator decomposes to produce nitrogen and alkyl free radicals, which become micron-sized bubbles to exert their effects. It has good tumor killing effect and ultrasonic imaging effect, and can be used to prepare tumor sonodynamic therapy drugs to realize the integrated diagnosis and treatment of tumors.

[0030] 3. The preparation method provided by the present invention has mild conditions, simple process, low requirements on equipment, operation, environment, etc., and is conducive to large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a transmission electron micrograph of an organic metal framework ultrasound contrast agent that activates tumor immunity.

[0032] Figure 2 This is the particle size distribution diagram of organic metal framework ultrasound contrast agents that activate tumor immunity.

[0033] Figure 3 Zeta potential map of organic metal framework ultrasound contrast agents that activate tumor immunity.

[0034] Figure 4 This is an ultrasound contrast image of an organic metal framework ultrasound contrast agent that activates tumor immunity.

[0035] Figure 5 These are ultrasound contrast images of the tumor sites in mice after different treatments.

[0036] Figure 6This is a picture of the distribution of organic metal framework ultrasound contrast agents that activate tumor immunity in tumor cells under different treatments.

[0037] Figure 7 is the apoptosis rate of 4T1 cells in different treatments.

[0038] Figure 8 CD8 infiltrating tumors in different treatments + The ratio of T cells. DETAILED DESCRIPTION

[0039] The technical solution of the present invention is further described below in conjunction with the embodiments and the accompanying drawings, but the protection scope of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the present invention are methods well known to those skilled in the art.

[0040] Materials and reagents involved in the examples are common commercially available products unless otherwise specified; experimental operations involved in the examples are performed according to routine operations in the art unless otherwise specified.

[0041] The breast cancer cell line 4T1 used in this example is available from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd.

[0042] Example 1

[0043] An organic metal framework ultrasound contrast agent for activating tumor immunity comprises zeolite imidazole skeleton-8, 2,2′-azabis(2-imidazoline) dihydrochloride (AIPH) loaded on the zeolite imidazole skeleton-8, and sodium hyaluronate modified on the surface of the zeolite imidazole skeleton-8, wherein the loading amount of AIPH is 80% and the molecular weight of the sodium hyaluronate is 10000Da.

[0044] The method for preparing the organic metal framework ultrasound contrast agent for activating tumor immunity comprises the following steps:

[0045] (1) Dissolving zinc nitrate, dimethylimidazole and AIPH in methanol respectively, stirring and mixing them evenly to obtain a zinc nitrate solution with a concentration of 20 mg / mL, a dimethylimidazole solution with a concentration of 180 mg / mL and an AIPH solution with a concentration of 6 mg / mL;

[0046] (2) 10 mL of zinc nitrate solution and 10 mL of AIPH solution were mixed to obtain a mixed solution; 10 mL of dimethyl imidazole solution was then added dropwise to the mixed solution. After the addition was completed, the mixture was stirred and reacted for 4 hours at 4° C. in a dark environment, and then allowed to stand for 4 hours, centrifuged at 8000 rpm for 20 minutes, and dried to obtain a zeolite imidazole framework-8 loaded with an azo initiator;

[0047] (3) 45 mg of zeolite imidazole framework-8 loaded with azo initiator was dispersed in 10 mL of 1 mg / mL sodium hyaluronate solution and stirred to mix evenly. The mixed solution was then transferred to a dialysis bag (molecular weight cutoff 10000 Da) and dialyzed and purified in deionized water for 48 h. The dialysate was freeze-dried to obtain an organic metal framework ultrasound contrast agent (HA / AIPH@ZIF-8, HAZ) that activates tumor immunity.

[0048] The organic metal framework ultrasound contrast agent for activating tumor immunity prepared in this example was observed using a transmission electron microscope, and the results were as follows: Figure 1 shown.

[0049] Depend on Figure 1 It can be seen that the organic metal framework ultrasound contrast agent for activating tumor immunity prepared in this embodiment was successfully prepared and appeared as a regular dodecahedron under a microscope.

[0050] Example 2

[0051] An organic metal framework ultrasound contrast agent for activating tumor immunity comprises zeolite imidazole skeleton-8, 2,2-azobis(2-methylpropionamidine) dihydrochloride (AIBA) loaded on the zeolite imidazole skeleton-8, and sodium hyaluronate modified on the surface of the zeolite imidazole skeleton-8, wherein the loading amount of AIBA is 75% and the molecular weight of the sodium hyaluronate is 8000Da.

[0052] The method for preparing the organic metal framework ultrasound contrast agent for activating tumor immunity comprises the following steps:

[0053] (1) Dissolving zinc nitrate, dimethylimidazole and AIBA in methanol respectively, stirring and mixing to obtain a zinc nitrate solution with a concentration of 15 mg / mL, a dimethylimidazole solution with a concentration of 175 mg / mL and an AIBA solution with a concentration of 5.5 mg / mL;

[0054] (2) 10 mL of zinc nitrate solution and 10 mL of AIBA solution were mixed to obtain a mixed solution; 10 mL of dimethylimidazole solution was then added dropwise to the mixed solution. After the addition was completed, the mixture was stirred for reaction at 4° C. in a dark environment for 3 h, then allowed to stand for 3 h, centrifuged at 8000 rpm for 20 min, and dried to obtain zeolite imidazole framework-8 loaded with an azo initiator;

[0055] (3) 36 mg of zeolite imidazole framework-8 loaded with azo initiator was dispersed in 10 mL of 0.8 mg / mL sodium hyaluronate solution and stirred to mix evenly. The mixed solution was then transferred to a dialysis bag (molecular weight cutoff 8000 Da) and dialyzed and purified in deionized water for 48 h. The dialysate was freeze-dried to obtain an organic metal framework ultrasound contrast agent that activates tumor immunity.

[0056] Example 3

[0057] An organic metal framework ultrasound contrast agent for activating tumor immunity comprises zeolite imidazole framework-8, 2,2'-azobis[2-(2-imidazoline)propane] dihydrochloride (AIBI) loaded on the zeolite imidazole framework-8, and sodium hyaluronate modified on the surface of the zeolite imidazole framework-8, wherein the loading amount of AIBI is 85% and the molecular weight of the sodium hyaluronate is 12000Da.

[0058] The method for preparing the organic metal framework ultrasound contrast agent for activating tumor immunity comprises the following steps:

[0059] (1) Dissolving zinc nitrate, dimethylimidazole and AIBI in methanol respectively, stirring and mixing to obtain a zinc nitrate solution with a concentration of 25 mg / mL, a dimethylimidazole solution with a concentration of 185 mg / mL and an AIBI solution with a concentration of 6.5 mg / mL;

[0060] (2) 10 mL of zinc nitrate solution and 10 mL of AIBI solution were mixed to obtain a mixed solution; 10 mL of dimethylimidazole solution was then added dropwise to the mixed solution. After the addition was completed, the mixture was stirred and reacted for 5 h at 4 °C in a dark environment, and then allowed to stand for 5 h, centrifuged at 8000 rpm for 20 min, and dried to obtain a zeolite imidazole framework-8 loaded with an azo initiator;

[0061] (3) 43.2 mg of zeolite imidazole framework-8 loaded with azo initiator was dispersed in 12 mL of 0.8 mg / mL sodium hyaluronate solution and stirred to mix evenly. The mixed solution was then transferred to a dialysis bag (molecular weight cutoff 12000 Da) and dialyzed and purified in deionized water for 48 h. The dialysate was freeze-dried to obtain an organic metal framework ultrasound contrast agent that activates tumor immunity.

[0062] Comparative Example 1

[0063] A zeolite imidazolate framework-8 (ZIF-8), the preparation method is as follows:

[0064] A zinc nitrate solution with a concentration of 20 mg / mL and a dimethylimidazole solution with a concentration of 180 mg / mL were prepared according to the method described in Example 1; then an equal volume of dimethylimidazole solution was added dropwise to the zinc nitrate solution. After the addition was completed, the mixture was stirred and reacted for 4 hours at 4°C in the dark, then allowed to stand for 4 hours, centrifuged at 8000 rpm for 20 minutes, and dried to obtain zeolite imidazole framework-8.

[0065] Comparative Example 2

[0066] A zeolite imidazole framework-8 (AIPH@ZIF-8, AZ) loaded with an azo initiator is prepared according to the method described in steps (1) to (2) of Example 1.

[0067] Comparative Example 3

[0068] A zeolite imidazolate framework-8 (HA / ZIF-8, HZ) modified with sodium hyaluronate, the preparation method is as follows:

[0069] According to the method described in Example 1, a zinc nitrate solution with a concentration of 20 mg / mL and a dimethyl imidazole solution with a concentration of 180 mg / mL were prepared; then an equal volume of dimethyl imidazole solution was added dropwise to the zinc nitrate solution, and after the addition was completed, the mixture was stirred and reacted for 4 hours at 4°C in the dark, then allowed to stand for 4 hours, centrifuged at 8000 rpm for 20 minutes, and dried to obtain zeolite imidazole framework-8. Zeolite imidazole framework-8 was dispersed in sodium hyaluronate solution, stirred and mixed evenly, and then the mixed solution was transferred to a dialysis bag (molecular weight cutoff 10000Da) and dialyzed and purified in deionized water for 48 hours, and the dialyzate was freeze-dried to obtain zeolite imidazole ester framework-8 (HA / ZIF-8, HZ) modified with sodium hyaluronate.

[0070] Example 4

[0071] 1. Determination of average particle size, dispersion index and Zeta potential of contrast agents

[0072] After the HA / AIPH@ZIF-8 prepared in Example 1 was diluted with deionized water, the particle size was measured by nano-laser particle size measurement. Figure 2 shown.

[0073] After HA / AIPH@ZIF-8 (HAZ) prepared in Example 1, ZIF8 prepared in Comparative Example 1, AIPH@ZIF-8 (AZ) prepared in Comparative Example 2 and AIPH were diluted to the same concentration with deionized water, their Zeta potentials were measured with a Zeta potential analyzer. The results are as follows: Figure 3 shown.

[0074] Depend on Figure 2 It can be seen that the average particle size of HA / AIPH@ZIF-8 (HAZ) prepared in Example 1 is about 165-201 nm, and the dispersion index is 0.091-0.246.

[0075] Depend on Figure 3 It can be seen that the Zeta potentials of ZIF8, AZ, HAZ and AIPH are -27.7 to -26.5 mV, among which only HA / AIPH@ZIF-8 (HAZ) prepared in Example 1 reaches -27.2 mV.

[0076] 2. Determination of ultrasound imaging capability of contrast agents

[0077] The 9L probe of a clinical diagnostic ultrasound instrument (LOGIQ E9; GE, USA) was used to detect the ultrasound imaging ability of the nano-ultrasound contrast agent. Specifically, the HA / AIPH@ZIF-8 prepared in Example 1 was diluted and placed in a pipette and clamped for ultrasound (US) imaging. The main parameters were: center frequency 9.0 MHz, transmission power 70%, dynamic range 60 dB, 1 MHz, 0.5 W / cm 2 , 2min; PBS buffer was used as the blank group and HA / AIPH@ZIF-8 without ultrasound was used as the control group. Figure 4 shown.

[0078] Depend on Figure 4 It can be seen that compared with the blank group and the control group, the HA / AIPH@ZIF-8 prepared in Example 1 achieved obvious ultrasound enhancement after ultrasound application, which indicates that the contrast agent has sufficient imaging capability.

[0079] Example 3

[0080] The specific steps of verifying the ultrasound contrast capability of the organic metal framework ultrasound contrast agent for activating tumor immunity of the present invention at the tumor site of mice are as follows:

[0081] (1) Two breast cancer cancer cell 4T1 subcutaneous tumor model mice were established. The specific construction method was as follows: 1×10 6 4T1 cells, wait until the tumor volume reaches 60mm 3 (Tumor volume = tumor long diameter * short diameter 2 / 2) to obtain breast cancer cancer cell 4T1 subcutaneous tumor model mice;

[0082] (2) Before injection, the tumor sites of two mice were detected using a 9L probe of a clinical diagnostic ultrasound instrument (LOGIQ E9; GE, USA) (Pre-injection). Then, 200 μL of 0.5 mg / mL HA / AIPH@ZIF-8 prepared in Example 1 and an equal volume of PBS buffer were injected into two mice bearing a 4T1 subcutaneous tumor model of breast cancer cells, respectively, as the experimental group and the control group. After injection, the tumor sites of the two mice were detected (Post-injection). Two hours after injection, the experimental group was subjected to ultrasound cavitation at 1 MHz, 1 W / cm 2 The tumor site was irradiated for 1 min under ultrasound. The tumor site of the mouse was detected immediately after ultrasound (US) (After US), and the tumor site of the mouse was detected 24 hours after ultrasound (After 24h). The specific images are as follows Figure 5 shown.

[0083] Depend on Figure 5It can be seen that compared with the control group and, the HA / AIPH@ZIF-8 prepared in Example 1 achieved obvious ultrasonic enhancement after applying ultrasound.

[0084] Example 4

[0085] The specific steps of verifying the ability of the organic metal framework ultrasound contrast agent for activating tumor immunity of the present invention to target tumor cells are as follows:

[0086] Breast cancer cancer cells 4T1 were cultured at 2×10 5 100 μg / ml of HA / AIPH@ZIF-8 (HAZ) prepared in Example 1 and AIPH@ZIF-8 (AZ) prepared in Comparative Example 2 were added after the cells adhered to the plate, with the final concentration of 40 μg / ml, as Experimental Group 1 and Experimental Group 2. At the same time, an equal volume of PBS buffer was added as the blank group (Control), and Zn was added as the control group. 2+ (final concentration 50mM) was used as the positive control group; each group was incubated for 4 hours, and the unbound contrast agent was washed away with PBS buffer. After incubation with the zinc ion probe N-(6-Methoxy-8-quinolyl)-p-toluenesulfonamide for 1 hour, the unbound probe was washed away with PBS buffer, and the cell targeting ability of each group was observed under a fluorescence microscope. The results are shown in Figure 6 shown.

[0087] Depend on Figure 6 It can be seen that compared with experimental group 2, the control group and the blank group, the targeting binding of HA / AIPH@ZIF-8 to 4T1 cells is significantly stronger.

[0088] Example 5

[0089] The ability of the organic metal framework ultrasound contrast agent for activating tumor immunity combined with ultrasound irradiation to activate anti-tumor immunotherapy was verified.

[0090] 1. The median lethal dose (IC50) of the contrast agent of the present invention was established by using the dose-cell activity curve. 4T1 breast cancer cells were cultured at 4×10 5 100 μg / ml HA / ZIF-8 (HZ) prepared in Comparative Example 3 and HA / AIPH@ZIF-8 (HAZ) prepared in Example 1 were added to the HZ group and the HAZ group, respectively. An equal volume of PBS buffer was added to the blank group (Control). After 4 h of co-incubation, the cells were irradiated with an ultrasonic cavitation instrument at 1 MHz, 0.5 W / cm 2 The cells were irradiated for 1 min at 4 °C, namely US group, HZ+US group and HAZ+US group. The apoptosis of breast cancer cell 4T1 in the above 6 groups was detected by flow cytometry. The results are as follows Figure 7 shown.

[0091] Depend on Figure 7 It can be seen that the cell apoptosis rate of the HAZ+US group reached 52.2%, which was much higher than the apoptosis rates of 13.7% in the HZ+US group, 12.5% ​​in the US group, 8.96% in the HAZ group, and 8.37% in the HZ group. This fully demonstrates that the organic metal framework ultrasound contrast agent for activating tumor immunity can specifically bind to CD44 highly expressed in tumors by targeting mucopolysaccharide hyaluronic acid, thereby enhancing the enrichment of the contrast agent in the tumor site. After ultrasonic irradiation, the azo initiator decomposes to produce nitrogen and alkyl free radicals, which become micron-sized bubbles to exert their effects. It has good tumor killing effect and ultrasonic imaging effect, and can be used to prepare tumor sonodynamic therapy drugs to achieve integrated tumor diagnosis and treatment.

[0092] 2. According to the method described in Example 3, 18 mice with 4T1 subcutaneous tumor model of breast cancer cells were established and divided into 3 groups, with 6 mice in each group. Then, on days 0, 2, 4, 6, 8 and 10, the HA / ZIF-8 (HZ) prepared in Comparative Example 3 and the HA / AIPH@ZIF-8 (HAZ) prepared in Example 1 were injected into the 3 groups of mice, respectively, with a final concentration of 40 μg / ml, which were the HZ group and the HAZ group. At the same time, an equal volume of PBS buffer was added as the blank group (Control); 2 hours after the injection, the 3 groups of mice were divided into two groups, and one group was extracted from each group and ultrasonic cavitation was performed at 1 MHz, 1 W / cm 2 The tumor site was irradiated for 1 min under the same conditions as the US group, HZ+US group, and HAZ+US group. On the 12th day, the tumor was removed, and immune cells were isolated. CD8 + T cell infiltration, the results are as follows Figure 8 Show.

[0093] Depend on Figure 8 It can be seen that CD8 in the HAZ+US group + The infiltration amount of T cells (16.9±2.51%) was 1.83 times that of the HZ+US group (9.3±0.79%), 2.22 times that of the HAZ group (7.6±3.37%), and 3.75 times that of the blank group (4.5±1.83%). This further illustrates that the organic metal framework ultrasound contrast agent for activating tumor immunity of the present invention has a good tumor killing effect and ultrasound imaging effect, and can be used to prepare tumor sonodynamic therapy drugs to achieve integrated tumor diagnosis and treatment.

[0094] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An organic metal framework ultrasound contrast agent for activating tumor immunity, characterized in that: The invention comprises zeolite imidazole skeleton-8, an azo initiator loaded on the zeolite imidazole skeleton-8, and a targeted mucopolysaccharide modified on the surface of the zeolite imidazole skeleton-8, and the particle size thereof is 165-201 nm.

2. The organic metal framework ultrasound contrast agent for activating tumor immunity according to claim 1, characterized in that: The azo initiator is one or more combinations of 2,2-azobis(2-methylpropionamidine) dihydrochloride, 2,2'-azobis[2-(2-imidazoline-2-yl)propane] dihydrochloride or 2,2'-azabis(2-imidazoline) dihydrochloride; More preferably, the azo initiator is 2,2′-azabis(2-imidazoline) dihydrochloride.

3. The organic metal framework ultrasound contrast agent for activating tumor immunity according to claim 1, characterized in that: The loading amount of the azo initiator is 75-85%.

4. The organic metal framework ultrasound contrast agent for activating tumor immunity according to claim 1, characterized in that: The targeted mucopolysaccharide is one or more combinations of chondroitin sulfate, keratan sulfate or hyaluronic acid; Further preferably, the targeting mucopolysaccharide is sodium hyaluronate, and the molecular weight of sodium hyaluronate is 8000 to 12000 Da.

5. The method for preparing the organic metal framework ultrasound contrast agent for activating tumor immunity according to claim 1, characterized in that: The steps include: (1) dissolving a zinc source, an organic ligand and an azo initiator in an organic solvent respectively to obtain a zinc source mother liquor, an organic ligand mother liquor and an azo initiator mother liquor; (2) mixing the zinc source mother liquor obtained in step (1) and the azo initiator mother liquor evenly to obtain a mixed solution; then dropping the organic ligand mother liquor into the mixed solution, stirring and reacting for 3 to 5 hours in a dark environment after the dropwise addition is completed, then standing for 3 to 5 hours, centrifuging and drying to obtain a zeolite imidazole framework-8 loaded with an azo initiator; (3) dispersing the zeolite imidazole framework-8 loaded with azo initiator obtained in step (2) in the targeted mucopolysaccharide solution, stirring and mixing evenly, and obtaining an organic metal framework ultrasound contrast agent that activates tumor immunity after dialysis purification and freeze drying.

6. The preparation method according to claim 5, characterized in that: In step (1), the zinc source is zinc nitrate, the organic ligand is dimethylimidazole, and the organic solvent is methanol; the concentration of the zinc source mother liquor is 15-25 mg / mL, the concentration of the organic ligand mother liquor is 175-185 mg / mL, and the concentration of the azo initiator mother liquor is 5.5-6.5 mg / mL.

7. The preparation method according to claim 5, characterized in that: In step (2), the volume ratio of the zinc source mother liquor, the organic ligand mother liquor and the azo initiator mother liquor is 1:1:

1.

8. The preparation method according to claim 5, characterized in that: In step (2), the stirring reaction temperature is 4° C., the stirring reaction time is 4 hours, and the standing time is 4 hours.

9. The preparation method according to claim 5, characterized in that: In step (3), the concentration of the targeted mucopolysaccharide solution is 0.5-2 mg / ml; the mass volume ratio of the zeolite imidazole framework-8 loaded with azo initiator to the targeted mucopolysaccharide solution is (3-5):1, unit: mg / mL.

10. Use of the organic metal framework ultrasound contrast agent for activating tumor immunity according to claim 1 in the preparation of tumor sonodynamic therapy drugs.

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