Magnetocaloric-metal immune embolism microsphere as well as preparation method and application thereof

Abstract

The invention discloses a magnetocaloric-metal immune embolism microsphere as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. Through component and structure optimization, the Zn-Fe3O4Co-Fe3O4 core-shell nanocube (ZnCo-Fe3O4CSNCs) with enhanced magnetocaloric performance is obtained, and the saturation magnetization (Ms) and the coercive force (Hc) of the Zn-Fe3O4Co-Fe3O4 core-shell nanocube (ZnCo-Fe3O4CSNCs) are remarkably improved. The nanocubes are further assembled into size-adjustable magnetic microspheres through a chip micro-fluidic technology, and embolism and magnetic thermal treatment functions are integrated. Under the action of an alternating magnetic field, ZnCo-Fe3O4MSs shows temperature-dependent ion release and local thermal therapy, and meanwhile, up-regulation of heat shock protein is inhibited through metabolic interference. The pyroptosis of cancer cells is effectively triggered, and then immune response is activated, so that the effect of magnetic metal immunotherapy is enhanced.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a magnetothermal-metal immunoembolization microsphere, its preparation method, and its application. Background Technology

[0002] Liver cancer is one of the most common malignant tumors in clinical practice. In recent years, with the continuous development of medical imaging equipment and technology, transarterial embolization (TAE) has been widely used in clinical practice and has achieved good clinical results. However, transarterial embolization treatment still faces the challenge of postoperative recurrence and metastasis.

[0003] Currently, materials used for carotid artery embolization mainly include iodized oil and drug-loaded microspheres. Iodized oil embolization is unstable, easily washed away by blood flow, leading to tumor recanalization. Furthermore, its combination with chemotherapy drugs is a simple mixture with a rapid release rate, lacking sustained release, which may reduce local drug concentration and increase systemic side effects. In addition, overuse may cause ectopic embolism (such as pulmonary embolism) or liver damage, and its high density may interfere with subsequent imaging assessments. Drug-loaded microspheres have complex preparation processes and are significantly more expensive than iodized oil, limiting their clinical application. They also have low loading rates for some drugs (such as oxaliplatin) and short release cycles, leading to limitations such as drug tolerance. Therefore, the development of novel multifunctional composite microspheres is a future direction for optimization.

[0004] Magnetothermia (MHT), first proposed by Gilchrist et al. in the 1960s, utilizes a high-frequency alternating magnetic field (AMF) to provide focal hyperthermia through Neel-Brownian relaxation, aiming to reduce or eradicate tumor activity. MHT relies on precise temperature control through the adjustment of magnetic field strength and magnetic medium concentration, inducing irreversible necrosis of tumor tissue without limitations on penetration depth. With advancements in materials science, nanomagnetic particles (NMPs) have been widely applied in MHT research. Currently, the EU and the US have approved NMPs for hyperthermia treatment of glioblastoma and prostate cancer, demonstrating good clinical efficacy.

[0005] Traditional tumor-targeting therapy (MHT) primarily involves introducing targeted magnetic nanomaterials into tumors via intravenous or direct intratumoral injection, followed by the application of an alternating magnetic field to generate heat for tumor treatment. However, insufficient accumulation of nanoparticles at the tumor site and low medium concentration limit their thermal effects. Furthermore, the safety of nano-formulations potentially entering the intravenous system and inducing heating under the influence of AMF requires further evaluation. Summary of the Invention

[0006] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0007] Here, we developed a novel magnetothermal-metal immunoembolization microsphere to enhance the efficacy of carotid artery embolization. Through compositional and structural optimization, we obtained Zn-Fe3O4@Co-Fe3O4 core-shell nanocubes (ZnCo-Fe3O4 CSNCs) with enhanced magnetothermal properties, exhibiting significantly improved saturation magnetization (Ms) and coercivity (Hc). These nanocubes were further assembled into tunably sized magnetic microspheres (ZnCo-Fe3O4 MSs) using chip microfluidics, integrating embolization and magnetothermal therapy functions. Under an alternating magnetic field (AMF), ZnCo-Fe3O4 MSs exhibited temperature-dependent ion release and local hyperthermia, while simultaneously inhibiting the upregulation of heat shock proteins (HSPs) through metabolic interference. This carefully designed therapeutic cascade effectively triggered pyroptosis in cancer cells, subsequently activating an immune response, thereby enhancing the efficacy of magnetic metal immunotherapy. Notably, ZnCo-Fe3O4 MSs exhibited a remarkable ability to modulate the immunosuppressive tumor microenvironment. The strategic combination of these microspheres with immune checkpoint blockade (ICB) therapy triggered comprehensive systemic immune activation, significantly improving therapeutic efficacy and effectively inhibiting the progression of metastatic tumors, thus greatly enhancing the effectiveness of arterial embolization therapy. Overall, this study opens new avenues for exploring the application of magnetic metal immunotherapy in cancer treatment and is applicable to other highly vascularized solid tumors, demonstrating promising clinical application prospects.

[0008] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0009] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing magnetothermal-metal immunoembolization microspheres.

[0010] To solve the above-mentioned technical problems, the present invention provides the following technical solutions, including:

[0011] Iron acetylacetonate and zinc acetylacetonate were mixed evenly, and then diphenyl ether and oleic acid were added. The mixture was heated under a nitrogen atmosphere and cooled to obtain Zn-Fe3O4 nanocrystals.

[0012] After uniformly mixing iron acetylacetonate and cobalt acetylacetonate, diphenyl ether and oleic acid were added, and then Zn-Fe3O4 nanocrystals were injected. The reaction was carried out under nitrogen atmosphere and cooled to obtain water-soluble ZnCo-Fe3O4CSNCs.

[0013] Water-soluble ZnCo-Fe3O4 CSNCs were dispersed in a methacrylamide gelatin-water solution, and a UV photoinitiator was added to form the aqueous phase. The oil phase was mineral oil containing 1-3% emulsifier. The aqueous and oil phases were injected into different channels of a microfluidic device at different flow rates using a micro-injection pump. Microdroplets were formed in the outflow channels and cured by UV crosslinking to obtain ZnCo-Fe3O4 CSNCs magnetothermal microspheres.

[0014] In a preferred embodiment of the preparation method of the magnetothermal-metal immunoembolization microspheres of the present invention, the molar volume ratio of the iron acetylacetonate, zinc acetylacetonate, diphenyl ether, and oleic acid is 0.9:1-2:15-25:2-3; and the molar volume ratio of the iron acetylacetonate, cobalt acetylacetonate, diphenyl ether, oleic acid, and Zn-Fe3O4 nanocrystals is 0.5:5-15:1-2:5.

[0015] In a preferred embodiment of the preparation method of the magnetothermal-metal immunoembolization microspheres of the present invention, the heating reaction is first stirred at 100-150°C for 20-40 min, and then stirred at 250-200°C for 20-40 min.

[0016] In a preferred embodiment of the preparation method of the magnetocaloric-metal immunoembolization microspheres of the present invention, the preparation method of the water-soluble ZnCo-Fe3O4 CSNCs includes,

[0017] After mixing 0.9 mmol of iron acetylacetonate and 1.35 mmol of zinc acetylacetonate evenly, 20 mL of diphenyl ether and 2.4 mL of oleic acid were added. The mixture was heated to 120 °C and stirred for 30 minutes under a nitrogen atmosphere, then heated to 290 °C and held for 30 minutes. After cooling, Zn-Fe3O4 nanocrystals were obtained.

[0018] After mixing 0.5 mmol of iron acetylacetonate and 0.33 mmol of cobalt acetylacetonate evenly, 10 mL of diphenyl ether and 1.2 mL of oleic acid were added, followed by the injection of 5 mL of Zn-Fe3O4 nanocrystals. The mixture was heated to 120 °C and stirred for 30 minutes under a nitrogen atmosphere, then heated to 290 °C and held for 30 minutes. After cooling, ZnCo-Fe3O4 CSNCs were obtained.

[0019] As a preferred embodiment of the preparation method of the magnetothermal-metal immunoembolization microspheres of the present invention, the aqueous phase contains 10 mg / mL of water-soluble ZnCo-Fe3O4 CSNCs, 10 wt.% of methacrylamide gelatin, and 0.5 wt.% of ultraviolet photoinitiator.

[0020] In a preferred embodiment of the preparation method of the magnetocaloric-metal immunoembolization microspheres of the present invention, the ultraviolet photoinitiator includes one or more of phenyl-2,4,6-trimethylbenzoyl lithium phosphite, 2,4,6-trimethylbenzoyl diphenylphosphine oxide, 1-hydroxycyclohexylbenzophenone, and benzophenone.

[0021] In a preferred embodiment of the preparation method of the magnetothermal-metal immunoembolization microspheres of the present invention, the emulsifier includes one or more of Span60, Span80, and lecithin.

[0022] In a preferred embodiment of the preparation method of the magnetothermal-metal immunoembolization microspheres of the present invention, the mineral oil includes isooctyl palmitate.

[0023] In a preferred embodiment of the preparation method of the magnetothermal-metal immunoembolization microspheres of the present invention, the flow rate ratio of the aqueous phase to the oil phase is 1:10 to 50.

[0024] In a preferred embodiment of the preparation method of the magnetothermal-metal immunoembolization microspheres of the present invention, the ultraviolet cross-linking curing is performed with a wavelength of 365 nm and a curing time of 10–60 s.

[0025] Another objective of this invention is to overcome the shortcomings of the prior art and provide a magnetocaloric-metal immunoembolization microsphere. The magnetocaloric-metal immunoembolization microsphere is a Zn-Fe3O4@Co-Fe3O4 core-shell nanocube with enhanced magnetocaloric properties, and has a particle size of 20–200 μm.

[0026] The third objective of this invention is to overcome the shortcomings of the prior art and provide an application of magnetothermal-metal immunoembolization microspheres in killing tumor cells.

[0027] Beneficial effects of this invention:

[0028] This invention utilizes chip microfluidic technology to fabricate tunable-size magnetothermal microspheres (ZnCo-Fe3O4 MSs), integrating embolization and magnetothermal therapy functions. By triggering pyroptosis in cancer cells, it subsequently activates an immune response, thereby enhancing the efficacy of magnetothermal-metal immunotherapy. The strategic combination of these microspheres with immune checkpoint blockade (ICB) therapy induces comprehensive systemic immune activation, significantly improving therapeutic efficacy. Furthermore, the ZnCo-Fe3O4 MSs are simple to prepare, readily available, and exhibit good biocompatibility in vivo. ZnCo-Fe3O4 MSs hold promise for further improving the efficacy of hepatic artery embolization therapy for liver malignancies, demonstrating promising clinical translational potential. Attached Figure Description

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0030] Figure 1 Characterization diagrams of ZnCo-Fe3O4 MSs are shown below; (a) bright-field micrographs of blank gelatin microspheres with four different diameters (20, 50, 100 and 200 μm), (b) bright-field micrographs of ZnCo-Fe3O4 composite nanocrystal supported magnetic microspheres with four different diameters (20, 50, 100 and 200 μm), and (c) histograms of particle size distribution of magnetic microspheres with diameters of 20, 50, 100 and 200 μm.

[0031] Figure 2 To assess the effectiveness and safety of in vitro heating; among them, (a) a schematic diagram of the magnetocaloric effect generated by ZnCo-Fe3O4 MS under the action of alternating magnetic fields (AMFs), and (b) ZnCo-Fe3O4 MSs with different particle sizes under alternating magnetic fields (AMFs, f appl =404kHz, H appl (c) Temperature rise curve under the action of 7.3kA / m, (d) Relative survival rate of H22 cells after different treatments (control group, AMF group, ZnCo-Fe3O4MSs group, ZnCo-Fe3O4MSs+AMF group), (e) Saturation magnetization curve of different microspheres, (f) Coercivity curve of ZnCo-Fe3O4 MSs.

[0032] Figure 3 This study evaluates the efficacy of magnetothermal ablation after intratumoral injection into subcutaneous tumors in mice. (a) Schematic diagram of mouse experimental design and magnetothermal treatment grouping, (b) Infrared thermographic images of mice during magnetothermal treatment, and (c) Temperature change curve of tumor tissue during magnetothermal treatment.

[0033] Figure 4 This study evaluates the efficacy of magnetothermal ablation after interventional embolization for in situ hepatocellular carcinoma in rabbits. (a) Schematic diagram of transcatheter arterial embolization combined with magnetothermal immunotherapy for rabbit hepatocellular carcinoma; (b) representative results before and after treatment. 18 (c) Schematic diagram of transcatheter arterial embolization (TAE) of liver tumors, and (d) Quantitative analysis of changes in tumor standardized uptake value (SUV). Detailed Implementation

[0034] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0035] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0036] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0037] Unless otherwise specified, all raw materials used in this invention are commercially available.

[0038] The microfluidic chip (ZX-LD-200) used in this invention was purchased from Suzhou Zhongxin Qiheng Scientific Instruments Co., Ltd.

[0039] The abbreviations of this invention and their full names are as follows:

[0040] Core-shell structured nanocubes (CSNCs), methacrylamide gelatin (GelMA), phenyl-2,4,6-trimethylbenzoyl lithium phosphite (LAP), blank gelatin microspheres (Gelatin MSs, or GMSs for short), 2,4,6-trimethylbenzoyl diphenylphosphine oxide (TPO), 1-hydroxycyclohexylbenzophenone (Irgacure184), benzophenone (Darocur BP), and ZnCo-Fe3O4 CSNCs magnetothermal microspheres (ZnCo-Fe3O4 MSs).

[0041] Example 1

[0042] This embodiment provides a method for preparing magnetocaloric-metal immunoembolization microspheres, specifically as follows:

[0043] 1) Preparation of Zn-Fe3O4 nanocrystals: 0.9 mmol of iron acetylacetonate and 1.35 mmol of zinc acetylacetonate were mixed thoroughly, and then 20 mL of diphenyl ether and 2.4 mL of oleic acid were added. The mixture was heated to 120 °C under a nitrogen atmosphere and stirred for 30 minutes to remove moisture and oxygen. The mixture was then heated to 290 °C and held at this temperature for 30 minutes. After cooling to room temperature, the mixture was washed three times with cyclohexane and ethanol to obtain Zn-Fe3O4 nanocrystals.

[0044] 2) Preparation of water-soluble ZnCo-Fe3O4 CSNCs: 0.5 mmol of iron acetylacetonate and 0.33 mmol of cobalt acetylacetonate were dispersed in a three-necked flask. 10 mL of diphenyl ether and 1.2 mL of oleic acid were added under magnetic stirring. Then, the previously prepared Zn-Fe3O4 nanocrystals (fixed at 5 mL) were injected into the three-necked flask. The mixture was heated to 120 °C under a nitrogen atmosphere and stirred for 30 minutes to remove moisture and oxygen. The mixture was then heated to 290 °C and held at this temperature for 30 minutes. After cooling to room temperature, the mixture was washed three times with cyclohexane and ethanol to obtain ZnCo-Fe3O4 CSNCs.

[0045] 3) Preparation of ZnCo-Fe3O4 MSs: First, water-soluble ZnCo-Fe3O4 CSNCs with a concentration of 10 mg / mL were dispersed in a solution containing 10% methacrylamide gelatin (GelMA) to form an aqueous phase. Then, 0.5% of the UV photoinitiator phenyl-2,4,6-trimethylbenzoyl lithium phosphite (LAP) was added to this system. The oil phase consisted of isooctyl palmitate containing 2% Span 80. The aqueous and oil phases were injected separately into a 30 μm channel microfluidic chip (ZX-LD-200) at a flow rate of 1:10 using a microinjection pump. Microdroplets formed in the outflow channels and were cross-linked and cured under 365 nm UV light for 30 s, ultimately yielding ZnCo-Fe3O4 CSNCs magnetocaloric microspheres (ZnCo-Fe3O4 MSs) with a particle size of 20 μm.

[0046] Example 2

[0047] The difference between this embodiment and Embodiment 1 is that the microfluidic chip channel in step 3) is adjusted to 50 μm, while the rest of the preparation process is the same as in Embodiment 1, and ZnCo-Fe3O4 MSs with a particle size of 50 μm are obtained.

[0048] Example 3

[0049] The difference between this embodiment and Embodiment 1 is that the microfluidic chip channel in step 3) is adjusted to 100 μm, while the rest of the preparation process is the same as in Embodiment 1, resulting in ZnCo-Fe3O4 MSs with a particle size of 100 μm.

[0050] Example 4

[0051] The difference between this embodiment and Embodiment 1 is that the microfluidic chip channel in step 3) is adjusted to 200 μm, while the rest of the preparation process is the same as in Embodiment 1, resulting in ZnCo-Fe3O4 MSs with a particle size of 200 μm.

[0052] Comparative Example 1

[0053] The difference between this comparative example and Example 1 is that the amount of ZnCo-Fe3O4 CSNCs added in the aqueous phase was adjusted to 0, while the rest of the preparation process was the same as in Example 1, and blank gelatin microspheres (GMSs) were obtained.

[0054] Microsphere characterization

[0055] The morphology of the prepared ZnCo-Fe3O4 MSs was observed and analyzed using an inverted microscope (Olympus IMT-2, Olympus Corporation, Japan) and a field emission scanning electron microscope (S-4700, Hitachi, Japan). The diameter of the microspheres was measured using ImageJ software, and a distribution map was created based on the percentage of each size. The results are as follows: Figure 1 As shown, both the blank gelatin microspheres and ZnCo-Fe3O4 MSs exhibited a regular spherical structure with precisely controllable dimensions. Figure 1 a&b), the particle size distribution histogram shows that it exhibits a narrow normal distribution characteristic ( Figure 1 c).

[0056] Magnetothermal properties and cell experiments

[0057] Microspheres of different sizes were placed in an alternating magnetic field (AMF, f appl =404kHz, H appl =7.3kA / m), the results show that it has a significant heating capacity ( Figure 2 a&b). Subsequently, a comprehensive controlled experiment was conducted to evaluate the cytotoxicity of different treatment groups (control group, AMF-only group, ZnCo-Fe3O4 magnetic microsphere group, and ZnCo-Fe3O4 magnetic microsphere + AMF combination group). H22 cells were typically cultured under suitable conditions, grown in RPMI 1640 medium (Procell, Wuhan Procell Biotechnology Co., Ltd., China) at 37°C in a humidified incubator containing 5% (v / v) CO2, supplemented with 10% (v / v) fetal bovine serum and antibiotics (100 U / mL streptomycin and 100 U / mL penicillin) to promote cell growth and prevent microbial contamination. The cytotoxicity of ZnCo-Fe3O4 MSs was assessed using the CCK-8 assay. Cell relative viability assays showed no statistically significant difference between the ZnCo-Fe3O4 MSs treatment group and the control group or the AMF treatment group, confirming the good biocompatibility of the microspheres and the significant improvement in saturation magnetization (Ms) and coercivity (Hc) of the microspheres. Figure 2 d, Figure 2 e).

[0058] animal experiments

[0059] Evaluation of the efficacy of magnetothermal ablation after intratumoral injection in mice.

[0060] Evaluation of the therapeutic effect of microspheres in H22 tumor-bearing mouse model Figure 3 a) The tumor-bearing mice were randomly divided into four groups (n=5 in each group):

[0061] ① Control group: 100 μL of physiological saline was injected into the tumor, and no magnetic heat treatment was performed.

[0062] ② Simple microsphere group (ZnCo-Fe3O4 MSs group): 100μL of physiological saline suspension containing 20mg / mL ZnCo-Fe3O4MSs was injected into the tumor (no magnetic field was applied after injection).

[0063] ③ Simple magnetic field group (in alternating magnetic field f) appl =404kHz, H appl (Magnetic field intervention treatment was carried out at 7.3 kA / m).

[0064] ④ Magnetothermal Therapy Group (ZnCo-Fe3O4 MSs + AMF Group): Injection was the same as in the simple microsphere group, followed immediately by application of an alternating magnetic field (AMF) for magnetothermal ablation. The tumor area of ​​the mice in the magnetothermal group was placed at the center of the magnetic field coil and treated with magnetothermal therapy for 20 minutes. Real-time infrared thermal imaging was used to monitor the tumor surface temperature, controlling it at 43±1℃ to avoid overheating and damaging surrounding tissues.

[0065] The results are as follows Figure 3 Real-time temperature monitoring showed that the tumor tissue in the combined treatment group remained in a stable high-temperature range below 45°C throughout the treatment process.

[0066] Evaluation of the efficacy of magnetothermal ablation after interventional embolization for in situ hepatocellular carcinoma in rabbits.

[0067] A VX2 orthotopic liver cancer model was established using 4-month-old New Zealand white rabbits (n=4 per group). The rabbits were divided into three groups: control group (PBS arterial perfusion), microsphere embolization group (5 mg / kg), and control group (PBS arterial perfusion). -1 Microsphere suspension arterial perfusion and magnetothermal therapy group (magnetic field applied after microsphere embolization). Under ultrasound guidance, a 20G puncture needle was used to insert a 1mm... 3 Tumor tissue blocks were injected into the left lobe of the liver of each rabbit. Postoperatively, rabbits received intramuscular injections of 400,000 units of sodium penicillin daily for three consecutive days to prevent infection. Tumor volume was monitored postoperatively by CT scan. All treatments were performed via arterial perfusion under digital subtraction angiography (DSA) guidance. Rabbits in each group underwent PET / CT examinations before treatment and 7 days after perfusion. The antitumor effect was assessed by comparing the tumor standardized uptake value (SUV) before and after treatment.

[0068] Magnetothermal therapy (MHT) effectively overcomes the depth limitations of percutaneous ablation techniques by uniformly distributing microspheres within the tumor after arterial embolization. Figure 4a, Figure 4 c). In the specific procedure, ZnCo-Fe3O4 MSs were slowly perfused into the left hepatic artery via a microcatheter, and digital subtraction angiography (DSA) imaging confirmed successful deposition of the microspheres in the tumor tissue. To comprehensively evaluate the treatment effect, [further details are needed]. 18 F-fluorodeoxyglucose positron emission tomography (PET) 18 FDG (F-FDG PET) imaging technology. This technology is based on the characteristic of malignant tumor cells overexpressing glucose transporter proteins, which promotes the specific accumulation of FDG in tumor tissue, thus serving as a reliable diagnostic biomarker. Pre-treatment PET scans showed significant FDG accumulation in liver tumor areas. 18 High intake of F-FDG ( Figure 4 b, Figure 4 d).

[0069] In summary, this invention develops a novel magnetocaloric-metal immunoembolization microsphere to enhance the efficacy of carotid artery embolization. Through composition and structural optimization, Zn-Fe3O4@Co-Fe3O4 core-shell nanocubes (ZnCo-Fe3O4 CSNCs) with enhanced magnetocaloric properties were obtained, exhibiting improved saturation magnetization (Ms) and coercivity (Hc). Figure 2 d, Figure 2 e) Significantly enhanced. These nanocubes were further assembled into tunably sized magnetic microspheres (ZnCo-Fe3O4 MSs) using chip microfluidics, integrating embolization and magnetothermal therapy functions. Under an alternating magnetic field (AMF), ZnCo-Fe3O4 MSs exhibited temperature-dependent ion release and localized hyperthermia, while simultaneously inhibiting the upregulation of heat shock proteins (HSPs) through metabolic interference. This carefully designed therapeutic cascade effectively triggered pyroptosis in cancer cells, subsequently activating the immune response and thus enhancing the efficacy of magnetic metal immunotherapy. Notably, ZnCo-Fe3O4 MSs demonstrated a remarkable ability to modulate the immunosuppressive tumor microenvironment. The strategic combination of these microspheres with immune checkpoint blockade (ICB) therapy triggered comprehensive systemic immune activation, significantly improving therapeutic efficacy and effectively inhibiting the progression of metastatic tumors, thus greatly enhancing the effectiveness of arterial embolization therapy. Overall, this study opens new avenues for exploring the application of magnetic metal immunotherapy in cancer treatment and is applicable to other highly vascularized solid tumors, showing promising clinical application prospects.

[0070] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing magnetocaloric-metal immunoembolization microspheres, characterized in that: include, Iron acetylacetonate and zinc acetylacetonate were mixed evenly, and then diphenyl ether and oleic acid were added. The mixture was heated under a nitrogen atmosphere and cooled to obtain Zn-Fe3O4 nanocrystals. After uniformly mixing iron acetylacetonate and cobalt acetylacetonate, diphenyl ether and oleic acid were added, and then Zn-Fe3O4 nanocrystals were injected. The reaction was carried out under nitrogen atmosphere and cooled to obtain water-soluble ZnCo-Fe3O4CSNCs. Water-soluble ZnCo-Fe3O4 CSNCs were dispersed in a methacrylamide gelatin-water solution, and a UV photoinitiator was added to form the aqueous phase. The oil phase was mineral oil containing 1-3% emulsifier. The aqueous and oil phases were injected into different channels of a microfluidic device at different flow rates using a micro-injection pump. Microdroplets were formed in the outflow channels and cured by UV crosslinking to obtain magnetocaloric microspheres, denoted as ZnCo-Fe3O4 MSs.

2. The method for preparing magnetocaloric-metal immunoembolization microspheres as described in claim 1, characterized in that: The molar volume ratio of the iron acetylacetone salt, zinc acetylacetone salt, diphenyl ether, and oleic acid is 0.9:1-2:15-25:2-3; the molar volume ratio of the iron acetylacetone salt, cobalt acetylacetone salt, diphenyl ether, oleic acid, and Zn-Fe3O4 nanocrystals is 0.5:5-15:1-2:

5.

3. The method for preparing magnetocaloric-metal immunoembolization microspheres as described in claim 1, characterized in that: The heating reaction is first stirred at 100-150°C for 20-40 minutes, and then stirred at 250-200°C for 20-40 minutes.

4. The method for preparing magnetocaloric-metal immunoembolization microspheres as described in claim 1, characterized in that: The aqueous phase contains 10 mg / mL of water-soluble ZnCo-Fe3O4 CSNCs, 10 wt.% of methacrylamide gelatin, and 0.5 wt.% of UV photoinitiator.

5. The method for preparing magnetocaloric-metal immunoembolization microspheres as described in claim 4, characterized in that: The ultraviolet photoinitiator includes one or more of phenyl-2,4,6-trimethylbenzoyl lithium phosphite, 2,4,6-trimethylbenzoyl diphenylphosphine oxide, 1-hydroxycyclohexylbenzophenone, and benzophenone.

6. The method for preparing magnetocaloric-metal immunoembolization microspheres as described in claim 1, characterized in that: The emulsifier includes one or more of Span60, Span80, and lecithin; the mineral oil includes isooctyl palmitate.

7. The method for preparing magnetocaloric-metal immunoembolization microspheres as described in claim 1, characterized in that: The flow rate ratio of the aqueous phase to the oil phase is 1:10 to 50.

8. The method for preparing magnetocaloric-metal immunoembolization microspheres as described in claim 1, characterized in that: The ultraviolet cross-linking curing process involves a wavelength of 365 nm and a curing time of 10–60 s.

9. A magnetocaloric-metal immunoembolization microsphere prepared by any one of the preparation methods described in claims 1 to 8, characterized in that: The magnetocaloric-metal immunoembolization microspheres are Zn-Fe3O4@Co-Fe3O4 core-shell nanocubes with enhanced magnetocaloric properties and a particle size of 20–200 μm.

10. The application of the magnetothermal-metal immunoembolization microspheres as described in claim 9 in killing tumor cells.

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