Hypoxia regulation type multi-stage embolism microsphere and application thereof to treatment of liver cancer

Abstract

The invention relates to the technical field of hepatocellular carcinoma interventional therapy, in particular to hypoxia regulation type multi-stage embolism microspheres and application of the hypoxia regulation type multi-stage embolism microspheres to hepatocellular carcinoma treatment, the microspheres are mSiO2 / Pt / aPD-1 (at) HAMA composite embolism agents, a multi-stage embolism structure is formed by 100 [mu] m HAMA microspheres and 3 [mu] m mSiO2 microspheres, a Pt NPs catalytic oxygen production module and an aPD-1 immunotherapy module are loaded, and the microspheres are prepared through the microfluidic technology. The hypoxia regulation type multi-stage embolism microsphere can realize targeted embolism of different levels of blood vessels, dynamically regulate hypoxia and cooperate with immunotherapy, and the hypoxia regulation type multi-stage embolism microsphere is applied to liver cancer treatment, realizes full-blood-vessel network coverage, improves the embolism coverage rate, relieves hypoxia to enhance the immunotherapy effect, is high in biological safety and good in clinical compatibility, and provides an efficient solution for liver cancer treatment.

Description

Technical Field

[0001] This invention relates to the field of interventional therapy for hepatocellular carcinoma, specifically to a hypoxia-regulated multi-stage embolization microsphere and its application in the treatment of liver cancer. Background Technology

[0002] Transarterial embolization (TAE) is a major interventional treatment for advanced hepatocellular carcinoma (HCC). It induces ischemic necrosis by blocking tumor blood supply, but its clinical efficacy is limited by two major challenges. Firstly, the vascular system of HCC exhibits significant heterogeneity, comprising multiple branches ranging from millimeter-scale feeding arteries to micrometer-scale capillaries. Traditional monodisperse embolic agents, due to their fixed size, can only block proximal large vessels, leaving distal microcirculation to maintain perfusion through collateral anastomosis, leading to tumor residue and recurrence. Furthermore, unembolized distal capillaries will re-establish blood flow through VEGF-mediated angiogenesis within 3-4 weeks post-procedure, resulting in a recurrence rate as high as 40%-60%. Secondly, embolization exacerbates tumor hypoxia. The combination of baseline hypoxia in HCC and post-embolization hypoxia creates "double hypoxia," causing the hypoxic area of ​​the tumor to expand 2.5-3 times, activating the HIF-1α / PD-L1 axis and increasing CD8+. + The T cell apoptosis rate increases by more than 50%, while hypoxia induces adenosine production, inhibits T cell receptor signaling, and increases the proportion of regulatory T cells (Tregs) to 35%-45%, forming an immunosuppressive microenvironment.

[0003] Currently, there are two main types of solutions to address the above problems, but both have limitations. One type is drug-loaded embolic microspheres (such as...). One approach involves loading chemotherapy drugs onto polyvinyl alcohol microspheres to enhance the killing effect through local release. However, their single size only allows for embolization of proximal vessels, failing to block distal microcirculation (residual perfusion rate >30%). Furthermore, drug release can further exacerbate hypoxia, leading to a 2-3 fold upregulation of PD-L1 expression, thus negating the effects of immunotherapy. Another type is oxygen-carrier composite embolizing agents (such as perfluorocarbon nanoemulsions), which alleviate tumor hypoxia by carrying oxygen through perfluorocarbons. However, their oxygen release relies on physical diffusion, failing to respond to the dynamic needs of the tumor microenvironment, and lacks integration of immune checkpoint inhibitors, thus failing to reverse hypoxia-mediated immunosuppression. These shortcomings stem from the existence of a single-level embolization architecture in the structural design, which cannot adapt to the heterogeneity of HCC vessels. The fragmentation of functional modules leads to poor spatiotemporal synergy. Additionally, the materials suffer from static oxygen release mechanisms, low oxygen supply efficiency, and poor embolization stability due to biological interface mismatch.

[0004] Therefore, in view of the above situation, there is an urgent need to develop a hypoxia-regulated multi-stage embolization microsphere and its application in the treatment of liver cancer, so as to overcome the shortcomings in current practical applications. Summary of the Invention

[0005] The purpose of this invention is to provide a hypoxia-regulated multi-stage embolization microsphere and its application in the treatment of liver cancer, so as to solve the problems mentioned in the background art.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A hypoxia-regulated multi-stage embolization microsphere comprises:

[0008] Outer carrier: large microspheres composed of biodegradable hydrogel, with an average diameter ranging from 50 to 200 μm;

[0009] Embedded functional unit: mesoporous microspheres loaded inside the outer carrier, with an average diameter ranging from 1 to 5 μm;

[0010] The embedded functional unit includes: a catalytic oxygen-generating component: nanoparticles that can catalyze the decomposition of hydrogen peroxide into oxygen in the tumor microenvironment; and an immunotherapy component: antibodies or agonists that block immune checkpoint pathways.

[0011] The outer carrier degrades in an acidic environment, triggering the release of the embedded functional units.

[0012] As a further aspect of the present invention: the outer carrier is a photocrosslinked HAMA microsphere, with a wavelength of 365 nm and an intensity of 10 mW / cm². 2 Cured under ultraviolet light.

[0013] As a further aspect of the present invention: the embedded functional unit is an mSiO2 microsphere with a pore size of 8-10 nm and a BET specific surface area of ​​300-400 m². 2 / g.

[0014] As a further aspect of the present invention: the catalytic oxygen-producing component is Pt NPs with a particle size of 3-5 nm, which is fixed in the pores of mSiO2 by in-situ reduction.

[0015] As a further aspect of the present invention: the immunotherapy component is aPD-1, which is loaded onto mSiO2 channels by electrostatic adsorption and coated with a pH-responsive chitosan coating.

[0016] A method for preparing the hypoxia-regulated multi-stage embolization microspheres described above includes the following steps:

[0017] (1) Synthesis of mSiO2 microspheres: The oil-water two-phase method was adopted, with cyclohexane / CTAB as the oil phase and tetraethyl silicate / ammonia as the aqueous phase, and the reaction was carried out at 60℃ for 12 hours.

[0018] (2) Loading Pt NPs: The mSiO2 microspheres obtained in step (1) were mixed with a platinum source solution and reacted for 2 hours at pH 8.5 and 60℃.

[0019] (3) Loading aPD-1: The aPD-1 solution was mixed with mSiO2 / Pt microspheres and shaken at 4℃ for 12 hours to form an mSiO2 / Pt / aPD-1 complex.

[0020] (4) Encapsulation of HAMA microspheres: The mSiO2 / Pt / aPD-1 composite is dispersed in HAMA prepolymer liquid and formed into droplets by a microfluidic device at a flow rate ratio of water phase: oil phase = 1:5. The droplets are then cured by irradiation with 365nm ultraviolet light for 60 seconds.

[0021] As a further aspect of the present invention: in step (4), the concentration of the HAMA prepolymer is 10% w / v, and the photoinitiator Irgacure 2959 is added.

[0022] The application of the aforementioned hypoxia-regulated multi-stage embolization microspheres in transarterial embolization therapy for hepatocellular carcinoma includes the following steps:

[0023] (1) The microsphere suspension was injected into the hepatic artery via a catheter;

[0024] (2) The outer HAMA microspheres are cross-linked in situ by ultraviolet light irradiation, which embolizes the blood vessels proximal to the tumor.

[0025] (3) In the acidic tumor microenvironment, HAMA degrades and releases embedded mesoporous silica microspheres, which embolize distal capillaries.

[0026] (4) Simultaneously achieve catalytic oxygen production and immune checkpoint blockade, reversing tumor hypoxia and immunosuppressive microenvironment.

[0027] As a further aspect of the present invention: the immune checkpoint blockade is achieved by slow-release of aPD-1 antibody, blocking the PD-1 / PD-L1 pathway to activate CD8. + T cells.

[0028] Compared with the prior art, the beneficial effects of the present invention are:

[0029] This invention has many significant advantages. Structurally, 100μm HAMA microspheres can target and block the third-order branches of the hepatic artery (80-150μm in diameter), and photocrosslinking and curing ensure embolization stability. 3μm mSiO2 microspheres, after being released by HAMA degradation, penetrate into 7-9μm capillaries, achieving full vascular network coverage and improving embolization coverage.

[0030] Functionally, Pt NPs can catalyze the decomposition of H2O2 overexpressed in the tumor microenvironment into O2, thereby increasing the local oxygen partial pressure, reducing hypoxic areas, and downregulating HIF-1α expression. Furthermore, HAMA degrades in the acidic environment of the tumor (pH 6.5), enabling catalytic oxygen production and aPD-1 release to be triggered simultaneously, thus avoiding the passive diffusion defects of traditional oxygen carriers.

[0031] In treatment, aPD-1 achieves sustained release through the mesoporous confinement effect of mSiO2, and combined with Pt NPs oxygen production, it can increase CD8. + The reduction of T cell infiltration, coupled with the decrease in PD-L1 expression due to hypoxia relief, relieved the inhibition of T cell function and significantly reduced tumor volume in a rabbit model.

[0032] In addition, in terms of materials, the degradation products of HAMA are non-toxic hyaluronic acid oligosaccharides. At a concentration of 200 μg / mL, the survival rate of Hepa1-6 cells is >95%, and its microspheres are completely degraded within 1-2 weeks, which can avoid long-term foreign body reactions and match the needs of clinical treatment cycles.

[0033] In terms of clinical translation, photocrosslinked HAMA microspheres can be delivered via standard catheters (with real-time DSA monitoring), achieving liver targeting accuracy >90%, and a single platform can simultaneously perform embolization, oxygen therapy, and immunotherapy, reducing the risk of multiple interventional procedures;

[0034] This invention systematically solves the three core defects of traditional TAE (transcatheter arterial embolization) – incomplete embolization, hypoxia resistance, and immunosuppression – through multi-level embolization structure design, catalytic-immune synergistic mechanism, and innovative biodegradable materials, providing an efficient, safe, and clinically feasible solution for interventional treatment of hepatocellular carcinoma. Attached Figure Description

[0035] Figure 1 This is a scanning electron microscope image of the overall structure of the hypoxia-regulated multi-stage embolization microsphere in an embodiment of the present invention.

[0036] Figure 2 This is a scanning electron microscope image of the surface morphology of a single hypoxia-regulated multi-stage embolization microsphere in an embodiment of the present invention.

[0037] Figure 3 This is a scanning electron microscope image of the overall structure of mesoporous silica microspheres in an embodiment of the present invention.

[0038] Figure 4 This is a magnified scanning electron microscope image of the edge of the mesoporous silica microspheres in an embodiment of the present invention.

[0039] Figure 5 This is a BET specific surface area diagram of the mesoporous silica microspheres in an embodiment of the present invention.

[0040] Figure 6This is a schematic diagram of HAMA degradation and release of mesoporous microspheres in an embodiment of the present invention.

[0041] Figure 7 This is a scanning electron microscope image of the overall structure of the mSiO2 / Pt composite material in an embodiment of the present invention.

[0042] Figure 8 This is a magnified scanning electron microscope image of the edge of the mSiO2 / Pt composite material in an embodiment of the present invention.

[0043] Figure 9 This is a mapping diagram of the distribution of Pt NPs within the mesoporous channels in an embodiment of the present invention.

[0044] Figure 10 The image shows the XRD pattern of the mSiO2 / Pt composite material in an embodiment of the present invention.

[0045] Figure 11 The UV-vis spectrum is used to verify the loading of aPD-1 antibody in this embodiment of the invention.

[0046] Figure 12 This is a load diagram of aPD-1 for verifying the Zeta potential in an embodiment of the present invention.

[0047] Figure 13 This is a graph showing the degradation kinetics of HAMA microspheres in an embodiment of the present invention.

[0048] Figure 14 This is a graph showing the catalytic oxygen production rate in an embodiment of the present invention.

[0049] Figure 15 This is a graph showing dissolved oxygen monitoring data in an embodiment of the present invention.

[0050] Figure 16 This is a graph showing the cytotoxic CCK-8 detection results in an embodiment of the present invention. Detailed Implementation

[0051] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0052] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0053] Please see Figures 1-16This invention provides a hypoxia-regulated multi-stage embolization microsphere and its application in the treatment of hepatocellular carcinoma. Specifically, it relates to the structural design, preparation process, working principle, and application effects of the hypoxia-regulated multi-stage embolization microsphere mSiO2 / Pt / aPD-1@HAMA. The aim is to address the problems of incomplete embolization, hypoxia resistance, and immunosuppression inherent in traditional transarterial embolization (TAE) in the treatment of hepatocellular carcinoma (HCC). The specific details are as follows:

[0054] I. Structural composition and spatial relationship of hypoxia-regulated multi-stage embolism microspheres;

[0055] The hypoxia-regulated multi-level embolization microspheres mSiO2 / Pt / aPD-1@HAMA are composed of a hierarchical embolization module, specifically including an outer carrier and embedded microspheres. The structure and spatial relationship of each part are as follows:

[0056] 1. Outer carrier (HAMA microspheres): These are sub-millimeter-sized spherical particles with a rough surface and an average diameter of approximately 100 μm (e.g., Figure 1-2 (As shown); its material is methacrylated hyaluronic acid (HAMA), prepared using microfluidic technology, and analyzed at a wavelength of 365 nm and an intensity of 10 mW / cm. 2 After being cross-linked by ultraviolet light, it forms a stable three-dimensional network structure; its function is positioned as a primary embolizing agent, which can target and embolize the proximal blood supply artery of the tumor (80-150μm in diameter);

[0057] The outer carrier can also be temperature-sensitive PLGA microspheres (50-200μm), which can be deformed by local heat therapy at 40-42℃ to adapt to different blood vessel diameters.

[0058] 2. Embedded microspheres (mSiO2 / Pt / aPD-1): These are mesoporous silica (mSiO2) microspheres with an average diameter of 3 μm and a pore size of 8.2 nm (e.g., Figure 3-4 As shown), the specific surface area of ​​BET is 386 m². 2 / g (e.g.) Figure 5 (As shown); it is embedded in the pores of HAMA microspheres using microfluidic co-encapsulation technology, with a loading rate of 25±3wt%, and can be released into distal capillaries (e.g., after HAMA degradation) after microfluidic co-encapsulation technology. Figure 6 (as shown); its function is positioned as a secondary embolizing agent, which can both block microvessels with a diameter of 7-9μm and serve as a drug carrier.

[0059] II. Functional modules and their connections;

[0060] The hypoxia-regulated multi-stage embolization microspheres integrate a catalytic oxygen production module and an immunotherapy module. The structural features and loading methods of each module are as follows:

[0061] 1. Catalytic oxygen production module: Platinum nanoparticles (Pt NPs) are loaded into mSiO2 channels via in-situ reduction, with a particle size of 3-5 nm and uniform distribution (e.g., ...). Figure 7-8 As shown); the mapping diagram confirms that it is uniformly loaded within the mSiO2 channels (as shown). Figure 9 As shown), the XRD pattern shows characteristic peaks of the Pt(111) crystal plane (as shown). Figure 10 As shown), the crystal structure is complete and can efficiently catalyze the decomposition of hydrogen peroxide; catalytic oxygen production: Pt NPs catalyze the decomposition of H2O2 overexpressed in the tumor microenvironment into O2 (reaction formula: 2H2O2→2H2O+O2↑). Dissolved oxygen monitoring showed that the oxygen production rate of 200 μg / mL mSiO2 / Pt / aPD-1 under 200 μmol H2O2 conditions reached 4.8±0.3 μmol / min / mg (e.g., ...). Figure 14-15 (as shown);

[0062] 2. Immunotherapy Module: Anti-PD-1 antibody (aPD-1) is loaded into mSiO2 channels via electrostatic adsorption and mesoporous confinement effect; UV-Vis spectroscopy shows a characteristic absorption peak at 259 nm, and FTIR confirms the presence of ν-C=O (1700 cm⁻¹). -1 ) and ν-NH (1560cm -1 ) key (such as Figure 11 (As shown); Zeta potential tests showed that the Zeta potential of mSiO2 / Pt / aPD-1 was +28.5mV, while that of mSiO2 / Pt / aPD-1 dropped to -15.2mV, verifying the successful loading of aPD-1 (as shown). Figure 12 (as shown);

[0063] The immunotherapy module can also be a STING agonist (such as cGAMP), or a co-loaded regimen of aPD-1 and a TGF-β inhibitor (Galunisertib), with the loading regimen being the same as that of aPD-1 loading.

[0064] III. Working principle and working process;

[0065] Hypoxia-regulated multi-stage embolization microspheres achieve liver cancer treatment through three stages: embolization, microsphere release, and functional synergy. The specific process is as follows:

[0066] 1. Embolization stage: mSiO2 / Pt / aPD-1@HAMA suspension is injected into the hepatic artery through a catheter. The outer HAMA microspheres (100μm) preferentially embolize the proximal large vessels. After ultraviolet irradiation, the HAMA microspheres solidify in situ within the target vessel, increasing the surface roughness and forming a stable embolism.

[0067] 2. Microsphere Release Phase: In the acidic tumor microenvironment (pH 6.5), HAMA is gradually degraded through ester bond hydrolysis, with 60.2% degradation in 5 days and 97.7% degradation in 9 days (e.g., Figure 13 As shown in the figure, embedded mSiO2 microspheres are released during the degradation process.

[0068] 3. Functional Coordination Phase:

[0069] Catalytic oxygen production: Pt NPs catalyze the decomposition of overexpressed H2O2 in the tumor microenvironment into O2 (reaction formula: 2H2O2→2H2O+O2↑). Dissolved oxygen monitoring showed that the oxygen production rate of 200 μg / mL mSiO2 / Pt / aPD-1 under 200 μmol H2O2 conditions reached 4.8 ± 0.3 μmol / min / mg (e.g., ...). Figure 14-15 (as shown);

[0070] Immune activation: aPD-1 is released slowly from the mSiO2 pores, blocking the PD-1 / PD-L1 pathway and restoring T cell killing activity.

[0071] IV. Dynamic morphological transformation mechanism;

[0072] The morphology transformation of hypoxia-regulated multi-stage embolization microspheres was triggered by H2O2 (100 μM), and the specific transformation process is as follows:

[0073] Stage 1: Intact HAMA microspheres (100μm) embolize proximal vessels to maintain primary embolism;

[0074] Stage 2: After HAMA degrades, it releases mSiO2 microspheres (3μm), which embolize distal microvessels and prevent collateral circulation reconstruction.

[0075] V. Preparation method;

[0076] The preparation method of hypoxia-regulated multi-stage embolization microspheres Pt / aPD-1 / mSiO2@HAMA includes the following steps:

[0077] 1. Synthesis and Functionalization of mSiO2 Microspheres: An oil-water two-phase method was adopted, using cyclohexane / CTAB as the oil phase and tetraethyl silicate / ammonia as the aqueous phase. Stirring at 60℃ for 12 hours yielded 3μm mesoporous mSiO2 (verified by TEM / SEM, e.g., ...). Figure 3-4 (As shown).

[0078] 2. Pt NPs loading: Pt NPs (3-5nm) are generated in mSiO2 channels through electrostatic adsorption and mesoporous confinement. The process conditions are pH 8.5, 60℃ reaction for 2 hours, followed by centrifugation, washing and drying.

[0079] 3. aPD-1 loading and controlled release: 1 mg / mL aPD-1 solution was mixed with mSiO2 / Pt and shaken at 4 °C for 12 hours to form a Pt / aPD-1 / mSiO2 complex;

[0080] 4. HAMA microsphere encapsulation: Using microfluidic co-encapsulation technology, the mSiO2 / Pt / aPD-1 composite is dispersed in a 10% w / v HAMA prepolymer solution containing the photoinitiator Irgacure2959 (dispersed phase), with mineral oil as the continuous phase. The flow rate ratio (water:oil) is controlled at 1:5 to form droplets, which are then cured by irradiation with 365nm ultraviolet light for 60 seconds.

[0081] In summary, the hypoxia-regulated multi-stage embolization microspheres of this invention and their application in the treatment of liver cancer have the following advantages:

[0082] 1. Multi-stage embolization enhancement: 100μm HAMA microspheres target and block the third-level branches of the hepatic artery (diameter 80-150μm), and photocrosslinking and curing ensure embolization stability; 3μm mSiO2 microspheres are released as HAMA degrades and penetrate into 7-9μm capillaries, significantly improving embolization coverage.

[0083] 2. Dynamic regulation of hypoxia: Pt NPs catalyze the decomposition of H2O2 overexpressed in the tumor microenvironment into O2, increasing local oxygen partial pressure, reducing hypoxic areas and downregulating HIF-1α expression; HAMA degrades in the acidic environment of the tumor (pH 6.5), enabling catalytic oxygen production and aPD-1 release to be triggered synchronously, avoiding the passive diffusion defects of traditional oxygen carriers.

[0084] 3. Synergistic effect of immune response: aPD-1 achieves sustained release through the mesoporous confinement effect of mSiO2, and combined with oxygen production by Pt NPs, it can increase the infiltration of CD8+ T cells; hypoxia relief reduces PD-L1 expression, relieves T cell function inhibition, and can significantly reduce tumor volume in rabbit models.

[0085] 4. Biosafety and Degradability: The degradation products of HAMA are non-toxic hyaluronic acid oligosaccharides. At a concentration of 200 μg / mL, the survival rate of Hepa1-6 cells is >95% (CCK-8 assay). Figure 16 (As shown); HAMA microspheres completely degrade within 1-2 weeks, avoiding long-term foreign body reactions and matching the clinical treatment cycle.

[0086] 5. Clinical translational advantages: Photocrosslinked HAMA microspheres can be delivered via standard catheters (with real-time DSA monitoring), achieving liver targeting accuracy >90%; a single platform can simultaneously perform embolization, oxygen therapy, and immunotherapy, reducing the risk of multiple interventional procedures.

[0087] Therefore, this invention systematically solves the core defects of traditional TAE through multi-level embolization structure design, catalysis-immune synergistic mechanism and biodegradable material innovation, providing an efficient, safe and clinically feasible solution for interventional treatment of hepatocellular carcinoma.

[0088] It should be noted that, in this invention, although the specification describes the embodiments, not every embodiment contains only one independent technical solution. This way of describing the specification is only for clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A hypoxia-regulated multi-stage embolization microsphere, characterized in that, include: Outer carrier: large microspheres composed of biodegradable hydrogel, with an average diameter ranging from 50 to 200 μm; Embedded functional unit: mesoporous microspheres loaded inside the outer carrier, with an average diameter ranging from 1 to 5 μm; The embedded functional unit includes: a catalytic oxygen-generating component: nanoparticles that can catalyze the decomposition of hydrogen peroxide into oxygen in the tumor microenvironment; and an immunotherapy component: antibodies or agonists that block immune checkpoint pathways. The outer carrier degrades in an acidic environment, triggering the release of the embedded functional units.

2. The hypoxia-regulated multi-stage embolization microspheres according to claim 1, characterized in that, The outer carrier is a photocrosslinked HAMA microsphere, with a wavelength of 365 nm and an intensity of 10 mW / cm². 2 Cured under ultraviolet light.

3. The hypoxia-regulated multi-stage embolization microspheres according to claim 1, characterized in that, The embedded functional unit is an mSiO2 microsphere with a pore size of 8-10 nm and a BET specific surface area of ​​300-400 m². 2 / g.

4. The hypoxia-regulated multi-stage embolization microsphere according to claim 1, characterized in that, The catalytic oxygen-producing component is Pt NPs with a particle size of 3-5 nm, which is fixed in the pores of mSiO2 by in-situ reduction.

5. The hypoxia-regulated multi-stage embolization microsphere according to claim 1, characterized in that, The immunotherapy component is aPD-1, which is loaded onto mSiO2 channels by electrostatic adsorption and coated with a pH-responsive chitosan coating.

6. A method for preparing hypoxia-regulated multi-stage embolization microspheres according to any one of claims 1-5, characterized in that, Includes the following steps: (1) Synthesis of mSiO2 microspheres: The oil-water two-phase method was adopted, with cyclohexane / CTAB as the oil phase and tetraethyl silicate / ammonia as the aqueous phase, and the reaction was carried out at 60℃ for 12 hours. (2) Loading Pt NPs: The mSiO2 microspheres obtained in step (1) were mixed with a platinum source solution and reacted for 2 hours at pH 8.5 and 60℃. (3) Loading aPD-1: Mix aPD-1 solution with mSiO2 / Pt microspheres and shake at 4℃ for 12 hours to form mSiO2 / Pt / aPD-1 complex; (4) Encapsulation of HAMA microspheres: The mSiO2 / Pt / aPD-1 composite is dispersed in HAMA prepolymer liquid and formed into droplets by a microfluidic device at a flow rate ratio of water phase: oil phase = 1:

5. The droplets are then cured by irradiation with 365nm ultraviolet light for 60 seconds.

7. The method for preparing hypoxia-regulated multi-stage embolization microspheres according to claim 6, characterized in that, In step (3), the aPD-1 is coated with a chitosan coating after loading, and the coating thickness is 50-100nm.

8. The method for preparing hypoxia-regulated multi-stage embolic microspheres according to claim 6, characterized in that, In step (4), the concentration of the HAMA prepolymer is 10% w / v, and the photoinitiator Irgacure 2959 is added.

9. The application of the hypoxia-regulated multi-stage embolization microspheres according to any one of claims 1-5 in the transarterial embolization treatment of hepatocellular carcinoma, characterized in that, Includes the following steps: (1) The microsphere suspension was injected into the hepatic artery via a catheter; (2) The outer HAMA microspheres are cross-linked in situ by ultraviolet light irradiation, which embolizes the blood vessels proximal to the tumor. (3) In the acidic tumor microenvironment, HAMA degrades and releases embedded mesoporous silica microspheres, which embolize distal capillaries. (4) Simultaneously achieve catalytic oxygen production and immune checkpoint blockade, reversing tumor hypoxia and immunosuppressive microenvironment.

10. The application of the hypoxia-regulated multi-stage embolization microspheres according to claim 9 in the transarterial embolization treatment of hepatocellular carcinoma, characterized in that, The immune checkpoint blockade is achieved through the sustained release of aPD-1 antibody, which blocks the PD-1 / PD-L1 pathway to activate CD8. + T cells.

Images