PNIPAM-based self-developing gel embolism system as well as preparation method and application thereof

Abstract

The invention relates to a double-framework self-developing embolism system based on iodine modified PNIPAM gel and cross-linked albumin microspheres, which simulates a brick wall-concrete effect through a microsphere-gel composite structure and synergistically improves the embolism strength. A novel TACE treatment system is designed and constructed, multiple effects of tracing, embolism, hypoxia enhanced chemotherapy and tumor microenvironment improvement are achieved through the self-developing temperature-sensitive gel-drug carrying microspheres, and the anti-tumor effect is cooperatively improved.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulations, specifically relating to a self-illuminating gel embolization system based on PNIPAM (poly(N-isopropylacrylamide)), its preparation method, and its application. Background Technology

[0002] Hepatocellular carcinoma (HCC) is a malignant tumor with leading incidence and mortality rates worldwide. It has an insidious onset and rapid progression, with most patients diagnosed at an advanced stage, thus losing the opportunity for surgical resection. Transcatheter arterial chemoembolization (TACE) is a first-line treatment for intermediate-to-advanced HCC, achieving a synergistic anti-tumor effect by blocking tumor blood supply and delivering chemotherapy drugs locally. However, TACE treatment still faces several challenges. For example, traditional embolization materials are difficult to effectively embolize small vessels at the tumor's distal end, and the burst release of chemotherapy drugs can easily cause systemic toxicity. Furthermore, the tumor microenvironment (such as the ECM and collagen) hinders the diffusion of chemotherapy drugs to distant tumors, and the hypoxia in hypoxic tumors is further exacerbated after TACE, leading to increased angiogenesis and other malignant changes in the tumor microenvironment.

[0003] The inventors previously developed a self-illuminating thermosensitive hydrogel composite embolization agent loaded with arsenic trioxide (ATO) nanomicelles for transcatheter arterial chemoembolization (TACE) in primary hepatocellular carcinoma. However, due to the use of a thermosensitive gel embolization framework, which is close to a liquid embolization agent, drug loading into nanoparticles can achieve sustained release, but the nanoparticles cannot support the embolization framework, and there is a risk of tumor drug resistance due to hypoxia.

[0004] Therefore, there is an urgent need to develop an embolization system that is more suitable for transcatheter arterial chemoembolization (TACE) for primary hepatocellular carcinoma. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a self-illuminating gel embolization system based on PNIPAM (poly(N-isopropylacrylamide)) to achieve multiple anti-tumor effects, including embolization, self-illumination, hypoxia-enhanced chemotherapy, and improvement of the tumor microenvironment.

[0006] In a first aspect, the present invention provides a self-developing temperature-sensitive gel, the preparation process of which is as follows:

[0007] (1) Triiodobenzoic acid was acrylic acidified to obtain acrylic triiodobenzoic acid;

[0008] (2) Synthesizing poly(N-isopropylacrylamide) modified with triiodobenzoic acid via soap-free emulsion polymerization;

[0009] (3) Poly(N-isopropylacrylamide) modified with triiodobenzoic acid acrylate is added to water and swollen to obtain the product.

[0010] In some embodiments, the acrylic acidification of triiodobenzoic acid in step (1) is performed as follows: Triiodobenzoic acid, 1,3-dicyclohexylcarbodiimide (DCC), and 4-dimethylaminopyridine (DMAP) are mixed and dispersed in an appropriate amount of dichloromethane, stirred, and 2-hydroxyethyl methacrylate (HEMA) is added under nitrogen atmosphere and the reaction is carried out at room temperature for 12-36 h. The mixture is filtered, and the residue is washed with dichloromethane. The organic phase is then washed with concentrated HCl, NaHCO3 aqueous solution, and pure water, respectively. The organic phase is extracted and separated, dried with anhydrous MgSO4, filtered to remove MgSO4, and then the organic solvent is removed by rotary evaporator to obtain a solid. The solid is dissolved in ethyl acetate, recrystallized twice, and dried to obtain the white product, triiodobenzoic acid acrylic acid (TIBN).

[0011] Preferably, the molar ratio of DCC to TIBA is 1-3:1, more preferably 1:1;

[0012] Preferably, the molar ratio of DMAP to TIBA is 0.2-0.5:1, more preferably 0.35:1;

[0013] Preferably, the molar ratio of HEMA to TIBA is 0.8-1.2:1, more preferably 1.025:1;

[0014] In some implementations, the acrylate reaction of triiodobenzoic acid in step (1) is performed as follows:

[0015] TIBA (1 g, 0.002 mol), DCC (0.412 g, 0.002 mol), and DMAP (0.085 g, 0.0007 mol) were mixed and dispersed in 30 mL of dichloromethane. After magnetic stirring, HEMA (0.242 mL, 0.00205 mol) was added, and nitrogen gas was purged for 10 min. The reaction was carried out under nitrogen atmosphere with magnetic stirring at room temperature for 24 h. Impurities were filtered out through filter paper, and the residue was washed with dichloromethane. The organic phase was then washed with concentrated HCl (2 mol / L, 100 mL), NaHCO3 (2 mol / L, 100 mL), and 100 mL of pure water, respectively. The organic phase was extracted and separated, dried over anhydrous MgSO4, filtered to remove MgSO4, and the organic solvent was removed by rotary evaporator to obtain a solid. The solid was dissolved in ethyl acetate in a water bath at 40 °C, recrystallized twice, and dried under vacuum at 50 °C for 12 h to obtain the white product triiodobenzoic acid acrylate (TIBN).

[0016] In some implementations, step (2) includes the following preparation process:

[0017] First, weigh out TIBN and dissolve it in acetone to prepare a TIBN solution.

[0018] Weigh an appropriate amount of PNIPAM purified by recrystallization from cyclohexane, add the crosslinking agent N,N-methylenebisacrylamide (MBA) and the initiator potassium persulfate (KPS) to a double-necked flask, and then add pure water to dissolve them evenly. After bubbling with nitrogen gas at a uniform rate, add tetramethylethylenediamine (TEDEM) and TIBN solution, raise the temperature to 60-80℃, and continue the reaction under a nitrogen atmosphere for 3-6 hours. After the reaction is completed, cool naturally to room temperature to obtain a gel dispersion. Dialyze the dispersion in pure water at room temperature for 1-5 days (MW = 8000-14000), changing the water 1-5 times a day. After dialysis, freeze-dry to obtain the iodine-modified thermosensitive material (PNIPAM-TIBN).

[0019] Preferably, the temperature is raised to 70°C and the reaction is continued for 4.5 hours under a nitrogen atmosphere;

[0020] Preferably, the dialysis duration is 3 days, with the water changed 3 times per day;

[0021] Preferably, the molar ratio of PNIPAM to TIBN is 2-20:1, more preferably 2-8:1;

[0022] Preferably, the molar ratio of MBA to PNIPAM is 5-10:1, more preferably 7:1;

[0023] Preferably, the molar ratio of KPS to PNIPAM is 8-15:1, more preferably 11:1;

[0024] In some implementations, step (2) includes the following preparation process:

[0025] First, weigh 0.1131g of TIBN and dissolve it in 10mL of acetone to prepare a TIBN solution.

[0026] Secondly, PNIPAM-TIBN was synthesized via addition-fragmentation chain transfer polymerization using a soap-free emulsion polymerization method. Specifically, the following steps were taken: 0.5658 g (5 mmol) of PNIPAM purified by recrystallization from cyclohexane, 0.03 g (35 mmol) of crosslinking agent MBA, and 0.04 g (55 mmol) of initiator KPS were added to a 100 mL double-necked flask. 30 mL of pure water was then added and dissolved uniformly under magnetic stirring. Nitrogen gas was bubbled through the flask at a constant rate for 20 min, followed by the addition of 15 µL of LTEDEM and TIBN solution. The reaction mixture was placed in an oil bath with stirring and heated to 70 °C. The reaction was continued under a nitrogen atmosphere for 4.5 h. After the reaction, the mixture was allowed to cool naturally to room temperature to obtain a gel dispersion. The dispersion was dialyzed in pure water at room temperature for 3 days (MW = 8000-14000), with the water changed 3 times daily. After dialysis, the iodine-modified thermosensitive material (PNIPAM-TIBN) was obtained by lyophilization.

[0027] In a second aspect, the present invention provides a composite embolizing agent of autoradiographic thermosensitive gel-microsphere gel, which is made of the autoradiographic thermosensitive gel and microsphere gel described in the first aspect, wherein the microsphere gel is albumin microspheres carrying hypoxia-responsive antitumor drugs and drugs that inhibit tumor angiogenesis.

[0028] In some embodiments, the composite embolic agent is prepared as follows:

[0029] The self-developing thermosensitive gel and microsphere gel are mixed in double-distilled water and stirred until homogeneous to obtain the final product.

[0030] Preferably, the mass ratio of the autoradiographic thermosensitive gel to the microsphere gel is 5-15:1, more preferably 10:1.

[0031] Preferably, the hypoxia-responsive antitumor drug is telatazamine, mitomycin C, doxorubicin, dacarbazine, temozolomide, or TH-302, more preferably telatazamine.

[0032] Preferably, the anti-tumor angiogenesis drug is nintedanib, apatinib, lenvatinib, anlotinib, axitinib, or sunitinib, and more preferably nintedanib.

[0033] In some embodiments, the microsphere gel is prepared by centrifugal emulsification, emulsion solvent evaporation, electrostatic spraying, or microfluidic methods, preferably by centrifugal emulsification.

[0034] In some embodiments, the microsphere gel is prepared as follows:

[0035] A certain amount of Span 80 was weighed and dissolved in n-hexane, and mixed thoroughly to prepare an oil phase solution. Then, a certain amount of Tween 80, bovine serum albumin (BSA), genipin (GNP), polyethylene oxide (PEO), teirazamin (TPZ), and nintedanib (NIB) were weighed and dissolved in pure water, and mixed thoroughly to prepare an aqueous phase solution. The oil and aqueous phases were mixed, centrifuged and emulsified using a degassing machine, and then removed. The mixture was stirred at room temperature for a period of time, followed by heating and stirring for a period of time. The sample was removed, washed with n-hexane, centrifuged to remove the supernatant, and then lyophilized after adding a certain amount of double-distilled water to obtain drug-loaded albumin microspheres.

[0036] Preferably, the mass ratio of Span 80 to Tween 80 is 18-30:1, more preferably 24:1;

[0037] Preferably, the volume ratio of n-hexane to water is 4-8:1, more preferably 6:1;

[0038] In some embodiments, the microsphere gel is prepared as follows:

[0039] Drug-loaded albumin microspheres were prepared using a centrifugal emulsification method, as follows: First, Span 80 was weighed and dissolved in 6 mL of n-hexane, and vortexed to prepare an oil phase solution (containing 2.88% Span 80). Then, Tween 80, BSA (40 mg), GNP (4 mg), PEO (4 mg), TPZ (0.5 mg), and NIB (0.5 mg) were weighed and dissolved in 1 mL of pure water, and vortexed to prepare an aqueous phase solution (containing 0.12% Tween 80). The oil and aqueous phases were mixed and centrifuged (1200 rpm, 3 min) using a defoamer. The mixture was then transferred to a vial and stirred at room temperature for 5 h, followed by heating and stirring at 50 °C for 3 h. The sample was washed with n-hexane, centrifuged at 3000 rpm for 5 min to remove the supernatant, and this process was repeated twice. After adding a certain amount of double-distilled water, the sample was lyophilized to obtain drug-loaded albumin microspheres (TPZ / NIB@BM).

[0040] Thirdly, the present invention provides the use of the autoradiographic thermosensitive gel described in the first aspect or the embolizing agent described in the second aspect in the preparation of a drug for treating tumors.

[0041] In some implementations, the tumor is liver cancer.

[0042] Preferably, the liver cancer is primary liver cancer.

[0043] The beneficial effects of this invention are:

[0044] ① This invention adopts a strategy of synergistic treatment with TPZ and NIB, providing a new approach to address the decline in the efficacy of chemotherapy drugs and angiogenesis caused by the hypoxic environment of TACE.

[0045] ②The designed TPZ-NIB co-loaded novel microsphere-gel dual-scaffold embolization formulation can fill the gaps between microspheres, forming a composite structure similar to a brick wall-concrete structure, effectively embolizing blood vessels.

[0046] ③Iodine-modified PNIPAM was used to prepare thermosensitive gels with autoradiography capabilities.

[0047] ④ TPZ is activated into highly active free radicals with cytotoxicity under hypoxic conditions, and exerts a synergistic anti-tumor therapeutic effect in conjunction with the synergistic effect of NIB in maintaining a hypoxic environment by inhibiting angiogenesis.

[0048] This invention is based on a PNIPAM (poly(N-isopropylacrylamide)) autoradiographic gel embolization system, which achieves multiple anti-tumor effects including embolization, autoradiography, hypoxia-enhanced chemotherapy, and improvement of the tumor microenvironment. Attached Figure Description

[0049] Figure 1 Thermosensitive phase transition of self-developing thermosensitive gel: (A) 20℃; (B) 30℃; (C) 37℃.

[0050] Figure 2. Embolization of the self-developing thermosensitive gel in a capillary tube under a water bath at 37℃.

[0051] Figure 3 (A) SEM characterization of autoradiography thermosensitive gel and microsphere-gel composite embolization agent; in vitro embolization of microsphere-gel composite embolization agent: (B) 20℃; (C) 37℃ water bath; (D) embolization in simulated blood vessels in vitro at 37℃.

[0052] Figure 4 Results of mechanical strength testing of materials by texture analyzer: (A) Autoradiographic gel and microsphere-gel composite plugs at different temperatures

[0053] Force-time curves of embolic agents; (B) force-distance curves and (C) force required for 10 mm of autoradiographic gel and microsphere-gel composite embolic agent at 37℃ (***p<0.001, n=3).

[0054] Figure 5 Visual images of tumors on day 14 of treatment in each group. Detailed Implementation

[0055] To enable those skilled in the art to better understand the technical solutions of the present invention, some non-limiting embodiments are further disclosed below to provide a more detailed description of the present invention.

[0056] The specific material ratios, process conditions, and results described in this invention are for illustrative purposes only and should not, and will not, limit the invention as described in the claims.

[0057] The following are the reagents and their manufacturers used in the examples.

[0058] name Specification factory 2,3,5-Triiodobenzoic acid (TIBA) Analytical Pure Macklin Biotechnology Co., Ltd. Tetramethylethylenediamine (TEMED) Analytical Pure Sinopharm Chemical Reagent Co., Ltd. 4-Dimethylaminopyridine (DMAP) Analytical Pure Aladdin Reagents (Shanghai) Co., Ltd. Hydroxyethyl methacrylate (HEMA) Analytical Pure Aladdin Reagents (Shanghai) Co., Ltd. 1,3-Dicyclohexylcarbodiimide (DCC) Analytical Pure Aladdin Reagents (Shanghai) Co., Ltd. N-Isopropylacrylamide (PNIPAM) Analytical Pure Aladdin Reagents (Shanghai) Co., Ltd. N,N-Methylenebisacrylamide (MBA) Analytical Pure Aladdin Reagents (Shanghai) Co., Ltd. Potassium persulfate (KPS) ≥98％ Sinopharm Chemical Reagent Co., Ltd. Hydrochloric acid (HCl) Analytical Pure Sinopharm Chemical Reagent Co., Ltd. Sodium bicarbonate (NaHCO3) ≥98％ Sinopharm Chemical Reagent Co., Ltd. Magnesium sulfate (MgSO4) ≥98％ Sinopharm Chemical Reagent Co., Ltd. Ethyl acetate Analytical Pure Sinopharm Chemical Reagent Co., Ltd. Bovine serum albumin (BSA) ≥98％ Aladdin Reagents (Shanghai) Co., Ltd. Tirazamin (TPZ) ≥98％ Hanxiang Biotechnology Co., Ltd. Nydanib (NIB) ≥98％ Nanjing Aosaikang Pharmaceutical Ginipin (GNP) ≥98％ Aladdin Reagents (Shanghai) Co., Ltd. Polyethylene oxide (PEO) ≥98％ Shanghai McLean Biochemical Technology Co., Ltd. n-Hexane Analytical Pure Sinopharm Chemical Reagent Co., Ltd. Span 80 ≥98％ Sinopharm Chemical Reagent Co., Ltd. Tween 80 ≥98％ Shanghai Myriel Chemical Technology Co., Ltd. PBS buffer self made

[0059] Example 1: Preparation of a self-illuminating thermosensitive gel-microsphere embolization system

[0060] 1.1 Synthesis of TIBN (triiodobenzoic acid acrylate)

[0061] TIBA (1 g, 0.002 mol), DCC (0.412 g, 0.002 mol), and DMAP (0.085 g, 0.0007 mol) were mixed and dispersed in 30 mL of dichloromethane. After magnetic stirring, HEMA (0.242 mL, 0.00205 mol) was added, and nitrogen gas was purged for 10 min. The reaction was carried out under nitrogen atmosphere with magnetic stirring at room temperature for 24 h. Impurities were filtered out through filter paper, and the residue was washed with dichloromethane. The organic phase was then washed with concentrated HCl (2 mol / L, 100 mL), NaHCO3 (2 mol / L, 100 mL), and 100 mL of pure water, respectively. The organic phase was extracted and separated, dried over anhydrous MgSO4, filtered to remove MgSO4, and the organic solvent was removed by rotary evaporator to obtain a solid. The solid was dissolved in ethyl acetate in a water bath at 40 °C, recrystallized twice, and dried under vacuum at 50 °C for 12 h to obtain the white product triiodobenzoic acid acrylate (TIBN).

[0062] 1.2 Synthesis of PNIPAM-TIBN

[0063] First, weigh 0.1131g of TIBN and dissolve it in 10mL of acetone to prepare a TIBN solution.

[0064] Secondly, PNIPAM-TIBN was synthesized via addition-fragmentation chain transfer polymerization using a soap-free emulsion polymerization method. Specifically, the following steps were taken: 0.5658 g (5 mmol) of PNIPAM purified by recrystallization from cyclohexane, 0.03 g (35 mmol) of crosslinking agent MBA, and 0.04 g (55 mmol) of initiator KPS were added to a 100 mL double-necked flask. 30 mL of pure water was then added and dissolved uniformly under magnetic stirring. Nitrogen gas was bubbled through the flask at a constant rate for 20 min, followed by the addition of 15 µL of LTEDEM and TIBN solution. The reaction mixture was placed in an oil bath with stirring and heated to 70 °C. The reaction was continued under a nitrogen atmosphere for 4.5 h. After the reaction, the mixture was allowed to cool naturally to room temperature to obtain a gel dispersion. The dispersion was dialyzed in pure water at room temperature for 3 days (MW = 8000-14000), with the water changed 3 times daily. After dialysis, the iodine-modified thermosensitive material (PNIPAM-TIBN) was obtained by lyophilization.

[0065] 1.3 Preparation of drug-loaded albumin microspheres

[0066] Drug-loaded albumin microspheres were prepared using a centrifugal emulsification method, as follows: First, Span 80 was weighed and dissolved in 6 mL of n-hexane, and vortexed to prepare an oil phase solution (containing 2.88% Span 80). Then, Tween 80, BSA (40 mg), GNP (4 mg), PEO (4 mg), TPZ (0.5 mg), and NIB (0.5 mg) were weighed and dissolved in 1 mL of pure water, and vortexed to prepare an aqueous phase solution (containing 0.12% Tween 80). The oil and aqueous phases were mixed and centrifuged (1200 rpm, 3 min) using a defoamer. The mixture was then transferred to a vial and stirred at room temperature for 5 h, followed by heating and stirring at 50 °C for 3 h. The sample was washed with n-hexane, centrifuged at 3000 rpm for 5 min to remove the supernatant, and this process was repeated twice. After adding a certain amount of double-distilled water, the sample was lyophilized to obtain drug-loaded albumin microspheres (TPZ / NIB@BM). (The encapsulation efficiencies of TPZ and NIB were 80.81±2.23% and 78.87±2.56%, respectively, and the drug loadings were 0.8±0.01% and 0.78±0.03%, respectively.)

[0067] 1.4 Preparation of self-illuminating thermosensitive gel and microsphere-gel composite embolizing agent

[0068] Weigh a certain amount of PNIPAM-TIBN, dissolve it in double-distilled water, stir, and allow it to fully swell and dissolve to obtain a thermosensitive gel (NMT) with self-developing function. Store it in a refrigerator at 4°C.

[0069] A certain amount of TPZ / NIB@BM and PNIPAM-TIBN were weighed and mixed in a certain amount of double-distilled water at a ratio of 10:1. The mixture was vortexed and stirred magnetically to obtain a microsphere-gel composite embolization agent (NMT-TPZ / NIB@BM).

[0070] Example 2: Characterization and in vitro embolization and release verification of the self-developing thermosensitive gel and the self-developing thermosensitive gel-microsphere embolization system.

[0071] 2.1 Morphological characteristics

[0072] After freeze-drying, the morphological characteristics of NMT and NMT-TPZ / NIB@BM were observed using SEM.

[0073] from Figure 3 As can be seen, NMT exhibits a dense, porous network structure, consistent with the microstructure of a gel. Microspheres with a diameter of approximately 50 μm can be observed in the NMT-TPZ / NIB@BM gel network structure; and smaller microspheres can be observed embedded in the gel framework structure in some areas.

[0074] 2.2 Phase transition temperature

[0075] The in vitro phase transition properties of NMT and NMT-TPZ / NIB@BM were investigated using the tilted bottle method. The specific operation was as follows: 5% NMT and NMT-TPZ / NIB@BM were prepared and placed in vials, and then placed in constant temperature water baths at 30℃ and 37℃ respectively. After observing the phase transition embolization of the samples in the vials, they were taken out and tilted to observe the in vitro phase transition.

[0076] Thermosensitive phase transition of NMT is as follows Figure 1 As shown, at 20°C, NMT is a uniform white, semi-transparent liquid with good flowability and needle penetration properties. Figure 1 A). Within 30 seconds of being placed in a 30°C water bath, NMT undergoes a phase transition, forming a solid gel at the bottom of the vial. Figure 1 B) indicates that NMT possesses certain adhesive properties after the phase transition. Increasing the water bath temperature to 37℃ accelerates the phase transition rate, but the embolization volume does not change significantly. Figure 1 C). The above results indicate that the self-developing thermosensitive gel can undergo a phase transition under body temperature conditions.

[0077] Thermosensitive phase transition of NMT-TPZ / NIB@BM as follows: Figure 3 B and Figure 3 As shown in Figure C, at 20°C, NMT-TPZ / NIB@BM is a blue suspension with good fluidity; however, in a water bath at 37°C, a phase transition occurs, and the microspheres condense to form a solid gel structure that adheres to the bottom of the vial, indicating that the addition of microspheres does not affect the temperature-sensitive phase transition of NMT.

[0078] 2.3 Extracorporeal embolization performance

[0079] An in vitro simulated vascular embolism model was established using the following method: Heat-shrink tubing was heated and stretched to create a series of smaller diameter tubes, simulating small to terminal blood vessels. The outlet end of a medical superselective catheter was inserted into the larger diameter section of the heat-shrink tubing to simulate vascular cannulation. The simulated vascular model was then placed in a beaker insulated at 37°C. 1 mL of 5% NMT or NMT-TPZ / NIB@BM was injected into the superselective catheter, and the embolism effect of NMT or NMT-TPZ / NIB@BM in the simulated vascular model was observed.

[0080] The results of NMT extracorporeal embolization simulation are as follows: Figure 2 As shown, after NMT is injected into the superselective catheter, it dehydrates and coagulates in a simulated distal vessel at 37°C (near the narrow end of the stretched thermoplastic tube), achieving a rapid phase transition. Notably, NMT did not undergo a significant phase transition during injection into the superselective catheter; instead, it formed an embolism near the narrow end of the stretched thermoplastic tube. This indicates that the thermosensitive gel possesses good fluidity and injectability, reducing the risk of catheter occlusion during TACE surgery and facilitating embolization in distal vessels.

[0081] The results of the NMT-TPZ / NIB@BM extracorporeal embolization simulation are as follows: Figure 3 As shown in Figure D, the NMT-TPZ / NIB@BM exhibits good needle penetration, allowing it to pass smoothly through the superselective catheter into the simulated blood vessel in vitro, and forming a stable embolism at the simulated terminal blood vessel (the narrow end of the stretched thermoplastic tube).

[0082] 2.4 Mechanical Strength

[0083] Two 30 mL samples of 5% NMT and NMT-TPZ / NIB@BM were placed in beakers for testing. One sample was placed at 20°C and the other was heated in a 37°C water bath for 30 s. The compression performance was measured using a texture analyzer at a test speed of 1 mm·s⁻¹ and a compression distance of 10 mm for 15 s.

[0084] The compressive properties of NMT and NMT-TPZ / NIB@BM were determined using a texture analyzer, and their mechanical strength was evaluated. Figure 4 Figure A shows the force-time curves of NMT and NMT-TPZ / NIB@BM at 20℃ and 37℃. Each peak represents the maximum force required to compress the gel sample by 10 mm. At 20℃, the required force for each gel group was less than 0.001 kg, while at 37℃, a force greater than 0.001 kg was required. This indicates that the embolic agent has strong fluidity at 20℃, while the gel undergoes a phase transition and its mechanical strength increases with increasing temperature. Figure 4 B indicates that NMT compression of 10mm requires 0.006kg of pressure, while NMT-TPZ / NIB@BM compression of 10mm requires 0.014kg of pressure. For example... Figure 4 As shown in Figure C, compared with NMT, NMT-TPZ / NIB@BM requires significantly more force to compress the same distance (p<0.001), and the mechanical strength is increased by about 133%.

[0085] It is speculated that the addition of microspheres forms a structure similar to a "concrete-brick wall," which increases the mechanical strength of the gel system and is beneficial to improving the arterial embolization effect.

[0086] Example 3: In vivo pharmacodynamic study of the autoradiographic thermosensitive gel-microsphere embolization system

[0087] A rat orthotopic liver cancer model was established, and tumor volume changes were monitored by MRI. The in vivo antitumor effect was evaluated by tumor inhibition experiments.

[0088] 3.1 Laboratory Animals

[0089] SD rats (male, 200-250g), 6-8 weeks old, were purchased from the Experimental Animal Center of Jiangsu University.

[0090] 3.2 Establishment of a rat liver cancer model

[0091] A rat hepatocellular carcinoma model was established using the in situ tumor-bearing method. The specific experimental method is as follows:

[0092] After anesthetizing and shaving the rats, a 2-3 cm incision was made along the midline of the abdomen towards the thoracic cavity to expose the left lobe of the liver. A 0.2 mL PBS suspension containing 1 × 10⁷ mouse-derived hepatocellular carcinoma cells (N1S1) was slowly injected into the left lobe of the liver. During the injection, a noticeable discoloration of the liver surface was observed. Then, a drop of biological fluid was applied to the wound dressing, and the injection site was manually compressed with a cotton swab for 2 minutes. Once the cell suspension stopped leaking out, the left lobe of the liver was repositioned, and the abdominal incision was sutured in layers.

[0093] 3.3 Evaluation of in vivo tumor suppression effect

[0094] A rat orthotopic liver cancer model was established according to method 3.2. Ten days after tumor bearing, MRI was used for observation, and the tumor size was approximately 150 mm. 3 The animals were randomly divided into five groups of three. Administered medication according to the following protocol:

[0095] Control group: physiological saline;

[0096] NMT group: 20 mg / mL NMT;

[0097] NMT-TPZ@BM group: a compound formulation of TPZ microspheres with a drug concentration of 2.7 mg / kg and NMT at a concentration of 20 mg / mL;

[0098] NMT-NIB@BM group: a compound formulation of NIB microspheres with a drug concentration of 2.7 mg / kg and NMT at a concentration of 20 mg / mL;

[0099] NMT-TPZ / NIB@BM group: a composite formulation of co-loaded microspheres of 2.7 mg / kg TPZ and 2.7 mg / kg NIB with 20 mg / mL NMT.

[0100] The preparation of NMT-TPZ@BM and NMT-NIB@BM is the same as that of NMT-TPZ / NIB@BM. The difference is that the former two use TPZ or NIB instead of TPZ and NIB as in NMT-TPZ / NIB@BM.

[0101] The procedure for hepatic artery embolization is as follows: After anesthetizing the tumor-bearing rats, an incision is made along the midline of the abdomen near the liver area to open the abdominal cavity. The liver is reversed to expose the blood vessels and bile ducts near the stomach and duodenum. The distal end of the gastroduodenal artery is clamped with a vascular clamp, and 1 mL of physiological saline or a preparation is slowly injected into the gastroduodenal artery using a syringe. The proximal end of the vessel is then clamped, hemostasis is achieved at the wound site, the vascular clamp is released, and the incision is sutured. Penicillin is administered intraperitoneally for three consecutive days. The liver and tumor status of the rats in each group are monitored using MRI before treatment and on days 7 and 14 after treatment.

[0102] 3.4 Results

[0103] An autopsy was performed 14 days after treatment to remove the tumor. Figure 5 As shown, compared with the control group, the tumor volume of each treatment group was significantly reduced, and the NMT-TPZ / NIB@BM group had the smallest tumor volume, showing the strongest tumor suppression effect, which is consistent with the MRI results.

[0104] The above results indicate that TAE embolization alone has a certain tumor-suppressing effect in TACE treatment. TACE treatment combined with NIB or TPZ embolization can improve the tumor-suppressing effect, while TPZ and NIB co-loaded microsphere-gel composite embolization treatment shows synergistic anti-tumor activity and exerts the best tumor-suppressing effect.

[0105] In summary, the TPZ-NIB co-loaded novel microsphere-gel dual-framework embolization formulation exhibits good in vivo tumor suppression effects.

[0106] Finally, it should be noted that the above embodiments are only used to illustrate and not limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention without departing from the spirit and scope of the present invention. Any modifications or partial substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A self-developing temperature-sensitive gel, characterized in that, The preparation process of the self-developing temperature-sensitive gel is as follows: (1) Triiodobenzoic acid was acrylic acidified to obtain acrylic triiodobenzoic acid; (2) Synthesizing poly(N-isopropylacrylamide) modified with triiodobenzoic acid via soap-free emulsion polymerization; (3) Poly(N-isopropylacrylamide) modified with triiodobenzoic acid acrylate is added to water and swollen to obtain the product.

2. The self-developing temperature-sensitive gel according to claim 1, characterized in that, The process of acrylate formation of triiodobenzoic acid in step (1) is as follows: Triiodobenzoic acid, 1,3-dicyclohexylcarbodiimide (DCC), and 4-dimethylaminopyridine (DMAP) were mixed and dispersed in an appropriate amount of dichloromethane, stirred, and 2-hydroxyethyl methacrylate (HEMA) was added and nitrogen gas was introduced. The reaction was carried out at room temperature under a nitrogen atmosphere for 12-36 h. The mixture was filtered, and the residue was washed with dichloromethane. The organic phase was then washed with concentrated HCl, NaHCO3 aqueous solution, and pure water, respectively. The organic phase was extracted and separated, dried with anhydrous MgSO4, filtered to remove MgSO4, and then the organic solvent was removed by rotary evaporator to obtain a solid. The solid was dissolved with ethyl acetate, recrystallized twice, and dried to obtain a white product, triiodobenzoic acid acrylate (TIBN). Preferably, the molar ratio of DCC to TIBA is 1-3:1, more preferably 1:1; and / or Preferably, the molar ratio of DMAP to TIBA is 0.2-0.5:1, more preferably 0.35:1; and / or Preferably, the molar ratio of HEMA to TIBA is 0.8-1.2:1, more preferably 1.025:

1.

3. The self-developing thermosensitive gel according to claim 1 or 2, characterized in that, The process of acrylate formation of triiodobenzoic acid in step (1) is as follows: TIBA (1 g, 0.002 mol), DCC (0.412 g, 0.002 mol), and DMAP (0.085 g, 0.0007 mol) were mixed and dispersed in 30 mL of dichloromethane. After magnetic stirring, HEMA (0.242 mL, 0.00205 mol) was added, and nitrogen gas was purged for 10 min. The reaction was carried out under nitrogen atmosphere with magnetic stirring at room temperature for 24 h. Impurities were filtered out through filter paper, and the residue was washed with dichloromethane. Then, the organic phase was washed with concentrated HCl (2 mol / L, 100 mL), NaHCO3 (2 mol / L, 100 mL), and 100 mL of pure water, respectively. The organic phase was extracted and separated, dried with anhydrous MgSO4, filtered to remove MgSO4, and then the organic solvent was removed by rotary evaporator to obtain a solid. The solid was dissolved in ethyl acetate in a water bath at 40°C, recrystallized twice, and dried under vacuum at 50°C for 12 h to obtain a white product, triiodobenzoic acid acrylate (TIBN).

4. The self-developing thermosensitive gel according to any one of claims 1-3, characterized in that, Step (2) includes the following preparation process: First, weigh out TIBN and dissolve it in acetone to prepare a TIBN solution; Weigh an appropriate amount of PNIPAM purified by recrystallization from cyclohexane, add the crosslinking agent N,N-methylenebisacrylamide (MBA) and the initiator potassium persulfate (KPS) to a double-necked flask, and then add pure water to dissolve evenly; after bubbling with nitrogen gas at a uniform rate, add tetramethylethylenediamine (TEDEM) and TIBN solution, raise the temperature to 60-80℃, and continue the reaction under a nitrogen atmosphere for 3-6 hours. After the reaction is completed, cool naturally to room temperature to obtain a gel dispersion; dialyze the dispersion in pure water at room temperature for 1-5 days (MW=8000-14000), changing the water 1-5 times a day. After dialysis, freeze-dry to obtain the iodine-modified thermosensitive material (PNIPAM-TIBN); Preferably, the temperature is raised to 70°C and the reaction is continued for 4.5 hours under a nitrogen atmosphere; and / or, Preferably, the dialysis duration is 3 days, with the water changed 3 times per day; and / or, Preferably, the molar ratio of PNIPAM to TIBN is 2-20:1, more preferably 2-8:1; and / or, Preferably, the molar ratio of MBA to PNIPAM is 5-10:1, more preferably 7:1; and / or, Preferably, the molar ratio of KPS to PNIPAM is 8-15:1, more preferably 11:

1.

5. The autoradiographic thermosensitive gel according to any one of claims 1-4, characterized in that, Step (2) includes the following preparation process: First, weigh 0.1131g of TIBN and dissolve it in 10mL of acetone to prepare a TIBN solution; Secondly, PNIPAM-TIBN was synthesized via addition-fragmentation chain transfer polymerization using a soap-free emulsion polymerization method. Specifically, the following steps were taken: 0.5658 g (5 mmol) of PNIPAM purified by recrystallization from cyclohexane, 0.03 g (35 mmol) of crosslinking agent MBA, and 0.04 g (55 mmol) of initiator KPS were added to a 100 mL double-necked flask. 30 mL of pure water was then added and dissolved uniformly under magnetic stirring. Nitrogen gas was bubbled through the flask at a constant rate for 20 min, followed by the addition of 15 μL of LTEDEM and TIBN solution. The reaction was carried out in an oil bath with stirring and heated to 70 °C. The reaction was continued under a nitrogen atmosphere for 4.5 h. After the reaction, the mixture was naturally cooled to room temperature to obtain a gel dispersion. The dispersion was dialyzed in pure water at room temperature for 3 days (MW = 8000-14000), with the water changed 3 times daily. After dialysis, the iodine-modified thermosensitive material (PNIPAM-TIBN) was obtained by lyophilization.

6. A composite embolic agent consisting of a self-illuminating thermosensitive gel and a microsphere gel, characterized in that, The composite embolizing agent is made from the autoradiographic thermosensitive gel and microsphere gel as described in any one of claims 1-5, wherein the microsphere gel is albumin microspheres carrying hypoxia-responsive antitumor drugs and drugs that inhibit tumor angiogenesis. Preferably, the composite embolic agent is prepared as follows: the autoradiographic thermosensitive gel and the microsphere gel are mixed in double-distilled water and stirred until homogeneous; and / or, Preferably, the mass ratio of the autoradiographic thermosensitive gel to the microsphere gel is 5-15:1, more preferably 10:1; and / or, Preferably, the hypoxia-responsive antitumor drug is telazamine, mitomycin C, doxorubicin, dacarbazine, temozolomide, or TH-302, more preferably telazamine; and / or, Preferably, the anti-tumor angiogenesis drug is nintedanib, apatinib, lenvatinib, anlotinib, axitinib, or sunitinib, and more preferably nintedanib.

7. The composite embolic agent according to claim 6, characterized in that, The microsphere gel can be prepared by centrifugal emulsification, emulsification solvent evaporation, electrostatic spraying, or microfluidics, with centrifugal emulsification being the preferred method.

8. The composite embolic agent according to claim 7, characterized in that, The microsphere gel is prepared as follows: A certain amount of Span 80 was weighed and dissolved in n-hexane, and mixed evenly to prepare an oil phase solution. Then, a certain amount of Tween 80, bovine serum albumin (BSA), genipin (GNP), polyethylene oxide (PEO), teirazamin (TPZ), and nintedanib (NIB) were weighed and dissolved in pure water, and mixed evenly to prepare an aqueous phase solution. The oil phase and aqueous phase were mixed, centrifuged and emulsified using a degassing machine, and then taken out. After stirring at room temperature for a period of time, the mixture was heated and stirred for a period of time. The sample was taken out, washed with n-hexane, centrifuged to remove the supernatant, and then a certain amount of double-distilled water was added before freeze-drying to obtain drug-loaded albumin microspheres. Preferably, the mass ratio of Span 80 to Tween 80 is 18-30:1, more preferably 24:1; and / or Preferably, the volume ratio of n-hexane to water is 4-8:1, more preferably 6:1; 9. The composite embolic agent according to claim 6 or 7, characterized in that, The microsphere gel is prepared as follows: Drug-loaded albumin microspheres were prepared using a centrifugal emulsification method, as follows: First, Span 80 was weighed and dissolved in 6 mL of n-hexane, and vortexed to prepare an oil phase solution (containing 2.88% Span 80); then, Tween 80, BSA (40 mg), GNP (4 mg), PEO (4 mg), TPZ (0.5 mg), and NIB (0.5 mg) were weighed and dissolved in 1 mL of pure water, and vortexed to prepare an aqueous phase solution (containing 0.12% Tween 80); the oil phase and aqueous phase were mixed, centrifuged and emulsified using a degassing machine (1200 rpm, 3 min), and then transferred to a vial. After stirring at room temperature for 5 h, the mixture was heated and stirred at 50 °C for 3 h; the sample was then washed with n-hexane, centrifuged at 3000 rpm for 5 min to remove the supernatant, and this process was repeated twice. After adding a certain amount of double-distilled water, the sample was freeze-dried to obtain drug-loaded albumin microspheres.

10. The use of the autoradiographic thermosensitive gel according to any one of claims 1-5 or the embolizing agent according to any one of claims 6-9 in the preparation of a drug for treating tumors, preferably, the tumor is liver cancer; more preferably, the liver cancer is primary liver cancer.

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