A thermosensitive tumor therapeutic agent containing an exosome inhibitor and its application

Abstract

This invention discloses a thermosensitive tumor therapeutic agent containing an exosome inhibitor and its application. The thermosensitive tumor therapeutic agent comprises a thermosensitive gel, within which antibodies, exosome inhibitors, and photosensitizers are encapsulated. This invention utilizes this tumor therapeutic agent to combine exosome inhibitors with thermo-effect synergistic immune checkpoint inhibitors for combined immunotherapy of tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a thermosensitive tumor therapeutic agent containing an exosome inhibitor and its application in tumor immunotherapy. Background Technology

[0002] Tumor immunotherapy is an emerging therapy that specifically kills tumor cells by activating the body's own immune response. Immune checkpoint blockade (ICB), represented by PD-1 / PD-L1, has achieved significant clinical breakthroughs in cancer treatment, making tumor immunotherapy a focus of attention. However, single immune checkpoint blockade therapy is only effective for a small number of cancer patients. Therefore, to further improve the response rate of patients to ICB treatment and expand the scope of indications, researchers are increasingly focusing on combination immunotherapy.

[0003] In previous studies, the inventors’ research group combined thermotherapy with ICB (Nature Communications, 2019, 10:4871.) for combined immunotherapy of cold tumors. Thermotherapy can effectively increase the infiltration of immune cells in the tumor microenvironment.

[0004] However, in previous studies, the inventors discovered that the thermotherapy effect increases PD-L1 expression in tumors both in vivo and in vitro. Simultaneously, thermotherapy accelerates the release of exosomes from tumor cells. Exosomes are vesicles released after the fusion of intracellular multivesicular bodies with the cell membrane. They contain complex proteins and nucleic acids and play a crucial role in intercellular interactions and regulation. Studies have shown that tumor cells can secrete exosomes carrying PD-L1 on their surface. This tumor self-defense mechanism, present in the tumor region and released into the bloodstream, not only depletes T cells and consumes therapeutic anti-PD-L1 antibodies at the tumor site but also binds to and inhibits T cell function in the circulatory system, thereby enabling tumor immune escape. Based on this, the inventors believe that the thermotherapy effect combined with exosome inhibitors can synergistically alleviate the immunosuppressive microenvironment of the tumor microenvironment, further improving the therapeutic response of ICB therapy. Summary of the Invention

[0005] To address the immunosuppressive microenvironment resulting from the combination of thermotherapy and ICB, this invention provides a thermosensitive tumor therapeutic agent containing an exosome inhibitor. This tumor therapeutic agent can release the drug while performing local thermotherapy, exerting an exosome inhibitory effect, and is combined with immune checkpoint blockade therapy.

[0006] The present invention adopts the following technical solution:

[0007] A thermosensitive tumor therapeutic agent includes a thermosensitive gel, wherein the thermosensitive gel encapsulates an antibody, an exosome inhibitor, and a photosensitizer;

[0008] The antibody is used as an immune checkpoint inhibitor and may be selected as anti-PDL1 antibody, anti-CTLA4 antibody, anti-Lag3 antibody, anti-VISTA antibody, anti-TIM3 antibody, anti-TIGIT antibody, anti-BTLA antibody, anti-CD40L antibody, or anti-CD47 antibody.

[0009] The exosome inhibitors may include GW4869, sulfamethoxazole, neteconazole, clotrimazole, tepifenesin, ketoconazole, clotrimazole, and shikonin.

[0010] The photosensitizer may be graphene nanoparticles or black phosphorus nanoparticles.

[0011] The thermosensitive gel described in this invention can be selected from commonly used thermosensitive gels in the art, such as poloxamer hydrogels, poly-N-isopropylacrylamide hydrogels, and phospholipid / oil ester lipid gels.

[0012] Further, the antibody accounts for 0.5-3% of the thermosensitive gel; the exosome inhibitor accounts for 0.1-0.3% of the thermosensitive gel; and the photosensitizer accounts for 0.2-0.5% of the thermosensitive gel.

[0013] In one embodiment of the present invention, the thermosensitive gel is a phospholipid / oil ester lipid gel, the antibody is an anti-PDL1 antibody, the exosome inhibitor is sulfamethoxazole, and the photosensitizer is graphene nanoparticles.

[0014] In one embodiment of the present invention, the preparation method of the above-mentioned thermosensitive tumor therapeutic agent includes the following steps:

[0015] Step 1: Mix the exosome inhibitor and photosensitizer at a mass ratio of 10:1, and sonicate to obtain a photosensitizer loaded with exosome inhibitor;

[0016] Step 2: The photosensitizer loaded with exosome inhibitor, antibody, phospholipid, dioleoyl glycerol, and surfactant are mixed in a mass ratio of 0.3:0.5:41:59:0.003, pre-frozen in liquid nitrogen, and lyophilized to prepare a lipid lyophilized powder loaded with exosome inhibitor / photosensitizer composition and antibody.

[0017] Step 3: Dissolve phospholipids and dioleoglycerides in anhydrous ethanol at a mass ratio of 41:59, where the mass of anhydrous ethanol is 10% of the sum of the masses of phospholipids and dioleoglycerides, to prepare a lipid precursor oil solution.

[0018] Step 4: Mix and dissolve an equal mass of the lipid lyophilized powder from Step 2 with the lipid precursor oil solution from Step 3 to obtain the lipid precursor oil phase formulation.

[0019] Step 5: Inject the lipid precursor oil phase formulation from step 4 into water to obtain the thermosensitive tumor therapeutic agent.

[0020] A tumor treatment drug, comprising the above-mentioned thermosensitive tumor treatment agent.

[0021] Thermosensitive gels serve as drug reservoirs. When the temperature is above their phase transition point, they can transform from a gel state to a sol state, accelerating drug release. When the temperature is below their phase transition point, they can transform from a sol state to a gel state, and can be used to encapsulate hydrophilic, hydrophobic, and amphiphilic drugs. The tumor therapeutic agent of this invention selects an in-situ gel that undergoes a phase transition within the thermotherapy range and releases drugs in response to temperature as a drug reservoir. Nanomaterials with a two-dimensional sheet structure serve as photosensitizers, carriers of exosome inhibitors, and drug release regulators for the thermosensitive gel. The two-dimensional sheet structure nanomaterials are first loaded with exosome inhibitors to form a composition, and then co-encapsulated with antibodies within the thermosensitive gel.

[0022] This invention encapsulates exosome inhibitors, photosensitizers, and immune checkpoint inhibitors in a thermosensitive gel, enabling the combined use of exosome inhibitors with thermo-effects and immune checkpoint inhibitors for combined immunotherapy of tumors. Attached Figure Description

[0023] Figure 1 The results are for the reversible temperature-sensitive properties of the blank lipid gel.

[0024] Figure 2 Photographs showing the formation process and phase transition properties of drug-loaded lipid gels.

[0025] Figure 3 The results show the in vitro photothermal conversion efficiency of the drug-loaded lipid gel.

[0026] Figure 4 The results show the in vivo thermodynamic effect of the drug-loaded lipid gel.

[0027] Figure 5 The results show the in vitro drug release from the drug-loaded lipid gel.

[0028] Figure 6 This is a curve showing the inhibitory effect of a tumor therapeutic agent on tumor cell-derived exosomes in vivo.

[0029] Figure 7 Tumor growth curves for the use of tumor therapeutic agents in the treatment of breast cancer in mice. Detailed Implementation

[0030] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0032] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Example 1

[0033] Determination of reversible thermosensitive properties of blank lipid gel

[0034] Phospholipids and dioleoglycerides were weighed at a mass ratio of 41:59 and dissolved in anhydrous ethanol at 10% of the total mass of the two lipids to prepare a lipid precursor oil solution. The precursor oil solution was then injected into purified water to prepare a blank lipid gel.

[0035] 200 mg of the above-mentioned lipid gel was placed on the sample pan of the rheometer and measured using a plate with a diameter of 20 mm. The temperature was set to 37 ℃ and 45 ℃, the angular frequency (ω) was 6.28 rad / s, and the strain constant (γ) was 1%. The storage modulus and loss modulus (G' and G") of the gel were measured in the frequency range of 0.1-100 Hz. The cycle was repeated twice to simulate the multiple thermotherapy processes of the gel in the body and the changes in the mechanical strength of the lipid gel were recorded.

[0036] The results of the reversible thermosensitive phase transition assay of the blank lipid gel are as follows: Figure 1 As shown, the changes in the rheological properties of the gel were measured during three cycles. At 37 °C, after three cycles, the measured storage modulus of the gel was still higher than the loss modulus, indicating that the gel could recover to the gel state after multiple heatings. At 45 °C, the measured storage modulus of the gel was lower than the loss modulus in all three cycles, indicating that the gel rapidly transformed into a sol state at this temperature. Example 2

[0037] Investigation on the formation process and phase transition properties of drug-loaded lipid gels

[0038] First, graphene nanoparticles, phospholipids, dioleoglycerate, and surfactant (Tween 80) were vortexed and mixed at a mass ratio of 0.1:41:59:0.003, then rapidly pre-frozen in liquid nitrogen and lyophilized to obtain a lipid lyophilized powder loaded with photosensitizer. Second, phospholipids and dioleoglycerate were weighed at a mass ratio of 41:59 and dissolved in anhydrous ethanol at 10% of the total mass of the two lipids to prepare a lipid precursor oil solution. Then, equal masses of the above two parts were mixed, dissolved, and homogenized to obtain the lipid precursor oil phase formulation.

[0039] To observe the formation process of lyotropic liquid crystal gels, the precursor oil phase formulation was drawn up with a syringe and slowly injected into water. The formation of the gel was photographed and recorded. Because this gel has thermosensitive properties, temperature changes from heating to cooling can induce a gel-sol phase transition. To further investigate the sol-gel phase transition process, 1 mL of the precursor oil phase formulation was first placed in a vial, and 2 mL of ultrapure water was slowly added along the vial wall to allow the precursor oil phase formulation to fully gel. The ultrapure water was discarded, and the gel formation was observed by inverting the vial. Then, the vial was irradiated with an 808 nm near-infrared laser to raise the temperature to 45 °C. The vial was tilted, and the gradual gelation of the gel was observed. The laser irradiation was then removed, and the vial was allowed to stand until the temperature dropped to room temperature. The vial was then inverted, and the gel formation was observed again.

[0040] like Figure 2 As shown in the middle left image, when the gel is injected into water, it immediately accumulates at the needle tip upon contact with water, forming a gel. (See image for details.) Figure 2 As shown in the right-middle figure, the vial inversion experiment demonstrates that the formed gel adheres to the bottom of the vial. When heated by laser irradiation to 45 °C, the gel undergoes a phase transition, becoming sol-like and exhibiting a certain degree of fluidity. When the laser is removed and the vial is allowed to return to room temperature, the sol transforms into a gel, losing its fluidity and adhering to the bottom. This process allows for direct observation of the drug-loaded lipid gel's shape retention and phase transition process, verifying its phase transition properties. Example 3

[0041] In vitro photothermal conversion efficiency of drug-loaded lipid gels

[0042] Preparation of drug-loaded lipid gel: First, graphene nanoparticles, phospholipids, dioleoylglycerol, and surfactant (Tween 80) were vortexed and mixed at a mass ratio of 0.3:41:59:0.003, rapidly pre-frozen in liquid nitrogen, and lyophilized to prepare a lyophilized lipid powder encapsulating a photosensitizer. Second, phospholipids and dioleoylglycerol were weighed at a mass ratio of 41:59 and dissolved in 10% anhydrous ethanol of the total mass of the two lipids to prepare a lipid precursor oil solution. Then, equal masses of the above two portions were mixed, dissolved, and homogenized to obtain the lipid precursor oil phase formulation. The above precursor oil phase formulation was injected into purified water to prepare the drug-loaded lipid gel.

[0043] Preparation of blank lipid gel: Phospholipids and dioleoglycerides were weighed at a mass ratio of 41:59, and dissolved in anhydrous ethanol at 10% of the total mass of the two lipids to prepare a lipid precursor oil solution. The precursor oil solution was then injected into purified water to prepare a blank lipid gel.

[0044] Drug-loaded lipogels were collected, with blank lipogels and PBS used as controls. The samples were irradiated with an 808 nm near-infrared laser, and the sample temperature was monitored in real time using an infrared imager. The samples were rapidly heated to 45 °C, and then the temperature was precisely controlled and maintained at 45 °C by adjusting the laser irradiation power. Throughout the process, temperature changes at different time points were recorded and photographed using an infrared imager, and temperature-time curves were plotted.

[0045] The in vitro photothermal conversion efficiency of drug-loaded lipid gels is as follows: Figure 3 As shown, the blank gel and PBS group showed no significant temperature fluctuations under near-infrared laser irradiation, while the drug-loaded lipid gel group could heat up rapidly, reaching 45℃ in about 100 s. This indicates that the drug-loaded lipid gel group has good photothermal conversion efficiency. Example 4

[0046] In vivo thermotherapy monitoring of drug-loaded lipid gels

[0047] Preparation of drug-loaded lipid gel: First, graphene nanoparticles, phospholipids, dioleoylglycerol, and surfactant (Tween 80) were vortexed and mixed at a mass ratio of 0.3:41:59:0.003, rapidly pre-frozen in liquid nitrogen, and lyophilized to prepare a lyophilized lipid powder encapsulating a photosensitizer. Second, phospholipids and dioleoylglycerol were weighed at a mass ratio of 41:59 and dissolved in 10% anhydrous ethanol of the total mass of the two lipids to prepare a lipid precursor oil solution. Then, equal masses of the above two portions were mixed, dissolved, and homogenized to obtain the lipid precursor oil phase formulation. The above precursor oil phase formulation was injected into purified water to prepare the drug-loaded lipid gel.

[0048] Preparation of blank lipid gel: Phospholipids and dioleoglycerides were weighed at a mass ratio of 41:59, and dissolved in anhydrous ethanol at 10% of the total mass of the two lipids to prepare a lipid precursor oil solution. The precursor oil solution was then injected into purified water to prepare a blank lipid gel.

[0049] Drug-loaded lipid gels were collected, with blank gels and PBS used as controls. 50 μL of each sample was injected into mice via intratumoral injection. Four hours after injection, the tumor site was irradiated with an 808 nm near-infrared laser, and the sample temperature was monitored in real time using an infrared imager. The tumor temperature was rapidly increased to 45 °C, and then precisely controlled to maintain the temperature at 45 °C by adjusting the laser irradiation power. Throughout the process, temperature changes at different time points were recorded and photographed using an infrared imager, and temperature-time curves were plotted.

[0050] The in vitro photothermal conversion efficiency of drug-loaded lipid gels is as follows: Figure 4 As shown, the blank gel and PBS groups only showed a slight increase in temperature under near-infrared laser irradiation, which may be due to the mice's own slight absorption of near-infrared light. The drug-loaded gel group reached 45 °C in about 100 seconds, indicating that it still has good photothermal conversion efficiency in vivo. Once the temperature reached 45 °C, adjusting the laser efficiency effectively stabilized the temperature at the tumor site at 45 °C, demonstrating that the photothermal effect and temperature can be precisely controlled in vivo, meeting the needs of subsequent thermotherapy. Example 5

[0051] In vitro drug release from drug-loaded lipid gels

[0052] Preparation of drug-loaded lipid gel: First, sulfamethoxazole was dissolved in DMSO (10 mg / mL). Then, graphene nanoparticles (0.1 mg / mL) solution was added at a sulfamethoxazole:graphene nanoparticle mass ratio of 10:1. An ultrasonic cell disruptor was used, with the ultrasonic power set to 30%, the ultrasonic mode set to 2 s sonication followed by 3 s pause, and the sonication time 60 min. The resulting reaction product was added to an ultrafiltration tube with a 100 kDa cutoff, centrifuged, resuspended in ultrapure water, and washed three times to remove excess sulfamethoxazole. The resulting product was filtered through a 0.22 μL filter. A μm microporous filter membrane was used to remove unstable nanoparticles, thus preparing sulfamethoxazole-loaded graphene nanoparticles (SG). Next, the sulfamethoxazole-loaded graphene nanoparticles, antibody drug (using immunoglobulin IgG as a structural analog of the anti-PD-L1 antibody), phospholipids, dioleoylglycerol, and surfactant (Tween 80) were vortexed at a mass ratio of 0.3:3:41:59:0.003, rapidly pre-frozen in liquid nitrogen, and lyophilized to prepare a photosensitizer-loaded lipid lyophilized powder. Then, phospholipids and dioleoylglycerol were weighed at a mass ratio of 41:59 and dissolved in 10% anhydrous ethanol of the total mass of the two lipids to prepare a lipid precursor oil solution. Finally, equal masses of the two portions were mixed, dissolved, and homogenized to obtain the drug-loaded lipid precursor oil phase formulation.

[0053] 50 μL of the drug-loaded gel precursor oil phase was injected into a centrifuge tube containing 2 mL of PBS containing 10% PEG2000. The tube was then placed in a 37 °C water bath with a shaker for release. 1 mL samples were taken at different time points (2 h, 4 h, 24 h, 48 h, 96 h, 168 h, 240 h, and 336 h), with an equal volume of the same temperature medium added simultaneously. For the thermotherapy setup, before sampling at 4 h and 48 h, the samples were irradiated with an 808 nm laser to raise the temperature to 45 °C and maintained for 10 min, followed by continued incubation in a 37 °C water bath with a shaker. The sulfamethoxazole content in the samples taken at each time point was determined by HPLC, and the IgG content was determined using a BCA kit. Cumulative release curves for both drugs were plotted to examine the drug release in both gel and sol states.

[0054] The drug release curve of the drug-loaded lipid gel is as follows: Figure 5 As shown ( Figure 5 a represents the IgG release curve. Figure 5 (b is the release curve of sulfamethoxazole). It can be seen that with periodic laser irradiation at different time points, controlled at 45 ℃, and photothermal for 10 min, the gel group (+L) irradiated with photothermal irradiation showed a significant burst release 4 h after release. Compared with the non-photothermal group, by 48 h, the release of IgG increased from 2.9% to 6.1%, and the release of sulfamethoxazole increased from 2.5% to 3.0%. By day 14, the release of IgG increased from 17.6% to 28.2%, and the release of sulfamethoxazole increased from 12.5% ​​to 15.5%. This indicates that photothermal-induced gel-sol phase transition can significantly improve drug release. Example 6

[0055] Tumor therapeutic agents for the inhibitory effect of tumor cell-derived exosomes in vivo

[0056] Preparation of sulfamethoxazole-loaded graphene nanoparticles (SG): Sulfamethoxazole was dissolved in DMSO (10 mg / mL). At a sulfamethoxazole:graphene nanoparticle mass ratio of 10:1, 0.1 mg / mL graphene nanoparticle solution was added. An ultrasonic cell disruptor was used, with the ultrasonic power set to 30%, the ultrasonic mode set to 2 s sonication followed by 3 s pauses, and the sonication time 60 min. The resulting reaction product was added to an ultrafiltration tube with a 100 kDa cutoff, centrifuged, resuspended in ultrapure water, and washed three times to remove excess sulfamethoxazole. The resulting product was then filtered through a 0.22 μm microporous membrane to remove unstable nanoparticles, thus obtaining sulfamethoxazole-loaded graphene nanoparticles.

[0057] Preparation of graphene nanoparticle-containing lipid gel (GO@LG): First, graphene nanoparticles, phospholipids, dioleoylglycerol, and surfactant (Tween 80) were vortexed and mixed at a mass ratio of 0.3:41:59:0.003, rapidly pre-frozen in liquid nitrogen, and lyophilized to prepare a lipid lyophilized powder loaded with photosensitizer; Second, phospholipids and dioleoylglycerol were weighed at a mass ratio of 41:59, and dissolved in anhydrous ethanol at 10% of the total mass of the two lipids to prepare a lipid precursor oil solution; Then, equal masses of the above two parts were mixed, dissolved, and homogenized to obtain the drug-loaded lipid precursor oil phase formulation.

[0058] Preparation of sulfamethoxazole / graphene nanoparticle-containing lipid gel (SG@LG): First, graphene nanoparticles loaded with sulfamethoxazole, phospholipids, dioleoylglycerol, and surfactant (Tween 80) were vortexed and mixed at a mass ratio of 0.3:41:59:0.003, rapidly pre-frozen in liquid nitrogen, and lyophilized to prepare a photosensitizer-loaded lipid lyophilized powder. Second, phospholipids and dioleoylglycerol were weighed at a mass ratio of 41:59 and dissolved in anhydrous ethanol at 10% of the total mass of the two lipids to prepare a lipid precursor oil solution. Then, equal masses of the above two parts were mixed, dissolved, and homogenized to obtain the drug-loaded lipid precursor oil phase formulation.

[0059] 45 °C was selected as the treatment temperature, and 100 μM sulfamethoxazole was selected as the effective concentration for exosome inhibition. The effects of single treatment methods and combined treatment with both on exosome release were investigated. Specifically, at a concentration of 1 × 10⁻⁶ μM... 7 A concentration of 4T1-CD63-GFP cells in logarithmic growth phase was seeded into the fat pad of the fourth pair of mammary glands in mice. The tumors were allowed to grow to 50-100 mm in size. 3 At the time of treatment, mice were injected with PBS (control group), GO@LG (single photothermal group), and SG@LG (no photothermal treatment: single exosome inhibition group; photothermal treatment: photothermal synergistic exosome inhibition group). For the photothermal group, photothermal therapy was administered at 4 h, 2 d, 4 d, and 6 d post-treatment. The temperature of the tumor site in mice was precisely controlled at 45℃ using an infrared imager. On day 8, mice were sacrificed, the tumors were dissected, and the tumor tissue was removed and weighed. The tumors were then minced, and exosomes were extracted from each group. The tissues were resuspended in 500 μL of PBS, and fluorescence values ​​were measured using a microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 530 nm. The fluorescence values ​​were then normalized to the tumor mass to evaluate the effect of different treatment methods on exosome release.

[0060] In vivo exosome inhibition effect such as Figure 6As shown, in vivo, single exosome inhibition effectively reduced exosome release from tumor cells to 56% of the control group; the photothermal group promoted exosome release from tumor cells, with the amount of exosomes released being approximately 1.3 times that of the control group; while the photothermal synergistic exosome inhibition group effectively inhibited the increase in exosome release caused by photothermal stimulation, with the amount of release being approximately 47% of the single photothermal group and 60% of the control group. This indicates that during combined treatment, administration of sulfamethoxazole effectively inhibits exosome release. Example 7

[0061] Tumor therapeutic agents for the treatment of breast cancer in mice.

[0062] Preparation of sulfamethoxazole-loaded graphene nanoparticles (SG): Sulfamethoxazole was dissolved in DMSO (10 mg / mL). At a sulfamethoxazole:graphene nanoparticle mass ratio of 10:1, 0.1 mg / mL graphene nanoparticle solution was added. An ultrasonic cell disruptor was used, with the ultrasonic power set to 30%, the ultrasonic mode set to 2 s sonication followed by 3 s pauses, and the sonication time 60 min. The resulting reaction product was added to an ultrafiltration tube with a 100 kDa cutoff, centrifuged, resuspended in ultrapure water, and washed three times to remove excess sulfamethoxazole. The resulting product was then filtered through a 0.22 μm microporous membrane to remove unstable nanoparticles, thus obtaining sulfamethoxazole-loaded graphene nanoparticles.

[0063] Preparation of sulfamethoxazole / graphene nanoparticle-containing lipid gel (SG@LG): First, graphene nanoparticles loaded with sulfamethoxazole, phospholipids, dioleoylglycerol, and surfactant (Tween 80) were vortexed and mixed at a mass ratio of 0.3:41:59:0.003, rapidly pre-frozen in liquid nitrogen, and lyophilized to prepare a photosensitizer-loaded lipid lyophilized powder. Second, phospholipids and dioleoylglycerol were weighed at a mass ratio of 41:59 and dissolved in anhydrous ethanol at 10% of the total mass of the two lipids to prepare a lipid precursor oil solution. Then, equal masses of the above two parts were mixed, dissolved, and homogenized to obtain the drug-loaded lipid precursor oil phase formulation.

[0064] Preparation of anti-PD-L1 antibody-containing lipid gel (aPDL1@LG): First, phospholipids, dioleoglycerides, aPDL1, and surfactant (Tween 80) were vortexed and mixed at a mass ratio of 41:59:0.5:0.003, rapidly pre-frozen in liquid nitrogen, and lyophilized to prepare aPDL1-loaded lipid lyophilized powder; Second, phospholipids and dioleoglycerides were weighed at a mass ratio of 41:59, and dissolved in 10% anhydrous ethanol of the total mass of the two lipids to prepare a lipid precursor oil solution; Then, equal masses of the above two parts were mixed, dissolved, and homogenized to obtain the drug-loaded lipid precursor oil phase formulation.

[0065] Preparation of lipid gel containing anti-PD-L1 antibody and sulfamethoxazole / graphene nanoparticle composition (aPDL1 / SG@LG): First, graphene nanoparticles loaded with sulfamethoxazole, phospholipids, dioleoylglycerol, aPDL1, and surfactant (Tween 80) were vortexed and mixed at a mass ratio of 0.3:41:59:0.5:0.003, rapidly pre-frozen in liquid nitrogen, and lyophilized to prepare a lipid lyophilized powder encapsulating the aPDL1 and sulfamethoxazole / graphene nanoparticle composition; Second, phospholipids and dioleoylglycerol (mass ratio of 41:59) were weighed and dissolved in 10% of the total mass of the two lipids in anhydrous ethanol to prepare a lipid precursor oil solution; Then, equal masses of the above two parts were mixed, dissolved, and homogenized to obtain the drug-loaded lipid precursor oil phase formulation.

[0066] 45 °C was selected as the treatment temperature. The tumor treatment agent contained 40 μg of sulfamethoxazole and 100 μg of anti-PD-L1 antibody. Specifically, it was administered at a dose of 1×10⁻⁶ μg / mL. 7 A concentration of 4T1 cells in logarithmic growth phase was seeded into the fat pad of the fourth pair of mammary glands in mice. The tumors were allowed to grow to 50-100 mm in size. 3 At the time of treatment, mice were injected with PBS (control group), aPDL1@LG (single immune checkpoint blockade therapy group), SG@LG+L (exosome inhibitor + thermotherapy group), and aPDL1 / SG@LG+L (exosome inhibitor + thermotherapy + immune checkpoint blockade therapy group). For the photothermal group, photothermal therapy was administered at 4 h, 2 d, 4 d, and 6 d after treatment. The temperature of the tumor site in the mice was precisely controlled at 45 ℃ using an infrared imager, and the size of the tumors was monitored to plot tumor growth curves.

[0067] Mouse tumor growth curve, such as Figure 7 As shown, the thermosensitive tumor therapeutic agent containing exosome inhibitors (aPDL1 / SG@LG+L) prepared in this invention has the best tumor inhibition rate, reaching 94% by day 30 after treatment; while the single immune checkpoint blockade group (aPDL1@LG) and the exosome inhibition + thermosensitive group (SG@LG+L) only showed weak tumor inhibition effects.

Claims

1. A thermosensitive tumor therapeutic agent, characterized in that, The product includes a thermosensitive gel containing antibodies, exosome inhibitors, and photosensitizers. The thermosensitive gel is a phospholipid / oil ester lipid gel, the antibody is an anti-PDL1 antibody, the exosome inhibitor is sulfamethoxazole, and the photosensitizer is graphene nanoparticles. The thermosensitive tumor therapeutic agent is prepared through the following steps: Step 1: Mix the exosome inhibitor and photosensitizer at a mass ratio of 10:1, and sonicate to obtain a photosensitizer loaded with exosome inhibitor; Step 2: The photosensitizer loaded with exosome inhibitor, antibody, phospholipid, dioleoyl glycerol, and surfactant are mixed in a mass ratio of 0.3:0.5:41:59:0.003, pre-frozen in liquid nitrogen, and lyophilized to prepare a lipid lyophilized powder loaded with exosome inhibitor / photosensitizer composition and antibody. Step 3: Dissolve phospholipids and dioleoglycerides in anhydrous ethanol at a mass ratio of 41:59, where the mass of anhydrous ethanol is 10% of the sum of the masses of phospholipids and dioleoglycerides, to prepare a lipid precursor oil solution. Step 4: Mix and dissolve an equal mass of the lipid lyophilized powder from Step 2 with the lipid precursor oil solution from Step 3 to obtain the lipid precursor oil phase formulation. Step 5: Inject the lipid precursor oil phase formulation from step 4 into water to obtain the thermosensitive tumor therapeutic agent.

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