Temperature-sensitive gel preparation containing glutaminase inhibitor as well as preparation method and application of temperature-sensitive gel preparation
By using a thermosensitive gel formulation under photothermal signal, it first releases glutaminase inhibitors to regulate the metabolism of the tumor microenvironment, and then releases immune checkpoint inhibitors to activate T cells, thus solving the problem of immunosuppression in the tumor microenvironment and achieving highly efficient anti-tumor immunotherapy.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHINA PHARM UNIV
- Filing Date
- 2026-01-16
- Publication Date
- 2026-04-21
AI Technical Summary
In current tumor immunotherapy, immune checkpoint inhibitors are not very effective due to immunosuppression caused by abnormally active glutamine metabolism in the tumor microenvironment.
Design a thermosensitive gel formulation comprising an outer gel layer and an inner gel layer, wherein the outer gel layer encapsulates the inner gel layer, the outer gel layer contains a glutaminase inhibitor and a photosensitizer, and the inner gel layer contains an immune checkpoint inhibitor. Drug release is controlled by photothermal signals, first releasing the glutaminase inhibitor to regulate metabolic state, and then releasing the immune checkpoint inhibitor to activate T cell immune response.
By regulating the metabolic state of the tumor microenvironment, immunosuppression can be alleviated, the effect of anti-tumor immunotherapy can be enhanced, and efficient tumor killing can be achieved.
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Figure CN121891291A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, specifically relating to a thermosensitive gel formulation containing a glutaminase inhibitor, its preparation method, and its application. Background Technology
[0002] Tumor immunotherapy, particularly inhibitors of immune checkpoints such as PD-1 / PD-L1, has achieved significant success in clinical applications. However, its efficacy is often limited by the presence of an immunosuppressive tumor microenvironment. Primary or secondary resistance to this therapy exhibited by patients remains a critical issue that urgently needs to be addressed in the field.
[0003] The formation of an immunosuppressive microenvironment is closely related to the abnormally active metabolic activities within the tumor microenvironment. Glutamine metabolism plays a central role in this process. On one hand, tumor cells exhibit an abnormal dependence on glutamine, highly expressing glutaminase (GLS) to take up and consume large amounts of glutamine in the microenvironment to support the biosynthesis and energy metabolism required for their rapid proliferation and to resist cell death. On the other hand, tumor cells also influence the glutamine metabolism of immune cells through various pathways. For example, tumor-associated macrophages, the most numerous immune cells in the tumor microenvironment, exhibit the M2 phenotype with high glutamine metabolic activity under the influence of metabolic interactions with tumor cells. They further exacerbate the immunosuppressive state of the tumor microenvironment by secreting immunosuppressive cytokines, thereby directly weakening the T-cell-mediated anti-tumor immune response and ultimately leading to poor efficacy of immune checkpoint inhibitors.
[0004] The effectiveness of immune checkpoint inhibitors depends on the presence and proper function of immune cells in the tumor microenvironment. Therefore, to maximize the efficacy of immunotherapy, preferentially modulating the immune status in the tumor microenvironment followed by immune checkpoint inhibitor treatment has been considered a feasible approach. Based on the above research, this invention designs a treatment strategy that preferentially delivers glutaminase inhibitors to overcome the immunosuppressive microenvironment caused by abnormal glutamine metabolism, followed by the delivery of immune checkpoint inhibitors. Summary of the Invention
[0005] One objective of this invention is to provide a thermosensitive gel formulation containing a glutaminase inhibitor, comprising an outer gel layer and an inner gel layer, wherein the outer gel layer covers the outside of the inner gel layer. The outer gel layer accounts for 40-75% of the total mass of the temperature-sensitive gel formulation, and the inner gel layer accounts for 25-60% of the total mass of the temperature-sensitive gel formulation. The outer gel is made from an outer gel matrix, a glutaminase inhibitor, a photosensitizer, and a stabilizer; the inner gel is made from an inner gel matrix and an immune checkpoint inhibitor.
[0006] Furthermore, the outer gel matrix and the inner gel matrix are selected from gelatin, carrageenan, poly(N-vinylcaprolactam) gel, and phospholipid-lipid liquid crystal gel. Phospholipid-lipid liquid crystal gel is preferred.
[0007] The glutaminase inhibitor is selected from CB-839, BPTES, and DON; the photosensitizer is selected from black phosphorus nanosheets, polydopamine nanoparticles, gold nanoparticles, and graphene materials; and the stabilizer is selected from serum albumin and mPEG. 2000 .
[0008] The immune checkpoint inhibitor is a drug that enhances the killing effect of T cells, selected from PD-1 inhibitors, CTLA-4 inhibitors, PD-L1 inhibitors, LAG-3 inhibitors, TIGIT inhibitors, and TIM-3 inhibitors.
[0009] Further, the mass of the glutaminase inhibitor accounts for 0.1-0.4% of the total mass of the thermosensitive gel formulation; the mass of the photosensitizer accounts for 0.05-0.2% of the total mass of the thermosensitive gel formulation; and the mass of the immune checkpoint inhibitor accounts for 0.05-0.2% of the total mass of the thermosensitive gel formulation.
[0010] Furthermore, for the preparation of the outer gel, a photosensitizer loaded with glutaminase inhibitor is first prepared by combining glutaminase inhibitor, photosensitizer and stabilizer. Then, the photosensitizer loaded with glutaminase inhibitor is dispersed into the outer gel matrix and finally injected into water to obtain the drug-loaded outer gel.
[0011] In a specific embodiment of the present invention, both the outer gel matrix and the inner gel matrix are phospholipid-dioleoglycerate lipid liquid crystal gels, the immune checkpoint inhibitor is an anti-PD-L1 antibody, the photosensitizer is black phosphorus nanosheets, the stabilizer is bovine serum albumin, and the glutaminase inhibitor is CB-839.
[0012] A second objective of this invention is to provide a method for preparing the above-mentioned thermosensitive gel formulation, comprising the following steps: Step 1: Mix the glutaminase inhibitor, photosensitizer, and stabilizer to obtain a photosensitizer loaded with the glutaminase inhibitor; Step 2: Add a photosensitizer loaded with a glutaminase inhibitor to part of the outer gel matrix, and freeze-dry to obtain the freeze-dried outer gel precursor. Step 3: Add the immune checkpoint inhibitor to part of the inner gel matrix, freeze-dry to obtain the inner gel precursor lyophilized product; Step 4: Mix the lyophilized outer lipid gel precursor with an equal mass of the outer gel matrix to obtain the outer gel precursor; mix the lyophilized inner lipid gel precursor with an equal mass of the inner gel matrix to obtain the inner gel precursor. Step 5: Load the outer gel precursor and the inner gel precursor into a double-barrel syringe to obtain the temperature-sensitive gel formulation.
[0013] In a specific embodiment of the present invention, the preparation method of the above-mentioned thermosensitive gel formulation containing glutaminase inhibitor includes the following steps: Step 1: Mix the glutaminase inhibitor, photosensitizer and stabilizer, shake at room temperature, centrifuge and wash to obtain the photosensitizer loaded with glutaminase inhibitor; Preferably, when the glutaminase inhibitor is CB-839, the photosensitizer is black phosphorus nanosheets, and the stabilizer is bovine serum albumin, the mass ratio of the glutaminase inhibitor, photosensitizer, and stabilizer is 2:1:50. Step 2: Mix the photosensitizer loaded with glutaminase inhibitor, phospholipid, dioleoyl glycerol and surfactant, quick freeze in liquid nitrogen, and then freeze dry to prepare the lyophilized outer lipid gel precursor containing the photosensitizer loaded with glutaminase inhibitor. Preferably, the mass ratio of the photosensitizer loaded with glutaminase inhibitor, phospholipid, dioleoyl glycerol and surfactant is 2:69:131:0.006; Step 3: Mix immune checkpoint inhibitors, phospholipids, dioleoyl glycerol and surfactants, freeze quickly in liquid nitrogen, and then freeze-dry to prepare an inner lipid gel precursor lyophilized product containing immune checkpoint inhibitors. Preferably, when the immune checkpoint inhibitor is an anti-PD-L1 antibody, the mass ratio of the immune checkpoint inhibitor, phospholipid, dioleoyl glycerol, and surfactant is 0.7:100:100:0.006. Step 4: Weigh out phospholipids and dioleoyl glycerol separately and dissolve them in anhydrous ethanol. Dissolve them by sonication to obtain an outer lipid oil solution and an inner lipid oil solution. Add the outer lipid oil solution and the inner lipid oil solution to the lyophilized outer lipid gel precursor and the lyophilized inner lipid gel precursor respectively, and vortex to dissolve them completely to obtain an outer lipid gel precursor solution and an inner lipid gel precursor solution. Preferably, for the outer lipid oil solution, the mass ratio of phospholipids to dioleoyl glycerol is 69:131; for the inner lipid oil solution, the mass ratio of phospholipids to dioleoyl glycerol is 100:100; and the amount of anhydrous ethanol used is 10% of the sum of the masses of phospholipids and dioleoyl glycerol. Step 5: Load the outer lipid gel precursor solution and the inner lipid gel precursor solution into a double-barrel syringe respectively to obtain the thermosensitive gel formulation; Preferably, the volume ratio of the outer lipid gel precursor solution to the inner lipid gel precursor solution is 2:1.
[0014] A third objective of this invention is to provide the application of the aforementioned thermosensitive gel formulation in the preparation of tumor therapeutic drugs. The tumor is various immunosuppressive tumors, including triple-negative breast cancer and melanoma.
[0015] In the inventors' previous research (Nano-Micro Letters, 2021, 13(1):141.), the focus was mainly on the feasibility of preparing thermosensitive bilayer lipid gels and their basic in vitro properties. However, the influence of structural parameters and the amount of inner and outer gel layers on the final in vivo biological effect was not thoroughly investigated. In particular, previous literature reports indicated that the immune status in the tumor microenvironment exhibits time-dynamic changes, with the proportion of inflammatory macrophages only reaching an ideal state from day 7 after treatment (Cell, 2018, 175(4):1014-1030.). Therefore, the compatibility between the thermosensitive bilayer lipid gel structure and changes in the in vivo microenvironment will significantly affect the synergistic effect of glutaminase inhibitors and immune checkpoint inhibitors. To this end, this invention, based on the time-dynamic process of the tumor microenvironment, further optimized and obtained a bilayer lipid gel with optimal performance, ultimately achieving the desired therapeutic effect.
[0016] The thermosensitive gel formulation of this invention has a bilayer lipid gel structure. Specifically, the outer lipid gel and the inner lipid gel have different gel-sol phase transition temperatures. The phase transition temperature of the outer lipid gel is 37-43°C, and the final mass ratio of phospholipids to dioleoglycerides in the outer gel is determined to be 69:131. The phase transition temperature of the inner lipid gel is significantly higher than that of the outer layer, above 50°C, and the final mass ratio of phospholipids to dioleoglycerides in the inner gel is determined to be 100:100. The outer lipid gel encapsulates a photosensitizer loaded with a glutaminase inhibitor, while the inner lipid gel encapsulates an immune checkpoint inhibitor. The dosage and volume ratio of the outer and inner gels in the thermosensitive gel formulation are determined by the in vivo time-kinetic changes in the tumor microenvironment, and the optimal dosage of the outer gel is determined to be 100 μl, and the optimal dosage of the inner gel is determined to be 50 μl.
[0017] The thermosensitive gel formulation of this invention sequentially releases a photosensitizer loaded with a glutaminase inhibitor and an immune checkpoint inhibitor. Under near-infrared light irradiation, the photosensitizer loaded on the outer layer generates a photothermal effect, raising the local temperature to the phase transition temperature of the outer lipid gel, causing it to transition from a gel state to a sol state, thereby preferentially releasing the photosensitizer loaded with the glutaminase inhibitor. Because the inner lipid gel has a higher phase transition temperature, it maintains structural integrity during this process, and the immune checkpoint inhibitor encapsulated in the inner layer is released with a delay as the gel slowly degrades.
[0018] This thermosensitive gel formulation aims to first regulate the metabolic state in the tumor microenvironment by using glutaminase inhibitors to alleviate immunosuppression, and then release immune checkpoint inhibitors that can effectively activate T-cell immune responses in this optimized microenvironment, ultimately achieving enhanced anti-tumor immunotherapy efficacy through synergistic effects.
[0019] The thermosensitive gel formulation provided by this invention can respond to photothermal signals and be used for the sequential delivery of glutaminase inhibitors and immune checkpoint inhibitors. Through photothermal control, this thermosensitive gel formulation first releases glutaminase inhibitors to alleviate immunosuppression, and then releases immune checkpoint inhibitors to kill tumors. The two work synergistically to achieve highly effective anti-tumor immunotherapy. Attached Figure Description
[0020] Figure 1 The results of the gel-sol phase transition assay are for a blank lipid gel. Figure 2 Photographs showing the gel-sol phase transition effect of drug-loaded lipid gels; Figure 3 To evaluate the in vitro photothermal conversion capacity of drug-loaded lipid gels; Figure 4 The results show the in vivo photothermal effect of the drug-loaded lipid gel; Figure 5 A structural photograph of a bilayer lipid gel; Figure 6 The results show the inhibitory effect of the active pharmaceutical ingredient in tumor therapeutic agents on glutaminase activity; Figure 7 Evaluation of the in vitro release behavior of thermosensitive tumor therapeutic agents under optimal prescription; Figure 8 Growth curve of tumors in mice after administration of a thermosensitive tumor therapeutic agent. Detailed Implementation
[0021] 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.
[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0023] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Example 1
[0024] Rheological behavior of blank lipid gels Phospholipids and dioleoglycerides were weighed at mass ratios of 69:131 and 100:100, respectively. Anhydrous ethanol was added at 10% of the total lipid mass. The mixture was sonicated until completely dissolved to obtain a blank lipid gel precursor solution. Then, 200 μl of each gel ball was injected into water. After the gel was fully formed, the blank lipid gels under the two formulations were obtained.
[0025] Take 200 mg of the above blank lipid gel and place it on a rheometer. Select a 20 mm flat plate clamp for measurement. Set the angular frequency (ω) to 6.28, the strain constant (γ) to 1%, the frequency to 1 Hz, and the heating rate to 10℃ / min. For a 69:131 ratio gel, set the measurement temperature to 25-55℃. For a 100:100 ratio gel, set the measurement temperature to 25-90℃. Measure the storage modulus (G') and loss modulus (G”) of the gel.
[0026] The gel-sol phase transition temperature of the blank lipid gel is as follows: Figure 1 As shown in the left-middle figure, the gel-sol phase transition temperature of a phospholipid:dioleoglycerate gel with a ratio of 69:131 is approximately 41.5℃. Below 41.5℃, the storage modulus is higher than the loss modulus; however, above 41.5℃, the loss modulus is higher than the storage modulus. This indicates that a gel structure can be successfully formed at normal body temperature, and the gel transforms into a sol state upon heating. For a phospholipid:dioleoglycerate gel with a ratio of 100:100, the gel-sol phase transition temperature is as high as 93.2℃, indicating that this gel formulation maintains its gel state during heating. Example 2
[0027] Evaluation of the gel-sol phase transition effect of drug-loaded lipid gels CB-839, black phosphorus nanosheets, and bovine serum albumin were mixed at a mass ratio of 2:1:50, shaken for 4 h, and centrifuged and washed to obtain black phosphorus nanosheets loaded with CB-839. The black phosphorus nanosheets loaded with CB-839, phospholipids, dioleoglyceride, and Tween 80 were mixed at a mass ratio of 2:69:131:0.006, rapidly frozen in liquid nitrogen, and then lyophilized to prepare a lyophilized precursor of the outer lipid gel containing black phosphorus nanosheets loaded with CB-839. IgG, phospholipids, dioleoglyceride, and surfactant were mixed at a mass ratio of 0.7:100:100:0.006, rapidly frozen in liquid nitrogen, and then lyophilized to prepare a lyophilized precursor of the inner lipid gel containing IgG. Phospholipids and dioleoglycerides were weighed at mass ratios of 69:131 and 100:100, respectively, and dissolved in anhydrous ethanol, with the mass of anhydrous ethanol being 10% of the combined mass of phospholipids and dioleoglycerides. The solutions were then sonicated to obtain an outer lipid oil solution and an inner lipid oil solution, respectively. These solutions were then added to the lyophilized outer and inner lipid gel precursors, respectively, and vortexed to ensure complete dissolution, yielding an outer lipid gel precursor solution and an inner lipid gel precursor solution, respectively.
[0028] To determine the response of the two gel formulations to thermal effects, 2 ml of water was slowly added to a vial containing the aforementioned lipid gel precursor solution. After the gel completely solidified, the upper aqueous phase was discarded, and the gel state was evaluated by inverting the vial. To determine whether the gel could accurately respond to thermal effects and produce changes in appearance, the gel was heated to 43°C in a water bath, and the fluidity of the gel was observed by inverting the vial. Finally, after the gel temperature returned to room temperature, the state of the gel was observed.
[0029] Figure 2 The gel states of two formulation gels under water, heating, and cooling treatments were demonstrated. Upon contact with water, the lipid gel precursor solution rapidly transformed into a gel state and solidified at the bottom of the vial. When the phospholipid:dioleoglycerate ratio was 69:131, heating to a 43°C water bath caused the gel to gradually transform into a flowable sol state, which then reverted to a gel state upon returning to room temperature. However, when the phospholipid:dioleoglycerate ratio was 100:100, the gel showed no significant thermosensitive properties between 25-43°C. These results indicate that the outer and inner lipid gels possess different thermosensitive properties and can be used to construct bilayer lipid gels with different drug release characteristics. Example 3
[0030] Evaluation of the in vitro photothermal conversion capacity of drug-loaded lipid gels CB-839, black phosphorus nanosheets, and bovine serum albumin were mixed at a mass ratio of 2:1:50, shaken for 4 h, and centrifuged and washed to obtain black phosphorus nanosheets loaded with CB-839. The black phosphorus nanosheets loaded with CB-839, phospholipids, dioleoylglycerol, and Tween 80 were mixed at a mass ratio of 2:69:131:0.006, rapidly frozen in liquid nitrogen, and then lyophilized to prepare a lyophilized lipid gel precursor containing black phosphorus nanosheets loaded with CB-839. Phospholipids and dioleoylglycerol were weighed at a mass ratio of 69:131 and dissolved in anhydrous ethanol (the mass of anhydrous ethanol being 10% of the sum of the masses of phospholipids and dioleoylglycerol). The solution was dissolved by sonication to obtain a lipid oil solution. The lipid oil solution was added to the lyophilized lipid gel precursor and vortexed to completely dissolve it, yielding the lipid gel precursor solution.
[0031] To determine the in vitro photothermal conversion capacity of the lipid gel, 50 μl of the above-mentioned gel precursor solution was slowly injected into 2 ml of water. After the gel completely solidified, the upper aqueous phase was discarded. Subsequently, an 808 nm near-infrared laser was used at a speed of 0.5 W / cm². 2 The sample was irradiated with high power, and the sample temperature was measured every 30 seconds using an infrared imager for a total of 10 minutes. The temperature change curve over time was plotted. A blank gel was used as a control.
[0032] Results of in vitro photothermal conversion capacity are as follows Figure 3 As shown, the lipid gel containing black phosphorus nanosheets loaded with CB-839 exhibits good photothermal conversion ability at 0.5 W / cm². 2 At low power, the gel temperature can be raised to 47℃ in just 30 s, with a maximum temperature reaching 90℃; while the temperature of the blank gel group remains almost unchanged. These results indicate that the lipid gel loaded with CB-839 black phosphorus nanosheets has good photothermal conversion ability. Example 4
[0033] Evaluation of the in vivo photothermal effect of drug-loaded lipid gel CB-839, black phosphorus nanosheets, and bovine serum albumin were mixed at a mass ratio of 2:1:50, shaken for 4 h, and centrifuged and washed to obtain black phosphorus nanosheets loaded with CB-839. The black phosphorus nanosheets loaded with CB-839, phospholipids, dioleoylglycerol, and Tween 80 were mixed at a mass ratio of 2:69:131:0.006, rapidly frozen in liquid nitrogen, and then lyophilized to prepare a lyophilized lipid gel precursor containing black phosphorus nanosheets loaded with CB-839. Phospholipids and dioleoylglycerol were weighed at a mass ratio of 69:131 and dissolved in anhydrous ethanol, where the mass of anhydrous ethanol was 10% of the sum of the masses of phospholipids and dioleoylglycerol. The solution was dissolved by sonication to obtain a lipid oil solution. The lipid oil solution was added to the above lyophilized lipid gel precursor, and vortexed to ensure complete dissolution, thus obtaining the lipid gel precursor solution.
[0034] To determine the in vivo photothermal effect of the lipid gel, 50 μl of the above-mentioned gel precursor solution was injected into the fat pad of the fourth pair of mammary glands in mice, and an 808 nm near-infrared laser was used at 0.5 W / cm². 2 The gel injection site was irradiated with high power, and the temperature was measured every 30 seconds using an infrared imager for a total of 10 minutes. The temperature change curve over time was plotted. Mice injected with phosphate-buffered saline (PBS) served as controls.
[0035] The photothermal effect was evaluated in mice, and the results were as follows: Figure 4 As shown, 0.5 W / cm 2 After irradiating the gel with the specified power for 2 minutes, the local temperature of the mouse mammary gland reached 43℃; after continuous irradiation for 10 minutes, the highest local temperature of the mouse mammary gland did not exceed 47℃, which is within the safe range. In contrast, the group injected with PBS only caused a slight increase in temperature at the injection site. These results indicate that the lipid gel containing black phosphorus nanosheets loaded with CB-839 can also produce ideal photothermal effects in vivo. To ensure the stability and consistency of the treatment temperature, the power of the near-infrared laser will be adjusted in real time to ensure that the photothermal temperature is controlled at 43±0.5℃. Example 5
[0036] Construction of bilayer lipid gel Phospholipids and dioleoglycerides were weighed at mass ratios of 69:131 and 100:100, respectively. Anhydrous ethanol, which is 10% of the total mass of lipids, was added and sonicated until completely dissolved to obtain an outer lipid gel precursor solution and an inner lipid gel precursor solution.
[0037] To distinguish the interface between the inner and outer gel layers, a small amount of methylene blue was added to the inner layer lipid gel precursor solution. Then, 100 μl of the outer layer and 50 μl of the inner layer lipid gel precursor solution were injected into water sequentially using a dual-channel syringe. After standing for 1 minute, the gel shape was photographed and recorded.
[0038] Front and side views of a bilayer lipid gel are shown below. Figure 5 As shown, the outer gel is transparent and the inner gel is blue. The structural boundary between the inner and outer bilayer gels is obvious, and the inner gel is located in the middle of the bilayer lipid gel, indicating the successful formation of the bilayer lipid gel. Example 6
[0039] The inhibitory effect of active pharmaceutical ingredients in tumor therapeutic agents on glutaminase activity CB-839, black phosphorus nanosheets, and bovine serum albumin were mixed at a mass ratio of 2:1:50, shaken for 4 h, and then centrifuged and washed to obtain black phosphorus nanosheets loaded with CB-839.
[0040] To verify that the tumor therapeutic agent can effectively block the metabolism of glutaminase, the active pharmaceutical ingredient CB-839 in the tumor therapeutic agent and black phosphorus nanosheets loaded with CB-839 (concentration based on CB-839, 400 ng / ml) were incubated with 4T1 cells and RAW 264.7 cells for 3 h, and then the enzyme activity was measured using a glutaminase activity assay kit.
[0041] Figure 6 This study showcases the glutaminase activity of 4T1 and RAW 264.7 cells after treatment with CB-839 and CB-839-loaded black phosphorus nanosheets. The results indicate that both CB-839 and CB-839-loaded black phosphorus nanosheets significantly reduced glutaminase activity, with the CB-839-loaded nanosheets showing a superior effect. This may be due to the easier uptake of the drug by cells after loading onto the black phosphorus nanosheets. These results demonstrate that CB-839 and CB-839-loaded black phosphorus nanosheets, as active pharmaceutical ingredients in the outer gel, can effectively inhibit glutaminase activity in tumor cells and macrophages. Example 7
[0042] Evaluation of the in vitro release behavior of thermosensitive tumor therapeutic agents under optimal prescription CB-839, black phosphorus nanosheets, and bovine serum albumin were mixed at a mass ratio of 2:1:50, shaken for 4 h, and centrifuged and washed to obtain black phosphorus nanosheets loaded with CB-839. The black phosphorus nanosheets loaded with CB-839, phospholipids, dioleoglyceride, and Tween 80 were mixed at a mass ratio of 2:69:131:0.006, rapidly frozen in liquid nitrogen, and then lyophilized to prepare a lyophilized precursor of the outer lipid gel containing black phosphorus nanosheets loaded with CB-839. IgG, phospholipids, dioleoglyceride, and surfactant were mixed at a mass ratio of 0.7:100:100:0.006, rapidly frozen in liquid nitrogen, and then lyophilized to prepare a lyophilized precursor of the inner lipid gel containing IgG. Phospholipids and dioleoglycerides were weighed at mass ratios of 69:131 and 100:100, respectively, and dissolved in anhydrous ethanol, with the mass of anhydrous ethanol being 10% of the combined mass of phospholipids and dioleoglycerides. The solutions were then sonicated to obtain an outer lipid oil solution and an inner lipid oil solution. These solutions were then added to their respective lyophilized outer and inner lipid gel precursors, and vortexed to ensure complete dissolution, yielding an outer lipid gel precursor solution and an inner lipid gel precursor solution.
[0043] To determine the drug release profile in the gel, the outer and inner lipogel precursor solutions were injected using a dual-channel syringe into a solution containing 10% PEG. 2000In PBS, 100 μl of the outer lipogel precursor solution and 50 μl of the inner lipogel precursor solution were injected. After gelation, the mixture was placed in a shaker at 37°C and shaken. 2 ml samples were taken at different time points (0.5 d, 1 d, 2 d, 4 d, 6 d, 8 d, 10 d, 12 d, 14 d), and an equal volume of solution containing 10% PEG was added. 2000 PBS was used. For the photothermal treatment group, samples were taken on days 1 and 2, and irradiated with an 808 nm near-infrared laser for 10 min at a controlled temperature of 43 ± 0.5 °C. The content of CB-839 was determined by HPLC, and the content of IgG was determined by the BCA method. Cumulative release curves of the two drugs under different experimental conditions were plotted.
[0044] The cumulative release curves of CB-839 and IgG are as follows: Figure 7 As shown, both CB-839 and IgG exhibited some degree of burst release during the initial gel formation stage, with approximately 10% of the drug released within one day. Consistent with expected results, photothermal treatment had different effects on the outer and inner gel layers: after photothermal treatment, the release of CB-839 in the outer lipid gel was significantly higher than in the non-photothermal group, while the release of IgG in the inner gel was similar to that in the non-photothermal group. Notably, the release of CB-839 essentially plateaued on day 8, while IgG showed no significant release for approximately seven days after day 1, only experiencing further rapid release after day 8. These differentiated release characteristics ensure that the two drugs have different sensitivities to photothermal signals, leading to the glutaminase inhibitor's effect preceding that of the immune checkpoint inhibitor. Furthermore, this release process conforms to the time-dynamic processes within the tumor microenvironment, enabling enhanced tumor immunotherapy. Example 8
[0045] Evaluation of the in vivo antitumor effect of thermosensitive tumor therapeutic agents CB-839, black phosphorus nanosheets, and bovine serum albumin were mixed at a mass ratio of 2:1:50, shaken for 4 h, and centrifuged and washed to obtain black phosphorus nanosheets loaded with CB-839. For the drug-containing outer lipid gel precursor and drug-containing inner lipid gel precursor solutions, black phosphorus nanosheets loaded with CB-839, phospholipids, dioleoylglycerol, and Tween 80 were mixed at a mass ratio of 2:69:131:0.006, rapidly frozen in liquid nitrogen, and then lyophilized to prepare the lyophilized outer lipid gel precursor containing black phosphorus nanosheets loaded with CB-839. Anti-PDL1 antibody, phospholipids, dioleoylglycerol, and surfactant were mixed at a mass ratio of 0.7:100:100:0.006, rapidly frozen in liquid nitrogen, and then lyophilized to prepare the lyophilized inner lipid gel precursor containing anti-PDL1 antibody. Phospholipids and dioleoglycerides were weighed at mass ratios of 69:131 and 100:100, respectively, and dissolved in anhydrous ethanol, with the mass of anhydrous ethanol being 10% of the combined mass of phospholipids and dioleoglycerides. The solutions were then sonicated to obtain an outer lipid oil solution and an inner lipid oil solution. These solutions were then added to the corresponding lyophilized outer and inner lipid gel precursors, respectively, and vortexed to ensure complete dissolution, yielding an outer lipid gel precursor solution and an inner lipid gel precursor solution.
[0046] For the CB-839-loaded black phosphorus nanosheet solution, CB-839, black phosphorus nanosheets, and bovine serum albumin were mixed at a ratio of 2:1:50, shaken for 4 h, and then centrifuged and washed to obtain the CB-839-loaded black phosphorus nanosheets. For the anti-PDL1 antibody solution, the anti-PDL1 antibody was directly diluted with PBS to an appropriate concentration for subsequent injection.
[0047] For drug-free blank lipid gels, phospholipids and dioleoglycerides were weighed at mass ratios of 69:131 and 100:100, respectively, and dissolved in anhydrous ethanol. The mass of anhydrous ethanol was 10% of the sum of the masses of phospholipids and dioleoglycerides. The solutions were then sonicated to obtain blank outer lipid gel precursor solutions and blank inner lipid gel precursor solutions.
[0048] 4T1 cells in logarithmic growth phase were inoculated at a rate of 1 million per mouse into the fat pad of the fourth pair of mammary glands. The tumors were allowed to grow to 50-100 mm. 3Mice were evenly divided into 7 groups. According to the groupings shown in Table 1, the outer and inner layers of the lipogel precursor solution were injected sequentially into the tumor using a dual-channel syringe. The injection volume of the outer lipogel precursor solution was 100 μl, and the injection volume of the inner lipogel precursor solution was 50 μl. For groups requiring photothermal therapy, photothermal treatment was performed at 4 h, 2 d, 4 d, and 8 d after drug injection. An 808 nm near-infrared laser was used to irradiate the injection site, and the photothermal temperature was controlled at 43 ± 0.5 ℃ using an infrared thermal imager. Tumor volume was measured every 4 days for a total of 28 days, and growth curves for each group of mice were plotted.
[0049] Table 1
[0050] The results are as follows Figure 8 As shown, the tumor therapeutic agent (BP / CB-839@gel + aPDL1@gel + L) prepared in this invention exhibits the best therapeutic efficacy, completely curing the orthotopic tumors in 5 / 7 mice. The free drug photothermal group (BP / CB-839 solution + aPDL1 solution + L) and the outer gel photothermal group (BP / CB-839@gel + Blankgel + L) both effectively controlled tumor occurrence and development, significantly slowing tumor growth. The double-layer gel non-photothermal group (BP / CB-839@gel + aPDL1@gel - L) and the inner gel non-photothermal group (Blank gel + aPDL1@gel - L) both showed weaker tumor-inhibiting effects, with tumors still growing relatively rapidly. These results demonstrate that the tumor therapeutic agent prepared in this invention has excellent therapeutic effects and can achieve enhanced tumor immunotherapy to eradicate tumors.
Claims
1. A temperature-sensitive gel formulation, characterized in that, It includes an outer gel and an inner gel, wherein the outer gel covers the outside of the inner gel; The outer gel layer accounts for 40-75% of the total mass of the temperature-sensitive gel formulation, and the inner gel layer accounts for 25-60% of the total mass of the temperature-sensitive gel formulation. The outer gel is made from an outer gel matrix, a glutaminase inhibitor, a photosensitizer, and a stabilizer; the inner gel is made from an inner gel matrix and an immune checkpoint inhibitor.
2. The thermosensitive gel formulation according to claim 1, characterized in that, The outer gel matrix and the inner gel matrix are selected from gelatin, carrageenan, poly(N-vinylcaprolactam) gel, phospholipid-lipid liquid crystal gel; preferably phospholipid-lipid liquid crystal gel. The glutaminase inhibitor is selected from CB-839, BPTES, and DON; the photosensitizer is selected from black phosphorus nanosheets, polydopamine nanoparticles, gold nanoparticles, and graphene materials; and the stabilizer is selected from serum albumin and mPEG. 2000 ; The immune checkpoint inhibitor is a drug that enhances the killing effect of T cells, selected from PD-1 inhibitors, CTLA-4 inhibitors, PD-L1 inhibitors, LAG-3 inhibitors, TIGIT inhibitors, and TIM-3 inhibitors.
3. The thermosensitive gel formulation according to claim 1, characterized in that, The mass of the glutaminase inhibitor accounts for 0.1-0.4% of the total mass of the thermosensitive gel formulation; the mass of the photosensitizer accounts for 0.05-0.2% of the total mass of the thermosensitive gel formulation; and the mass of the immune checkpoint inhibitor accounts for 0.05-0.2% of the total mass of the thermosensitive gel formulation.
4. The thermosensitive gel formulation according to claim 1, characterized in that, For the preparation of the outer gel, a photosensitizer loaded with glutaminase inhibitor is first prepared by combining glutaminase inhibitor, photosensitizer and stabilizer. Then, the photosensitizer loaded with glutaminase inhibitor is dispersed into the outer gel matrix and finally injected into water to obtain the drug-loaded outer gel.
5. The method for preparing the thermosensitive gel formulation according to any one of claims 1 to 4, characterized in that, Includes the following steps: Step 1: Mix the glutaminase inhibitor, photosensitizer, and stabilizer to obtain a photosensitizer loaded with the glutaminase inhibitor; Step 2: Add a photosensitizer loaded with a glutaminase inhibitor to part of the outer gel matrix, and freeze-dry to obtain the freeze-dried outer gel precursor. Step 3: Add the immune checkpoint inhibitor to part of the inner gel matrix, freeze-dry to obtain the inner gel precursor lyophilized product; Step 4: Mix the lyophilized outer lipid gel precursor with an equal mass of the outer gel matrix to obtain the outer gel precursor; mix the lyophilized inner lipid gel precursor with an equal mass of the inner gel matrix to obtain the inner gel precursor. Step 5: Load the outer gel precursor and the inner gel precursor into a double-barrel syringe to obtain the temperature-sensitive gel formulation.
6. The use of the thermosensitive gel formulation according to any one of claims 1 to 4 in the preparation of tumor therapeutic drugs.
7. The application according to claim 6, characterized in that, The tumors mentioned are immunosuppressive tumors, including triple-negative breast cancer and melanoma.