Thermo-sensitive double-layer gel containing PARP inhibitor and application of thermo-sensitive double-layer gel

Abstract

The invention discloses temperature-sensitive double-layer gel containing a PARP inhibitor and application of the temperature-sensitive double-layer gel, and belongs to the technical field of biological medicine. The temperature-sensitive double-layer gel comprises inner-layer gel and outer-layer gel, the inner-layer gel is coated with the outer-layer gel, the inner-layer gel comprises an inner-layer gel matrix and a CD47 inhibitor, and the outer-layer gel comprises an outer-layer gel matrix, PARPi and a photosensitizer. As the inner-layer gel and the outer-layer gel have different gel-sol phase transition temperatures, sequential release of PARPi and aCD47 antibodies can be realized. The PARPi, the photosensitizer and the CD47 inhibitor are jointly entrapped in the temperature-sensitive gel, so that the CD47 inhibitor, the mild photothermal therapy and the PARPi are combined for combined treatment of tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a thermosensitive bilayer gel containing a PARP inhibitor and its application in the preparation of tumor immunotherapy drugs. Background Technology

[0002] Triple-negative breast cancer is a highly aggressive subtype of breast cancer with a poor prognosis, accounting for approximately one-fifth of all breast cancer cases. Due to the negative expression of multiple tumor-targeting receptors such as estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2), patients are unlikely to benefit from endocrine therapy and anti-HER2 targeted drugs, resulting in a low overall survival rate and an extremely high risk of recurrence and metastasis.

[0003] Recent studies have revealed a high proportion of homologous recombination repair gene defects in triple-negative breast cancer, particularly BRCA1 / 2 gene mutations. This deficiency leads tumor cells to rely on alternative DNA repair pathways, such as the poly(ADP-ribose) polymerase pathway, to maintain genomic stability, making tumors sensitive to poly(ADP-ribose) polymerase inhibitors (PARPi). However, the highly immunosuppressive tumor microenvironment results in high PD-L1 expression and difficulty in T cell infiltration, often leading to poor efficacy of PARPi.

[0004] Related studies have shown that tumor-associated macrophages (TAMs) not only establish immunosuppressive barriers through cytokine secretion, but also directly mediate resistance to PARPi. In previous studies, the inventors' research group found that thermotherapy (Nature Communications, 2019, 10:4871.) can effectively promote the reprogramming of TAMs, polarizing them from M2-like macrophages to M1-type macrophages. This not only effectively increases the infiltration of immune cells in the tumor immune microenvironment, but also increases the phagocytosis of tumor cells by macrophages and promotes autoimmune circulation.

[0005] However, the reprogramming induced by hyperthermia leads to the high expression of CD47 on tumor cells. Mechanistic studies have shown that the expression of CD47 can counteract the reprogramming effect. CD47 binds to the SIRPα protein on macrophages, transmitting escape signals to avoid being phagocytosed by macrophages. It is evident that blocking CD47 is crucial for macrophages to perform their phagocytic function.

[0006] Based on this, the inventors designed a combination of PARPi and CD47 inhibitors to alleviate the immunosuppressive effects of the tumor immune microenvironment and enhance the therapeutic effect of PARPi. Summary of the Invention

[0007] One objective of this invention is to provide a temperature-sensitive bilayer gel agent, comprising an inner gel layer and an outer gel layer, wherein the outer gel layer covers the inner gel layer, and the volume ratio of the inner gel layer to the outer gel layer is 1:2. The inner gel comprises an inner gel matrix and a CD47 inhibitor, wherein the mass of the CD47 inhibitor is 0.1-0.3% of the inner gel matrix. The outer gel comprises an outer gel matrix, PARPi, and a photosensitizer. The mass of PARPi is 0.3-3% of the outer gel matrix, and the mass of the photosensitizer is 0.2-0.5% of the outer gel matrix.

[0008] Furthermore, the inner gel matrix and the outer gel matrix are thermosensitive gels, selected from phospholipid-oleic acid lipid gels, chitosan-glycerophosphate gels, poloxamer hydrogels, and poly-N-isopropylacrylamide hydrogels.

[0009] Furthermore, the inner gel matrix and the outer gel matrix are phospholipid-oleic acid lipid gels, preferably phospholipids and dioleoglycerides.

[0010] In order to achieve the goal of a phase transition temperature of 37-43°C for the outer gel and a phase transition temperature of over 50°C for the inner gel, the amount of phospholipid to dioleoglyceride in the outer gel matrix is ​​33:67-34:66, and the amount of phospholipid to dioleoglyceride in the inner gel matrix is ​​35:65-70:30.

[0011] In this invention, the photosensitizer in the outer gel absorbs light energy and converts it into heat energy, causing the outer gel to heat up above the phase transition temperature, changing from a gel state to a sol state, thereby preferentially releasing PARPi.

[0012] In this invention, the CD47 inhibitor is selected from Magrolimab, Lemzoparlimab, Evorpacept, HX009, and anti-CD47 antibody; the PARPi is selected from fluzoparib, tapazoli, rucaparib, niraparib, and olaparib; and the photosensitizer is selected from graphene nanoparticles and black phosphorus nanoparticles.

[0013] In this invention, the photosensitizer is used as a carrier for PARPi. First, PARPi and photosensitizer are combined, and then the photosensitizer loaded with PARPi is dispersed into the gel matrix.

[0014] The photosensitizer selected in this invention is a two-dimensional layered nanosheet, which not only serves as a drug carrier but also as a switch for photosensitizer and mild photothermal therapy. The nanosheet is first encapsulated with the drug and then co-encapsulated in a thermosensitive gel. In addition, the nanosheet has good biocompatibility and can degrade into harmless phosphate in vivo.

[0015] A second objective of this invention is to provide a method for preparing the aforementioned temperature-sensitive bilayer gel, comprising the following steps: Step 1: Mix the PARPi solution and the photosensitizer solution, centrifuge, and resuspend in water to obtain the PARPi-loaded photosensitizer; Step 2: Add the photosensitizer loaded with PARPi to part of the outer gel matrix, and freeze-dry to obtain the freeze-dried outer gel precursor. Step 3: Add CD47 inhibitor to part of the inner layer gel matrix, freeze-dry to obtain the inner layer gel precursor freeze-dried product; Step 4: Mix the lyophilized outer gel precursor with an equal mass of the outer gel matrix to obtain the outer gel precursor; mix the lyophilized inner 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 bilayer gel agent.

[0016] In a specific embodiment of the present invention, the method for preparing the temperature-sensitive bilayer gel agent includes the following steps: Step 1: Mix PARPi and photosensitizer at a mass ratio of 2:1 under continuous stirring in the dark, centrifuge, and resuspend in ultrapure water to obtain photosensitizer loaded with PARPi; Step 2: Mix 30-40 parts by weight of phospholipids, 60-68 parts by weight of glycerides and 2-12 parts by weight of solubilizer to obtain an outer lipid gel matrix. Then add a photosensitizer loaded with PARPi to the outer lipid gel matrix and freeze to obtain a lyophilized outer lipid gel precursor loaded with PARPi. Step 3: Mix 40-60 parts by weight of phospholipids, 45-65 parts by weight of glycerides and 4-14 parts by weight of solubilizer to obtain an inner lipid gel matrix. Then add aCD47 antibody to the inner lipid gel matrix and freeze-dry to obtain a CD47 antibody-loaded inner lipid gel precursor freeze-dried product. Step 4: Mix the lyophilized outer lipid gel precursor with an equal mass of the outer lipid gel matrix to obtain an outer lipid oil solution; mix the lyophilized inner lipid gel precursor with an equal mass of the inner lipid gel matrix to obtain an inner lipid oil solution. Step 5: The outer lipid oil solution and the inner lipid oil solution are respectively loaded into the syringe barrel of the double-barrel syringe, and the inner lipid oil solution and the outer lipid oil solution are pushed into the aqueous phase in sequence. After the inner gel precursor gels upon contact with water, the outer gel precursor gels upon contact with water and encapsulates the inner gel, thereby forming a double-layer drug-loaded gel.

[0017] A third objective of this invention is to provide the application of the above-mentioned thermosensitive bilayer gel in the preparation of tumor therapeutic drugs. Preferably, the tumor is triple-negative breast cancer.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The thermosensitive bilayer gelling agent of the present invention has a bilayer lipid gel structure, specifically, the inner gel and the outer gel have different gel-sol phase transition temperatures. The phase transition temperature of the outer gel is 37-45℃, while the phase transition temperature of the inner gel is higher than 50℃, which is much higher than that of the outer gel.

[0019] The thermosensitive bilayer gel of this invention can sequentially release PARPi and aCD47 antibody. Specifically, under near-infrared local irradiation, the photosensitizer in the outer gel absorbs light energy and converts it into heat energy, causing the outer gel to heat up above its phase transition temperature, changing from a gel state to a sol state, thereby preferentially releasing PARPi; while the inner gel is unaffected by photothermal effects, and the aCD47 antibody encapsulated therein is slowly released as the gel structure gradually degrades in vivo.

[0020] This invention encapsulates PARPi, a photosensitizer, and a CD47 inhibitor in a thermosensitive gel, enabling the combined treatment of tumors, particularly triple-negative breast cancer, with CD47 inhibitors and mild photothermal therapy in conjunction with PARPi. Attached Figure Description

[0021] Figure 1 Results of screening for the proportion of lipid gel formulation in the inner layer.

[0022] Figure 2 The results of screening the formulation ratio of the outer lipid gel.

[0023] Figure 3 The result is the construction of a bilayer lipid gel.

[0024] Figure 4 This serves as verification of the lipid gel bilayer structure.

[0025] Figure 5 The results show the stability of the in vitro photothermal conversion efficiency of drug-loaded black phosphorus nanoparticles.

[0026] Figure 6 For monitoring in vivo thermotherapy of drug-loaded lipid gels.

[0027] Figure 7 This refers to the in vitro drug release from drug-loaded lipid gels.

[0028] Figure 8 This refers to the in vivo drug release from drug-loaded lipid gels.

[0029] Figure 9 To evaluate the in vivo pharmacokinetic processes of different prescription thermosensitive tumor therapeutic agents.

[0030] Figure 10The results show the effect of aCD47 antibody on the phagocytic activity of macrophages in vitro.

[0031] Figure 11 This is the result of treating triple-negative breast cancer in mice with a tumor therapeutic agent. Detailed Implementation

[0032] To overcome the suppressive tumor immune microenvironment and utilize PARPi for targeted therapy in patients with BRCA1 / 2 gene mutations and triple-negative breast cancer, this invention provides a bilayer tumor therapeutic agent containing a CD47 inhibitor. This therapeutic agent can sequentially release PARPi and aCD47 antibody under thermotherapy. Under near-infrared local irradiation, the PARPi contained in the outer gel is rapidly released, while the CD47 inhibitor contained in the inner gel is slowly released. This enhances the phagocytic activity of macrophages under thermotherapy reprogramming, optimizes antigen cross-presentation, and thereby activates T-cell immunity.

[0033] Thermosensitive gels serve as excellent drug reservoirs. When the ambient temperature is higher than the phase transition temperature of the gel, they exhibit a sol state, which can accelerate drug release. When the ambient temperature is lower than the phase transition temperature of the gel, they exhibit a gel state, which can store hydrophilic, hydrophobic, and amphiphilic drugs for a long time and release them slowly, thus achieving the purpose of sustained-release and controlled-release.

[0034] In previous studies, the inventors' research group had initially focused on the feasibility of preparing thermosensitive bilayer lipid gels and their basic in vitro properties (Nano-Micro Letters, 2021, 13(1):141.), but had not thoroughly investigated the influence of their structural parameters and the amount of inner and outer gel layers on the final in vivo biological effects. Related studies have shown that the immune status in the tumor microenvironment exhibits time-dynamic changes (Cell, 2018, 175(4):e19), especially the proportion of inflammatory macrophages, which only reaches an ideal state one week after treatment. Therefore, the matching between the thermosensitive bilayer lipid gel structure and changes in the in vivo microenvironment will greatly affect the synergistic effect of RARPi and CD47 inhibitors. Based on the time-dynamic process of the tumor microenvironment, this invention further optimizes and screens the ratio of bilayer lipid gels with the best performance, ultimately achieving the ideal therapeutic effect.

[0035] Through optimization and screening, this invention determined the optimal volume ratio of the inner and outer gel layers to be 1:2. This ratio is crucial for achieving a high degree of synergy between the drug release behavior of the inner and outer layers and the body's biological metabolic processes, ensuring time-matched therapeutic kinetics. Specifically, the PARPi rapidly released by the bilayer lipid gel under photothermal triggering initially induces DNA damage and immunogenic death in tumor cells, creating preconditions for macrophage recognition and phagocytosis; the inner gel continuously releases aCD47 antibody, further relieving the inhibition of macrophage phagocytosis. Its release kinetics coincide with the innate immune activation process of macrophage activation, phagocytosis, and subsequent antigen presentation, more efficiently reshaping the tumor immune microenvironment and activating the system's anti-tumor immune response, thereby achieving the best therapeutic effect.

[0036] 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.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0038] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Example 1

[0039] Screening of Inner Lipid Gel Formulation Ratio To determine the gel-forming properties of lipids with different composition ratios, lipids with different compositions and mass ratios of phospholipids to dioleoglycerides (PGEs) of 10:90-90:10 were studied. Phospholipids and dioleoglycerides were weighed separately according to the mass ratio, and dissolved in anhydrous ethanol at 10% of the total mass of the two lipids to prepare lipid precursor oil solutions. The precursor oil solutions were then injected into purified water to prepare blank lipid gels.

[0040] The gelation properties of blank lipid gels with different proportions are as follows: Figure 1 As shown in Figure A, a good lipid gel system can be formed when the lipid ratio is between 30:70 and 70:30.

[0041] Phospholipids and dioleoglycerides were weighed at a mass ratio of 35:65, and dissolved in anhydrous ethanol at 10% of the total mass of the two lipids to prepare an inner layer lipid precursor oil solution. The precursor oil solution was then injected into purified water to prepare an inner layer blank lipid gel.

[0042] A 200 mg sample of inner-layer blank lipid gel was placed in the center of a 20 mm rheometer sample plate and equilibrated at 25 °C for 10 min. Then, an oscillating temperature gradient test was performed by heating from 25 °C to 100 °C at a rate of 1 °C / min. The strain constant (γ) was controlled at 5%, and the angular frequency (ω) at 2 rad / s. The storage modulus (G') and loss modulus (G") were monitored within the frequency range of 0.1–100 Hz to characterize the thermoresponsive sol-gel transition behavior.

[0043] The results of the inner gel temperature-sensitive property test are as follows: Figure 1 As shown in Figure B, its phase transition temperature is 58.003 ℃, indicating that under mild photothermal therapy conditions (around 45 ℃), the inner gel always remains in a gel state. Example 2

[0044] Screening of outer lipid gel formulation ratio To determine the ratio range of phospholipids and dioleoglycerides in the outer gel carrier, phospholipids and dioleoglycerides were weighed at mass ratios of 30:70, 31:69, 32:68, 33:67, and 34:66, respectively. They were dissolved in anhydrous ethanol at 10% of the total mass of the two lipids to prepare outer lipid precursor oil solutions with different ratios. The above precursor oil solutions were injected into purified water to prepare outer blank lipid gels.

[0045] 200 mg of the above lipid gel samples were placed in the center of a 20 mm rheometer sample plate and equilibrated at 25 °C for 10 min. Then, the temperature was increased from 25 °C to 100 °C at a heating rate of 1 °C / min to perform oscillating temperature gradient tests. The strain constant (γ) was controlled at 5% and the angular frequency (ω) was 2 rad / s. The storage modulus (G') and loss modulus (G") were monitored in the frequency range of 0.1-100 Hz to characterize the thermoresponsive sol-gel transition behavior.

[0046] The results of the reversible thermosensitive properties determination of outer gels with different proportions are as follows: Figure 2 As shown in Figure A, the phase transition temperature of the outer gel carrier with a dosage of 33:67-34:66 is within the range of mild photothermal therapy conditions (37-45 ℃).

[0047] Furthermore, the formation of a stable bilayer gel structure by encapsulating the inner gel within this ratio range was verified. Phospholipids and dioleoglycerides were weighed at a mass ratio of 50:50, and 1 mg of Oil Red chromogenic agent was added to better distinguish the inner and outer layer boundaries. The mixture was also dissolved in 10% anhydrous ethanol (based on the total mass of the two lipids) to prepare an inner layer lipid precursor oil solution. The outer and inner layer lipid precursor oil solutions (33:67 or 34:66) were respectively loaded into the syringe barrel of a double-barrel syringe. 100 μl and 50 μl of the outer and inner layer solutions were then injected into the aqueous phase, respectively. The outer gel precursor gelled upon contact with water, encapsulating the inner gel and forming a bilayer lipid gel structure.

[0048] The formed lipid bilayer gel structure was observed after standing for 10 min, and the results are as follows: Figure 2 As shown in Figure B, both ratios of the outer gel carrier can effectively encapsulate the inner gel to form a bilayer lipid gel structure. Therefore, the ratio of phospholipids to dioleoglycerides in the outer gel carrier is determined to be 33:67-34:66; and the ratio of phospholipids to dioleoglycerides in the inner gel carrier is determined to be 35:65-70:30. Example 3

[0049] Construction of bilayer lipid gel Preferably, the lipid composition ratio of phospholipids and dioleoglycerides is selected as 34:66 and 50:50, respectively.

[0050] Phospholipids and dioleoglycerides were weighed in ratios of 34:66 and 50:50, respectively, and 10% by mass of anhydrous ethanol was added as a co-solvent. After sonication until completely dissolved, an outer layer gel precursor oil solution and an inner layer lipid gel precursor oil solution were obtained.

[0051] 1 mg of oil red dye was added to the inner gel to better facilitate visual differentiation between the inner and outer gel layers. Then, the two gel precursor oil solutions were loaded into a dual-channel syringe, and the outer and inner gel precursor oil solutions were injected sequentially by adjusting the three-way valve. After standing for 10 minutes, the gel morphology was photographed and recorded.

[0052] Front and side views of a bilayer lipid gel are shown below. Figure 3 As shown, the outer gel is pale yellow and the inner gel is dark red. The two layers of gel structure are clearly separated, and the inner gel is well located in the center of the outer gel, indicating the successful construction of the bilayer gel. Example 4

[0053] Validation of lipid gel bilayer structure Preferably, egg phosphatidylcholine (EPC) and glycerol dioleate (GDO) are selected as the constituent lipids of the phospholipid / oleic acid lipid gel.

[0054] Preparation of blank lipid bilayer gels: Phospholipids and dioleoglycerides were weighed at a mass ratio of 34:66 and dissolved thoroughly in anhydrous ethanol (10% of the total mass of the two lipids) to prepare an outer lipid precursor oil solution. Phospholipids and dioleoglycerides were weighed at a mass ratio of 50:50 and dissolved thoroughly in anhydrous ethanol (10% of the total mass of the two lipids) to prepare an inner lipid precursor oil solution. The two gel precursor oil solutions were loaded into a dual-channel syringe. By controlling the three-way valve, the outer and inner gel precursor oil solutions were injected sequentially into the aqueous phase. The outer gel precursor gelled upon contact with water, encapsulating the inner gel to form a lipid bilayer gel structure.

[0055] Preparation of frozen sections of lipid bilayer gel: A blank lipid bilayer gel was placed and suspended in OCT frozen section embedding medium and frozen solidified at -80 ℃. The solidified block was sectioned at -30 ℃ using a cryostat, with a section thickness of 15 μm. The surface embedding medium was washed away with PBS buffer, and a coverslip was placed on the section to complete the preparation of frozen sections of lipid bilayer gel.

[0056] Preparation of reference slide: Place 1 drop of reference substance GDO or EPC on the slide, press it flat with a coverslip to avoid air bubbles and ensure that the reference substance is on the same horizontal plane to complete the preparation of the reference slide.

[0057] Establishment of Raman spectroscopy reference library: The confocal Raman spectrometer was pre-cooled to -60 °C. Reference slides were mounted under a 20x objective lens. A 532 nm laser was used, with laser intensity set to 50%, scanning time to 0.5 s, and one scan. Raman spectra of the reference materials GDO and EPC were obtained, as shown below. Figure 4 C.

[0058] Raman scanning of sections of bilayer lipogels: The confocal Raman spectrometer was pre-cooled to -60 °C. Sections of the bilayer lipogels were mounted under a 20x objective lens. A 532 nm laser was used, with a laser intensity of 70%, a scanning time of 0.2 s, and one scan. Scanning was performed at 20x20 resolution over a region with a diameter of 200 μm to obtain the confocal Raman spectrum of the lipogels. Scanning was performed at different sites on the same section to obtain the Raman spectra of the inner and outer gel layers. Using GDO and EPC Raman spectra as reference materials, the Raman spectra of the inner and outer gel layers were analyzed using an algorithm to obtain the compositional distribution map of the bilayer gels. The results are shown below. Figure 4 As shown in Figure A. ImageJ was used to perform grayscale quantitative analysis on the component distribution map, and the results are as follows. Figure 4 As shown in Figure B, the quantitative results indicate that the EPC:GDO ratio in the outer gel is approximately 34:66, and the EPC:GDO ratio in the inner gel is approximately 50:50, which is consistent with the mass ratio of the outer and inner gels. This verifies the bilayer structure in the gelling agent. Example 5

[0059] In vitro photothermal conversion efficiency and stability of drug-loaded black phosphorus nanoparticles Preparation of blank black phosphorus nanoparticles (BP-NSs): Bulk black phosphorus and N-methyl-2-pyrrolidone (NMP) were thoroughly ground in a mortar until a homogeneous suspension was formed. The supernatant containing black phosphorus flakes was transferred to an amber glass bottle, and a probe sonicator was set to pulse mode with 60% power and an on-off-off cycle of 5 s. The mixture was sonicated in an ice bath for 8 h. The resulting dispersion was centrifuged at 3000 rpm for 20 min at 4 ℃ to remove unexfoliated black phosphorus aggregates. The supernatant was centrifuged at 12000×g for 30 min at 4 ℃, washed twice with ultrapure water, pre-cooled with liquid nitrogen, freeze-dried, and stored at -80 ℃.

[0060] Preparation of olaparib-loaded black phosphorus nanoparticles (OL@BP-NSs): First, olaparib was weighed and dissolved in DMSO (10 mg / mL). Then, black phosphorus nanoparticle solution was added at a mass ratio of olaparib to black phosphorus nanoparticles of 2:1. The mixture was stirred continuously for 4 h under light-protected conditions. The resulting mixture was centrifuged at 12000×g for 30 min at 4 °C to remove unloaded olaparib from the supernatant. The drug-loaded black phosphorus nanoparticles were washed twice with ultrapure water, resuspended in ultrapure water, pre-cooled with liquid nitrogen, lyophilized, and stored at -80 °C.

[0061] The photothermal conversion efficiency of BP-NSs and OL@BP-NSs was evaluated using an 808 nm near-infrared (NIR) laser.

[0062] 50 μg / mL of OL@BP-NSs was used, and BP-NSs at the same concentration was applied. The mixture was subjected to four consecutive heating / cooling cycles (1.0 W / cm² irradiation for 5 min, followed by natural cooling for 7 min), and the temperature change was monitored using an infrared imager. Throughout the process, temperature changes at different time points were recorded and photographed using an infrared imager, and a temperature-time curve was plotted.

[0063] The stability results of in vitro photothermal conversion efficiency of drug-loaded black phosphorus nanoparticles are as follows: Figure 5As shown, BP-NSs and OL@BP-NSs can rapidly heat up after laser irradiation and maintain a stable heating capacity throughout the four heating cycles, always exceeding the phase transition temperature of the outer gel (>41.922 ℃). This indicates that the drug-loaded black phosphorus nanoparticles have excellent photothermal conversion efficiency stability in vitro. Example 6

[0064] In vivo thermotherapy monitoring of drug-loaded lipid gels Preparation of blank black phosphorus nanoparticles (BP-NSs): Bulk black phosphorus and N-methyl-2-pyrrolidone (NMP) were thoroughly ground in a mortar until a homogeneous suspension was formed. The supernatant containing black phosphorus flakes was transferred to an amber glass bottle, and a probe sonicator was set to pulse mode with 60% power and an on-off-off cycle of 5 s. The mixture was sonicated in an ice bath for 8 h. The resulting dispersion was centrifuged at 3000 rpm for 20 min at 4 ℃ to remove unexfoliated black phosphorus aggregates. The supernatant was centrifuged at 12000×g for 30 min at 4 ℃, washed twice with ultrapure water, pre-cooled with liquid nitrogen, freeze-dried, and stored at -80 ℃.

[0065] Preparation of a bilayer lipid gel loaded with black phosphorus nanoparticles (DLG(BP-NSs)): Black phosphorus nanoparticles, phospholipids, dioleoylglycerol, and surfactant (Tween 80) were vortexed and mixed at a mass ratio of 0.5:34:66:0.007, rapidly pre-frozen in liquid nitrogen, and lyophilized to prepare an outer lipid lyophilized powder loaded with photosensitizer. Next, phospholipids and dioleoylglycerol were weighed at a mass ratio of 34:66, and dissolved thoroughly in anhydrous ethanol, a co-solvent accounting for 10% of the total mass of the two lipids, to prepare an outer lipid precursor oil solution. Finally, equal masses of the outer lipid lyophilized powder and the outer lipid precursor oil solution were mixed, dissolved, and homogenized to obtain an outer lipid precursor oil phase formulation. Phospholipids and dioleoylglycerol were weighed at a mass ratio of 50:50, and dissolved thoroughly in anhydrous ethanol, a co-solvent accounting for 10% of the total mass of the two lipids, to prepare an inner lipid precursor oil solution.

[0066] Preparation of Blank DLG ​​precursor oil solution: Phospholipids and dioleoglycerides were weighed at a mass ratio of 34:66, and dissolved thoroughly in anhydrous ethanol, a co-solvent accounting for 10% of the total mass of the two lipids, to prepare the outer lipid precursor oil solution; Phospholipids and dioleoglycerides were weighed at a mass ratio of 50:50, and dissolved thoroughly in anhydrous ethanol, a co-solvent accounting for 10% of the total mass of the two lipids, to prepare the inner lipid precursor oil solution.

[0067] DLG (BP-NSs) was used, with PBS, Blank DLG, and BP-NSs as controls. Two lipid precursor oil solutions (or lipid precursor oil-phase formulations) were separately loaded into the syringe barrels of a double-barreled syringe, and 100 μl and 50 μl of each were injected intratumorally into 4T1 tumor-bearing mice, respectively. Four hours after injection, the tumor site was irradiated with an 808 nm near-infrared laser, and the tumor temperature was monitored using the laser. When the tumor temperature rapidly reached 45 °C, the irradiation power of the near-infrared laser was adjusted to maintain the temperature of the irradiated area at 45 °C. Throughout the process, temperature changes at different time points were recorded and photographed using the near-infrared laser, and temperature-time curves were plotted.

[0068] In vivo thermotherapy monitoring results of drug-loaded lipid gels are as follows: Figure 6 As shown, the temperature of the double-layer blank gel and PBS group only increased slightly under near-infrared laser irradiation, which is because mice themselves can absorb some near-infrared laser light. For the drug-loaded gel and blank black phosphorus nanoparticle group, both could reach 45 °C in about 3 minutes, indicating that the drug-loaded gel and blank black phosphorus nanoparticles have excellent photothermal conversion efficiency in vivo. Once the irradiated site reached 45 °C, the temperature of the irradiated site could be effectively controlled at 45 °C by adjusting the irradiation power of the near-infrared laser, demonstrating that the photothermal effect and temperature can be precisely controlled in vivo, meeting the experimental requirements for subsequent photothermal therapy. Example 7

[0069] In vitro drug release from drug-loaded lipid gels Preparation of the outer drug-loaded lipid bilayer gel (DLG(OL)): Olaparib, phospholipids, dioleoylglycerol, and surfactant (Tween 80) were vortexed and mixed at a mass ratio of 0.25:34:66:0.007, rapidly pre-frozen in liquid nitrogen, and lyophilized to prepare the lyophilized outer lipid powder loaded with olaparib. Next, phospholipids and dioleoylglycerol were weighed at a mass ratio of 34:66, and dissolved thoroughly in anhydrous ethanol (10% of the total mass of the two lipids) to prepare the outer lipid layer. Precursor oil solution; Finally, equal masses of outer lipid lyophilized powder and outer lipid precursor oil solution are mixed, dissolved and mixed evenly to obtain outer lipid precursor oil phase formulation; phospholipids and dioleoglycerides are weighed at a mass ratio of 50:50, and anhydrous ethanol, a cosolvent of 10% of the total mass of the two lipids, is added to fully dissolve them to prepare inner lipid precursor oil solution; the outer lipid precursor oil phase formulation and the inner lipid precursor oil solution are respectively loaded into the syringe barrel of a double-barrel syringe, and 33.3 μl and 16.7 μl of the outer and inner layers are respectively pushed into the aqueous phase; the outer gel precursor gels upon contact with water and encapsulates the inner gel, thereby forming an outer drug-loaded bilayer lipid gel (DLG(OL)).

[0070] Preparation of the inner drug-loaded bilayer lipid gel (DLG(IgG)): Phospholipids and dioleoglycerides were weighed at a mass ratio of 34:66 and dissolved thoroughly in anhydrous ethanol, a co-solvent accounting for 10% of the total mass of the two lipids, to prepare an outer lipid precursor oil solution; IgG, phospholipids, dioleoglycerides, and surfactant (Tween 80) were vortexed at a mass ratio of 0.25:50:50:0.007, rapidly pre-frozen in liquid nitrogen, and lyophilized to prepare an inner lipid lyophilized powder encapsulating IgG. Finally, phospholipids and dioleoglycerides were weighed at a mass ratio of 50:50 and dissolved thoroughly in anhydrous ethanol, a co-solvent accounting for 10% of the total mass of the two lipids, to prepare an inner lipid precursor oil solution. Then, an equal mass ratio of lyophilized inner lipid powder and the inner lipid precursor oil solution were mixed, dissolved, and thoroughly mixed to obtain an inner lipid precursor oil phase formulation. The outer lipid precursor oil solution and the inner lipid precursor oil phase formulation were respectively loaded into the syringe barrel of a double-barrel syringe, and 33.3 μl and 16.7 μl of the outer and inner layers were respectively injected into the aqueous phase. The outer gel precursor gelled upon contact with water, encapsulating the inner gel, thus forming an inner drug-loaded bilayer lipid gel (DLG (IgG)).

[0071] A 50 μl bilayer lipid gel sample was immersed in 3 mL of PBS solution (with 10% PEG2000 added) and placed in a 37°C water bath shaker for continuous release, maintaining the leak conditions throughout. 1 mL of release solution was collected at different time points (1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 1 d, 2 d, 4 d, 6 d, 8 d, 10 d), and an equal volume of isothermal medium was added. For the near-infrared laser-enhanced drug release group (+L), samples were treated with an 808 nm laser (1 W / cm²) at 6 h, 2 d, 4 d, and 6 d after incubation. 2 Irradiation was performed for 10 minutes. For samples taken at each time point, the olaparib content was determined by HPLC, and the IgG content was determined by a BCA kit.

[0072] In vitro drug release monitoring results of drug-loaded lipid gels are as follows: Figure 7 As shown, the outer gel exhibited slow release of olaparib under non-irradiation conditions, with a cumulative release of 35% over 10 days. However, olaparib release accelerated significantly after laser irradiation for 4 h, 48 h, 96 h, and 144 h, reaching a cumulative release of 55% over 10 days. IgG encapsulated in the inner gel consistently maintained slow release (<10% over 10 days), and photothermal intervention had no significant effect on its release kinetics. Example 8

[0073] In vivo drug release from drug-loaded lipid gels Preparation of black phosphorus nanoparticles loaded with dihydroporphyrin E6 (Chlorin E6, Ce6) (Ce6@BP-NSs): First, Ce6 (in place of olaparib) was weighed and dissolved in DMSO (10 mg / mL). The black phosphorus nanoparticle solution was added at a mass ratio of Ce6 to black phosphorus nanoparticles of 2:1. The mixture was stirred continuously for 4 h under light-protected conditions. The resulting mixture was centrifuged at 12000×g for 30 min at 4 °C to remove unloaded Ce6 from the supernatant. The drug-loaded black phosphorus nanoparticles were washed twice with ultrapure water, resuspended in ultrapure water, pre-cooled with liquid nitrogen, lyophilized, and stored at -80 °C.

[0074] Preparation of the oil phase formulation of the drug-loaded lipid gel (DLG(Ce6@BP-NSs + IgG)): Ce6-loaded black phosphorus nanoparticles, phospholipids, dioleoylglycerol, and surfactant (Tween 80) were vortexed at a mass ratio of 0.5:34:66:0.007, rapidly pre-frozen in liquid nitrogen, and lyophilized to prepare an outer lipid lyophilized powder encapsulating the drug-loaded photosensitizer. Next, phospholipids and dioleoylglycerol were weighed at a mass ratio of 34:66 and dissolved thoroughly in anhydrous ethanol (10% of the total mass of the two lipids) to prepare an outer lipid precursor oil solution. Finally, equal masses of the outer lipid lyophilized powder and the outer lipid precursor oil solution were mixed together. The outer lipid precursor oil phase formulation is prepared by mixing, dissolving, and homogenizing the ingredients. IgG, phospholipids, and dioleoglycerides are vortexed at a mass ratio of 0.5:50:50, rapidly pre-frozen in liquid nitrogen, and lyophilized to prepare an inner lipid lyophilized powder encapsulating IgG. Next, phospholipids and dioleoglycerides are weighed at a mass ratio of 50:50 and dissolved thoroughly in anhydrous ethanol, a co-solvent accounting for 10% of the total mass of the two lipids, to prepare an inner lipid precursor oil solution. Finally, equal masses of the inner lipid lyophilized powder and the inner lipid precursor oil solution are mixed, dissolved, and homogenized to obtain the inner lipid precursor oil phase formulation.

[0075] Preparation of the oil phase formulation of the drug-loaded lipid gel (DLG(BP-NSs + AFIgG)) precursor: Black phosphorus nanoparticles, phospholipids, dioleoylglycerol, and surfactant (Tween 80) were vortexed and mixed at a mass ratio of 0.5:34:66:0.007, rapidly pre-frozen in liquid nitrogen, and lyophilized to prepare an outer lipid lyophilized powder encapsulating a photosensitizer. Next, phospholipids and dioleoylglycerol were weighed at a mass ratio of 34:66 and dissolved thoroughly in anhydrous ethanol (10% of the total mass of the two lipids) to prepare an outer lipid precursor oil solution. Finally, equal masses of the outer lipid lyophilized powder and the outer lipid precursor oil solution were mixed, dissolved, and... The mixture was thoroughly mixed to obtain the outer lipid precursor oil phase formulation. AFIgG, phospholipids, and dioleoglycerides were vortexed at a mass ratio of 0.5:50:50, rapidly pre-frozen in liquid nitrogen, and lyophilized to prepare an inner lipid lyophilized powder encapsulating AFIgG. Next, phospholipids and dioleoglycerides were weighed at a mass ratio of 50:50, and dissolved thoroughly in anhydrous ethanol, a co-solvent accounting for 10% of the total mass of the two lipids, to prepare an inner lipid precursor oil solution. Finally, equal masses of the inner lipid lyophilized powder and the inner lipid precursor oil solution were mixed, dissolved, and thoroughly mixed to obtain the inner lipid precursor oil phase formulation.

[0076] To verify the release behavior of olaparib in mice, the outer and inner layers of the drug-loaded lipogel (DLG(Ce6@BP-NSs+IgG)) precursor oil phase formulation were taken and loaded into the syringe barrel of a double-barreled syringe. 100 μl and 50 μl of the outer and inner layers, respectively, were injected intratumorally into 4T1 tumor-bearing BAL B / c mice, with free Ce6 as a control. To verify the release behavior of the aCD47 inhibitor in mice, the outer and inner layers of the drug-loaded lipogel (DLG(BP-NSs+AFIgG)) precursor oil phase formulation were taken and loaded into the syringe barrel of a double-barreled syringe. 100 μl and 50 μl of the outer and inner layers, respectively, were injected intratumorally into 4T1 tumor-bearing BAL B / c mice, with free AFIgG as a control. For the near-infrared light-triggered group (+L), the tumor was irradiated with an 808 nm laser for 10 minutes each time on days 0, 2, 4, and 6 post-injection, with the irradiated site maintained at 45 °C. On days 0, 1, 3, 5, 7, 14, and 21, fluorescence images were acquired under isoflurane anesthesia using a small animal in vivo imaging system, and the signal intensity was quantitatively analyzed using in vivo imaging software to evaluate the drug release from the bilayer lipid gel in vivo.

[0077] In vivo drug release monitoring results of drug-loaded lipid gels are as follows: Figure 8 As shown ( Figure 8 Images A and B in the image show the fluorescence in vivo imaging and fluorescence intensity of Ce6 in the outer gel layer. Figure 8In the images (C and D), AFIgG fluorescence in vivo imaging and fluorescence intensity are shown. Compared with the free Ce6 control group, the retention time of Ce6 in the un-laser-irradiated bilayer lipid gel (DLG-L(Ce6@BP-NSs + IgG)) group was significantly prolonged. Compared with the un-laser-irradiated bilayer lipid gel (DLG-L(Ce6@BP-NSs + IgG)) group, the mean fluorescence intensity of the laser-irradiated bilayer lipid gel (DLG+L(Ce6@BP-NSs + IgG)) group was significantly reduced on day 21, indicating that light irradiation triggered accelerated release of the outer layer drug. Compared with the free AFIgG group, the bilayer drug-loaded lipid gel (DLG(BP-NSs + AFIgG)) showed the characteristics of sustained and slow release, with a fluorescence signal retention rate of over 30% within 21 days. Moreover, there was no statistically significant difference in release rate between the two groups regardless of whether they were irradiated. In contrast, the free AFIgG almost lost its fluorescence signal by day 14. In summary, the bilayer lipid gel can achieve controlled and orderly release in time and space. Example 9

[0078] Evaluation of in vivo pharmacokinetic processes of different prescription thermosensitive tumor therapeutic agents Preparation of DIR-loaded black phosphorus nanoparticles (DIR@BP-NSs): First, DIR (in place of olaparib) was weighed and dissolved in DMSO (10 mg / mL). The black phosphorus nanoparticle solution was added at a mass ratio of DIR to black phosphorus nanoparticles of 2:1. The mixture was stirred continuously for 4 h under light-protected conditions. The resulting mixture was centrifuged at 12000×g for 30 min at 4 °C to remove unloaded DIR from the supernatant. The drug-loaded black phosphorus nanoparticles were washed twice with ultrapure water, resuspended in ultrapure water, pre-cooled with liquid nitrogen, lyophilized, and stored at -80 °C.

[0079] Preparation of Cy5-loaded IgG (IgG-Cy5): First, Cy5 was prepared into a 0.8 mg / ml solution with DMF, and IgG was prepared into a 1 mg / ml solution with PBS. Then, the IgG solution and Cy5 solution were mixed at a volume ratio of 4:1 and stirred for 2 h. After purification by ultrafiltration, PBS was added and the mixture was vortexed for 1 min. The IgG-Cy5 on the membrane was then collected.

[0080] Preparation of drug-containing outer lipid gel precursor oil phase formulation and drug-containing inner lipid gel precursor oil phase formulation: Black phosphorus nanoparticles loaded with DIR, phospholipids, dioleoyl glycerol, and surfactant (Tween 80) were vortexed and mixed at a mass ratio of 0.5:34:66:0.007, rapidly pre-frozen in liquid nitrogen, and lyophilized to prepare an outer lipid lyophilized powder loaded with a drug-loaded photosensitizer. Next, phospholipids and dioleoyl glycerol were weighed at a mass ratio of 34:66, and dissolved thoroughly in anhydrous ethanol (10% of the total mass of the two lipids) to prepare an outer lipid precursor oil solution. Finally, equal masses of the outer lipid lyophilized powder and the outer lipid precursor oil were mixed... The solutions are mixed, dissolved, and homogenized to obtain the outer lipid precursor oil phase formulation. IgG-Cy5, phospholipids, and dioleoglycerides are vortexed at a mass ratio of 0.5:50:50, rapidly pre-frozen in liquid nitrogen, and lyophilized to prepare an inner lipid lyophilized powder encapsulating IgG-Cy5. Next, phospholipids and dioleoglycerides are weighed at a mass ratio of 50:50, and dissolved thoroughly in anhydrous ethanol (10% of the total mass of the two lipids) to prepare an inner lipid precursor oil solution. Finally, equal masses of the inner lipid lyophilized powder and the inner lipid precursor oil solution are mixed, dissolved, and homogenized to obtain the inner lipid precursor oil phase formulation.

[0081] Healthy mice were evenly divided into four groups. 0 μl, 50 μl, 100 μl, and 200 μl of the outer layer lipid gel precursor oil phase preparation were injected into the fat pad of the fourth mammary gland, respectively. The amount of the inner layer lipid gel precursor oil phase preparation was maintained at 50 μl in each group. Photothermal therapy was administered at 4 h, 2 d, 4 d, and 8 d post-injection. The injection site was irradiated with an 808 nm near-infrared laser, and the photothermal temperature was controlled at 43 ± 0.5 ℃ using an infrared thermal imager. Fluorescence changes of Cy5 and DIR were monitored using in vivo imaging of small animals at 2 h, 1 d, 3 d, 5 d, 7 d, 14 d, 21 d, and 28 d post-injection, and fluorescence curves were plotted.

[0082] The results are as follows Figure 9 As shown ( Figure 9 Image A shows fluorescence in vivo imaging of patients treated with different amounts of gel. Figure 9(Mean B is for in vivo imaging fluorescence quantification). Different amounts of the outer lipid gel lead to different DIR and Cy5 release behaviors: when the amount of outer gel is 50 μl and 100 μl, the release process of DIR in the outer layer is similar, with about 30% of the drug remaining after 7 days. However, when the amount of outer gel is 200 μl, 60% of DIR remains in the outer layer after 7 days. The fluorescence intensity of Cy5 in the inner layer is also related to the amount of outer gel, especially IgG-Cy5, which exhibits a burst release phenomenon. When the amount of outer gel is 0 μl, the burst release of IgG-Cy5 occurs from 0 to 3 days. As the amount of outer gel increases, the burst release time of IgG-Cy5 is continuously delayed. In particular, when the amount of outer gel is 100 μl, the burst release of IgG-Cy5 occurs from 7 to 14 days. This indicates that under this formulation, the release time of the immune checkpoint inhibitor can be precisely in the optimal tumor microenvironment state. Therefore, the optimal amount of the bilayer lipid gel is determined to be 100 μl. μl of outer layer gel and 50 μl of inner layer gel. Example 10

[0083] Effect of aCD47 antibody on macrophage phagocytic activity in vitro Preparation of the PARPi pretreated cell death model: First, 4T1Brca1-KD cells in the logarithmic growth phase were labeled with carboxyfluorescein acetoacetate. Then, 4T1Brca1-KD cells were treated with 1 μg / mL olaparib for 12 h to induce a cell death state.

[0084] Pretreated or untreated tumor cells were co-cultured with eFluor-670-labeled M1-like BMDMs at a 1:2 ratio in Fc receptor blocking buffer. The co-cultured cells were then exposed to aCD47 antibody at concentrations of 0, 2, 5, and 10 μg / mL for 6 h. Phagocytosis was finally analyzed by flow cytometry and CLSM. The effect of aCD47 antibody on macrophage phagocytic activity in vitro is shown in the following figures. Figure 10 As shown ( Figure 10 Figure A shows the experimental procedure, and Figure B shows the effect of aCD47 antibody on the phagocytic activity of macrophages in vitro. As the dose of aCD47 antibody increases, the phagocytic efficiency is significantly enhanced, indicating that it effectively blocks the CD47-SIRPα axis and can be effectively used in combination with PARPi to enhance the killing effect on tumor cells. Example 11

[0085] The therapeutic effect of tumor therapeutic agents on triple-negative breast cancer in mice. Preparation of olaparib-loaded black phosphorus nanoparticles (OL@BP-NSs): First, olaparib was weighed and dissolved in DMSO (10 mg / mL). Olaparib and black phosphorus nanoparticles were added to the black phosphorus nanoparticle solution at a mass ratio of 2:1. The mixture was stirred continuously for 4 h under light-protected conditions. The resulting mixture was centrifuged at 12000×g for 30 min at 4 °C to remove unloaded olaparib from the supernatant. The drug-loaded black phosphorus nanoparticles were washed twice with ultrapure water, resuspended in ultrapure water, pre-cooled with liquid nitrogen, lyophilized, and stored at -80 °C.

[0086] Preparation of blank lipid bilayer gel precursor oil solution: Phospholipids and dioleoglycerides were weighed at a mass ratio of 34:66, and dissolved completely in anhydrous ethanol, a co-solvent accounting for 10% of the total mass of the two lipids, to prepare the outer lipid precursor oil solution; Phospholipids and dioleoglycerides were weighed at a mass ratio of 50:50, and dissolved completely in anhydrous ethanol, a co-solvent accounting for 10% of the total mass of the two lipids, to prepare the inner lipid precursor oil solution.

[0087] Preparation of the oil phase formulation and oil solution of olaparib-loaded bilayer lipid gel (OL@(BP-NSs) precursor: Black phosphorus nanoparticles loaded with olaparib, phospholipids, dioleoylglycerol, and surfactant (Tween 80) were vortexed and mixed at a mass ratio of 0.5:34:66:0.007, rapidly pre-frozen in liquid nitrogen, and lyophilized to prepare an outer lipid lyophilized powder loaded with photosensitizer; Next, phospholipids and dioleoylglycerol were weighed at a mass ratio of 34:66, and dissolved thoroughly in anhydrous ethanol, a co-solvent accounting for 10% of the total mass of the two lipids, to prepare an outer lipid precursor oil solution; Finally, equal masses of the outer lipid lyophilized powder and the outer lipid precursor oil solution were mixed, dissolved, and homogenized to obtain the outer lipid precursor oil phase formulation; Phospholipids and dioleoylglycerol were weighed at a mass ratio of 50:50, and dissolved thoroughly in anhydrous ethanol, a co-solvent accounting for 10% of the total mass of the two lipids, to prepare an inner lipid precursor oil solution.

[0088] Preparation of the oil phase formulation of the drug-loaded bilayer lipid gel (OL@BP-NSs + aCD47) lipid precursor: First, black phosphorus nanoparticles loaded with olaparib, phospholipids, dioleoylglycerol, and surfactant (Tween 80) were vortexed and mixed at a mass ratio of 0.5:34:66:0.007, rapidly pre-frozen in liquid nitrogen, and lyophilized to prepare the photosensitizer-loaded outer lipid lyophilized powder; Second, phospholipids and dioleoylglycerol were weighed at a mass ratio of 34:66, and dissolved thoroughly in anhydrous ethanol, a co-solvent accounting for 10% of the total mass of the two lipids, to prepare the outer lipid precursor oil solution; Finally, equal masses of the outer lipid lyophilized powder and the outer lipid precursor oil solution were mixed... Mix, dissolve, and thoroughly mix to obtain the outer lipid precursor oil phase formulation; vortex mix aCD47, phospholipids, and dioleoglycerides at a mass ratio of 0.5:50:50, rapidly pre-freeze in liquid nitrogen, and lyophilize to prepare an inner lipid lyophilized powder encapsulating aCD47; next, weigh phospholipids and dioleoglycerides at a mass ratio of 50:50, add 10% of the total mass of the two lipids to anhydrous ethanol as a co-solvent and dissolve thoroughly to prepare an inner lipid precursor oil solution; finally, mix, dissolve, and thoroughly mix equal masses of the inner lipid lyophilized powder and the inner lipid precursor oil solution to obtain the inner lipid precursor oil phase formulation.

[0089] 45 °C was selected as the treatment temperature for mild photothermal therapy, 500 μg of olaparib was selected as the effective concentration of PARPi, and 50 μg of aCD47 antibody was selected as the effective concentration of CD47 inhibitor. The therapeutic effects of individual treatments and the combination of bilayer gel and photothermal therapy (+L) were investigated. 1×10 8 4T1Brca1-KD tumor cells in the logarithmic growth phase were inoculated into the fat pads of the mammary glands of female BAL B / c mice and randomly divided into six groups: PBS, blank bilayer gel, bilayer gel (BP-NSs) + L, bilayer gel (OL@BP-NSs) + L, bilayer gel (OL@BP-NSs + aCD47) + L, and free drug (OL@BP-NSs + aCD47) + L. One week after tumor implantation, the outer and inner layers of lipid gel precursor solution or lipid precursor oil phase preparation (or PBS solution) were respectively loaded into the syringe barrel of a double-barreled syringe, and 100 μl and 50 μl of the outer and inner layers were injected into the tumor of the tumor-bearing mice, respectively. For the photothermal group, photothermal treatment was performed on the 4th h, 2nd d, 4th d, and 6th d after implantation, and the temperature of the mouse tumor site was precisely controlled at 45 ℃ using an infrared imager. On the 8th day, the mice were sacrificed, the mouse tumors were dissected, and the mouse tumor tissue was removed and weighed.

[0090] The results of using tumor therapeutic agents to treat triple-negative breast cancer in mice are as follows: Figure 11As shown, significant differences in tumor volume were observed after injection and treatment. The double-layer gel (OL@BP-NSs + aCD47) + L group showed tumor volume reduction and decreased anatomical tumor mass as early as 3 days after treatment. Figure 11 In the treatment of BD, 20% of mice (1 / 5) achieved complete remission at the end of the treatment, while the free drug (OL@BP-NSs + aCD47) + L group showed tumors several times larger, indicating the importance of the bilayer gel in controlling drug release in time and space. Comparing the combination therapy group with other single therapy groups in terms of survival, the 60-day survival rate of the combination therapy group was 80%, significantly better than the other treatment groups. Figure 11 (E).

Claims

1. A temperature-sensitive bilayer gel agent, characterized in that, It includes an inner gel and an outer gel, wherein the outer gel covers the inner gel, and the volume ratio of the inner gel to the outer gel is 1:2; The inner gel comprises an inner gel matrix and a CD47 inhibitor, wherein the mass of the CD47 inhibitor is 0.1-0.3% of the inner gel matrix. The outer gel comprises an outer gel matrix, PARPi, and a photosensitizer. The mass of PARPi is 0.3-3% of the outer gel matrix, and the mass of the photosensitizer is 0.2-0.5% of the outer gel matrix.

2. The temperature-sensitive bilayer gel agent according to claim 1, characterized in that, The inner and outer gel matrices are selected from phospholipid-oleic acid lipid gels, chitosan-glycerophosphate gels, poloxamer hydrogels, and poly-N-isopropylacrylamide hydrogels.

3. The temperature-sensitive bilayer gel agent according to claim 2, characterized in that, The inner gel matrix and the outer gel matrix are phospholipid-oleic acid lipid gels, preferably phospholipids and dioleoglycerides.

4. The temperature-sensitive bilayer gel agent according to claim 3, characterized in that, The outer gel carrier contains phospholipids in a mass ratio of 33:67 to 34:66, while the inner gel matrix contains phospholipids in a mass ratio of 35:65 to 70:

30.

5. The temperature-sensitive bilayer gel agent according to claim 1, characterized in that, The CD47 inhibitor is selected from Magrolimab, Lemzoparlimab, Evorpacept, HX009, and anti-CD47 antibody; the PARPi is selected from fluzoparib, tapazoli, rucaparib, niraparib, and olaparib; and the photosensitizer is selected from graphene nanoparticles and black phosphorus nanoparticles.

6. The temperature-sensitive bilayer gel agent according to claim 1, characterized in that, First, PARPi and photosensitizer are combined, and then the photosensitizer loaded with PARPi is dispersed into the outer gel matrix.

7. The method for preparing the temperature-sensitive bilayer gel agent according to claim 1, characterized in that, Includes the following steps: Step 1: Mix the PARPi solution and the photosensitizer solution, centrifuge, and resuspend in water to obtain the PARPi-loaded photosensitizer; Step 2: Add the photosensitizer loaded with PARPi to part of the outer gel matrix, and freeze-dry to obtain the freeze-dried outer lipid gel precursor; Step 3: Add CD47 inhibitor to part of the inner layer gel matrix, freeze dry to obtain the inner layer lipid gel precursor freeze-dried 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 bilayer gel agent.

8. The use of the thermosensitive bilayer gel agent according to claim 1 in the preparation of tumor therapeutic drugs.

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