A ROS-responsive protein-crosslinked liposome and its application

Abstract

The present invention discloses a ROS-responsive protein-crosslinked liposome and its application. The protein-crosslinked liposome of the present invention is a co-delivery system formed by crosslinking protein drugs electrostatically adsorbed on the surface of drug-loaded liposomes using a ROS-responsive crosslinking agent. This delivery system can not only achieve the co-loading of protein drugs and small molecule drugs with different physicochemical properties, but also respond to ROS in the tumor microenvironment to controllably release protein drugs, improve the therapeutic effect of protein drugs and avoid the toxic and side effects caused by the systemic distribution of protein drugs. At the same time, it can also play a synergistic anti-tumor role with the small molecule drugs loaded in the liposomes. The present invention will provide a novel ROS-responsive crosslinking agent and a new delivery system for the combined delivery of protein drugs and small molecule drugs, and has broad application prospects in the treatment of solid tumors, especially pancreatic cancer.

Description

Technical Field

[0001] The present invention relates to the fields of chemistry and pharmaceuticals, and particularly relates to a ROS-responsive protein-crosslinked liposome and its application. Background Art

[0002] Immunotherapy has become the mainstream research direction for the treatment of solid tumors, but the key problems it faces are immunosuppression and immune escape. On the one hand, there are a large number of immunosuppressive cells in the solid tumor microenvironment, represented by tumor-associated macrophages (TAMs) with the M2 tumor-promoting phenotype, which secrete a large amount of immunosuppressive factors, severely weakening the effect of immunotherapy. On the other hand, tumor cells can also produce immune escape by downregulating the expression of major histocompatibility complex class I molecules (MHC-I). Therefore, drug combination, especially the combination of protein drugs with immunomodulatory effects and small molecule drugs, is an effective strategy to solve this limitation. For example, the protein drug interferon-γ (IFN-γ) has immunomodulatory and anti-tumor effects and has been approved for enhancing immune function. IFN-γ can not only regulate the transformation of TAMs into the M1 anti-tumor phenotype, but also upregulate the expression of MHC-I molecules on tumor cells, but single treatment still has poor efficacy. Chloroquine (CQ), as an anti-malaria drug used clinically for many years, has been found to have anti-tumor-related effects in recent years; and as a type of lysosomal alkalizer, CQ can inhibit the binding of autophagosomes to lysosomes, reduce the degradation of MHC-I proteins, and restore antigen presentation; at the same time, CQ has been proven to enhance the activation of p38 / NF-κB downstream of IFN-γ and the expression of the transcription factor TFEB, enhancing the IFN-γ-mediated phenotypic conversion of TAMs. Therefore, the combined application of CQ and IFN-γ can play a good role in the treatment of solid tumors.

[0003] However, the combination of protein drugs and small molecule drugs still faces challenges in delivery technology. On the one hand, their blood half-lives are short, tumor targeting is poor, and they have dose-dependent side effects when administered conventionally. On the other hand, there are large differences in their physicochemical properties and action sites. The action sites of protein drugs are often the corresponding receptors on the cell membrane surface, while the action sites of small molecule drugs are often intracellular. Moreover, protein drugs are prone to inactivation due to conformational changes. Therefore, achieving the stable co-loading and controllable release of the two drugs is an urgent problem to be solved. Summary of the Invention

[0004] The purpose of the present invention is to overcome the defects existing in the prior art and provide a ROS-responsive protein-crosslinked liposome and its preparation method.

[0005] Another object of the present invention is to provide a novel ROS-responsive crosslinking agent, which can not only rapidly crosslink protein drugs, but also rapidly respond to ROS, release the prototype protein drugs, and does not affect their physiological activities.

[0006] Another object of the present invention is to provide the application of ROS-responsive protein-crosslinked liposomes in the treatment of solid tumors, especially pancreatic cancer.

[0007] A kind of ROS-responsive protein-crosslinked liposome is formed by crosslinking a protein drug on the surface of a liposome loaded with a small molecule drug by using a ROS-responsive crosslinking agent. Its preparation method is as follows: first, prepare a liposome loaded with a small molecule drug, then adsorb a protein drug with an opposite charge onto the surface of the liposome, and crosslink it by using a ROS-responsive crosslinking agent.

[0008] As a preferred embodiment of the present invention, the ROS-responsive crosslinking agent is bis(2,5-dioxopyrrolidin-1-yl) ((propane-2,2-diylbis(sulfanediyl))bis(ethane-2,1-diyl) bis(carbonate)), and its structural formula is:

[0009]

[0010] As a preferred embodiment of the present invention, the liposome is composed of a cationic lipid or an anionic lipid, and a common auxiliary lipid.

[0011] As a preferred embodiment of the present invention, the cationic lipid is selected from trimethyl-2,3-dioleyloxypropylammonium chloride (DOTMA), trimethyl-2,3-dioleoyloxypropylammonium bromide (DOTAP), or tertiary amine-based cationic lipid derivatives in Patent ZL2018113048420, etc., and preferably the tertiary amine-based cationic lipid derivatives described in Patent ZL2018113048420.

[0012] The chemical structure of the tertiary amine-based cationic lipid derivative can be represented by the general formula (I) or (II):

[0013]

[0014] Among them,

[0015] n = 1 or 2;

[0016] m represents an integer from 1 to 4;

[0017]

[0018] p represents an integer from 2 to 4, q represents an integer from 1 to 3, r represents an integer from 1 to 2, s represents an integer from 0 to 3, R 5represents methyl, hydroxymethyl, ethyl, hydroxyethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, R 6 represents methyl, ethyl or benzyl, R 7 represents pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, pyrazolyl or imidazolyl;

[0019] u represents an integer from 7 to 19;

[0020]

[0021] As a preference of the present invention, the anionic lipid is selected from dioleoyl phosphatidylserine (DOPS), dioleoyl phosphatidylglycerol (DOPG), 4-((1,5-dioxo-1,5-bis(tetradecyloxy)pentan-2-yl)amino)-4-oxobutanoic acid (TA-COOH), preferably TA-COOH, and the structural formula is:

[0022]

[0023] As a preference of the present invention, the common co-lipids are selected from soy phosphatidylcholine (SPC), dioleoyl phosphatidylethanolamine (DOPE), distearoyl phosphatidylethanolamine (DSPE), distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dierucoyl phosphatidylcholine (DEPC), dipalmitoyl phosphatidylcholine (DPPC) or cholesterol, etc., preferably SPC and / or cholesterol.

[0024] As a preference of the present invention, the small molecule drug is selected from tumor chemotherapy drugs or small molecule immunomodulatory drugs; the tumor chemotherapy drugs are selected from paclitaxel, doxorubicin, chloroquine phosphate (CQ), preferably CQ; the small molecule immunomodulatory drugs are selected from TGF-β inhibitors.

[0025] As a preference of the present invention, the protein drug is selected from positively charged protein drugs and negatively charged protein drugs; the positively charged protein drugs are selected from IFN-γ, tumor necrosis factor-related apoptosis-inducing ligand, and the negatively charged protein drugs are selected from IL-2, IL-15.

[0026] As a further preference of the present invention, the protein drug is selected from IFN-γ.

[0027] As a preference of the present invention, the molar ratio of the crosslinking agent to the protein drug is 50:1.

[0028] Application of the ROS-responsive crosslinking agent of the present invention as a protein crosslinking agent.

[0029] The application of the ROS-responsive protein-crosslinked liposomes described in the present invention in the preparation of drugs for treating solid tumors, preferably pancreatic cancer, breast cancer, lung cancer, liver cancer, and more preferably pancreatic cancer.

[0030] A ROS-responsive crosslinker, characterized in that its chemical structure is as follows:

[0031]

[0032] The application of the ROS-responsive crosslinker in the preparation of liposomes and protein-crosslinked drugs.

[0033] The application of the ROS-responsive crosslinker in the preparation of protein crosslinkers.

[0034] The present invention discloses a synthesis method of a ROS-responsive crosslinker, and the specific steps are as follows:

[0035] (1) Dissolve thioglycolic acid in acetone and trifluoroacetic acid, and stir and react at room temperature for 4-8 h. After the reaction is completed, filter by suction to obtain a white powdery solid (product 1) 2,2'-(propane-2,2-diylbis(sulfanediyl))diacetic acid.

[0036] (2) Dissolve product 1 in anhydrous tetrahydrofuran, slowly add lithium aluminum hydride under an ice-water bath condition, and then transfer it to room temperature and stir overnight. After the reaction is completed, quench the reaction with pure water under an ice-water bath condition, add a saturated aqueous solution of potassium sodium tartrate tetrahydrate, and stir at room temperature for 2-6 h. Finally, extract with ethyl acetate 4 times, dry with anhydrous sodium sulfate, rotary evaporate and concentrate to obtain an oily crude product, and purify it by column chromatography (petroleum ether:ethyl acetate = 2:1) to obtain an oily product (product 2) 2,2'-(propane-2,2-diylbis(sulfanediyl))bis(ethane-1-ol).

[0037] (3) Dissolve N,N-disuccinimidyl carbonate, product 2, and N,N-diisopropylethylamine in DMSO, and stir at 40 °C for 2-4 h. After the reaction is completed, add an appropriate amount of pure water and extract with ethyl acetate. Then collect the organic phase and wash it with a saturated saline solution. After drying with anhydrous sodium sulfate, purify the product by column chromatography (petroleum ether:ethyl acetate = 10:1) to obtain a white solid end product bis(2,5-dioxopyrrolidin-1-yl)((propane-2,2-diylbis(sulfanediyl))bis(ethane-2,1-diyl) bis(carbonate).

[0038] The synthesis reaction formula is as follows:

[0039]

[0040] Beneficial effects:

[0041] The ROS-responsive crosslinker disclosed by the present invention has mild and efficient ROS responsiveness (see Example 1). The ROS-responsive protein-crosslinked liposomes of the present invention have good encapsulation efficiency for small molecule drugs and protein drug crosslinking efficiency, and the particle size is about 161 nm. It is spherical under the TEM image, and has a gel-like layer wrapping on the surface compared with the liposomes loaded with small molecule drugs (see Example 2). It can effectively regulate the phenotypic transformation of macrophages (see Example 3), promote the MHC-I expression of tumor cells (see Example 4), and effectively inhibit the growth of tumor cells (see Example 5).

[0042] The innovation of the present invention is to provide a novel ROS-responsive protein crosslinker, which realizes the mild co-loading and controlled release of protein drugs and small molecule drugs, and provides a new combined delivery strategy and delivery system for solid tumor immunotherapy. Brief Description of the Drawings

[0043] Figure 1 is the 1 H-NMR spectrum of the ROS-responsive crosslinker of the present invention

[0044] Figure 2 is the mass spectrum of the ROS-responsive crosslinker of the present invention

[0045] Figure 3 is the ROS responsiveness experiment of the ROS-responsive crosslinker of the present invention

[0046] Figure 4 is the transmission electron microscope (TEM) image and particle size diagram of CQ-Lip of the present invention

[0047] Figure 5 is the TEM image and particle size diagram of IFN-γ / CQ-Lip of the present invention

[0048] Figure 6 is the expression of CD80 and CD206 after treating M2 macrophages with IFN-γ / CQ-Lip of the present invention

[0049] Figure 7 is the MHC-I expression after treating murine pancreatic cancer cell PANC02 with IFN-γ / CQ-Lip of the present invention

[0050] Figure 8 is the apoptosis situation after treating murine pancreatic cancer cell PANC02 with IFN-γ / CQ-Lip of the present invention Detailed Description of the Invention

[0051] The present invention is further explained by the following examples, but these examples do not constitute any limitation to the present invention.

[0052] Example 1

[0053] Synthesis and Characterization of ROS-responsive Crosslinker, with the chemical structural formula as follows:

[0054]

[0055] (1) Weigh accurately thioglycolic acid (10.0 g, 108.6 mmol, 0.1 eq.) and acetone (3.8 g, 65.1 mmol, 1.2 eq.), and place them in a eggplant-shaped flask. Add trifluoroacetic acid (618.9 mg, 0.1 eq.), and stir the reaction at room temperature for 6 h. After the reaction is completed, filter by suction to obtain 11.2 g of white powdery solid (Product 1), which is 2,2'-(propane-2,2-diylbis(sulfanediyl))diacetic acid.

[0056] (2) Weigh Product 1 (10.0 g, 44.6 mmol, 1 eq.), and place it in a eggplant-shaped flask. Add 200 mL of anhydrous tetrahydrofuran to dissolve it completely. Slowly add lithium aluminum hydride powder (4.2 g, 111.5 mmol, 2.5 eq.) under ice-water bath conditions. After reacting for 30 min, transfer the reaction system to room temperature and stir overnight. After the reaction is completed, slowly add water with the same mass as lithium aluminum hydride to quench the reaction under ice-water bath conditions. After quenching, add 150 mL of water and 100 mL of saturated aqueous sodium potassium tartrate solution to the reaction system, and stir at room temperature for 4 h. The reaction system gradually changes from a gray suspension to a dark brown emulsion. After 4 h, extract the reaction solution with ethyl acetate 4 times. After drying with anhydrous sodium sulfate, concentrate the reaction solution by rotary evaporation to obtain an oily crude product, which is purified by column chromatography (petroleum ether:ethyl acetate = 2:1) to obtain 4 g of oily product (Product 2), which is 2,2'-(propane-2,2-diylbis(sulfanediyl))bis(ethane-1-ol).

[0057] (3) Weigh N,N-disuccinimidyl carbonate (1.2 g, 4.6 mmol, 4 eq.) and dissolve it in 20 mL of dimethyl sulfoxide. Add Product 2 (224.2 mg, 1.1 mmol, 1 eq.) and N,N-diisopropylethylamine (590.3 mg, 4.6 mmol, 4 eq.) in sequence, and stir at 40 °C for 3 h. After the reaction is completed, transfer the reaction solution to a separatory funnel, add about 10 times the volume of water of the reaction solution, shake well and extract with ethyl acetate 2 times. Collect the upper organic phase, and wash the organic phase with saturated saline solution twice. After drying with anhydrous sodium sulfate, purify the product by column chromatography (petroleum ether:ethyl acetate = 10:1) to obtain 200 mg of white solid (Product 3), bis(2,5-dioxopyrrolidin-1-yl)((propane-2,2-diylbis(sulfanediyl))bis(ethane-2,1-diyl) bis(carbonate).

[0058] (4) Characterization by hydrogen nuclear magnetic resonance and mass spectrometry

[0059] The hydrogen spectrum of the ROS-responsive crosslinker is as follows Figure 1 shown. The characteristic peak of -CH at δ = 1.65 ppm (d) is 3 The characteristic peak of -CH at δ = 3.02 ppm (c) is 2 The characteristic peak of -CH next to the carbonate bond at δ = 4.49 ppm (a) is 2 The characteristic peak of -CH on the lactam at δ = 2.86 ppm (a) is 2 .

[0060] The high-resolution mass spectrometry results of the ROS-responsive crosslinker are as follows Figure 2 shown. The molecular weight should be 478.06, and the [M+Na] + peak should be 501.06. The above results prove the successful synthesis of the crosslinker.

[0061] (5) Investigation of ROS responsiveness

[0062] Prepare a 1 mg / mL ROS-responsive crosslinker solution, 3% H 2 O 2 solution and 30% H 2 O 2 solution. Then, add 10 μL of 3% H 2 O 2 solution and 30% H 2 O 2 solution to 1 mL of the crosslinker solution respectively, so that the H 2 O 2 concentrations in the solutions are 5 mM and 50 mM respectively. After incubation for 15 min, measure the absorbance change using a UV spectrophotometer.

[0063] The results are as follows Figure 3 shown. The crosslinker produces an absorption peak of the response product N-hydroxysuccinimide (NHS) at 260 nm. It is proved by the absorbance change that the crosslinker undergoes about 50% responsive cleavage in 15 min under the condition of 5 mM H 2 O 2 , and about 100% responsive cleavage in 15 min under the condition of 50 mM H 2 O 2 . This indicates that the crosslinker has good ROS responsiveness.

[0064] Example 2

[0065] Preparation and characterization of ROS-responsive protein-crosslinked liposomes

[0066] (1) Preparation of CQ liposomes

[0067] Weigh 40 mg of SPC, 8 mg of Chol and 12 mg of TA-COOH, dissolve them in a mixed solution of methanol and chloroform (1:4, v:v), and transfer the solution to a round-bottom flask. Perform vacuum distillation at 37 °C to form a lipid film. Then dry it under vacuum at room temperature for 12 h to remove the organic solvents. Subsequently, add 5 mL of citrate buffer (pH 3.5, 0.3 M), hydrate it at 37 °C for 15 min, and sonicate it at 30% power for 15 min. Finally, filter it through a 0.22 μm microporous membrane to obtain a blank liposome solution.

[0068] Dissolve CQ in a disodium hydrogen phosphate solution, mix it with the blank liposome solution at a drug-lipid ratio of 1:12 (w:w), and adjust the pH to 7.4 using 5% NaOH. Subsequently, transfer the reaction solution to a round-bottom flask and stir it at 37 °C for 15 min. Finally, transfer the reaction solution to a dialysis bag and dialyze it in PBS buffer for 4 h, changing the dialysis solution every 1 h. After dialysis is completed, filter it through a 0.22 μm microporous membrane to obtain CQ-Lip. Observe its morphology using TEM.

[0069] (2) Preparation of ROS-responsive protein-crosslinked liposomes

[0070] Mix CQ-Lip and IFN-γ solution at a mass ratio of 150:1, and incubate it with shaking at 4 °C for 15 min. Subsequently, add a ROS-responsive crosslinker such that the mass ratio of crosslinker:IFN-γ is 50:1. Continue to incubate it with shaking at 4 °C for 15 min, and then use an ultrafiltration centrifugal tube with a molecular weight cut-off of 100 k to remove free IFN-γ to obtain IFN-γ / CQ-Lip. Observe its morphology using TEM.

[0071] (3) Characterization of ROS-responsive protein-crosslinked liposomes

[0072] Measure the particle size and zeta potential of CQ-Lip and IFN-γ / CQ-Lip respectively, and observe the morphology using TEM. The TEM images and particle size diagrams of the two are shown in Figure 4 、 Figure 5 respectively. The surface of CQ-Lip is smooth, while IFN-γ / CQ-Lip is spherical and has a gel-like layer on its surface. Determine the content of CQ in CQ-Lip and IFN-γ / CQ-Lip using high-performance liquid chromatography, and calculate the encapsulation efficiency. At the same time, detect the content of uncrosslinked IFN-γ using an ELISA kit and calculate the crosslinking efficiency of IFN-γ. The results are shown in Table 1.

[0073] Table 1 Particle size, zeta potential and encapsulation efficiency of CQ-Lip and IFN-γ / CQ-Lip

[0074]

[0075] Example 3

[0076] Phenotypic conversion effect of ROS-responsive protein-crosslinked liposomes on M2 macrophages

[0077] Bone marrow-derived macrophages (BMDMs) were extracted from C57 mice as model cells and induced into the M2 phenotype using IL-4. They were adjusted to a cell suspension with a density of 1×10 6 cells / mL, and 500 μL per well was seeded in a 12-well plate. After the cells adhered, the supernatant was discarded, and the cells were washed with PBS. Then, 1 mL of PBS, CQ (4.5 μg / mL), IFN-γ (30 ng / mL), and IFN-γ / CQ-Lip (30 ng / mL + 4.5 μg / mL) were added to each well, with 3 replicate wells set. After incubating with the above groups for 24 h respectively, the expressions of CD80 and CD206 on the surface of macrophages were detected using a flow cytometer. The results were as Figure 6 shown. The IFN-γ / CQ-Lip group effectively upregulated the expression of the M1 macrophage marker CD80 and downregulated the expression of the M2 macrophage marker CD206, demonstrating that IFN-γ / CQ-Lip can fully exert and enhance the phenotypic conversion effect of IFN-γ and CQ on macrophages.

[0078] Example 4

[0079] Effect of ROS-responsive protein-crosslinked liposomes on MHC-I expression in tumor cells

[0080] Mouse pancreatic cancer cell line PANC02 was selected as the model cell and adjusted to a cell suspension with a density of 1×10 6 cells / mL. 500 μL per well was seeded in a 12-well plate. After the cells adhered, the supernatant was discarded, and the cells were washed with PBS. Then, 1 mL of PBS, CQ (4.5 μg / mL), IFN-γ (30 ng / mL), and IFN-γ / CQ-Lip (30 ng / mL + 4.5 μg / mL) were added to each well, with 3 replicate wells set. After incubating with the above groups for 24 h respectively, the MHC-I expression of PANC02 was detected using a flow cytometer. The results were as Figure 7 shown. The IFN-γ / CQ-Lip group effectively upregulated the MHC-I expression of PANC02 cells, and was significantly better than the IFN-γ group and the CQ group. This demonstrated that IFN-γ / CQ-Lip can fully exert and enhance the upregulation effect of IFN-γ and CQ on MHC-I in tumor cells.

[0081] Example 5

[0082] Pro-apoptotic effect of ROS-responsive protein-crosslinked liposomes on tumor cells

[0083] PANC02 cells were adjusted to a density of 1×106 A cell suspension of cells / mL was added at 500 μL per well to a 12-well plate. After the cells adhered to the wall, the supernatant was discarded, and the cells were washed with PBS. Then, 1 mL of PBS, CQ (4.5 μg / mL), IFN-γ (30 ng / mL), and IFN-γ / CQ-Lip (30 ng / mL + 4.5 μg / mL) were added to each well, with 3 replicate wells set up. After incubation with each of the above groups for 24 h, flow cytometry was used to detect the apoptosis of PANC02. The results are as Figure 8 shown. The IFN-γ / CQ-Lip group effectively promoted the apoptosis of tumor cells.

[0084] The above examples are only illustrative of the technical solutions and effects of the present invention, and thus cannot limit the protection scope of the present invention. The present invention has also prepared ROS-responsive protein cross-linked liposomes using other small molecule drugs and protein drugs, and has extremely similar effects to those of the above examples, showing good therapeutic effects on solid tumors.

Claims

1. A ROS-responsive protein-crosslinked liposome, characterized in that, it is formed by crosslinking a drug-loaded liposome with a protein drug electrostatically adsorbed on the surface of the drug-loaded liposome using a ROS-responsive crosslinking agent; the chemical structure of the ROS-responsive crosslinking agent is as follows: ， The protein drug is selected from positively charged protein drugs and negatively charged protein drugs; the positively charged protein drugs are selected from IFN-γ and tumor necrosis factor-related apoptosis-inducing ligand, and the negatively charged protein drugs are selected from IL2 and IL15.

2. The ROS-responsive protein-crosslinked liposome according to claim 1, characterized in that, first prepare a liposome loaded with a small molecule drug, then adsorb a protein drug with an opposite charge onto the surface of the liposome, and finally crosslink it using a ROS-responsive crosslinking agent.

3. The ROS-responsive protein-crosslinked liposome according to claim 2, characterized in that, the liposome is composed of a cationic lipid or an anionic lipid, and a common auxiliary lipid; the cationic lipid is selected from trimethyl-2,3-dioleyloxypropylammonium chloride, trimethyl-2,3-dioleoyloxypropylammonium bromide, or a tertiary amine cationic lipid derivative, and the chemical structure of the tertiary amine cationic lipid derivative is shown in the general formula (I): ， Wherein, n = 1 or 2; m represents an integer from 1 to 4; R 1 = ， p represents an integer from 2 to 4, q represents an integer from 1 to 3, r represents an integer from 1 to 2, s represents an integer from 0 to 3, R 5 represents methyl, hydroxymethyl, ethyl, hydroxyethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl, R 6 represents methyl, ethyl or benzyl, R 7 represents pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, pyrrolyl, pyrazolyl or imidazolyl; R 2 = , where u represents an integer from 7 to 19; the anionic lipid is selected from dioleoyl phosphatidylserine, dioleoyl phosphatidylglycerol, 4-((1,5-dioxo-1,5-bis(tetradecyloxy)pentan-2-yl)amino)-4-oxobutyric acid; the common auxiliary lipid is selected from lecithin, dioleoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, distearoyl phosphatidylcholine, dioleoyl lecithin, dierucoyl lecithin, dipalmitoyl lecithin or cholesterol.

4. The ROS-responsive protein-crosslinked liposome according to claim 3, characterized in that, the anionic lipid is 4-((1,5-dioxo-1,5-bis(tetradecyloxy)pentan-2-yl)amino)-4-oxobutyric acid, and the structural formula is: 。 5. The ROS-responsive protein-crosslinked liposome according to claim 3, characterized in that, the common auxiliary lipid is selected from lecithin or cholesterol.

6. The ROS-responsive protein-crosslinked liposome according to claim 2, characterized in that, the small molecule drug is selected from tumor chemotherapy drugs or small molecule immunomodulatory drugs; the tumor chemotherapy drugs are selected from paclitaxel, doxorubicin, chloroquine phosphate; the small molecule immunomodulatory drugs are selected from TGF-β inhibitors.

7. The ROS-responsive protein-crosslinked liposome according to claim 6, characterized in that, the small molecule drug is chloroquine phosphate.

8. The ROS-responsive protein-crosslinked liposome according to claim 1, characterized in that, the protein drug is IFN-γ.

9. Use of the ROS-responsive protein-crosslinked liposome according to any one of claims 1-8 in the preparation of a drug for treating solid tumors.

10. The use according to claim 9, characterized in that, The solid tumors described above are selected from pancreatic cancer, breast cancer, lung cancer, and liver cancer.

11. A ROS-responsive crosslinker, characterized in that its chemical structure is as follows: 。 12. Use of the ROS-responsive crosslinker according to claim 11 in the preparation of liposome and protein crosslinking drugs.

13. Use of the ROS-responsive crosslinker according to claim 11 in the preparation of protein crosslinkers.

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