Agglomerate for intracellular delivery of protein as well as preparation method and application of agglomerate

Abstract

The invention relates to the technical field of protein delivery, and particularly discloses a condensation body for protein intracellular delivery and a preparation method and application thereof.The preparation method comprises the steps that methoxy polyethylene glycol is dissolved to obtain a methoxy polyethylene glycol solution, and protein is dissolved to obtain a protein solution; uniformly mixing the methoxy polyethylene glycol solution with the protein solution, then carrying out amidation reaction, and after the reaction is finished, carrying out dialysis and freeze-drying to obtain polyethylene glycol modified protein; dissolving the polyethylene glycol modified protein to obtain a polyethylene glycol modified protein solution, uniformly mixing the polyethylene glycol modified protein solution with a polyphenol solution, and assembling to obtain a protein-loaded aggregate. The protein is treated by methoxy polyethylene glycol, and polyphenol is used for treatment and assembly to obtain the aggregate, so that the loading efficiency of the protein in the aggregate and the stability of the aggregate are remarkably improved, and the loading efficiency of the protein is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of protein delivery, and in particular to an aggregate for intracellular protein delivery and a preparation method and application thereof. Background Art

[0002] In recent years, protein therapeutics have emerged as highly effective and specific treatments for a wide range of diseases, including cancer, autoimmune diseases, and metabolic diseases, which account for a large proportion of the pharmaceutical market. However, due to the large and complex molecular structure, hydrophilicity, and membrane impermeability of proteins, the intracellular uptake and bioavailability of proteins are complicated, resulting in clinically used protein drugs targeting only extracellular targets. Therefore, the development of powerful and efficient intracellular protein delivery systems remains a key challenge in the biomedical field.

[0003] A coacervate is a delivery vehicle formed by self-assembly of charged polymers through liquid-liquid phase separation. It can be used as a "protocell" to simulate membraneless organelles and explore cell functions. However, coacervates are easily affected by the surrounding environment such as pH and salt concentration, which affects the coacervate-gel / sol transition and destroys the microstructure of the coacervate. The formed coacervate will inevitably diffuse and exchange rapidly with the surrounding dilute solution, which makes it impossible to quickly separate the coacervate loaded with macromolecules, affecting the encapsulation efficiency of the macromolecules. Therefore, it is extremely important to provide a method for preparing a stable protein intracellular delivery coacervate. Summary of the invention

[0004] In order to develop a method for preparing a stable protein intracellular delivery aggregate, the present invention provides an aggregate for protein intracellular delivery and its preparation method and application. The present invention treats the protein with methoxy polyethylene glycol and treats the assembled aggregate with polyphenols, which significantly improves the loading efficiency of the protein in the aggregate and the stability of the aggregate, thereby improving the protein loading efficiency.

[0005] The present invention provides a method for preparing an aggregate for intracellular delivery of a protein, comprising the following steps:

[0006] The methoxy polyethylene glycol is dissolved to obtain a methoxy polyethylene glycol solution, the protein is dissolved to obtain a protein solution, the methoxy polyethylene glycol solution and the protein solution are mixed evenly to cause an amidation reaction, and after the reaction is completed, dialysis and freeze-drying are performed to obtain a polyethylene glycol-modified protein; the polyethylene glycol-modified protein is dissolved to obtain a polyethylene glycol-modified protein solution, the polyethylene glycol-modified protein solution is mixed evenly with a polyphenol solution, and a protein-loaded aggregate is assembled.

[0007] The present invention uses methoxy polyethylene glycol solution and protein solution to mix and then undergo amidation reaction, uses methoxy polyethylene glycol to modify protein, and uses polyphenol to treat and assemble the obtained coacervate, effectively encapsulates proteins of different sizes and isoelectric points into the coacervate, significantly improves the loading efficiency of the protein in the coacervate and the stability of the coacervate. The coacervate obtained by the preparation method of the present invention has a higher protein loading efficiency, can effectively deliver proteins of different properties into cells, and overcomes the limitations of traditional delivery methods.

[0008] Furthermore, the methoxy polyethylene glycol is any one of methoxy polyethylene glycol succinimidyl succinate, methoxy polyethylene glycol carboxyl and methoxy polyethylene glycol amino.

[0009] Furthermore, the protein is any one of bovine serum albumin, glucose oxidase, casein, catalase, superoxide dismutase, lysozyme and horseradish peroxidase.

[0010] Further, the polyphenol solution is obtained by dissolving polyphenol;

[0011] The polyphenol is any one of tannic acid, gallic acid, epigallocatechin gallate, catechin and anthocyanin, or a combination of several of them.

[0012] Furthermore, the solvents of the methoxy polyethylene glycol solution, protein solution and polyphenol solution are all selected from any one of ultrapure water, physiological saline and PBS buffer.

[0013] Furthermore, the concentration of the methoxypolyethylene glycol solution is 100 mg / mL to 150 mg / mL, the concentration of the protein solution is 10 mg / mL to 25 mg / mL, the concentration of the polyethylene glycol-modified protein solution is 3 mg / mL to 10 mg / mL, and the concentration of the polyphenol solution is 0.5 mg / mL to 10 mg / mL.

[0014] Furthermore, the concentration of the polyethylene glycol-modified protein solution is 3 mg / mL to 5 mg / mL.

[0015] Furthermore, the mixing volume ratio of the polyethylene glycol-modified protein solution to the polyphenol solution is 0.1 to 10:1.

[0016] The present invention also provides an aggregate for intracellular protein delivery, which is prepared by the above-mentioned method for preparing the aggregate for intracellular protein delivery.

[0017] The present invention also provides a use of the aggregate for intracellular protein delivery in the preparation of a drug for treating acute liver injury.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The invention uses a methoxypolyethylene glycol solution and a protein solution to undergo an amidation reaction, utilizes a PEGylation protein method, and uses polyphenol treatment for assembly, and effectively encapsulates proteins of different sizes and isoelectric points into coacervates through hydrogen bond interactions and the like, thereby significantly improving the loading efficiency of the protein in the coacervates and the stability of the coacervates; the coacervates obtained by the preparation method of the invention have a higher protein loading efficiency, can effectively deliver proteins of different properties into cells, and overcome the limitations of traditional delivery methods.

[0020] The coacervate obtained by the preparation method of the present invention has a better efficacy in scavenging ROS and rescuing hepatocyte sepsis in a mouse model of acute liver injury induced by acetaminophen than the clinical drug N-acetylcysteine. The present invention utilizes the advantages of PEGylation and coacervate technology and is expected to solve key problems related to protein therapy, thereby potentially providing more effective treatments for various diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 Schematic diagram of the preparation method of PEG-CAT@Coa aggregates prepared in Example 1 of the present invention.

[0023] Figure 2 TEM photo of the agglomerate prepared by the present invention;

[0024] In the figure, a is the TEM image of PEG-CAT@Coa aggregates;

[0025] b is the TEM image of PEG-SOD@Coa aggregates;

[0026] c is the TEM image of PEG-BSA@Coa aggregates.

[0027] Figure 3 The particle size results of the agglomerates prepared in Example 1, Example 5, Example 6, Comparative Example 1, Comparative Example 2 and Comparative Example 3 of the present invention;

[0028] In the figure, a is the particle size result of CAT@Coa and PEG-CAT@Coa immersed in water or PBS;

[0029] b shows the particle size results of SOD@Coa and PEG-SOD@Coa immersed in water or PBS;

[0030] c is the particle size results of BSA@Coa and PEG-BSA@Coa immersed in water or PBS.

[0031] Figure 4 The protein loading amount of the coacervates prepared in Example 1, Example 5, Example 6, Comparative Example 1, Comparative Example 2 and Comparative Example 3 of the present invention.

[0032] Figure 5 The enzyme activity of the aggregate prepared by the present invention can;

[0033] In the figure, a is the enzyme activity energy of PEG-CAT@Coa;

[0034] b is the enzyme activity of PEG-SOD@Coa.

[0035] Figure 6 The biocompatibility of the coacervate prepared by the present invention;

[0036] In the figure, a shows the effect of PEG-CAT@Coa aggregates on the survival rate of RAW 264.7 cells;

[0037] b shows the effect of PEG-CAT@Coa aggregates on the survival rate of L02 cells;

[0038] c is the effect of PEG-SOD@Coa aggregates on the survival rate of RAW 264.7 cells;

[0039] d is the effect of PEG-SOD@Coa aggregates on the survival rate of L02 cells;

[0040] e is the effect of PEG-BSA@Coa aggregates on the survival rate of RAW 264.7 cells;

[0041] f is the effect of PEG-BSA@Coa aggregates on the survival rate of L02 cells.

[0042] Figure 7 The cell endocytosis ability of the aggregate prepared in Example 1 of the present invention;

[0043] In the figure, a is a photograph of the endocytosis of the aggregates;

[0044] b shows the quantitative data of endocytosis of aggregates.

[0045] Figure 8 The test results of the intracellular active oxygen scavenging ability of the aggregates prepared by the present invention;

[0046] In the figure, a is the test result of PEG-CAT@Coa and PEG-SOD@Coa on the ability of scavenging reactive oxygen species in RAW 264.7 cells;

[0047] b shows the test results of PEG-CAT@Coa and PEG-SOD@Coa on the reactive oxygen species scavenging ability in L02 cells.

[0048] Fig. 9 This is the in vivo distribution diagram of the coacervate prepared in Example 1 of the present invention;

[0049] In the figure, a is a photo of the distribution of aggregates in the body;

[0050] b is the quantitative distribution diagram in vivo.

[0051] Fig.10 The present invention demonstrates the therapeutic effects of the PEG-CAT@Coa aggregates and the PEG-SOD@Coa aggregates prepared by the present invention on acute liver injury in mice.

[0052] Fig.11 Schematic diagram of the principle of using the aggregates in an embodiment of the present invention to treat acute liver injury, wherein a is the preparation process of the aggregates for intracellular protein delivery; b is a schematic diagram of the mechanism of the aggregates for intracellular protein delivery to treat acute liver injury by releasing catalase or superoxide dismutase to remove intracellular reactive oxygen species. DETAILED DESCRIPTION

[0053] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0054] Example 1: A polymer for intracellular protein delivery and a method for preparing the same.

[0055] (1) Accurately weigh 100 mg of catalase in 5 mL of ultrapure water, stir and dissolve at 25°C and 500 rpm for 3 min to obtain a dissolved catalase solution. Accurately weigh 625 mg of methoxy polyethylene glycol succinimidyl succinate (Mw = 5000) in 5 mL of ultrapure water, ultrasonically dissolve at 25°C for 10 min to obtain a dissolved methoxy polyethylene glycol succinimidyl succinate solution. After mixing the methoxy polyethylene glycol succinimidyl succinate solution and the catalase solution in equal volumes for 4 hours, dialyze the solution for 24 hours using a dialysis bag with a molecular weight cutoff of 3500 Da, and then place it in a freeze dryer at -80°C for 48 hours to obtain polyethylene glycol-modified catalase.

[0056] (2) Accurately weigh 3 mg of polyethylene glycol-modified catalase in 1 mL of ultrapure water, stir at 25° C. and 500 rpm for 5 min to obtain a dissolved polyethylene glycol-modified catalase solution with a concentration of 3 mg / mL; accurately weigh 1 mg of epigallocatechin gallate in 1 mL of ultrapure water, stir at 25° C. and 500 rpm to obtain a dissolved epigallocatechin gallate solution with a concentration of 1 mg / mL.

[0057] (3) 0.5 mL of the prepared polyethylene glycol-modified catalase solution and epigallocatechin gallate solution were taken and mixed evenly, and stirred at 25° C. and 100 rpm for 5 min to obtain a protein-loaded aggregate, which was recorded as PEG-CAT@Coa.

[0058] Example 1 Schematic diagram of preparing PEG-CAT@Coa Figure 1 shown.

[0059] Example 2: A polymer for intracellular protein delivery and a method for preparing the same.

[0060] The preparation steps of this example are basically the same as those of Example 1, except that the concentration of the polyethylene glycol-modified catalase solution is 10 mg / mL.

[0061] Example 3: A polymer for intracellular protein delivery and a method for preparing the same.

[0062] The preparation steps of this embodiment are basically the same as those of embodiment 1, except that the concentration of epigallocatechin gallate is 5 mg / mL.

[0063] Example 4: A polymer for intracellular protein delivery and a method for preparing the same.

[0064] The preparation steps of this example are basically the same as those of Example 1, except that: the polyethylene glycol-modified catalase solution and the epigallocatechin gallate solution are mixed in equal volumes and stirred at 25° C. and 100 rpm for 1 h.

[0065] Example 5: A polymer for intracellular protein delivery and a method for preparing the same.

[0066] The preparation steps of this example are basically the same as those of Example 1, except that catalase is replaced by superoxide dismutase. The aggregate prepared in Example 5 is denoted as PEG-SOD@Coa.

[0067] Example 6: A polymer for intracellular protein delivery and a method for preparing the same.

[0068] The preparation steps of this example are basically the same as those of Example 1, except that catalase is replaced by bovine serum albumin. The aggregate prepared in Example 6 is denoted as PEG-BSA@Coa.

[0069] Example 7: A polymer for intracellular protein delivery and a method for preparing the same.

[0070] The preparation steps of this embodiment are basically the same as those of embodiment 1, except that epigallocatechin gallate is replaced by tannic acid.

[0071] Example 8: A polymer for intracellular protein delivery and a method for preparing the same.

[0072] The preparation steps of this embodiment are basically the same as those of embodiment 1, except that methoxy polyethylene glycol succinimidyl succinate is replaced by the carboxyl group of methoxy polyethylene glycol.

[0073] Example 9: A polymer for intracellular protein delivery and a method for preparing the same.

[0074] The preparation steps of this embodiment are basically the same as those of embodiment 1, except that methoxy polyethylene glycol succinimide succinate is replaced by methoxy polyethylene glycol amino.

[0075] Example 10: A polymer for intracellular protein delivery and a method for preparing the same.

[0076] The preparation steps of this example are basically the same as those of Example 1, except that: epigallocatechin gallate is replaced by an equal volume mixed solution of epigallocatechin gallate and tannic acid, and the concentration of the mixed solution is consistent with the concentration of the epigallocatechin gallate solution. The polyethylene glycol-modified catalase solution is replaced by an equal volume mixed solution of a polyethylene glycol-modified catalase solution and a polyethylene glycol-modified superoxide dismutase solution, and the concentration of the mixed solution is consistent with the polyethylene glycol-modified catalase solution.

[0077] Comparative Example 1

[0078] In order to compare the effects of PEGylated protein and direct blending of PEG and protein on the formation of coacervates, Comparative Example 1 was set up. The difference between Comparative Example 1 and Example 1 is that methoxypolyethylene glycol carboxyl, catalase and epigallocatechin gallate were directly and evenly blended to obtain coacervates. The coacervates prepared in Comparative Example 1 are recorded as CAT@Coa.

[0079] Comparative Example 2:

[0080] The preparation steps of this comparative example are basically the same as those of comparative example 1, except that catalase is replaced by superoxide dismutase. The aggregate prepared in comparative example 2 is denoted as SOD@Coa.

[0081] Comparative Example 3:

[0082] The preparation steps of this comparative example are basically the same as those of comparative example 1, except that catalase is replaced by bovine serum albumin. The aggregates prepared in comparative example 3 are denoted as BSA@Coa.

[0083] Taking the PEG-CAT@Coa aggregates prepared in Example 1, the PEG-SOD@Coa aggregates prepared in Example 5, and the PEG-BSA@Coa aggregates prepared in Example 6 as examples, TEM tests, stability performance tests, protein loading capacity tests, enzyme activity tests, biocompatibility tests, cell endocytosis ability tests, intracellular reactive oxygen level tests, and therapeutic effects on acute liver injury in mice were performed.

[0084] 1. TEM test

[0085] The morphology of the agglomerates prepared in Example 1, Example 5 and Example 6 was characterized by TEM.

[0086] The results are as follows Figure 2 As shown, the agglomerates in Example 1, Example 5 and Example 6 were successfully prepared and showed good dispersibility in the solution.

[0087] 2. Stability performance test

[0088] The stability of the coacervates prepared in Example 1, Example 5, Example 6, Comparative Example 1, Comparative Example 2 and Comparative Example 3 was tested. The three coacervates were immersed in water and PBS for 0 h to 24 h, respectively, and the particle size of the coacervates was measured using a nanoparticle size analyzer.

[0089] The results are as follows Figure 3 As shown, the coacervates prepared in Example 1, Example 5 and Example 6 were maintained in the range of 200nm to 300nm in water and PBS, showing good stability. However, the coacervates prepared in Comparative Examples 1 to Comparative Examples 3 showed an increase in particle size in water and PBS, increasing to >1000nm within 6 hours, indicating that the coacervates prepared in the examples of the present invention have better stability.

[0090] 3. Protein loading capacity test

[0091] The protein loading capacity of the coacervates prepared in Example 1, Example 5, Example 6, Comparative Example 1, Comparative Example 2 and Comparative Example 3 was tested, and the protein content in the coacervates was quantified using fluorescence spectroscopy.

[0092] The results are as follows Figure 4 As shown, the loading amounts of CAT, SOD and BSA in the coacervates prepared in Comparative Examples 1, 2 and 3 were 9.6%, 18.2% and 10.2%, respectively, while the loading amount of CAT in the PEG-CAT@Coa coacervate prepared in Example 1 was 31.8%, the loading amount of SOD in the PEG-SOD@Coa coacervate prepared in Example 5 was 58.2%, and the loading amount of BSA in the PEG-BSA@Coa coacervate prepared in Example 6 was 69.1%, indicating that the coacervates prepared in the examples have higher loading efficiency for proteins.

[0093] 4. Enzyme activity test

[0094] The enzyme activity of the aggregates prepared in Example 1 and Example 5 can be tested by measuring the enzyme activity using a catalase detection kit (purchased from Biotime Biotechnology Co., Ltd.) and a superoxide dismutase detection kit (purchased from Biotime Biotechnology Co., Ltd.).

[0095] The results are as follows Figure 5 As shown in the figure, with the increase of CAT concentration, its ability to scavenge hydrogen peroxide increased significantly, reaching the highest point when the CAT concentration exceeded 12 μg / mL. PEG-SOD@Coa also had a similar trend, with a superoxide anion scavenging rate of about 60%.

[0096] 5. Biocompatibility testing

[0097] The biocompatibility test was performed on the coacervates prepared in Example 1, Example 5 and Example 6, and the biocompatibility of the coacervates on mouse mononuclear macrophages (RAW 264.7) and mouse hepatocytes (L02) was characterized by using a cell proliferation and toxicity detection kit (CCK-8, purchased from Bio-Tech Biotech Co., Ltd.). The specific steps are as follows:

[0098] A 96-well cell culture plate was selected, and 5000 cells were inoculated in each well. After overnight culture, 100 μL of PEG-CAT@Coa prepared in Example 1, PEG-SOD@Coa prepared in Example 5, and PEG-BSA@Coa prepared in Example 6 were added to the cells and cultured for 24 hours. After the culture was completed, 10 μL of CCK-8 solution was added to each well. After 4 hours of action, the absorbance was detected using an ELISA instrument with an excitation wavelength of 480 nm. The biocompatibility curve is shown in FIG. Figure 6 As shown, different types of aggregates can maintain cell growth very well, and the cell survival rate can reach more than 95%.

[0099] 6. Cell endocytosis ability test

[0100] The cell endocytosis ability of the aggregates prepared in Example 6 was tested. A 24-well cell culture plate was selected and 50,000 cells were inoculated in each well. After overnight culture, 500 μL of the PEG-BSA@Coa aggregates prepared in Example 6 were taken out and incubated with the cells for 24 hours. The nuclei of RAW 264.7 cells were stained with Hoechst 33342, and the cytoskeleton was stained with wheat germ agglutinin 594 (WGA594).

[0101] The results are as follows Figure 7 PEG-BSA@Coa aggregates can effectively deliver proteins into cells, confirming their potential in drug delivery and therapeutic applications.

[0102] 7. Test of intracellular reactive oxygen levels

[0103] The intracellular reactive oxygen levels were tested for the aggregates prepared in Example 1 and Example 5. A 24-well cell culture plate was selected, 50,000 cells were inoculated in each well, and after overnight culture, 500 μL of aggregates were taken out and incubated with cells for 24 h, and then hydrogen peroxide was added to continue the treatment for 24 h. The intracellular reactive oxygen levels were then stained with a cell-permeable intracellular reactive oxygen probe (DCFH-DA) and the results were observed using an inverted fluorescence microscope.

[0104] The results are as follows Figure 8As shown, the level of intracellular reactive oxygen species increased after hydrogen peroxide treatment, while the ROS levels in RAW 264.7 cells and L02 cells treated with PEG-CAT@Coa and PEG-SOD@Coa were comparable to those in the normal cell group, highlighting their effective ROS scavenging ability.

[0105] 8. Treatment of acute liver injury in mice using protein intracellular delivery of aggregates

[0106] In order to further demonstrate the therapeutic potential of the aggregates obtained in Example 1 for acute liver injury, the distribution of the aggregates in mice was analyzed. First, mice were anesthetized with isoflurane, and 200 μL of cy5.5-labeled PEG-CAT@Coa was injected into the tail vein. The mice were killed after 0, 1, 2, 4, 6, and 8 hours, and the main organs (heart, liver, spleen, lung, and kidney) were collected. The tissue distribution was analyzed by small animal in vivo imaging, and the fluorescence intensity was evaluated using the ROI tool.

[0107] The results are as follows Fig. 9 As shown, PEG-CAT@Coa accumulated mainly in the liver and reached a peak after 4 h. This liver accumulation is critical for targeting liver diseases and alleviating liver oxidative stress and damage.

[0108] 9. Treatment of acute liver injury in mice using protein intracellular delivery of aggregates

[0109] To further demonstrate the potential of the agglomerates in treating acute liver injury obtained in Examples 1 and 5, 6-week-old BALB / C female mice were intraperitoneally injected with acetaminophen solution (APAP, 60 mg / kg) after overnight fasting, and an APAP-induced mouse liver injury model was successfully established 3 hours after injection. Subsequently, PEG-CAT@Coa and PEG-SOD@Coa were injected into the tail vein of the mice, and 12 hours later, the mouse liver tissues were collected for H&E staining analysis.

[0110] The results are as follows Fig.10 As shown in Figure 2, APAP-induced liver injury mice showed extensive liver tissue damage, severe tissue destruction, sinus congestion and bleeding, while mice treated with PEG-CAT@Coa and PEG-SOD@Coa suffered only mild damage. Fig.11 .

[0111] Although preferred embodiments of the present invention have been described, additional changes and modifications may occur to these embodiments once those skilled in the art are aware of the basic inventive concepts.

[0112] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing an aggregate for intracellular delivery of a protein, characterized in that: The steps include: The methoxy polyethylene glycol is dissolved to obtain a methoxy polyethylene glycol solution, the protein is dissolved to obtain a protein solution, the methoxy polyethylene glycol solution and the protein solution are mixed evenly to cause an amidation reaction, and after the reaction is completed, dialysis and freeze-drying are performed to obtain a polyethylene glycol-modified protein; the polyethylene glycol-modified protein is dissolved to obtain a polyethylene glycol-modified protein solution, the polyethylene glycol-modified protein solution is mixed evenly with a polyphenol solution, and a protein-loaded aggregate is assembled.

2. The method for preparing an aggregate for intracellular delivery of a protein according to claim 1, characterized in that: The methoxy polyethylene glycol is any one of methoxy polyethylene glycol succinimidyl succinate, methoxy polyethylene glycol carboxyl and methoxy polyethylene glycol amino.

3. The method for preparing an aggregate for intracellular delivery of a protein according to claim 1, characterized in that: The protein is any one of bovine serum albumin, glucose oxidase, casein, catalase, superoxide dismutase, lysozyme and horseradish peroxidase.

4. The method for preparing an aggregate for intracellular delivery of a protein according to claim 1, characterized in that: The polyphenol solution is obtained by dissolving polyphenol; The polyphenol is any one of tannic acid, gallic acid, epigallocatechin gallate, catechin and anthocyanin, or a combination of several of them.

5. The method for preparing an aggregate for intracellular delivery of a protein according to claim 1, characterized in that: The solvents of the methoxy polyethylene glycol solution, protein solution and polyphenol solution are all selected from any one of ultrapure water, physiological saline and PBS buffer.

6. The method for preparing an aggregate for intracellular delivery of protein according to claim 1, characterized in that: The concentration of the methoxypolyethylene glycol solution is 100 mg / mL to 150 mg / mL, the concentration of the protein solution is 10 mg / mL to 25 mg / mL, the concentration of the polyethylene glycol-modified protein solution is 3 mg / mL to 10 mg / mL, and the concentration of the polyphenol solution is 0.5 mg / mL to 10 mg / mL.

7. The method for preparing an aggregate for intracellular delivery of a protein according to claim 6, characterized in that: The concentration of the polyethylene glycol-modified protein solution is 3 mg / mL to 5 mg / mL.

8. The method for preparing an aggregate for intracellular delivery of protein according to claim 6, characterized in that: The mixing volume ratio of the polyethylene glycol-modified protein solution to the polyphenol solution is 0.1 to 10:

1.

9. An aggregate for intracellular delivery of proteins, characterized in that: The protein aggregate is prepared by the method for preparing the protein aggregate for intracellular delivery according to any one of claims 1 to 8.

10. Use of the aggregate for intracellular protein delivery according to claim 9 in the preparation of a drug for treating acute liver injury.

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