A coacervate for intracellular delivery of proteins and methods of making and using the same

Abstract

The present application relates to the technical field of protein delivery, and specifically discloses a condensate for intracellular delivery of protein as well as a preparation method and application thereof. After methoxypolyethylene glycol is dissolved, a methoxypolyethylene glycol solution is obtained. After protein is dissolved, a protein solution is obtained. After the methoxypolyethylene glycol solution and the protein solution are uniformly mixed, an amidation reaction occurs. After the reaction is completed, dialysis and freeze-drying are performed to obtain polyethylene glycol modified protein. After the polyethylene glycol modified protein is dissolved, a polyethylene glycol modified protein solution is obtained. After the polyethylene glycol modified protein solution and a polyphenol solution are uniformly mixed, a condensate loaded with protein is assembled. The present application processes protein by using methoxypolyethylene glycol and assembles a condensate by using polyphenol, thereby significantly improving the loading efficiency of protein in the condensate and the stability of the condensate and improving the protein loading efficiency.

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 become highly effective and specific treatments for a wide range of diseases, including cancer, autoimmune diseases, and metabolic disorders, which together represent a significant portion of the pharmaceutical market. However, due to their large and complex molecular structures, hydrophilicity, and membrane impermeability, the intracellular uptake and bioavailability of proteins are complicated, resulting in clinically used protein drugs targeting only extracellular targets. Therefore, the development of robust and efficient intracellular protein delivery systems remains a key challenge in the biomedical field.

[0003] Coacervates are a type of delivery vehicle formed by self-assembly of charged polymers through liquid-liquid phase separation. They can serve as "protocells" 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 thus 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 stable coacervates for intracellular protein delivery. Summary of the Invention

[0004] To develop a method for preparing stable protein aggregates for intracellular delivery, the present invention provides aggregates for protein intracellular delivery, as well as their preparation and application. By treating proteins with methoxypolyethylene glycol and then assembling the aggregates with polyphenols, the present invention significantly improves the protein loading efficiency and aggregate stability within the aggregates, thereby enhancing 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 method comprises dissolving methoxy polyethylene glycol to obtain a methoxy polyethylene glycol solution, dissolving protein to obtain a protein solution, mixing the methoxy polyethylene glycol solution and the protein solution, performing an amidation reaction, and performing dialysis and freeze-drying after the reaction to obtain a polyethylene glycol-modified protein; dissolving the polyethylene glycol-modified protein to obtain a polyethylene glycol-modified protein solution, mixing the polyethylene glycol-modified protein solution with a polyphenol solution, and assembling to obtain a protein-loaded aggregate.

[0007] The present invention involves mixing a methoxypolyethylene glycol solution with a protein solution, followed by an amidation reaction, modifying the protein with methoxypolyethylene glycol, and treating the resulting aggregates with polyphenols. This effectively encapsulates proteins of varying sizes and isoelectric points within the aggregates, significantly improving both the protein loading efficiency and the stability of the aggregates. The aggregates obtained using the preparation method of the present invention have a higher protein loading efficiency and can effectively deliver proteins of varying properties into cells, overcoming the limitations of traditional delivery methods.

[0008] Furthermore, the methoxy polyethylene glycol is any one of methoxy polyethylene glycol succinimide 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 methoxypolyethylene 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 present invention uses a methoxypolyethylene glycol solution to dissolve protein to produce an amidation reaction, utilizes a PEGylation protein method, and assembles it with polyphenol treatment, and effectively encapsulates proteins of different sizes and isoelectric points into coacervates through hydrogen bond interactions and other methods, 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 present invention have higher protein loading efficiency, can effectively deliver proteins of different properties into cells, and overcome the limitations of traditional delivery methods.

[0020] The coacervates produced by the preparation method of the present invention demonstrated superior efficacy in scavenging ROS and rescuing hepatocellular sepsis in a mouse model of acetaminophen-induced acute liver injury compared to the clinical drug N-acetylcysteine. By leveraging the advantages of PEGylation and coacervation technologies, the present invention is expected to address key challenges associated with protein therapeutics, potentially providing more effective treatments for a variety of 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 following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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 any 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 photos of the aggregates prepared in 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 are as follows;

[0028] In the figure, a is the particle size results 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 viability 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 shows the effect of PEG-BSA@Coa aggregates on the viability 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 cellular endocytosis ability of the aggregates prepared in Example 1 of the present invention;

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

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

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

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

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

[0048] Figure 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] Figure 10 The present invention demonstrates the therapeutic effects of the PEG-CAT@Coa aggregates and the PEG-SOD@Coa aggregates prepared in the present invention on acute liver injury in mice.

[0052] Figure 11 Figure 1 is a schematic diagram of the principle of using aggregates for treating acute liver injury in an embodiment of the present invention, wherein a is the preparation process of aggregates for intracellular protein delivery; b is a schematic diagram of the mechanism by which aggregates for intracellular protein delivery treat acute liver injury by releasing catalase or superoxide dismutase to clear 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 scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within 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 sources unless otherwise specified.

[0054] Example 1: A polymer for intracellular protein delivery and a preparation method thereof.

[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 minutes 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 minutes to obtain a dissolved methoxy polyethylene glycol succinimidyl succinate solution. After mixing equal volumes of methoxy polyethylene glycol succinimidyl succinate solution and catalase solution for 4 hours, dialyze the solution using a dialysis bag with a molecular weight cutoff of 3500 Da for 24 hours, 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. After stirring at 25°C and 100 rpm for 5 min, a protein-loaded aggregate was obtained, which was recorded as PEG-CAT@Coa.

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

[0059] Example 2: A polymer for intracellular protein delivery and a preparation method thereof.

[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 preparation method thereof.

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

[0063] Example 4: A polymer for intracellular protein delivery and a preparation method thereof.

[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 preparation method thereof.

[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 aggregates prepared in Example 5 are denoted as PEG-SOD@Coa.

[0067] Example 6: A polymer for intracellular protein delivery and a preparation method thereof.

[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 aggregates prepared in Example 6 are denoted as PEG-BSA@Coa.

[0069] Example 7: A polymer for intracellular protein delivery and a preparation method thereof.

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

[0071] Example 8: A polymer for intracellular protein delivery and a preparation method thereof.

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

[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 Example 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 preparation method thereof.

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

[0077] Comparative Example 1

[0078] To compare the effects of PEGylated protein and direct blending of PEG and protein on coacervate formation, Comparative Example 1 was prepared. This differed from Example 1 in that methoxypolyethylene glycol carboxyl groups, catalase, and epigallocatechin gallate were directly and uniformly blended to form coacervates. The coacervates prepared in Comparative Example 1 were designated 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 aggregates prepared in comparative example 2 are 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 species level tests, and therapeutic effects on acute liver injury in mice were performed.

[0084] 1. TEM test

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

[0086] The results are as follows Figure 2 As shown, the coacervates 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 Examples 1, 5, and 6 maintained a size range of 200 nm to 300 nm in both water and PBS, demonstrating good stability. However, the coacervates prepared in Comparative Examples 1 to 3 showed an increase in particle size in both water and PBS, reaching >1000 nm 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 was tested by using a catalase detection kit (purchased from Beyotime Biotechnology Co., Ltd.) and a superoxide dismutase detection kit (purchased from Beyotime Biotechnology Co., Ltd.).

[0095] The results are as follows Figure 5 As shown in Figure 3, as the concentration of CAT increases, its ability to scavenge hydrogen peroxide increases significantly, reaching its highest point when the CAT concentration exceeds 12 μg / mL. PEG-SOD@Coa also has a similar trend, with a superoxide anion scavenging rate of approximately 60%.

[0096] 5. Biocompatibility testing

[0097] Biocompatibility testing was performed on the aggregates prepared in Examples 1, 5, and 6. The biocompatibility of the aggregates in mouse monocytes (RAW 264.7) and mouse hepatocytes (L02) was characterized using a cell proliferation and toxicity assay kit (CCK-8, purchased from Biyuntian Biotechnology Co., Ltd.). The specific steps are as follows:

[0098] A 96-well cell culture plate was used, and 5000 cells were seeded per 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 another 24 hours. After the culture was completed, 10 μL of CCK-8 solution was added to each well. After 4 hours, the absorbance was measured using a microplate reader with an excitation wavelength of 480 nm. The biocompatibility curve is shown in Figure 2. 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 endocytic ability of the aggregates prepared in Example 6 was tested. A 24-well cell culture plate was selected and 50,000 cells were inoculated per 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 for drug delivery and therapeutic applications.

[0102] 7. Intracellular reactive oxygen species level test

[0103] The aggregates prepared in Examples 1 and 5 were tested for intracellular reactive oxygen species (ROS) levels. A 24-well cell culture plate was used, with 50,000 cells seeded per well. After overnight culture, 500 μL of aggregates were removed and incubated with the cells for 24 hours. Hydrogen peroxide was then added for an additional 24 hours. The intracellular ROS levels were then stained with a cell-permeable intracellular ROS probe (DCFH-DA) and 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] 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 injected via the tail vein with 200 μL of Cy5.5-labeled PEG-CAT@Coa. Mice were sacrificed 0, 1, 2, 4, 6, and 8 hours later. Major organs (heart, liver, spleen, lung, and kidney) were harvested, and tissue distribution was analyzed using in vivo small animal imaging. Fluorescence intensity was assessed using the ROI tool.

[0107] The results are as follows Figure 9 As shown, PEG-CAT@Coa accumulated primarily in the liver and peaked after 4 hours. This liver accumulation is crucial 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 therapeutic potential of the aggregates obtained in Examples 1 and 5 for acute liver injury, 6-week-old BALB / C female mice were fasted overnight and intraperitoneally injected with acetaminophen (APAP, 60 mg / kg). Three hours after injection, an APAP-induced liver injury model was successfully established in mice. Subsequently, PEG-CAT@Coa and PEG-SOD@Coa were injected into the tail vein of the mice. Twelve hours later, liver tissues were harvested for H&E staining.

[0110] The results are as follows Figure 10 As shown, 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. Figure 11 .

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

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

Claims

1. A method for preparing an aggregate for intracellular delivery of a protein, characterized in that: The steps include: dissolving methoxy polyethylene glycol to obtain a methoxy polyethylene glycol solution, dissolving a protein to obtain a protein solution, mixing the methoxy polyethylene glycol solution and the protein solution, and performing an amidation reaction. After the reaction, dialyzing and lyophilizing are performed to obtain a polyethylene glycol-modified protein. Dissolving the polyethylene glycol-modified protein to obtain a polyethylene glycol-modified protein solution, mixing the polyethylene glycol-modified protein solution with a polyphenol solution, and assembling to obtain a protein-loaded aggregate. The methoxy polyethylene glycol is any one of methoxy polyethylene glycol succinimide succinate, methoxy polyethylene glycol carboxyl and methoxy polyethylene glycol amino.

2. The method for preparing an aggregate for intracellular protein delivery 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.

3. The method for preparing an aggregate for intracellular protein delivery 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.

4. The method for preparing an aggregate for intracellular protein delivery 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.

5. The method for preparing an aggregate for intracellular protein delivery 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.

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

7. The method for preparing an aggregate for intracellular protein delivery according to claim 5, characterized in that: The mixing volume ratio of the polyethylene glycol-modified protein solution to the polyphenol solution is 0.1 to 10:

1.

8. 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 7.

9. Use of the aggregate for intracellular protein delivery according to claim 8 in the preparation of a drug for treating acute liver injury, characterized in that: The protein is catalase or superoxide dismutase.

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