A protein nanodelivery system, its construction method and application

Abstract

The application discloses a protein nano delivery system and a construction method and application thereof, and belongs to the field of biological medicine materials. The construction method comprises the following steps: using natural polysaccharide modified with dopamine as a carrier skeleton, adding phenylboronic acid and proteins to assemble, and obtaining the protein nano delivery system. The application constructs a universal platform for delivery of various intracellular proteins, obtains a protein nano delivery system with uniform nanoparticle size and stable system, the protein nano delivery system can make proteins effectively enter cells to play functions, through experimental verification, it is found that the protein nano delivery system realizes the functions of improving the intracellular protein delivery efficiency and guaranteeing the biological activity of proteins entering cells. The method of the protein nano delivery system has the advantages of simple process, cheap and easily available raw materials and the like, realizes intracellular protein delivery and maintains the activity of the proteins, and can be applied to the fields of intracellular disease treatment and gene coding.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine materials, in particular to a protein nano delivery system and a construction method and application thereof. BACKGROUND

[0002] Protein drugs have an important position in the clinic. Compared with small molecule drugs, protein drugs have the characteristics of high efficiency, high selectivity and low side effects. However, at present, only protein drugs acting on extracellular targets have been used in clinical use, and there is no related clinical case for protein drugs acting on intracellular targets to play a role. This is because proteins have the characteristics of large size and hydrophilicity, and the three-dimensional structure of proteins is complex, which can easily lead to loss of activity, so it is difficult to pass through the cell membrane into the intracellular. However, most of the protein encoded by genes and protein therapy need to deliver the protein into the intracellular to play a role. Therefore, it is of great significance to take appropriate measures to deliver the protein into the intracellular and play the activity of the protein.

[0003] The current frontier research in this field mainly loads the protein by constructing a carrier. The combination of the carrier and the protein mainly includes two ways: one is to combine through covalent bond, and the other is to combine through intermolecular force. Both ways have some problems. The method of combining through covalent bond is complex and needs to modify or transform the protein, which may have the risk of destroying the activity of the protein; and the covalent bond combination is too strong, which is not conducive to the escape of the protein in the endosome in the intracellular. The combination method of intermolecular interaction is simple, but the action strength is relatively weak, and the combination is unstable, which can easily lead to the early release of the protein in the delivery process. Therefore, the solution strategy for this problem is to develop a new type of protein carrier with appropriate binding force.

[0004] Dynamic covalent bond is a covalent bond that can be broken under responsive conditions and can be formed under appropriate conditions. The application of this moderate strength bond system to protein delivery can realize efficient loading, targeted delivery and responsive release, which provides a possibility for the delivery of intracellular protein drugs. SUMMARY

[0005] The purpose of the present application is to provide a protein nano delivery system and a construction method and application thereof, so as to solve the problems existing in the prior art. By constructing the protein nano delivery system, the problems of too strong or too weak binding force between the carrier and the protein can be solved. The assembly body with strong combination can be formed under the assembly condition, and the binding force is weakened after being delivered into the intracellular, which promotes the release of the protein, thereby improving the efficiency of the intracellular protein delivery and ensuring the biological activity of the protein entering the intracellular.

[0006] In order to achieve the above purpose, the present application provides the following scheme:

[0007] The application provides a construction method for preparing an assembly by using natural polysaccharide to load proteins, comprising the following steps: using natural polysaccharide modified with dopamine as a carrier skeleton, adding phenylboronic acid and proteins to assemble, and obtaining a protein nano delivery system.

[0008] Preferably, the construction method specifically comprises the following steps:

[0009] (1) mixing and dissolving natural polysaccharide, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, adjusting the pH value to be acidic, then adding dopamine, stirring at room temperature for 20-30 hours, and obtaining dopamine-modified natural polysaccharide through dialysis and freeze-drying;

[0010] (2) respectively preparing protein, 2-acetylphenylboronic acid and dopamine-modified natural polysaccharide stock solutions;

[0011] (3) sequentially adding the stock solutions of the 2-acetylphenylboronic acid, the protein and the dopamine-modified natural polysaccharide, stirring at low temperature for 20-30 hours, and obtaining the protein nano delivery system through ultrafiltration purification.

[0012] Preferably, in step (1), the mass ratio of the natural polysaccharide, the 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and the N-hydroxysuccinimide is 100:(50-80):(20-60).

[0013] The mass ratio of the natural polysaccharide and the dopamine is 100:(40-60).

[0014] Preferably, in step (1), the pH value is adjusted to be 4.0-5.5.

[0015] Preferably, in step (3), the final concentrations of the protein, the 2-acetylphenylboronic acid and the dopamine-modified natural polysaccharide in the mixed solution in the reactor are respectively 0.1-1.0 mg / mL, 2-4 mg / mL and 0.01-0.1 mg / mL.

[0016] Preferably, in step (3), the low-temperature condition is 1-4 DEG C, and the molecular weight cut-off is 100 kDa during ultrafiltration.

[0017] Preferably, the natural polysaccharide comprises hyaluronic acid, and the protein comprises bovine serum albumin or horseradish peroxidase.

[0018] The application also provides a protein nano delivery system prepared by the construction method.

[0019] The application also provides application of the protein nano delivery system in preparing an antitumor drug.

[0020] The application discloses the following technical effects:

[0021] The application uses a long chain of a natural polysaccharide (hyaluronic acid) modified dopamine as a carrier skeleton, adds 2-acetylphenylboronic acid and a protein to assemble, constructs a protein nano delivery system, and then uses the protein nano delivery system for intracellular delivery. It can be seen that the application constructs a universal platform for intracellular protein delivery, and the process of constructing the protein nano delivery system is simple, and raw materials are cheap and easy to obtain. It can be seen from lens observation that the formed nanoparticles are uniform in size, the system is stable, and the protein can effectively enter the intracellular to play a function. Through experimental verification, it is found that the protein nano delivery system can improve the intracellular protein delivery efficiency, ensure the biological activity of the protein entering the intracellular, and can be applied to the field of intracellular disease treatment and gene coding.

[0022] The protein nano delivery system constructed by the application uses a natural polysaccharide to load various proteins through a dynamic covalent bond to perform intracellular delivery and maintain the activity of the intracellular protein. Through experimental verification, it is found that the protein nano delivery system realizes the functions of improving the intracellular protein delivery efficiency and ensuring the biological activity of the protein entering the intracellular. The application can solve the problems of too strong or too weak binding force between the carrier and the protein. The protein nano delivery system with strong binding force can be formed under the assembly condition, and the binding force is weakened after being delivered into the intracellular, so as to promote the release of the protein, which is beneficial to the efficient use of clinical protein drugs, and can be applied to the field of intracellular disease treatment and gene coding. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor.

[0024] Figure 1 It is a transmission electron microscope (TEM) photo of BSA-APBA-HA;

[0025] Figure 2 It is a confocal image result of protein delivery on HeLa cells by BSA-APBA-HA;

[0026] Figure 3 It is an endosome escape effect of BSA-APBA-HA in HeLa cells;

[0027] Figure 4 It is a protein activity detection of intracellular delivery of HRP-APBA-HA by using the carrier. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present application will now be described in detail, with reference to the drawings, which are by way of illustration, and are not intended to be limiting of the present application. Further, the description of the present application is not intended to limit the scope of the application, but rather to provide an explanation of certain aspects, features, and embodiments of the application.

[0029] It should be understood that the terms used herein are for the purpose of describing particular embodiments and are not intended to limit the present application. Additionally, for numerical ranges that are expressed in a range format, it is intended that any numerical value implicitly recited within the range is also expressly stated to be the end point of the range. For example, a range of "1 to 5" is intended to specifically include the end points of 1 and 5. Any numerical values, however, are expressly intended to include the end points of the ranges.

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All patents, patent applications, publications, and descriptions mentioned herein are incorporated by reference in their entirety for the disclosure and

[0031] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof, and it is to be understood that all such modifications and variations warrant the patentable subject matter under the patent statutes. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. For example, the specification can be used as a textbook for preparing like systems, devices, and methods.

[0032] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional elements or steps.

[0033] Example 1 Construction of the protein nanodelivery system BSA-APBA-HA

[0034] (1) Hyaluronic acid (HA, 100 mg, 0.15 μmol) was dissolved in 14 mL of deionized water, then 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC-HCl, 80 mg, 417 μmol) and N-hydroxysuccinimide (NHS, 60 mg, 522 μmol) were dissolved in 20 mL of DMSO and added to the aqueous solution. After adjusting the pH of the entire reaction system to 5.5 with HC1 (1 M), 10 mL of DMSO containing dopamine (DA, 60 mg, 316 μmol) was added to the above mixture. The resulting mixture was stirred at room temperature for 30 hours. Then the reaction product was dialyzed against deionized water for 3 days (MWCO = 3500 Da), and dopamine-modified hyaluronic acid (DA-HA) was obtained after lyophilization.

[0035] (2) Preparation of protein, 2-acetylphenylboronic acid (2-APBA), DA-HA stock solution: The stock solutions of protein, 2-APBA, and DA-HA were prepared at concentrations of 5 mg / mL, 10 mg / mL, and 2 mg / mL in carbonate buffer (0.1 M, pH 8.5).

[0036] (3) In a clean glass vial, 2-APBA, protein solution, and DA-HA were added dropwise in sequence, so that the final protein concentration was 1.0 mg / mL, the 2-APBA concentration was 4 mg / mL, and the DA-HA concentration was 0.1 mg / mL. The resulting mixture was stirred at 4°C for 30 hours, and then a protein nanodelivery system was obtained by purification through ultrafiltration (MWCO = 100 kDa).

[0037] Example 2 Construction of protein nanodelivery system BSA-APBA-HA

[0038] (1) Hyaluronic acid (HA, 100 mg, 0.15 μmol) was dissolved in 14 mL of deionized water, then 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC-HCl, 80 mg, 417 μmol) and N-hydroxysuccinimide (NHS, 60 mg, 522 μmol) were dissolved in 20 mL of DMSO and added to the aqueous solution. After adjusting the pH of the entire reaction system to 5.5 with HC1 (1 M), 10 mL of DMSO containing dopamine (DA, 60 mg, 316 μmol) was added to the above mixture. The resulting mixture was stirred at room temperature for 30 hours. Then the reaction product was dialyzed against deionized water for 3 days (MWCO = 3500 Da), and dopamine-modified hyaluronic acid (DA-HA) was obtained after lyophilization.

[0039] (2) Preparation of protein, 2-acetylphenylboronic acid (2-APBA), DA-HA stock solution: Prepare the stock solution of protein, 2-APBA, DA-HA at a concentration of 5 mg / mL, 10 mg / mL, 2 mg / mL in carbonate buffer (0.1 M, pH 8.5).

[0040] (3) In a clean glass vial, add 2-APBA, protein solution, DA-HA in sequence, so that the final protein concentration is 0.67 mg / mL, the 2-APBA concentration is 3 mg / mL, and the DA-HA concentration is 0.03 mg / mL. The resulting mixture is stirred at 4°C for 24 hours, and then purified by ultrafiltration (MWCO = 100 kDa) to obtain a protein nanodelivery system.

[0041] Example 3 Construction of protein nanodelivery system BSA-APBA-HA

[0042] (1) Hyaluronic acid (HA, 100 mg, 0.15 μmol) was dissolved in 8.5 mL of deionized water, and then 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC-HCl, 50 mg, 261 μmol) and N-hydroxysuccinimide (NHS, 20 mg, 174 μmol) were dissolved in 6 mL of DMSO and added to the aqueous solution. After adjusting the pH of the entire reaction system to 4 with HCl (1 M), 3.5 mL of DMSO containing dopamine (DA, 40 mg, 348 μmol) was added to the above mixture. The resulting mixture was stirred at room temperature for 20 hours. Then the reaction product was dialyzed against deionized water for 3 days (MWCO = 3500 Da), and dopamine-modified hyaluronic acid (DA-HA) was obtained after lyophilization.

[0043] (2) Preparation of protein, 2-acetylphenylboronic acid (2-APBA), DA-HA stock solution: Prepare the stock solution of protein, 2-APBA, DA-HA at a concentration of 5 mg / mL, 10 mg / mL, 2 mg / mL in carbonate buffer (0.1 M, pH 8.5).

[0044] (3) In a clean glass vial, add 2-APBA, protein solution, DA-HA in sequence, so that the final protein concentration is 0.1 mg / mL, the 2-APBA concentration is 2 mg / mL, and the DA-HA concentration is 0.01 mg / mL. The resulting mixture is stirred at 1°C for 20 hours, and then purified by ultrafiltration (MWCO = 100 kDa) to obtain a protein nanodelivery system.

[0045] Example 4 TEM test of assembly BSA-APBA-HA

[0046] The protein nanodelivery system BSA-APBA-HA formed by the above construction method of Example 2 was dropped onto a copper mesh, and after standing for 24 h and drying, re-staining was performed by dropping phosphotungstic acid solution, and after drying, TEM testing was performed, and the TEM photo shown in FIG. 2 was obtained. Figure 1 The TEM photo shown in FIG. 2, Figure 1 The morphology of the assembly is regular, the particle size is uniform, and the particle size is nanoscale, which provides a possibility for intracellular protein delivery.

[0047] Example 5: Evaluation of the protein nanodelivery system BSA-APBA-HA for protein delivery experiments on HeLa cells

[0048] (1) BSA-FITC synthesis

[0049] Bovine serum albumin (BSA, 200 mg, 0.0029 mmol) and fluorescein isothiocyanate (FITC, 5.15 mg, 0.013 mmol) were dissolved in 30 mL of sodium carbonate-sodium bicarbonate buffer (pH 7.5, 0.1 M). The reaction was carried out at room temperature for 24 h in the dark. After the reaction was completed, dialysis was performed in distilled water, and the freeze-dried product was weighed and stored at -20 °C. The entire process was carried out in the dark.

[0050] (2) Protein nanodelivery system BSA-APBA-HA

[0051] 2-APBA, BSA-FITC, and DA-HA were sequentially added to 1.5 mL of sodium carbonate-sodium bicarbonate buffer at pH 8.5, and the reaction was carried out at 4 °C for 24 h.

[0052] (3) Protein delivery. HeLa cells were cultured in a confocal dish at a concentration of 4 x 10 5 / mL for 12 h, and after the cells adhered, the culture solution was removed, washed with PBS 3 times, and BSA-APBA-HA was added for incubation for 12 h. After the culture solution was removed, PBS was washed 3 times, and laser confocal imaging was performed to evaluate the protein endocytosis efficiency.

[0053] The results are shown in FIG. 3, and the fluorescence intensity of the image obtained by laser confocal imaging after the material was applied to the cells was statistically analyzed. Compared with free BSA, the protein nanodelivery system can effectively improve the delivery efficiency of intracellular proteins. Figure 2

[0054] Example 6: Evaluation of whether the protein delivered into the cell by BSA-APBA-HA can escape from the endosome

[0055] (1) BSA-FITC synthesis

[0056] ​BSA (200 mg, 0.0029 mmol) was dissolved with FITC (5.15 mg, 0.013 mmol) in 30 mL of sodium carbonate-sodium bicarbonate buffer (pH 7.5, 0.1 M). The reaction was carried out at room temperature for 24 h in the dark. After the reaction was completed, the impurities were removed by dialysis in distilled water, freeze-dried and weighed, and stored at -20 °C. The whole process was carried out in the dark.

[0057] (2) Protein nanodelivery system BSA-APBA-HA

[0058] 2-APBA, BSA-FITC, DA-HA were added to 1.5 mL of sodium carbonate-sodium bicarbonate buffer at pH 8.5 in turn, and the reaction was carried out at 4 °C for 24 h.

[0059] (3) HeLa cells were pre-cultured in a laser confocal dish, and after 12 h the cells were adherent for protein delivery experiments. Before adding the material, the culture medium in the dish was aspirated, and the cells were gently washed three times with PBS buffer, and then new culture medium was added. Then BSA-APBA-HA was added to the dish, and the BSA protein concentration in the dish was 20 μg / mL. The cells were incubated in an incubator for 12 h. Then the culture medium containing the material was removed, and PBS buffer was added for washing twice. Lysotracker Red was used for staining at 37 °C for 20 min, CLSM was measured, and the protein and lysosome co-localization of each sample was analyzed by image J.

[0060] As shown in Figure 3 , this experiment proves that after the protein is delivered into the cell, it can escape from the endosome and avoid being hydrolyzed by the acidic conditions of the endosome, thereby ensuring that the protein activity is not lost. From the confocal image, it can be seen that the two are basically not coincident, and the co-localization analysis shows that the correlation between the two is weak, proving that the protein has successfully escaped from the acidic organelle.

[0061] Example 7 Evaluation of the protein nanodelivery system for intracellular delivery of protein activity

[0062] (1) Synthesis of HRP-APBA-HA assembly

[0063] APBA, horseradish peroxidase (HRP), and HA were added to 0.8 mL of pH 8.5 buffer in turn, with HRP concentration of 0.67 mg / mL, APBA concentration of 3.33 mg / mL, and HA concentration of 0.0268 mg / mL. The reaction was carried out at 4 °C.

[0064] (2) In situ staining experiment

[0065] The cells were cultured in a 48-well plate at a cell density of 50,000 cells / well. After the cells adhered, the culture medium was aspirated, and the plates were gently washed three times with PBS. Culture medium containing HRP and HRP-APBA-HA was added, with the HRP concentration in each well being 20 μg / mL. The plates were incubated at 37°C for 12 hours. The culture medium was aspirated, and the plates were gently washed three times with PBS. 200 μL of pH 5.0 acetate buffer containing 10 μg / mL TMB and 3 mM hydrogen peroxide was added to each well, and the plates were reacted at room temperature for 10 minutes. The color change in each well was observed, and the UV absorbance at 450 nm was tested.

[0066] like Figure 4 As shown, this protein nanoparticle delivery system demonstrated that horseradish peroxidase could enter cells and exert enzymatic activity. Compared to delivering HRP into cells using a carrier, delivering HRP into cells using a carrier ensured that HRP could exert its catalytic effect inside the cells and maintain enzyme activity. Free HRP, on the other hand, was essentially unable to enter cells and had no catalytic effect.

[0067] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for constructing a protein nanodelivery system, characterized by, The method comprises the following steps: (1) mixing and dissolving natural polysaccharide, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, adjusting the pH value to 4.0-5.5, adding dopamine, stirring at room temperature for 20-30 h, and obtaining dopamine-modified natural polysaccharide through dialysis and lyophilization; (2) respectively preparing protein, 2-acetylphenylboronic acid and dopamine-modified natural polysaccharide stock solutions; (3) sequentially adding the stock solutions of 2-acetylphenylboronic acid, protein and dopamine-modified natural polysaccharide, and stirring at low temperature for 20-30 h to perform self-assembly, and obtaining the protein nanodelivery system through ultrafiltration purification; The mass ratio of the natural polysaccharide to the dopamine is 100:(40-60); In step (3), the low-temperature condition is 1-4℃; and the ultrafiltration is performed with a molecular weight cut-off of 100 kDa; The natural polysaccharide is hyaluronic acid; In step (1), the mass ratio of the natural polysaccharide, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide is 100:(50-80):(20-60); In step (3), the final concentrations of the protein, 2-acetylphenylboronic acid and dopamine-modified natural polysaccharide in the mixed solution in the reactor are 0.1-1.0 mg / mL, 2-4 mg / mL and 0.01-0.1 mg / mL, respectively; The protein is bovine serum albumin or horseradish peroxidase.

2. A protein nanodelivery system prepared by the construction method of claim 1.

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