Nanometer carrier and nanometer medicine carrying system based on self-assembly of protein and hydrophobic polymer and preparation method and application of nanometer carrier and nanometer medicine carrying system

Abstract

The invention relates to a nano-carrier based on self-assembly of protein and hydrophobic polymer, a nano-drug delivery system and a preparation method and application thereof. The nano-carrier is a nano-particle formed by self-assembly of amphiphilic protein and hydrophobic polymer by using hydrophobic interaction as driving force, wherein the hydrophobic polymer is wrapped in the nanoparticles by the amphiphilic protein. The nano-carrier has the advantages of high drug loading rate and encapsulation rate, high colloidal stability, good uniformity and production repeatability, good dispersibility, good biocompatibility, simple formula, low energy consumption and the like. The nano-carrier can be used for drug delivery and can also be used for contrast imaging of tumor parts. And the drug molecules can be loaded at different positions of the nano-carrier according to different requirements, and the nano-carrier has diversity in structure and component, and is suitable for the requirements of different application occasions. The invention provides a new scheme for in-vivo delivery of drug molecules, and has a good clinical transformation prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and relates to a nano - carrier based on the self - assembly of proteins and hydrophobic polymers, a nano - drug delivery system, and their preparation methods and applications. Background Art

[0002] Nano - drugs refer to nanoparticles that use nanomaterials as carriers and load drug molecules onto the nanomaterials, which have functions such as diagnosis, treatment, or imaging. Nano - drugs have many advantages such as small particle size, large specific surface area, and easy modification and design. They are used to improve the solubility of hydrophobic drug molecules, solve drug delivery problems, reduce biological toxicity, and enhance the effect of targeted therapy.

[0003] Since the 1960s, extensive research has been carried out on nano - drug delivery systems, but only a few systems have been successfully translated into clinical applications. Most small - molecule drugs lack affinity for nano - carriers, resulting in low drug - loading rates (usually less than 10%), poor colloidal stability, and complex synthesis processes. Key challenges in nano - drug research and clinical practice need to be addressed: (1) simple and controllable nano - drug production methods; (2) drug - loading rate problems of nano - drugs; (3) in - vivo safety problems of nano - drugs; (4) stability of nano - drugs in a complex in - vivo environment; (5) in - vivo biological effects of nano - drugs. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a nano - carrier based on the self - assembly of proteins and hydrophobic polymers, a nano - drug delivery system, and their preparation methods and applications.

[0005] To achieve the purpose of this invention, the following technical solutions are adopted:

[0006] In the first aspect, the present invention provides a nano - carrier based on the self - assembly of proteins and hydrophobic polymers. The nano - carrier is a nanoparticle formed by the self - assembly of amphiphilic proteins and hydrophobic polymers driven by hydrophobic interaction, wherein the hydrophobic polymer is wrapped inside the nanoparticle by the amphiphilic protein.

[0007] The nano - carrier based on the self - assembly of proteins and hydrophobic polymers involved in the present invention has the advantages of high drug - loading rate and encapsulation efficiency, high colloidal stability, good homogeneity and production repeatability, good dispersibility, good biocompatibility, simple formulation, and low energy consumption. After loading drugs or other bioactive components, it has a good slow - release effect, can effectively solve the toxic and side effects of drug molecules (or other bioactive components) on the body, and meet the needs of long - term and stable release of drug molecules (or other bioactive components). The present invention provides a new solution for the in - vivo delivery of drug molecules (or other bioactive components).

[0008] Preferably, the hydrophobic polymer includes any one or a combination of at least two of polylactic-glycolic acid, polylactic acid, poly(p-dioxanone), or polycaprolactone.

[0009] Preferably, the amphiphilic protein is an unmodified protein, including any one or a combination of at least two of bovine serum albumin, human serum albumin, bovine α-lactalbumin, β-casein, ovalbumin, β-lactoglobulin, lactoferrin, or transferrin.

[0010] Preferably, the molecular weight of the hydrophobic polymer is 10 - 500KDa, such as 10KDa, 30KDa, 50KDa, 100KDa, 200KDa, 300KDa, 400KDa, 500KDa, etc.

[0011] The molar ratio of the hydrophobic polymer to the amphiphilic protein is 1:5 - 10:1, such as 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, etc.

[0012] In a second aspect, the present invention provides a method for preparing a nanocarrier based on self-assembly of a protein and a hydrophobic polymer according to the first aspect, and the preparation method includes:

[0013] (1) Dissolve the hydrophobic polymer in an organic solvent to obtain solution A; dissolve the amphiphilic protein in a phosphate buffer to obtain solution B;

[0014] (2) Mix and stir solution A and solution B to obtain solution C;

[0015] (3) Centrifuge solution C to obtain a centrifugation product, and obtain the nanocarrier based on self-assembly of the protein and the hydrophobic polymer.

[0016] The nanocarrier based on self-assembly of a protein and a hydrophobic polymer involved in the present invention has the characteristics of simple formulation, easy preparation, low energy consumption, low cost, and high yield, is suitable for large-scale production, and is convenient for production, storage, transportation, carrying, and use.

[0017] Preferably, the organic solvent includes any one or a combination of at least two of tetrahydrofuran, N,N-dimethylformamide, or dimethyl sulfoxide.

[0018] Preferably, the concentration of solution A is 1 - 20mg / mL, such as 2mg / mL, 4mg / mL, 6mg / mL, 8mg / mL, 10mg / mL, 12mg / mL, 15mg / mL, 20mg / mL, etc.

[0019] Preferably, the concentration of the solution B is 0.01 - 5 mg / mL, such as 0.01 mg / mL, 0.05 mg / mL, 0.08 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 5 mg / mL, etc.

[0020] Preferably, the mixing and stirring in step (2) are carried out at 4 - 35 °C (such as 4 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, etc.) for 0.1 - 40 min (such as 0.1 min, 0.5 min, 1 min, 2 min, 3 min, 5 min, 8 min, 10 min, 15 min, 20 min, 25 min, 30 min, 40 min, etc.).

[0021] Preferably, the centrifugation is carried out at 10000 - 15000 rpm (such as 10000 rpm, 11000 rpm, 12000 rpm, 13000 rpm, 14000 rpm, 15000 rpm, etc.) at 2 - 8 °C (such as 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, etc.) for 20 - 50 min (such as 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, etc.).

[0022] Preferably, after obtaining the centrifuged product, it is resuspended or further freeze - dried.

[0023] In a third aspect, the present invention provides a nano - drug delivery system, which includes the nano - carrier self - assembled based on proteins and hydrophobic polymers described in the first aspect and bioactive components loaded in the nano - carrier;

[0024] The bioactive components include any one or a combination of at least two of hydrophobic drugs, fluorescent materials, magnetic resonance materials, or photothermal - sensitive materials.

[0025] The nano - drug delivery system involved in the present invention uses proteins as the main component of the carrier, endowing the drug delivery system with good biocompatibility and providing many modifiable sites for subsequent further applications; this nano - drug delivery system is bound by non - covalent bonds, without destroying the structures of each component and retaining its biological functions; it has a small particle size, good dispersibility in water - soluble liquids, good stability in physiological environments, and a long biological half - life; and it has a high drug - loading rate, high encapsulation rate, and at the same time retains high colloidal stability; this nano - drug delivery system has a sustained - release effect, avoiding frequent injection drug administration, with good patient experience and being easy to accept; it has the EPR effect, can passively target tumor sites, greatly improving the drug utilization rate, while avoiding the intake of drugs by normal tissues and reducing toxic side effects; after loading tumor drugs, it has excellent in vitro cancer cell killing ability and in vivo anti - tumor effect.

[0026] In addition to being used for loading drugs, the nanocarrier based on the self-assembly of proteins and hydrophobic polymers described in the present invention can also be used for loading other hydrophobic compounds such as fluorescent materials, magnetic resonance materials, photothermal sensitive materials, etc.

[0027] In addition to being used for drug delivery, the nanocarrier based on the self-assembly of proteins and hydrophobic polymers described in the present invention can also be used for contrast imaging of tumor sites. The growth rate of tumor sites is significantly higher than that of normal tissues. There are defects between tumor vascular endothelial cells, the arrangement is not tight, it has permeability, and there is insufficient lymphatic drainage inside the tumor and low blood flow rate. Therefore, the enhanced permeability and retention effect (EPR) appears. Once the nanoparticles enter, they will be retained in the tumor site and accumulate in the tumor site. If it is loaded with fluorescent materials such as semiconductor quantum dots, fluorescent proteins, metal nanoclusters, magnetic resonance materials such as superparamagnetic iron oxide nanoparticles, manganese oxide nanoparticles, etc., and photothermal sensitive materials such as gold nanoparticles, polypyrrole, etc., it can be used for contrast imaging of tumor sites.

[0028] Preferably, the molar ratio of the hydrophobic polymer to the bioactive component is 1:1000 - 1:10000, such as 1:1000, 1:2000, 1:4000, 1:6000, 1:7000, 1:8000, 1:10000, etc.

[0029] Preferably, the hydrophobic drug includes any one or a combination of at least two of anticancer drugs, anti-inflammatory drugs, analgesic drugs, antihypertensive drugs, hypoglycemic drugs, lipid-lowering drugs or anesthetic drugs.

[0030] Preferably, the anticancer drug includes any one or a combination of at least two of doxorubicin, paclitaxel, cisplatin, ursolic acid, curcumin, dexamethasone, celecoxib, docetaxel, fenretinide, dihydroartemisinin, vinblastine, camptothecin or apatinib mesylate.

[0031] Preferably, the loading method includes loading inside the nanocarrier by hydrophobic interaction and / or loading on the surface of the nanocarrier by diffusion.

[0032] According to different needs, drug molecules (or other bioactive components) can be loaded at different positions of the self-assembled nanoparticles, and the structure and composition are diverse, meeting the requirements of different application scenarios.

[0033] Fourthly, the present invention provides a preparation method of the nanodrug delivery system as described in the third aspect.

[0034] The nano-drug delivery system involved in the present invention is characterized by simple formulation, easy preparation, low energy consumption, low cost, and high yield. It is suitable for large-scale production and is convenient for production, storage, transportation, carrying, and use. The specific preparation methods can be as follows:

[0035] In the present invention, the method of loading inside the nano-carrier by hydrophobic interaction includes the following steps:

[0036] (1) Co-dissolve the hydrophobic polymer and the bioactive component in an organic solvent to obtain solution A; dissolve the amphiphilic protein in phosphate buffer to obtain solution B;

[0037] (2) Mix and stir solution A and solution B to obtain solution C;

[0038] (3) Centrifuge solution C to obtain a centrifugation product, and obtain the nano-carrier based on the self-assembly of protein and hydrophobic polymer.

[0039] Preferably, the organic solvent includes any one or a combination of at least two of tetrahydrofuran, N,N-dimethylformamide, or dimethyl sulfoxide.

[0040] Preferably, the concentration of the hydrophobic polymer in solution A is 1-20 mg / mL, such as 2 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL, 12 mg / mL, 15 mg / mL, 20 mg / mL, etc.; the concentration of the bioactive component in solution A is 0.5-20 mg / mL, such as 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL, 12 mg / mL, 15 mg / mL, 20 mg / mL, etc.

[0041] Preferably, the concentration of solution B is 0.01-5 mg / mL, such as 0.01 mg / mL, 0.05 mg / mL, 0.08 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 5 mg / mL, etc.

[0042] Preferably, the mixing and stirring in step (2) is carried out at 4-35 °C (4 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, etc.) for 0.1-40 min (such as 0.1 min, 0.5 min, 1 min, 2 min, 3 min, 5 min, 8 min, 10 min, 15 min, 20 min, 25 min, 30 min, 40 min, etc.).

[0043] Preferably, the centrifugation is carried out at 10,000 - 15,000 rpm (such as 10,000 rpm, 11,000 rpm, 12,000 rpm, 13,000 rpm, 14,000 rpm, 15,000 rpm, etc.) at 2 - 8 °C (such as 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, etc.) for 20 - 50 min (such as 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, etc.).

[0044] Preferably, after obtaining the centrifuged product, it is resuspended or further lyophilized.

[0045] In the present invention, the method of loading on the surface of the nanocarrier by diffusion includes the following steps:

[0046] (1) Dissolve the hydrophobic polymer in an organic solvent to obtain solution A; dissolve the amphiphilic protein in a phosphate buffer to obtain solution B;

[0047] (2) Mix and stir solution A and solution B to obtain solution C;

[0048] (3) Centrifuge solution C to obtain a centrifuged product, resuspend the centrifuged product and mix and stir it with the bioactive component, and then centrifuge to obtain a centrifuged product.

[0049] Preferably, the organic solvent includes any one or a combination of at least two of tetrahydrofuran, N,N-dimethylformamide or dimethyl sulfoxide.

[0050] Preferably, the concentration of solution A is 1 - 20 mg / mL, such as 2 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL, 12 mg / mL, 15 mg / mL, 20 mg / mL, etc.

[0051] Preferably, the concentration of solution B is 0.01 - 5 mg / mL, such as 0.01 mg / mL, 0.05 mg / mL, 0.08 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 5 mg / mL, etc.

[0052] Preferably, the mixing and stirring in step (2) is carried out at 4 - 35 °C (4 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, etc.) for 0.1 - 40 min (such as 0.1 min, 0.5 min, 1 min, 2 min, 3 min, 5 min, 8 min, 10 min, 15 min, 20 min, 25 min, 30 min, 40 min, etc.).

[0053] Preferably, the mixing and stirring in step (3) are carried out at 4 - 35 °C (such as 4 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, etc.) for 0.1 - 10 min (such as 0.1 min, 0.5 min, 1 min, 2 min, 4 min, 5 min, 6 min, 8 min, 10 min, etc.).

[0054] Preferably, each centrifugation is independently selected to be carried out at 10000 - 15000 rpm (such as 10000 rpm, 11000 rpm, 12000 rpm, 13000 rpm, 14000 rpm, 15000 rpm, etc.) at 2 - 8 °C (such as 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, etc.) for 20 - 50 min (such as 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, etc.).

[0055] Preferably, after obtaining the centrifuged product, it is resuspended or further lyophilized.

[0056] In a fifth aspect, the present invention provides the application of the nanocarrier based on the self - assembly of protein and hydrophobic polymer described in the first aspect in the preparation of chemotherapeutic drugs, immunotherapeutic drugs, contrast imaging agents, cosmetics, skin care products, health care products, plant growth regulators, agricultural fungicides or agricultural insecticides.

[0057] The nanocarrier based on the self - assembly of protein and hydrophobic polymer described in the present invention has a very wide range of applications. In addition to being used to load drugs, fluorescent materials, magnetic resonance materials, photothermal - sensitive materials, it can also be used to load cosmetic active ingredients, nutritional regulatory ingredients, agricultural bactericidal and insecticidal ingredients, etc., that is, it is applied to the preparation of chemotherapeutic drugs, immunotherapeutic drugs, contrast imaging agents, cosmetics, skin care products, health care products, plant growth regulators, agricultural fungicides or agricultural insecticides, etc.

[0058] All other specific values not listed within the numerical ranges involved in the present invention are within the protection scope of the present invention. Considering space and brevity of description, they will not be elaborated one by one here.

[0059] Compared with the prior art, the present invention has the following beneficial effects:

[0060] (1) The nano-carrier based on the self-assembly of protein and hydrophobic polymer involved in the present invention has the advantages of high drug loading rate and encapsulation rate, high colloidal stability, good homogeneity and production repeatability, good dispersibility, good biocompatibility, simple formulation, and low energy consumption. This nano-carrier can be used for drug delivery and also for contrast imaging of tumor sites. Moreover, drug molecules (or other bioactive components) can be loaded at different positions of the nano-carrier according to different requirements, and the structure and composition are diverse, meeting the needs of different application scenarios. The present invention provides a new solution for the in vivo delivery of drug molecules and has good prospects for clinical translation.

[0061] (2) The nano-drug delivery system involved in the present invention uses protein as the main component of the carrier, endowing the drug delivery system with good biocompatibility and providing many modifiable sites for subsequent further applications; this nano-drug delivery system is bound by non-covalent bonds, without destroying the structures of each component and retaining its biological functions; it has a small particle size, good dispersibility in water-soluble liquids, good stability in physiological environments, and a long biological half-life; moreover, it has a high drug loading rate and encapsulation rate, while retaining high colloidal stability; this nano-drug delivery system has a sustained release effect, avoiding frequent injection drug administration, with good patient experience and being easy to accept; and it has the EPR effect, capable of passively targeting tumor sites, greatly improving the drug utilization rate, while avoiding the intake of drugs by normal tissues and reducing toxic and side effects; after loading tumor drugs, it has excellent in vitro cancer cell killing ability and in vivo anti-tumor effect.

[0062] (3) The nano-carrier and nano-drug delivery system based on the self-assembly of protein and hydrophobic polymer involved in the present invention have the characteristics of simple formulation, easy preparation, low energy consumption, low cost, and high yield, are suitable for large-scale production, and are convenient for production, storage, transportation, carrying, and use. Description of the Drawings

[0063] Figure 1 is the transmission electron microscope observation diagram of PLGA@BSA prepared in Example 1;

[0064] Figure 2 is the particle size detection result diagram of PLGA@Zein prepared in Comparative Example 1;

[0065] Figure 3 is the particle size detection result diagram of PVA@BSA prepared in Comparative Example 2;

[0066] Figure 4 is the change result diagram of the particle size of self-assembled nanoparticles PLGA@BSA with time in ultrapure water;

[0067] Figure 5 is the change result diagram of the particle size of self-assembled nanoparticles PLGA@BSA with time in different pH buffer solutions;

[0068] Figure 6 It is a graph showing the change of particle size of self-assembled nanoparticles PLGA@BSA over time in a physiologically approximate solvent;

[0069] Figure 7 It is a graph showing the change of particle size of self-assembled nanoparticles PCL@BSA over time in ultrapure water;

[0070] Figure 8 It is a graph showing the change of particle size of self-assembled nanoparticles PLGA@OVA over time in ultrapure water;

[0071] Figure 9 It is a standard curve graph of Dox·HCl;

[0072] Figure 10 It is PLGA@BSA-Dox in 、PLGA@BSA-Dox s 、PLGA@BSA-Dox c Graph showing the change of particle size over time in ultrapure water;

[0073] Figure 11 It is PLGA@BSA-Dox in 、PLGA@BSA-Dox s 、PLGA@BSA-Dox c Drug release curve graph in simulated body fluid (SBF);

[0074] Figure 12 It is PLGA@BSA-Dox in 、PLGA@BSA-Dox s 、PLGA@BSA-Dox c Drug release curve graph in acetic acid-EDTA;

[0075] Figure 13 It is a graph showing the in vitro cytotoxicity evaluation results of nanoparticles PLGA@BSA against MCF-7 cells;

[0076] Figure 14 It is a graph showing the in vitro cytotoxicity evaluation results of doxorubicin-loaded nanoparticles PLGA@BSA-Dox against MCF-7 cells;

[0077] Figure 15 It is a graph showing the change of tumor size in mice after treatment;

[0078] Figure 16 It is a graph showing the change of body weight in mice after treatment. Specific implementation manners

[0079] The technical solution of the present invention will be further described below through specific embodiments. Those skilled in the art should understand that the embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0080] In the following embodiments, the experimental methods are all conventional methods unless otherwise specified. The experimental materials used in the following embodiments are all commercially available through conventional commercial channels unless otherwise specified.

[0081] Example 1

[0082] Construction of bovine serum albumin and poly(lactic-co-glycolic acid) self-assembled nanoparticles (PLGA@BSA):

[0083] (1) Prepare a 5 mg / mL PLGA solution dissolved in THF (the molar ratio of polylactic acid to polyglycolic acid is 1:1, and the molecular weight is 107 kDa), and a 2 mg / mL BSA solution dissolved in 0.01 M PBS with pH = 7.2.

[0084] (2) Use a pipette to take 200 μL of the above PLGA solution and add it to 3 mL of the above BSA solution, and stir at 25 °C for 30 min to obtain uniformly mixed PLGA@BSA nanoparticles.

[0085] (3) Then centrifuge for purification. The centrifugation conditions are 13300 rpm, 45 min, and 4 °C. After centrifugation, discard the supernatant. Finally, resuspend every 6 mL of the PLGA@BSA centrifugation product with 1 mL of ultrapure water.

[0086] Characterization of the particle size, potential, and microscopic morphology of the product:

[0087] The prepared PLGA@BSA appears white and translucent, has good dispersibility in aqueous solution, the particle size measured by DLS is 112.9 ± 4.8 nm, and the zeta potential is -39.4 ± 0.2 mV.

[0088] Take 10 μL of the self-assembled nanoparticle PLGA@BSA stock solution and drop it onto a copper grid covered with a support film. After natural drying, observe and take pictures with a transmission electron microscope, as Figure 1 shown. The prepared nanoparticles are spherical, the hydrophobic polymer is successfully encapsulated by the protein, and they have good monodispersity, with a particle size of about 68.4 nm.

[0089] Example 2

[0090] Construction of bovine serum albumin and polycaprolactone self-assembled nanoparticles (PCL@BSA):

[0091] (1) Prepare a 3 mg / mL PCL solution (with a molecular weight of 50 kDa) dissolved in DMF, and a 1 mg / mL BSA solution dissolved in 0.01 M PBS with pH = 7.2.

[0092] (2) Use a pipette to take 200 μL of the above PCL solution and add it to 3 mL of the above BSA solution. Stir at 20 °C for 30 min to obtain uniformly mixed PCL@BSA nanoparticles.

[0093] (3) Then perform centrifugation for purification. The centrifugation conditions are 15000 rpm, 35 min, and 4 °C. After centrifugation, discard the supernatant. Finally, resuspend every 6 mL of the PCL@BSA centrifugation product with 1 mL of ultrapure water.

[0094] Characterization of the particle size and potential of the product:

[0095] The prepared PCL@BSA is white and translucent in appearance, has good dispersibility in aqueous solution, and the particle size measured by DLS is 81.4 ± 1.33 nm, and the zeta potential is -15.4 ± 1.0 mV.

[0096] Example 3

[0097] Construction of ovalbumin and poly (lactic - glycolic acid) self - assembled nanoparticles (PLGA@OVA):

[0098] (1) Prepare a 5 mg / mL PLGA solution (with a molecular weight of 107 kDa) dissolved in DMSO, and a 3 mg / mL OVA solution dissolved in 0.01 M PBS with pH = 7.2.

[0099] (2) Use a pipette to take 200 μL of the above PLGA solution and add it to 3 mL of the above OVA solution. Stir at 30 °C for 30 min to obtain uniformly mixed PLGA@OVA nanoparticles.

[0100] (3) Then perform centrifugation for purification. The centrifugation conditions are 12000 rpm, 50 min, and 4 °C. After centrifugation, discard the supernatant. Finally, resuspend every 6 mL of the PLGA@OVA centrifugation product with 1 mL of ultrapure water.

[0101] Characterization of the particle size and potential of the product:

[0102] The prepared PLGA@OVA is white and translucent in appearance, has good dispersibility in aqueous solution, and the particle size measured by DLS is 171.8 ± 2.19 nm, and the zeta potential is -28.8 ± 1.2 mV.

[0103] Comparative Example 1

[0104] This comparative example attempted to construct zein and poly(lactic-co-glycolic acid) self-assembled nanoparticles (PLGA@Zein) as follows:

[0105] (1) Prepare a 5 mg / mL PLGA solution dissolved in THF (the molar ratio of polylactic acid to polyglycolic acid is 1:1, and the molecular weight is 107 kDa), and a 2 mg / mL zein solution dissolved in 0.01 M PBS with pH = 7.2.

[0106] (2) Use a pipette to take 200 μL of the above PLGA solution and add it to 3 mL of the above zein solution. Stir at 25 °C for 30 min. The solution becomes turbid and a large amount of precipitation appears. The DLS test results are as Figure 2 shown.

[0107] From Figure 2 the results, it can be seen that the nanocarriers involved in the present invention need to use amphiphilic proteins and hydrophobic polymers as raw materials, and other types of proteins, such as hydrophobic proteins, cannot produce products with good monodispersity and high colloidal stability.

[0108] Comparative Example 2

[0109] This comparative example attempted to construct bovine serum albumin and polyvinyl alcohol self-assembled nanoparticles (PVA@BSA) as follows:

[0110] (1) Prepare a 5 mg / mL PVA solution dissolved in THF (the molecular weight is 100 kDa), and a 2 mg / mL BSA solution dissolved in 0.01 M PBS with pH = 7.2.

[0111] (2) Use a pipette to take 200 μL of the above PVA solution and add it to 3 mL of the above BSA solution. Stir at 25 °C for 30 min. The solution becomes turbid and a large amount of precipitation appears. The DLS test results are as Figure 3 shown.

[0112] From Figure 3 the results, it can be seen that the nanocarriers involved in the present invention need to use amphiphilic proteins and hydrophobic polymers as raw materials, and other types of polymers, such as amphiphilic polymers, cannot produce products with good monodispersity and high colloidal stability.

[0113] Test Example 1

[0114] Stability of self-assembled nanoparticles in different environments:

[0115] (1) The self-assembled nanoparticles PLGA@BSA prepared in Example 1 were centrifuged and then resuspended with ultrapure water, different pH buffers (pH = 3, 7, 9, 11), and physiological approximation solvents (fetal bovine serum FBS, DMEM high-glucose medium containing 10% FBS). Among them, the DMEM group was stored at 37 °C, and the rest were stored at 4 °C. Samples were taken at regular intervals and the particle size changes were measured by DLS to investigate their stability in different environments.

[0116] The results are shown in Figure 4 (ultrapure water), Figure 5 (different pH buffers), and Figure 6 (physiological approximation solvents). PLGA@BSA has good colloidal stability in aqueous solution and can be stored for at least 7 months, while it can be stored for 3 months in different pH buffers and about 1 month in physiological environment.

[0117] (2) The self-assembled nanoparticles PCL@BSA and PLGA@OVA prepared in Examples 2-3 were centrifuged and then resuspended with ultrapure water and stored at 4 °C. Samples were taken at regular intervals and the particle size changes were measured by DLS to investigate their stability in different environments.

[0118] The results are shown in Figure 7 (Example 2), Figure 8 (Example 3). Each group of samples has good colloidal stability in aqueous solution.

[0119] Example 6

[0120] Construction of a bovine serum albumin and poly(lactic-co-glycolic acid) self-assembled nanoparticle system loaded with doxorubicin by the internal encapsulation method:

[0121] (1) Take 200 μL of a 5 mg / mL PLGA solution dissolved in THF (the molar ratio of polylactic acid to polyglycolic acid is 1:1, and the molecular weight is 107 kDa) and premix it with 50 μL of a 5 mg / mL Dox solution dissolved in DMF to obtain Solution A;

[0122] (2) Then take 200 μL of Solution A and add it to 3 mL of a 2 mg / mL BSA PBS solution (pH = 7.2, 0.01 M) for co-self-assembly, and stir at 25 °C for 5 min;

[0123] (3) Centrifuge (13300 rpm, 45 min, 4 °C) and resuspend with ultrapure water to obtain the internal encapsulation group doxorubicin-loaded nanoparticles (PLGA@BSA-Dox in ), and the volume ratio of the solvent before centrifugation to the resuspended solvent is 6:1.

[0124] Characterization of the particle size and potential of the product:

[0125] The particle size of PLGA@BSA-Dox measured by the DLS method in was 276.2±19.1 nm, and the Zeta potential was -24.7±0.6 mV.

[0126] Example 7

[0127] Construction of a bovine serum albumin and poly(lactic-co-glycolic acid) self-assembled nanoparticle system loaded with doxorubicin by the diffusion method:

[0128] (1) Use a pipette to take 200 μL of a 5 mg / mL PLGA solution dissolved in THF (the molar ratio of polylactic acid to polyglycolic acid is 1:1, and the molecular weight is 107 kDa), and add it to 3 mL of a 2 mg / mL BSA solution dissolved in 0.01 M PBS at pH = 7.2. Stir at 25 °C for 30 min;

[0129] (2) Centrifuge (13300 rpm, 45 min, 4 °C) and resuspend with ultrapure water. The volume ratio of the solvent before centrifugation to that after resuspension is 6:1 to obtain a PLGA@BSA nanoparticle solution;

[0130] (3) Take 100 μL of a 10 mg / mL Dox solution dissolved in DMF, and make up to 200 μL with DMF to obtain a drug solution. Then take 1 mL of the PLGA@BSA nanoparticle solution, and gradually add the drug solution dropwise thereto under magnetic stirring, and react at 4 °C for 24 h. Then centrifuge (13300 rpm, 45 min, 4 °C) and resuspend with ultrapure water to obtain the diffusion group doxorubicin-loaded nanoparticles (PLGA@BSA-Dox s ).

[0131] Characterization of the particle size and potential of the product:

[0132] The particle size of PLGA@BSA-Dox measured by the DLS method in was 193.1±7.4 nm, and the Zeta potential was 20.4±1.2 mV.

[0133] Example 8

[0134] Construction of a bovine serum albumin and poly(lactic-co-glycolic acid) self-assembled nanoparticle system loaded with doxorubicin by the complex method:

[0135] (1) Take 200 μL of a 5 mg / mL PLGA solution dissolved in THF (the molar ratio of polylactic acid to polyglycolic acid is 1:1, and the molecular weight is 107 kDa) and premix it with 50 μL of 5 mg / mL Dox dissolved in DMF to obtain solution A;

[0136] (2) Then, take 200 μL of Solution A and add it to 3 mL of PBS solution (pH = 7.2, 0.01 M) containing 2 mg / mL BSA for co - self - assembly, and stir at 25 °C for 5 min;

[0137] (3) Centrifuge (13300 rpm, 45 min, 4 °C) and resuspend with ultrapure water to obtain the inner - encapsulated doxorubicin - loaded nanoparticles (PLGA@BSA - Dox in ) solution. The volume ratio of the solvent before centrifugation to that after resuspension is 6:1.

[0138] (4) Take 100 μL of Dox solution dissolved in DMF at 10 mg / mL, and make up to 200 μL with DMF to obtain the drug solution. Then take 1 mL of PLGA@BSA - Dox in nanoparticle solution, and drop - wise add the drug solution thereto under magnetic stirring, and react at 25 °C for 24 h. Then centrifuge (13300 rpm, 45 min, 4 °C) and resuspend with ultrapure water to obtain the composite doxorubicin - loaded nanoparticles (PLGA@BSA - Dox c ).

[0139] Characterization of the particle size and potential of the product:

[0140] The particle size of PLGA@BSA - Dox in measured by the DLS method is 222.5 ± 22.3 nm, and the Zeta potential is 20.6 ± 0.6 mV.

[0141] Test Example 2

[0142] Determination of the drug - loading rate and encapsulation efficiency of the self - assembled doxorubicin - loaded nanoparticles:

[0143] Centrifuge the products prepared in Examples 6 - 8 (13300 rpm, 45 min, 4 °C). The supernatant obtained by centrifugation is subjected to ultraviolet - visible spectrophotometric testing, and a standard curve of Dox·HCl is plotted. The correlation curve equation between the characteristic absorbance of Dox·HCl at 480 nm and its concentration is y = 0.01826x + 0.03239, R 2 = 0.99865 (the linear equation range is 0 - 200 μg / mL), as Figure 9 shown, having a high correlation and a wide detection range. Collect the drug - loaded supernatant, measure the absorbance of the supernatant at 480 nm, and the amount of drug not loaded by the nanoparticles can be known according to the standard curve, and finally the drug - loading rate and drug encapsulation efficiency of the nanoparticles are calculated. The calculation formulas for the drug - loading rate and encapsulation efficiency are as follows:

[0144]

[0145]

[0146] The results showed that the PLGA@BSA-Dox in The drug loading rate of PLGA@BSA-Doxs in Example 7 was 14.4±0.2%, and the encapsulation efficiency was 67.3±0.9%; the drug loading rate of PLGA@BSA-Doxs in Example 7 was 32.1±0.1%, and the encapsulation efficiency was 94.4±0.2%; the drug loading rate of PLGA@BSA-Doxs in Example 8 was 39.4±0.4%, and the encapsulation efficiency was 86.5±1.3%.

[0147] Example 9

[0148] Construction of curcumin-loaded bovine serum albumin and polylactic-co-glycolic acid self-assembled nanoparticle system by endogenous inclusion method:

[0149] (1) 200 μL of a 5 mg / mL PLGA solution (polylactic acid and polyglycolic acid molar ratio of 1:1, molecular weight of 107 kDa) dissolved in THF was premixed with 25 μL of curcumin of different concentrations (2 mg / ml, 5 mg / ml, 10 mg / ml) dissolved in DMF to obtain solution A;

[0150] (2) Take another 200 μL of solution A and add it to 3 mL of 2 mg / mL BSA in PBS (pH = 7.2 0.01 M) for co-self-assembly, and stir at 25 °C for 5 min;

[0151] (3) Centrifugation (13300 rpm, 45 min, 4°C) and resuspending with ultrapure water to obtain three types of curcumin-loaded nanoparticles (PLGA@BSA-Cur). The solvent volume ratio before centrifugation and after resuspension was 6:1.

[0152] Characterization of product particle size:

[0153] The particle sizes of three curcumin-loaded nanoparticles (PLGA@BSA-Cur) measured by DLS method were 86.02±2.22nm, 86.51±11.44nm, and 98.84±6.22nm, respectively.

[0154] Test Example 3

[0155] Determination of drug loading rate of self-assembled Cur-loaded nanoparticles:

[0156] The three products obtained in Example 9 were centrifuged (13300 rpm, 45 min, 4° C.), and the supernatant obtained by centrifugation was subjected to UV-visible spectrophotometry test, and a standard curve of curcumin was drawn to obtain a correlation curve equation of the characteristic absorbance of curcumin at 430 nm and its concentration: y=0.12709x-0.00376, R 2= 0.99821 (linear equation range is 0 - 20 μg / mL), with high correlation and a wide detection range. The drug-loaded supernatant was collected, and the absorbance of the supernatant at 430 nm was measured. According to the standard curve, the amount of drug not loaded by the nanoparticles could be known, and finally the drug loading rate of the nanoparticles was calculated.

[0157] The results showed that the drug loading rates of the three curcumin-loaded nanoparticles (PLGA@BSA-Cur) were 15.62 ± 0.11%, 34.30 ± 0.68%, and 52.63 ± 0.01% respectively.

[0158] Test Example 4

[0159] Stability of the self-assembled Dox-loaded nanoparticles in aqueous solution:

[0160] The doxorubicin-loaded nanoparticles (PLGA@BSA-Dox in ) prepared in Example 6, the diffusive doxorubicin-loaded nanoparticles (PLGA@BSA-Dox s ) prepared in Example 7, and the composite doxorubicin-loaded nanoparticles (PLGA@BSA-Dox c ) prepared in Example 8 were resuspended with ultrapure water respectively after centrifugation and stored at 4°C. Their stability in aqueous solution was investigated by DLS, and the particle size changes were measured by sampling at regular intervals. Figure 10 The stability situation is shown. The particle size fluctuated within about one month, but no significant aggregation occurred, indicating that the nanoparticles had good stability before and after drug loading.

[0161] Test Example 5

[0162] Drug release of the self-assembled Dox-loaded nanoparticles in simulated body fluid (SBF):

[0163] The doxorubicin-loaded nanoparticles (PLGA@BSA-Dox in ) prepared in Example 6, the diffusive doxorubicin-loaded nanoparticles (PLGA@BSA-Dox s ) prepared in Example 7, and the composite doxorubicin-loaded nanoparticles (PLGA@BSA-Dox cAfter centrifugation, resuspend with SBF buffer at a colloid concentration of 2 mg / mL and calculate using the PLGA concentration as an equivalent. Place the sample in a centrifuge tube, wrap it with sealing film and tin foil to ensure airtight and light-proof, and incubate it in a constant temperature shaker. The shaker parameters are 150 rpm and 37 °C to simulate the in vivo dynamic environment. Collect the centrifuged supernatant of the drug-loaded nanoparticles at regular intervals. The centrifugation conditions are 13,300 rpm, 45 min, and 4 °C. By performing ultraviolet-visible spectrophotometric tests on the supernatant, the amount of drug released by the drug-loaded nanoparticles during this period can be obtained, and the cumulative results can obtain the long-term drug release pattern of the drug-loaded nanoparticles in SBF buffer.

[0164] The results are as Figure 11 shown. All three showed a drug sustained-release phenomenon, effectively preventing the situation of a large amount of drug release in the early stage and little drug release in the later stage during in vivo application, which is very helpful for diseases that require long-term drug administration. Among them, PLGA@BSA-Dox in had a relatively fast drug release rate, releasing 25.6% in the first 10 days and a total of 39.3% by the 52nd day. PLGA@BSA-Dox s and PLGA@BSA-Dox c had similar drug release rates, releasing 17.2% and 16.6% respectively in the first 10 days and 28.0% and 27.4% respectively by the 52nd day.

[0165] Test Example 6

[0166] Drug release of self-assembled Dox-loaded nanoparticles in acetic acid-EDTA (HAc-EDTA, pH = 5.5) buffer:

[0167] The drug-loaded nanoparticles encapsulating doxorubicin (PLGA@BSA-Dox in ) prepared in Example 6, the drug-loaded nanoparticles with diffused doxorubicin (PLGA@BSA-Dox s ) prepared in Example 7, and the composite drug-loaded nanoparticles with doxorubicin (PLGA@BSA-Dox c ) prepared in Example 8 were resuspended with HAc-EDTA after centrifugation at a colloid concentration of 2 mg / mL and calculated using the PLGA concentration as an equivalent. Place the sample in a centrifuge tube, wrap it with sealing film and tin foil to ensure airtight and light-proof, and incubate it in a constant temperature shaker. The shaker parameters are 150 rpm and 37 °C to simulate the in vivo dynamic environment. Collect the centrifuged supernatant of the drug-loaded nanoparticles at regular intervals. The centrifugation conditions are 13,300 rpm, 45 min, and 4 °C. By performing ultraviolet-visible spectrophotometric tests on the supernatant, the amount of drug released by the drug-loaded nanoparticles during this period can be obtained, and the cumulative results can obtain the long-term drug release pattern of the drug-loaded nanoparticles in HAc-EDTA buffer.

[0168] The results are as Figure 12 shown. All three also showed drug sustained release, and the release rate slowed down with the increase of time. PLGA@BSA-Dox in showed an increased drug release rate under acidic conditions, releasing 34.7% in the first 10 days and a total of 50.0% by the 52nd day. PLGA@BSA-Dox s and PLGA@BSA-Dox c were obtained by dropping Dox into the PLGA@BSA solution, and Dox diffused into PLGA@BSA. The diffusion drug loading method filled the gaps on the surface of PLGA@BSA to a certain extent, making it difficult for the solvent to penetrate, so the PLGA@BSA structure was not easily damaged and the drug release was slower. PLGA@BSA-Dox s and PLGA@BSA-Dox c showed basically no affected drug release, releasing 18.8% and 17.1% respectively in the first 10 days and 25.2% and 23.1% respectively by the 52nd day.

[0169] Test Example 7

[0170] In vitro cytotoxicity evaluation of nanoparticles PLGA@BSA and doxorubicin-loaded nanoparticles PLGA@BSA-Dox:

[0171] The cytotoxicity of nanoparticles PLGA@BSA and the inhibitory effect of doxorubicin-loaded nanoparticles PLGA@BSA-Dox prepared by different drug loading methods on cancer cells were detected by MTT colorimetric method. Human breast cancer cells MCF-7 were selected for the MTT experiment. Among them, the concentration range of PLGA@BSA nanoparticles was 0 - 160 μg / mL, and the concentration range of Dox in PLGA@BSA-Dox was 1 - 10 μg / mL, including free doxorubicin hydrochloride (Dox·HCl), the encapsulation group (PLGA@BSA-Dox in ), the diffusion group (PLGA@BSA-Dox s ), and the composite group (PLGA@BSA-Dox c ). The specific operation steps are as follows:

[0172] The logarithmically growing MCF-7 cells were digested with trypsin, centrifuged, resuspended in 1640 medium containing serum, and cell counting was performed. 3000 cells were seeded in each well of a 96-well plate, with a volume of 200 μL per well. Five replicates were set for each group and cultured in an incubator at 37 °C and 5% CO2. Sampling was carried out 24 hours after seeding. After culturing for 24 hours, 200 μL of a 0.5% MTT solution freshly prepared in 1640 medium was added to each well and incubated for 4 hours. Then the medium was discarded, and 150 μL of DMSO was added to each well to dissolve the precipitate. The absorbance value of each well at 490 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The parallel experiment was repeated at least three times to obtain the results of the cancer cell killing by the drug-loaded nanoparticles with different concentration gradients.

[0173] Figure 13 The 24-hour MTT results of PLGA@BSA nanoparticles showed that cell viability was not greatly affected, indicating that PLGA@BSA had good biocompatibility and could be administered within this concentration range. Figure 14 The 24-hour MTT results of doxorubicin-loaded nanoparticles showed that the doxorubicin-loaded nanoparticles inhibited cell survival to varying degrees.

[0174] Test Example 8

[0175] Evaluation of the in vivo anti-cancer effect of doxorubicin-loaded nanoparticles PLGA@BSA-Dox:

[0176] (1) Construction of the tumor model:

[0177] Considering that MCF-7 cells are related to gender, female BALB / c Nude mice were selected for modeling, and the initial age of the mice was 4 weeks. The MCF-7 cells were digested with trypsin and resuspended in PBS and mixed evenly. 100 μl of the MCF-7 cell suspension (cell number 1.0×10 6 cells / mouse) was aspirated and injected into the subcutaneous tissue on the right side of the mouse. The growth of the subcutaneous tumor in the mouse was observed daily, and the short diameter a (mm) and long diameter b (mm) of the tumor tissue were measured with a vernier caliper. From this, the tumor volume V (mm 3 ) of the mouse was calculated, and the calculation formula was as follows: V = a 2 ×b / 2.

[0178] (2) Experimental grouping and drug intervention method:

[0179] When the tumor in the mouse grew to 80 mm 3Start dosing based on body size. The tumor-bearing mice were randomly divided into 6 groups, namely the low-dose negative control group Low saline (normal saline, 100 μL), the low-dose positive control group Low free Dox (free Dox·HCl, 5 mg / kg, 100 μL), the low-dose drug group Low PLGA@BSA-Dox c (PLGA@BSA-Dox described in Example 8 c , 5 mg / kg, 100 μL), the high-dose negative control group High saline (normal saline, 200 μL), the high-dose positive control group High free Dox (free Dox·HCl, 15 mg / kg, 200 μL), and the high-dose drug group High PLGA@BSA-Dox c (PLGA@BSA-Dox described in Example 8 c , 15 mg / kg, 200 μL). The solvent was normal saline for all groups, with 5 mice in each group. The mice were injected via the tail vein according to the above doses. The low-dose group was dosed every other day for a total of 4 times, and the high-dose group was dosed daily for a total of 10 times.

[0180] (3) Index detection and analysis:

[0181] Measure the body weight and tumor size changes of the mice every 2 days and make corresponding records. Figure 15 is the change in the tumor size of the mice after treatment. Figure 16 is the change in the body weight of the mice after treatment.

[0182] The results showed that although free Dox·HCl could effectively inhibit tumor growth, it caused serious toxic side effects to the mice, and the body weight showed a continuous decline. While the body weights of the high- and low-dose normal saline groups and the PLGA@BSA-Dox c group of nude mice remained stable, fluctuating in the range of 18 - 20 g. It can be seen that PLGA@BSA-Dox c will not cause damage to the body and has no systemic toxicity. The low-dose PLGA@BSA-Dox c group had a long dosing interval and a small single-dose administration, and did not show the effect of inhibiting the growth of tumor volume. While the high-dose PLGA@BSA-Dox c group showed a significant inhibition of the tumor volume growth 6 days after dosing.

[0183] The applicant declares that the technical solution of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent substitution of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc. all fall within the protection scope and the disclosure scope of the present invention.

[0184] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0185] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above specific embodiments can be combined in any appropriate way. In order to avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

Claims

1. A nanocarrier based on the self-assembly of proteins and hydrophobic polymers, characterized in that, The nanocarrier is a nanoparticle formed by the self-assembly of an amphiphilic protein and a hydrophobic polymer driven by hydrophobic interaction, where the hydrophobic polymer is wrapped by the amphiphilic protein inside the nanoparticle.

2. The nano-carrier based on the self-assembly of proteins and hydrophobic polymers according to claim 1, characterized in that The hydrophobic polymer includes any one or a combination of at least two of polylactic-co-glycolic acid, polylactic acid, poly(p-dioxanone), or polycaprolactone.

3. The nano-carrier based on the self-assembly of proteins and hydrophobic polymers according to claim 1, characterized in that, The amphiphilic protein includes any one or a combination of at least two of bovine serum albumin, human serum albumin, bovine α-lactalbumin, β-casein, ovalbumin, β-lactoglobulin, lactoferrin, or transferrin.

4. The nano-carrier based on the self-assembly of proteins and hydrophobic polymers according to any one of claims 1-3, characterized in that, The molecular weight of the hydrophobic polymer is 10 - 500 KDa; The molar ratio of the hydrophobic polymer to the amphiphilic protein is 1:5 - 10:

1.

5. The preparation method of the nanocarrier based on the self-assembly of protein and hydrophobic polymer according to any one of claims 1-4, characterized in that, The preparation method includes: (1) Dissolve the hydrophobic polymer in an organic solvent to obtain solution A; dissolve the amphiphilic protein in phosphate buffer to obtain solution B; (2) Mix and stir solution A and solution B to obtain solution C; (3) Centrifuge solution C to obtain a centrifugation product, thereby obtaining the nanocarrier based on the self-assembly of protein and hydrophobic polymer.

6. The preparation method of the nano-carrier based on the self-assembly of proteins and hydrophobic polymers according to claim 5, characterized in that, The organic solvent includes any one or a combination of at least two of tetrahydrofuran, N,N-dimethylformamide, or dimethyl sulfoxide; Preferably, the concentration of solution A is 1 - 20 mg / mL; Preferably, the concentration of solution B is 0.01 - 5 mg / mL; Preferably, the mixing and stirring in step (2) is carried out at 4 - 35 °C for 0.1 - 40 min; Preferably, the centrifugation is carried out at 10000 - 15000 rpm at 2 - 8 °C for 20 - 50 min; Preferably, after obtaining the centrifugation product, it is resuspended or further freeze-dried.

7. A nano drug delivery system, characterized in that, The nano-drug delivery system includes the nanocarrier based on the self-assembly of protein and hydrophobic polymer according to any one of claims 1 - 4 and a bioactive component loaded in the nanocarrier; The bioactive component includes any one or a combination of at least two of hydrophobic drugs, fluorescent materials, magnetic resonance materials, or photothermal sensitive materials.

8. The nano drug delivery system according to claim 7, characterized in that, The molar ratio of the hydrophobic polymer to the bioactive component is 1:1000 - 1:10000; Preferably, the hydrophobic drug includes any one or a combination of at least two of anti-cancer drugs, anti-inflammatory drugs, analgesic drugs, antihypertensive drugs, hypoglycemic drugs, lipid-lowering drugs, or anesthetic drugs; Preferably, the anti-cancer drug includes any one or a combination of at least two of doxorubicin, paclitaxel, cisplatin, ursolic acid, curcumin, dexamethasone, celecoxib, docetaxel, fenretinide, dihydroartemisinin, vinblastine, camptothecin, or apatinib mesylate.

9. The nano-drug delivery system according to claim 7, characterized in that, The loading method includes loading inside the nanocarrier by hydrophobic interaction and / or loading on the surface of the nanocarrier by diffusion; Preferably, the method of loading inside the nanocarrier by hydrophobic interaction includes the following steps: (1) Co-dissolve the hydrophobic polymer and the bioactive component in an organic solvent to obtain solution A; dissolve the amphiphilic protein in phosphate buffer to obtain solution B; (2) Mix solution A and solution B and stir to obtain solution C; (3) Centrifuge solution C to obtain a centrifugation product, thereby obtaining the nano-carrier self-assembled from protein and hydrophobic polymer; Preferably, the method for loading on the surface of the nano-carrier by diffusion action comprises the following steps: (1) Dissolve the hydrophobic polymer in an organic solvent to obtain solution A; dissolve the amphiphilic protein in a phosphate buffer solution to obtain solution B; (2) Mix solution A and solution B and stir to obtain solution C; (3) Centrifuge solution C to obtain a centrifugation product. Resuspend the centrifugation product, mix it with the bioactive component and stir, then centrifuge to obtain a centrifugation product.

10. Use of the nano-carrier self-assembled from protein and hydrophobic polymer according to any one of claims 1-4 in the preparation of chemotherapeutic drugs, immunotherapeutic drugs, contrast imaging agents, cosmetics, skin care products, health care products, plant growth regulators, agricultural fungicides or agricultural insecticides.

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