Protein-based nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses protein-based nanoparticles as well as a preparation method and application thereof. The protein-based nanoparticles have a core-shell structure, the core is zein particles loaded with active drugs, and the shell comprises a trypsin inhibitor. The preparation method of the protein-based nanoparticles comprises the following steps: 1) preparing a zein dispersion liquid loaded with an active drug and a shell material aqueous solution; and 2) slowly adding the zein dispersion liquid loaded with the active drug into the shell material aqueous solution in a stirring state, and then carrying out rotary evaporation and freeze drying. The protein-based nano-particle has an excellent gastrointestinal digestion resisting effect, various hydrophobic active drugs can be effectively delivered to a lower digestive system, the stability and slow release efficiency of the loaded drugs are remarkably enhanced, and the protein-based nano-particle is good in biocompatibility, safe, non-toxic, simple in preparation process and suitable for industrial production. The method is suitable for large-scale industrial production and application.

Description

Technical Field

[0001] The present invention relates to the technical field of targeted therapy, and particularly relates to a protein-based nanoparticle, a preparation method thereof, and an application thereof. Background Art

[0002] Among the numerous treatment approaches for diseases, the oral route is generally considered the most common, convenient, and preferred administration route, which is convenient, minimally invasive, cost-effective, and has patient compliance in both short-term and long-term treatments. Research shows that many hydrophobic natural active drugs have pharmacological activities such as antioxidant, anti-inflammatory, anti-cancer, anti-allergic, and blood pressure lowering, but the solubility of these active drugs in water is generally very small, and direct oral administration will have the problem of low bioavailability, which limits their practical applications. Therefore, it is necessary to develop appropriate delivery systems to improve the solubility, stability, bioavailability, and biological efficacy of natural active drugs.

[0003] Nanoparticles are a common type of nanoscale delivery system, which usually use polymer as the wall material and can effectively encapsulate and carry natural active ingredients. Natural polymer materials have the advantages of good biocompatibility, non-toxic and harmless degradation products, rich sources, and strong affinity with drugs, and have become an ideal choice for preparing drug delivery carriers. However, most natural polymer materials are difficult to overcome the multiple absorption barriers of the gastrointestinal tract, especially protein-based polymer materials cannot resist the digestion of proteases at all. Specifically, in the upper digestive system, after oral administration, the drug will first interact with strongly acidic gastric juice with a pH value of 1-2 and a large amount of pepsin, and then will fully react with intestinal juice containing various enzymes such as trypsin, carboxypeptidase, amylase, lactase, and lipase. The complex gastrointestinal environment is likely to cause the structure and efficacy of the drug to be destroyed prematurely, and ultimately will seriously affect the actual efficacy of the drug.

[0004] Therefore, it is of great significance to develop a drug-loaded nanoparticle with excellent anti-gastrointestinal digestion effect. Summary of the Invention

[0005] The purpose of the present invention is to provide a protein-based nanoparticle, a preparation method thereof, and an application thereof.

[0006] The technical solution adopted by the present invention is:

[0007] A protein-based nanoparticle having a core-shell structure, wherein the core is a zein particle loaded with an active drug, and the shell comprises a trypsin inhibitor.

[0008] Preferably, the shell further comprises an active polysaccharide.

[0009] Preferably, the active polysaccharide is at least one of fucoidan, astragalus polysaccharide, bupleurum polysaccharide, dendrobium polysaccharide, chlorella polysaccharide, xanthium polysaccharide, wolfberry polysaccharide, lotus root polysaccharide, arctium polysaccharide, orange peel polysaccharide, ginseng polysaccharide, cordyceps polysaccharide.

[0010] More preferably, the active polysaccharide is fucoidan. Fucoidan is a water-soluble anionic polysaccharide, which can be adsorbed onto the surface of positively charged zein self-assembled particles loaded with active drugs through electrostatic interaction. Adding this active polysaccharide, fucoidan, to the trypsin inhibitor / zein system can show good synergistic effects, further enhancing the stability and redispersibility of the loaded drug, and providing synergistic functional effects such as antioxidant and anti-inflammatory effects.

[0011] Preferably, the active drug is at least one of quercetin, curcumin, polydatin, phenethyl isothiocyanate, naringenin, hesperetin, metformin, chrysin, rutin, resveratrol, apigenin, genistein, paclitaxel, picric acid.

[0012] Preferably, the trypsin inhibitor is extracted from plant raw materials.

[0013] Preferably, the plant raw materials are at least one of potato, tamarind, soybean, buckwheat, bran, pumpkin, mung bean.

[0014] More preferably, the plant raw material is potato.

[0015] Note: Trypsin inhibitor (TI) refers to a class of proteins or polypeptides that can inhibit the activity of proteolytic enzymes, including various proteolytic enzymes such as trypsin, plasmin, and lysosomal enzymes. It is widely present in plants and is commonly distributed in plant seeds, tubers and other parts. Legumes, Solanaceae, Gramineae and Cruciferae and other plant sources are the richest. The functions of trypsin inhibitor in physiological and biochemical activities include anti-insect and anti-pathogen infection, anti-cancer, satiety effect, anti-radiation, antibacterial and anti-inflammatory, hypoglycemic, etc. Potatoes are rich in protease inhibitors, which can be specifically divided into the following seven categories: potato protease inhibitor I, potato protease inhibitor II, potato Kunitz-type protease inhibitor, potato aspartic protease inhibitor, potato carboxypeptidase, potato cysteine protease inhibitor and other serine potato protease inhibitors. Except for potato carboxypeptidase, the remaining protease inhibitors have good inhibitory activity against trypsin. In particular, potato protease inhibitor II (PI-2) has 82% total trypsin inhibitory activity, the strongest inhibitory activity against trypsin, and is the most abundant protease inhibitor in potato tubers, accounting for about 44.2% of the total amount of potato protease inhibitors.

[0016] Preferably, the extraction of the trypsin inhibitor comprises the following steps:

[0017] a) Adding plant raw materials into an aqueous sodium sulfite solution for crushing and cell wall breaking, and then filtering to obtain a plant stock solution;

[0018] b) Adjusting the pH value of the plant stock solution to 2 - 6, then centrifuging to obtain the supernatant, mixing the supernatant with an aqueous inorganic salt solution, then centrifuging to obtain the precipitate, and freeze-drying the precipitate to obtain the trypsin inhibitor.

[0019] Preferably, the mass fraction of the aqueous sodium sulfite solution in step a) is 1% - 5%.

[0020] Preferably, the inorganic salt in the aqueous inorganic salt solution in step b) is at least one of sodium chloride, ammonium carbonate, and sodium sulfate.

[0021] Preferably, the mass fraction of the aqueous inorganic salt solution in step b) is 10% - 50%.

[0022] Preferably, the particle size of the protein-based nanoparticles is 80 nm - 400 nm.

[0023] A method for preparing the protein-based nanoparticles as described above comprises the following steps:

[0024] 1) Dissolving zein and an active drug in an aqueous ethanol solution to prepare a zein dispersion loaded with the active drug, and dissolving the trypsin inhibitor in water to prepare an aqueous solution of the shell material, or dissolving the trypsin inhibitor and an active polysaccharide in water to prepare an aqueous solution of the shell material;

[0025] 2) Slowly adding the zein dispersion loaded with the active drug to the aqueous solution of the shell material under stirring, then performing rotary evaporation to remove ethanol, and then performing freeze-drying to obtain the protein-based nanoparticles.

[0026] Preferably, the mass fraction of the aqueous ethanol solution in step 1) is 60% - 90%.

[0027] Preferably, the concentration of zein in the zein dispersion loaded with the active drug in step 1) is 5 mg / mL - 40 mg / mL.

[0028] Preferably, the dosage of the trypsin inhibitor in step 1) is 5% - 50% of the weight of zein.

[0029] Preferably, the concentration of the trypsin inhibitor in the aqueous solution of the shell material in step 1) is 0.003 mg / mL - 0.8 mg / mL.

[0030] Preferably, the dosage of the active polysaccharide in step 1) is 5% to 50% of the weight of zein.

[0031] Preferably, the concentration of the active polysaccharide in the aqueous solution of the shell material in step 1) is 0.003 mg / mL to 0.8 mg / mL.

[0032] A colon-targeted therapeutic drug, which comprises the above-mentioned protein-based nanoparticles.

[0033] The technical principle of the present invention is as follows: Zein contains a large number of hydrophobic amino acids and has unique self-assembly characteristics, and can be used to encapsulate hydrophobic active drugs (such as quercetin, curcumin, polydatin, etc.) to form positively charged zein self-assembled particles loaded with active drugs. Trypsin inhibitor can be adsorbed on the surface of the self-assembled particles through electrostatic interaction, and finally protein-based nanoparticles with a core-shell structure, excellent anti-gastrointestinal digestion effect, good biocompatibility, safety and non-toxicity are obtained.

[0034] The beneficial effects of the present invention are: The protein-based nanoparticles of the present invention have excellent anti-gastrointestinal digestion effect, can effectively deliver various hydrophobic active drugs to the lower digestive system, significantly enhance the stability and sustained-release efficiency of the loaded drugs, and the protein-based nanoparticles have good biocompatibility, safety and non-toxicity, and the preparation process is simple, which is suitable for large-scale industrial production and application.

[0035] Specifically:

[0036] 1) The protein-based nanoparticles of the present invention use zein as the delivery core and trypsin inhibitor as the shell, and utilize the anti-gastrointestinal digestion characteristics of the trypsin inhibitor to overcome the complex gastrointestinal barriers, so that the hydrophobic active drugs can be delivered to the lower digestive system in a time-delayed manner, realizing the sustained release of the active drugs in the gastrointestinal tract, slowing down the metabolism of the active drugs, and effectively improving the bioavailability of the active drugs;

[0037] 2) By further adding active polysaccharide to the shell in the present invention, while improving the loading efficiency, maintaining the consistent stability, storage property and sustained-release property of the system, the carrier itself also obtains strong biological activities such as antioxidant and anti-inflammatory effects, providing a synergistic therapeutic effect;

[0038] 3) The trypsin inhibitor and active polysaccharide in the protein-based nanoparticles of the present invention are both of natural origin and have good biocompatibility. While ensuring the ability to deliver drugs into the lower digestive system, the biocompatibility and use safety of the product are greatly improved;

[0039] 4) The protein-based nanoparticles of the present invention improve the encapsulation rate of the active drug and effectively solve the problem of low bioavailability of the active drug existing in common nanoscale delivery systems;

[0040] 5) The protein-based nanoparticles of the present invention use trypsin inhibitor as the outer shell, providing excellent redispersion stability and storage stability for the delivery system. Description of the Drawings

[0041] Figure 1 SEM images of the protein-based nanoparticles of Comparative Example 1, Comparative Example 3 and Comparative Example 5, and trypsin inhibitor.

[0042] Figure 2 Test result graphs of the average particle size, particle size distribution and Zeta potential of the protein-based nanoparticles of Example 1, Example 2, Comparative Example 2 and Comparative Example 6.

[0043] Figure 3 Test result graphs of the average particle size, particle size distribution and Zeta potential of the protein-based nanoparticles of Example 1, Example 2 and Comparative Examples 1-6.

[0044] Figure 4 Test result graphs of the pharmacokinetics of the protein-based nanoparticles of Example 1, Example 2 and Comparative Example 2 in animals.

[0045] Figure 5 Test result graphs of the hemolysis inhibition ability of the protein-based nanoparticles of Example 1, Example 2, Comparative Example 3 and Comparative Example 5 on red blood cells.

[0046] Figure 6 Test result graphs of the anti-inflammatory activity of the protein-based nanoparticles of Example 1 and Example 2. Detailed Embodiments

[0047] The present invention will be further explained and described below in conjunction with specific embodiments.

[0048] The extraction process of the trypsin inhibitor in the examples is as follows:

[0049] a) Wash and cut the potatoes, then add them to a sodium sulfite aqueous solution with a mass fraction of 2% - 5% and crush and break the cells with a blender for 10 minutes. The weight ratio of potatoes to the sodium sulfite aqueous solution is 1:5. Then filter with gauze to remove potato starch to obtain potato juice;

[0050] b) Adjust the pH value of the potato juice to 4 with hydrochloric acid, stir for another 4 h, then let it stand for 12 h. Centrifuge to obtain the supernatant, add the supernatant to an aqueous sodium chloride solution with a mass fraction of 30%, stir for 4 h, then let it stand for 12 h. Centrifuge to obtain the precipitate, and then perform freeze-drying on the precipitate to obtain the trypsin inhibitor (denoted as Que; store in the dark).

[0051] Example 1:

[0052] A protein-based nanoparticle having a core-shell structure, wherein the core is a zein particle loaded with quercetin, and the shell is composed of a trypsin inhibitor.

[0053] The preparation method of the above protein-based nanoparticle is as follows:

[0054] 1) Stir and disperse 0.1 g of zein powder and 0.008 g of quercetin in 10 mL of an aqueous ethanol solution with a mass fraction of 70% to prepare a zein dispersion loaded with quercetin, and stir and dissolve 0.03 g of trypsin inhibitor in 50 mL of deionized water to prepare a shell material aqueous solution;

[0055] 2) Slowly add the zein dispersion loaded with quercetin to the stirring shell material aqueous solution at a stirring speed of 600 rpm. The zein dispersion loaded with quercetin is added within 2 min, then perform rotary evaporation to remove ethanol, and then perform freeze-drying to obtain the protein-based nanoparticle (denoted as ZTQs).

[0056] Example 2:

[0057] A protein-based nanoparticle having a core-shell structure, wherein the core is a zein particle loaded with quercetin, and the shell is composed of a trypsin inhibitor and fucoidan sulfate.

[0058] The preparation method of the above protein-based nanoparticle is as follows:

[0059] 1) Stir and disperse 0.1 g of zein powder and 0.008 g of quercetin in 10 mL of an aqueous ethanol solution with a mass fraction of 70% to prepare a zein dispersion loaded with quercetin, and stir and dissolve 0.01 g of trypsin inhibitor and 0.03 g of fucoidan sulfate in 50 mL of deionized water to prepare a shell material aqueous solution;

[0060] 2) Slowly add the zein dispersion loaded with quercetin to the stirring shell material aqueous solution at a stirring speed of 600 rpm. The zein dispersion loaded with quercetin is added within 2 min, then perform rotary evaporation to remove ethanol, and then perform freeze-drying to obtain the protein-based nanoparticle (denoted as ZTFQs).

[0061] Comparative Example 1:

[0062] A protein-based nanoparticle, and its preparation method is as follows:

[0063] 1) Stir and disperse 0.1 g of zein powder in 10 mL of an ethanol aqueous solution with a mass fraction of 70% to prepare a zein dispersion;

[0064] 2) Slowly add the zein dispersion to 50 mL of deionized water under stirring, with a stirring speed of 600 rpm. The zein dispersion is added within 2 minutes, then rotary evaporation is carried out to remove ethanol, and then freeze-drying is carried out to obtain the protein-based nanoparticle (denoted as Zein or Z).

[0065] Comparative Example 2:

[0066] A protein-based nanoparticle, and its preparation method is as follows:

[0067] 1) Stir and disperse 0.1 g of zein powder and 0.008 g of quercetin in 10 mL of an ethanol aqueous solution with a mass fraction of 70% to prepare a zein dispersion loaded with quercetin;

[0068] 2) Slowly add the zein dispersion loaded with quercetin to 50 mL of deionized water under stirring, with a stirring speed of 600 rpm. The zein dispersion loaded with quercetin is added within 2 minutes, then rotary evaporation is carried out to remove ethanol, and then freeze-drying is carried out to obtain the protein-based nanoparticle (denoted as ZQs).

[0069] Comparative Example 3:

[0070] A protein-based nanoparticle, and its preparation method is as follows:

[0071] 1) Stir and disperse 0.1 g of zein powder in 10 mL of an ethanol aqueous solution with a mass fraction of 70% to prepare a zein dispersion, and dissolve 0.01 g of trypsin inhibitor in 50 mL of deionized water to prepare an aqueous solution of the shell material;

[0072] 2) Slowly add the zein dispersion to the aqueous solution of the shell material under stirring, with a stirring speed of 600 rpm. The zein dispersion is added within 2 minutes, then rotary evaporation is carried out to remove ethanol, and then freeze-drying is carried out to obtain the protein-based nanoparticle (denoted as ZT).

[0073] Comparative Example 4:

[0074] A protein-based nanoparticle, and its preparation method is as follows:

[0075] 1) Stir and disperse 0.1 g of zein powder in 10 mL of ethanol aqueous solution with a mass fraction of 70% to prepare a zein dispersion, and stir and dissolve 0.03 g of fucoidan in 50 mL of deionized water to prepare an aqueous solution of the shell material;

[0076] 2) Slowly add the zein dispersion to the aqueous solution of the shell material under stirring at a stirring speed of 600 rpm. The zein dispersion is added within 2 min, then rotary evaporation is carried out to remove ethanol, and then freeze-drying is carried out to obtain protein-based nanoparticles (denoted as ZF).

[0077] Comparative Example 5:

[0078] A protein-based nanoparticle, and its preparation method is as follows:

[0079] 1) Stir and disperse 0.1 g of zein powder in 10 mL of ethanol aqueous solution with a mass fraction of 70% to prepare a zein dispersion, and stir and dissolve 0.01 g of trypsin inhibitor and 0.03 g of fucoidan in 50 mL of deionized water to prepare an aqueous solution of the shell material;

[0080] 2) Slowly add the zein dispersion to the aqueous solution of the shell material under stirring at a stirring speed of 600 rpm. The zein dispersion is added within 2 min, then rotary evaporation is carried out to remove ethanol, and then freeze-drying is carried out to obtain protein-based nanoparticles (denoted as ZTF).

[0081] Comparative Example 6:

[0082] A protein-based nanoparticle, and its preparation method is as follows:

[0083] 1) Stir and disperse 0.1 g of zein powder and 0.008 g of quercetin in 10 mL of ethanol aqueous solution with a mass fraction of 70% to prepare a zein dispersion loaded with quercetin, and stir and dissolve 0.03 g of fucoidan in 50 mL of deionized water to prepare an aqueous solution of the shell material;

[0084] 2) Slowly add the zein dispersion loaded with quercetin to the aqueous solution of the shell material under stirring at a stirring speed of 600 rpm. The zein dispersion loaded with quercetin is added within 2 min, then rotary evaporation is carried out to remove ethanol, and then freeze-drying is carried out to obtain protein-based nanoparticles (denoted as ZFQs).

[0085] Performance test:

[0086] 1) Scanning electron microscopy (SEM) images of the protein-based nanoparticles of Comparative Example 1 (Zein), Comparative Example 3 (ZT), Comparative Example 5 (ZTF), and trypsin inhibitor (PTI; derived from potato) are as Figure 1 shown.

[0087] It can be seen from Figure 1 that:

[0088] a) The average particle size of the protein-based nanoparticles of Comparative Example 1 is about 400 nm, while the average particle size of the protein-based nanoparticles of Comparative Example 3 is significantly smaller, indicating that the addition of trypsin inhibitor will significantly change the particle size of the obtained protein-based nanoparticles;

[0089] b) Compared with the protein-based nanoparticles of Comparative Example 3, the average particle size of the protein-based nanoparticles of Comparative Example 5 is significantly larger, indicating that the addition of fucoidan will increase the particle size of the obtained protein-based nanoparticles (trypsin inhibitor and fucoidan are used as a composite shell to modify zein together, and a small amount of adhesion occurs).

[0090] 2) Take the fresh samples (not freeze-dried) and freeze-dried samples prepared in Example 1 (ZTQs), Example 2 (ZTFQs), Comparative Example 2 (ZQs), and Comparative Example 6 (ZFQs), dilute / disperse them with deionized water to a protein concentration of 1%, and then take 1 mL of the sample to measure the average particle size, particle size distribution (PDI), and Zeta (ζ) potential of the protein-based nanoparticles with a particle size and zeta potential analyzer. The test results are as Figure 2 (Figure a is the test result graph of the average particle size, Figure b is the particle size distribution graph, Figure c is the ζ-potential test result graph, and Figure d is the physical picture of the sample) shown.

[0091] It can be seen from Figure 2 that: The particle size change of the protein-based nanoparticles after reconstitution in Example 1 and Example 2 is very small, indicating that the present invention perfectly solves the problem of poor reconstitution of the freeze-dried powder, which is beneficial to industrial production and application.

[0092] 3) Take the fresh samples (not freeze-dried) and refrigerated samples (samples stored at 4 °C for 30 days; not freeze-dried) prepared in Example 1 (ZTQs), Example 2 (ZTFQs), and Comparative Examples 1-6 (Z, ZQs, ZT, ZF, ZTF, and ZFQs), dilute them with deionized water to a protein concentration of 1%, and then take 1 mL of the sample to measure the average particle size, particle size distribution (PDI), and Zeta (ζ) potential of the protein-based nanoparticles with a particle size and zeta potential analyzer. The test results are as Figure 3 (Figure a is the test result graph of the average particle size, Figure b is the particle size distribution graph, Figure c is the ζ-potential test result graph, and Figure d is the physical picture of the sample) shown.

[0093] It can be seen from Figure 3 that

[0094] a) A large amount of precipitation occurred in the protein-based nanoparticles (Z, ZT, ZF, and ZTF) without loaded quercetin, indicating that the lipophilic active drug is the main factor affecting the storage stability of the protein-based nanoparticles;

[0095] b) After storing the protein-based nanoparticles modified with quercetin at 4 °C for 30 days, no precipitation particles were found at the bottom of the storage container. The particle size and particle size distribution showed almost no change compared with the fresh particles. Only the Zeta potential decreased slightly, but still remained below -20 mV. The test results were consistent with those observed from the physical pictures;

[0096] In summary, it can be seen that the co-assembly of a small amount of trypsin inhibitor and zein can effectively solve the problem of poor redissolution of protein-based nanoparticles. The additional addition of a small amount of fucoidan sulfate for compounding can synergistically act with the trypsin inhibitor to maintain the storage stability and redissolution stability at a satisfactory level, which is an ability that cannot be achieved by only modifying with fucoidan sulfate.

[0097] 4) The protein-based nanoparticles of Example 1 (ZTQs), Example 2 (ZTFQs), and Comparative Example 2 (ZQs) were used to feed rats. Blood was collected at fixed points within 24 h. The plasma was extracted with acetonitrile containing 5 wt% acetic acid for 5 min, and then redissolved by nitrogen blowing. The mobile phase was selected as acetonitrile-formic acid aqueous solution. The content of quercetin was determined by high performance liquid chromatography (HPLC) at a wavelength of 373 nm. The pharmacokinetic test results of the protein-based nanoparticles in animals are as Figure 4 (a quercetin solution with the same concentration equivalent as the control group) shown.

[0098] It can be seen from Figure 4 that

[0099] a) Compared with free quercetin (1 h), the peak time T of the protein-based nanoparticles (nanonized quercetin) in rats max was significantly prolonged. The peak time of ZQs was extended to 4 h, while that of ZTQS and ZTFQs was extended to 8 h, indicating that the modification with trypsin inhibitor significantly delayed the rate of quercetin entering the blood, which means that the trypsin inhibitor played an effective protective role in gastrointestinal digestion;

[0100] b) The mean residence time (MRT) of ZQs in vivo was not much different from that of free quercetin, while the mean residence time of ZTQs and ZTFQs was almost doubled compared with the former two, indicating that the modification with trypsin inhibitor effectively prolonged the retention time of quercetin in vivo;

[0101] c) Compared with ZQs, ZTQs and ZTFQs had a Cmax Significantly decreased, MRT increased, indicating that the quercetin blood drug concentrations of ZTQs and ZTFQs were relatively low, reducing the release and metabolism rates of quercetin, and enabling the release mainly through the colonic route. For free quercetin, it was rapidly metabolized in the body after intragastric administration, with its relatively short absorption half-life t 1 / 2 and overall clearance rate C Lz / F also corroborating this point.

[0102] 5) Dilute the protein-based nanoparticles (ZTQs) of Example 1, the protein-based nanoparticles (ZTFQs) of Example 2, the protein-based nanoparticles (ZT) of Comparative Example 3, the protein-based nanoparticles (ZTF) of Comparative Example 5, and quercetin (Que) with PBS buffer into samples of different concentrations. Add erythrocyte working solution and 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH). Measure the absorbance of the supernatant at a wavelength of 540 nm using an enzyme-linked immunosorbent assay reader (the erythrocytes without adding samples and AAPH are recorded as the Control group, and the erythrocytes without adding samples but adding AAPH are recorded as the Model group). Wash the erythrocytes treated with different samples 3 times with PBS buffer and then dilute them to an appropriate concentration. Fix them with 2.5% glutaraldehyde, and then wash the erythrocytes successively with ethanol solutions with mass fractions of 30%, 50%, 70%, 90%, and 100%. Place them on a mica sheet, then paste the mica sheet onto the conductive adhesive of a scanning electron microscopy disk, and finally sputter gold to observe the morphology of the erythrocytes through a field emission scanning electron microscope. The test results of the hemolysis inhibition ability of the protein-based nanoparticles on erythrocytes are as Figure 5 (A quercetin solution with the same concentration equivalent is used as the control group; a is the SEM image of the treated erythrocytes, and b is the test result image of the hemolysis inhibition ability) shown.

[0103] From Figure 5 it can be seen that:

[0104] a) The hemolysis inhibition rate of the Model group induced by AAPH on erythrocytes was 41.10%, and that of the Control group was 88.47%, indicating that the erythrocyte model of oxidative hemolysis was successfully established;

[0105] b) Compared with the Model group induced by AAPH on erythrocytes, adding free quercetin had little effect on the hemolysis inhibition rate of the sample, only increasing it by 4.54%, indicating that the solubility of quercetin free in aqueous solution was poor and it could not maximize the release of its high antioxidant performance;

[0106] c) The hemolysis inhibition rate of the sample containing trypsin inhibitor was 42.60%, showing little difference compared with the Model group, indicating that trypsin inhibitor itself did not possess strong antioxidant properties;

[0107] d) Compared with the sample of trypsin inhibitor alone, the hemolysis inhibition rate of blank nanoparticles ZT increased to 55.55%, and that of blank nanoparticles ZTF continued to increase to 62.82%, indicating that the inhibition of oxidative hemolysis of red blood cells by blank particles may stem from the characteristics of the particle composition itself, and blank nanoparticles ZTF exhibit strong antioxidant capacity;

[0108] e) Based on blank nanoparticles, quercetin with the same equivalent was loaded to obtain composite nanoparticles ZTQs and ZTFQs, and the hemolysis inhibition rates reached 57.79% and 72.95% respectively. At the same time, the hemolysis inhibition rate of red blood cells by blank particle ZTF (62.82%) exceeded that of composite nanoparticle ZTQs loaded with quercetin (57.79%);

[0109] f) The SEM observation results are consistent with the experimental results of the hemolysis inhibition rate of red blood cells;

[0110] In summary, the quercetin-loaded composite nanoparticles co-assembled by trypsin inhibitor and Zein effectively improve the bioavailability of quercetin, exhibit a synergistic inhibitory activity against red blood cell hemolysis, and also indicate that the high hemolysis inhibition rate of ZTFQs close to the Control group is mainly due to the synergistic effect of trypsin inhibitor and nano-sized quercetin.

[0111] 6) Take the protein-based nanoparticles (ZTQs) of Example 1 and the protein-based nanoparticles (ZTFQs) of Example 2 as the sample group, free quercetin (Que) as the positive control group, and use CuSO4 as the inducer of the zebrafish inflammation model. The typical feature of the CuSO4-induced zebrafish inflammation model is the migration of neutrophils to the vicinity of the lateral line neuromasts. The anti-inflammatory activity was evaluated by observing the number of cells migrating to the lateral line neuromasts, and the obtained anti-inflammatory activity test results are as Figure 6 (The fluorescence image of the lateral line neuromast is shown in a, and the test result image of neutrophils is shown in b).

[0112] It can be seen from Figure 6 that:

[0113] a) Most neutrophils in the blank group are located in the ventral rod and the tail hematopoietic tissue of the tail, while obvious changes occur in the model group exposed to the CuSO4 solution, that is, a large number of neutrophils migrate to the lateral line neuromasts and form clustered neurohypertrophy near the lateral line, thus proving that the CuSO4 inflammation model has been successfully established;

[0114] b) Combining the number of neutrophils migrating and the fluorescence image of the lateral line neuromasts, it can be seen that compared with the model group with clustered neurohypertrophy, the neuroinflammation of zebrafish is slightly alleviated after ingesting free quercetin, and the number of neutrophils in the lateral line neuromasts decreases by 10.8%, indicating that free quercetin has poor solubility and extremely low bioavailability;

[0115] c) After zebrafish ingested two kinds of protein-based nanoparticles, ZTQs and ZTFQs, compared with free quercetin, neuroinflammation was effectively alleviated, the symptom of clustered nerve hypertrophy disappeared, and the number of neutrophils within the dotted line decreased significantly, by 43.21% and 63.71% respectively, indicating that the present invention significantly improved the solubility of quercetin, promoted the absorption of quercetin by zebrafish, effectively improved the bioavailability of quercetin, and also indicating that the outer materials, trypsin inhibitor and fucoidan, participated in the anti-inflammatory pathway to varying degrees (numerous studies have shown that the active polysaccharide fucoidan has good anti-inflammatory activity against inflammatory diseases such as pancreatitis, colitis, osteoarthritis, skin inflammation, etc.);

[0116] d) Compared with ZTQs, ZTFQs containing trypsin inhibitor showed a strong synergistic enhancement effect in anti-inflammatory ability.

[0117] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A protein-based nanoparticle, characterized in that, It has a core-shell structure, where the core is zein particles loaded with an active drug, and the composition of the shell includes trypsin inhibitor.

2. The protein-based nanoparticle according to claim 1, wherein: The composition of the shell further includes an active polysaccharide.

3. The protein-based nanoparticle according to claim 2, characterized in that: The active polysaccharide is at least one of fucoidan, astragalus polysaccharide, bupleurum polysaccharide, dendrobium polysaccharide, chlorella polysaccharide, xanthium polysaccharide, wolfberry polysaccharide, lotus root polysaccharide, arctium polysaccharide, orange peel polysaccharide, ginseng polysaccharide, cordyceps polysaccharide.

4. The protein-based nanoparticle according to any one of claims 1 to 3, characterized in that: The active drug is at least one of quercetin, curcumin, polydatin, phenethyl isothiocyanate, naringenin, hesperetin, metformin, chrysin, rutin, resveratrol, apigenin, genistein, paclitaxel, picric acid.

5. The protein-based nanoparticle according to any one of claims 1 to 3, characterized in that: The trypsin inhibitor is extracted from plant raw materials; the plant raw materials are at least one of potato, tamarind, soybean, buckwheat, bran, pumpkin, mung bean.

6. The protein-based nanoparticle according to claim 5, wherein: The extraction of the trypsin inhibitor includes the following steps: a) Adding the plant raw materials into an aqueous sodium sulfite solution for pulverization and cell wall breaking, followed by filtration to obtain a plant stock solution; b) Adjusting the pH value of the plant stock solution to 2-6, followed by centrifugation to collect the supernatant, mixing the supernatant with an inorganic salt aqueous solution, followed by centrifugation to collect the precipitate, and then freeze-drying the precipitate to obtain the trypsin inhibitor.

7. The protein-based nanoparticle according to claim 6, wherein: In step a), the mass fraction of the aqueous sodium sulfite solution is 1%-5%; in step b), the inorganic salt in the inorganic salt aqueous solution is at least one of sodium chloride, ammonium carbonate, sodium sulfate; in step b), the mass fraction of the inorganic salt aqueous solution is 10%-50%.

8. The protein-based nanoparticle according to any one of claims 1 to 3, characterized in that: The particle size of the protein-based nanoparticles is 80 nm to 400 nm.

9. A method for preparing the protein-based nanoparticles according to any one of claims 1 to 8, characterized in that, It includes the following steps: 1) Dispersing zein and an active drug in an aqueous ethanol solution to prepare a zein dispersion loaded with the active drug, and dissolving the trypsin inhibitor in water to prepare an aqueous solution of the shell material, or dissolving the trypsin inhibitor and an active polysaccharide in water to prepare an aqueous solution of the shell material; 2) Slowly adding the zein dispersion loaded with the active drug to the aqueous solution of the shell material under stirring, then performing rotary evaporation to remove ethanol, and then performing freeze-drying to obtain the protein-based nanoparticles.

10. A colon-targeted therapeutic drug, characterized in that, It contains the protein-based nanoparticles described in any one of claims 1 to 8.

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