Composite nanoparticles, carrier delivery system, pharmaceutical composition, cosmetic, nutritional health product, preparation method and application

Abstract

The present invention discloses a composite nanoparticle, a carrier delivery system, a pharmaceutical composition, a cosmetic, a nutritional health product, and a preparation method and application thereof, belonging to the technical field of nano-carrier delivery systems. The composite nanoparticle includes: a core particle formed of zein; and a coating layer formed of fructan and an ionic polymer. The present invention for the first time develops a zein core / shell nanoparticle co-modified with fructan and casein, which can be used for efficiently encapsulating hydrophobic active ingredients such as astilbin, can significantly enhance the redispersibility of the zein composite nanoparticle and the stability under different environmental pressures (pH, temperature, ionic strength, etc.), improve the encapsulation and drug loading efficiency of the active ingredient and the absorption and distribution in vivo, thereby improving the bioavailability of the active ingredient. The nanoparticles prepared by the present invention are expected to become an effective carrier for hydrophobic active ingredients for food, health products, cosmetics and drug development.

Description

Technical Field

[0001] The present invention relates to the technical field of nano - carrier delivery systems, and specifically relates to a composite nanoparticle, a carrier delivery system, a pharmaceutical composition, a cosmetic, a nutritional health product, and a preparation method and application thereof. Among them, it relates to the method and application of surface modification of zein to increase the stability of nanoparticle carriers. More specifically, it relates to a composite nanoparticle of fructan (P - FOS) - modified astilbin (Ast) / zein / sodium caseinate (NaCas), a carrier delivery system, a pharmaceutical composition, a cosmetic, a nutritional health product, and a preparation method and application thereof. Background Art

[0002] Zein is a widely used nano - delivery biomaterial. Due to its unique advantages such as biodegradability, biocompatibility, and its oral application, it has become increasingly popular in the fields of food, cosmetics, health food, and pharmaceuticals. Zein encapsulating bioactive ingredients or zein micro / nanoparticles contribute to the implementation of many nutritional and health strategies in the above - mentioned fields. However, this kind of zein - based micro / nanoparticles has very poor stability and redispersibility under various environmental stress challenges, such as pH, ionic strength, and thermal processing. Especially in thermal processing, zein nanoparticles will denature and aggregate, which will have a negative impact on the production control and quality of products, restricting their practical applications.

[0003] Therefore, there is an urgent need for new materials and strategies to modify zein nanoparticles to solve these problems and expand the application of zein nanocarrier systems in the pharmaceutical, functional food, cosmetic, and nutritional supplement industries. Summary of the Invention

[0004] In view of the above problems, the present invention provides a zein composite nanoparticle co - modified by fructan and an ionic polymer, its carrier delivery system, and the preparation methods and applications of both.

[0005] To achieve these objects of the present invention, in a first aspect, a composite nanoparticle provided by the present invention includes:

[0006] A core particle formed of zein;

[0007] A coating layer formed of fructan and an ionic polymer.

[0008] Specifically, the ionic polymer can be a protein, a polypeptide / oligopeptide, an ionic polysaccharide / oligosaccharide, and related synthetic ionic polymers. Preferably, it is sodium caseinate.

[0009] Specifically, the average molecular weight of the fructan is 800 - 150,000 Da, for example, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 80,000, 90,000, 100,000, 110,000, 150,000 Da, especially 800 - 5,000 Da. In some embodiments of the present invention, the average molecular weight of the fructan is 1,600 Da.

[0010] Specifically, the particle size of the composite nanoparticles can be 100, 120, 130, 150, 160, 170, 180, 190, 200, 220, 250, 300 nm), especially 100 - 180 nm.

[0011] Specifically, the thickness of the coating layer can be 1 - 100 nm. For example, 10, 18, 20, 22, 24, 25, 26, 28, 30, 40, 50, 60, 70, 80, 90, 100 nm, especially 10 - 50 nm.

[0012] Specifically, the particle size of the core particles in the composite nanoparticles can be 50 - 120 nm (for example, 50, 60, 70, 80, 85, 86, 88, 90, 92, 94, 96, 98, 100 nm), especially 85 - 95 nm.

[0013] Specifically, the composite nanoparticles are spherical.

[0014] Specifically, the composite nanoparticles are negatively charged.

[0015] In the second aspect of the present invention, a method for preparing the composite nanoparticles is provided, including:

[0016] (1) Forming the core particles;

[0017] (2) Forming the coating layer.

[0018] Specifically, the core particles are formed by an anti-solvent co-assembly precipitation method. More specifically, step (1) includes: dissolving zein in an ethanol aqueous solution; adjusting the pH to acidic. Preferably, the ethanol aqueous solution can be 70 - 95% (v / v) ethanol aqueous solution, especially 75% ethanol aqueous solution. Preferably, adjusting the pH to acidic is to adjust the pH to 3.5 - 6.5 (for example, 3.5, 4.5, 5, 5.2, 5.5, 5.7, 6, 6.5). Preferably, the reagent used to adjust the pH to acidic is an inorganic acid solution, such as hydrochloric acid solution.

[0019] Specifically, step (1) further includes a stirring step; more specifically, the stirring speed can be 500 - 5000 rpm (such as 500, 1000, 2000, 3000, 4000, 5000 rpm), and the stirring time can be 10 - 60 minutes (such as 10, 20, 30, 40, 50, 60 minutes).

[0020] In one embodiment of the present invention, step (1) includes: dissolving zein in an aqueous ethanol solution, stirring, adjusting the pH to acidic, and stirring to obtain a core particle solution.

[0021] Specifically, the zein concentration in the core particle solution can be 1 - 70% (w / v) (such as 1%, 5%, 7.5%, 10%, 20%, 25%, 40%, 50%, 60%), especially 20 - 60%, more especially 20 - 40%; in some embodiments of the present invention, the zein concentration is 20%.

[0022] Specifically, step (2) includes: dissolving fructan and an ionic polymer in an aqueous solution; in one embodiment of the present invention, step (2) includes: dissolving fructan and sodium caseinate in an aqueous solution, stirring, adjusting the pH to acidic, and stirring to obtain a coating solution.

[0023] Specifically, the pH of the coating solution is acidic, for example, pH 2 - 6 (such as 2, 3, 4, 5, 6).

[0024] Specifically, step (2) further includes a stirring step; more specifically, the stirring speed can be 500 - 5000 rpm (such as 500, 1000, 2000, 3000, 4000, 5000 rpm), and the stirring time can be 30 - 720 minutes (such as 30, 60, 120, 240, 360, 720 minutes).

[0025] Specifically, the fructan concentration in the coating solution can be 1 - 70% (w / v) (such as 1%, 5%, 7.5%, 10%, 20%, 25%, 40%, 50%, 60%), especially 20 - 60%, more especially 30 - 50%; in some embodiments of the present invention, the fructan concentration is 50%.

[0026] Specifically, the concentration of the ionic polymer in the coating solution can be 1 - 80% (w / v) (such as 1%, 5%, 7.5%, 10%, 20%, 25%, 40%, 50%, 60%, 70%, 80%), especially 20 - 60%, more especially 30 - 50%; in some embodiments of the present invention, the concentration of sodium caseinate is 50%.

[0027] Specifically, the average molecular weight of the fructan is 800 - 150,000 Da (such as 800, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 80,000, 90,000, 100,000, 110,000, 150,000 Da), especially 800 - 5,000 Da. In some embodiments of the present invention, the average molecular weight of the fructan is 1,600 Da.

[0028] Specifically, it further includes step (3): mixing the core particle solution obtained in step (1) with the coating layer solution obtained in step (2).

[0029] Optionally, impurity removal is also performed in step (3).

[0030] Specifically, step (3) further includes a stirring step; more specifically, the stirring speed can be 500 - 5,000 rpm (such as 500, 1,000, 2,000, 3,000, 4,000, 5,000 rpm), and the stirring time can be 30 - 240 minutes (such as 30, 40, 60, 80, 100, 120, 240 minutes).

[0031] Specifically, step (3) includes: adding the core particle solution obtained in step (1) to the coating layer solution obtained in step (2), adjusting the pH to acidic, such as pH 2 - 6 (such as 2, 3, 4, 5, 6), and stirring to obtain a solution system. Preferably, ethanol is further removed.

[0032] Specifically, step (3) further includes impurity removal, and centrifuging the solution system obtained in step (3) to remove impurities.

[0033] In the third aspect of the present invention, there is provided the use of the said nanoparticles as a drug carrier or for preparing a drug or for preparing a cosmetic or for preparing a health food.

[0034] In the fourth aspect of the present invention, there is provided a carrier delivery system which has sustained release and targeting properties, and which is the said nanoparticles or contains the said nanoparticles. The nanoparticles have a core particle and a coating layer coated on the outer surface of the core particle. Among them, the core particle contains zein, and the coating layer contains fructan and an ionic polymer; and, the core particle is loaded with one or more active ingredients.

[0035] In one embodiment of the present invention, the core particle is formed by zein and an active ingredient.

[0036] In one embodiment of the present invention, the coating layer is formed by fructan and sodium caseinate.

[0037] Specifically, the average molecular weight of the fructan is 800 - 150,000 Da (e.g., 800, 1,000, 2,000, 5,000, 10,000, 20,000, 50,000, 80,000, 90,000, 100,000, 110,000, 150,000 Da), particularly 800 - 5,000 Da. In some embodiments of the present invention, the average molecular weight of the fructan is 1,600 Da.

[0038] Specifically, the particle size of the nanoparticles can be 100, 120, 130, 150, 160, 170, 180, 190, 200, 220, 250, 300 nm, particularly 100 - 180 nm.

[0039] Specifically, the thickness of the coating layer can be 1 - 100 nm (e.g., 10, 18, 20, 22, 24, 25, 26, 28, 30, 40, 50, 60, 70, 80, 90, 100 nm), particularly 10 - 50 nm.

[0040] Specifically, the core particles in the nanoparticles can be 50 - 120 nm (e.g., 50, 60, 70, 80, 85, 86, 88, 90, 92, 94, 95, 96, 98, 100 nm), particularly 85 - 95 nm.

[0041] Specifically, the nanoparticles are spherical.

[0042] Specifically, the nanoparticles are negatively charged.

[0043] In one embodiment of the present invention, the above active ingredient can be an active ingredient in food or nutraceuticals. For example, curcumin, vitamin E, lutein, β - carotene, etc.

[0044] In one embodiment of the present invention, the above active ingredient can be a cosmetic active ingredient. For example, glutathione, kojic acid, tranexamic acid, salicylic acid, niacinamide, fruit acid, azelaic acid, vitamin A and its derivatives, vitamin C, glycerol, hyaluronic acid, sodium pyrrolidone carboxylate, natural moisturizing factor, ceramide, collagen, lactic acid, urea, chitin derivatives, aloe vera, seaweed extract, polyglutamic acid, jojoba oil, squalene, shea butter, etc.

[0045] In one embodiment of the present invention, the above active ingredient can be a pharmaceutically active ingredient, such as doxorubicin, epirubicin, pirarubicin, idarubicin; mitoxantrone; topotecan, irinotecan, exatecan, aminocamptothecin; paclitaxel, docetaxel; gefitinib, imatinib, nilotinib, sunitinib, lapatinib, tofacitinib, crizotinib, masitinib, entrectinib, ibrutinib, afatinib, flumatinib, erlotinib, neratinib, alectinib, apatinib, Talazoparib, lorlatinib, TPX-0005; cisplatin, carboplatin, nedaplatin, cycloplatin, oxaliplatin, lobaplatin; vinblastine, vincristine, vinorelbine, berberine, berbamine; honokiol (HNK); uracil mustard, nitrogen mustard, ifosfamide, melphalan, chlorambucil, pipobroman, tretamine, thiotepa, busulfan, carmustine, lomustine, streptozocin, dacarbazine, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate; and so on.

[0046] Specifically, in the core particle, the weight ratio of zein to the active ingredient can be from 1:1 to 100:1 (such as 1:1, 2:1, 5:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1), particularly from 1:1 to 5:1.

[0047] In one embodiment of the present invention, the active ingredient is astilbin (Ast); more specifically, in the core particle, the weight ratio of zein to astilbin is 2:1.

[0048] In the fifth aspect of the present invention, there is provided a method for preparing the carrier delivery system, comprising the following steps:

[0049] (1) Forming a core particle;

[0050] (2) Forming a coating layer.

[0051] Specifically, the core particles are formed by anti-solvent co-assembly precipitation method. More specifically, step (1) includes: dissolving zein and the active ingredient (separately or together) in an aqueous ethanol solution and adjusting the pH to acidic. Specifically, the aqueous ethanol solution can be 70-95% (v / v) aqueous ethanol solution, especially 75% ethanol aqueous solution. Specifically, adjusting the pH to acidic means adjusting the pH to 3.5-6.5 (such as 3.5, 4.5, 5, 5.2, 5.5, 5.7, 6, 6.5). Specifically, the reagent used to adjust the pH to acidic is an inorganic acid solution, such as hydrochloric acid solution. Specifically, step (1) also includes a stirring step; more specifically, the stirring speed can be 500-5000 rpm (such as 500, 1000, 2000, 3000, 4000, 5000 rpm), and the stirring time can be 10-60 minutes (such as 10, 20, 30, 40, 50, 60 minutes).

[0052] In one embodiment of the present invention, step (1) includes: dissolving zein and the active ingredient (separately or together) in an aqueous ethanol solution, stirring, adjusting the pH to acidic, and stirring to obtain a core particle solution.

[0053] Specifically, the concentration of zein can be 1-70% (w / v) (such as 1%, 5%, 7.5%, 10%, 20%, 25%, 40%, 50%, 60%), especially 20-60%, more especially 10-40%; in some embodiments of the present invention, the concentration of zein is 20%.

[0054] Specifically, step (2) includes: dissolving fructan and an ionic polymer in an aqueous solution; in one embodiment of the present invention, step (2) includes: dissolving fructan and sodium caseinate in an aqueous solution, stirring, adjusting the pH to acidic, and stirring to obtain a coating solution.

[0055] Specifically, the pH of the fructan solution is acidic, such as pH 2-6 (such as 2, 3, 4, 5, 6). Specifically, the stirring speed can be 500-5000 rpm (such as 500, 1000, 2000, 3000, 4000, 5000 rpm), and the stirring time can be 30-720 minutes (such as 30, 60, 120, 240, 360, 720 minutes).

[0056] Specifically, the concentration of fructan can be 1-70% (w / v) (such as 1%, 5%, 7.5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%), especially 20-60%, more especially 30-50%; in some embodiments of the present invention, the concentration of the fructan solution is 50%.

[0057] Specifically, the concentration of the ionic polymer solution can be 1-80% (w / v) (e.g., 1%, 5%, 7.5%, 10%, 20%, 25%, 40%, 50%, 60%, 80%), particularly 20-60%, more particularly 30-50%; in some embodiments of the present invention, the concentration of the sodium caseinate solution is 50%.

[0058] Specifically, the average molecular weight of the fructan is 800-150000 Da (e.g., 800, 1000, 2000, 5000, 10000, 20000, 50000, 80000, 90000, 100000, 110000, 150000 Da), particularly 800-5000 Da, and in some embodiments of the present invention, the average molecular weight of the fructan is 1600 Da.

[0059] Specifically, it further includes step (3): adding the core particle solution obtained in step (1) to the coating solution obtained in step (2). Preferably, step (3) further includes adjusting the pH to acidic, e.g., pH 2-6 (e.g., 2, 3, 4, 5, 6). Preferably, step (3) further includes a stirring step; more specifically, the stirring speed can be 500-5000 rpm (e.g., 500, 1000, 2000, 3000, 4000, 5000 rpm), and the stirring time can be 30-240 minutes (e.g., 30, 40, 60, 80, 100, 120, 240 minutes). Preferably, step (3) further includes removing ethanol.

[0060] In some embodiments of the present invention, step (3) includes: adding the zein ethanol solution (core particle solution) obtained in step (1) to the aqueous solution of fructan and ionic polymer (coating solution) obtained in step (2), adjusting the pH to acidic, stirring to obtain a solution system, and removing ethanol.

[0061] Optionally, step (3) further includes impurity removal. Specifically, the solution system obtained in step (3) is centrifuged to remove impurities.

[0062] In the sixth aspect of the present invention, there is provided an application of the carrier delivery system in improving any one or more of the properties of drug loading component stability, redispersibility, loading rate, release efficiency, or pharmacological activity.

[0063] Or, the application of the carrier delivery system in improving any one or more of the properties of food health products, cosmetics loading component stability, redispersibility, loading rate, release efficiency, or activity.

[0064] Specifically, the activity refers to the activity or physiological functionality of additives permitted in health foods such as vitamin supplements, mineral supplements, etc.

[0065] Specifically, the pharmacological activity may be antioxidant, anti-inflammatory, immunomodulatory, antibacterial, etc.

[0066] Specifically, the medicament may be one or more of a medicament for improving the stability of the encapsulated component (such as a stabilizer), a medicament for improving redispersibility (such as a dispersant), a medicament for improving the encapsulation rate and release efficiency (such as a drug carrier product), and a medicament for improving pharmacological activity (such as a synergist).

[0067] In the seventh aspect of the present invention, there is provided a pharmaceutical composition comprising the carrier delivery system described in the fourth aspect, and one or more pharmaceutically acceptable excipients.

[0068] Specifically, the pharmaceutically acceptable excipients refer to conventional excipients in the pharmaceutical field, especially pharmaceutically acceptable injection excipients, such as isotonic sterile salt solutions (sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, etc., or a mixture of the above salts), or dry compositions, such as freeze-dried compositions, which are appropriately formed into injectable solutes by adding sterile water or physiological saline.

[0069] Specifically, the pharmaceutical composition can be administered by any suitable route, such as gastrointestinal administration or parenteral administration (e.g., intravenous, intramuscular, subcutaneous, intra-organ, intranasal, intradermal, infusion, intracerebral, rectal, etc.) routes; the above drugs can be in any suitable dosage form, such as gastrointestinal dosage forms, for example, including but not limited to, tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, suspensions, etc.; parenteral dosage forms, for example, injection dosage forms: such as injections (e.g., for subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection), respiratory dosage forms: such as sprays, aerosols, powder aerosols, etc., skin dosage forms, such as topical solutions, lotions, ointments, plasters, pastes, patches, etc., mucosal dosage forms: such as eye drops, ophthalmic ointments, nasal drops, gargles, sublingual tablets, etc., cavity dosage forms: such as suppositories, aerosols, effervescent tablets, drops, dripping pills, etc., for rectum, vagina, urethra, nasal cavity, ear canal, etc. Preferably, the pharmaceutical composition is for oral administration.

[0070] Specifically, the various dosage forms of the pharmaceutical composition can be prepared according to the conventional production methods in the pharmaceutical and food fields, for example, by mixing the carrier delivery system with one or more pharmaceutically acceptable excipients, and then forming them into the required dosage forms.

[0071] In the eighth aspect of the present invention, there is provided a food nutritional health product comprising the carrier delivery system described in the fourth aspect, and one or more food nutritional health product acceptable excipients.

[0072] The acceptable excipients on the food nutritional health products refer to the conventional excipients in the field of food nutritional health products. For example, starch, lactose, cellulose, sweeteners, colorants, and so on.

[0073] In the ninth aspect of the present invention, there is provided a cosmetic, which comprises the carrier delivery system described in the fourth aspect, and one or more cosmetic-acceptable excipients.

[0074] The cosmetic-acceptable excipients refer to the conventional excipients in the field of cosmetics. For example, glycerol, hyaluronic acid, vegetable oils, emulsifiers, thickeners, preservatives, pigments, fragrances, and so on.

[0075] The present invention has developed for the first time a zein core / shell nanoparticle co-modified with fructan (P-FOS) and protein, which can be used for efficiently encapsulating hydrophobic active ingredients (such as astilbin (Ast)). It can significantly enhance the stability of zein under different environmental pressures (pH, temperature, ionic strength), improve the encapsulation and drug loading efficiency of active ingredients and their absorption and distribution in vivo, thereby improving the bioavailability of active ingredients. Taking Ast as an example, experiments have proved that Ast / zein / fructan / sodium caseinate nanoparticles have significantly enhanced stability and redispersibility under different environmental pressures (pH, temperature, ionic strength). Moreover, Ast / zein / fructan / sodium caseinate nanoparticles have significantly improved the dissolution and release of Ast in simulated gastrointestinal fluids and produced stronger antioxidant activity. Therefore, the nanoparticles prepared by the present invention are expected to become an effective carrier for hydrophobic active ingredients for the development of foods, health products, cosmetics and pharmaceuticals.

[0076] The present invention has at least the following beneficial effects:

[0077] 1. The present invention overcomes the deficiencies of the prior art. For the first time, it utilizes the self-assembly structural characteristics of natural neutral polysaccharide - fructan to develop a novel physical cross-linking stabilizer for zein composite nanoformulations, and adopts a new strategy of anti-solvent co-assembly precipitation. Without introducing chemical cross-linking agents, it can significantly improve the stability of zein composite nanoparticles. The preparation method is simple, which expands the application of zein-based biological carrier systems in the fields of functional foods, cosmetics, nutritional supplements and the pharmaceutical industry.

[0078] 2. Compared with the zein composite nanoparticles formed by only protein modification, the zein composite nanoparticles modified with fructan have significantly improved physical and chemical stability, that is, in the temperature range of 40 - 90 °C, pH value of 2 - 8, and NaCl concentration range of 40 mM to 2 M, they exhibit excellent stability to temperature, pH, and ionic strength. In addition, the zein composite nanoparticles with dual fructan / protein modification show excellent redispersibility after freeze-drying. They can improve the encapsulation and drug-loading efficiency of active ingredients, antioxidant activity, and absorption and distribution in vivo, thereby improving the bioavailability of active ingredients.

[0079] Other advantages, objectives, and features of the present invention will be partially reflected by the following description and partially understood by those skilled in the art through the research and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 It is the determination results of the drug encapsulation rate and drug-loading amount of the composite nanoparticles in Example 1 of the present invention;

[0081] Figure 2 It is the detection results of the particle size distribution and ζ potential of the composite nanoparticles in Example 1 of the present invention;

[0082] Figure 3 It is the characterization results of the FT-IR spectrum structure and XRD spectrum structure of the composite nanoparticles in Example 1 of the present invention;

[0083] Figure 4 It is the SEM and TEM profile structure characterization results of the composite nanoparticles in Example 1 of the present invention;

[0084] Figure 5 It is the in vitro release curve of different Ast nanoparticles measured by the dynamic dialysis method in the release medium simulating gastrointestinal fluid in Example 1 of the present invention;

[0085] Figure 6 It is the redispersibility test results of the Zein composite nanoparticles loaded with Ast with different modification methods in Example 2 of the present invention;

[0086] Figure 7 It is the stability test results of the Zein composite nanoparticles loaded with Ast with different modification methods in Example 2 of the present invention;

[0087] Figure 8 It is the encapsulation rate of the Zein / P-FOS / NaCas composite nanoparticles loaded with Ast prepared with different P-FOS composition ratios in Example 3 of the present invention;

[0088] Figure 9Particle sizes and ζ potentials of freeze-dried Zein / P-FOS / NaCas composite nanoparticles encapsulating Ast prepared with different P-FOS composition ratios in Example 3 of the present invention before and after freeze-drying;

[0089] Figure 10 Test results of the effects of AZS and AZPS nanoparticles in Example 4 of the present invention on the antioxidant activity of Ast;

[0090] Figure 11 Cytotoxicity detection results of Ast preparations with different concentrations in Example 4 of the present invention;

[0091] Figure 12 Ast serum drug concentration-time curves corresponding to the pharmacokinetic experiment results of different Ast preparations in Example 5 of the present invention. Detailed implementation manners

[0092] The present invention will be further described in detail below with reference to examples, so that those skilled in the art can implement it according to the description in the specification.

[0093] It should be understood that terms such as "having", "comprising" and "including" used herein do not exclude the presence or addition of one or more other elements or their combinations. It should be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial sources unless otherwise specified.

[0094] Some of the reagents, materials and experimental animals used in the experiments of the examples are as follows:

[0095] Astilbin (Ast, >98%) was purchased from Purifa Biotechnology Co., Ltd. (Chengdu, China). Food-grade zein, fructan and sodium caseinate were purchased from Macklin Technology Co., Ltd. (Shanghai, China). Polysialic acid (PSA) was produced by Zhenjiang Changxing Pharmaceutical Co., Ltd. (Zhenjiang, China). The CCK8 kit was provided by TargetMol (Shanghai, China). 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and reactive oxygen species (ROS) detection kits were purchased from Beyotime Biotechnology Co., Ltd. (Beijing, China). Unless otherwise specified, all other chemical reagents were used as received.

[0096] The mouse monocyte macrophage RAW 264.7 cell line was obtained from Zhejiang Meisen Cell Technology Co., Ltd. (Zhejiang, China) and cultured in DMEM medium (Gibco, Thermofisher, USA) supplemented with 10% fetal bovine serum (Zhejiang Meisen Cell Technology Co., Ltd., Zhejiang, China), 100 U / mL penicillin and 100 μg / mL streptomycin at 37 °C in a humid atmosphere containing 5% CO 2 2.

[0097] Statistical analysis:

[0098] All experimental results in the examples are expressed as mean ± standard deviation. Student's t-test was used to determine statistical differences. p < 0.05 was considered significantly different, and p < 0.01 was considered extremely significantly different.

[0099] Example 1: Preparation and characterization of Ast-loaded Zein composite nanoparticles with different modification methods

[0100] 1. Preparation of nanoparticles

[0101] Ast-loaded zein composite nanoparticles were prepared using P-FOS / NaCas dual modification and NaCas single modification respectively. The specific steps are as follows:

[0102] Preparation of Ast / Zein mixed solution: Dissolve Zein (100 mg) in 5.0 mL of ethanol / water (75:25, v / v) and stir magnetically (1000 rpm) for 30 min. Dissolve Ast (50 mg) in 5.0 mL of ethanol / water (75:25, v / v) and stir magnetically (1000 rpm) for 30 min. Add 5 mL of the AST solution dropwise to 5 mL of the Zein solution and stir for 0.5 h to obtain 10 mL of the AST / Zein mixture.

[0103] Preparation of P-FOS / NaCas mixed solution: Dissolve P-FOS (100 mg) in 5.0 mL of ultrapure water and stir magnetically (1000 rpm) for 30 min. Dissolve NaCas (200 mg) in 30.0 mL of ultrapure water and stir magnetically (1000 rpm) for 1 h. Add 5 mL of the P-FOS aqueous solution dropwise to 30 mL of the NaCas solution and stir for 1 h to obtain 35 mL of the P-FOS / NaCas mixed solution.

[0104] For the method of coating Zein nanoparticles with P-FOS and NaCas dual modification, inject the obtained Ast / Zein mixed solution (10.0 mL) into 35.0 mL of the P-FOS / NaCas mixed solution, continuously stir (1000 rpm) for 1 hour, and evaporate and concentrate the suspension on a rotary evaporator (45 °C) to remove ethanol to obtain an Ast / Zein / P-FOS / NaCas nano-suspension. Centrifuge the prepared sample suspension at a speed of 5000 r / min for 10 min to remove large particles and free Ast. Freeze-dry the prepared sample to obtain nanoparticle powder, which is abbreviated as Ast / Zein / P-FOS / NaCas nanoparticles (AZPS) in the figures.

[0105] For the method of modifying and coating Zein nanoparticles with NaCas, the obtained Ast / Zein mixed solution (10.0 mL) was injected into 30.0 mL of NaCas solution, and continuously stirred (1000 rpm) for 1 hour. The suspension was evaporated and concentrated on a rotary evaporator (45 °C) to remove ethanol, obtaining an Ast / Zein / NaCas nano-suspension. The prepared sample suspension was centrifuged at a speed of 5000 r / min for 10 min to remove large particles and free Ast. The prepared sample was freeze-dried to obtain nanoparticle powder, which is simply referred to as Ast / Zein / NaCas nanoparticles (AZS) in the attached figure.

[0106] The molecular weights of the above-mentioned P-FOS used are all 1600 - 2000 Da.

[0107] The mass composition ratio of the above-mentioned Ast / Zein / P-FOS / NaCas used is 1:2:3:4.

[0108] 2. Experimental Results

[0109] 2.1 Encapsulation Efficiency, Drug Loading Capacity and Particle Size

[0110] The drug encapsulation efficiency (Encapsulation efficiency, abbreviated as EE) and drug loading capacity (loading capacity, abbreviated as LC) of Zein composite nanoparticles were determined by the HPLC-MS / MS system. The calculation formula of EE is as follows: EE = (weight of the loaded Ast / total weight of the fed Ast) × 100%. The calculation formula of LC is as follows: LC = (weight of the loaded Ast / weight of the nanoparticles) × 100%. The average particle size (Particle Diameter), polydispersity index (PDI) and ζ potential value (Zeta) of different nanoparticles were analyzed by a dynamic light scattering instrument (DLS, Zetasizer NANO ZSP, Malvern).

[0111] The experimental results are as Figure 1 shown. The encapsulation efficiency and drug loading capacity of NaCas-modified Zein nanoparticles (AZS) for Ast were 73.56 ± 1.62% and 13.3 ± 1.13% respectively. When fructan P-FOS and NaCas were used for double modification, the encapsulation efficiency of Zein nanoparticles (AZPS) for Ast was significantly improved, with an encapsulation efficiency of 92.34 ± 1.28%, while the drug loading capacity was 12.4 ± 0.93%, indicating that the crosslinking of fructan P-FOS can promote the encapsulation efficiency of Zein / NaCas composite nanoparticles for hydrophobic active ingredients.

[0112] As Figure 2As shown, the particle size distributions obtained by dynamic light scattering detection show that both AZPS and AZS have narrow particle size distributions, and there is no obvious difference in the particle diameter between AZS and AZPS. The average size is 160 nm, and the particle size distribution index (PDI) is less than 0.2, indicating that the obtained zein nanoparticles have good dispersibility. At the same time, the crosslinking of fructan P-FOS has little effect on the particle size of zein / NaCas composite nanoparticles, indicating that P-FOS has been successfully introduced into the interior of zein / NaCas composite nanoparticles.

[0113] The ζ potential of AZS was measured to be -25.2 ± 2.2 mV. After modification with P-FOS, the ζ potential of AZPS was -27.8 ± 2.8 mV. Since P-FOS belongs to neutral polysaccharide and there are no carboxyl or amino groups in its chemical structure, the addition of P-FOS did not significantly change the charge of the nanoparticles, but the absolute value increased slightly, indicating that P-FOS has been successfully introduced into the interior of zein / NaCas composite nanoparticles. It increases the steric repulsion force through hydrogen bonding and hydrophobic interaction between zein and NaCas, weakens the electrostatic adsorption force between zein and NaCas, thereby increasing the absolute value of the ζ potential. The relatively high ζ potential is a key factor to improve the stability of nanoparticles in aqueous media through electrostatic repulsion. Generally, compared with positively charged nanoparticles, negatively charged nanoparticles may exhibit improved nanocolloid stability, increased blood circulation time, and reduced toxicity to normal cells.

[0114] 2.2. Characterization of Nanoparticles

[0115] The FT-IR spectral structure characterization of zein composite nanoparticles is as Figure 3 shown in -A. For zein, the main characteristic peaks in the FT-IR spectrum show a broadband at 3292 cm -1 , which is attributed to the overlapping stretching vibrations of -NH and -OH. The peaks at 1640.7 cm -1 , 1515.1 cm -1 and 1446.1 cm -1 are related to the C-O stretching vibration of amide I, the N-H bending vibration of amide II, and the -CH 2 stretching vibration, respectively. In the AZS and AZPS samples, the zein peaks at 3292 cm -1 shifted to 3273.80 cm -1 and 3280.23 cm -1 , respectively, while the peaks related to amide I shifted to 1637.8 and 1638.9 cm -1 , and the peaks related to amide II shifted to 1514.7 and 1525.5 cm -1。The movement of zein at 3292 cm -1 is related to the formation of intermolecular hydrogen bonds, while the peaks moving at 1640 cm -1 and 1515 cm -1 correspond to the intermolecular electrostatic interactions. The results show that electrostatic interactions and intermolecular hydrogen bonds are involved in the co-assembly process of zein / P-FOS / NaCas composite nanoparticles. In the FT-IR spectrum of free Ast, the peaks at 1631.8 cm -1 , 1601.3 cm -1 , 1524.5 cm -1 , 1503.8 cm -1 and 1473.2 cm -1 are consistent with the presence of aromatic rings and the stretching of -C=O groups. The stretching vibrations of the C-C-C, C-C-H and C-O-C in the ring-chain part and aromatic rings of Ast are reflected in the peaks at 1350, 1290.75 and 1173.74 cm cm -1 , while these peaks disappear in the FT-IR spectra of AZS and AZPS, indicating that Ast is encapsulated in the zein composite nanoparticles.

[0116] The XRD spectrum structural characterization of zein composite nanoparticles is shown in Figure 3 -B. The XRD pattern of Ast monomers shows characteristic peaks at diffraction angles (2θ) of 5 - 50°, which is related to its crystalline state. However, these characteristic peaks disappear in the XRD spectra of AZS and AZPS. It indicates that Ast is encapsulated in the zein composite nanoparticles in an amorphous state. P-FOS shows a broad peak around 19.5, while zein and NaCas show two broad peaks at 10.5 and 22.9, indicating that zein, NaCas and P-FOS are all amorphous. After the formation of composite nanoparticles, the intensity of the zein diffraction peak decreases, indicating an interaction between zein, NaCas and P-FOS. It is worth noting that AZS and AZPS NPs have similar XRD behaviors, which indicates that the crosslinking of P-FOS in AZPS nanoparticles has no significant effect on the amorphous structure of zein / NaCas composite nanoparticles, consistent with the experimental results observed by DLS.

[0117] The size, structure and morphology of the nanoparticles were confirmed by field emission scanning electron microscopy (SEM, Zeiss Gemini 300, Japan) and transmission electron microscopy (TEM, FEI Tecnai G2 F30, Japan).

[0118] The SEM and TEM profile structural characterization of zein composite nanoparticles is shown in Figure 4As shown, the TEM images of AZPS and AZS exhibit a distinct core-shell spherical structure with an average size of 140 nm (there is a deviation between the particle size measured by TEM and that measured by a nanoparticle sizer, which is within a reasonable error range), consistent with the experimental results observed by DLS.

[0119] 2.3 Simulated Gastrointestinal Fluid Release

[0120] The in vitro release curves of different Ast nanoparticles were determined using the dynamic dialysis method in a release medium consisting of simulated gastrointestinal fluids.

[0121] Simulated Gastric Fluid: The sample was dispersed in 2 mL of simulated gastric fluid (SGF, pH 2.0, containing pepsin) to achieve an Ast concentration of 0.8 mg / mL. Then the sample was transferred into a dialysis bag with SGF as the dialysis medium (molecular weight cut-off: 3500 Da), dialyzed in 30 mL of the release medium, placed in a water bath, and subsequently the liquid was stirred at 110 rpm at 37 °C. During digestion, 100 μL of the external medium was collected at regular intervals and replenished with the same volume of fresh medium to maintain a constant volume. The gastric digestion stage lasted for 120 minutes.

[0122] Simulated Intestinal Fluid: After the gastric fluid stage ended, the remaining liquid was mixed with an equal volume of small intestinal fluid (SIF, pH 7.0, containing 8.5 mg / mL trypsin), then the pH value was adjusted to 7.0 with NaOH solution (2 M), and the resulting liquid was dialyzed in 30 mL of the release medium, followed by stirring the liquid at 110 rpm in a 37 °C water bath. During digestion, 100 μL of the external medium was collected at regular intervals and replenished with the same volume of fresh medium to maintain a constant volume. The simulated intestinal fluid digestion stage lasted for 120 minutes.

[0123] The samples collected at different digestion stages were heated to 90 °C for 5 minutes to inactivate the digestive enzymes, then diluted with 1 mL of methanol and centrifuged at 13000 rpm / min for 10 minutes. The Ast content in the external medium was determined using an HPLC-MS / MS system within 2 hours.

[0124] As Figure 5As shown in the figure, the release of different Ast preparations gradually increased steadily during the simulated gastric juice stage. Among them, the efficiency of AZS and AZPS in releasing Ast was significantly higher than that of free Ast. After 60 minutes, free Ast, AZS, and AZPS released approximately 32.5%, 55.6%, and 76.3% of Ast, respectively. Among them, AZPS had nearly 100% release at 2 h. The release rate of free Ast was relatively low because its crystal structure hindered its dispersion in gastrointestinal fluids. In contrast, in AZS and AZPS nanoparticles, Ast exists in an amorphous form, which contributes to its release behavior. It should be noted that compared with AZPS, the release rate of Ast in AZS was delayed. This may be because in AZS, the electrostatic interaction between Zein and NaCas is stronger, and Ast is embedded in the hydrophobic region of Zein, resulting in a more compact microstructure of the particles and lower hydration efficiency. On the contrary, introducing hydrophilic P-FOS into the Zein matrix in AZPS can generate multiple intermolecular hydrogen bonds between Zein and NaCas, reduce the hydrophobicity of the particle matrix, and improve its hydration efficiency, thus ultimately making Ast have the greatest stability and the most effective release behavior in gastrointestinal fluids, indicating that AZPS has better bioavailability in gastrointestinal fluids.

[0125] Example 2: Investigation of the stability and redispersibility of Zein composite nanoparticles loaded with Ast with different modification methods

[0126] 1. Stability and redispersibility

[0127] The challenges faced by Zein-based nanocarriers are redispersibility and stability under different environmental conditions, such as high temperature, ionic strength, and pH challenges during the manufacturing processes of food, cosmetics, health foods, and pharmaceutical products. Therefore, the present invention comparatively tested the redispersibility of Zein composite nanoparticles loaded with Ast with different modification methods and the effects of various environmental pressures on the stability of the composite particles.

[0128] 1.1 Redispersibility

[0129] Samples of Zein composite nanoparticles with different modification methods before and after freeze-drying were redispersed into ultrapure water, and the average particle size and PDI value were measured. As Figure 6 shown, AZPS generally maintained the particle size unchanged after freeze-drying, while the ζ potential remained at about -25 mV and the PDI value was less than 0.2. In contrast, the particle size of AZS increased from 179 nm to 250 nm after freeze-drying. This shows that the redispersibility of AZPS nanoparticles before and after freeze-drying is good, and at the same time, it has a narrow particle size distribution. It indicates that P-FOS crosslinking can significantly improve the redispersibility of Zein / NaCas NPs. In the figure, freeze-drying before is before freeze-drying, and freeze-drying later is after freeze-drying.

[0130] 1.2, Stability investigation

[0131] Ionic strength stability

[0132] Sodium ions are usually added during the manufacturing process. Sodium ions can shield the net surface charge of protein particles, thereby reducing the electrostatic repulsion between particles, resulting in the instability and easy aggregation of Zein nanoparticles. As Figure 7 shown, AZS nanoparticles showed a high sensitivity to NaCl solution. After adding different concentrations of NaCl solution, a large number of AZS nanoparticles aggregated and settled. This indicates that due to the shielding effect of sodium ions, the electrostatic repulsion between Zein and NaCas in AZS weakened. On the contrary, the particle size of AZPS nanoparticles was not significantly affected in a wide range of NaCl ionic strengths (40 mM to 2 M), the particle size change was not obvious, and the distribution was uniform (PDI < 0.25), although the ζ potential decreased to near the net zero electrorepulsion force ( Figure 7 ). Sodium ions will shield the surface charge of protein particles, resulting in a decrease in ζ potential, while AZPS in this study showed good ionic strength resistance, indicating that the cross-linking of P-FOS can significantly enhance the steric repulsion effect of zein composite nanoparticles, thereby obtaining more stable and uniform zein composite nanoparticles.

[0133] pH stability

[0134] Zein nanoparticles may experience different pH environments during manufacturing, storage, and administration. However, protein particles are usually sensitive to pH changes. Generally speaking, the surface charge of particles will decrease near the isoelectric point of the protein, which minimizes the electrostatic repulsion between protein particles and promotes aggregation. As Figure 7It is shown that due to the high sensitivity of zein and NaCas to pH changes, with their isoelectric points being pH 6.2 and 4.2 respectively, obvious aggregation of AZS nanoparticles occurs in the pH range of 3 - 8. The ζ potential of particles close to the isoelectric point is relatively low, and the electrostatic repulsion is very weak, resulting in obvious particle aggregation. However, AZPS nanoparticles can still maintain a relatively stable state even near the isoelectric points of zein and NaCas (pH 6.2 and 4.2). When the nanoparticles are incubated at pH 3, a slight increase in particle size is observed. This may be due to the weakening of electrostatic interactions between proteins and the protonation of P-FOS, where the three polymers repel each other, leading to the swelling of the polymer network and thus significantly increasing its hydrodynamic diameter. However, at pH 3, the PDI value of AZPS nanoparticles is still small (<0.2), indicating that the particles have a stable and relatively narrow particle size distribution. Near the isoelectric point of Zein nanoparticles, the electrostatic repulsion weakens, but the steric repulsion can be not significantly affected by changes in environmental conditions. Therefore, it further shows that P-FOS stabilizes the Zein / NaCas composite nanoparticles by increasing the steric repulsion force, thus showing a more stable and more uniform colloidal solution in the pH range of 2 - 8.

[0135] Thermal stability

[0136] To verify the stability of the composite particles under heating conditions, AZS and AZPS composite nanoparticles were suspended in ultrapure water at a concentration of 1.0 mg / mL and incubated at temperatures (Temperature) of 40, 50, 60, 70, 80, and 90 °C for 30 min. As Figure 7 shown, obvious aggregation occurred in the AZS group when the temperature exceeded 50 °C. In contrast, the AZPS group showed good colloidal stability with a narrow particle size distribution (PDI < 0.3), and no obvious aggregation was observed even when the temperature was raised to 90 °C. This indicates that AZPS has significantly improved thermal stability compared to AZS. At 90 °C, the particle size of AZPS increased, from the original 205 nm to about 320 nm, which may be due to the partial unfolding of zein molecules at high temperatures. In the present invention, heating the AZPS nanoparticles at 90 °C for 30 minutes only caused a certain degree of increase in the particle size of the nanoparticles, but still maintained a stable colloidal solution with good uniformity, which further verified the stabilizing effect of P-FOS on Zein composite nanoparticles.

[0137] Overall, the above stability study results indicate that P-FOS can significantly improve the stability of Zein / NaCas nanoparticles against various environmental stresses (i.e., pH changes, high temperature, and ionic strength). P-FOS increases the steric repulsion between Zein and NaCas through the cross-network of the hydrophilic carbohydrate layer, enhances the hydration of hydrophobic Zein nanoparticles, and thus improves the stability and dispersibility in the aqueous environment.

[0138] Example 3: Investigation of the Composition Ratio of P-FOS

[0139] 1. Preparation of Nanoparticles

[0140] Zein / P-FOS / NaCas composite nanoparticles encapsulating Ast were prepared using different composition ratios of P-FOS, and the specific steps are as follows:

[0141] Preparation of the Ast / Zein mixed solution: Dissolve Zein (100 mg) in 5.0 mL of ethanol / water (75:25, v / v) and stir magnetically (1000 rpm) for 30 min. Dissolve Ast (50 mg) in 5.0 mL of ethanol / water (75:25, v / v) and stir magnetically (1000 rpm) for 30 min. Drop 5 mL of the AST solution into 5 mL of the Zein solution and stir for 0.5 h to obtain 10 mL of the AST / Zein mixture.

[0142] Preparation of the P-FOS / NaCas mixed solution: Dissolve P-FOS (100 mg) in 5.0 mL of ultrapure water and stir magnetically (1000 rpm) for 30 min. Dissolve NaCas (200 mg) in 30.0 mL of ultrapure water and stir magnetically (1000 rpm) for 1 h. Drop 5 mL of the P-FOS aqueous solution into 30 mL of the NaCas solution and stir for 1 h to obtain 35 mL of the P-FOS / NaCas mixed solution.

[0143] For the method of modifying Zein composite nanoparticles with P-FOS, inject the obtained Ast / Zein mixed solution (10.0 mL) into 35.0 mL of the P-FOS / NaCas mixed solution, continuously stir (1000 rpm) for 1 hour, and evaporate and concentrate the suspension on a rotary evaporator (45 °C) to remove ethanol to obtain the Ast / Zein / P-FOS / NaCas nano-suspension. Centrifuge the prepared sample suspension at a speed of 5000 r / min for 10 min to remove large particles and free Ast. Freeze-dry the prepared sample to obtain nanoparticle powder, which is called Ast / Zein / P-FOS / NaCas nanoparticles.

[0144] The above composition ratios of Ast / Zein / P-FOS / NaCas are 1:2:1:4, 1:2:2:4, 1:2:3:4, 1:2:4:4, and 1:2:5:4 respectively.

[0145] The size, potential, and distribution of the nanoparticles were confirmed by a dynamic light scattering instrument (DLS, Zetasizer NANO ZSP, Malvern).

[0146] 2. Experimental results

[0147] The experimental results are as Figure 8 shown. When the relative mass ratio of P-FOS was increased, the particle size slightly increased (170 - 200 nm) with the increase in the P-FOS ratio, but the increase trend of the encapsulation efficiency of the nanoparticles was not obvious.

[0148] Meanwhile, by comparing the particle sizes of the nanoparticles before and after freeze-drying ( Figure 9 A), the Zein / NaCas composite nanoparticles with different ratios of P-FOS all had good redispersibility, and the absolute value of the ζ potential of the nanoparticles was between 20 - 25 mV ( Figure 9 B), indicating good stability of the nanoparticles. In addition, P-FOS belongs to neutral polysaccharides and has the characteristic of self-assembly. The multi-hydroxyl and β(1-2) flexible fructosyl glycosidic bond structure determine that the cross-linking process between P-FOS, cationic Zein, and anionic NaCas belongs to non-covalent bond (mainly through hydrogen bonds and hydrophobic interactions) co-assembly physical cross-linking. For the Zein / NaCas composite nanoparticles cross-linked with different mass ratios of P-FOS, the negative ζ potential of the nanoparticles was not reversed, and the absolute value was 20 - 25 mV, with no obvious change, indicating that the cross-linking of P-FOS was mainly located inside zein and NaCas. In the figure, freeze-dryingbefore is before freeze-drying, and freeze-drying later is after freeze-drying.

[0149] Example 4: Investigation of the antioxidant activity of different Ast preparations

[0150] Ast has good antioxidant activity. However, due to its poor water solubility, its in vivo activity and bioavailability are limited. To study the enhancing effect of P-FOS cross-linked Zein / NaCas composite nanoparticles on the biological activity of encapsulated active substances, the present invention evaluated the effects of AZS and AZPS nanoparticles on the antioxidant activity of Ast.

[0151] DPPH free radical scavenging test

[0152] 1. Experimental method

[0153] Briefly, DPPH (2 mg) was dissolved in ethanol (5 mL) to obtain a DPPH stock solution. Then, the stock solution was diluted with ethanol to obtain DPPH working solutions with concentrations between 0.1 - 2.0 mM. Then, 100 μL of the diluted working solution was added to the designated wells in a 96 - well microplate, and then 100 μL of the sample or control solution was added. The microplate was placed in the dark for 30 minutes of reaction, and then the absorbance was read at 517 nm using a Thermo Scientific Varioskan Flash microplate reader (Thermo, USA).

[0154] 2. Experimental Results

[0155] As Figure 10 shown in Figure A, when the Ast concentrations were 50, 100, 150, and 200 μg / ml, AZPS nanoparticles were able to inhibit 28.7%, 62.7%, 80.5%, and approximately 90% of superoxide radicals. At the same time, AZPS exhibited a more significant free - radical scavenging effect than free Ast monomers and AZS. This can be explained by the good dispersibility and bio - accessibility of AZPS. The free - radical scavenging efficiency is related to the dissolution and release rate of Ast. The cross - linking of hydrophilic P - FOS promoted the hydration and dispersibility of the hydrophobic Zein matrix in the aqueous environment, thus enhancing the dissolution of Ast and achieving a higher equilibrium concentration of Ast, enabling better contact between superoxide radicals and Ast. On the other hand, due to the electrostatic interaction between Zein and NaCas, AZS particles had a more compact core - shell structure, which hindered the release of Ast in aqueous solution. Therefore, AZPS had a more significant free - radical scavenging efficiency than AZS nanoparticles.

[0156] Intracellular Reactive Oxygen Species (ROS) Test

[0157] Furthermore, a cell model was used to evaluate the ability of AZPS to regulate intracellular ROS levels. Human macrophages are key coordinators in the initiation and resolution phases of inflammation and are also sentinel cells for maintaining homeostasis and preventing infection. The stimulation of macrophages induces an inflammatory response, which causes various cellular changes, among which the production of reactive oxygen species (ROS) plays a key role in the differentiation and activation of macrophages. In this invention, lipopolysaccharide (LPS) of Gram - negative bacteria was applied to mouse mononuclear macrophages (RAW 264.7) to promote oxidative stress and generate ROS, and the ability of different Ast preparations (Ast monomers, AZS, and AZPS nanoparticles) to regulate intracellular ROS levels was detected.

[0158] 1. Experimental Method

[0159] Cytotoxicity Assay

[0160] The CCK8 assay was used to evaluate the viability of RAW 264.7 cells after treatment with different concentrations of Ast preparations (free Ast, AZS, and AZPS) for 24 hours. Briefly, RAW 264.7 cells were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin solution in an incubator at 37°C and 5% CO2.

[0161] RAW 264.7 cells were seeded in 96-well plates at a density of approximately 1×10 4 per well and incubated for 24 h. Serial Ast preparation solutions with different Ast concentrations (1 - 20 μg / mL) were prepared using serum-free medium.

[0162] Cells were treated with different concentrations of Ast preparations. After incubation for 24 h, the medium was discarded and the cells were washed twice with phosphate buffer system (PBS).

[0163] According to the manufacturer's instructions, the CCK-8 method was used to determine the cytotoxicity of the three Ast preparations. Cells in each well were treated with 10% CCK8 and incubated at 37°C for another 2 hours, and the absorbance of the final solution representing cell viability was measured using a Thermo Scientific Varioskan Flash microplate reader (Thermo, USA). Viability was expressed as a percentage of the control group.

[0164] Determination of intracellular ROS

[0165] The intracellular ROS level was detected using the fluorescent probe DCFH-DA. Briefly, RAW 264.7 cells were seeded in 6-well cell culture plates at a density of 1×10 5 cells per well and incubated for 24 h.

[0166] Cells were treated with three gradually increasing concentrations of Ast preparations (5, 10, 20 μg / mL) for 2 h and then stimulated with LPS (5 μg / mL). The positive group was cells stimulated with LPS (5 μg / mL) without drug pretreatment, and the control group was treated with normal medium.

[0167] After incubation for 24 h, all the medium was carefully removed, and then the cells were incubated with 10 μM DCFH-DA at 37°C for 30 min. The cells were rinsed twice with PBS to completely remove the DCFH-DA that did not enter the cells.

[0168] Cells were collected and analyzed by flow cytometry (Guava In the United States), the fluorescence intensity was analyzed. Meanwhile, the fluorescence intensity was detected and recorded using a microplate reader (Infinite M200 Pro, Tecan, Austria), with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The relative fluorescence intensity was calculated compared to the control (cells not treated with LPS).

[0169] 2. Experimental Results

[0170] As Figure 11 shown, when the concentration range of Ast was 1 to 20 μg / mL, no obvious cytotoxicity was observed in all groups. Therefore, this dose range can be used for further research.

[0171] DCFH-DA was used as a fluorescent probe to monitor the intracellular ROS level.

[0172] As Figure 10 B and Figure 10 C shown, compared with the untreated group, LPS stimulation led to an increase in the fluorescence intensity of DCF in RAW 264.7 cells, indicating an enhanced ROS level. While compared with the Ast monomer and AZS treatment groups, the AZPS treatment group significantly reduced the fluorescence intensity of DCF, indicating that AZPS pretreatment was more effective in reducing the upregulation of ROS generation in RAW 264.7 cells induced by LPS stimulation. This result was consistent with the results of the in vitro DPPH free radical scavenging assay. Therefore, the ability of AZPS to inhibit the ROS level in LPS-stimulated RAW 264.7 macrophages may be related to its free radical scavenging ability. These results indicate that P-FOS cross-linked zein / NaCas composite nanoparticles can effectively enhance the antioxidant activity of hydrophobic bioactive substances.

[0173] Example 5: Pharmacokinetic Experiment on Promoting Oral Absorption of Different Ast Preparations

[0174] 1. Experimental Method

[0175] Sprague-Dawley (SD) rats, weighing 250 - 300 g, male, were randomly divided into an Ast monomer group, an AZS group, and an AZPS nanoparticle group, with 6 rats in each group. The gavage administration methods for the two groups of rats were as follows:

[0176] Before the experiment, the rats were adaptively fed. They were fasted but allowed to drink water for 12 h before administration and fasted from food and water for 2 h after administration. Each group was gavaged at an equal dose of Ast 20 mg / kg.

[0177] After oral administration, 0.5 mL of orbital blood was collected at 0.167 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 24 h, placed in an EP centrifuge tube. After standing at room temperature for stratification (about 2 h), centrifugation was carried out (2000 r·min -1, (15 min), repeat 2 times, take the upper serum, seal it and store it in a -80 °C refrigerator for later use.

[0178] The plasma concentration of Ast was detected by HPLC-MS / MS method, and the area under the drug concentration-time curve and relative bioavailability were calculated.

[0179] 2. Experimental results:

[0180] As Figure 12 shown, it is the serum drug concentration-time curve of Ast corresponding to the pharmacokinetic experiment results.

[0181] The pharmacokinetic parameters corresponding to the three groups of experiments of the Ast monomer group, AZS group and AZPS nanoparticles group are shown in Table 1 of the results.

[0182] Table 1 Pharmacokinetic parameters of the AST group, AZS group and AZPS group at an oral dose of 20 mg / kg (results are expressed as mean ± SD)

[0183] Group <![CDATA[T 1 / 2 (h)]]> <![CDATA[C max (ng / mL)]]> <![CDATA[T max (h)]]> <![CDATA[AUC (0-24) (ng / ml*h)]]> AST Group 8.455±7.265 31.982±19.645 1.564±2.673 101.137±63.269 AZS Group 4.276±2.794 39.769±17.081 1.918±1.818 149.777±40.991 AZPS Group 4.272±2.317 <![CDATA[94.214±58.177 △△ > 1.105±1.311 <![CDATA[299.150±109.662 △△ >

[0184] Note: AST vs AZS P < 0.05: *; P < 0.01: **

[0185] AST vs AZPS P < 0.05: △ ; P < 0.01: △△

[0186] It can be seen from Table 1 that compared with the Ast monomer and AZS, the AZPS nanoparticles provided by the present invention can significantly improve the oral absorption of Ast. The oral relative bioavailability of Ast is increased by about 3 times compared with the Ast monomer and about 2 times compared with AZS without adding the fructan P-FOS crosslinking.

[0187] In summary, the inventors have successfully prepared for the first time a zein core / shell nanoparticle co-modified with a fructan (P-FOS) and a protein using the anti-solvent co-assembly precipitation technique, which can be used for efficient encapsulation of hydrophobic active ingredients (such as astilbin (Ast)), and can significantly enhance the stability of zein under different environmental stresses (pH, temperature, ionic strength), improve the encapsulation and drug-loading efficiency of active ingredients, as well as their absorption and distribution in vivo, thereby improving the bioavailability of active ingredients. Taking Ast as an example, experiments have proven that Ast / Zein / P-FOS / NaCas (AZPS) nanoparticles have excellent stability and redispersibility under different environmental stresses (pH, temperature, ionic strength). Moreover, AZPS nanoparticles significantly improve the dissolution and release of Ast in simulated gastrointestinal fluids, thereby generating stronger antioxidant activity. AZPS is a biosafe and biocompatible nanodrug delivery system, which is non-toxic to mouse mononuclear macrophages and can regulate the level of reactive oxygen species stimulated by inflammation. Therefore, the zein core / shell nanoparticle co-modified with a fructan (P-FOS) and a protein prepared in the present invention is expected to become an effective carrier for hydrophobic active ingredients for food, health products, cosmetics and drug development.

[0188] The foregoing is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

[0189] The foregoing embodiments and methods described in the present invention may vary based on the capabilities, experience and preferences of those skilled in the art.

[0190] The mere listing of the steps of the method in a certain order in the present invention does not constitute any limitation on the order of the method steps.

Claims

1. A composite nanoparticle, characterized in that: include: core particles, formed by zein; a coating layer formed of fructan and ionic polymer; The ionic polymer is sodium caseinate, the average molecular weight of the fructan is 800-5000 Da; the particle size of the composite nanoparticles is 100-300 nm; the thickness of the coating layer is 1-100 nm; the particle size of the core particles is 50-120 nm; and the composite nanoparticles are negatively charged.

2. A method for preparing the composite nanoparticles according to claim 1, characterized in that: The process includes forming core particles and forming a coating layer, specifically including: Dissolving zein in an ethanol aqueous solution, stirring, adjusting the pH to acidic, and stirring to obtain a zein ethanol mixed solution; Dissolving fructan and sodium caseinate in an aqueous solution, stirring, adjusting the pH to acidic, and stirring to obtain a fructan and sodium caseinate mixed solution; The obtained zein ethanol mixed solution is mixed with the obtained fructan and sodium caseinate mixed solution, the pH is adjusted to acidic, stirred to obtain a solution system, ethanol and impurities are removed, and dried to obtain composite nanoparticles.

3. Use of the composite nanoparticles according to claim 1 or the composite nanoparticles prepared by the preparation method according to claim 2 as a carrier in the preparation of drugs, cosmetics or nutritional health products.

4. A vector delivery system, characterized in that: The composite nanoparticles according to claim 1 or containing the composite nanoparticles, wherein the core particles of the composite nanoparticles are loaded with one or more active ingredients.

5. The vector delivery system according to claim 4, characterized in that The active ingredient is astilbin; in the core particles, the weight ratio of zein to astilbin is 2:

1.

6. A method for preparing the carrier delivery system according to claim 4, characterized in that: include: 1) Forming core particles with zein and active ingredients; 2) Forming a coating layer.

7. A pharmaceutical composition for improving the stability, redispersibility, encapsulation rate, release efficiency or pharmacological activity of drug-encapsulated components, characterized in that: The invention comprises the carrier delivery system according to claim 4, and one or more pharmaceutically acceptable excipients.

8. A nutritional health product, characterized in that: The invention comprises the carrier delivery system according to claim 4, and one or more excipients acceptable for nutritional health products.

9. A cosmetic, characterized in that: The invention comprises the carrier delivery system according to claim 4, and one or more cosmetically acceptable excipients.

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