Beta-lactoglobulin-lipoic acid hydrogel material as well as preparation method and application thereof

Abstract

The invention provides a beta-lactoglobulin-lipoic acid hydrogel material as well as a preparation method and application thereof, and belongs to the technical field of molecular biomedicine. Dissolving lipoic acid powder in alkali liquor, mixing an obtained lipoic acid salt solution, a beta-lactoglobulin solution and a carboxyl activating agent, adjusting the pH value, and carrying out chemical modification reaction, so as to obtain a modified product; sequentially dialyzing and drying the modified product to obtain beta-lactoglobulin-lipoic acid powder; mixing the beta-lactoglobulin-lipoic acid powder with water to obtain a beta-lactoglobulin-lipoic acid solution; enabling the beta-lactoglobulin-lipoic acid solution to be in contact with the oil phase solution, and performing ultraviolet irradiation to obtain the beta-lactoglobulin-lipoic acid hydrogel material. The lipoic acid is converted into the water-soluble lipoic acid salt, so that the solubility of the lipoic acid in a water phase is improved, and the reaction efficiency of the lipoic acid and beta-lactoglobulin is improved; oil-water interface mediation is adopted, so that photo-crosslinking sites are effectively exposed, and the photo-crosslinking efficiency is further improved.

Description

Technical Field

[0001] The invention relates to the technical field of molecular biomedicine, and in particular to a beta-lactoglobulin-lipoic acid hydrogel material and a preparation method and application thereof. Background Art

[0002] Protein has become a carrier material of great concern in drug delivery systems due to its natural biological properties and multifunctionality. Among them, β-lactoglobulin (BLG) is one of the main components of whey protein in milk, accounting for about 50% to 60% of the total whey protein. BLG belongs to the lipocalin protein family and has a unique combination of hydrophilic and hydrophobic surfaces and multiple amino acid residues that are easy to modify. Compared with most protein drug delivery carriers, BLG has the advantages of wide sources, low cost, relatively simple extraction and purification process, and the ability to achieve large-scale production.

[0003] Existing studies have shown that BLG can bind and load drugs, and can protect the active ingredients of drugs from degradation by gastric juice during oral administration, thereby improving the bioavailability of drugs and reducing the occurrence of side effects. Existing studies have mostly focused on using BLG monomers to bind and transport small molecule drugs. These studies have shown that BLG has appropriate binding ability and excellent carrying capacity for small molecule drugs. However, due to its small molecular weight (only 18.3kDa), the BLG monomer has relatively weak binding ability to macromolecular drugs such as proteins and nucleic acids or some special drugs, which makes BLG less efficient in loading macromolecular drugs and difficult to meet the clinical requirements for high drug loading and targeted delivery.

[0004] Hydrogel / hydrogel microspheres are a three-dimensional network structure with high water content, good biocompatibility, controllable degradability and excellent environmental responsiveness, and show significant advantages in drug delivery systems. Compared with traditional small molecule carriers, hydrogel / hydrogel microspheres as drug carriers can not only load large molecular drugs (including proteins, nucleic acids, peptide drugs and even cells), but also can achieve precise regulation of drug release rate and release time by changing chemical composition, network density or cross-linking mode. At present, the preparation methods of hydrogel / hydrogel microspheres with BLG as raw material include physical cross-linking, chemical cross-linking, thermal induced gelation, etc. These methods generally have problems such as long cycle, complex process and poor controllability.

[0005] Photocrosslinking is a process that promotes crosslinking polymerization between molecular chains through photoinitiators / photoactive groups under light. It has the advantages of fast speed, simple operation, time and space controllability and good versatility, and has important application value in the construction of drug delivery carriers. At present, the existing BLG photocrosslinked hydrogels usually rely on photoinitiators to achieve crosslinking reactions. For example, in related studies on grafting glycidyl methacrylate or methacrylic anhydride onto BLG molecules, photoinitiators induced crosslinking of BLG molecular chains and formed a stable three-dimensional network structure. However, the introduction of photoinitiators may bring some potential problems, such as:

[0006] (1) Some toxic or irritating photoinitiators may pose safety risks to tissues and cells, such as Irgacue 2959 and benzophenone (BP);

[0007] (2) The price of photoinitiators is generally high, and the use of photoinitiators will increase the cost of preparing hydrogels / hydrogel microspheres;

[0008] (3) When using a photoinitiator, its concentration and lighting conditions need to be precisely controlled, which increases the complexity and uncertainty of preparing hydrogels / hydrogel microspheres.

[0009] In summary, it is crucial to provide a new type of photo-cross-linked hydrogel material to overcome the problems caused by the current introduction of photoinitiators. Summary of the invention

[0010] The purpose of the present invention is to provide a β-lactoglobulin-lipoic acid hydrogel material and a preparation method and application thereof, so as to solve the problems in the current preparation process of β-lactoglobulin photo-crosslinked hydrogel.

[0011] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0012] The present invention provides a method for preparing a β-lactoglobulin-lipoic acid hydrogel material, comprising the following steps:

[0013] dissolving lipoic acid powder in alkaline solution to obtain lipoic acid salt solution;

[0014] The lipoic acid salt solution, the β-lactoglobulin solution and the carboxyl activator are mixed, the pH of the solution is adjusted to 5.5-6.0, and a chemical modification reaction is performed to obtain a modified product;

[0015] The modified product is dialyzed and dried in sequence to obtain β-lactoglobulin-lipoic acid powder;

[0016] mixing the β-lactoglobulin-lipoic acid powder with water to obtain a β-lactoglobulin-lipoic acid solution;

[0017] The β-lactoglobulin-lipoic acid solution is contacted with an oil phase solution, and after ultraviolet light exposure, a β-lactoglobulin-lipoic acid hydrogel material is obtained;

[0018] The contacting method is: attaching the oil phase solution to the β-lactoglobulin-lipoic acid solution; or using the oil phase solution as a continuous phase and the β-lactoglobulin-lipoic acid solution as a dispersed phase, and dispersing by a microfluidic method.

[0019] Preferably, the alkali solution includes sodium hydroxide solution or potassium hydroxide solution, the concentration of the alkali solution is 1.0-1.5 mol / L, the mass volume ratio of the thioctic acid powder to the alkali solution is 1.38-5.5 g:6.67-53.32 mL, the pH value of the thioctic acid salt solution is 9.0-11.0, and the molar concentration is 1.0-1.5 mol / L.

[0020] Preferably, the β-lactoglobulin solution is obtained by mixing β-lactoglobulin powder with water, and the concentration of the β-lactoglobulin solution is 4-8wt%; the volume ratio of the β-lactoglobulin solution to the lipoic acid salt solution is 50-100:10-30.

[0021] Preferably, the carboxyl activator comprises 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide; the molar ratio of the lipoic acid powder, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide is 1:1:0.2-1; the temperature of the chemical modification reaction is 25-30°C, and the time is 16-24h.

[0022] Preferably, the molecular weight cutoff of the dialysis bag used for the dialysis is 1000 to 3500 Da, the dialysis temperature is 2 to 6° C., and the time is 2 to 4 days; the drying method is vacuum freeze drying, the drying temperature is -80 to -60° C., and the time is 8 to 16 hours.

[0023] Preferably, the mass concentration of the β-lactoglobulin-lipoic acid solution is 4-12wt%.

[0024] Preferably, the oil phase solution comprises paraffin oil and Span 80, the concentration of Span 80 in the oil phase solution is 10-20wt%; the volume ratio of the oil phase solution to the β-lactoglobulin-lipoic acid solution is 0.5-1:1; the ultraviolet irradiation conditions are: the illumination intensity is 100-300mW / cm 2 , wavelength is 365nm, time is 1 to 5min.

[0025] Preferably, the flow rate of the dispersed phase is 0.8 to 1.2 mL / h, and the flow rate of the continuous phase is 18 to 108 mL / h.

[0026] The invention provides a beta-lactoglobulin-lipoic acid hydrogel material prepared by the preparation method.

[0027] The present invention provides application of the beta-lactoglobulin-lipoic acid hydrogel material in preparing an oral drug delivery carrier.

[0028] Beneficial effects of the present invention:

[0029] The invention introduces lipoic acid, and the lipoic acid molecule contains a disulfide bond five-membered ring structure of dynamic ring-opening polymerization, which gives beta-lactoglobulin the characteristic of photo-crosslinking. The disulfide bond serves as both a light absorption unit and a dynamic covalent crosslinking unit, and can be broken and reorganized under ultraviolet light irradiation, thereby realizing a crosslinking reaction, and the formation of a photo-crosslinked polymer can be realized without introducing a photoinitiator, thereby providing a possibility for beta-lactoglobulin to construct a photoresponsive hydrogel; by converting lipoic acid into a water-soluble lipoic acid salt solution, the solubility of lipoic acid in the aqueous phase is improved, thereby improving the efficiency of the reaction between lipoic acid and beta-lactoglobulin; at the same time, the introduction of lipoic acid also avoids the safety hazard of using a traditional photoinitiator, reduces the potential harm to operators and the environment, and significantly reduces the cost in the preparation process.

[0030] The present invention adopts an oil-water interface-mediated strategy, that is, the interfacial tension of the oil-water interface is used to regulate the arrangement of β-lactoglobulin-lipoic acid molecules, so that the hydrophobic disulfide bond five-membered ring structure in the molecule is exposed to the outer surface of the molecule. After ultraviolet light irradiation, the disulfide bond five-membered ring structures of different molecules are ring-opened and polymerized, and a cross-linking reaction occurs, which further improves the efficiency of the photocross-linking process and realizes a safer, more economical and efficient preparation of hydrogels / hydrogel microspheres.

[0031] The raw material for preparing the β-lactoglobulin-lipoic acid hydrogel / hydrogel microspheres of the present invention is β-lactoglobulin derived from milk, which has a wide source and low cost. The prepared β-lactoglobulin-lipoic acid hydrogel / hydrogel microspheres have good biocompatibility and biosafety, and can be used to prepare oral drug delivery carriers; the preparation method is simple, cheap, efficient and sustainable, the intermediate products in each reaction stage are easy to control, and the method has the advantages of short gelation time, low energy consumption in the gelation process, stable product performance, easy large-scale preparation, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the H NMR spectrum of the β-lactoglobulin-lipoic acid powder obtained in Example 1;

[0033] Figure 2 is the Fourier transform infrared spectrum of the β-lactoglobulin-lipoic acid powder obtained in Example 1;

[0034] Figure 3The physical pictures of the products of Example 1, Comparative Example 1 and Comparative Example 2 are shown in FIG. Figure 3 A is the product obtained by directly irradiating the β-lactoglobulin-lipoic acid solution in Comparative Example 1 without adding an oil phase. Figure 3 B is the β-lactoglobulin-lipoic acid hydrogel material obtained in Comparative Example 2, Figure 3 C is the β-lactoglobulin-lipoic acid hydrogel material obtained in Example 1;

[0035] Figure 4 The optical microscope image of the β-lactoglobulin-lipoic acid hydrogel microspheres obtained in Example 3 and the particle size statistics thereof;

[0036] Figure 5 The optical microscope images of the β-lactoglobulin-lipoic acid hydrogel microspheres obtained in Example 2 in PBS solution, simulated gastric fluid and intestinal fluid for 0 to 24 hours (scale bar is 300 μm);

[0037] Figure 6 The optical microscope image and the fluorescence microscope image of the curcumin-loaded β-lactoglobulin-lipoic acid hydrogel microspheres obtained in Application Example 1 are shown. DETAILED DESCRIPTION

[0038] The present invention provides a method for preparing a β-lactoglobulin-lipoic acid hydrogel material, comprising the following steps:

[0039] dissolving lipoic acid powder in alkaline solution to obtain lipoic acid salt solution;

[0040] The lipoic acid salt solution, the β-lactoglobulin solution and the carboxyl activator are mixed, the pH of the solution is adjusted to 5.5-6.0, and a chemical modification reaction is performed to obtain a modified product;

[0041] The modified product is dialyzed and dried in sequence to obtain β-lactoglobulin-lipoic acid powder;

[0042] mixing the β-lactoglobulin-lipoic acid powder with water to obtain a β-lactoglobulin-lipoic acid solution;

[0043] The β-lactoglobulin-lipoic acid solution is contacted with an oil phase solution, and after ultraviolet light exposure, a β-lactoglobulin-lipoic acid hydrogel material is obtained;

[0044] The contacting method is: attaching the oil phase solution to the β-lactoglobulin-lipoic acid solution; or using the oil phase solution as a continuous phase and the β-lactoglobulin-lipoic acid solution as a dispersed phase, and dispersing by a microfluidic method.

[0045] In the present invention, unless otherwise specified, the required raw materials for preparation are all commercially available products well known to those skilled in the art.

[0046] The present invention preferably dissolves thioctic acid powder in alkali solution to obtain a thioctic acid salt solution; in the present invention, the alkali solution preferably includes a sodium hydroxide solution or a potassium hydroxide solution, and more preferably includes a sodium hydroxide solution; the concentration of the alkali solution is preferably 1.0-1.5 mol / L, and more preferably 1.0 mol / L; the mass volume ratio of the thioctic acid powder to the alkali solution is 1.38-5.5 g:6.67-53.32 mL, and more preferably 2.75 g:13.33 mL; the pH value of the thioctic acid salt solution is preferably 9.0-11.0, and more preferably 10.0-11.0, and the molar concentration of the thioctic acid salt solution is preferably 1.0-1.5 mol / L, and more preferably 1.0 mol / L.

[0047] The present invention preferably mixes beta-lactoglobulin powder with water to obtain a beta-lactoglobulin solution; mixes the lipoic acid salt solution with the beta-lactoglobulin solution, slowly adds a carboxyl activator to the obtained mixed solution in sequence, uses a 1 mol / L HCl solution to adjust the pH of the solution to 5.5-6.0, and performs a chemical modification reaction in a stirring state in the dark to obtain a modified product.

[0048] In the present invention, the β-lactoglobulin powder is preferably derived from cow's milk, and the purity is preferably >93%, more preferably 93%; the concentration of the β-lactoglobulin solution is preferably 4-8wt%, more preferably 6-8wt%.

[0049] In the present invention, the volume ratio of the β-lactoglobulin solution to the lipoic acid salt solution is preferably 50-100:10-30, and more preferably 69-100:18.4-30.

[0050] In the present invention, the carboxyl activator preferably includes 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, and the molar ratio of the lipoic acid powder, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide is preferably 1:1:0.2-1, and more preferably 1:1:0.5-1; the purpose of adding the carboxyl activator is to activate the carboxyl group of lipoic acid, so that lipoic acid is more likely to react with β-lactoglobulin to undergo amide reaction, that is, to graft lipoic acid onto the β-lactoglobulin molecule.

[0051] In the present invention, after the β-lactoglobulin solution, lipoic acid salt solution and carboxyl activator are mixed, the pH of the solution is preferably adjusted to 5.5-6.0, and more preferably to 6.0. Under this pH condition, the activation efficiency of the carboxyl group of lipoic acid can be guaranteed without causing precipitation and aggregation of β-lactoglobulin.

[0052] In the present invention, the stirring speed is preferably 150 to 450 rpm, more preferably 300 to 450 rpm; the temperature of the chemical modification reaction is preferably 25 to 30° C., and the time is preferably 16 to 24 h, more preferably 18 to 24 h.

[0053] In the present invention, the modified product is preferably dialyzed and dried in sequence to obtain β-lactoglobulin-lipoic acid powder.

[0054] In the present invention, the dialysis method is preferably dialysis with a dialysis bag, and the molecular weight cutoff of the dialysis bag is preferably 1000 to 3500 Da, and more preferably 3500 Da; the dialysis agent used for the dialysis is preferably deionized water, and the pH of the deionized water is 7 to 9, and more preferably 7.5 to 9; the dialysis temperature is preferably 2 to 6°C, and more preferably 4 to 6°C, and the time is preferably 2 to 4 days, and more preferably 3 days. During the dialysis, the deionized water is preferably changed 3 to 4 times a day; the drying method is preferably vacuum freeze drying, and the drying temperature is preferably -80 to -60°C, and more preferably -75 to -60°C, and the time is preferably 8 to 16 hours, and more preferably 12 to 16 hours.

[0055] The present invention preferably mixes the β-lactoglobulin-lipoic acid powder with water to obtain a β-lactoglobulin-lipoic acid solution; in the present invention, the mass concentration of the β-lactoglobulin-lipoic acid solution is preferably 4-12wt%, more preferably 8-12wt%.

[0056] The present invention preferably mixes paraffin oil and Span 80 to obtain an oil phase solution; the β-lactoglobulin-lipoic acid solution is contacted with the oil phase solution to form an oil-water interface, and after ultraviolet light irradiation, the β-lactoglobulin-lipoic acid hydrogel material is obtained; the contacting method is: the oil phase solution is attached to the β-lactoglobulin-lipoic acid solution; or the oil phase solution is used as a continuous phase and the β-lactoglobulin-lipoic acid solution is used as a dispersed phase, and a microfluidic method is used for dispersion.

[0057] In the present invention, the concentration of Span 80 in the oil phase solution is preferably 10 to 20 wt %, and more preferably 10 to 15 wt %.

[0058] In the present invention, the volume ratio of the oil phase solution to the β-lactoglobulin-lipoic acid solution is preferably 0.5 to 1:1, and more preferably 0.5 to 0.8:1.

[0059] In the present invention, the intensity of the ultraviolet light is preferably 100 to 300 mW / cm 2 , more preferably 200 to 300 mW / cm 2The wavelength is preferably 365 nm, the time is preferably 1 to 5 min, and more preferably 3 to 5 min.

[0060] In the present invention, the flow rate of the dispersed phase is preferably 0.8 to 1.2 mL / h, more preferably 1.0 to 1.2 mL / h, and the flow rate of the continuous phase is preferably 18 to 108 mL / h, more preferably 30 to 108 mL / h.

[0061] In the present invention, when the contacting method is to attach the oil phase solution to the β-lactoglobulin-lipoic acid solution, it is preferably as follows: placing the β-lactoglobulin-lipoic acid solution in a cylindrical polytetrafluoroethylene mold with a diameter of 1.1 cm, covering the surface of the β-lactoglobulin-lipoic acid solution with the oil phase solution, and obtaining a cylindrical β-lactoglobulin-lipoic acid hydrogel with a bottom diameter of about 1.1 cm after ultraviolet light exposure.

[0062] In the present invention, when the contacting method is to use the oil phase solution as a continuous phase and the β-lactoglobulin-lipoic acid solution as a dispersed phase, and to use a microfluidic method for dispersion, specifically: using the oil phase solution as a continuous phase and the β-lactoglobulin-lipoic acid solution as a dispersed phase, using microfluidic technology to cover the oil phase solution on the surface of β-lactoglobulin-lipoic acid droplets, and obtaining β-lactoglobulin-lipoic acid hydrogel microspheres with a particle size range of 100 to 500 μm after ultraviolet light exposure.

[0063] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0064] The β-lactoglobulin powder used in Examples 1 to 3 is derived from cow's milk and has a purity of 93%.

[0065] Example 1

[0066] Dissolve 3.8 g of lipoic acid powder in 18.4 mL of 1.0 mol / L sodium hydroxide solution, adjust the solution pH to 10.0, and obtain a sodium lipoate solution with a concentration of 1.0 mol / L;

[0067] 6 g of β-lactoglobulin powder was mixed with 69 mL of ultrapure water to obtain a β-lactoglobulin solution with a mass concentration of 8 wt %;

[0068] The β-lactoglobulin solution and the sodium lipoate solution were mixed in a volume ratio of 50:13.33, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added to the obtained mixed solution in sequence, the molar ratio of lipoic acid powder, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide was 1:1:0.5, and the pH was adjusted to 6.0 with 1 mol / L HCl, and the mixture was stirred at 300 rpm in the dark at 25° C. for 24 hours to obtain a modified product;

[0069] The modified product was placed in a dialysis bag with a molecular weight cutoff of 3500Da, and dialyzed with deionized water with a pH value of 7.5 at 4°C for 3 days, with the deionized water being changed 3 times a day, and then the dialysate was freeze-dried in a vacuum at -75°C for 12h to obtain β-lactoglobulin-lipoic acid powder;

[0070] Dissolve 4 g of β-lactoglobulin-lipoic acid powder in 46 mL of ultrapure water to obtain a β-lactoglobulin-lipoic acid solution with a mass concentration of 8 wt %;

[0071] Paraffin oil and Span 80 were mixed to obtain an oil phase solution with a Span 80 mass concentration of 10 wt%. 0.8 mL of β-lactoglobulin-lipoic acid solution was placed in a cylindrical polytetrafluoroethylene mold with a diameter of 1.1 cm. 0.4 mL of the oil phase solution was covered on the surface of the β-lactoglobulin-lipoic acid solution and allowed to stand for 15 to 30 minutes to form an oil-water interface. Then, an intensity of 200 mW / cm 2 , irradiate with ultraviolet light of wavelength 365nm for 3min, and initiate the photo-crosslinking reaction of the β-lactoglobulin-lipoic acid solution after ultraviolet light irradiation to obtain the β-lactoglobulin-lipoic acid hydrogel material of Example 1.

[0072] Example 2

[0073] The only difference from Example 1 is:

[0074] After the β-lactoglobulin-lipoic acid solution is prepared, paraffin oil and Span 80 are mixed to obtain an oil phase solution with a Span 80 mass concentration of 10wt%, the β-lactoglobulin-lipoic acid solution is used as a dispersed phase, the oil phase solution is used as a continuous phase, and microfluidic technology is used to control the flow rate of the β-lactoglobulin-lipoic acid solution to 1.2mL / h and the flow rate of the oil phase solution to 99mL / h in a microfluidic device so that the two meet in a microchannel to form droplets;

[0075] The intensity is 200mW / cm 2The droplets were irradiated with ultraviolet light at a wavelength of 365 nm for 3 minutes to induce a photo-crosslinking reaction of the β-lactoglobulin-lipoic acid solution to obtain β-lactoglobulin-lipoic acid hydrogel microspheres.

[0076] Example 3

[0077] The only difference from Example 1 is:

[0078] After preparing the β-lactoglobulin-lipoic acid solution, paraffin oil and Span 80 are mixed to obtain an oil phase solution with a Span 80 mass concentration of 10 wt %, the β-lactoglobulin-lipoic acid solution is used as a dispersed phase, the oil phase solution is used as a continuous phase, and microfluidic technology is used to control the flow rate of the β-lactoglobulin-lipoic acid solution to 1.2 mL / h and the flow rate of the oil phase solution to 18 mL / h, 30 mL / h, 45 mL / h, 99 mL / h, and 108 mL / h in a microfluidic device, so that the two meet in a microchannel to form droplets;

[0079] The intensity is 200mW / cm 2 The droplets were irradiated with ultraviolet light at a wavelength of 365 nm for 3 minutes to induce a photo-crosslinking reaction of the β-lactoglobulin-lipoic acid solution to obtain β-lactoglobulin-lipoic acid hydrogel microspheres of different sizes.

[0080] Comparative Example 1

[0081] The only difference from Example 1 is:

[0082] After the β-lactoglobulin-lipoic acid solution is prepared, no oil phase solution is added, but it is directly irradiated with light;

[0083] 0.8 mL of β-lactoglobulin-lipoic acid solution was placed in a cylindrical polytetrafluoroethylene mold with a diameter of 1.1 cm and directly heated with an intensity of 200 mW / cm 2 , irradiate with ultraviolet light with a wavelength of 365nm for 3 minutes to carry out cross-linking, and obtain the product of the β-lactoglobulin-lipoic acid solution of Comparative Example 1 after direct illumination.

[0084] Comparative Example 2

[0085] The only difference from Example 1 is:

[0086] After the β-lactoglobulin-lipoic acid solution is prepared, the oil phase solution is not added, but the β-lactoglobulin-lipoic acid solution is treated by adjusting the pH;

[0087] The pH of the β-lactoglobulin-lipoic acid solution was adjusted to 4.8 with 1 mol / L HCl solution. 0.8 mL of the pH-adjusted β-lactoglobulin-lipoic acid solution was placed in a cylindrical polytetrafluoroethylene mold with a diameter of 1.1 cm. The intensity was 200 mW / cm 2 , irradiate with ultraviolet light with a wavelength of 365 nm for 3 minutes for cross-linking to obtain the β-lactoglobulin-lipoic acid hydrogel material of Comparative Example 2.

[0088] Application Example 1

[0089] The only difference from Example 1 is:

[0090] Adding 2 mg / mL of curcumin to the obtained β-lactoglobulin-lipoic acid solution, mixing, and obtaining a β-lactoglobulin-lipoic acid solution containing curcumin;

[0091] paraffin oil and Span 80 are mixed to obtain an oil phase solution having a Span 80 mass concentration of 10 wt %;

[0092] The β-lactoglobulin-lipoic acid solution containing curcumin is used as the dispersed phase and the oil phase solution is used as the continuous phase. The microfluidic technology is used. In the microfluidic device, the flow rates of the β-lactoglobulin-lipoic acid solution containing curcumin and the oil phase solution are controlled to be 1.2 mL / h and 99 mL / h, respectively, so that the two meet in the microchannel to form droplets.

[0093] The intensity is 200mW / cm 2 The droplets were irradiated with ultraviolet light at a wavelength of 365 nm for 3 minutes to induce a photo-crosslinking reaction of the β-lactoglobulin-lipoic acid solution to obtain β-lactoglobulin-lipoic acid hydrogel microspheres loaded with curcumin.

[0094] Characterization and performance determination

[0095] 1. Replace D with deuterium 2 O is the solvent, and the nuclear magnetic resonance hydrogen spectrum of the β-lactoglobulin-lipoic acid powder obtained in Example 1 is analyzed by nuclear magnetic resonance spectrometer. The results are shown in Figure 1 .

[0096] like Figure 1 As shown in the figure, after modification with lipoic acid, the methylene protons (-H 2 C-, a / b / d / e / f / g) signal peaks were significantly enhanced, indicating that lipoic acid had been successfully grafted onto the β-lactoglobulin chain (a: 3.113ppm, b: 2.842ppm, c: 3.696ppm, d: 1.026ppm, e: 1.860ppm, f: 2.611ppm, g: 2.150ppm).

[0097] 2. The changes in the functional groups of the β-lactoglobulin-lipoic acid powder obtained in Example 1 were analyzed by Fourier transform infrared spectroscopy. Figure 2 .

[0098] like Figure 2 As shown, at 3284cm -1 、1652cm -1 、1525cm -1 、1230cm -1 The peaks of amide A, amide I, II and III are shown at . There are significant differences in the intensity of amide I, amide II and amide III bands of β-lactoglobulin after lipoic acid modification, which indicates that the protein structure has changed after lipoic acid modification.

[0099] 3. Figure 3 The physical pictures of the products of Example 1, Comparative Example 1 and Comparative Example 2 are shown in FIG. Figure 3 A is the product obtained by directly irradiating the β-lactoglobulin-lipoic acid solution in Comparative Example 1 without adding the oil phase solution. Figure 3 B is the β-lactoglobulin-lipoic acid hydrogel material obtained in Comparative Example 2, Figure 3 C in the middle is the β-lactoglobulin-lipoic acid hydrogel material obtained in Example 1.

[0100] like Figure 3 As shown, Figure 3 A in the figure indicates that no hydrogel material was prepared. Figure 3 Combined with Comparative Example 1, it can be seen that the β-lactoglobulin-lipoic acid hydrogel material cannot be prepared by directly using the β-lactoglobulin-lipoic acid solution for photocrosslinking without any post-treatment steps;

[0101] Figure 3 The transparency of the hydrogel in Example B is relatively low. By comparing Comparative Example 1 and Comparative Example 2, it can be seen that after the pH of the β-lactoglobulin-lipoic acid solution was adjusted, the β-lactoglobulin-lipoic acid hydrogel material was successfully prepared, indicating that the β-lactoglobulin-lipoic acid solution needs to be subjected to some post-treatment methods to effectively expose the photo-crosslinking sites; however, the pH adjustment process can easily cause changes in protein solubility, which can easily lead to local precipitation of β-lactoglobulin-lipoic acid, so the transparency of the obtained hydrogel material is relatively low;

[0102] Figure 3 The C hydrogel material has good transparency. Figure 3 In combination with Example 1, it can be seen that the tension difference formed at the oil-water interface can mediate the exposure of photo-crosslinking sites of β-lactoglobulin-lipoic acid, which can be quickly cross-linked under light; this method can avoid the problem of solution instability caused by pH adjustment, so the obtained β-lactoglobulin-lipoic acid hydrogel material has good transparency.

[0103] 4. The morphology of the β-lactoglobulin-lipoic acid hydrogel microspheres obtained in Example 3 was characterized using an optical microscope. Figure 4 .

[0104] like Figure 4 As shown, under the condition that the dispersed phase solution flow rate is 1.2 mL / h, by adjusting the continuous phase flow rate (18 mL / h to 108 mL / h), β-lactoglobulin-lipoic acid hydrogel microspheres of different sizes can be prepared, and the particle size range is 100 to 500 μm.

[0105] 5. Stability test

[0106] The β-lactoglobulin-lipoic acid hydrogel microspheres of Example 2 were placed in 1.5 mL of PBS solution, simulated gastric juice and intestinal juice, respectively. The PBS solution was used as the control group. The morphological changes of the β-lactoglobulin-lipoic acid hydrogel microspheres in the simulated gastric juice and intestinal juice were observed under an optical microscope for 0 to 24 h. The results are shown in Figure 5 ; Simulated gastric juice and intestinal juice were prepared according to the following steps:

[0107] Simulated gastric fluid: weigh 0.02 g NaCl and 0.032 g pepsin and dissolve them in 10 mL ultrapure water, and adjust the pH of the solution to 1.2;

[0108] Simulated intestinal fluid: weigh 0.068 g of potassium dihydrogen phosphate and 0.1 g of trypsin and dissolve them in 10 mL of ultrapure water, and adjust the pH of the solution to 6.8.

[0109] like Figure 5 As shown in the figure, in PBS solution, β-lactoglobulin-lipoic acid hydrogel microspheres have good stability and can maintain their complete shape; in gastric simulated fluid, β-lactoglobulin-lipoic acid hydrogel microspheres have better stability, not only can they maintain their complete shape, but also have a certain densification trend, and can better resist the degradation of gastric simulated fluid; in intestinal simulated fluid, β-lactoglobulin-lipoic acid hydrogel microspheres are almost completely degraded within 4 hours, and then completely disappear, leaving only microsphere fragments.

[0110] Depend on Figure 5 It can be seen that β-lactoglobulin-lipoic acid hydrogel microspheres have good stability in gastric juice and can be effectively degraded in intestinal juice, and have the potential to be used as an oral drug delivery carrier.

[0111] 6. The curcumin-loaded β-lactoglobulin-lipoic acid hydrogel microspheres obtained in Example 1 were characterized using optical microscopy and fluorescence microscopy. The results are shown in Figure 6 ,in Figure 6 A in the figure is an optical microscope image. Figure 6 B is a fluorescence microscopy image.

[0112] like Figure 6 As shown, Figure 6 The optical microscope image in Figure A clearly shows the morphology of the microspheres. Figure 6 In the fluorescence microscope image (B), an obvious fluorescence signal can be observed. This signal originates from the intrinsic fluorescence properties of curcumin, and the fluorescence signal coincides with the morphology of the microspheres, proving that curcumin has been successfully loaded inside the β-lactoglobulin-lipoic acid hydrogel microspheres, rather than just adsorbed on the surface.

[0113] Depend on Figure 6 It can be seen that the β-lactoglobulin-lipoic acid hydrogel microspheres prepared by the present invention have good curcumin loading capacity, which lays a foundation for subsequent drug delivery applications.

[0114] It can be seen from the above embodiments and Application Example 1 that the present invention provides a β-lactoglobulin-lipoic acid hydrogel material and a preparation method and application thereof, which utilizes oil-water interface drive to effectively solve the problem that photocrosslinking sites are difficult to expose after lipoic acid modifies β-lactoglobulin, thereby improving the crosslinking efficiency and making the preparation of hydrogel microspheres more efficient; the obtained β-lactoglobulin-lipoic acid hydrogel microspheres have good stability and can be used to prepare oral drug delivery carriers.

[0115] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a β-lactoglobulin-lipoic acid hydrogel material, characterized in that: The following steps are involved: dissolving lipoic acid powder in alkaline solution to obtain lipoic acid salt solution; The lipoic acid salt solution, the β-lactoglobulin solution and the carboxyl activator are mixed, the pH of the solution is adjusted to 5.5-6.0, and a chemical modification reaction is performed to obtain a modified product; The modified product is dialyzed and dried in sequence to obtain β-lactoglobulin-lipoic acid powder; mixing the β-lactoglobulin-lipoic acid powder with water to obtain a β-lactoglobulin-lipoic acid solution; The β-lactoglobulin-lipoic acid solution is contacted with an oil phase solution, and after ultraviolet light exposure, a β-lactoglobulin-lipoic acid hydrogel material is obtained; The contacting method is: attaching the oil phase solution to the β-lactoglobulin-lipoic acid solution; or using the oil phase solution as a continuous phase and the β-lactoglobulin-lipoic acid solution as a dispersed phase, and dispersing by a microfluidic method.

2. The preparation method according to claim 1, characterized in that: The alkali solution includes sodium hydroxide solution or potassium hydroxide solution, and the concentration of the alkali solution is 1.0-1.5 mol / L; the mass volume ratio of the thioctic acid powder to the alkali solution is 1.38-5.5 g:6.67-53.32 mL; the pH value of the thioctic acid salt solution is 9.0-11.0, and the molar concentration is 1.0-1.5 mol / L.

3. The preparation method according to claim 1, characterized in that: The beta-lactoglobulin solution is obtained by mixing beta-lactoglobulin powder with water. The concentration of the beta-lactoglobulin solution is 4-8wt%. The volume ratio of the beta-lactoglobulin solution to the lipoic acid salt solution is 50-100:10-30.

4. The preparation method according to claim 1, characterized in that: The carboxyl activator comprises 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide; the molar ratio of the lipoic acid powder, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide is 1:1:0.2-1; the temperature of the chemical modification reaction is 25-30°C, and the time is 16-24h.

5. The preparation method according to claim 1, characterized in that: The molecular weight cutoff of the dialysis bag used for the dialysis is 1000-3500Da, the dialysis temperature is 2-6°C, and the time is 2-4d; the drying method is vacuum freeze drying, the drying temperature is -80--60°C, and the time is 8-16h.

6. The preparation method according to claim 1, characterized in that: The mass concentration of the β-lactoglobulin-lipoic acid solution is 4-12wt%.

7. The preparation method according to claim 1, characterized in that: The oil phase solution comprises paraffin oil and Span 80, wherein the concentration of Span 80 in the oil phase solution is 10-20 wt %; the volume ratio of the oil phase solution to the β-lactoglobulin-lipoic acid solution is 0.5-1:1; the ultraviolet irradiation conditions are: the illumination intensity is 100-300 mW / cm 2 , wavelength is 365nm, time is 1 to 5min.

8. The preparation method according to claim 7, characterized in that: The flow rate of the dispersed phase is 0.8 to 1.2 mL / h, and the flow rate of the continuous phase is 18 to 108 mL / h.

9. The β-lactoglobulin-lipoic acid hydrogel material prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the β-lactoglobulin-lipoic acid hydrogel material according to claim 9 in preparing an oral drug delivery carrier.

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