A β-lactoglobulin-lipoic acid hydrogel material and its preparation method and application

Abstract

The present invention provides a β-lactoglobulin lipoic acid hydrogel material and its preparation method and application, belonging to the field of molecular biomedical technology. Lipoic acid powder is dissolved in alkali solution, the obtained lipoic acid salt solution, β-lactoglobulin solution and carboxyl activator are mixed, pH is adjusted to carry out chemical modification reaction, and a modified product is obtained; after the modified product is dialyzed and dried in sequence, β-lactoglobulin lipoic acid powder is obtained; β-lactoglobulin lipoic acid powder is mixed with water to obtain β-lactoglobulin lipoic acid solution; β-lactoglobulin lipoic acid solution is contacted with an oil phase solution, and β-lactoglobulin lipoic acid hydrogel material is obtained after ultraviolet light irradiation. The present invention converts lipoic acid into water-soluble lipoic acid salt, increases the solubility of lipoic acid in the aqueous phase, thereby improving the efficiency of the reaction with β-lactoglobulin; adopts oil-water interface mediation, effectively exposes the photocrosslinking site, and further improves the photocrosslinking efficiency.

Description

Technical Field

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

[0002] Proteins, due to their natural biological properties and multifunctionality, have become highly sought-after carrier materials in drug delivery systems. β-lactoglobulin (BLG) is a major component of whey protein in milk, accounting for approximately 50% to 60% of the total whey protein content. BLG, a member of the lipocalin family of proteins, possesses a unique combination of hydrophilic and hydrophobic surfaces and multiple easily modifiable amino acid residues. Compared to most protein-based drug delivery carriers, BLG offers advantages such as widespread availability, low cost, relatively simple extraction and purification processes, and the ability to achieve scalable production.

[0003] Previous studies have shown that BLG can bind and load drugs, protecting active drug ingredients from degradation in gastric fluid during oral administration, thereby improving drug bioavailability and reducing the occurrence of side effects. Current research has focused on using BLG monomers to bind and transport small-molecule drugs. These studies have shown that BLG has appropriate binding and excellent transport capacity for small-molecule drugs. However, due to its small molecular weight (only 18.3 kDa), the BLG monomer has relatively weak binding capacity for macromolecular drugs such as proteins and nucleic acids, as well as some special drugs. This makes BLG less efficient in loading macromolecular drugs, making it difficult to meet 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 control of drug release rate and release time by changing the chemical composition, network density or cross-linking method. At present, the preparation methods of hydrogel / hydrogel microspheres using BLG as raw material include physical cross-linking, chemical cross-linking, thermally induced gelation, etc. These methods generally have problems such as long cycle time, complex process and poor controllability.

[0005] Photocrosslinking is a process in which photoinitiators / photoactive groups promote crosslinking polymerization between molecular chains under light. It has the advantages of fast speed, simple operation, spatiotemporal control, and good versatility, and has important application value in the construction of drug delivery carriers. Currently, existing BLG photocrosslinking 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 photocross-linked hydrogel material to overcome the problems caused by the introduction of current 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 photocrosslinked 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 alkali 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] contacting the β-lactoglobulin-lipoic acid solution with an oil phase solution, and irradiating the solution with ultraviolet light to obtain a β-lactoglobulin-lipoic acid hydrogel material;

[0018] The contacting method is: attaching the oil phase solution to the β-lactoglobulin-lipoic acid solution; or using the oil phase solution as the continuous phase and the β-lactoglobulin-lipoic acid solution as the 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 4-8 wt %; the volume ratio of the β-lactoglobulin solution to the lipoic acid salt solution is 50-100:10-30.

[0021] Preferably, the carboxyl activator includes 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 16-24 hours.

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

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

[0024] Preferably, the oil phase solution comprises paraffin oil and Span 80, 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 5 minutes.

[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 present invention provides a beta-lactoglobulin-lipoic acid hydrogel material prepared by the above 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 present invention introduces lipoic acid, which contains a disulfide bond five-membered ring structure for dynamic ring-opening polymerization in the lipoic acid molecule, thereby imparting the photocrosslinking property to beta-lactoglobulin. The disulfide bond serves as both a light absorption unit and a dynamic covalent crosslinking unit, and can be broken and reassembled under ultraviolet light irradiation, thereby realizing a crosslinking reaction. The formation of a photocrosslinked polymer can be realized without introducing a photoinitiator, thereby providing the possibility of constructing a light-responsive hydrogel with beta-lactoglobulin. By converting lipoic acid into a water-soluble lipoic acid salt solution, the solubility of lipoic acid in the aqueous phase is increased, thereby improving the efficiency of its reaction with beta-lactoglobulin. At the same time, the introduction of lipoic acid avoids the safety hazards of using traditional photoinitiators, reduces potential hazards 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 undergo ring-opening polymerization 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 is widely available and low in 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, inexpensive, efficient and sustainable, and the intermediate products in each reaction stage are easy to control. The method has the advantages of short gelation time, low energy consumption in the gelation process, stable product performance, and easy large-scale preparation. 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 This 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 in the middle is the β-lactoglobulin-lipoic acid hydrogel material obtained in Example 1;

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

[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: 300 μm);

[0037] Figure 6 These are optical microscope images and fluorescence microscope images of curcumin-loaded β-lactoglobulin-lipoic acid hydrogel microspheres obtained in Application Example 1. 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 alkali 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] contacting the β-lactoglobulin-lipoic acid solution with an oil phase solution, and irradiating the solution with ultraviolet light to obtain a β-lactoglobulin-lipoic acid hydrogel material;

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

[0045] In the present invention, unless otherwise specified, the raw materials required 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 sodium hydroxide solution or potassium hydroxide solution, and more preferably includes 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. 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, adjusts the pH of the solution to 5.5-6.0 using a 1 mol / L HCl solution, 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 its 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 undergo an amide reaction with β-lactoglobulin, 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, 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 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; brings the β-lactoglobulin-lipoic acid solution into contact with the oil phase solution to form an oil-water interface; and obtains the β-lactoglobulin-lipoic acid hydrogel material after ultraviolet irradiation. The contacting method comprises: adhering 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 adopting a microfluidic method for dispersion.

[0057] In the present invention, the concentration of Span 80 in the oil phase solution is preferably 10 to 20 wt %, 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, 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 particularly preferred that the β-lactoglobulin-lipoic acid solution is placed in a cylindrical polytetrafluoroethylene mold with a diameter of 1.1 cm, the oil phase solution is covered on the surface of the β-lactoglobulin-lipoic acid solution, and after ultraviolet light irradiation, a cylindrical β-lactoglobulin-lipoic acid hydrogel with a bottom diameter of about 1.1 cm is obtained.

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

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

[0064] The β-lactoglobulin powder used in Examples 1 to 3 was derived from cow's milk and had 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 and adjust the pH of the solution to 10.0 to obtain a 1.0 mol / L sodium lipoate solution;

[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 resulting mixed solution in sequence, wherein 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 using 1 mol / L HCl. The mixture was stirred at 300 rpm in the dark at 25° C. for 24 h to obtain a modified product.

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

[0070] 4 g of β-lactoglobulin-lipoic acid powder was dissolved 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 optical microscope with an intensity of 200 mW / cm 2 , irradiated with ultraviolet light of 365 nm for 3 min, and the photocrosslinking reaction of the β-lactoglobulin-lipoic acid solution was initiated after ultraviolet 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 preparing a 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, and the oil phase solution is used as a continuous phase. 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 99 mL / 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 photocrosslinking 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 a 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 and the oil phase solution is used as a continuous phase. Microfluidic technology is used to control the flow rates of the β-lactoglobulin-lipoic acid solution to 1.2 mL / h and 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 photocrosslinking 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 the solution 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 365 nm for 3 minutes for cross-linking to 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, no oil phase solution is 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 solution was heated at an intensity of 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] 2 mg / mL of curcumin was added to the obtained β-lactoglobulin-lipoic acid solution, and the mixture was mixed to obtain a β-lactoglobulin-lipoic acid solution containing curcumin;

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

[0092] A β-lactoglobulin-lipoic acid solution containing curcumin was used as the dispersed phase, and an oil phase solution was used as the continuous phase. Microfluidic technology was used to control the flow rates of the β-lactoglobulin-lipoic acid solution containing curcumin and the oil phase solution at 99 mL / h in a microfluidic device, so that the two phases met in a 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 photocrosslinking reaction of the β-lactoglobulin-lipoic acid solution to obtain curcumin-loaded β-lactoglobulin-lipoic acid hydrogel microspheres.

[0094] Characterization and performance determination

[0095] 1. Using deuterated D2O as solvent, the nuclear magnetic resonance (NMR) spectrum of the β-lactoglobulin-lipoic acid powder obtained in Example 1 was analyzed by nuclear magnetic resonance spectrometer. The results are shown in FIG. Figure 1 .

[0096] like Figure 1 As shown in the figure, after modification with lipoic acid, the signal peaks of each methylene proton (-H2C-, a / b / d / e / f / g) of the β-lactoglobulin-lipoic acid powder 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 of functional groups of 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 in Figure 3. The intensities of amide I, amide II and amide III bands of β-lactoglobulin modified with lipoic acid are significantly different, indicating that the protein structure has changed after modification with lipoic acid.

[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-processing steps;

[0101] Figure 3 The transparency of the hydrogel in Example B is low. Comparison of Comparative Example 1 and Comparative Example 2 shows that after the pH of the β-lactoglobulin-lipoic acid solution is adjusted, the β-lactoglobulin-lipoic acid hydrogel material is successfully prepared, indicating that the β-lactoglobulin-lipoic acid solution needs to undergo some post-treatment to effectively expose the photocrosslinking 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 resulting hydrogel material is 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 photocrosslinking sites of β-lactoglobulin-lipoic acid, which can be rapidly crosslinked under light; this method can avoid the problem of solution instability caused by pH adjustment, and thus 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 in the figure, under the condition of a dispersed phase solution flow rate of 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, with a particle size range of 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 using an optical microscope for 0 to 24 hours. The results are shown in FIG. 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. Adjust the pH of the solution to 1.2.

[0108] Simulated intestinal fluid: Weigh 0.068 g of dipotassium hydrogen phosphate and 0.1 g of trypsin and dissolve them in 10 mL of ultrapure water. 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 Table 6. Figure 6 ,in Figure 6 A in the middle 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 middle 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 in the present invention have good curcumin loading capacity, which lays a foundation for subsequent drug delivery applications.

[0114] As can be seen from the above embodiments and Application Example 1, the present invention provides a β-lactoglobulin-lipoic acid hydrogel material, a preparation method, and an application thereof. By utilizing oil-water interface drive, the problem of difficulty in exposing photocrosslinking sites after lipoic acid modification of β-lactoglobulin is effectively solved, the crosslinking efficiency is improved, and the preparation of hydrogel microspheres is 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 principles of the present invention. These improvements and modifications should also be regarded as within 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 alkali 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; contacting the β-lactoglobulin-lipoic acid solution with an oil phase solution, and irradiating the solution with ultraviolet light to obtain a β-lactoglobulin-lipoic acid hydrogel material; The carboxyl activator includes 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 16-24 hours; The oil phase solution includes paraffin oil and Span80, and the concentration of Span80 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 contacting method is: attaching the oil phase solution to the β-lactoglobulin-lipoic acid solution; or using the oil phase solution as the continuous phase and the β-lactoglobulin-lipoic acid solution as the 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 4-8 wt %. 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 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 2-4 days; the drying method is vacuum freeze drying, the drying temperature is -80--60°C, and the time is 8-16 hours.

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

6. The preparation method according to claim 1, characterized in that The conditions of the ultraviolet light are: the light intensity is 100-300mW / cm 2 , wavelength is 365nm, time is 1 to 5 minutes.

7. The preparation method according to claim 1, 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.

8. The β-lactoglobulin-lipoic acid hydrogel material prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the β-lactoglobulin-lipoic acid hydrogel material according to claim 8 in preparing an oral drug delivery carrier.

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