Preparation method and application of bioactive substance Pickering emulsion temperature-controlled release hydrogel

Abstract

The invention relates to a preparation method and application of a temperature-controlled release hydrogel of a bioactive substance Pickering emulsion, and belongs to the field of high polymer materials. The preparation method comprises the following steps: by taking modified starch obtained by modifying starch as an emulsifier, uniformly mixing the emulsifier with an oil phase containing a bioactive substance to obtain a Pickering emulsion of the bioactive substance embedded in the modified starch, and carrying out free radical polymerization on the Pickering emulsion and an N-isopropylacrylamide monomer under the action of an initiator, a cross-linking agent and a catalyst to prepare the hydrogel. According to the preparation method, a free radical polymerization method is adopted, the modified starch in the emulsion and N-isopropylacrylamide hydrogel are polymerized, three-dimensional cross-linked network hydrogel is formed, and the mechanical property of the hydrogel is enhanced. Meanwhile, the hydrogel has unique temperature response performance, phase change is generated when the temperature exceeds 32 DEG C, macromolecules are curled and shrunk, extrusion deformation occurs in hydrogel molecules, and demulsification release of the Pickering emulsion is controlled.

Description

Technical Field

[0001] The present invention relates to a preparation method and application of a thermo-responsive release hydrogel of a bioactive substance Pickering emulsion, belonging to the field of polymer materials. Background Art

[0002] Natural bioactive substances are widely distributed in various animals, plants, marine organisms and microorganisms, and have physiological activities such as anti-inflammatory, anti-cancer and antioxidant properties. Due to their unique physiological activities and functions, they have been widely studied and applied in the fields of food and medical production. Bioactive substances are highly recognized for their benefits to human health. Many studies have elucidated their antioxidant, anti-inflammatory and antibacterial activities, which are beneficial to wound healing. However, poor water solubility, chemical instability, low bioavailability and delivery efficiency limited by a rapid metabolic rate severely limit their application in wound healing.

[0003] Pickering emulsions have good stability. The stabilization of conventional emulsions is mainly achieved by electrostatic stabilization, reducing the interfacial tension and combining with steric stabilization by surfactants or soluble macromolecules. Pickering emulsions, on the other hand, form a physical barrier through particles adsorbed at the oil-water interface, which can block interfacial interactions and droplet contact by volume. Utilizing the excellent stability and good safety of Pickering emulsions, preparing oil-soluble curcumin into an O / W Pickering emulsion is an effective method to protect the activity of curcumin.

[0004] Starch is a rich and inexpensive material for preparing Pickering stabilizers. However, due to the strong hydrophilicity of starch granules, hydrophobic modification is required. Octenyl succinic anhydride (OSA)-modified starch (OS-starch) is the only hydrophobic starch legally allowed to be used as a food emulsifier. For better application in emulsification, starch modification adopts the method of lipase nanoflower-catalyzed modification. Compared with chemical modification, enzyme-catalyzed modification has higher efficiency and degree of substitution. Due to the unique spatial structure of lipase nanoflowers, when catalyzing the esterification of starch molecules, steric hindrance is generated, resulting in the modification of starch molecules into amphiphilic starch with one end grafted and the other end ungrafted, which can better maintain stability at the oil-water interface to maintain the stability of Pickering emulsions.

[0005] The controlled release of Pickering emulsions on wounds is another problem that urgently needs to be solved. Nowadays, Pickering emulsions are widely used in the food industry, and the demulsification effect is achieved through gastric juice digestion. Due to its extremely high stability, in vitro release usually adopts drastic means such as high heat and centrifugation, which is not suitable for application on wounds. Hydrogels that shrink based on temperature response are ideal matrices that can be further used as drug delivery methods. Utilizing the property that the intermolecular contraction and extrusion of hydrogels are affected by temperature, the controlled release of Pickering emulsions can be achieved. Summary of the Invention

[0006] The purpose of the present invention is to provide a preparation method and application of a temperature-controlled release hydrogel of a bioactive substance Pickering emulsion. With the unique spatial structure of lipase nanoflowers, starch molecules are catalytically modified into amphiphilic starches with one end grafted and the other end ungrafted, which can better maintain stability at the oil-water interface to maintain the stability of Pickering emulsions; by using the free radical polymerization method, a bioactive substance Pickering emulsion with modified starch as an emulsifier and a poly(N-isopropylacrylamide) hydrogel are polymerized to form a three-dimensional cross-linked network hydrogel. The present invention utilizes the property that the intermolecular contraction and extrusion of hydrogels are affected by temperature to achieve the demulsification of Pickering emulsions and the site-specific controlled release of bioactive substances, endowing the hydrogel dressing with temperature-controlled release on wounds.

[0007] In order to achieve the above purpose, the technical solution of the present invention is as follows:

[0008] In the first aspect of the present invention, a temperature-controlled release hydrogel of a bioactive substance Pickering emulsion is provided. A Pickering emulsion in which a modified starch obtained by modifying starch is used as an emulsifier and an oil phase containing a bioactive substance are mixed and homogenized to obtain a Pickering emulsion in which the modified starch encapsulates the bioactive substance. The Pickering emulsion in which the modified starch encapsulates the bioactive substance and N-isopropylacrylamide monomers are subjected to free radical polymerization under the action of an initiator, a cross-linking agent, and a catalyst to be prepared.

[0009] In the above technical solution, the initiator is ammonium persulfate; the cross-linking agent is N,N'-methylenebisacrylamide; the catalyst is tetramethylethylenediamine.

[0010] In the above technical solution, the modified starch is prepared by using lipase nanoflowers as an esterification reaction catalyst; the starch is glutinous rice flour.

[0011] In the above technical solution, the bioactive substance includes one of curcumin, gallic acid, flavonol, tannic acid, lutein, anthocyanin, and astaxanthin.

[0012] In a second aspect of the present invention, there is provided a method for preparing the above-mentioned Pickering emulsion thermo-responsive hydrogel of bioactive substances, comprising the following steps:

[0013] (1) Add starch to a mixed solution of water and DMSO, heat and stir to obtain a gelatinized starch solution;

[0014] (2) Add 2-octenyl succinic anhydride to the gelatinized starch solution obtained in step (1), add lipase nanoflowers, and carry out an esterification reaction. During the addition process, the pH value of the solution system is maintained at 7.5 - 8.5. After the addition, continue stirring to obtain a paste-like product;

[0015] (3) Centrifuge the paste-like product obtained in step (2) to remove the supernatant, and wash the precipitate with absolute ethanol to obtain modified starch;

[0016] (4) Use the modified starch obtained in step (3) as an emulsifier, dissolve it in water as the aqueous phase, dissolve the bioactive substance in olive oil as the oil phase, and homogenize through a homogenizer to obtain a Pickering emulsion in which the bioactive substance is encapsulated by the modified starch;

[0017] (5) Dissolve the Pickering emulsion obtained in step (4), N-isopropylacrylamide, a cross-linking agent, and an initiator in water to obtain a mixed solution. Remove the oxygen in the solution, add a catalyst and carry out a reaction to obtain the above-mentioned Pickering emulsion thermo-responsive hydrogel of bioactive substances.

[0018] In the above technical solution, in step (1), the volume ratio of water to DMSO is 1:1 - 1:2, preferably 1:1; the heating temperature is 90 - 110°C, preferably 100°C; the mass ratio of starch to the mixed solution is 1:5 - 15, preferably 1:10.

[0019] In the above technical solution, in step (2), the method for preparing the lipase nanoflowers comprises the following steps:

[0020] 1) Dissolve potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, and potassium chloride in deionized water to obtain a phosphate buffer solution; the pH of the phosphate buffer solution is 7.4;

[0021] 2) Add lipase and a copper chloride solution to the phosphate buffer solution obtained in step 1) for cultivation to obtain a suspension; in the suspension, the concentration of lipase is 0.25 - 1.0 mg / ml, preferably 0.25 mg / ml, and the concentration of copper chloride is 100 - 150 mM, preferably 110 mM;

[0022] 3) Centrifuge the suspension obtained in step 2), collect the precipitate, and after washing, centrifuging, and freeze-drying, obtain the lipase nanoflowers.

[0023] In the above technical solution, in step (2), the mass ratio of the lipase nanoflowers to the starch is 1:7 to 3:7, preferably 1.5:7.

[0024] In the above technical solution, in step (2), after completely dissolving 2-octenyl succinic anhydride in absolute ethanol, add it to the gelatinized starch solution. 2-Octenyl succinic anhydride is grafted onto the starch through an esterification reaction to obtain modified starch; the temperature of the esterification reaction is 30 - 40 °C, preferably 35 °C.

[0025] In the above technical solution, in step (4), in the oil phase, the concentration of the bioactive substance is 0.5 - 1 mg / mL; in the aqueous phase, the concentration of the modified starch is 5 wt% - 10 wt%; the volume ratio of the oil phase to the aqueous phase is 1:2 to 1:1, preferably 1:1.

[0026] In the above technical solution, in step (5), the volume ratio of the Pickering emulsion to water is 1:1 to 9, preferably 1:1; in the mixed solution, the concentration of N-isopropylacrylamide is 0.16 g / mL, the concentration of the cross-linking agent is 0.2 - 0.8 mg / mL, preferably 0.4 mg / mL, and the concentration of the initiator is 2 - 8 mg / mL, preferably 4 mg / mL; the reaction temperature is 4 - 10 °C, preferably 4 °C.

[0027] In the third aspect of the present invention, there is provided the application of the above bioactive substance Pickering emulsion thermo-responsive hydrogel in medical dressings. That is, the bioactive substance Pickering emulsion - poly(N-isopropylacrylamide) thermo-responsive hydrogel with modified starch as the emulsifier of the present invention has good temperature responsiveness and can be applied to wound protection, anti-inflammatory and antibacterial, and accelerated healing devices. Moreover, the bioactive substance Pickering emulsion - poly(N-isopropylacrylamide) thermo-responsive hydrogel with modified starch as the emulsifier of the present invention has good antioxidant and antibacterial properties and has wide application value in wound dressings.

[0028] In the present invention, starch is dissolved in a mixed solution of water and DMSO in a specific ratio and heated to 100 °C to completely gelatinize the starch. After cooling, under the condition of adding lipase nanoflowers as a catalyst, a solution of 2-octenyl succinic anhydride is gradually added dropwise to the gelatinized starch. During the whole process, the pH of the reaction system is always maintained at 7.5 - 8.5. After the addition is completed, the reaction is carried out for 2 - 5 h under stirring to prepare the modified starch. Due to the unique steric hindrance caused by the special petal structure of lipase nanoflowers, it can only catalyze the starch part in contact with the active center, thereby realizing the directional catalysis of starch and preparing amphiphilic starch.

[0029] In the present invention, the starch modified by lipase nanoflowers is dissolved in deionized water at an appropriate concentration (5 wt% - 10 wt%) as the aqueous phase, and a bioactive substance at an appropriate concentration (0.5 mg / mL - 1 mg / mL) is dissolved in olive oil as the oil phase; the oil phase and the aqueous phase are mixed in a specific ratio and homogenized by a homogenizer to prepare a Pickering emulsion. Using the amphiphilic modified starch modified by lipase nanoflowers as an emulsifier, the prepared Pickering emulsion has stronger stability and the ability to protect bioactive substances.

[0030] In the present invention, a specific amount of Pickering emulsion and N-isopropylacrylamide are subjected to free radical polymerization at an appropriate temperature (the above step (5)) under the action of an initiator ammonium persulfate and a catalyst tetramethylethylenediamine. In this way, the polymer formed by the modified starch and N-isopropylacrylamide can be more stable through covalent bond connection compared with a simple copolymerization method. In the hydrogel system, the carboxyl groups (-COOH) of N-isopropylacrylamide and the carboxyl groups of 2-octenyl succinic anhydride can form a large number of hydrogen bonds with the hydroxyl groups (-OH) on the molecular chain of the modified starch. These hydrogen bonds form a three-dimensional network for the hydrogel, thereby endowing the hydrogel with good mechanical properties. At the same time, due to the characteristic that N-isopropylacrylamide undergoes phase transition and curls and shrinks above 32 °C, the hydrogel can undergo phase transition and shrinkage at a higher temperature, and the pressure generated between molecules causes the Pickering emulsion to demulsify, and the bioactive substance is released from the hydrogel. The hydrogel prepared in the present invention has good antibacterial properties and biocompatibility, providing a safe condition for the hydrogel to be used as a medical dressing on the wound surface. At the same time, the hydrogel shows good wound healing effects in the rat wound experiment, demonstrating the application potential of the hydrogel in medical dressings.

[0031] Compared with the prior art through the above technical solutions of the present invention, the following beneficial effects can be achieved:

[0032] (1) By using lipase nanoflowers for directional catalysis, the prepared modified starch has amphiphilicity, and the prepared Pickering emulsion has higher stability and the ability to protect bioactivity.

[0033] (2) By using the free radical polymerization method, the Pickering emulsion and N-isopropylacrylamide monomer are polymerized. Under the action of ammonium persulfate as the initiator and tetramethylethylenediamine as the catalyst, a hydrogel material is prepared. As a hydrogel medical dressing, it can adhere to the skin surface by itself.

[0034] (3) Due to the introduction of the modified starch Pickering emulsion, the tensile property of the hydrogel of the present invention is greatly improved. Its maximum tensile strain rate can reach 137%, which is 4.9 times that of the poly-N-isopropylacrylamide hydrogel.

[0035] (4) The hydrogel of the present invention has excellent adhesion performance and can adhere to the surfaces of various materials.

[0036] (5) The hydrogel of the present invention has unique temperature-responsive performance. A phase change occurs at about 32 °C, the hydrogel is extruded and shrunk, causing the Pickering emulsion to demulsify and releasing the active substance.

[0037] (6) The hydrogel of the present invention has excellent antibacterial ability and biocompatibility, and shows good healing ability in the application of rat wounds.

[0038] (7) The preparation method of the hydrogel of the present invention is simple, time-consuming is short, and the raw material cost is low, and it has a development prospect of large-scale application in industrial production. Description of the Drawings

[0039] Figure 1 Infrared absorption spectra of starch obtained in Comparative Example 1, chemically modified starch obtained in Comparative Example 2, and lipase nanoflower-modified starch obtained in Example 2;

[0040] Figure 2 Scanning electron microscope pictures of starch obtained in Comparative Example 1, chemically modified starch obtained in Comparative Example 2, and lipase nanoflower-modified starch obtained in Example 2. a is Comparative Example 1, b is Comparative Example 2, and c is Example 2;

[0041] Figure 3 Storage stability test results of Pickering emulsions encapsulating curcumin with starch obtained in Comparative Example 1, Pickering emulsions encapsulating curcumin with chemically modified starch obtained in Comparative Example 2, and Pickering emulsions encapsulating curcumin with lipase nanoflower-modified starch obtained in Example 2;

[0042] Figure 4 Mechanical property results of the hydrogels obtained in Examples 12 - 14;

[0043] Figure 5 Release amount and antioxidant activity results of the hydrogels obtained in Examples 12 - 14. Detailed implementation manners

[0044] To make the technical solutions of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0045] In the following embodiments, unless otherwise specified, the experimental methods used are all conventional methods, and the reagents used can be purchased from chemical or biological reagent companies.

[0046] Reagents used in the following embodiments:

[0047] Lipase: Shanghai Yuanye Bio-Technology Co., Ltd.

[0048] Copper chloride dihydrate: Guangdong Guanghua Sci-Tech Co., Ltd.

[0049] N-Isopropylacrylamide: Anhui Zesheng Technology Co., Ltd.

[0050] N,N′-Methylenebisacrylamide: Shanghai Aladdin Biochemical Technology Co., Ltd.

[0051] Tetramethylethylenediamine: Shanghai Aladdin Biochemical Technology Co., Ltd.

[0052] Ammonium persulfate: Shanghai Aladdin Biochemical Technology Co., Ltd.

[0053] Curcumin: Shanghai Aladdin Biochemical Technology Co., Ltd.

[0054] Gallic acid: Shanghai Yuanye Bio-Technology Co., Ltd.

[0055] Flavonol: Shanghai Aladdin Biochemical Technology Co., Ltd.

[0056] Tannic acid: Shanghai Macklin Biochemical Co., Ltd.

[0057] Lutein: Anhui Zesheng Sci-Tech Co., Ltd.

[0058] Anthocyanin: Shanghai Macklin Biochemical Co., Ltd.

[0059] Astaxanthin: Anhui Zesheng Sci-Tech Co., Ltd.

[0060] Water-milled glutinous rice flour: Xinxiang Liangrun Whole Grain Food Co., Ltd.

[0061] Olive oil: Sinopharm Chemical Reagent Co., Ltd.

[0062] Comparative Example 1

[0063] (1) Weigh 7 g of glutinous rice flour and dissolve it in a mixed solution of 35 mL of water and 35 mL of DMSO, and heat and stir in an oil bath at 100 °C for 30 min to obtain a gelatinized starch solution;

[0064] (2) Centrifuge the gelatinized starch solution at 6000 r / min, remove the supernatant, and repeatedly rinse the lower solid precipitate with absolute ethanol. After drying, gelatinized starch is obtained.

[0065] (3) Dissolve 20 g of gelatinized starch in 200 mL of deionized water as the aqueous phase, and dissolve 200 mg of curcumin in 200 mL of olive oil as the oil phase. Mix the aqueous phase and the oil phase, and homogenize at 20000 rpm for 5 min to prepare a Pickering emulsion of starch-embedded curcumin.

[0066] Comparative Example 2

[0067] (1) Weigh 7 g of glutinous rice flour and dissolve it in a mixed solution of 35 mL of water and 35 mL of DMSO. Heat and stir in an oil bath at 100 °C for 30 min to obtain a gelatinized starch solution.

[0068] (2) Completely dissolve 2.1 g of 2-octenyl succinic anhydride in 20 mL of absolute ethanol.

[0069] (3) After the temperature of the oil bath drops to 35 °C, gradually add the solution in step (2) dropwise to the solution in step (1). During the addition process, use 3 wt% NaOH solution to adjust the pH value to keep the pH value of the solution between 7.5 and 8.5 all the time. After all the addition, continue mechanical stirring at 35 °C for 5 h.

[0070] (4) Centrifuge the product prepared in step (3) at 6000 r / min, remove the supernatant, and repeatedly rinse the lower solid precipitate with absolute ethanol. After drying, chemically modified starch is obtained.

[0071] (5) Dissolve 20 g of chemically modified starch in 200 mL of deionized water as the aqueous phase, and dissolve 200 mg of curcumin in 200 mL of olive oil as the oil phase. Mix the aqueous phase and the oil phase, and homogenize at 20000 rpm for 5 min to prepare a Pickering emulsion of chemically modified starch-embedded curcumin.

[0072] Example 1

[0073] (1) Weigh 7 g of glutinous rice flour and dissolve it in a mixed solution of 35 mL of water and 35 mL of DMSO. Heat and stir in an oil bath at 100 °C for 30 min to obtain a gelatinized starch solution.

[0074] (2) Completely dissolve 2.1 g of 2-octenyl succinic anhydride in 20 mL of absolute ethanol.

[0075] (3) After the temperature of the oil bath pot drops to 35 °C, add 1 g of lipase nanoflowers, and gradually add the solution in step (2) dropwise into the solution in step (1). During the addition process, use 3 wt% NaOH solution to adjust the pH value to keep the pH value of the solution at 7.5 - 8.5 all the time. After all the addition, continue mechanical stirring at 35 °C for 5 h;

[0076] (4) Centrifuge the product prepared in step (3) at 6000 r / min to remove the supernatant. The lower solid precipitate is rinsed with absolute ethanol multiple times and dried to obtain lipase nanoflower-modified starch;

[0077] (5) Use 20 g of lipase nanoflower-modified starch as an emulsifier, dissolve it in 200 mL of deionized water as the aqueous phase, dissolve 200 mg of curcumin in 200 mL of olive oil as the oil phase, mix the aqueous phase and the oil phase, and homogenize at 20000 rpm for 5 min to prepare a Pickering emulsion with curcumin encapsulated by lipase nanoflower-modified starch.

[0078] Example 2

[0079] (1) Weigh 7 g of glutinous rice flour and dissolve it in a mixed solution of 35 mL of water and 35 mL of DMSO. Heat and stir in an oil bath pot at 100 °C for 30 min to obtain a gelatinized starch solution;

[0080] (2) Completely dissolve 2.1 g of 2-octenyl succinic anhydride in 20 mL of absolute ethanol;

[0081] (3) After the temperature of the oil bath pot drops to 35 °C, add 1.5 g of lipase nanoflowers, and gradually add the solution in step (2) dropwise into the solution in step (1). During the addition process, use 3 wt% NaOH solution to adjust the pH value to keep the pH value of the solution at 7.5 - 8.5 all the time. After all the addition, continue mechanical stirring at 35 °C for 5 h;

[0082] (4) Centrifuge the product prepared in step (3) at 6000 r / min to remove the supernatant. The lower solid precipitate is rinsed with absolute ethanol multiple times and dried to obtain lipase nanoflower-modified starch;

[0083] (5) Dissolve 20 g of lipase nanoflower-modified starch in 200 mL of deionized water as the aqueous phase, dissolve 200 mg of curcumin in 200 mL of olive oil as the oil phase, mix the aqueous phase and the oil phase, and homogenize at 20000 rpm for 5 min to prepare a Pickering emulsion with curcumin encapsulated by lipase nanoflower-modified starch, denoted as.

[0084] Example 3

[0085] (1) Weigh 7 g of glutinous rice flour and dissolve it in a mixed solution of 35 mL of water and 35 mL of DMSO. Heat and stir it in an oil bath at 100 °C for 30 min to obtain a gelatinized starch solution;

[0086] (2) Completely dissolve 2.1 g of 2-octenyl succinic anhydride in 20 mL of absolute ethanol;

[0087] (3) After the temperature of the oil bath drops to 35 °C, add 2 g of lipase nanoflowers. Dropwise add the solution in step (2) into the solution in step (1). During the addition process, use 3 wt% NaOH solution to adjust the pH value to keep the solution pH value always between 7.5 and 8.5. After all additions, continue mechanical stirring at 35 °C for 5 h;

[0088] (4) Centrifuge the product prepared in step (3) at 6000 r / min to remove the supernatant. Wash the lower solid precipitate with absolute ethanol multiple times and dry it to obtain lipase nanoflower-modified starch;

[0089] (5) Dissolve 20 g of lipase nanoflower-modified starch in 200 mL of deionized water as the aqueous phase, and dissolve 200 mg of curcumin in 200 mL of olive oil as the oil phase. Mix the aqueous phase and the oil phase and homogenize at 20000 rpm for 5 min to prepare a Pickering emulsion of lipase nanoflower-modified starch entrapping curcumin.

[0090] Example 4

[0091] (1) Weigh 7 g of glutinous rice flour and dissolve it in a mixed solution of 35 mL of water and 35 mL of DMSO. Heat and stir it in an oil bath at 100 °C for 30 min to obtain a gelatinized starch solution;

[0092] (2) Completely dissolve 2.1 g of 2-octenyl succinic anhydride in 20 mL of absolute ethanol;

[0093] (3) After the temperature of the oil bath drops to 35 °C, add 2.5 g of lipase nanoflowers. Dropwise add the solution in step (2) into the solution in step (1). During the addition process, use 3 wt% NaOH solution to adjust the pH value to keep the solution pH value always between 7.5 and 8.5. After all additions, continue mechanical stirring at 35 °C for 5 h;

[0094] (4) Centrifuge the product prepared in step (3) at 6000 r / min to remove the supernatant. Wash the lower solid precipitate with absolute ethanol multiple times and dry it to obtain lipase nanoflower-modified starch;

[0095] (5) Dissolve 20 g of lipase nanoflower-modified starch in 200 mL of deionized water as the aqueous phase, dissolve 200 mg of curcumin in 200 mL of olive oil as the oil phase, mix the aqueous phase and the oil phase, and homogenize at 20,000 rpm for 5 min with a homogenizer to prepare a Pickering emulsion of curcumin encapsulated by lipase nanoflower-modified starch.

[0096] Example 5

[0097] (1) Weigh 7 g of glutinous rice flour and dissolve it in a mixed solution of 35 mL of water and 35 mL of DMSO, and heat and stir in an oil bath at 100 °C for 30 min to obtain a gelatinized starch solution;

[0098] (2) Completely dissolve 2.1 g of 2-octenyl succinic anhydride in 20 mL of absolute ethanol;

[0099] (3) After the temperature of the oil bath drops to 35 °C, add 3 g of lipase nanoflowers, and gradually add the solution in step (2) to the solution in step (1). During the addition process, use 3 wt% NaOH solution to adjust the pH value to keep the pH value of the solution at 7.5 - 8.5 all the time. After all the addition, continue mechanical stirring at 35 °C for 5 h;

[0100] (4) Centrifuge the product prepared in step (3) at 6000 r / min, remove the supernatant, and repeatedly rinse the lower solid precipitate with absolute ethanol, and dry it to obtain lipase nanoflower-modified starch;

[0101] (5) Dissolve 20 g of lipase nanoflower-modified starch in 200 mL of deionized water as the aqueous phase, dissolve 200 mg of curcumin in 200 mL of olive oil as the oil phase, mix the aqueous phase and the oil phase, and homogenize at 20,000 rpm for 5 min with a homogenizer to prepare a Pickering emulsion of curcumin encapsulated by lipase nanoflower-modified starch.

[0102] Example 6

[0103] (1) Weigh 7 g of glutinous rice flour and dissolve it in a mixed solution of 35 mL of water and 35 mL of DMSO, and heat and stir in an oil bath at 100 °C for 30 min to obtain a gelatinized starch solution;

[0104] (2) Completely dissolve 2.1 g of 2-octenyl succinic anhydride in 20 mL of absolute ethanol;

[0105] (3) After the temperature of the oil bath drops to 35 °C, add 1.5 g of lipase nanoflowers, and gradually add the solution in step (2) to the solution in step (1). During the addition process, use 3 wt% NaOH solution to adjust the pH value to keep the pH value of the solution at 7.5 - 8.5 all the time. After all the addition, continue mechanical stirring at 35 °C for 5 h;

[0106] (4) Centrifuge the product obtained in step (3) at 6000 r / min, remove the supernatant, and repeatedly rinse the lower solid precipitate with absolute ethanol. After drying, lipase nanoflower-modified starch is obtained.

[0107] (5) Dissolve 20 g of lipase nanoflower-modified starch in 200 mL of deionized water as the aqueous phase, and dissolve 200 mg of gallic acid in 200 mL of olive oil as the oil phase. Mix the aqueous phase and the oil phase, and homogenize at 20000 rpm for 5 min to prepare a Pickering emulsion with lipase nanoflower-modified starch encapsulating gallic acid.

[0108] Example 7

[0109] (1) Weigh 7 g of glutinous rice flour and dissolve it in a mixed solution of 35 mL of water and 35 mL of DMSO. Heat and stir in an oil bath at 100 °C for 30 min to obtain a gelatinized starch solution.

[0110] (2) Completely dissolve 2.1 g of 2-octenyl succinic anhydride in 20 mL of absolute ethanol.

[0111] (3) After the temperature of the oil bath drops to 35 °C, add 1.5 g of lipase nanoflowers. Dropwise add the solution in step (2) into the solution in step (1). During the addition process, use 3 wt% NaOH solution to adjust the pH value to keep the solution pH value at 7.5 - 8.5 all the time. After all additions, continue mechanical stirring at 35 °C for 5 h.

[0112] (4) Centrifuge the product obtained in step (3) at 6000 r / min, remove the supernatant, and repeatedly rinse the lower solid precipitate with absolute ethanol. After drying, lipase nanoflower-modified starch is obtained.

[0113] (5) Dissolve 20 g of lipase nanoflower-modified starch in 200 mL of deionized water as the aqueous phase, and dissolve 200 mg of flavonol in 200 mL of olive oil as the oil phase. Mix the aqueous phase and the oil phase, and homogenize at 20000 rpm for 5 min to prepare a Pickering emulsion with lipase nanoflower-modified starch encapsulating flavonol.

[0114] Example 8

[0115] (1) Weigh 7 g of glutinous rice flour and dissolve it in a mixed solution of 35 mL of water and 35 mL of DMSO. Heat and stir in an oil bath at 100 °C for 30 min to obtain a gelatinized starch solution.

[0116] (2) Completely dissolve 2.1 g of 2-octenyl succinic anhydride in 20 mL of absolute ethanol.

[0117] (3) After the temperature of the oil bath pan drops to 35 °C, add 1.5 g of lipase nanoflowers, and dropwise add the solution in step (2) into the solution in step (1). During the addition process, use 3 wt% NaOH solution to adjust the pH value to keep the pH value of the solution at 7.5 - 8.5 all the time. After all the addition, continue mechanical stirring at 35 °C for 5 h;

[0118] (4) Centrifuge the product prepared in step (3) at 6000 r / min using a centrifuge, remove the supernatant, and repeatedly rinse the lower solid precipitate with absolute ethanol. After drying, lipase nanoflower-modified starch is obtained;

[0119] (5) Dissolve 20 g of lipase nanoflower-modified starch in 200 mL of deionized water as the aqueous phase, and dissolve 200 mg of tannic acid in 200 mL of olive oil as the oil phase. Mix the aqueous phase and the oil phase, and homogenize at 20000 rpm for 5 min to prepare a Pickering emulsion of lipase nanoflower-modified starch entrapping tannic acid.

[0120] Example 9

[0121] (1) Weigh 7 g of glutinous rice flour and dissolve it in a mixed solution of 35 mL of water and 35 mL of DMSO. Heat and stir in an oil bath pan at 100 °C for 30 min to obtain a gelatinized starch solution;

[0122] (2) Completely dissolve 2.1 g of 2-octenyl succinic anhydride in 20 mL of absolute ethanol;

[0123] (3) After the temperature of the oil bath pan drops to 35 °C, add 1.5 g of lipase nanoflowers, and dropwise add the solution in step (2) into the solution in step (1). During the addition process, use 3 wt% NaOH solution to adjust the pH value to keep the pH value of the solution at 7.5 - 8.5 all the time. After all the addition, continue mechanical stirring at 35 °C for 5 h;

[0124] (4) Centrifuge the product prepared in step (3) at 6000 r / min using a centrifuge, remove the supernatant, and repeatedly rinse the lower solid precipitate with absolute ethanol. After drying, lipase nanoflower-modified starch is obtained;

[0125] (5) Dissolve 20 g of lipase chemically modified starch in 200 mL of deionized water as the aqueous phase, and dissolve 200 mg of lutein in 200 mL of olive oil as the oil phase. Mix the aqueous phase and the oil phase, and homogenize at 20000 rpm for 5 min to prepare a Pickering emulsion of lipase nanoflower-modified starch entrapping lutein.

[0126] Example 10

[0127] (1) Weigh 7 g of glutinous rice flour and dissolve it in a mixed solution of 35 mL of water and 35 mL of DMSO. Heat and stir it in an oil bath at 100 °C for 30 min to obtain a gelatinized starch solution;

[0128] (2) Completely dissolve 2.1 g of 2-octenyl succinic anhydride in 20 mL of absolute ethanol;

[0129] (3) After the temperature of the oil bath drops to 35 °C, add 1.5 g of lipase nanoflowers. Dropwise add the solution in step (2) into the solution in step (1). During the addition process, use 3 wt% NaOH solution to adjust the pH value to keep the solution pH value always between 7.5 - 8.5. After all additions, continue mechanical stirring at 35 °C for 5 h;

[0130] (4) Centrifuge the product prepared in step (3) at 6000 r / min to remove the supernatant. Wash the lower solid precipitate with absolute ethanol multiple times and dry it to obtain lipase nanoflower-modified starch;

[0131] (5) Dissolve 20 g of lipase nanoflower-modified starch in 200 mL of deionized water as the aqueous phase, and dissolve 200 mg of anthocyanin in 200 mL of olive oil as the oil phase. Mix the aqueous phase and the oil phase and homogenize at 20000 rpm for 5 min to prepare a Pickering emulsion with lipase nanoflower-modified starch encapsulating anthocyanin.

[0132] Example 11

[0133] (1) Weigh 7 g of glutinous rice flour and dissolve it in a mixed solution of 35 mL of water and 35 mL of DMSO. Heat and stir it in an oil bath at 100 °C for 30 min to obtain a gelatinized starch solution;

[0134] (2) Completely dissolve 2.1 g of 2-octenyl succinic anhydride in 20 mL of absolute ethanol;

[0135] (3) After the temperature of the oil bath drops to 35 °C, add 1.5 g of lipase nanoflowers. Dropwise add the solution in step (2) into the solution in step (1). During the addition process, use 3 wt% NaOH solution to adjust the pH value to keep the solution pH value always between 7.5 - 8.5. After all additions, continue mechanical stirring at 35 °C for 5 h;

[0136] (4) Centrifuge the product prepared in step (3) at 6000 r / min to remove the supernatant. Wash the lower solid precipitate with absolute ethanol multiple times and dry it to obtain lipase nanoflower-modified starch;

[0137] (5) Dissolve 20 g of lipase nanoflower modified starch in 200 mL of deionized water as the aqueous phase, dissolve 200 mg of astaxanthin in 200 mL of olive oil as the oil phase, mix the aqueous phase and the oil phase, and homogenize at 20000 rpm for 5 min to prepare a Pickering emulsion with lipase nanoflower modified starch encapsulating astaxanthin.

[0138] Characterize the starches, chemically modified starches, and lipase nanoflower modified starches and the corresponding Pickering emulsion properties of the above comparative examples and examples as follows:

[0139] 1. Figure 1 The infrared absorption spectra of the starches, chemically modified starches, and lipase nanoflower modified starches of Comparative Examples 1 and 2 and Example 2 are shown. From Figure 1 it can be seen that a broad and strong peak appears at 3440 cm -1 , which is caused by the stretching vibration of -OH (hydroxyl group). The characteristic peak at 2926 cm -1 is attributed to the stretching vibration of -CH 2 . The characteristic peak at 1737 cm -1 is attributed to -COOH in 2-octenyl succinic anhydride in the structure.

[0140] 2. Figure 2 The scanning electron microscope pictures of the starches, chemically modified starches, and lipase nanoflower modified starches of Comparative Examples 1 and 2 and Example 2 are shown. Among them, a is starch, b is chemically modified starch, and c is lipase nanoflower modified starch. From Figure 2 a, it can be seen that the starch is a relatively regular hexagon with a relatively smooth surface; after chemical modification, Figure 2 from b, it can be seen that the surface of the chemically modified starch becomes rough and there are grooves; Figure 2 c, the lipase nanoflower modified starch is rougher than the chemically modified starch, proving that the lipase nanoflower modified starch has a higher degree of substitution.

[0141] 3. Storage stability of Pickering emulsion:

[0142] The specific method is as follows: Determine by spectrophotometry. Use an olive oil solution containing 1 mg / mL curcumin as the oil phase of the control group, denoted as Oil. Store the control group, the starch curcumin Pickering emulsions (NS), chemically modified starch curcumin Pickering emulsions (OSAS), and lipase nanoflower modified starch curcumin Pickering emulsions (Cu-OSAS) prepared in Comparative Examples 1-2 and Example 2 for 1 d, 3 d, 5 d, 7 d, and 9 d respectively. Extract curcumin in the remaining emulsion with absolute ethanol and measure its absorbance at 425 nm to calculate the retention rate of the curcumin emulsion.

[0143] Figure 3 The storage stability test results of starch-curcumin Pickering emulsion, chemically modified starch-curcumin Pickering emulsion, and lipase nanoflower-modified starch-curcumin Pickering emulsion are shown in the figure. It can be seen that the curcumin olive oil solution in the blank control remained stable within 10 days, with only a slight decrease in activity; the retention rates of starch-curcumin Pickering emulsion and chemically modified starch-curcumin Pickering emulsion gradually decreased with time; the lipase nanoflower-modified starch-curcumin Pickering emulsion maintained almost the same stability as the control group, and due to its activity protection ability, the retention rate exceeded that of the control group after 6 days.

[0144] Comparative Example 3

[0145] Preparation of poly(N-isopropylacrylamide) temperature-responsive release hydrogel, and the specific method is as follows:

[0146] Dissolve 1.6 g of N-isopropylacrylamide, 0.004 g of N,N-dimethylbisacrylamide, and 0.04 g of ammonium persulfate in 10 mL of water, remove the oxygen in the solution, and add 250 μL of tetramethylethylenediamine to obtain poly(N-isopropylacrylamide) temperature-responsive release hydrogel.

[0147] Comparative Example 4

[0148] Preparation of curcumin Pickering emulsion-poly(N-isopropylacrylamide) temperature-responsive release hydrogel using chemically modified starch as an emulsifier, and the specific method is as follows:

[0149] Add 5 mL of the Pickering emulsion prepared in Comparative Example 2 with curcumin encapsulated by chemically modified starch, 1.6 g of N-isopropylacrylamide, 0.004 g of N,N-dimethylbisacrylamide, and 0.04 g of ammonium persulfate to 5 mL of water and dissolve them. Remove the oxygen in the solution, and add 250 μL of tetramethylethylenediamine to obtain curcumin Pickering emulsion-poly(N-isopropylacrylamide) temperature-responsive release hydrogel using chemically modified starch as an emulsifier.

[0150] Example 12

[0151] Preparation of curcumin Pickering emulsion-poly(N-isopropylacrylamide) temperature-responsive release hydrogel with a volume ratio of Pickering emulsion to water of 1:9:

[0152] The specific method is as follows: 1 mL of the lipase nanoflower-modified starch curcumin Pickering emulsion prepared in Example 2, 1.6 g of N-isopropylacrylamide, 0.004 g of N,N-dimethylbisacrylamide, and 0.04 g of ammonium persulfate are added to 9 mL of water and dissolved. After removing the oxygen in the solution, 250 μL of tetramethylethylenediamine is added to obtain a curcumin Pickering emulsion-poly(N-isopropylacrylamide) temperature-responsive hydrogel with lipase nanoflower-modified starch as the emulsifier.

[0153] Example 13

[0154] Preparation of curcumin Pickering emulsion-poly(N-isopropylacrylamide) temperature-responsive hydrogel with a volume ratio of Pickering emulsion to water of 3:7:

[0155] The specific method is as follows: 3 mL of the lipase nanoflower-modified starch curcumin Pickering emulsion prepared in Example 2, 1.6 g of N-isopropylacrylamide, 0.004 g of N,N-dimethylbisacrylamide, and 0.04 g of ammonium persulfate are added to 7 mL of water and dissolved. After removing the oxygen in the solution, 250 μL of tetramethylethylenediamine is added to obtain a curcumin Pickering emulsion-poly(N-isopropylacrylamide) temperature-responsive hydrogel with lipase nanoflower-modified starch as the emulsifier.

[0156] Example 14

[0157] Preparation of curcumin Pickering emulsion-poly(N-isopropylacrylamide) temperature-responsive hydrogel with a volume ratio of Pickering emulsion to water of 1:1:

[0158] The specific method is as follows: 5 mL of the lipase nanoflower-modified starch curcumin Pickering emulsion prepared in Example 2, 1.6 g of N-isopropylacrylamide, 0.004 g of N,N-dimethylbisacrylamide, and 0.04 g of ammonium persulfate are added to 5 mL of water and dissolved. After removing the oxygen in the solution, 250 μL of tetramethylethylenediamine is added to obtain a curcumin Pickering emulsion-poly(N-isopropylacrylamide) temperature-responsive hydrogel with lipase nanoflower-modified starch as the emulsifier.

[0159] Mechanical properties of curcumin Pickering emulsion-poly(N-isopropylacrylamide) temperature-responsive hydrogel with modified starch as the emulsifier:

[0160] The mechanical properties of the hydrogels prepared in Comparative Examples 3-4 and Examples 12-14 were determined by tensile tests conducted at room temperature. The tensile test samples were molded into a cuboid shape (length: 70 mm, width: 15 mm, thickness: 10 mm), and a constant tensile rate (10 mm / min) was maintained.

[0161] Figure 4 Results of the mechanical properties of curcumin Pickering emulsion-poly(N-isopropylacrylamide) thermosensitive hydrogel with lipase nanoflower-modified starch as the emulsifier. Among them, PNM is the poly(N-isopropylacrylamide) hydrogel of Comparative Example 3, which is the control group of the experiment; PNM0 is the poly(N-isopropylacrylamide) thermosensitive hydrogel containing chemically modified starch curcumin Pickering emulsion of Comparative Example 4; C-PNM1 is the N-isopropylacrylamide hydrogel containing 1 mL of curcumin Pickering emulsion in Example 12; C-PNM2 is the N-isopropylacrylamide hydrogel containing 3 mL of curcumin Pickering emulsion in Example 13; C-PNM3 is the N-isopropylacrylamide hydrogel containing 5 mL of curcumin Pickering emulsion in Example 14.

[0162] As Figure 4 shown, the mechanical strengths of PNM, PNM0, C-PNM1, C-PNM2, and C-PNM3 increase in turn. The tensile strain of C-PNM3 reaches about 137%, which is about 4.9 times that of the control group. This proves that the mechanical properties of the hydrogel are greatly improved due to the introduction of lipase nanoflower-modified starch Pickering emulsion.

[0163] Release amount and antioxidant capacity of curcumin Pickering emulsion-poly(N-isopropylacrylamide) thermosensitive hydrogel with modified starch as the emulsifier:

[0164] The hydrogels prepared in Examples 12-14 were placed in a water bath at 35 °C and taken out at 10 min, 30 min, 1 h, 2 h, 3 h, 4 h, and 5 h respectively. 5 mL of absolute ethanol was added to the leachate of the hydrogel for extraction, and its absorbance was measured at 425 nm to calculate the release amount of the hydrogel.

[0165] The antioxidant capacity was determined by spectrophotometry through the DPPH free radical scavenging experiment. 5 mL of DPPH free radicals were added to the above hydrogel release products respectively, and the reaction was carried out in the dark for 1 h. Its absorbance was measured at 517 nm to calculate the antioxidant activity of curcumin Pickering emulsion-poly(N-isopropylacrylamide) thermosensitive hydrogel with modified starch as the emulsifier.

[0166] Figure 5Results of the release amount and antioxidant activity of the curcumin Pickering emulsion-poly(N-isopropylacrylamide) thermosensitive hydrogel with modified starch as the emulsifier obtained in Examples 12-14. It can be seen from the figure that as the addition amount of the curcumin Pickering emulsion increases, the release amounts of C-PNM1, C-PNM2, and C-PNM3 increase in turn; the DPPH radical scavenging rates all reach more than 85%, proving that the hydrogel has good antioxidant activity, and the antioxidant activity of C-PNM3 reaches 90%. It is proved that the curcumin Pickering emulsion-poly(N-isopropylacrylamide) hydrogel with modified starch as the emulsifier has good thermosensitive release characteristics and can ensure the antioxidant activity of the active substance, and can be used as a wound healing dressing.

[0167] The above embodiments are only the preferred embodiments of the present invention and do not limit the implementation manners. The protection scope of the present invention should be subject to the scope defined by the claims. Other different forms of changes or modifications can be made on the basis of the above description. The obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A bioactive substance Pickering emulsion temperature-controlled release hydrogel, characterized in that: The modified starch obtained by modifying starch is used as an emulsifier and is mixed with an oil phase containing a bioactive substance to obtain a Pickering emulsion of the modified starch encapsulating the bioactive substance. The Pickering emulsion of the modified starch encapsulating the bioactive substance and an N-isopropylacrylamide monomer are prepared by free radical polymerization under the action of an initiator, a crosslinking agent and a catalyst.

2. The bioactive substance Pickering emulsion temperature-controlled release hydrogel according to claim 1, characterized in that: The initiator is ammonium persulfate; the cross-linking agent is N,N-methylenebisacrylamide; the catalyst is tetramethylethylenediamine; and the bioactive substance includes one of curcumin, gallic acid, flavonol, tannic acid, lutein, anthocyanin, and astaxanthin.

3. The bioactive substance Pickering emulsion temperature-controlled release hydrogel according to claim 1, characterized in that: The modified starch is prepared by using lipase nanoflowers as an esterification reaction catalyst; and the starch is glutinous rice flour.

4. A method for preparing the temperature-controlled release hydrogel of a bioactive substance Pickering emulsion according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: (1) adding starch to a mixed solution of water and DMSO, heating and stirring, to obtain a gelatinized starch solution; (2) adding 2-octenylsuccinic anhydride and lipase nanoflowers to the gelatinized starch solution obtained in step (1) to carry out an esterification reaction, wherein the pH value of the solution system is maintained at 7.5 to 8.5 during the addition process, and stirring is continued after the addition is completed to obtain a paste product; (3) centrifuging the paste product obtained in step (2) to remove the supernatant, and rinsing the precipitate with anhydrous ethanol to obtain modified starch; (4) using the modified starch obtained in step (3) as an emulsifier, dissolving it in water as the aqueous phase, dissolving the bioactive substance in olive oil as the oil phase, and homogenizing by a homogenizer to obtain a Pickering emulsion in which the modified starch encapsulates the bioactive substance; (5) dissolving the Pickering emulsion obtained in step (4), N-isopropylacrylamide, a crosslinking agent, and an initiator in water to obtain a mixed solution, removing oxygen from the solution, and adding a catalyst to react to obtain the bioactive substance Pickering emulsion temperature-controlled release hydrogel.

5. The preparation method according to claim 4, characterized in that: In step (1), the volume ratio of water to DMSO is 1:1 to 1:2; The heating temperature is 90-110° C.; the mass ratio of starch to the mixed solution is 1:5-15.

6. The preparation method according to claim 4, characterized in that: In step (2), the mass ratio of the lipase nanoflowers to starch is 1:7 to 3:

7.

7. The preparation method according to claim 4, characterized in that: In step (2), 2-octenyl succinic anhydride is completely dissolved in anhydrous ethanol and then added to the gelatinized starch solution, and 2-octenyl succinic anhydride is grafted onto the starch through an esterification reaction, thereby obtaining modified starch; The temperature of the esterification reaction is 30-40°C.

8. The preparation method according to claim 4, characterized in that: In step (4), the concentration of the bioactive substance in the oil phase is 0.5 to 1 mg / mL; In the aqueous phase, the concentration of the modified starch is 5wt% to 10wt%; The volume ratio of the oil phase to the water phase is 1:2 to 1:

1.

9. The preparation method according to claim 4, characterized in that: In step (5), the volume ratio of the Pickering emulsion to water is 1:1 to 9; In the mixed solution, the concentration of N-isopropylacrylamide is 0.16 g / mL, the concentration of the crosslinking agent is 0.2-0.8 mg / mL, and the concentration of the initiator is 2-8 mg / mL; The reaction temperature is 4-10°C.

10. Use of the bioactive substance Pickering emulsion temperature-controlled release hydrogel according to any one of claims 1 to 3 or the bioactive substance Pickering emulsion temperature-controlled release hydrogel prepared by the preparation method according to any one of claims 4 to 9 in medical dressings.

Images