Preparation method and application of an enzyme-regulated injectable alginate-based hydrogel

Abstract

The present invention provides a preparation method and application of an enzyme-regulated injectable alginate-based hydrogel, wherein the injectable hydrogel comprises the following raw materials: water-soluble alginate, insoluble or slightly soluble phytate particles, phytase and functional polymers; after the above raw materials are dissolved and dispersed in water, they do not gel at low temperature (<20°C) or have extremely low gelation and are fluid and injectable, and the suspension can be slowly cross-linked into a hydrogel at 37±5°C. In the present invention, based on natural polysaccharides, the phytate catalytic properties of phytase are utilized to make the insoluble phytate particles slowly release divalent metal ions to chelate alginate for cross-linking to obtain a hydrogel.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical materials and relates to a preparation method and application of an enzyme-regulated injectable alginate-based hydrogel. Background Art

[0002] Injectable hydrogel is a water-soluble polymer solution that undergoes in situ gelation when applied to a target environment (such as a human or animal body). This type of material can be used for cell culture matrices, tissue engineering repair, biosensors, drug delivery carriers, etc. Therefore, it has a wide range of application value and potential in the biomedical field and has become a research hotspot in the field of materials in recent years.

[0003] Existing injectable hydrogel products mainly include: temperature-sensitive hydrogels, pH-sensitive hydrogels, and temperature- and pH-responsive hydrogels. The commercialized Corning Matrigel® is a representative of temperature-sensitive hydrogels. This product is a naturally extracted mixed polysaccharide with high biocompatibility and safety, but the product has complex components and poor quality controllability. In addition, there are chemically synthesized temperature-sensitive hydrogels, such as polyacrylic acid or polyacrylate (CN202310685215.0) to achieve temperature sensitivity. The presence of monomer residues in the preparation process of this polymer leads to potential biological toxicity risks, thereby limiting its wide application in the medical field. pH-sensitive hydrogels are mainly artificially synthesized hydrogel materials, such as composite hydrogels represented by polyethyleneimine (PEI) (CN202110397161.9). This type of polymer is not biodegradable, which limits its application in the medical field.

[0004] Utilize free divalent metal ions such as Ca 2+ The carboxyl groups (-COO - ) to produce hydrogels by electrostatic interaction, which meets the requirements of mild conditions, degradability, and biosafety. 2+ The cross-linking reaction with alginate is rapid, which cannot meet the requirements of injectability and slow gelation. In the application document of CN104893010A, insoluble CaCO3 is used as a cross-linking agent, and gluconic acid lactone is hydrolyzed to produce gluconic acid, which reacts with CaCO3 to release Ca 2+ Cross-linking alginate, but the reaction requires pH < 4 to generate free Ca 2+ , and this pH environment will cause cell apoptosis or inflammatory response in the body's microenvironment.

[0005] In addition, single alginate hydrogel is biologically inert, so current research on alginate-based hydrogels is adding some functional excipients with better biocompatibility to improve the function of the hydrogel. Summary of the invention

[0006] In view of the problems existing in existing injectable hydrogels, the present invention designs a method for preparing an enzyme-regulated injectable alginate-based hydrogel, which can be used in the fields of organoid culture, tissue repair, drug delivery carrier, biosensor, etc.

[0007] The technical solution of the present invention is as follows:

[0008] A method for preparing an enzyme-regulated injectable alginate-based hydrogel, the injectable alginate-based hydrogel comprising the following raw materials: water-soluble alginate, poorly soluble or slightly soluble phytate particles, phytase and water, and functional excipients added to improve the function of the injectable hydrogel;

[0009] Wherein, the poorly soluble phytate particles refer to the phytate particles whose solubility is less than 1 gram at room temperature 20°C;

[0010] Slightly soluble phytate particles refer to phytate particles having a solubility of 0.01 g to 1 g per 100 g at room temperature 20°C;

[0011] The above raw materials are dissolved and dispersed in water at <20℃ to obtain an aqueous solution which does not gel or has a low gel degree and has fluidity and injectability. When the aqueous solution is placed in or injected into an environment of 30-100℃, the aqueous solution can slowly cross-link to form a hydrogel.

[0012] Phytase hydrolyzes phytate particles into inositol, phosphate, and divalent metal ions at a suitable temperature; the divalent metal ions cross-link the polymers in the solution to form a hydrogel through electrostatic action. Inositol belongs to the vitamin B group, and all enzymatic hydrolysis products also meet the requirements of biosafety, making the hydrogel safe and non-toxic.

[0013] Preferably, every 100 parts of the injectable alginate-based hydrogel comprises the following raw materials in parts by weight:

[0014] 0.2-10 parts of water-soluble alginate;

[0015] 0.02-10 parts of sparingly soluble or slightly soluble phytate particles;

[0016] Functional excipients 0-5 parts;

[0017] Phytase 0.01-10 parts (50000U / part);

[0018] Water 65-99.77 parts.

[0019] Preferably, the water-soluble alginate is a mixture of one or more of sodium alginate, potassium alginate, ammonium alginate, sodium alginate, and alginate derivatives.

[0020] Preferably, the alginate derivative is propylene glycol sodium alginate.

[0021] Preferably, the poorly soluble or slightly soluble phytate particles are divalent metal phytates, and the particle size is 0.1-200 μm.

[0022] Preferably, the poorly soluble or slightly soluble phytate includes, but is not limited to, a mixture of one or more of calcium phytate, zinc phytate, magnesium phytate or ferrous phytate, barium phytate, and calcium-magnesium phytate.

[0023] Preferably, the phytase contains phytase from various sources, including low-temperature phytase with a temperature range of 30-50°C and high-temperature phytase with a temperature range of 50-100°C, or a mixture of the two. The cross-linking temperature is related to the type of phytase. The active temperature of the low-temperature phytase is 30-50°C, and the optimal temperature is 37±5°C (in line with the temperature requirements of biomedical injectable hydrogels); the active temperature of the high-temperature phytase is 50-100°C, and the optimal temperature is 80±5°C.

[0024] Preferably, the functional excipients include, but are not limited to, water-soluble pectin, carrageenan, xanthan gum, carboxymethyl polysaccharide, chitosan, protein, and bioactive glass particles.

[0025] Preferably, the carboxymethyl polysaccharide includes but is not limited to one or more combinations of carboxymethyl chitosan, carboxymethyl cellulose, and carboxymethyl starch; and the protein includes but is not limited to soy protein isolate.

[0026] Disclosed is an application of an injectable alginate-based hydrogel, including application in preparing organoid culture materials, application in preparing tissue repair materials, application in preparing drug delivery carriers, and application in preparing biosensors.

[0027] Preferably, when the hydrogel is used for organoid culture, the hydrogel raw material is dissolved or dispersed in water, and after inoculating cells, the aqueous solution is injected into the animal body or co-cultured in vitro to obtain organoids.

[0028] Preferably, the cells include but are not limited to stem cells, somatic cells, and engineered cell lines.

[0029] Preferably, the hydrogel is used as a drug delivery carrier, and the above hydrogel raw material and the drug are dissolved or dispersed in water together, injected into the body or in vitro to form a hydrogel, and achieve drug delivery or sustained release.

[0030] Preferably, the hydrogel is used for tissue repair, and the hydrogel raw material is dissolved and dispersed in water, and then injected into the defective part of human or animal tissue to achieve tissue repair.

[0031] Compared with the prior art, the present invention has the following characteristics:

[0032] 1. The hydrogel product designed in the present invention has the advantages of safety, non-toxicity, good biocompatibility and low price.

[0033] 2. The preparation method of the present invention has the advantages of simple process and strong operability. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0035] Figure 1 The hydrogel is prepared using calcium phytate, sodium alginate, carboxymethyl chitosan and phytase as raw materials in Example 1.

[0036] Figure 2 The hydrogel products obtained in Example 1 and Example 2 are shown in FIG.

[0037] Figure 3 These are microscopic images of mouse intestinal stem cell organoid culture in Example 1. In the figure, 3-1 is cultured for 1 day; 3-2 is cultured for 2 days; and 3-3 is cultured for 5 days. DETAILED DESCRIPTION

[0038] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0039] The phytase of Examples 1 to 5 was purchased from Longkote Enzyme Preparation Company, which was a low-temperature liquid type, 30-50° C., 50,000 U / g;

[0040] The phytase in Example 6 was purchased from Longkote Enzyme Preparation Company, and was a high temperature resistant type, 50-100°C, 50000 U / g.

[0041] Example 1

[0042] Combination Figure 1 , an enzyme-regulated injectable alginate-based hydrogel, the preparation method process is as follows:

[0043] 1.5 g of sodium alginate, 0.1 g of calcium phytate with a particle size of 5-10 μm, 0.2 g of carboxymethyl chitosan and 0.2 g of low-temperature phytase were weighed and dissolved in 98 g of water at 7°C, and then the solution was placed in an oven at 37°C for 15 min to obtain a hydrogel; Figure 2 .

[0044] Example 2

[0045] An enzyme-regulated injectable alginate-based hydrogel, the preparation method of which is as follows:

[0046] 5 g of potassium alginate, 5 g of zinc phytate with a particle size of 20-50 μm, and 1 g of low-temperature phytase were measured and dissolved in 89 g of water at 15°C. The solution was then placed in an oven at 42°C for 8 min to obtain a hydrogel. Figure 2 .

[0047] Example 3

[0048] An enzyme-regulated injectable alginate-based hydrogel, the preparation method of which is as follows:

[0049] 3 g of ammonium alginate, 0.5 g of magnesium phytate with a particle size of 50-100 μm, 0.1 g of soy protein isolate, and 5 g of low-temperature phytase were measured and dissolved in 91.4 g of water at 13° C., and then the solution was placed in an oven at 32° C. for 5 min to obtain a hydrogel.

[0050] Example 4

[0051] An enzyme-regulated injectable alginate-based hydrogel, the preparation method of which is as follows:

[0052] 9.8 g of sodium alginate, 10 g of magnesium phytate with a particle size of 50-100 μm, 0.1 g of xanthan gum, 0.1 g of carboxymethyl starch, and 10 g of low-temperature phytase were measured and dissolved in 69.9 g of water at 3°C. The solution was then placed in an oven at 30°C for 2 min to obtain a hydrogel.

[0053] Example 5

[0054] An enzyme-regulated injectable alginate-based hydrogel, the preparation method of which is as follows:

[0055] 2 g of sodium alginate, 1 g of potassium alginate, 0.05 g of barium phytate with a particle size of 0.1-10 μm, 0.05 g of ferrous phytate with a particle size of 150-200 μm, 1 g of pectin, 1 g of carrageenan, 0.5 g of soy protein isolate, and 2 g of low-temperature phytase were measured and dissolved in 92.4 g of water at 13° C., and the solution was placed in an oven at 32° C. for 8 min to obtain a hydrogel.

[0056] Example 6

[0057] An enzyme-regulated injectable alginate-based hydrogel, the preparation method of which is as follows:

[0058] 3 g of sodium alginate, 0.2 g of sodium alginate propylene glycol ester, 0.3 g of calcium phytate with a particle size of 100-150 μm, 0.5 g of chitosan, 0.5 g of carboxymethyl cellulose, 1 g of bioactive glass particles, and 3 g of high-temperature resistant phytase were measured and dissolved in 91.5 g of water at 18°C. The solution was then placed in an oven at 42°C for 12 h to obtain a hydrogel.

[0059] Example 7

[0060] The raw material ratio of Example 1 was dissolved and dispersed in water at 4°C to form 5 g of injectable hydrogel solution, which was inoculated with mouse intestinal stem cells and cultured at 37°C. The microscopic examination results showed that the mouse intestinal stem cells could grow and differentiate normally in the hydrogel, as shown in the microscopic examination photos. Figure 3 shown.

[0061] Although the present invention has been described in detail by reference to the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection of the claims.

Claims

1. A method for preparing an enzyme-regulated injectable alginate-based hydrogel, characterized in that: The injectable alginate-based hydrogel comprises the following raw materials: water-soluble alginate, poorly soluble or slightly soluble phytate particles, phytase, functional excipients and water; The above raw materials are dissolved and dispersed in water at a temperature of <20°C to obtain an aqueous solution; when the aqueous solution is placed or injected into a temperature environment of 37±5°C, it is slowly cross-linked to form a hydrogel; Each 100 parts of injectable alginate-based hydrogel includes the following raw materials in parts by weight: 0.2-10 parts of water-soluble alginate; 0.02-10 parts of sparingly soluble or slightly soluble phytate particles; Functional excipients 0-5 parts; Phytase 0.01-10 parts; Water 65-99.77 parts.

2. The method for preparing an enzyme-regulated injectable alginate-based hydrogel according to claim 1, characterized in that: The water-soluble alginate is a mixture of one or more of sodium alginate, potassium alginate, ammonium alginate, sodium alginate and alginate derivatives; the alginate derivative is sodium alginate propylene glycol ester.

3. The method for preparing the enzyme-regulated injectable alginate-based hydrogel according to claim 1, characterized in that: The insoluble or slightly soluble phytate particles are divalent metal phytates, and the insoluble phytate particles have a size of 0.1-200 μm; the insoluble or slightly soluble phytates include but are not limited to a mixture of one or more of calcium phytate, zinc phytate, magnesium phytate, ferrous phytate, barium phytate, and calcium-magnesium phytate.

4. The method for preparing an enzyme-regulated injectable alginate-based hydrogel according to claim 1, characterized in that: The phytase contains phytase from various sources, including one or a mixture of low-temperature phytase with a temperature range of 30-50°C and high-temperature phytase with a temperature range of 50-80°C.

5. The method for preparing an enzyme-regulated injectable alginate-based hydrogel according to claim 1, characterized in that: The functional excipients include but are not limited to pectin, carrageenan, xanthan gum, carboxymethyl polysaccharide, chitosan and protein, and bioactive glass particles; the carboxymethyl polysaccharide is one or more combinations of carboxymethyl chitosan, carboxymethyl cellulose, and carboxymethyl starch; the protein includes but is not limited to soy protein isolate.

6. Use of the hydrogel prepared by the method for preparing an enzyme-regulated injectable alginate-based hydrogel as claimed in claim 1, characterized in that: The application includes, but is not limited to, application in the preparation of organoid culture materials, application in the preparation of tissue repair materials, application in the preparation of drug delivery carriers, and application in the preparation of biosensors.

7. The use according to claim 6, characterized in that When the hydrogel is used in the preparation of an organoid culture system, the hydrogel raw material is dissolved or dispersed in water, and after inoculating cells, the aqueous solution is injected into an animal body or co-cultured in vitro to obtain organoids; the cells include but are not limited to stem cells, somatic cells, and engineered cell lines.

8. The use according to claim 6, characterized in that When the hydrogel is used in the preparation of a drug delivery carrier, the hydrogel raw material and the drug are dissolved or dispersed in water and injected into the body or in vitro to form the hydrogel.

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