A multi-functional injectable tissue adhesive and a method for preparing the same

Abstract

This invention provides a multifunctional injectable tissue adhesive, characterized by comprising component A: an active protein with chemically modified reduced polyphenol groups and a surface rich in RGD sequences, lysine, aspartic acid, and / or glutamic acid; component B: a natural polysaccharide with chemically modified active ester groups; and gelation buffers: an alkaline buffer solution and an acidic solution. The tissue adhesive provided by this invention combines good biocompatibility and adhesion, exhibits antibacterial activity, and can promote tissue healing, showing great promise for clinical application.

Description

Technical Field

[0001] This invention belongs to the field of clinical application materials, specifically relating to a multifunctional injectable tissue adhesive and its preparation method. Background Technology

[0002] Tissue adhesives, as a new generation of clinical application materials, can quickly close wounds, effectively prevent fluid penetration, and minimize additional trauma. Clinically commonly used adhesives are mainly divided into three categories: bio-extracted, synthetic, and semi-synthetic. However, currently commercially available adhesives have gradually shown the following two major shortcomings: (1) it is difficult to achieve both excellent biocompatibility and good tissue adhesion strength; (2) the required biological functions are lacking, which can easily lead to wound infection, slow healing, and other problems.

[0003] Bio-extracted adhesives, such as crossseed glue TM CryoSeal TM , Collagen-based protein binders While these materials exhibit excellent biocompatibility and a reaction process that mimics their own cross-linking and curing mechanism, making them mild and safe, and capable of degradation as wounds heal, thus possessing some tissue-healing activity, they suffer from low cohesion and lack stable connectivity with the tissue interface. Consequently, their adhesive strength is generally insufficient, and their hemostatic effect is limited by the wound environment, resulting in a very limited range of applications.

[0004] Synthetic tissue adhesives, such as α-cyanoacrylate adhesives Polyethylene glycol adhesive ( CoSeal TM , Although substances such as α-hydroxylamine (α-hydroxylamine) can establish stable covalent bonds within materials and between tissue interfaces and have high adhesive strength, the reaction process is often accompanied by exothermic reactions, the dissolution of toxic byproducts, and the use of toxic catalysts, resulting in poor biosafety. At the same time, the degradation rate is limited by the type of reaction and usually cannot degrade as needed with wound healing, which may occupy the site for a long time and affect healing.

[0005] Building upon bio-extracted and synthetic tissue adhesives, semi-synthetic adhesives combining synthetic and natural molecules have emerged to achieve higher adhesive strength and biocompatibility. However, current semi-synthetic tissue adhesives, such as gelatin-based adhesives... Bovine serum albumin-based binders In pursuit of high cross-linking efficiency and strength, highly active but toxic cross-linking agents such as formaldehyde and glutaraldehyde are often used, but this still cannot fundamentally solve the contradiction between biosafety and adhesive strength.

[0006] Chinese Patent Application No. CN202011631269.1 discloses a composite tissue adhesive, its preparation method, and its application. This composite tissue adhesive comprises a three-dimensional porous matrix and an adhesion-enhancing factor adsorbed on the matrix surface. The three-dimensional porous matrix is ​​obtained by freeze-drying a composite hydrogel of methacrylic anhydride-modified gelatin and N-hydroxysuccinimide-grafted polyacrylic acid. The adhesion-enhancing factor is polydopamine. This invention has two components that can bond with amino groups on wound tissue. Although the adhesion strength to moist wounds can reach over 180 kPa, it does not exhibit good antibacterial properties or biological activity against wound healing. Furthermore, its lack of injectability limits its application to sealing complex wounds, resulting in significant limitations.

[0007] Therefore, designing a multifunctional injectable tissue adhesive that is easy to prepare, has excellent biocompatibility, high adhesive strength, anti-swelling, antibacterial properties, and promotes tissue healing is of great research and practical significance. This would further expand its clinical application and improve wound healing quality and shorten hospital stay. Summary of the Invention

[0008] To address the aforementioned problems, this invention provides a multifunctional injectable tissue adhesive and its preparation method. The biomimetic design mimics the components of the natural extracellular matrix: using hyaluronic acid (HA) and type III recombinant collagen (Col) as the main components, it fully leverages the advantages of natural molecules, such as high biocompatibility, anti-inflammatory properties, pro-angiogenic effects, and high cell affinity.

[0009] Functionalization of natural macromolecules: HA and Col were modified with active esters (NHS) and reduced polyphenol functional groups (Dopa), respectively. red Modification with HA allows for nucleophilic addition reactions between the NHS groups on HA and amino or thiol groups on the Col and tissue surfaces, enabling in-situ sealing of tissue wounds. The gelation time can be controlled by altering the type and concentration of inorganic salts in the buffer solution. Subsequently, the reduced polyphenol groups in the network undergo slow oxidation and self-polymerization, reacting with surrounding residual amino or thiol groups via Schiff base reactions or Michael additions to enhance the material's cohesion and adhesion, providing self-reinforcing and anti-swelling effects. Simultaneously, the in-situ generation of appropriate amounts of H2O2 imparts long-lasting antibacterial activity to the adhesive.

[0010] On the one hand, the present invention provides a multifunctional tissue adhesive.

[0011] The multifunctional tissue adhesive includes:

[0012] (1) Component A: Chemically modified reduced polyphenol groups and active protein with RGD sequence, lysine, aspartic acid and / or glutamic acid on the surface;

[0013] (2) Component B: Natural polysaccharide with chemically modified active ester groups;

[0014] (3) Gel-forming buffer: alkaline buffer solution and acidic solution.

[0015] Specifically, the surface of the active protein in component A includes: an RGD sequence to provide a cell-adhesive active site; a lysine segment to provide a reactive active amino group; and an aspartic acid and / or glutamate segment to provide a reactive active carboxyl group.

[0016] Preferably, the active protein is selected from one or more of the following: recombinant type I collagen, recombinant type III collagen, gelatin, and lysozyme.

[0017] Specifically, the polyphenolic groups in component A are selected from catechol, pyrogallol, or combinations thereof.

[0018] The degree of reduction of the polyphenol groups can be controlled by a reducing agent, and the ratio of phenol to quinone is between 2:8 and 8:2.

[0019] Preferably, component A is reduced dopamine-functionalized recombinant type III collagen (Col-Dopamine). red ).

[0020] The active ester groups of the natural polysaccharide in component B are selected from one or more of the following: succinimide carbonate, succinimide acetate, succinimide propionate, succinimide succinate, and succinimide glutarate.

[0021] The modification rate of the active ester groups is 10-50%.

[0022] Preferably, the natural polysaccharide side chain in component B contains a carboxyl group.

[0023] Preferably, the molecular weight of the natural polysaccharide in component B is 10-500 kDa.

[0024] Preferably, component B is active ester-functionalized hyaluronic acid (HA-NHS).

[0025] In the gel-forming buffer solution:

[0026] The inorganic salts in the alkaline buffer solution are selected from: sodium tetraborate, sodium carbonate, disodium hydrogen phosphate, or combinations thereof;

[0027] The acidic substance in the acidic solution is selected from: hydrochloric acid, acetic acid, sodium dihydrogen phosphate, or a combination thereof.

[0028] The reaction principle of the present invention:

[0029] Gel formation and adhesion principle: The active ester groups modified on the side chains of hyaluronic acid can spontaneously undergo nucleophilic addition reactions with the amino groups on the lysine residues in recombinant type III collagen and tissue surface proteins to form stable amide bonds, thereby achieving the curing of the precursor solution and wound sealing.

[0030] The principle of controllable gelation time: The amidation reaction between the active ester and the amino group is a nucleophilic addition process. Its reaction rate is closely related to the degree of deprotonation of the amino component. The higher the degree of deprotonation of the amino component, the stronger the nucleophilicity and the faster the reaction rate. The degree of deprotonation of the amino component is positively correlated with the pH value of the system environment. Therefore, by adjusting the type and concentration of strong base weak acid salt in the buffer solution, precise control of gelation time can be achieved.

[0031] Self-reinforcing principle: The reduced dopamine groups modified on recombinant type III collagen have self-oxidation properties. Through a slow self-polymerization reaction, they form a double cross-link inside the gel. The oxidized benzoquinone groups can undergo Michael addition or Schiff base reaction with the surrounding amino and thiol groups, further enhancing the adhesive strength and the stability of the colloid.

[0032] Antibacterial Mechanism: During the self-oxidation process, the reduced dopamine groups modified on recombinant type III collagen can transfer the electrons stored in the phenolic hydroxyl groups to the surrounding oxygen, thereby generating hydrogen peroxide in situ, a highly efficient and clean antibacterial agent that endows the material with good antibacterial activity.

[0033] Mechanism of promoting tissue healing: The RGD sequence of the active protein itself and the modified reduced polyphenol groups can promote cell adhesion. At the same time, the degradation of the material also provides space for the growth of new tissue, thereby accelerating tissue healing.

[0034] On the other hand, the present invention provides an adhesive medical device package.

[0035] The adhesive medical device package includes one of the aforementioned multifunctional injectable tissue adhesives.

[0036] The components of the tissue adhesive are packaged separately, wherein component A and component B are powders; and the alkaline buffer and acidic solution are liquids.

[0037] The adhesive medical device kit includes a dual-barrel syringe or applicator.

[0038] Preferably, the multifunctional tissue adhesive comprises active ester-functionalized hyaluronic acid (HA-NHS) and reduced dopamine-functionalized recombinant type III collagen (Col-Dopamine). red The solution comprises an acidic solution and an alkaline buffer solution; more preferably, the acidic solution is a diluted hydrochloric acid solution, and the alkaline buffer solution is a Na₂B₄O₇ solution; even further, the hydrochloric acid is diluted by a factor of 10. 5The concentration of the Na2B4O7 solution is 0.1M.

[0039] In some embodiments, the HA-NHS is dissolved in hydrochloric acid dilution solution (10 mg / mL) at a concentration of 60 mg / mL. 5 (times); the aforementioned Col-Dopa red Dissolved in 0.1M Na₂B₄O₇ solution at a concentration of 120 mg / mL.

[0040] In another aspect, the present invention provides a method for preparing the aforementioned tissue adhesive.

[0041] The steps include:

[0042] (1) Dissolve the powders of components A and B in alkaline buffer and acidic solution respectively to obtain gelation precursor solution A and gelation precursor solution B.

[0043] (2) Using a double-barrel syringe or a spreading stick, mix the gel precursor liquid A and the gelation precursor liquid B evenly to form the multifunctional tissue adhesive.

[0044] Preferably, the method is as follows: functionalizing reduced dopamine with recombinant type III collagen (Col-Dopamine). red A gel-forming precursor A is dissolved in an alkaline buffer solution, and a gel-forming precursor B is dissolved in a weakly acidic solution. The two solutions are then mixed and delivered to form the gel adhesive (HA-Col) in situ.

[0045] In another aspect, the present invention provides the application of the aforementioned multifunctional injectable tissue adhesive.

[0046] The application is for sealing soft / hard tissues such as blood vessels, heart, and dura mater after surgery.

[0047] The beneficial effects of this invention are:

[0048] 1. This adhesive solves the problem that current adhesives cannot simultaneously achieve excellent biocompatibility and good tissue adhesion strength, and are prone to excessive swelling due to absorption of body fluids. This adhesive can establish a stable connection with tissues and can effectively resist excessive swelling caused by body fluid infiltration through subsequent self-crosslinking within the gel network. This reduces the risk of compressing surrounding tissues and nerves, as well as weakening adhesion or even adhesive detachment, ensuring its safety and stability during application.

[0049] 2. This adhesive addresses the shortcomings of current adhesives, which lack antibacterial activity and cannot prevent wound infection. It mimics the antibacterial mechanism of organisms, killing bacteria by releasing clean, efficient, and safe antibacterial agents in situ.

[0050] 3. It addresses the shortcomings of current adhesives that lack bioactivity and cannot promote tissue healing, and simulates the composition and structure of the natural extracellular matrix, providing a favorable microenvironment for tissue regeneration. Attached Figure Description

[0051] Figure 1 The images show the detection of active ester-functionalized sodium hyaluronate (HA-NHS), where (a) is the ultraviolet spectrum and (b) is the NMR spectrum. 1 H spectrum.

[0052] Figure 2 The images show the detection of dopamine-functionalized recombinant type III collagen (Col-Dopa), where (a) is the UV spectrum and (b) is the NMR spectrum. 1 H spectrum.

[0053] Figure 3 Dopamine-functionalized recombinant type III collagen (Col-Dopa) and reduced dopamine-functionalized recombinant type III collagen (Col-Dopa) red XPS spectra of ), where (a) is Col-Dopa and (b) is Col-Dopa. red .

[0054] Figure 4 This demonstrates the adhesive properties of the adhesive on the surface of pigskin.

[0055] Figure 5 In this context, (a) represents (HA-NHS)-Col and (HA-NHS)-(Col-Dopa). red (a) Images of pigskin bonded with gel and soaked in PBS for 7 days before and after; (b) shows the bonding strength.

[0056] Figure 6 (HA-NHS)-(Col-Dopa) red The H2O2 generation curve of ) Detailed Implementation

[0057] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0058] Example 1: A method for preparing a multifunctional tissue adhesive

[0059] Includes the following steps:

[0060] (1) Synthesis of active ester-functionalized sodium hyaluronate (HA-NHS)

[0061] Low molecular weight hyaluronic acid (Mw 50kDa) obtained by enzymatic digestion (purchased from Freda Biotechnology Co., Ltd.) was dissolved in water to obtain a 2wt% HA solution. Following a molar ratio of HA repeating unit: carbodiimide (EDC): N-hydroxysuccinimide (NHS) of 1:2:4, EDC was first added to activate the carboxyl group for 1 hour, followed by the addition of NHS for functional group grafting. After reacting at room temperature for 6 hours, 2% sodium chloride was added sequentially for dissolution, followed by cooling (4℃). Then, 5 volumes of ice-cold ethanol (-20℃) were added to precipitate the product. After filtration, the product was washed multiple times with anhydrous ice-cold ethanol and then freeze-dried under vacuum to obtain HA-NHS. The chemical structure of HA-NHS was analyzed using nuclear magnetic resonance spectroscopy and ultraviolet absorption spectroscopy, and the grafting rate of the NHS group was quantitatively determined.

[0062] like Figure 1 As shown in (a), HA-NHS exhibits a distinct absorption peak at 259 nm, which coincides with the absorption peak position corresponding to the addition of NHS to the HA solution. Meanwhile, as... Figure 1 As shown in (b), by NMR characterization, a proton peak on the methylene group of succinimide ester appeared at the 4H chemical shift of 2.75-2.90 ppm. By integrating with the proton peak on the methyl group of HA (1.80-2.00 ppm, 3H), the grafting rate can be calculated to be 23.25%, indicating the successful synthesis of HA-NHS.

[0063] (2) Synthesis of dopamine-functionalized recombinant type III collagen (Col-Dopa)

[0064] Type III recombinant collagen (Col) was used as the raw material (purchased from Jiangsu Chuangjian Biotechnology Co., Ltd.). It was dissolved at a concentration of 1 wt% in anoxic MES buffer (pH 6), and 2 mg / mL of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl (DMTMM) chloride and 30 mg / mL of dopamine hydrochloride (Dopa) solution were added. After reacting at room temperature for 24 h, the mixture was purified by dialysis at low temperature for 2 days. The resulting dry Col-Dopa powder was obtained by vacuum freeze-drying and stored at -20 °C. The chemical structure of Col-Dopa was analyzed using nuclear magnetic resonance spectroscopy and ultraviolet absorption spectroscopy.

[0065] like Figure 2 As shown in (a), Col-Dopa exhibits a distinct absorption peak at 280 nm, which coincides with the absorption peak position corresponding to the addition of Dopa to the Col solution. Meanwhile, as... Figure 2 In the NMR spectrum of (b) in the figure, the absorption peak at 2.75 ppm of the Col-Dopa group is the methyl proton peak of the dopamine group near the benzene ring, while there is no such absorption peak in the Col group, which proves the successful synthesis of Col-Dopa.

[0066] (3) Reduced dopamine-functionalized recombinant type III collagen (Col-Dopamine) red Preparation of )

[0067] A 1M ascorbic acid solution was prepared, and Col-Dopa was dissolved in it at a concentration of 1 wt%. After reacting at room temperature for 24 hours, the solution was dialyzed in deoxygenated water, and then freeze-dried under vacuum to obtain reduced Col-Dopa. red The proportion of phenolic quinone groups was quantitatively determined using X-ray photoelectron spectroscopy (XPS).

[0068] Depend on Figure 3 The XPS results in (a) show that in the initial synthesized Col-Dopa, the ratio of CO to C=O in the dopamine group modified on Col was approximately 1:1. After reduction with ascorbic acid, the CO ratio increased to 70%, indicating that the benzoquinone group in Col-Dopa could be reduced to a phenol group, thus proving that Col-Dopa... red Successful preparation.

[0069] (4) Preparation of multifunctional tissue adhesive and demonstration of its adhesion properties

[0070] Col-Dopa red A gelation precursor solution A was obtained by dissolving HA-NHS in 0.1M Na2B4O7 solution at a concentration of 120 mg / mL. HA-NHS was then dissolved in hydrochloric acid diluted with a small amount of brilliant blue at a concentration of 60 mg / mL (10... 5 To obtain the pre-adhesive liquid B, use a double-barrel syringe as the delivery tool to mix equal amounts and inject it into the pigskin surface in situ. After it has cured, stretch, bend, and twist the pigskin to observe the adhesion of the adhesive on the pigskin surface.

[0071] Depend on Figure 4 The results show that, since the active ester groups modified on HA can spontaneously undergo amidation reactions with amino or thiol groups in Col and tissue surface proteins, the precursor solution can be rapidly solidified on the surface of pigskin after injection and stably adhere to the surface, and can be resisted by stretching, bending, twisting and other destructive actions without falling off.

[0072] (5) Adhesion strength test

[0073] Col-Dopa red A gelation precursor solution A was obtained by dissolving HA-NHS in 0.1M Na2B4O7 solution at a concentration of 120 mg / mL. HA-NHS was then dissolved in hydrochloric acid dilution solution (10... 5A pre-adhesive solution B was obtained by applying a double-barreled syringe and injected between two pieces of porcine dermal tissue (2.5cm × 7.5cm) adhered to a glass slide to achieve bonding (overlap area 2.5cm × 1.0cm). The overlap shear strength was tested using a biomechanical testing instrument, and the adhesive strength was calculated. To quantitatively characterize the self-reinforcing effect of the adhesive, after bonding, the two pieces of porcine skin were immersed in phosphate-buffered saline (PBS), and the adhesive strength was evaluated after 7 days.

[0074] Depend on Figure 5 As shown in result (a), after soaking the bonded pigskin in PBS for 7 days, due to the large-scale oxidative cross-linking reaction of the reduced Dopa groups in the gel network, (HA-NHS)-(Col-Dopa) red The adhesive has turned noticeably darker and shows no significant swelling, while its cohesive strength and interfacial adhesion to the tissue have been further enhanced. Figure 5 As shown in (b), the Pa increased from the initial 23 kPa to approximately 35 kPa. In contrast, the (HA-NHS)-(Col) component without modified Dopa groups showed significant gel swelling and partial extrusion, and a significant decrease in adhesive strength.

[0075] (6) Determination of hydrogen peroxide (H2O2) generation

[0076] Col-Dopa red A gelation precursor solution A was obtained by dissolving HA-NHS in 0.1M Na2B4O7 solution at a concentration of 120 mg / mL. HA-NHS was then dissolved in hydrochloric acid dilution solution (10... 5 The gelation precursor solution B was obtained by mixing equal volumes using a double-barrel syringe and then injected into a cylindrical mold with a diameter of 15 mm and a height of 7.5 mm. After demolding, the sample was immersed in 5 times its volume of PBS and incubated at 37°C. Samples were taken continuously for 7 days, and the H2O2 release content was determined using an H2O2 kit.

[0077] Depend on Figure 6 The results showed that the adhesive could continuously release H2O2 in situ. After 7 days, the H2O2 concentration in the solution was about 190 μM. In contrast, no H2O2 was detected in the adhesive without the modified Dopa group. This proved that the H2O2 generation ability of the adhesive was entirely due to the reduced Dopa group modified on Col, which generated H2O2 in situ through the electron transfer mechanism during the self-oxidation process.

[0078] Example 2: Synthesis of functionalized sodium hyaluronate with active esters - Condition testing

[0079] Referring to Example 1, when synthesizing active ester-functionalized sodium hyaluronate (HA-NHS), five HA repeating units were set up with molar ratios of carbodiimide (EDC) to N-hydroxysuccinimide (NHS) as follows: Group A 1:1:1; Group B 1:2:2; Group C 1:2:3; Group D 1:3:4; and Group E 1:3:6.

[0080] Project / Group A B C D E Grafting rate of active ester groups 10.2±1.6％ 9.4±1.8％ 15.4±2.1％ 24.5±2.3％ 23.2±2.8％

[0081] Example 3: Recombinant Type III Collagen Reduction Condition Test

[0082] Referring to Example 1, when reducing recombinant type III collagen, five ascorbic acid concentration groups were set up: Group A: 0.01M; Group B: 0.10M; Group C: 0.50M; Group D: 1.00M; Group E: 2.00M, with the rest remaining unchanged.

[0083] Project / Group A B C D E phenolic hydroxyl ratio 52.9±3.2％ 57±4.1％ 64±4.5％ 70.2±5.2％ 71.5±5.5％

[0084] Example 4: Precursor Concentration Condition Test

[0085] Referring to Example 1, four precursor concentration groups were set up during the preparation of the adhesive: Group A: HA-NHS: 30 mg / mL, Col-Dopa red Group A: 60 mg / mL; Group B: HA-NHS: 50 mg / mL, Col-Dopa red Group C: HA-NHS: 70 mg / mL, Col-Dopa red Group D: HA-NHS: 80 mg / mL, Col-Dopa red 160 mg / mL.

[0086]

[0087] Example 5

[0088] Referring to Example 1, sodium hyaluronate in component B was replaced with sodium heparin (purchased from Shanghai Maclean Biochemical Technology Co., Ltd.), while the rest remained unchanged.

[0089] Project / Group Example 5 Bond strength (0d) 32±4.1 (kPa) Bond strength (7d) 47±9.2 (kPa) Hydrogen peroxide concentration (7 days) 185±20.8(μM)

[0090] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for preparing a multifunctional injectable tissue adhesive, characterized in that, Includes the following steps: (1) Preparation of component A: Active protein containing chemically modified reduced polyphenol groups and having RGD sequence, lysine, aspartic acid and / or glutamic acid on its surface is chemically modified with dopamine, and then reduced to obtain reduced dopamine functionalized active protein. Benzoquinone in the reduced dopamine functionalized active protein is reduced to phenol. (2) Preparation of component B: Natural polysaccharide is modified by active esterification to obtain natural polysaccharide containing chemically modified active ester groups; (3) Prepare gelation buffer solution: alkaline buffer solution and acidic solution; (4) Dissolve the powders of components A and B in alkaline buffer and acidic solution respectively to obtain gelation precursor solution A and gelation precursor solution B; (5) Using a double-barrel syringe or a spreading stick, mix the gelation precursor liquid A and the gelation precursor liquid B evenly to form the multifunctional tissue adhesive; The active protein is selected from one or more of the following: recombinant type I collagen, recombinant type III collagen, gelatin, and lysozyme; The active ester group is selected from one or more of the following: succinimide carbonate, succinimide acetate, succinimide propionate, succinimide succinate, and succinimide glutarate; In the gel-forming buffer solution: the inorganic salt in the alkaline buffer solution is selected from sodium tetraborate, sodium carbonate, disodium hydrogen phosphate, or a combination thereof; the acidic substance in the acidic solution is selected from hydrochloric acid, acetic acid, sodium dihydrogen phosphate, or a combination thereof.

2. The preparation method according to claim 1, characterized in that, The ratio of the reduction of the polyphenol groups to phenolic quinones is between 2:8 and 8:

2.

3. The preparation method according to claim 1, characterized in that, The active ester group modification rate is 10-50%.

4. The preparation method according to claim 1, characterized in that, The protein concentration in the gelation precursor solution A is 5-20% (w / v).

5. The preparation method according to claim 1, characterized in that, In the gelation precursor solution B, the mass concentration of natural polysaccharides is 2-10% (w / v).

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