An ultrabionic artificial matrix glue and construction method and application thereof

Abstract

The application discloses an ultrabiomimetic artificial matrix glue and a construction method and application thereof. The ultrabiomimetic artificial matrix glue is a stress stiffening / stress relaxation interpenetrating double network hydrogel composed of spiral polyisocyanide polypeptide (PIC) and alginate (SA), wherein the PIC self-assembles to form a fibrous network structure, and the alginate penetrates into the fibrous structure and pore diameter of the PIC to form an interpenetrating network under the action of an ionic crosslinking agent. The application adopts a method of rapidly mixing two solutions at low temperature and using calcium sulfate instead of calcium chloride, thereby avoiding the phenomenon that the calcium chloride rapidly gels with the alginate. The mixing is performed by means of a connecting lock, the interpenetration of the two networks is enhanced, and a structure similar to an ECM is formed, and meanwhile, the stress stiffening and stress relaxation interactions are exhibited.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials, specifically relating to a super-bionic artificial matrix adhesive, its construction method, and its application. Background Technology

[0002] Biological tissues and organs contain both cellular and noncellular components, which intertwine to form a stable and optimal complex structure. This provides essential biophysical and biochemical signals for cell growth, creating the local microenvironment upon which cells depend for survival, known as the extracellular matrix (ECM). The ECM is the noncellular component of tissues, composed of an intricate 3D network structure. It provides cells with the necessary three-dimensional space, physical support, and signal regulation, and also influences cell development, including migration, proliferation, and differentiation. The ECM comprises thousands of different components, primarily consisting of rigid fibrous structures such as collagen and polysaccharides. Furthermore, the physical and mechanical properties of the ECM affect cell behavior, including cell adhesion, migration, differentiation, and gene expression. Currently, most research focuses on the influence of ECM linear stiffness. However, due to its inherent properties, natural ECM is not a simple linear elastic material, but rather a material with complex mechanical properties, including viscoelastic stress relaxation and nonlinear elastic stress stiffening. These mechanical properties, such as those of nonlinear elastic materials, show exponential changes in stress and strain even under very small external forces; while viscoelasticity is a time-dependent mechanical property of materials, exhibiting stress relaxation behavior under constant deformation or strain increase or creep under constant stress. ECM simultaneously exhibits the phenomenon that the stress decreases over time when a constant strain is applied, which is the stress relaxation characteristic, and the phenomenon that its own hardness increases after being subjected to external forces, which is the stress stiffening characteristic.

[0003] Currently, most single-component artificial hydrogels do not simultaneously possess stress stiffening and stress relaxation properties. In recent years, the emergence of dual-network hydrogels has changed the function of single-component hydrogels. Dual-network hydrogels consist of a rigid first network and a flexible second network; the synergistic effect of these two networks gives them excellent mechanical properties. The high mechanical strength, toughness, and modulus of dual-network hydrogels have led to their wider research scope. Furthermore, significant research progress in dual-network hydrogels in recent years has not only solved the problem of the fragility and breakage of traditional hydrogels but also expanded their application range and extended their service life to some extent. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a method for constructing a stress-rigidifying / stress-relaxing interpenetrating network hydrogel, also known as a super-biomimetic artificial matrix gel. The interpenetrating network hydrogel integrates stress-rigidifying and stress-relaxing properties and can be used as a cell culture substrate for cellular biomaterials to simulate the effects of different mechanical properties on cell behavior in vitro and for two-dimensional and three-dimensional cell culture.

[0005] The biomimetic artificial matrix provided by this invention is a stress-rigidified / stress-relaxed interpenetrating double network hydrogel composed of helical polyisocyanate peptide (PIC) and alginate (SA). In this hydrogel, PIC self-assembles to form a fibrous network structure, and alginate penetrates the fibrous structure and pores of PIC under the action of an ionic crosslinking agent to form an interpenetrating network.

[0006] The spiral polyisocyanate polypeptide (PIC) has the structural formula shown in formula (I):

[0007]

[0008] In equation (I), n represents the degree of polymerization, n = 50 to 50000, specifically 500-5000; x = 2-4, specifically 3;

[0009] The molecular weight of the alginate is 5kDa-3000kDa;

[0010] The mass ratio of helical polyisocyanate peptide (PIC) to alginate can be 1:200-20:1, specifically 2:5;

[0011] The alginate may specifically be sodium alginate (SA);

[0012] The ionic crosslinking agent may be Ba. 2+ and / or Ca 2+ Specifically, it can be CaSO4.

[0013] Furthermore, the biomimetic artificial matrix gel can also be functionalized, that is, bioactive molecules can be modified into the interpenetrating double network hydrogel composed of helical polyisocyanate peptides (PIC) and alginate.

[0014] The bioactive molecule may be selected from at least one of peptides, nucleic acids, and growth factors, specifically the adhesion peptide RGD.

[0015] The present invention also provides a method for constructing the above-mentioned biomimetic artificial matrix adhesive.

[0016] The method for constructing a biomimetic artificial matrix provided by this invention includes the following steps:

[0017] 1) Dissolve the helical polyisocyanate polypeptide (PIC) in a solvent, add an ionic crosslinking agent, and obtain solution 1;

[0018] 2) Dissolve alginate in a solvent to obtain solution 2;

[0019] 3) Mix solution 1 and solution 2 rapidly and heat to obtain PIC / alginate interpenetrating network hydrogel. PIC assembles into a fibrous network structure after heating, and alginate penetrates the fibrous structure and pores of PIC under the crosslinking of an ionic crosslinking agent, forming an interpenetrating network.

[0020] In step 1) of the above method, the solvent can be at least one of water, PBS buffer solution, and DMEM, specifically DMEM;

[0021] PIC dissolves in solvents at low temperatures of 0℃-15℃;

[0022] The ionic crosslinking agent may be Ba. 2+ and / or Ca 2+ Specifically, it can be CaSO4;

[0023] The concentration of PIC in solution 1 is 0.1-20.0 mg / mL, and the concentration of the ionic crosslinking agent is 0-100 mM, with the endpoint 0 being unacceptable;

[0024] In step 2), the solvent may be at least one of water, PBS buffer solution, and DMEM, specifically DMEM;

[0025] The concentration of alginate in solution 2 can be 0.1-40 mg / mL (the endpoint value of 0 is not acceptable);

[0026] In step 3), the solution mixing is achieved by connecting syringe 1 containing alginate hydrogel to syringe 2 containing PIC solution through a Luer lock connector;

[0027] In the mixed system, the concentration of PIC can be 0.1-20 mg / mL, the concentration of the ionic crosslinking agent can be 0.1-100 mM (endpoint 0 is not acceptable), and the concentration of alginate can be 0.1-40 mg / mL.

[0028] The temperature for the heating can be 15-50℃, specifically 37℃.

[0029] In the PIC / alginate interpenetrating network hydrogel system, stress stiffening properties can be controlled by adjusting the chain length of the PIC polymer, while stress relaxation properties can be controlled by adjusting the molecular weight of the alginate. Therefore, by adjusting the concentration, chain length, and molecular weight of different components, individual mechanical properties such as matrix hardness, stress stiffening, and stress relaxation can be independently regulated.

[0030] The present invention also provides a functionalized modified biomimetic artificial matrix adhesive, specifically a biomimetic artificial matrix adhesive modified with adhesion peptide RGD.

[0031] The biomimetic artificial matrix modified with the adhesion peptide RGD is prepared by a method comprising the following steps:

[0032] 1) Preparation of PIC-RGD

[0033] Diphenylcyclooctyne-tetraethylene glycol-active ester (DBCO-PEG4-NHS) was reacted with adhesive peptide (GGGGRGDS) to obtain DBCO-GRGDS. Then, PIC with azide was reacted with DBCO-GRGDS by click chemistry to obtain PIC-RGD.

[0034] The PIC structure with azide is shown below:

[0035]

[0036] n represents the degree of polymerization, n = 50 to 50000, specifically 500-5000;

[0037] 2) Preparation of SA-RGD

[0038] The lyophilized alginate powder was dissolved in MES buffer solution, and then reacted with sulfo-NHS, EDC, and adhesion peptides (GRGDS) via carbodiimide and click chemistry to obtain alginate-RGD;

[0039] 3) Preparation of PIC-RGD / alginate-RGD hydrogel

[0040] PIC-RGD and calcium ions were dissolved in a culture medium, mixed with alginate-RGD, and cross-linked to obtain a PIC / alginate hydrogel containing the adhesive peptide RGD.

[0041] In step 1) of the above method, the ratio of DBCO-PEG4-NHS to adhesion peptide (GRGDS) and PIC with azide can be 0-20mg: 0-15mg: 0-200mg, with the endpoint value of 0 being unacceptable. Specifically, it can be 1.3mg: 1.0mg: 15mg.

[0042] The reaction temperature of DBCO-PEG4-NHS with adhesion peptides (GRGDS) can be room temperature, and the reaction time can be 1-28h.

[0043] The temperature of the click chemical reaction can be room temperature, and the time can be 18-28 hours.

[0044] In step 2), 1 g of lyophilized alginate was added to every 100 mL of MES buffer. After dissolution, Soulf-NHS, EDC and RGD powder were quickly added and reacted for 20 h. The reaction was quenched with 18 mg of hydroxylamine hydrochloride per g of alginate. The resulting system was dialyzed in deionized water and sodium chloride solution to remove unreacted alginate. After dialyzing, alginate-RGD was obtained, which was then freeze-dried to obtain alginate-RGD.

[0045] In step 3) of the above method, the calcium concentration in PIC-RGD and calcium ion culture medium solution can be 488mM.

[0046] Fill syringe 1 with PIC-RGD and calcium ion culture medium solution, and fill syringe 2 with alginate-RGD solution. Connect syringe 1 and 2 with Luer lock connector, mix and quickly dispense into the rheometer Pelplate or cell culture plate to obtain PIC / alginate hydrogel.

[0047] The application of the aforementioned biomimetic artificial matrix gel and functionalized biomimetic artificial matrix gel as in vitro simulated extracellular matrix materials in two-dimensional and three-dimensional cell culture also falls within the scope of protection of this invention.

[0048] This invention rapidly mixes a stress-stiffened PIC hydrogel with a stress-relaxed SA hydrogel to form a PIC / SA interpenetrating double-network hydrogel with stress stiffening and relaxation, for the three-dimensional culture of human mesenchymal stem cells (hMSCs). The PIC and SA hydrogels mimic the fibrous structure and mechanical properties of natural ECM materials. The PIC hydrogel is heated to a solution above its critical temperature T. gel Gel formation occurs at 20°C. Cooling the gel below 20°C reverses this process, turning it into a solution. Due to their fibrous and porous structure, PIC hydrogels are soft at low concentrations, but their stiffness increases with material deformation or stress, a process known as stress stiffening. This process is also present in biopolymer-based gels, such as actin and collagen, which are key components of the ECM that coordinate cell movement and differentiation. Therefore, PIC hydrogels can serve as an easily aggregated alternative scaffold to mimic the fibrous structure and mechanical properties of natural ECM materials such as collagen and fibrin.

[0049] Sodium alginate (SA) is a linear polysaccharide formed by the linkage of repeating sugar units β-D-mannuronic acid (M fragment) and α-L-guluronic acid (G fragment) via 1,4-glycosidic bonds. The G fragment can interact with calcium ions, i.e., sodium ions exchange with calcium ions. Therefore, sodium alginate, as a modifiable viscoelastic material, can be used as a cell culture medium for cell culture and research.

[0050] The stress-stiffening / stress-relaxation interpenetrating network hydrogel composed of SA hydrogel and PIC hydrogel exhibits a synergistic effect, which allows for independent adjustment of stress stiffening and stress relaxation while maintaining consistent modulus.

[0051] The present invention is characterized by a biomaterial system, such as a hydrogel, composed of an interpenetrating network of alginate and helical polyisocyanate, which separates gel stiffness from the effects of gel mechanical properties such as stiffness and stress stiffening and stress relaxation.

[0052] The PIC hydrogel of this invention exhibits a structure and mechanical properties very similar to collagen and fibroin, displaying a characteristic hardening response under high strain and reversible thermal response behavior for easy application and removal. The SA hydrogel's structure and mechanical properties give it a stress relaxation effect, similar to the polysaccharides in ECM. This invention employs a method of rapid mixing of the two solutions at low temperature and uses calcium sulfate instead of calcium chloride to avoid the rapid gelation of calcium chloride with alginate. Mixing via interlocking enhances the interpenetration of the two networks and forms an ECM-like structure, while simultaneously exhibiting the interaction of stress stiffening and stress relaxation.

[0053] This invention utilizes the constructed interpenetrating network to conduct two-dimensional and three-dimensional cell culture studies on the influence of mechanical properties on cell behavior.

[0054] This invention designs a PIC / SA interpenetrating network hydrogel based on the cross-linking mechanism of dual hydrogels. Spiral polyisocyanate (PIC) hydrogel is a reversible, temperature-sensitive hydrogel capable of mimicking the fibrous structure and mechanical properties of the natural extracellular matrix. PIC is liquid below its gelation temperature; as the temperature rises to the gelation temperature, multiple polymer chains aggregate and bind together to form a fibrous network that mimics collagen in the extracellular matrix and exhibits similar mechanical properties, such as stress stiffening. Sodium alginate (SA) is an ionic natural polymer hydrogel where divalent cations (e.g., calcium ions) can ionically cross-link different alginate chains, forming a gel. These weak ionic cross-links can break under stress and then reform, causing local matrix flow to dissipate stress, resulting in macroscopic stress relaxation in the hydrogel. However, the stress relaxation of high-molecular-weight SA is slow, taking about kiloseconds, while the relaxation of biological tissues and organs takes only a few hundred seconds. Therefore, by adjusting the molecular weight of SA and the chain length of PIC, an interpenetrating network hydrogel with high or low stress stiffening / fast or slow stress relaxation can be constructed to mimic the mechanical properties of the extracellular matrix. Attached Figure Description

[0055] Figure 1 The storage modulus, stress stiffening mechanical properties, stress relaxation mechanical properties, and plasticity of the PIC / SA interpenetrating network hydrogel in Example 1 of this invention were measured using a rheometer.

[0056] Figure 2 The modulus of the hydrogel prepared in Example 2 of this invention was stabilized at 700 Pa, and the results of nonlinear elasticity test and viscoelasticity test were subsequently performed on a rheometer.

[0057] Figure 3 The cell proliferation experiment and cell death results were obtained after 7 days of cell culture of the composite hydrogel prepared in Example 3 of this invention.

[0058] Figure 4 The results of cell culture experiments and morphological analysis after 7 days were obtained for the composite hydrogel prepared in Example 3 of this invention. Detailed Implementation

[0059] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0060] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0061] The hydrogel cell cultures used in the following examples were prepared according to the literature Diba M, Spaans S, Hendrikse SIS, Bastings MMC, Schottman MJG, van Sprang JF, Wu DJ, Hoeben FJM, Janssen HM, Dankers PYW. Engineering the Dynamics of Cell Adhesion Cues in Supramolecular Hydrogels for Facile Control over Cell Encapsulation and Behavior. Adv Mater. 2021 Sep; 33(37):e2008111.

[0062] The reference for the preparation of PIC with azide is Synthetic Extracellular Matrices as a Toolbox to Tune Stem Cell Secretome, Kaizheng Liu, ACS Appl. Mater. Interfaces 2020, 12, 56723-56730.

[0063] Example 1: Preparation of PIC / SA interpenetrating network hydrogel and rheological testing of PIC / SA interpenetrating network hydrogel

[0064] A calcium sulfate dihydrate solution was first prepared for crosslinking the alginate network. For each PIC / SA interpenetrating network hydrogel, 0.2 mL of PIC (chain length n = 5000, x = 3) (0.2 mg / mL, dissolved in DMEM) solution containing 5 μL of 10 mM calcium sulfate and 0.2 mL of SA solution (SA molecular weight 300 kDa, 0.5 mg / mL, dissolved in DMEM) was rapidly crosslinked and mixed under Luer lock linkage and then deposited on a pre-cooled Peltier plate of a rotationally controlled rheometer (TA instrument). A 20 mm aluminum plate was immediately brought into contact with the PIC / SA before gelation began, forming a 20 mm PIC / SA interpenetrating network hydrogel disk. The plate was heated to 37 °C, and the storage modulus at 1 Hz under 1% strain was measured and recorded over time. The nonlinear properties of the PIC / SA interpenetrating network hydrogel were determined using a prestressing method. The PIC / SA interpenetrating network hydrogel exhibited stress stiffening properties, with the critical stress denoted as σ. c Under constant strain, the relaxation time is defined as the time when the stress decreases to half of the initial stress over time. The results show that the stress relaxation time of the PIC / SA interpenetrating network hydrogel is ~1000 seconds. At a frequency of 1 rad / s, pressures of 1 Pa, 10 Pa, and 40 Pa were applied for 300 s, and the pressure was released for one hour to test the plasticity of the hydrogel under different pressures.

[0065] Figure 1 Figure a shows the modulus of the PIC / SA interpenetrating network hydrogel. Figure 1 Figure b shows the nonlinear properties of the PIC / SA interpenetrating network hydrogel, with a critical stress of approximately 10 Pa. Figure 1 Figure c shows the stress relaxation of the PIC / SA interpenetrating network hydrogel, with a stress relaxation time of 1000 s. Figure 1 Figure d shows the plasticity of the PIC / SA interpenetrating network hydrogel at 1 Pa, 10 Pa, and 40 Pa.

[0066] Example 2: Independently controlling the nonlinear mechanical properties of PIC / SA interpenetrating network hydrogels

[0067] According to the method for preparing PIC / SA interpenetrating network hydrogels in Example 1, by changing the chain length of PIC, the critical stress value of long-chain PIC is higher, while that of short-chain PIC is lower. Adjusting the chain length of PIC can regulate the stress stiffening properties of the interpenetrating network. When the molecular weight of SA is high, its stress relaxation time is longer; when the molecular weight is low, the stress relaxation time is shorter. Changing the molecular weight of SA can control the mechanical properties of stress relaxation. Therefore, the mechanical properties of the PIC / SA interpenetrating network can be controlled and adjusted. PIC / SA1 uses short-chain PIC with a degree of polymerization n = 1000, and the molecular weight of SA is 300 kDa; PIC / SA2 uses long-chain PIC with a degree of polymerization n = 5000, and the molecular weight of SA is 300 kDa; PIC / SA3 uses long-chain PIC with a degree of polymerization n = 5000, and the molecular weight of SA is 50 kDa.

[0068] The results showed Figure 2 In interpenetrating networks, by fixing the stiffness and concentration, the mechanical properties of PIC / SA interpenetrating network hydrogels can be adjusted by controlling the chain length of PIC and the molecular weight of SA to achieve the purpose of biomimetic extracellular matrix.

[0069] Figure 2 In the figure, 'a' represents the modulus result of different combinations of interpenetrating networks with constant uniform stiffness. Figure 2 Figure b shows the stress stiffening results after mechanical property adjustment. PIC / SA-1 and PIC / SA-2 exhibit different stress stiffening, while PIC / SA-3 exhibits the same stress stiffening. PIC / SA-1 and PIC / SA-2 exhibit the same stress relaxation, while PIC / SA-3 exhibits different stress relaxation.

[0070] Example 3: PIC / SA interpenetrating network hydrogel for 3D cell culture

[0071] Add 1 g of lyophilized sodium alginate (molecular weight 50 kDa) to every 100 mL of LME S buffer, dissolve, and then quickly add 0.3 g of Soulf-NHS, 0.5 g of EDC, and 0.15 g of RGD powder. React for 20 h. Quench the reaction with 18 mg of hydroxylamine hydrochloride per g of sodium alginate. Dialyze in deionized sodium chloride solution for 3 days to remove unreacted sodium alginate. Use the following amounts of NaCl per liter of deionized water for dialysis: 30 g; 25 g; 20 g; 15 g; 10 g; 5 g, dialyze for half a day for each. Freeze SA-RGD overnight at -20°C to -30°C. Lyophilize for 3 days until SA-RGD is completely dry.

[0072] Preparation of PIC-RGD, i.e., GRGDS linked to PIC (n=5000) hydrogel: 1 mg of GRGDS (adhesive peptide) was dissolved in 167 μL of borate buffer (pH=8.4). 1.3 mg of DBCO-PEG4-NHS was dissolved in 217 μL of DMSO, and after complete dissolution, it was added to the peptide solution in borate buffer and stirred on a stir plate at room temperature for 24 hours. Separately, 15 mg of azide-containing PIC was dissolved in 6 mL of acetonitrile, and then 82 μL of the synthesized DBCO-GRGDS was added, and stirred at room temperature for 24 hours. The solution was then precipitated in diisopropyl ether, collected by centrifugation, and air-dried for 24 hours.

[0073] The SA-RGD solution is loaded into syringe 2. High molecular weight alginate is viscous, so when using a pipette to pipette small volumes, it is necessary to let it linger for a while to achieve the required volume.

[0074] To remove any trapped air bubbles within the alginate: For highly viscous alginate (high molecular weight): Pull the syringe plunger down and then back up several times to remove air bubbles. Introducing air bubbles into the hydrogel may interfere with cell experiments or affect local mechanical properties. For less viscous alginate (low molecular weight): Pull the syringe plunger down, cover the syringe opening with your gloved finger, and firmly tap the syringe (e.g., with a marker). Air bubbles should dislodge and rise to the surface. After removing your finger from the syringe opening, pull the syringe plunger down first to avoid expelling any alginate droplets.

[0075] PIC-RGD and calcium ion culture medium solution are loaded into syringe 1 (where the calcium sulfate concentration is 488mM), syringe 1 and 2 are connected with Luer lock connector, mixed and then quickly dispensed into the rheometer Pelplate or 96-well cell culture plate to obtain PIC / sodium alginate hydrogel.

[0076] PIC / SA interpenetrating network hydrogels with different mechanical properties but the same stiffness were prepared in standard tissue culture 96-well plates to encapsulate cells. First, cells were encapsulated in 1×10⁶ wells. 5 The PIC / SA hydrogel was mixed with an equal volume of PIC chain length hydrogels (4℃) at a density of PIC / SA, and then mixed with SA via Luerlock and deposited into 96-well plates for culture. The plates were divided into three groups: PIC / SA-1, PIC / SA-2, and PIC / SA-3, with 100 μL per well and three replicates per group. The plates were then transferred to an incubator at 37℃ and 5% CO2 for equilibration for 20 minutes. After gel formation, 100 μL of medium was added. The plates were cultured for 7 days, with the medium replaced every two days.

[0077] according to Figure 3As shown, cell proliferation tests were performed on cells cultured in an interpenetrating network for 7 days, revealing a small but not significant proliferation. Live / dead staining on day 7 showed that dead cells were spherical, while extended spindle-shaped cells remained intact.

[0078] Figure 4 Images of interpenetrating network three-dimensional cell cultures on day 7 were selected. All groups showed some degree of elongation. The PIC / SA-1 group, with rapid stress relaxation and high stress stiffening, exhibited a longer elongation. The PIC / SA-2 group, with low stress stiffening and slow stress relaxation, showed a moderate elongation and branching. The PIC / SA-3 hydrogel, with low stress stiffening and rapid stress relaxation, showed a shorter elongation. Scale bar: 100 μm.

[0079] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for constructing a super-biomimetic artificial matrix adhesive, The biomimetic artificial matrix gel is a stress-rigidifying / stress-relaxing interpenetrating double-network hydrogel composed of helical polyisocyanate peptide (PIC) and alginate. PIC self-assembles to form a fibrous network structure, and alginate penetrates the fibrous structure and pores of PIC under the action of ionic crosslinking agents to form an interpenetrating network; The structure of the helical polyisocyanate polypeptide is shown in formula (I): In formula (I), n represents the degree of polymerization, n = 50 to 50000; x = 2-4; The molecular weight of the alginate is 5kDa-3000kDa; The mass ratio of helical polyisocyanate peptide to alginate is 1:200 - 20:1; The ionic crosslinking agent is CaSO4; The method includes the following steps: 1) Dissolve the helical polyisocyanate peptide in a solvent, add an ionic crosslinking agent, and obtain solution 1; 2) Dissolve alginate in a solvent to obtain solution 2; 3) Mix solution 1 and solution 2 rapidly and heat to obtain a helical polyisocyanate / alginate interpenetrating network hydrogel. The helical polyisocyanate assembles into a fibrous network structure after heating, and the alginate penetrates the fibrous structure and pores of the helical polyisocyanate fibrous structure under the cross-linking of the ionic cross-linking agent to form an interpenetrating network. In step 1), the helical polyisocyanate peptide is dissolved in a solvent at a low temperature of 0℃-15℃; In step 3), solution mixing is achieved by connecting syringe 1 containing alginate hydrogel to syringe 2 containing spiral polyisocyanate peptide solution via Luer lock connector; The temperature for the heating is 15-50 °C.

2. The method according to claim 1, characterized in that, The alginate is sodium alginate.

3. The method according to claim 1, characterized in that, The super-bionic artificial matrix gel is also functionalized by modifying the interpenetrating double network hydrogel composed of helical polyisocyanate peptides and alginate with bioactive molecules. The bioactive molecule is selected from at least one of polypeptides, nucleic acids, and growth factors.

4. The method according to claim 1, characterized in that, In step 1), the solvent is at least one of water, PBS buffer solution, and DMEM. The concentration of the helical polyisocyanate polypeptide in solution 1 is 0.1-20 mg / mL, and the concentration of the ionic crosslinking agent is 0-100 mM, with the endpoint 0 being undesirable; In step 2), the solvent is at least one of water, PBS buffer solution, and DMEM. The concentration of alginate in solution 2 is 0.1-40 mg / mL; In the mixed system, the concentration of the helical polyisocyanate peptide is 0.1-20 mg / mL, the concentration of the ionic crosslinking agent is 0.1-100 mM, and the concentration of alginate is 0.1-40 mg / mL.

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