A method for improving room temperature fast setting properties of gelatin-based hydrogels

Abstract

The present application relates to gelatin production technical field, disclose a kind of method for improving the room temperature easy solidification characteristics of gelatin-based hydrogel.A kind of gelatin-based hydrogel, including the following preparation raw materials: gelatin, hydrogen bond competitive material and water;The hydrogen bond competitive material is selected from at least one of metformin, L-arginine, amide small molecule crosslinking agent, polyarginine, polyurea compound.The gelatin-based hydrogel provided by the present application, gelation temperature is less than 25 DEG C, not easy to solidify at room temperature (20-25 DEG C), compared with ordinary gelatin solution, gel room temperature easy solidification characteristics are improved;The preparation method of gelatin-based hydrogel provided by the present application, by simple physical blending, introduce the material with competitive hydrogen bond into gelatin aqueous solution, to improve the room temperature easy solidification of gelatin aqueous solution, compared with traditional covalent modification method, the present application is simple in operation, applicability is strong, and does not cause damage to original structure of gelatin, and repeatability is good.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of gelatin production, in particular to a method for improving the room-temperature easy-solidification property of gelatin-based hydrogel. BACKGROUND

[0002] Gelatin is a colloidal substance widely used in food industry, medicine and biological materials, etc. It is mainly derived from the connective tissue of animals and is partially hydrolyzed from animal collagen. In the biomedical field, gelatin, as a natural polymer material, is widely used due to its good biocompatibility, degradability and modifiable functionality. For example, in the field of tissue engineering, gelatin can promote cell growth and new tissue formation as a scaffold material; in the field of drug carriers, gelatin microspheres and nanoparticles can achieve drug release and targeted delivery, reducing side effects; gelatin can also be used for surgical sutures, which have biodegradability and can reduce the need for secondary surgery; in addition, gelatin-based adhesives can be used for tissue adhesion in minimally invasive surgery, which can improve surgical efficiency.

[0003] However, the gelatin in the above application scenarios is mostly in the form of gelatin aqueous solution. At low temperature or even room temperature (20-25℃), the gelatin aqueous solution is prone to "sol-gel" transformation, which brings great inconvenience to use. For example, in the case of injection, it is easy to block the pipeline. In addition, in the preparation and storage process of gelatin aqueous solution, in order to maintain its fluidity, specific temperature control is required, which increases the complexity and cost of operation.

[0004] The existing technology mainly overcomes the room-temperature easy-solidification property of gelatin by changing the molecular structure of gelatin. Common methods include biological modification and chemical modification. Biological modification is a relatively mild method that uses enzymes or microorganisms to change the molecular structure of gelatin. However, the cost is relatively high, and the dependence on enzymes or microorganisms may limit its application in large-scale production. Chemical modification method changes the chemical properties of gelatin molecules by introducing new chemical groups or cross-linking agents. For example, special groups (such as catechol) are modified on the side chain of gelatin, thereby reducing the "sol-gel" transformation temperature of gelatin aqueous solution to below room temperature, and improving its injectability. However, covalent chemical modification method is complex to operate, and due to the difficulty in accurately controlling the modification process, it may lead to poor uniformity of the chemical structure of modified gelatin between different batches, and may also cause the residual of toxic substances. SUMMARY

[0005] The present application aims to solve at least one of the above technical problems in the prior art. To this end, one of the purposes of the present application is to provide a gelatin-based hydrogel; the second purpose of the present application is to provide a preparation method of the gelatin-based hydrogel; the third purpose of the present application is to provide an application of the gelatin-based hydrogel; and the fourth purpose of the present application is to provide a gelatin-based biological ink.

[0006] To achieve the above object, the technical scheme adopted by the present application is:

[0007] The first aspect of the present application provides a gelatin-based hydrogel, comprising the following raw materials: gelatin, hydrogen bond competitive material and water.

[0008] The hydrogen bond competitive material is selected from at least one of metformin, L-arginine, an amide small molecule crosslinking agent, polyarginine, and a polyurea compound.

[0009] The basic principle of the present application is explained as follows:

[0010] The property of easy solidification of gelatin aqueous solution at room temperature is closely related to its secondary structure. The gelatin molecules are long chains composed of amino acids. These long chains can form a three-dimensional network structure through strong hydrogen bond interaction at a certain temperature (25-35℃), thereby forming a gel. Hydrogen bond is a special intermolecular force, stronger than ordinary van der Waals force, but weaker than covalent bond and ionic bond, and usually occurs between atoms with high electronegativity (such as oxygen, nitrogen) and hydrogen atoms. In the present application, materials with competitive hydrogen bond interaction are introduced into the gelatin hydrogel network. The introduction of these materials can form new hydrogen bonds with the hydrogen bond donors or acceptors in the gelatin molecules, thereby replacing the original intermolecular hydrogen bond interaction in the gelatin hydrogel network, achieving the purpose of reducing the "sol-gel" transition temperature of gelatin, and thus improving the room temperature easy solidification property of gelatin aqueous solution.

[0011] In some embodiments of the present application, the mass ratio of gelatin to water is 1:(3-20).

[0012] In some specific embodiments of the present application, the mass ratio of gelatin to water is 1:(10-20).

[0013] In some embodiments of the present application, the mass ratio of hydrogen bond competitive material to gelatin is 1:(0.25-6).

[0014] In some specific embodiments of the present application, the mass ratio of hydrogen bond competitive material to gelatin is 1:(0.5-5).

[0015] In some embodiments of the present application, the amide small molecule crosslinking agent is selected from at least one of a small molecule diamine solution, a carboxylic acid derivative, and a polyamino compound.

[0016] In some embodiments of the present application, the polyamino compound is selected from at least one of 1,4-bis(3-aminopropyl)piperazine, N,N-bis(3-aminopropyl)methylamine, tris(2-aminoethyl)amine, tris(3-aminopropyl)amine, and N,N,N',N'-tetra(3-aminopropyl)-1,4-butanediamine.

[0017] In some embodiments of the present application, the weight average molecular weight of the polyarginine is 3000-5000 Da.

[0018] In some embodiments of the present application, the weight average molecular weight of the polyurea compound is 1000-5000 Da.

[0019] In some embodiments of the present application, the gelation temperature of the gelatin-based hydrogel is less than 25℃.

[0020] In some embodiments of the present application, the gelation temperature of the gelatin-based hydrogel is less than 25℃ and greater than or equal to 15℃.

[0021] The second aspect of the present application provides a preparation method of the gelatin-based hydrogel of the first aspect of the present application, comprising the following steps:

[0022] The gelatin is dissolved in water to form a gelatin aqueous solution, and then the hydrogen bond competitive material is added and uniformly mixed to obtain the gelatin-based hydrogel.

[0023] In some embodiments of the present application, the hydrogen bond competitive material is uniformly mixed with the gelatin aqueous solution by stirring.

[0024] In some embodiments of the present application, the stirring speed is 1000-3000 rpm.

[0025] In some embodiments of the present application, after the hydrogen bond competitive material is uniformly mixed with the gelatin aqueous solution, the method further comprises the step of testing the rheological viscosity and / or thermal stability and / or macroscopic coagulation of the gelatin aqueous solution.

[0026] In the present application, after introducing the hydrogen bond competitive material into the gelatin aqueous solution, the rheological viscosity, thermal stability and macroscopic coagulation of the gelatin aqueous solution are tested to verify the performance changes of the gelatin solution after introducing the hydrogen bond competitive material, so as to prolong the time for the gelatin aqueous solution to remain in liquid state at room temperature, thereby improving the use flexibility and convenience in various applications.

[0027] The third aspect of the present application provides the use of the gelatin-based hydrogel of the first aspect of the present application in the preparation of gelatin-based bio-ink and / or injectable gelatin-based hydrogel microspheres and / or injectable gelatin-based sustained-release system and / or gelatin-based biological composite material.

[0028] The gelatin-based hydrogel provided by the present application has a gelation temperature of less than 25℃, which is lower than that of ordinary gelatin aqueous solution. When used for preparing gelatin-based bio-ink, injectable gelatin-based hydrogel microspheres, injectable gelatin-based sustained-release system or gelatin-based biological composite material, it can ensure that the product is in a stable state at room temperature and is not easy to change into gel.

[0029] The fourth aspect of the present invention provides a gelatin-based bio-ink, comprising the gelatin-based hydrogel described in the first aspect of the present invention.

[0030] In some embodiments of the present invention, the gelatin-based bio-ink is applied to biological 3D printing.

[0031] In some embodiments of the present invention, the bio-3D printing temperature of the gelatin-based bio-ink is less than or equal to 20°C.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1) The gelatin-based hydrogel provided by the present invention has a gelation temperature of less than 25°C and is not easy to solidify at room temperature (20-25°C). Compared with ordinary gelatin solutions, the easy solidification property at room temperature is improved;

[0034] 2) The present invention provides a method for preparing gelatin-based hydrogels. By introducing a material with competitive hydrogen bonds into a gelatin aqueous solution through simple physical blending, the room-temperature coagulation properties of the gelatin aqueous solution are improved, and the gelation temperature of the gelatin-based hydrogel is reduced to below 25°C, allowing it to be used normally at room temperature. Compared with traditional covalent modification methods, this method is simple to operate, has strong applicability, does not damage the original structure of the gelatin, has good reproducibility, and is suitable for promotion.

[0035] 3) The gelatin-based hydrogel provided by the present invention is not easy to solidify at room temperature and can be used to prepare gelatin-based bio-inks, injectable gelatin-based hydrogel microspheres, injectable gelatin-based sustained-release systems or gelatin-based biological composite materials, ensuring that the product is stable at room temperature and is not easy to turn into a gel;

[0036] 4) The gelatin-based bio-ink provided by the present invention can be applied to biological 3D printing, the printing temperature can be less than or equal to 20°C, and the printed pattern fidelity is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The rheological viscosity test results of the gelatin-based hydrogel in Example 3;

[0038] Figure 2 The rheological viscosity test results of the gelatin-based hydrogel in Example 7;

[0039] Figure 3 The rheological viscosity test results of the gelatin-based hydrogel in Example 11;

[0040] Figure 4 Comparison of the fluidity of the gelatin-based hydrogels in Examples 1-4 and the gelatin aqueous solution in Comparative Example 1 after standing at 25°C for 30 minutes and then inverting;

[0041] Figure 5 To compare the flowability of the gelatin-based hydrogel in Examples 5-8 with the gelatin aqueous solution in Comparative Example 1 after being left to stand at 25°C for 30 min and then being inverted;

[0042] Figure 6 To compare the flowability of the gelatin-based hydrogel in Examples 9-12 with the gelatin aqueous solution in Comparative Example 1 after being left to stand at 25°C for 30 min and then being inverted;

[0043] Figure 7 To provide a biological 3D printing real scene photo in Application Example 1. DETAILED DESCRIPTION

[0044] The present application will be further described in details by specific examples. The raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial channels or can be obtained by prior art methods, unless otherwise specified. Unless otherwise specified, the test or test method is a conventional method in the art.

[0045] Example 1

[0046] In this example, a gelatin-based hydrogel was prepared using metformin as a hydrogen bond competitive material, and the steps were as follows:

[0047] 15 g of gelatin powder was dissolved in 100 mL of water to prepare a gelatin aqueous solution with a concentration of 15 wt%, and then 5 g of metformin was added to the gelatin aqueous solution. The metformin and the gelatin aqueous solution were mixed uniformly at a stirring speed of 2000 rpm to obtain a gelatin-based hydrogel. The gelatin-based hydrogel was left to stand at room temperature, and the solidification of the gelatin-based hydrogel at room temperature was observed.

[0048] Example 2

[0049] In this example, a gelatin-based hydrogel was prepared using metformin as a hydrogen bond competitive material, and the steps were as follows:

[0050] 15 g of gelatin powder was dissolved in 100 mL of water to prepare a gelatin aqueous solution with a concentration of 15 wt%, and then 10 g of metformin was added to the gelatin aqueous solution. The metformin and the gelatin aqueous solution were mixed uniformly at a stirring speed of 2000 rpm to obtain a gelatin-based hydrogel. The gelatin-based hydrogel was left to stand at room temperature, and the solidification of the gelatin-based hydrogel at room temperature was observed.

[0051] Example 3

[0052] In this example, a gelatin-based hydrogel was prepared using metformin as a hydrogen bond competitive material, and the steps were as follows:

[0053] Dissolve 15 g of gelatin powder in 100 mL of water to prepare a gelatin aqueous solution with a concentration of 15 wt%, and then add 15 g of metformin to the gelatin aqueous solution, and fully stir at a rotation speed of 2000 rpm to mix the metformin and the gelatin aqueous solution uniformly, to obtain a gelatin-based hydrogel. Place the gelatin-based hydrogel at room temperature, and observe the gelation of the gelatin-based hydrogel at room temperature.

[0054] Example 4

[0055] In this example, a gelatin-based hydrogel is prepared by using metformin as a hydrogen bond competitive material, and the steps are as follows:

[0056] Dissolve 15 g of gelatin powder in 100 mL of water to prepare a gelatin aqueous solution with a concentration of 15 wt%, and then add 20 g of metformin to the gelatin aqueous solution, and fully stir at a rotation speed of 2000 rpm to mix the metformin and the gelatin aqueous solution uniformly, to obtain a gelatin-based hydrogel. Place the gelatin-based hydrogel at room temperature, and observe the gelation of the gelatin-based hydrogel at room temperature.

[0057] Example 5

[0058] In this example, a gelatin-based hydrogel is prepared by using L-arginine as a hydrogen bond competitive material, and the steps are as follows:

[0059] Dissolve 15 g of gelatin powder in 100 mL of water to prepare a gelatin aqueous solution with a concentration of 15 wt%, and then add 5 g of L-arginine to the gelatin aqueous solution, and fully stir at a rotation speed of 2000 rpm to mix the L-arginine and the gelatin aqueous solution uniformly, to obtain a gelatin-based hydrogel. Place the gelatin-based hydrogel at room temperature, and observe the gelation of the gelatin-based hydrogel at room temperature.

[0060] Example 6

[0061] In this example, a gelatin-based hydrogel is prepared by using L-arginine as a hydrogen bond competitive material, and the steps are as follows:

[0062] Dissolve 15 g of gelatin powder in 100 mL of water to prepare a gelatin aqueous solution with a concentration of 15 wt%, and then add 10 g of L-arginine to the gelatin aqueous solution, and fully stir at a rotation speed of 2000 rpm to mix the L-arginine and the gelatin aqueous solution uniformly, to obtain a gelatin-based hydrogel. Place the gelatin-based hydrogel at room temperature, and observe the gelation of the gelatin-based hydrogel at room temperature.

[0063] Example 7

[0064] In this example, a gelatin-based hydrogel is prepared by using L-arginine as a hydrogen bond competitive material, and the steps are as follows:

[0065] Dissolve 15 g of gelatin powder in 100 mL of water to prepare a gelatin aqueous solution with a concentration of 15 wt%, and then add 15 g of L-arginine to the gelatin aqueous solution, and fully stir at a rotation speed of 2000 rpm to mix the L-arginine and the gelatin aqueous solution uniformly, thereby obtaining a gelatin-based hydrogel. Place the gelatin-based hydrogel at room temperature and observe the gelation of the gelatin-based hydrogel at room temperature.

[0066] Example 8

[0067] In this example, a gelatin-based hydrogel is prepared by using L-arginine as a hydrogen bond competitive material, and the steps are as follows:

[0068] Dissolve 15 g of gelatin powder in 100 mL of water to prepare a gelatin aqueous solution with a concentration of 15 wt%, and then add 20 g of L-arginine to the gelatin aqueous solution, and fully stir at a rotation speed of 2000 rpm to mix the L-arginine and the gelatin aqueous solution uniformly, thereby obtaining a gelatin-based hydrogel. Place the gelatin-based hydrogel at room temperature and observe the gelation of the gelatin-based hydrogel at room temperature.

[0069] Example 9

[0070] In this example, a gelatin-based hydrogel is prepared by using polyarginine (with a weight average molecular weight of 4000) as a hydrogen bond competitive material, and the steps are as follows:

[0071] Dissolve 15 g of gelatin powder in 100 mL of water to prepare a gelatin aqueous solution with a concentration of 15 wt%, and then add 5 g of polyarginine to the gelatin aqueous solution, and fully stir at a rotation speed of 2000 rpm to mix the polyarginine and the gelatin aqueous solution uniformly, thereby obtaining a gelatin-based hydrogel. Place the gelatin-based hydrogel at room temperature and observe the gelation of the gelatin-based hydrogel at room temperature.

[0072] Example 10

[0073] In this example, a gelatin-based hydrogel is prepared by using polyarginine (with a weight average molecular weight of 4000) as a hydrogen bond competitive material, and the steps are as follows:

[0074] Dissolve 15 g of gelatin powder in 100 mL of water to prepare a gelatin aqueous solution with a concentration of 15 wt%, and then add 10 g of polyarginine to the gelatin aqueous solution, and fully stir at a rotation speed of 2000 rpm to mix the polyarginine and the gelatin aqueous solution uniformly, thereby obtaining a gelatin-based hydrogel. Place the gelatin-based hydrogel at room temperature and observe the gelation of the gelatin-based hydrogel at room temperature.

[0075] Example 11

[0076] In this example, a gelatin-based hydrogel is prepared by using polyarginine (with a weight average molecular weight of 4000) as a hydrogen bond competitive material, and the steps are as follows:

[0077] Dissolve 15g of gelatin powder in 100mL of water to prepare a 15wt% gelatin aqueous solution. Add 15g of polyarginine to the gelatin aqueous solution and stir thoroughly at 2000rpm to uniformly mix the polyarginine and gelatin aqueous solution to obtain a gelatin-based hydrogel. Allow the gelatin-based hydrogel to stand at room temperature and observe its solidification at room temperature.

[0078] Example 12

[0079] In this example, gelatin-based hydrogels were prepared using polyarginine (weight-average molecular weight of 4000) as a hydrogen bond competitive material, and the steps were as follows:

[0080] Dissolve 15g of gelatin powder in 100mL of water to prepare a 15wt% gelatin aqueous solution. Add 20g of polyarginine to the gelatin aqueous solution and stir thoroughly at 2000rpm to uniformly mix the polyarginine and gelatin aqueous solution to obtain a gelatin-based hydrogel. Allow the gelatin-based hydrogel to stand at room temperature and observe its solidification at room temperature.

[0081] Comparative Example 1

[0082] 15 g of gelatin powder was dissolved in 100 mL of water to prepare a gelatin aqueous solution with a concentration of 15 wt %. The gelatin aqueous solution was allowed to stand at room temperature and its coagulation at room temperature was observed.

[0083] Performance Testing

[0084] 1. The rheological viscosity of the gelatin-based hydrogels in Examples 3, 7, and 11 was tested:

[0085] The rheological properties of the gelatin-based hydrogels in Examples 3, 7, and 11 were measured at 25°C using an Anton Paar MCR302e rheometer equipped with a 25 mm diameter conical plate. The apparent viscosity of the hydrogels was measured at a constant shear rate of 0.1 s -1 Here are the changes over time:

[0086] Figure 1 The rheological viscosity test results of the gelatin-based hydrogel in Example 3 are as follows: Figure 2 The rheological viscosity test results of the gelatin-based hydrogel in Example 7 are as follows: Figure 3 The rheological viscosity test results of the gelatin-based hydrogel in Example 11 are as follows. Figures 1-3 It can be seen that after metformin, L-arginine and polyarginine were introduced into the gelatin aqueous solution in Examples 3, 7 and 11, respectively, the rheological viscosity of the gelatin-based hydrogel in Example 3 changed the least over time, followed by Example 11, and finally Example 7, indicating that at the same dosage, metformin has the best effect on improving the room temperature solidification properties of the gelatin-based hydrogel.

[0087] 2. The gelatin-based hydrogels in Examples 1-12 and the gelatin aqueous solution in Comparative Example 1 were placed in a constant temperature mixer, and after reaching the set temperature, they were placed for 30 min (25℃, 500 rpm). After the constant temperature program ended, they were taken out and inverted to observe the gelation of the gelatin at this temperature:

[0088] Figure 4 To compare the flowability of the gelatin-based hydrogels in Examples 1-4 and the gelatin aqueous solution in Comparative Example 1 after being inverted after 30 min at 25℃, Figure 5 To compare the flowability of the gelatin-based hydrogels in Examples 5-8 and the gelatin aqueous solution in Comparative Example 1 after being inverted after 30 min at 25℃, Figure 6 To compare the flowability of the gelatin-based hydrogels in Examples 9-12 and the gelatin aqueous solution in Comparative Example 1 after being inverted after 30 min at 25℃. From Figures 4-6 It can be seen that the gelatin aqueous solution in Comparative Example 1 has become a gel after being placed at 25℃ for 30 min and does not flow when inverted, while in the gelatin-based hydrogels in Examples 1-4, when the amount of metformin added is 5wt%-20wt%, the gelatin-based hydrogels all have good flowability after being placed at 25℃ for 30 min; in the gelatin-based hydrogels in Examples 5-8, when the concentration of L-arginine is 5wt%-10wt%, the room temperature easy gelation property of the gelatin aqueous solution cannot be improved, but when the concentration of L-arginine is greater than 15wt%, the room temperature easy gelation property of the gelatin aqueous solution can be improved; in the gelatin-based hydrogels in Examples 9-12, when the amount of polyarginine added is 5wt%-20wt%, the gelatin-based hydrogels all have good flowability after being placed at 25℃ for 30 min. This shows that the gelatin-based hydrogels provided by the present application can all reduce the hydrogen bonding in the gelatin aqueous solution and lower the gelation temperature to below 25℃ after introducing hydrogen bond competitive materials such as metformin, L-arginine, and polyarginine into the gelatin aqueous solution, and among them, metformin and polyarginine can achieve good improvement effects at a small amount of addition.

[0089] Application Example

[0090] The gelatin-based hydrogels in Example 2 and the gelatin aqueous solution in Comparative Example 1 were prepared into gelatin-based bio-inks at room temperature, and were used for bio-3D printing at 20℃. To facilitate observation, 1% of rhodamine B dye was added to the gelatin-based bio-inks.

[0091] Figure 7 The bio-3D printing real scene photos in Application Example 1, wherein, Figure 7 (a) is a photo of the printing process of the gelatin-based bio-ink prepared from the gelatin aqueous solution in Comparative Example 1, and Figure 7(a) It can be seen that the gelatin-based bio-ink prepared from the gelatin aqueous solution in Comparative Example 1 has become gel-like at 20°C and cannot be extruded; Figure 7 (b) is a photo of the printing process of the gelatin-based bio-ink prepared from the gelatin-based hydrogel in Example 2, Figure 7 (c) is a photo of the printed gelatin-based bio-ink prepared from the gelatin-based hydrogel in Example 2, which is Figure 7 (b) and Figure 7 (c) It can be seen that the addition of 10 wt% metformin as a hydrogen bond competitive material in the gelatin aqueous solution in Example 2 improves the room-temperature easy-setting property of the gelatin-based hydrogel, so that the gelation temperature of the gelatin-based hydrogel is reduced to below 25°C, and the gelatin-based bio-ink prepared therefrom still has good fluidity at 20°C, can be extruded to form uniform lines, and a high-fidelity "SYSU" pattern can be printed.

Claims

1. A gelatin-based hydrogel, characterized in that, The preparation raw materials comprise: gelatin, a hydrogen bond competitive material, and water, wherein the mass ratio of the hydrogen bond competitive material to the gelatin is 1: (0.25-6), and the hydrogen bond competitive material is metformin.

2. The gelatin-based hydrogel according to claim 1, wherein The mass ratio of the gelatin to the water is 1: (3-20).

3. The gelatin-based hydrogel according to claim 1 or 2, characterized in that, The gelation temperature of the gelatin-based hydrogel is less than 25 DEG C.

4. Process for the preparation of a gelatin-based hydrogel according to any one of claims 1 to 3, characterized in that, The method comprises the following steps: The gelatin is dissolved in water to form a gelatin aqueous solution, and then the hydrogen bond competitive material is added and uniformly mixed to obtain the gelatin-based hydrogel.

5. Use of the gelatin-based hydrogel according to any one of claims 1-3 in the preparation of a gelatin-based biological compound material.

6. Use according to claim 5, characterized in that, The gelatin-based biological compound material is a gelatin-based biological ink.

7. Use according to claim 5, characterized in that, The gelatin-based biological compound material is injectable gelatin-based hydrogel microspheres and / or injectable gelatin-based sustained-release systems.

8. A gelatin-based bioink, characterized by, The gelatin-based hydrogel is according to any one of claims 1-3.

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