An alkaline mucopolysaccharide, modified gelatin, and their preparation methods and applications

Abstract

The present invention relates to the field of biomedicine, in particular to an alkaline mucopolysaccharide, modified gelatin, and their preparation methods and applications. The alkaline mucopolysaccharide is a polymer with the following structural formula, where m, n, and l all represent the number of each polymerization monomer. At the same time, modified gelatin based on the alkaline mucopolysaccharide, hydrogels based on the modified gelatin, and the applications of the hydrogels, as well as their corresponding preparation methods, are also disclosed. The alkaline mucopolysaccharide of the present invention can modify and modify gelatin, and then prepare a hydrogel through the modified gelatin. The prepared hydrogel has the microscopic structural kinetic characteristics of ECM and can be used for the culture of organoids.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, in particular to alkaline mucopolysaccharide, modified gelatin and preparation methods and applications thereof. Background Art

[0002] Hydrogels are highly hydrated polymer networks composed of macromolecules. Due to their ability to mimic many properties of natural tissues, they have received extensive attention in basic and translational biomedical research and have become one of the preferred carriers for therapeutic drugs and cells. Previously, most common synthetic hydrogels were formed by stable covalent cross-linking of polymer chains. This static network structure may limit the interaction between cells and substrates and hinder the normal function of cells or tissues (for example, affecting cell diffusion and migration, or causing the failure of mitosis). Currently, the hydrogels used in organoid culture applications cannot simulate the microstructural dynamics of natural ECM in order to support the various activities of the cells encapsulated in it in 3D space.

[0003] Gelatin is a pure natural polymer compound and a type of natural hydrogel. It is homologous to collagen, and the amino acid composition of gelatin is similar to that of collagen. Gelatin is used in tissue engineering and drug delivery systems because of its high biocompatibility, biodegradability, no other by-products after degradation in the body, no immunogenicity and blood compatibility, and the same components and biological properties as collagen. However, there is currently no suitable method to modify gelatin so that gelatin can not only have macroscopic stability but also produce viscoelastic behaviors including stress relaxation, creep and hysteresis. These dynamic properties allow the ECM to respond and store / release the traction generated by cells and transmit external forces to cells. Therefore, there is currently no gelatin as a matrix material for organoid culture on the market. Summary of the invention

[0004] In view of the existing deficiencies, the present invention provides an alkaline mucopolysaccharide, modified gelatin and a preparation method and application thereof.

[0005] The technical solution adopted by the present invention to solve the technical problem is: an alkaline mucopolysaccharide, wherein the alkaline mucopolysaccharide is a polymer having the following structural formula: Wherein m, n, and l represent the number of each polymerized monomer.

[0006] A method for preparing alkaline mucopolysaccharide, characterized in that the steps are as follows:

[0007] S1. Mix polylysine, triethylamine, and methyl methacrylate to obtain Product 1. The molar ratio of polylysine to triethylamine is 1:0.1 - 1:0.2, and the molar ratio of polylysine to methyl methacrylate is 1:10.0 - 1:15.0. The reaction temperature is 60 °C, and the reaction time is 4 - 6 hours.

[0008] S2. Adjust the pH value of Product 1 to alkaline, and then mix it with (2R,3S,4S,5R)-2,3,4,5,6-pentahydroxyhexanal to prepare Product 2. Product 2 is a polymer with the following structural formula, where m, n, and l all represent the number of each polymerization monomer. The pH value is 8 - 10, and the mass of (2R,3S,4S,5R)-2,3,4,5,6-pentahydroxyhexanal added to Product 1 per 1 mol of polylysine is 1.8 - 3.6 grams. The reaction temperature is 50 °C, and the reaction time is 48 - 72 hours.

[0009] Preferably, Product 1 is a polymer with the following structural formula, , where m and n both represent the number of each polymerization monomer.

[0010] An alkaline mucopolysaccharide modified gelatin, including gelatin, on which alkaline mucopolysaccharide groups are polymerized. The alkaline mucopolysaccharide groups are polymer groups with the following structural formula, where m, n, and l all represent the number of each polymerization monomer.

[0011] A preparation method of an alkaline mucopolysaccharide modified gelatin, the steps are as follows:

[0012] S3. Activate gelatin with carbodiimide and N-hydroxysulfosuccinimide, and then react it with Product 2 prepared by the above-mentioned preparation method of alkaline mucopolysaccharide to obtain alkaline mucopolysaccharide modified gelatin.

[0013] A hydrogel based on alkaline mucopolysaccharide modified gelatin, the matrix of the hydrogel is the above-mentioned alkaline mucopolysaccharide modified gelatin.

[0014] A preparation method of a hydrogel based on alkaline mucopolysaccharide modified gelatin, the steps are as follows:

[0015] S4. Dissolve the above-mentioned alkaline mucopolysaccharide modified gelatin in PBS solution, and then add a photoinitiator and crosslink it under ultraviolet light to form Hydrogel 1. The mass-volume ratio of the alkaline mucopolysaccharide modified gelatin to PBS is 0.3 - 1 g / 10 ml. The wavelength of the ultraviolet light used for crosslinking is 250 - 420 nm, and the crosslinking time is 3 - 40 seconds.

[0016] S5. The hydrogel 1 is crosslinked for the second time in a sodium tetraborate solution to obtain the hydrogel 2. The mass-to-volume ratio of the sodium tetraborate to the PBS in the previous step is 0.2 - 0.4 g / 20 ml, and the time for the second crosslinking is 5 - 30 seconds.

[0017] Application of a hydrogel based on alkaline mucopolysaccharide-modified gelatin. The aforementioned hydrogel based on alkaline mucopolysaccharide-modified gelatin is used for the culture of organoids.

[0018] The beneficial effects of the present invention are as follows: The alkaline mucopolysaccharide of this invention can be used for the modification of gelatin. The modified gelatin is used to prepare a hydrogel to change the structural characteristics of traditional hydrogels. The hydrogel 1 first prepared by modifying gelatin is a chemical crosslinking based on the modified gelatin as a long-chain compound through double bonds, which can provide structural stability and elasticity for the hydrogel. Subsequently, the hydrogel 2 is based on the hydrogel 1 and forms a reversible complexation reaction through hydroxyl groups and borate groups, which is responsible for retaining moisture and providing dynamic properties, and at the same time enhances the mechanical properties of the hydrogel 2. This binding property of the hydrogel 2 mimics the microstructural kinetic characteristics of natural ECM and can effectively support various activities of the cells encapsulated therein in the 3D space formed by the hydrogel. Description of the Drawings

[0019] Figure 1 It is a schematic structural diagram of the hydrogel 2 in the embodiment of the present invention;

[0020] Figure 2 It is a nuclear magnetic resonance hydrogen spectrum diagram of the product 2 in the embodiment of the present invention; Detailed Embodiments

[0021] To more clearly illustrate the purpose, technical solutions, and advantages of the embodiments of the present invention, the present invention will be further described below in conjunction with the drawings and embodiments. A clear and complete description is made. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the protection scope of the present invention.

[0022] The "embodiments" mentioned herein mean that the specific features described in conjunction with the embodiments can be included in at least one embodiment of the present invention. The appearance of this phrase in various positions in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment mutually exclusive with other embodiments. Those skilled in the art explicitly and implicitly understand that the embodiments described herein can be combined with other embodiments. At the same time, the "stirring, purification", etc. mentioned herein all belong to the operation methods of the prior art.

[0023] An alkaline mucopolysaccharide and its preparation method, the alkaline mucopolysaccharide is a polymer with the following structural formula, where m, n, and l each represent the number of each polymerization monomer.

[0024] The steps of the preparation method are as follows:

[0025] S1, Dissolve polylysine in deionized water, stir until completely dissolved, add triethylamine, continue stirring, then dropwise add methyl methacrylate, and continue stirring. After the reaction is completed, product 1 is obtained. Product 1 is a polymer with the following structural formula, , where m and n each represent the number of each polymerization monomer,

[0026] At this time, the molar ratio of polylysine to triethylamine is 1:0.1 - 1:0.2, the molar ratio of polylysine to methyl methacrylate is 1:10.0 - 1:15.0, the reaction temperature is 60 °C, and the reaction time is 4 - 6 hours.

[0027] S2, After cooling product 1 to room temperature, adjust the pH value to 10 with 5M sodium hydroxide solution, add 3.6 grams of (2R,3S,4S,5R)-2,3,4,5,6-pentahydroxyhexanal, stir in a 50° constant temperature water bath, and the reaction lasts for 48 hours to obtain product 2. Product 2 is a polymer with the following structural formula, where m, n, and l each represent the number of each polymerization monomer; its nuclear magnetic resonance spectrum is as shown in Figure 2 as follows, and the hydrogen spectrum attribution: 1 H NMR (400 MHz,D2O): δ 5.73 (s, 4 H), 5.65 (s, 1 H), 5.50 (s, 4 H), 5.33 (s, 1 H),

[0028] 4.69 - 4.62 (m, 14 H), 4.47 - 4.15 (m, 28 H), 3.95 - 3.86 (m, 35 H), 3.72 - 3.64 (m, 17 H), 3.44 - 3.39 (m, 4 H), 3.20 - 3.00 (m, 30 H), 2.79 - 2.65 (m, 8 H),2.35 - 2.16 (m, 8 H), 2.09 - 2.03 (m, 20 H), 1.93 (s, 20 H), 1.87 (s, 6 H), 1.77 - 1.68 (m, 30 H), 1.53 (br s 25 H), 1.42 - 1.32 (m, 36 H), 1.22 (br s, 4 H),0.93 - 0.88 (m, 14 H);

[0029] At this time, the pH value can be within the range of 8 - 10, and the reaction time can be within the range of 48 - 72 hours. The mass of (2R,3S,4S,5R)-2,3,4,5,6-pentahydroxyhexanal added to Product 1 corresponding to every 1 mol of polylysine is 1.8 - 3.6 grams, and the reaction temperature is 50 °C.

[0030] An alkaline mucopolysaccharide-modified gelatin and its preparation method, including gelatin, on which alkaline mucopolysaccharide groups are polymerized, that is, the aforementioned Product 2. The alkaline mucopolysaccharide group is a polymer group with the following structural formula where m, n, and l all represent the number of each polymerization monomer.

[0031] The steps of the preparation method are as follows:

[0032] S3. Dissolve gelatin in deionized water, warm and stir to dissolve it, cool to room temperature after dissolution, add carbodiimide and N-hydroxysulfosuccinimide to activate the carboxyl group for a period of time, then add Product 2, stir, and after the reaction ends, obtain alkaline mucopolysaccharide-modified gelatin through dialysis purification. At this time, the concentration of the gelatin solution is 1.5% - 2%, the mass ratio of gelatin to carbodiimide is 1:4.0 - 1:4.5, the mass ratio of carbodiimide to N-hydroxysulfosuccinimide is 1:0.9 - 1:1.0, the reaction temperature is room temperature, and the reaction time is 24 - 48 hours.

[0033] A hydrogel based on alkaline mucopolysaccharide-modified gelatin and its preparation method. The matrix of the hydrogel is the aforementioned alkaline mucopolysaccharide-modified gelatin, and its structure is as Figure 1 shown.

[0034] The steps of the preparation method are as follows:

[0035] S4. Dissolve the aforementioned alkaline mucopolysaccharide-modified gelatin in PBS solution, then add a light initiator, that is, a photoinitiator, and crosslink it under ultraviolet light to form Hydrogel 1. At this time, the mass-volume ratio of alkaline mucopolysaccharide-modified gelatin to PBS is 0.3 - 1 g / 10 ml, the wavelength of the ultraviolet light used for crosslinking is 250 - 420 nm, and the crosslinking time is 3 - 40 seconds;

[0036] S5. Immerse Hydrogel 1 in a sodium borate solution for secondary crosslinking, and remove the precipitated sodium borate to obtain Hydrogel 2. At this time, the mass-volume ratio of sodium borate to the PBS solution in the previous step (Step S4) is 0.2 - 0.4 g / 20 ml, and the time for secondary crosslinking is 5 - 30 seconds.

[0037] Application of a hydrogel based on alkaline mucopolysaccharide-modified gelatin, using the aforementioned hydrogel based on alkaline mucopolysaccharide-modified gelatin for the culture of organoids, that is, the application of the alkaline mucopolysaccharide-modified gelatin hydrogel as a matrix material in the culture of organoids.

[0038] Such alkaline mucopolysaccharides can be used for the modification of gelatin. The modified gelatin is then used to prepare a hydrogel to change the structural properties of traditional hydrogels. The first hydrogel 1 prepared from the modified gelatin is a chemical cross-linking based on the modified gelatin as a long-chain compound through double bonds, which can provide structural stability and elasticity for the hydrogel. Subsequently, hydrogel 2 is based on hydrogel 1 and forms a reversible complexation reaction through hydroxyl and borate groups, responsible for retaining moisture and providing dynamic properties, while also enhancing the mechanical properties of hydrogel 2. This binding property of hydrogel 2 mimics the microstructural kinetic characteristics of natural ECM and can effectively support various activities of the cells it encapsulates in the 3D space formed by the hydrogel.

[0039] It should be understood that those of ordinary skill in the art can make improvements or transformations based on the above description, and all such improvements and transformations should fall within the protection scope of the appended claims of the present invention.

Claims

1. An alkaline mucopolysaccharide, characterized in that: The alkaline mucopolysaccharide is a polymer with the following structural formula, wherein m, n, and l each represent the number of each polymerization monomer.

2. A method for preparing an alkaline mucopolysaccharide, characterized in that: The steps are as follows: S1. Mix polylysine, triethylamine, and methyl methacrylate to obtain Product 1; the molar ratio of polylysine to triethylamine is 1:0.1 - 1:0.2, the molar ratio of polylysine to methyl methacrylate is 1:10.0 - 1:15.0, the reaction temperature is 60°C, and the reaction time is 4 - 6 hours; S2, adjust the pH value of Product 1 to alkaline, and then mix it with (2R,3S,4S,5R)-2,3,4,5,6-pentahydroxyhexanal to prepare Product 2, and the Product 2 is a polymer with the following structural formula, where m, n, and l all represent the number of each polymerization monomer; the pH value is 8-10, the mass of (2R,3S,4S,5R)-2,3,4,5,6-pentahydroxyhexanal added to Product 1 per 1 mol of polylysine is 1.8-3.6 grams, the reaction temperature is 50 °C, and the reaction time is 48-72 hours.

3. The preparation method of the alkaline mucopolysaccharide according to claim 2, wherein : The product 1 is a polymer with the following structural formula, , where m and n both represent the number of each polymerized monomer.

4. An alkaline mucopolysaccharide-modified gelatin, characterized in that : Comprising gelatin, on which basic mucopolysaccharide groups are polymerized, and the basic mucopolysaccharide groups are polymer groups having the following structural formula, wherein m, n, and l each represent the number of respective polymerized monomers.

5. A preparation method of alkaline mucopolysaccharide-modified gelatin, characterized in that : The steps are as follows: S3. Activate gelatin with carbodiimide and N-hydroxysuccinimide, and then react it with Product 2 prepared by the method for preparing the basic mucopolysaccharide according to any one of claims 2 - 3 to obtain basic mucopolysaccharide-modified gelatin.

6. A hydrogel based on alkaline mucopolysaccharide-modified gelatin, characterized in that : The matrix of the hydrogel is the basic mucopolysaccharide-modified gelatin described in claim 4.

7. A preparation method of a hydrogel based on alkaline polysaccharide modified gelatin, characterized in that : The steps are as follows: S4. Dissolve the basic mucopolysaccharide-modified gelatin described in claim 4 in PBS solution, then add a light initiator and crosslink it under ultraviolet light to prepare Hydrogel 1. The mass-volume ratio of the basic mucopolysaccharide-modified gelatin to PBS is 0.3 - 1 g / 10 ml, the wavelength of the ultraviolet light used for crosslinking is 250 - 420 nm, and the crosslinking time is 3 - 40 seconds; S5. Perform secondary crosslinking on Hydrogel 1 in sodium borate solution to obtain Hydrogel 2. The mass-volume ratio of sodium borate to PBS in the previous step is 0.2 - 0.4 g / 20 ml, and the time for secondary crosslinking is 5 - 30 seconds.

8. Application of a hydrogel based on alkaline polysaccharide-modified gelatin, characterized in that : Use the hydrogel based on basic mucopolysaccharide-modified gelatin described in claim 6 for the culture of organoids.

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