Composition for bio-based hydrogel, bio-based hydrogel as well as preparation method and application of bio-based hydrogel

Abstract

The invention relates to the field of bio-based hydrogel, and discloses a composition for bio-based hydrogel, the bio-based hydrogel as well as a preparation method and application of the bio-based hydrogel. The composition contains myricetin, glucan, polyvinyl alcohol and a buffer solution, the content mass ratio of the myricetin to the glucan to the polyvinyl alcohol is 1: (2-20): (200-300); the alcoholysis degree of the polyvinyl alcohol is greater than or equal to 90%; the pH (Potential of Hydrogen) of the buffer solution is 6.0 to 8.5. The bio-based hydrogel prepared from the composition for the bio-based hydrogel provided by the invention not only can meet the rheological property requirements of remarkable shear thinning characteristic, rapid thixotropic recovery and the like, but also has excellent biocompatibility, and can avoid the problem of biotoxicity possibly caused in traditional chemical modification; the preparation method has an excellent application prospect in the fields of 3D printing bio-ink and antibiosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bio-based hydrogel, in particular to a bio-based hydrogel composition, a bio-based hydrogel and a preparation method and application thereof. BACKGROUND

[0002] Direct ink writing (DIW) is one of the common 3D printing technologies, which has been widely used in tissue engineering and regenerative medicine due to its precise extrusion capability and high resolution. In addition, DIW also shows great potential in drug delivery systems, by customizing the printing of drug carriers, achieving precise drug release and targeted therapy, which improves the treatment effect and reduces side effects.

[0003] However, existing bio-inks still have many challenges in rheological properties, especially in high yield stress, significant shear thinning characteristics and rapid thixotropic recovery, which are difficult to meet the requirements of DIW technology.

[0004] Current technical solutions usually rely on device optimization or physical constraints, such as multi-stage micro-nozzles, temperature-controlled bath systems, and adjustment of printing parameters. Although these methods can improve the printing effect to some extent, they often come with significant increases in research and development costs, low design efficiency, and other issues.

[0005] Another common solution strategy is to improve the performance of bio-inks through material modification, mainly by introducing dynamic covalent bonds, chemical cross-linking or chemical structure modification to regulate the shear response behavior of the ink.

[0006] Although the above-mentioned solutions can improve the printing performance, there are still some limitations.

[0007] Firstly, the inherent chemical properties of the material limit its modification space, making many modification methods rely on empirical trial and error optimization, and the regulation process is complex and time-consuming; secondly, some chemical modifications may introduce unnecessary biological toxicity problems, affecting the biocompatibility and biological safety of the printing material.

[0008] Therefore, how to maintain the excellent printing performance of hydrogel ink while ensuring its biological safety is still a technical problem to be solved. SUMMARY

[0009] The purpose of the present application is to overcome the problems of poor rheological properties and poor biocompatibility of existing hydrogel bio-inks.

[0010] To achieve the above-mentioned purpose, the first aspect of the present application provides a bio-based hydrogel composition, which contains myricetin, dextran, polyvinyl alcohol and a buffer solution; The content mass ratio of the myricetin, the dextran and the polyvinyl alcohol is 1:2-20:200-300. The alcoholysis degree of the polyvinyl alcohol is greater than or equal to 90%, and the viscosity at 20 DEG C is 20-50 mPa S; the pH of the buffer solution is 6.0-8.5.

[0011] The second aspect of the present application provides a method for preparing a bio-based hydrogel, which comprises: contacting and mixing the components in the composition of the aforementioned first aspect.

[0012] The third aspect of the present application provides a bio-based hydrogel prepared by the method of the aforementioned second aspect.

[0013] The fourth aspect of the present application provides an application of the bio-based hydrogel of the aforementioned third aspect in the fields of 3D printing biological ink and antibiosis.

[0014] The bio-based hydrogel prepared by using the bio-based hydrogel composition provided by the present application not only meets the rheological performance requirements such as shear thinning characteristics and rapid thixotropic recovery, but also has excellent biocompatibility, can avoid the biological toxicity problems possibly caused by traditional chemical modification, and has excellent application prospect in the fields of 3D printing biological ink and antibiosis.

[0015] The method for preparing a bio-based hydrogel provided by the present application is simple, has strong operability, and is suitable for industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is an appearance and micro-morphology diagram of the bio-based hydrogel prepared in Example 1 of the present application; Figure 2 is a rheological test diagram of the bio-based hydrogel prepared in Example 1 of the present application; Figure 3 is a 3D printing schematic diagram of the bio-based hydrogel prepared in Example 1 of the present application as a biological ink; Figure 4 is a biocompatibility test diagram of the bio-based hydrogel prepared in Example 1 of the present application; Figure 5 is an antibiosis test diagram of the bio-based hydrogel prepared in Example 1 of the present application. DETAILED DESCRIPTION

[0017] The endpoints of the ranges and any values disclosed herein are not limited to the precise values stated. The endpoints of the ranges and any values are provided as approximations. Any numerical value, however, can inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Also, the endpoints of the ranges and any values are only approximate, as some variations can occur. Thus, the endpoints of the ranges and any values are not to be understood as being stated exactly at the maximum or minimum values. It should also be understood that the numerical properties forming the preface of the claims are intended to function merely as concretizing examples of the ranges and values recited in the claims.

[0018] As previously stated, a first aspect of the present invention provides a composition for use in bio-based hydrogels, the composition comprising myricetin, dextran, polyvinyl alcohol and a buffer solution; The mass ratio of the myricetin, the dextran, and the polyvinyl alcohol is 1:2-20:200-300; The polyvinyl alcohol has a degree of alcoholysis ≥90% and a viscosity of 20-50 mPa·s at 20°C; the buffer solution has a pH of 6.0-8.5.

[0019] According to a preferred embodiment, the dextran has a weight-average molecular weight of 300,000 to 500,000. The inventors of this invention have discovered that, under this preferred condition, the obtained bio-based hydrogel exhibits superior rheological properties, making it more suitable for 3D printing.

[0020] In a preferred embodiment, the mass ratio of myricetin to the buffer solution is 1:1600-2000.

[0021] Preferably, the buffer solution is selected from at least one of bicarbonate solution, phosphate solution and hydrogen phosphate solution, and more preferably a phosphate solution.

[0022] As previously stated, a second aspect of the present invention provides a method for preparing a bio-based hydrogel, the method comprising: contact mixing the components of the composition described in the first aspect.

[0023] According to a preferred embodiment, the step of contact mixing the components in the composition includes: (1) In the presence of a buffer solution, myricetin, dextran and polyvinyl alcohol are heated to obtain a mixture; (2) The mixture is subjected to gelation treatment to obtain the bio-based hydrogel; The temperature of the gel treatment is at least 30°C lower than the temperature of the heat treatment.

[0024] In a preferred embodiment, in step (1), the conditions for the heat treatment include: a temperature of 80-100°C and a time of 30-80 min.

[0025] To obtain a more uniformly dispersed mixture, the method further includes: before the heat treatment, stirring and mixing the myricetin, the dextran, and the polyvinyl alcohol in the presence of the buffer solution to obtain a mixed solution; and then subjecting the mixed solution to the heat treatment to obtain the mixture.

[0026] In a preferred embodiment, the mixing conditions include a temperature of 20-30°C and a time of 20-30 hours.

[0027] It should be noted that the present invention does not have any special requirements for the stirring speed, and those skilled in the art can select it as needed. The present invention will not elaborate further here, and those skilled in the art should not understand that this is a limitation of the present invention.

[0028] In a preferred embodiment, the heat treatment is performed under ultrasound, and the power of the ultrasound is 100-400W.

[0029] According to a preferred embodiment, in step (2), the gel treatment conditions include a temperature of 20-30°C and a time of 60-120 min. The inventors of this invention have found that, under this preferred condition, the obtained bio-based hydrogel has better mechanical strength and a more uniform microstructure.

[0030] As previously stated, the third aspect of the present invention provides a bio-based hydrogel prepared by the method described in the second aspect above.

[0031] As previously stated, the fourth aspect of the present invention provides the application of the bio-based hydrogel described in the third aspect above in the fields of 3D printing bio-inks and antibacterial agents.

[0032] The present invention will be described in detail below through examples. In the following examples, unless otherwise specified, the raw materials are all commercially available products.

[0033] Myricetin: analytical grade, CAS No. 529-44-2, purchased from Macklin.

[0034] Glucan: Dextran I: weight average molecular weight 500,000, purchased from Macklin, brand name D992677.

[0035] Dextran II: weight average molecular weight 250,000, purchased from Macklin, brand name D992650.

[0036] Sodium hyaluronate: weight average molecular weight 400,000-800,000, purchased from Macklin, brand name H909937.

[0037] Polyvinyl alcohol: Polyvinyl alcohol I: with a degree of alcoholysis of 99% and a viscosity of 23-31 mPa·s at 20°C, purchased from Macklin, grade P815725.

[0038] Polyvinyl alcohol (DI): with a degree of hydrolysis of 88% and a viscosity of 20.5-24.5 mPa·s at 20°C, purchased from Macklin, grade P815723.

[0039] Buffer solution: Buffer solution I: phosphate solution, pH=7.4.

[0040] Buffer solution DI: phosphate solution, pH=10.0.

[0041] Example 1 This embodiment illustrates the preparation of bio-based hydrogels according to the formulation and process parameters in Table 1, following the steps outlined below: (1) In the presence of a buffer solution, myricetin, dextran and polyvinyl alcohol were stirred and mixed at 25°C for 24 h to obtain a mixed solution; The mixed solution was heated under ultrasound (ultrasound power of 200W) to obtain a mixture; (2) The mixture is subjected to gelation treatment to obtain the bio-based hydrogel; Example 2 This embodiment uses a similar method to Example 1, except that the formulation and process are as shown in Table 1, and a bio-based hydrogel is prepared. All parts not listed are the same as in Example 1.

[0042] Example 3 This embodiment uses a method similar to that of Example 1, except that: an equal mass of dextran II is used to replace dextran I in Example 1, as shown in Table 1, to prepare a bio-based hydrogel; All parts not listed are the same as in Example 1.

[0043] Example 4 This embodiment uses a similar method to Example 1, except that the process parameters are shown in Table 1, and a bio-based hydrogel is prepared. All parts not listed are the same as in Example 1.

[0044] Comparative Example 1 This comparative example was prepared using a similar method to Example 1, except that the formulation and process were as shown in Table 1, and a bio-based hydrogel was obtained. All parts not listed are the same as in Example 1.

[0045] Table 1

[0046] Comparative Example 2 This comparative example was conducted using a method similar to that of Example 1, except that an equal mass of sodium hyaluronate was used to replace dextran I in Example 1 to prepare a bio-based hydrogel. All parts not listed are the same as in Example 1.

[0047] Comparative Example 3 This comparative example was prepared using a method similar to that of Example 1, except that: in this comparative example, polyvinyl alcohol DI of equal mass was used to replace polyvinyl alcohol I in Example 1 to prepare a bio-based hydrogel. All parts not listed are the same as in Example 1.

[0048] Comparative Example 4 This comparative example was prepared using a method similar to that of Example 1, except that: in this comparative example, buffer solution DI of equal mass was used to replace buffer solution I in Example 1 to prepare a bio-based hydrogel. All parts not listed are the same as in Example 1.

[0049] Test Example 1 The morphology of the dextran / polyvinyl alcohol complex (preparation method: similar to that in Example 1, except that myricetin was not added in step (1), and all other parts not listed are the same as in Example 1) and the bio-based hydrogel prepared in Example 1 were tested using scanning electron microscopy. The results are as follows: Figure 1 As shown: Figure 1 Image (A) shows the appearance of the dextran / polyvinyl alcohol complex. Figure 1 (B) is a SEM image of the dextran / polyvinyl alcohol complex; Figure 1 Image C shows the appearance of the bio-based hydrogel prepared in Example 1. Figure 1 Image (D) is a SEM image of the bio-based hydrogel prepared in Example 1; Depend on Figure 1 It is known that the dextran / polyvinyl alcohol complex cannot form a hydrogel, and its SEM image shows an irregular sheet-like network; while the bio-based hydrogel prepared in Example 1 forms a three-dimensional interconnected network structure.

[0050] Test Example 2 The rheological properties of the bio-based hydrogel prepared in Example 1 were tested (the bio-based hydrogel was placed on a rheometer, and the parallel plates used had a diameter of 50 mm and a gap of 0.1 mm). (1) For dynamic strain scanning: the temperature was fixed at 25°C, the frequency was fixed at 1.0 rad / s, and the strain test range was 0.1%~1000%. The results are as follows: Figure 2 As shown in (A): pass Figure 2As can be seen in (A), within the low strain scanning range of 1-100%, G' (storage modulus) is much greater than G" (loss modulus), and the sample remains in a gel state. When the strain increases to 120%, G' begins to be less than G" and the difference increases with the increase of strain, and the sample is in a solution state. Again, under low strain, the sample returns to a gel state, which indicates that the hydrogel exhibits stability at room temperature (25℃) and shear thinning behavior, which is a prerequisite for realizing 3D printing. (2) For temperature change scanning: the frequency was fixed at 1.0 rad / s, the strain was fixed at 0.1%, and the temperature test range was 25~80 ℃. The results are shown in Figure 2(B): pass Figure 2 As can be seen in (B), the G' and G" values ​​of the bio-based hydrogel prepared in Example 1 are relatively stable in the range of 25~65°C; after 65°C, G' decreases with increasing temperature until it is lower than G", indicating that its gel state is gradually destroyed. Sustained high temperature will destroy the stability of most gels, thereby triggering the gel-solution transition; during this process, the gel-solution transition point of the bio-based hydrogel is about 68°C; it was found in the experiment that when it was cooled to room temperature, the destroyed gel state could be restored. (3) For frequency scanning, the temperature was fixed at 25°C, the strain was fixed at 0.1%, and the frequency test range was 1.0~100 rad / s; the results are as follows Figure 2 As shown in (C): pass Figure 2 As can be seen from (C), the G' value of all samples in this range is always greater than G", which indicates that the bio-based hydrogel exhibits a weak frequency dependence and maintains elastic properties.

[0051] Test Example 3 The bio-based hydrogel prepared in Example 1 was used for direct-write 3D printing. Specifically, the prepared bio-based hydrogel was loaded into a sterile printing cylinder, and the printing parameters included: temperature 25°C, pressure 100 Pa, and speed 3 m / s. During the printing process, the bio-based hydrogel was precisely deposited layer by layer onto a temperature-controlled printing platform using a BioX6 3D bioprinter under program control, ultimately forming a stable biological scaffold with a specific three-dimensional structure. The results are as follows: Figure 3 As shown: pass Figure 3As shown in Figure (A), thanks to its excellent rheological properties, this bio-based hydrogel successfully achieved in-situ molding with a minimum linewidth of 100 μm and could print stable porous scaffolds with no structural breakage or collapse. In the results of bio-ink diameter fidelity (the ratio of the actual diameter to the theoretical diameter of the bio-ink filaments), the bio-ink diameter fidelity is close to 1, indicating good diameter fidelity. Furthermore, the printed hydrogel scaffold can maintain its intact structure for a long time (24 h). In subsequent printing tests, the hydrogel can print various customized shapes with supporting meshes, such as pentagons and hexagons. Figure 3 (B) Furthermore, the support did not fracture or break during the tensile test, demonstrating excellent structural stability. Figure 3 (C) These results demonstrate that the bio-based hydrogels prepared in the examples have great potential to become 3D printing bio-inks.

[0052] Test Example 4 Referring to the method in Test Example 2, the temperature was fixed at 25°C, the strain at 0.1%, and the frequency at 1.0 rad / s. The mechanical properties of the bio-based hydrogels prepared in the aforementioned examples and comparative examples were analyzed, and the results are shown in Table 2: Table 2

[0053] The results above show that the bio-based hydrogel prepared using the composition for bio-based hydrogel provided by the present invention has excellent mechanical strength (the storage modulus is significantly greater than the loss modulus, and the storage modulus is in the range of 120-300 Pa), making it very suitable for application in the field of 3D printing bio-inks.

[0054] Test Example 5 This test example exemplarily evaluates the effect of the bio-based hydrogel prepared in Example 1 above on normal cell activity in the following manner; References (A self-fused peptide-loaded hydrogel with injectability and tissue-adhesiveness for preventing postoperative peritoneal adhesion[J], Mater Today Bio The study (2024, 28:101205) used the live-cell calcein / PI staining method. Mouse fibroblasts (L929) and hydrogels were co-incubated for 24 hours, followed by the addition of green fluorescent nuclear dye to observe the viability of the live cells. The results are as follows: Figure 4 As shown: pass Figure 4As can be seen, compared with the blank group without any sample, the L929 cells in the bio-based hydrogel experimental group had a higher survival rate at 24 h, which indicates that the prepared bio-based hydrogel has good biocompatibility. For the control group containing only myricetin (content 0.05wt%), the direct contact and release of the drug caused a significant decrease in the survival rate of L929 cells. The inventors speculate that this is because myricetin can maintain a stable state for a long time after combining with dextran and polyvinyl alcohol to form a gel, thereby reducing its toxicity to normal cells. Therefore, this hydrogel has the potential to be used as a 3D printing bio-ink.

[0055] Test Example 6 References (Multifunctional Oxidized Dextran–Metformin as a Tissue-Adhesive Hydrogel to Prevent Postoperative Peritoneal Adhesions in Patients with Metabolic Syndrome. Advance Sciences. (2023, 2303767) The antibacterial properties of the bio-based hydrogel prepared in Example 1 above were tested. Specifically, the bio-based hydrogel was cultured with Escherichia coli for 48 hours to evaluate its antibacterial ability, and the results were measured as follows. Figure 5 As shown: Compared to the E. coli control group, no significant bacterial growth was observed on the agar plates treated with the bio-based hydrogel. However, a certain number of colonies appeared on the agar plates treated with the dextran / polyvinyl alcohol complex (prepared in Test Example 1), indicating that these polymers themselves have weak antibacterial ability. Nevertheless, the bio-based hydrogel prepared in the embodiments of this invention has a prominent antibacterial effect against E. coli.

[0056] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A composition for use in bio-based hydrogels, characterized in that, The composition contains myricetin, dextran, polyvinyl alcohol, and a buffer solution; The mass ratio of the myricetin, the dextran, and the polyvinyl alcohol is 1:2-20:200-300; The polyvinyl alcohol has a degree of alcoholysis ≥90% and a viscosity of 20-50 mPa·s at 20°C; the buffer solution has a pH of 6.0-8.

5.

2. The composition according to claim 1, characterized in that, The weight-average molecular weight of the dextran is 300,000 to 500,000.

3. The composition according to claim 1, characterized in that, The mass ratio of myricetin to the buffer solution is 1:1600-2000.

4. The composition according to any one of claims 1-3, characterized in that, The buffer solution is selected from at least one of bicarbonate solution, phosphate solution and biphosphate solution.

5. A method for preparing bio-based hydrogels, characterized in that, The method includes: contact mixing of the components in the composition according to any one of claims 1-4.

6. The method according to claim 5, characterized in that, The step of contact mixing the components in the composition includes: (1) In the presence of a buffer solution, myricetin, dextran and polyvinyl alcohol are heated to obtain a mixture; (2) The mixture is subjected to gelation treatment to obtain the bio-based hydrogel; The temperature of the gel treatment is at least 30°C lower than the temperature of the heat treatment.

7. The method according to claim 6, characterized in that, In step (1), the conditions for the heat treatment include: a temperature of 80-100℃ and a time of 30-80min.

8. The method according to claim 6, characterized in that, In step (2), the conditions for gel treatment include: a temperature of 20-30°C and a time of 60-120 min.

9. A bio-based hydrogel prepared by the method according to any one of claims 5-8.

10. The application of the bio-based hydrogel of claim 9 in the fields of 3D printing bio-inks and antibacterial agents.

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