Injectable hydrogel as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of hydrogel, and discloses injectable hydrogel as well as a preparation method and application thereof. The injectable hydrogel disclosed by the invention is prepared from the following components: sulfhydrylated microspheres and dopamine modified hyaluronic acid, the preparation raw materials of the sulfhydrylated microspheres comprise a main monomer, a pH response monomer, a redox response monomer, an emulsifier, an initiator and a reducing agent. The dopamine modified hyaluronic acid is prepared from the following raw materials: dopamine hydrochloride and hyaluronic acid. The injectable hydrogel provided by the invention can form hydrogel with certain modulus and better stability under the condition of human body temperature, and shows excellent mechanical properties and biocompatibility; in addition, the mechanical property of the hydrogel formed in a human body can be adjusted by adjusting the ratio of the sulfhydrylated microspheres to the hyaluronic acid and the pH value of the injectable hydrogel, and the hydrogel has obvious advantages.

Description

Technical Field

[0001] The invention belongs to the technical field of hydrogels, and particularly relates to an injectable hydrogel and a preparation method and application thereof. Background Art

[0002] In recent years, with the development of biomedical engineering, injectable hydrogels, as a new type of biomaterial, have gradually attracted widespread attention. Injectable hydrogels not only have excellent biocompatibility and biodegradability, but can also achieve precise positioning and release through simple injection. These characteristics make injectable hydrogels show great application potential in the fields of tissue engineering, drug delivery, wound healing and regenerative medicine.

[0003] Traditional hydrogels formed by monomer polymerization are not injectable due to the fluidity of the monomers; although the fluidity and shear thinning properties of hydrogels based on physical cross-linking between polymers ensure their injectability, the mechanical strength and stability of this type of hydrogel are relatively weak; chemically cross-linked hydrogels form more stable networks through chemical reactions, however, their preparation process is complicated, the regulation of functions lacks flexibility, and there may be biosafety issues.

[0004] Therefore, it is necessary to develop an injectable hydrogel with superior mechanical properties and biocompatibility. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides an injectable hydrogel and a preparation method and application thereof. The injectable hydrogel of the present invention can form a hydrogel with a certain modulus and better stability under human body temperature conditions, and exhibits excellent mechanical properties and biocompatibility.

[0006] In a first aspect of the present invention, an injectable hydrogel is provided, wherein the injectable hydrogel comprises the following components: thiolated microspheres and dopamine-modified hyaluronic acid;

[0007] The raw materials for preparing the thiolated microspheres include a main monomer, a pH-responsive monomer, a redox-responsive monomer, an emulsifier, an initiator and a reducing agent, wherein the main monomer includes at least one of 2-methyl-2-acrylic acid-2-(2-methoxyethoxy)ethyl ester, methyl methacrylate, ethyl acrylate and N-isopropylacrylamide, the pH-responsive monomer includes methacrylic acid and / or acrylic acid, and the redox-responsive monomer includes disulfanediylbis(ethane-2,1-diyl)bis(2-methylacrylate) and / or N,N'-bis(acryl)cystamine;

[0008] The raw materials for preparing the dopamine-modified hyaluronic acid include dopamine hydrochloride and hyaluronic acid.

[0009] Specifically, the injectable hydrogel of the present invention can undergo Michael addition reaction at 35-40°C for 3-5 hours to further generate a hydrogel with a certain modulus and better stability, showing excellent mechanical properties and biocompatibility.

[0010] In some embodiments of the present invention, the raw materials for preparing the thiol microspheres include, by weight, 1-100 parts of a main monomer, 0.1-20 parts of a pH-responsive monomer, 0.1-10 parts of a redox-responsive monomer, 1-5 parts of an emulsifier, 0.1-0.5 parts of an initiator, and 0.1-5 parts of a reducing agent.

[0011] In some embodiments of the present invention, the raw materials for preparing the thiol microspheres include, by weight, 4-90 parts of a main monomer, 0.5-15 parts of a pH-responsive monomer, 0.4-9 parts of a redox-responsive monomer, 2-4 parts of an emulsifier, 0.15-0.3 parts of an initiator, and 0.5-4 parts of a reducing agent.

[0012] In some embodiments of the present invention, the raw materials for preparing the dopamine-modified hyaluronic acid include 0.1-1 parts of dopamine hydrochloride and 1-10 parts of hyaluronic acid, measured by weight.

[0013] In some embodiments of the present invention, the raw materials for preparing the dopamine-modified hyaluronic acid include 0.1-1 parts of dopamine hydrochloride and 3-7 parts of hyaluronic acid, measured by weight.

[0014] In some embodiments of the invention, the emulsifier comprises sodium lauryl sulfate.

[0015] In some embodiments of the present invention, the initiator comprises ammonium persulfate and / or potassium persulfate.

[0016] In some embodiments of the present invention, the reducing agent comprises tris(2-carboxyethyl)phosphine and / or dithiothreitol.

[0017] In some embodiments of the present invention, the mass ratio of the thiolated microspheres to hyaluronic acid is 5:(0.1-2).

[0018] In some embodiments of the present invention, the mass ratio of the thiolated microspheres to hyaluronic acid is 5:(0.1-0.5).

[0019] In some embodiments of the present invention, the pH value of the injectable hydrogel is 7-10.

[0020] The second aspect of the present invention provides a method for preparing the injectable hydrogel according to the first aspect of the present invention, comprising the following steps:

[0021] The thiolated microspheres and the dopamine-modified hyaluronic acid are uniformly mixed to obtain the injectable hydrogel.

[0022] In some embodiments of the present invention, the method for preparing the injectable hydrogel further comprises the step of adjusting the pH value of the injectable hydrogel to 7-10.

[0023] In some embodiments of the present invention, the method for preparing the thiol-modified microspheres comprises the following steps:

[0024] The main monomer, the pH responsive monomer, the redox responsive monomer and the solvent are stirred and mixed to obtain a comonomer solution;

[0025] After the emulsifier and the solvent are stirred and mixed, an initiator is added under an inert gas atmosphere at 70-90° C. to carry out a polymerization reaction to obtain a microsphere dispersion;

[0026] The comonomer solution is added into the microsphere dispersion, and then a reducing agent is added to react at room temperature to obtain the thiolated microspheres.

[0027] In some embodiments of the present invention, the polymerization reaction time is 5-8 hours.

[0028] In some embodiments of the present invention, the reaction time at room temperature is 30-60 minutes.

[0029] In some embodiments of the present invention, the inert gas comprises argon and / or nitrogen.

[0030] In some embodiments of the present invention, the method for preparing dopamine-modified hyaluronic acid comprises the following steps:

[0031] Dopamine hydrochloride, hyaluronic acid and a solvent are stirred and mixed, and the pH is adjusted to alkaline to obtain the dopamine-modified hyaluronic acid.

[0032] In some embodiments of the present invention, in the method for preparing dopamine-modified hyaluronic acid, the stirring time is 4-6 hours.

[0033] In some embodiments of the present invention, in the method for preparing dopamine-modified hyaluronic acid, the pH is adjusted to 8-9.

[0034] In some embodiments of the present invention, the solvent is water.

[0035] The third aspect of the present invention provides use of the injectable hydrogel described in the first aspect of the present invention in the preparation of biomedical materials.

[0036] In some embodiments of the present invention, the biomedical material includes a tissue repair and regeneration material.

[0037] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0038] (1) The injectable hydrogel provided by the present invention comprises thiol-functionalized swellable macromolecular microspheres and dopamine-modified hyaluronic acid, which can be prepared by simply physically mixing the two components. At human body temperature (about 37°C), the system can generate a hydrogel with a certain modulus and better stability through a Michael addition reaction between the thiol and catechol groups, showing excellent mechanical properties and biocompatibility. This new type of hydrogel is expected to open up new application prospects in the field of biomedical materials.

[0039] (2) The two-component polymer (thiolated microspheres and dopamine-modified hyaluronic acid) of the present invention has strong biocompatibility and can form an injectable physical hydrogel after mixing. It can further form a hydrogel with a certain modulus through chemical cross-linking under human body temperature. In addition, the present invention can adjust the mechanical properties of the hydrogel subsequently formed in the human body by adjusting the ratio of thiolated microspheres to hyaluronic acid and the pH value of the injectable hydrogel, which has obvious advantages. DETAILED DESCRIPTION

[0040] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0041] Unless otherwise specified, the raw materials, reagents or devices used in the present invention can be obtained from conventional commercial channels or by existing known methods.

[0042] Example 1: Thiolated microspheres 1

[0043] This example provides a thiolated microsphere 1, the raw material components of which are shown in Table 1.

[0044] Table 1

[0045]

[0046]

[0047] The preparation method of the thiol microspheres 1 of this example comprises the following steps:

[0048] (1) stirring and mixing 2-(2-methoxyethoxy)ethyl 2-methyl-2-acrylate, methacrylic acid, disulfanediylbis(ethane-2,1-diyl)bis(2-methylacrylate) and deionized water to obtain a comonomer solution;

[0049] (2) After sodium dodecyl sulfate and deionized water were stirred and mixed, ammonium persulfate was added under a nitrogen atmosphere at 80° C. to carry out polymerization reaction for 6.5 hours to obtain a microsphere dispersion;

[0050] (3) uniformly adding the comonomer solution of step (1) into the microsphere dispersion of step (2), and then adding tri(2-carboxyethyl)phosphine, and reacting at room temperature for 45 minutes to obtain thiolated microspheres.

[0051] Example 2: Thiolated microspheres 2

[0052] This example provides a thiolated microsphere 2, the raw material components of which are shown in Table 2.

[0053] Table 2

[0054] Raw material components Mass Methyl Methacrylate 87 Methacrylic acid 15 Disulfanediylbis(ethane-2,1-diyl)bis(2-methacrylate) 8.7 Sodium Lauryl Sulfate 3.3 Ammonium Persulfate 0.3 Deionized water 600 Tris(2-carboxyethyl)phosphine 3.55

[0055] The preparation method of the thiol microspheres 2 of this example comprises the following steps:

[0056] (1) stirring and mixing methyl methacrylate, methacrylic acid, disulfanediylbis(ethane-2,1-diyl)bis(2-methacrylate) and deionized water to obtain a comonomer solution;

[0057] (2) After sodium dodecyl sulfate and deionized water were stirred and mixed, ammonium persulfate was added under a nitrogen atmosphere at 80° C. to carry out polymerization reaction for 6.5 hours to obtain a microsphere dispersion;

[0058] (3) uniformly adding the comonomer solution of step (1) into the microsphere dispersion of step (2), and then adding tri(2-carboxyethyl)phosphine, and reacting at room temperature for 45 minutes to obtain thiolated microspheres.

[0059] Example 3: Thiolated microspheres 3

[0060] This example provides a thiol-modified microsphere 3, the raw material components of which are shown in Table 3.

[0061] Table 3

[0062] Raw material components Mass Ethyl acrylate 87 acrylic acid 15 Disulfanediylbis(ethane-2,1-diyl)bis(2-methacrylate) 8.7 Sodium Lauryl Sulfate 3.3 Ammonium Persulfate 0.3 Deionized water 600 Tris(2-carboxyethyl)phosphine 3.55

[0063] The preparation method of the thiol microspheres 3 in this example comprises the following steps:

[0064] (1) stirring and mixing ethyl acrylate, acrylic acid, disulfanediylbis(ethane-2,1-diyl)bis(2-methylacrylate) and deionized water to obtain a comonomer solution;

[0065] (2) After sodium dodecyl sulfate and deionized water were stirred and mixed, ammonium persulfate was added under a nitrogen atmosphere at 80° C. to carry out polymerization reaction for 6.5 hours to obtain a microsphere dispersion;

[0066] (3) uniformly adding the comonomer solution of step (1) into the microsphere dispersion of step (2), and then adding tri(2-carboxyethyl)phosphine, and reacting at room temperature for 45 minutes to obtain thiolated microspheres.

[0067] Example 4: Thiolated microspheres 4

[0068] This example provides a thiol-modified microsphere 4, the raw material components of which are shown in Table 4.

[0069] Table 4

[0070]

[0071]

[0072] The preparation method of the thiol microsphere 4 in this example comprises the following steps:

[0073] (1) stirring and mixing N-isopropylacrylamide, acrylic acid, N,N'-bis(acryl)cystamine and deionized water to obtain a comonomer solution;

[0074] (2) After sodium dodecyl sulfate and deionized water were stirred and mixed, potassium persulfate was added under a nitrogen atmosphere at 80° C. to carry out polymerization reaction for 6.5 hours to obtain a microsphere dispersion;

[0075] (3) The comonomer solution of step (1) is uniformly added to the microsphere dispersion of step (2), and then dithiothreitol is added, and the mixture is reacted at room temperature for 45 minutes to obtain thiolated microspheres.

[0076] Example 5: Dopamine modified hyaluronic acid 1

[0077] This example provides a dopamine-modified hyaluronic acid 1, whose raw material components are shown in Table 5.

[0078] Table 5

[0079] Raw material components Mass Dopamine Hydrochloride 0.5 Hyaluronic acid 3 Deionized water 45

[0080] The preparation method of dopamine-modified hyaluronic acid 1 in this example comprises the following steps:

[0081] Dopamine hydrochloride, hyaluronic acid and deionized water were stirred for 5 hours, and a 2 mol / L NaOH solution was added to adjust the pH to 8.3 to obtain dopamine-modified hyaluronic acid.

[0082] Example 6: Dopamine modified hyaluronic acid 2

[0083] This example provides a dopamine-modified hyaluronic acid 2, the raw material components of which are shown in Table 6.

[0084] Table 6

[0085] Raw material components Mass Dopamine Hydrochloride 0.5 Hyaluronic acid 5 Deionized water 45

[0086] The preparation method of dopamine-modified hyaluronic acid 2 in this example comprises the following steps:

[0087] Dopamine hydrochloride, hyaluronic acid and deionized water were stirred for 5 hours, and a 2 mol / L NaOH solution was added to adjust the pH to 8.3 to obtain dopamine-modified hyaluronic acid.

[0088] Example 7: Dopamine modified hyaluronic acid 3

[0089] This example provides a dopamine-modified hyaluronic acid 3, the raw material components of which are shown in Table 7.

[0090] Table 7

[0091] Raw material components Mass Dopamine Hydrochloride 0.5 Hyaluronic acid 7 Deionized water 45

[0092] The preparation method of dopamine-modified hyaluronic acid 3 in this example comprises the following steps:

[0093] Dopamine hydrochloride, hyaluronic acid and deionized water were stirred for 5 hours, and a 2 mol / L NaOH solution was added to adjust the pH to 8.3 to obtain dopamine-modified hyaluronic acid.

[0094] Example 8: Injectable Hydrogel

[0095] This example provides an injectable hydrogel, the raw material components of which are shown in Table 8.

[0096] Table 8

[0097] Raw material components Mass Thiolated microspheres 1 50 Dopamine-modified hyaluronic acid 1 20

[0098] The method for preparing the injectable hydrogel of this example comprises the following steps:

[0099] The thiolated microspheres 1 and the dopamine-modified hyaluronic acid 1 were stirred and mixed, and a 2 mol / L NaOH solution was added to adjust the pH to 7.4 to obtain an injectable hydrogel.

[0100] Example 9: Injectable Hydrogel

[0101] This example provides an injectable hydrogel, the raw material components of which are shown in Table 9.

[0102] Table 9

[0103] Raw material components Mass Thiolated microspheres 1 50 Dopamine modified hyaluronic acid 2 20

[0104] The method for preparing the injectable hydrogel of this example comprises the following steps:

[0105] The thiolated microspheres 1 and the dopamine-modified hyaluronic acid 2 were stirred and mixed, and a 2 mol / L NaOH solution was added to adjust the pH to 7.4 to obtain an injectable hydrogel.

[0106] Example 10: Injectable Hydrogel

[0107] This example provides an injectable hydrogel, the raw material components of which are shown in Table 10.

[0108] Table 10

[0109] Raw material components Mass Thiolated microspheres 1 50 Dopamine modified hyaluronic acid 3 20

[0110] The method for preparing the injectable hydrogel of this example comprises the following steps:

[0111] The thiolated microspheres 1 and dopamine-modified hyaluronic acid 3 were stirred and mixed, and a 2 mol / L NaOH solution was added to adjust the pH to 7.4 to obtain an injectable hydrogel.

[0112] Example 11: Injectable Hydrogel

[0113] This example provides an injectable hydrogel, the raw material components of which are shown in Table 11.

[0114] Table 11

[0115] Raw material components Mass Thiolated microspheres 1 50 Dopamine modified hyaluronic acid 2 20

[0116] The method for preparing the injectable hydrogel of this example comprises the following steps:

[0117] The thiolated microspheres 1 and the dopamine-modified hyaluronic acid 2 were stirred and mixed, and a 2 mol / L NaOH solution was added to adjust the pH to 8.4 to obtain an injectable hydrogel.

[0118] Example 12: Injectable Hydrogel

[0119] This example provides an injectable hydrogel, the raw material components of which are shown in Table 12.

[0120] Table 12

[0121] Raw material components Mass Thiolated microspheres 1 50 Dopamine modified hyaluronic acid 2 20

[0122] The method for preparing the injectable hydrogel of this example comprises the following steps:

[0123] The thiolated microspheres 1 and the dopamine-modified hyaluronic acid 2 were stirred and mixed, and a 2 mol / L NaOH solution was added to adjust the pH to 9.6 to obtain an injectable hydrogel.

[0124] Mechanical properties test

[0125] Rheological test methods:

[0126] The products prepared in Examples 1, 7 and Examples 8-12 were placed in an oven at 37°C for 3 hours, taken out as test samples, and then the dynamic rheology of each sample was tested using a rheometer (TA HR30). The samples were fixed with parallel plates with a diameter of 20 mm and a gap between the plates of 1000 μm. The strain scan was tested at a frequency of 1 Hz, and the storage modulus (G′) and loss modulus (G″) were recorded; the rupture strain value was the strain when G″≥G′.

[0127] The test results are shown in Table 13.

[0128] Table 13

[0129]

[0130]

[0131] As can be seen from Table 13, the ratio of thiolated microspheres to hyaluronic acid in the injectable hydrogel of the present invention and the pH value of the injectable hydrogel can affect the mechanical properties of the hydrogel formed by further reaction at 37°C, and can be adjusted according to actual needs, which has obvious advantages.

[0132] Biocompatibility testing

[0133] Cytotoxicity test method:

[0134] Human breast cancer cells (MCF-7) were used and the cells were grown in Invitrogen α-minimal essential medium (α-MEM) in an incubator (37°C, 5% CO 2 ) were cultured at a density of 5×10 3 Cells / well plate, MCF-7 cells were inoculated into 1.5mL of α-MEM in 24-well plates and cultured for 1 day. In the next few days, cells containing new 1.5mL of α-MEM were exposed to 5mm gel discs (30-40mg) for 1, 2 and 3 days. Subsequently, 40μL MTS analysis (2mg / mL) was performed to test the cytotoxicity of the hydrogel. As a positive control, cells were grown only in α-MEM and PBS buffer. After incubation at 37°C for 1 hour, the absorbance at 490nm was measured using a microplate reader to calculate cell viability.

[0135] The test results are shown in Table 14.

[0136] Table 14

[0137] Cell survival rate (%) Control group Example 12 Day 1 99 97 Day 2 98 96 Day 3 98 96

[0138] As can be seen from Table 14, the cell compatibility of the injectable hydrogel of the present invention is similar to that of the PBS physiological buffer, demonstrating its excellent biocompatibility.

[0139] The preferred embodiments of the present invention are specifically described above, but the invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. An injectable hydrogel, characterized in that The injectable hydrogel comprises the following components: thiolated microspheres and dopamine-modified hyaluronic acid; The raw materials for preparing the thiolated microspheres include a main monomer, a pH-responsive monomer, a redox-responsive monomer, an emulsifier, an initiator and a reducing agent, wherein the main monomer includes at least one of 2-methyl-2-acrylic acid-2-(2-methoxyethoxy)ethyl ester, methyl methacrylate, ethyl acrylate and N-isopropylacrylamide, the pH-responsive monomer includes methacrylic acid and / or acrylic acid, and the redox-responsive monomer includes disulfanediylbis(ethane-2,1-diyl)bis(2-methylacrylate) and / or N,N'-bis(acryl)cystamine; The raw materials for preparing the dopamine-modified hyaluronic acid include dopamine hydrochloride and hyaluronic acid.

2. The injectable hydrogel according to claim 1, characterized in that The raw materials for preparing the thiolated microspheres include, by weight, 1-100 parts of a main monomer, 0.1-20 parts of a pH-responsive monomer, 0.1-10 parts of a redox-responsive monomer, 1-5 parts of an emulsifier, 0.1-0.5 parts of an initiator, and 0.1-5 parts of a reducing agent.

3. The injectable hydrogel according to claim 1, characterized in that In terms of weight, the raw materials for preparing the dopamine-modified hyaluronic acid include 0.1-1 parts of dopamine hydrochloride and 1-10 parts of hyaluronic acid.

4. The injectable hydrogel according to claim 1 or 2, characterized in that: The emulsifier includes sodium dodecyl sulfate; and / or the initiator includes ammonium persulfate and / or potassium persulfate; and / or the reducing agent includes tris(2-carboxyethyl)phosphine and / or dithiothreitol.

5. The injectable hydrogel according to claim 1, characterized in that The mass ratio of the thiolated microspheres to the hyaluronic acid is 5:(0.1-2).

6. The injectable hydrogel according to claim 1, characterized in that The pH value of the injectable hydrogel is 7-10.

7. The method for preparing the injectable hydrogel according to any one of claims 1 to 6, characterized in that: The following steps are involved: The thiolated microspheres and the dopamine-modified hyaluronic acid are uniformly mixed to obtain the injectable hydrogel.

8. The preparation method according to claim 7, characterized in that: The method for preparing the thiol-modified microspheres comprises the following steps: The main monomer, the pH responsive monomer, the redox responsive monomer and the solvent are stirred and mixed to obtain a comonomer solution; After the emulsifier and the solvent are stirred and mixed, an initiator is added under an inert gas atmosphere at 70-90° C. to carry out a polymerization reaction to obtain a microsphere dispersion; The comonomer solution is added into the microsphere dispersion, and then a reducing agent is added to react at room temperature to obtain the thiolated microspheres.

9. The preparation method according to claim 7, characterized in that: The preparation method of dopamine-modified hyaluronic acid comprises the following steps: Dopamine hydrochloride, hyaluronic acid and a solvent are stirred and mixed, and the pH is adjusted to alkaline to obtain the dopamine-modified hyaluronic acid.

10. Use of the injectable hydrogel according to any one of claims 1 to 6 in the preparation of biomedical materials.

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