阅读说明：本技术 一种生物组织水凝胶粘合剂及其制备方法 (Biological tissue hydrogel adhesive and preparation method thereof ) 是由 周应山 周鼎 万婷婷 杨红军 刘欣 顾绍金 叶德展 陶咏真 徐卫林 于 2020-02-22 设计创作，主要内容包括：本发明公开了一种生物组织水凝胶粘合剂及其制备方法,通过合成接枝多巴胺的马来酰化透明质酸,并对其进行紫外光聚合成型和氧化剂增强,制备了生物组织水凝胶粘合剂。通过上述方式,本发明无需进行醛基化,不仅能简化制备过程,还能够使制得的前驱体溶液粘度高、不易分散,在紫外光的照射下易于原位成型,能够良好地贴合生物组织。同时,本发明通过提高马来酰基的取代度,大幅提高了多巴胺的取代度,使制得的生物组织水凝胶粘合剂具有更高的粘结强度；并利用高碘酸钠对水凝胶进行氧化增强,进一步提高其在潮湿环境下的持久粘附性能。且本发明制备的生物组织水凝胶具有优异的止血封闭性能和较好的柔韧性,能够满足实际应用的需求。(The invention discloses a biological tissue hydrogel adhesive and a preparation method thereof. Through the mode, the preparation method does not need to carry out hydroformylation, not only can simplify the preparation process, but also can ensure that the prepared precursor solution has high viscosity and is difficult to disperse, is easy to form in situ under the irradiation of ultraviolet light, and can be well attached to biological tissues. Meanwhile, the substitution degree of the maleic acyl is improved, so that the substitution degree of the dopamine is greatly improved, and the prepared biological tissue hydrogel adhesive has higher bonding strength; and the sodium periodate is utilized to carry out oxidation enhancement on the hydrogel, so that the lasting adhesion performance of the hydrogel in a humid environment is further improved. The biological tissue hydrogel prepared by the invention has excellent hemostatic sealing performance and better flexibility, and can meet the requirements of practical application.)

1. A method for preparing a biological tissue hydrogel adhesive is characterized by comprising the following steps:

s1, preparation of maleylated hyaluronic acid: dispersing hyaluronic acid in an aprotic solvent according to a preset mass-volume ratio, and fully stirring to obtain a hyaluronic acid solution; dissolving a predetermined amount of maleic anhydride in the aprotic solvent, adding the maleic anhydride into the hyaluronic acid solution to enable the mass ratio of the hyaluronic acid to the maleic anhydride to be 1 (1-5), reacting at 40-60 ℃ for 24-48 h, centrifuging the reaction solution, adjusting the pH of the centrifuged upper layer liquid to 8-9 by using an aqueous sodium bicarbonate solution, precipitating by using acetone, and performing suction filtration to obtain a precipitate; dialyzing the precipitate by using a dialysis membrane, and freeze-drying after dialysis to obtain maleylation hyaluronic acid with the molar substitution degree of maleoyl of 0.5-3;

s2, preparation of dopamine grafted maleylation hyaluronic acid: dissolving the maleylation hyaluronic acid obtained in the step S1 in a phosphate buffer solution, fully stirring and dissolving, adding N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in a predetermined amount, fully mixing, adding dopamine hydrochloride to enable the mass ratio of the maleylation hyaluronic acid to the dopamine hydrochloride to be 1 (0.5-1.5), reacting at 25-30 ℃ for 12-36 hours, dialyzing the obtained reaction liquid by using the dialysis membrane, and freeze-drying after dialysis to obtain the dopamine grafted maleylation hyaluronic acid with the dopamine molar substitution degree of 0.7-1.5;

s3, preparation of biological tissue hydrogel adhesive: adding deionized water into the dopamine grafted maleylation hyaluronic acid obtained in the step S2 to prepare a precursor solution with a preset concentration, adding a predetermined amount of photoinitiator into the precursor solution, fully mixing, and irradiating by ultraviolet light to obtain hydrogel; and dropwise adding sodium periodate aqueous solution on the surface of the hydrogel to fully oxidize the hydrogel so as to obtain the biological tissue hydrogel adhesive with enhanced oxidation.

2. A method of preparing a biological tissue hydrogel adhesive according to claim 1, wherein: the cut-off molecular weight of the dialysis membrane is 8000-14000 Da, and the dialysis time is 2-3 days.

3. The method of claim 1, wherein in step S1, the predetermined ratio of hyaluronic acid to aprotic solvent is 1g (100-150) m L.

4. A method of preparing a biological tissue hydrogel adhesive according to claim 1, wherein: in step S2, the mass ratio of the maleylated hyaluronic acid to the N-hydroxysuccinimide to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 1 (0.5-1) to (1-2).

5. A method of preparing a biological tissue hydrogel adhesive according to claim 1, wherein: in step S3, the concentration of the dopamine-grafted maleylated hyaluronic acid in the precursor solution is 3% to 7%; the concentration of the photoinitiator is 0.3-0.7%.

6. A method of preparing a biological tissue hydrogel adhesive according to claim 1, wherein: in step S3, the photoinitiator is 2-hydroxy-2-methyl-1-p-hydroxyethyl ether phenyl acetone, the illumination time of the precursor solution containing the photoinitiator under ultraviolet light is 10-20 min, and the wavelength of the ultraviolet light is 365 nm.

7. A method of preparing a biological tissue hydrogel adhesive according to claim 1, wherein: in step S3, the concentration of the sodium periodate aqueous solution is 2% to 5%, and the time of oxidation is 30 to 60 seconds.

8. A method of preparing a biological tissue hydrogel adhesive according to claim 1, wherein: in step S1, the aprotic solvent is dimethyl sulfoxide or dimethylformamide.

9. A method of preparing a biological tissue hydrogel adhesive according to claim 1, wherein: in step S2, the phosphate buffer solution is Na with pH 5.0₂HPO₄-NaH₂PO₄Buffer solution or K₂HPO₄-KH₂PO₄And (4) buffer solution.

10. A biological tissue hydrogel adhesive characterized by: the biological tissue hydrogel adhesive is prepared by the preparation method of any one of claims 1 to 9.

Technical Field

The invention relates to the technical field of biological tissue adhesives, in particular to a biological tissue hydrogel adhesive and a preparation method thereof.

Background

A biological tissue adhesive is a biomedical material used for biological tissue adhesion, local tissue hemorrhage closure and repair, can replace or assist sutures in surgery, can be applied more quickly, reduces pain and eliminates the need for stitches to be removed. The ideal biological tissue adhesive has the following requirements: good biocompatibility, high bonding strength, high bonding speed, biodegradability and the like.

Currently, the commercially available adhesives used clinically are primarily cyanoacrylate and fibrin adhesives. The cyanoacrylate adhesive has certain bonding strength, but is generally poor in water resistance, and simultaneously inevitably generates formaldehyde to stimulate biological tissues to generate side effects; and the cyanoacrylate adhesive has brittle mechanical property and poor adaptability to soft tissues, thereby further limiting the application range of the adhesive. Although the fibrin adhesive can play a certain sealing effect on the damage of biological tissues, the durable sealing performance of the fibrin adhesive in a humid environment is weak, and the biological tissues can generate immune reaction on fibrinogen to cause certain side effect, thereby limiting the application range of the adhesive. Therefore, there is a need to develop other types of adhesives to meet the application requirements.

The hydrogel is an ideal biological tissue adhesive material as a polymer with a three-dimensional network crosslinking structure, and the structure of the hydrogel is similar to that of a human extracellular matrix. In various synthetic materials of hydrogel, hyaluronic acid is one of main components of extracellular matrix, has specific cell recognition sites, can interact with cell receptors, has important effects on growth, migration and adhesion of cells, and has good biocompatibility and biodegradability, so the hyaluronic acid is widely applied to the field of biomedical materials. Meanwhile, as the structural unit of the hyaluronic acid has rich functional group sites such as carboxyl, hydroxyl and the like, the grafting reaction can be designed according to the application, and various grafting modifications aiming at the hyaluronic acid are widely concerned by researchers.

Patent publication No. CN107158453A provides a preparation method of a hyaluronic acid tissue adhesive, wherein maleic acyl, aldehyde and dopamine hydrochloride are adopted to modify sodium hyaluronate molecules, and the content of grafted dopamine is increased through a hyaluronic acid hydroformylation process, so that the bonding strength of the hyaluronic acid tissue adhesive is increased. Although the introduction amount of dopamine can be increased by utilizing the Schiff base rapid condensation reaction principle between aldehyde groups and amino groups of dopamine, the dopamine capable of being introduced by the method is limited, and the molar substitution degree of the dopamine in the patent is 0.1-0.6 and still needs to be increased; meanwhile, the hydroformylation can reduce the molecular weight of the material, so that the obtained hyaluronic acid tissue adhesive becomes thin and has high fluidity, and the difficulty of in-situ molding is increased. In addition, the hyaluronic acid tissue adhesive prepared by the patent is brittle, and limits the application range of the hyaluronic acid tissue adhesive.

In view of the above, there is still a need to provide a biological tissue hydrogel adhesive with high dopamine substitution degree, easy in-situ forming, and more flexible and suitable for use, so that the biological tissue hydrogel adhesive has excellent biological tissue adhesion performance and hemostatic sealing performance to meet the requirements of practical applications.

Disclosure of Invention

In view of the above problems, an object of the present invention is to provide a biological tissue hydrogel adhesive which is easily formed in situ and has excellent adhesive properties, and a method for preparing the same, by synthesizing dopamine-grafted maleylated hyaluronic acid without aldehyde group formation, and performing ultraviolet polymerization formation and sodium periodate oxidation on the synthesized product.

In order to achieve the above object, the present invention provides a method for preparing a hydrogel adhesive for biological tissue, comprising the steps of:

s1, preparation of maleylated hyaluronic acid: dispersing hyaluronic acid in an aprotic solvent according to a preset mass-volume ratio, and fully stirring to obtain a hyaluronic acid solution; dissolving a predetermined amount of maleic anhydride in the aprotic solvent, adding the maleic anhydride into the hyaluronic acid solution to enable the mass ratio of the hyaluronic acid to the maleic anhydride to be 1 (1-5), reacting at 40-60 ℃ for 24-48 h, centrifuging the reaction solution, adjusting the pH of the centrifuged upper layer liquid to 8-9 by using a sodium bicarbonate solution, precipitating by using acetone, and performing suction filtration to obtain a precipitate; dialyzing the precipitate by using a dialysis membrane, and freeze-drying after dialysis to obtain maleylation hyaluronic acid with the molar substitution degree of maleoyl of 0.5-3;

s2, preparation of dopamine grafted maleylation hyaluronic acid: dissolving the maleylation hyaluronic acid obtained in the step S1 in a phosphate buffer solution, fully stirring and dissolving, adding N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in a predetermined amount, fully mixing, adding dopamine hydrochloride to enable the mass ratio of the maleylation hyaluronic acid to the dopamine hydrochloride to be 1 (0.5-1.5), reacting at 25-30 ℃ for 12-36 hours, dialyzing the obtained reaction liquid by using the dialysis membrane, and freeze-drying after dialysis to obtain the dopamine grafted maleylation hyaluronic acid with the dopamine molar substitution degree of 0.7-1.5;

s3, preparation of biological tissue hydrogel adhesive: adding deionized water into the dopamine grafted maleylation hyaluronic acid obtained in the step S2 to prepare a precursor solution with a preset concentration, adding a predetermined amount of photoinitiator into the precursor solution, fully mixing, and irradiating by ultraviolet light to obtain hydrogel; and dropwise adding sodium periodate aqueous solution on the surface of the hydrogel to fully oxidize the hydrogel so as to obtain the biological tissue hydrogel adhesive with enhanced oxidation.

Furthermore, the cut-off molecular weight of the dialysis membrane is 8000-14000 Da, and the dialysis time is 2-3 days.

Further, in step S1, the predetermined mass-to-volume ratio of the hyaluronic acid to the aprotic solvent is 1g (100-150) m L.

Further, in step S2, the mass ratio of the maleylated hyaluronic acid to the N-hydroxysuccinimide to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 1 (0.5-1) to 1-2.

Further, in step S3, the concentration of the dopamine-grafted maleylated hyaluronic acid in the precursor solution is 3% to 7%; the concentration of the photoinitiator is 0.3-0.7%.

Further, in step S3, the photoinitiator is 2-hydroxy-2-methyl-1-p-hydroxyethyl ether phenyl acetone, the illumination time of the precursor solution containing the photoinitiator under ultraviolet light is 10-20 min, and the wavelength of the ultraviolet light is 365 nm.

Further, in step S3, the concentration of the aqueous sodium periodate solution is 2% to 5%, and the time for oxidation is 30 to 60 seconds.

Further, in step S1, the aprotic solvent is dimethyl sulfoxide or dimethylformamide.

Further, in step S2, the phosphate buffer solution is Na with pH 5.0₂HPO₄-NaH₂PO₄Buffer solution or K₂HPO₄-KH₂PO₄And (4) buffer solution.

In order to achieve the above object, the present invention further provides a biological tissue hydrogel adhesive prepared according to any one of the above technical solutions.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention prepares the biological tissue hydrogel adhesive which is easy to form in situ and has excellent adhesive property by synthesizing the dopamine-grafted maleylation hyaluronic acid and carrying out ultraviolet polymerization forming and oxidant enhancement on the maleic acylation hyaluronic acid. Compared with the prior art, the preparation method does not carry out hydroformylation, not only can simplify the preparation process, but also can effectively avoid the problems of reduction of the molecular weight of the substance, thinning of the adhesive and increase of the fluidity caused by the hydroformylation, so that the precursor solution prepared by the preparation method has high viscosity and is not easy to disperse, and is easy to form in situ under the irradiation of ultraviolet light, and the adhesive can be well adapted to the shape of the biological tissue and attached to the surface of the biological tissue.

2. In the process of preparing the maleylation hyaluronic acid, hydroxyl is not required to be reserved for hydroformylation, and the substitution degree of the maleyl on hydroxyl on a hyaluronic acid molecule can be effectively improved by regulating and controlling the dosage of the hyaluronic acid and maleic anhydride, so that more carboxyl is introduced into the hyaluronic acid molecule by utilizing the maleyl. A large amount of carboxyl brought by introduction of maleyl can be subjected to condensation reaction with amino on dopamine molecules under the catalytic action of N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, so that the substitution degree of dopamine is greatly improved, and the prepared biological tissue hydrogel adhesive has higher bonding strength. In addition, the dopamine group has a benzene ring structure, so that the rigidity of a polymer chain segment can be improved, the volume shrinkage phenomenon caused by high-content carbon-carbon double bonds introduced by maleic acyl can be effectively relieved, and the prepared hydrogel adhesive can be well attached to the surface of a tissue.

3. The method utilizes the sodium periodate to rapidly oxidize dopamine groups in the hydrogel, so that the structure of the hydrogel can be enhanced and the adhesion performance of the hydrogel can be further improved. In addition, the biological tissue hydrogel adhesive prepared by the invention has better flexibility, can be firmly attached to the surface of a damaged biological tissue and has better hemostatic sealing effect; meanwhile, abundant dopamine groups contained in the hydrogel can react with chemical substances on the surface of the tissue, new chemical bonds are formed between the tissue and the hydrogel interface, water molecules are removed, and the wet adhesion performance is improved, so that the hydrogel adhesive can still maintain the adhesion performance in a wet environment. Meanwhile, the biological tissue hydrogel binding agent can be naturally degraded in a physiological environment, has good affinity to biological tissues, and can meet the requirements of practical application.

Drawings

FIG. 1 is a schematic diagram of the synthetic route for synthesizing a maleated hyaluronic acid grafted with dopamine according to the present invention;

FIG. 2 is a pictorial representation of a biological tissue hydrogel adhesive prepared in accordance with example 1 of the present invention in various states of tension;

FIG. 3 is a stress-strain diagram of a biological tissue hydrogel adhesive prepared in example 1 of the present invention during compression.

Detailed Description

The following detailed description of the preferred embodiments of the present invention, taken in conjunction with the accompanying drawings, will make the advantages and features of the invention easier to understand by those skilled in the art, and thus will clearly and clearly define the scope of the invention. It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without any inventive step, are within the scope of the present invention.

The invention provides a preparation method of a biological tissue hydrogel adhesive, which comprises the following steps:

s1, preparation of maleylated hyaluronic acid: dispersing hyaluronic acid in an aprotic solvent according to a preset mass-volume ratio, and fully stirring to obtain a hyaluronic acid solution; dissolving a predetermined amount of maleic anhydride in the aprotic solvent, adding the maleic anhydride into the hyaluronic acid solution to enable the mass ratio of the hyaluronic acid to the maleic anhydride to be 1 (1-5), reacting at 40-60 ℃ for 24-48 h, centrifuging the reaction solution, adjusting the pH of the centrifuged upper layer liquid to 8-9 by using a sodium bicarbonate solution, precipitating by using acetone, and performing suction filtration to obtain a precipitate; dialyzing the precipitate by using a dialysis membrane, and freeze-drying after dialysis to obtain maleylation hyaluronic acid with the molar substitution degree of maleoyl of 0.5-3;

s2, preparation of dopamine grafted maleylation hyaluronic acid: dissolving the maleylation hyaluronic acid obtained in the step S1 in a phosphate buffer solution, fully stirring and dissolving, adding N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in a predetermined amount, fully mixing, adding dopamine hydrochloride to enable the mass ratio of the maleylation hyaluronic acid to the dopamine hydrochloride to be 1 (0.5-1.5), reacting at 25-30 ℃ for 12-36 hours, dialyzing the obtained reaction liquid by using the dialysis membrane, and freeze-drying after dialysis to obtain the dopamine grafted maleylation hyaluronic acid with the dopamine molar substitution degree of 0.7-1.5;

s3, preparation of biological tissue hydrogel adhesive: adding deionized water into the dopamine grafted maleylation hyaluronic acid obtained in the step S2 to prepare a precursor solution with a preset concentration, adding a predetermined amount of photoinitiator into the precursor solution, fully mixing, and irradiating by ultraviolet light to obtain hydrogel; and dropwise adding sodium periodate aqueous solution on the surface of the hydrogel to fully oxidize the hydrogel so as to obtain the biological tissue hydrogel adhesive with enhanced oxidation.

The cut-off molecular weight of the dialysis membrane is 8000-14000 Da, and the dialysis time is 2-3 days.

In step S1, the predetermined mass-to-volume ratio of the hyaluronic acid to the aprotic solvent is 1g (100-150) m L.

In step S2, the mass ratio of the maleylated hyaluronic acid to the N-hydroxysuccinimide to the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is 1 (0.5-1) to (1-2).

In step S3, the concentration of the dopamine-grafted maleylated hyaluronic acid in the precursor solution is 3% to 7%; the concentration of the photoinitiator is 0.3-0.7%.

In step S3, the photoinitiator is 2-hydroxy-2-methyl-1-p-hydroxyethyl ether phenyl acetone, the illumination time of the precursor solution containing the photoinitiator under ultraviolet light is 10-20 min, and the wavelength of the ultraviolet light is 365 nm.

In step S3, the concentration of the sodium periodate aqueous solution is 2% to 5%, and the time of oxidation is 30 to 60 seconds.

In step S1, the aprotic solvent is dimethyl sulfoxide or dimethylformamide.

In step S2, the phosphate buffer solution is Na with pH 5.0₂HPO₄-NaH₂PO₄Buffer solution or K₂HPO₄-KH₂PO₄And (4) buffer solution.

The invention also provides a biological tissue hydrogel adhesive which is prepared according to any one of the technical schemes.

In the preparation method of the biological tissue hydrogel adhesive provided by the invention, the synthetic route of the dopamine-grafted maleylated hyaluronic acid is shown in figure 1. As can be seen from FIG. 1, when the maleylation hyaluronic acid is prepared, the four hydroxyl sites on the hyaluronic acid molecule can be substituted by the maleic acyl through regulating the dosage of the hyaluronic acid and the maleic anhydride, so that the maleylation hyaluronic acid with high maleylation content is obtained. Then, under the catalytic action of N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, the amino group on the dopamine molecule and the carboxyl group on the hyaluronic acid molecule are subjected to condensation reaction, so that the dopamine is grafted on the maleylated hyaluronic acid. In the traditional process of grafting dopamine by hyaluronic acid, because each hyaluronic acid structure has only one carboxyl, the substitution degree of grafted dopamine is very low, and in order to improve the substitution degree of dopamine, the prior art generally adopts an aldehyde group method, and utilizes the Schiff base rapid condensation reaction principle between aldehyde groups and amino groups of dopamine to improve the introduction amount of dopamine, but the introduction amount of dopamine in the method is limited, so that the molecular weight of a substance is reduced, the fluidity of an adhesive is increased, and the difficulty of in-situ forming is increased. The invention utilizes the high-substitution maleic acyl to add four carboxyl groups to each hyaluronic acid molecule, thereby adding four reaction sites for the grafting reaction of the dopamine, greatly improving the substitution degree of the dopamine and leading the prepared adhesive to have higher bonding strength.

The following describes a biological tissue hydrogel adhesive and a method for preparing the same according to the present invention with reference to examples and drawings.