阅读说明：本技术 一种基于改性透明质酸的超分子纳米纤维的制备方法 (Preparation method of supermolecule nanofiber based on modified hyaluronic acid ) 是由 陈苏 贺福坤 于 2020-06-02 设计创作，主要内容包括：本发明提供了一种基于改性透明质酸的超分子纳米纤维的制备方法。由甲基丙烯酸酐和Phe-Cys-Cys-Phe四肽改性的透明质酸与葫芦脲发生主客体相互作用形成超分子结构再结合静电纺丝技术制得。本发明通过对透明质酸改性而赋予的共价键和非共价键,可以解决透明质酸在组织工程和医药领域中极易溶于水、在组织中停留时间短、降解速度快等涉及稳定性和机械强度的问题。另外改性透明质酸与静电纺丝技术相结合制备的纳米纤维,对于像神经修复领域需要的高比表面积纤维状结构也是具有重大意义的,因此,有望替代神经修复领域常用的如PCL,PLA,PU等高分子聚合物材料。(The invention provides a preparation method of a supermolecule nanofiber based on modified hyaluronic acid. The hyaluronic acid super-molecular nano-fiber is prepared by performing host-guest interaction between hyaluronic acid modified by methacrylic anhydride and Phe-Cys-Cys-Phe tetrapeptide and cucurbituril to form a super-molecular structure and combining an electrostatic spinning technology. The invention can solve the problems of stability and mechanical strength, such as high solubility in water, short retention time in tissues, high degradation speed and the like of hyaluronic acid in the fields of tissue engineering and medicine through covalent bonds and non-covalent bonds endowed by modifying hyaluronic acid. In addition, the nanofiber prepared by combining the modified hyaluronic acid and the electrostatic spinning technology has great significance for fibrous structures with high specific surface areas, such as needed in the nerve repair field, and therefore, the nanofiber is expected to replace high polymer materials, such as PCL, PLA, PU and the like, commonly used in the nerve repair field.)

1. A preparation method of supermolecule nano-fiber based on modified hyaluronic acid comprises the following specific steps:

(1) preliminary modification of hyaluronic acid: carrying out esterification reaction on hyaluronic acid and methacrylic anhydride under an alkaline condition, separating out the reaction product by using an organic solvent after the reaction is finished, and carrying out freeze drying and collection to obtain primarily modified hyaluronic acid with double bonds, and storing the primarily modified hyaluronic acid at a low temperature;

(2) final modification of hyaluronic acid: dissolving the primarily modified hyaluronic acid prepared in the step (1) in deionized water, adding Phe-Cys-Cys-Phe tetrapeptide under the protection of inert gas, adding a reducing agent for reaction, dialyzing a reaction product after the reaction is finished, and freeze-drying to obtain the final modified hyaluronic acid with double bonds and Phe-Cys dipeptide;

(3) dissolving the final modified hyaluronic acid prepared in the step (2) in water, adding cucurbituril and a photoinitiator, and stirring and mixing uniformly to obtain a spinning solution for preparing the nanofiber;

(4) and (4) preparing the spinning solution prepared in the step (3) into nano fibers by an electrostatic spinning method, and then placing the nano fibers under ultraviolet light for crosslinking to obtain the supermolecule nano fibers.

2. The method of claim 1, wherein: the molecular weight of the hyaluronic acid in the step (1) is between 100 and 150 ten thousand.

3. The method of claim 1, wherein: in the step (1), the addition amount of the methacrylic anhydride is 10-15% of the mass of the hyaluronic acid; the alkaline condition in the step (1) is to control the pH of the reaction to be maintained between 8 and 12.

4. The method of claim 1, wherein: the organic solvent in the step (1) is absolute ethyl alcohol, acetone or dimethyl sulfoxide.

5. The method of claim 1, wherein: the dosage of the Phe-Cys-Cys-Phe tetrapeptide in the step (2) is 0.1-1% of the mass of the primary modified hyaluronic acid; the reducing agent in the step (2) is dithiothreitol, 2-mercaptoethanol or tris (2-carbonylethyl) phosphate; the addition amount of the reducing agent is 10-15% of the mass of the first modified hyaluronic acid.

6. The method of claim 1, wherein: the reaction time in the step (2) is 3 to 4 hours.

7. The method of claim 1, wherein: in the step (3), the addition amount of cucurbituril is 0.1-0.5% of the mass of the final modified hyaluronic acid; and (3) dissolving the finally modified hyaluronic acid in water in the step (3) to obtain the finally modified hyaluronic acid with the mass concentration of 1-5%.

8. The method of claim 1, wherein: the photoinitiator in the step (3) is one of potassium persulfate, azobisisobutyramidine hydrochloride, azobisisobutyrimidazoline hydrochloride, ammonium persulfate or 2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone; the addition amount of the photoinitiator is 0.25-1% of the mass of the final modified hyaluronic acid.

9. The method of claim 1, wherein: the parameters of the electrostatic spinning method in the step (5) are as follows: the voltage is 15-20 kv, the flow rate of the spinning solution is 0.5-2ml/h, and the receiving distance is 15-20 cm.

10. The method of claim 1, wherein: the wavelength of the ultraviolet light is 365-; the irradiation time is 1-3 min.

Technical Field

The invention relates to the technical field of preparation of biomedical materials such as tissue engineering, drug release and the like, in particular to a preparation method of supermolecule nano-fibers based on modified hyaluronic acid.

Background

Hyaluronic acid, also known as hyaluronic acid, is a natural high-molecular acidic mucopolysaccharide, widely exists in synovial fluid of human tissues, eyeball and joint head, and plays a role in adhesion, moisture retention and lubrication. Meanwhile, the hydrogel material has excellent properties such as inherent biocompatibility, high water absorption, injectability and similarity with a natural extracellular matrix structure, and the like, and has attracted much attention in the biomedical field in recent years. (Giovanna Pitarresi, Paola Pierro. biomacromolecules2006,7,1302-1310) indicates that the traditional hyaluronic acid hydrogel is a bulk gel formed by randomly crosslinking hyaluronic acid macromolecules, and has the defects of low mechanical property, high hydrolysis speed, short retention time in tissues and the like, so that the application of tissue engineering is greatly limited. To solve this problem, antuenes J C, Oliveira J, Reis R, etc. journal biological materials Research PartA,2010,94, 856-869; ibrahim S, Kang Q K, Ramamurthi A. journal of biological materials Reserch partA,2010,94, 355-; ohri R, Hahn S K, HoffmanA S, etc. research PartA,2004,70,328-334, the literature teaches the use of cross-linking agents including glutaraldehyde, divinyl sulfone (DVS), Adipic Dihydrazide (ADH), etc. to enhance the properties of hyaluronic acid, although this has been shown to enhance mechanical properties. However, there are biocompatibility problems, and references [ Dong C-M, WuX, Caves J, etc. photomedicated cross linking of C6-cinannamate derivative type Icolegen. biomaterials,2005,26, 4041-. In addition, the surface of the hyaluronic acid is rich in carboxyl and hydroxyl, and the purpose of self-crosslinking can be achieved by modifying functional groups on the surface of the hyaluronic acid, so that the defect of poor stability of the hyaluronic acid is overcome. In addition, the nanofiber structure shows better cell adhesion, proliferation and differentiation effects in fields such as nerve repair, wound healing and the like.

Disclosure of Invention

The invention aims to improve the defects of the prior art and provides a preparation method of supermolecular nano-fiber based on modified hyaluronic acid; the method has the advantages of universality, high efficiency, mature technology and the like, provides a path for realizing the rapid preparation of the modified nanofiber, and has higher application value in the fields of tissue engineering and biomedicine.

The technical scheme of the invention is as follows: a preparation method of supermolecule nano-fiber based on modified hyaluronic acid comprises the following specific steps:

(1) preliminary modification of hyaluronic acid: carrying out esterification reaction on hyaluronic acid and methacrylic anhydride under an alkaline condition, separating out the reaction product by using an organic solvent after the reaction is finished, and carrying out freeze drying and collection to obtain primarily modified hyaluronic acid with double bonds, and storing the primarily modified hyaluronic acid at a low temperature;

(2) final modification of hyaluronic acid: dissolving the primarily modified hyaluronic acid in deionized water, adding Phe-Cys-Cys-Phe tetrapeptide under the protection of inert gas, adding a reducing agent for reaction, dialyzing a reaction product after the reaction is finished, and freeze-drying to obtain the final modified hyaluronic acid with double bonds and Phe-Cys dipeptide;

(3) dissolving the final modified hyaluronic acid prepared in the step (2) in water, adding cucurbituril and a photoinitiator, and stirring and mixing uniformly to obtain a spinning solution for preparing the nanofiber;

(4) and (4) preparing the spinning solution prepared in the step (3) into nano fibers by an electrostatic spinning method, and then placing the nano fibers under ultraviolet light for crosslinking to obtain the supermolecule nano fibers.

The Phe-Cys-Cys-Phe tetrapeptide in step (1) of the present invention can be prepared by referring to the references [ Matthew J.Rowland, Marina Atgie, Dominique Hoogland, and Oren A.Scherman.Biomacrolecules 2015,16,2436 and 2443 ] and optimizing some parameters; the preparation method comprises the following specific steps: amidating L-cystine and BOC-Phe-OSU to obtain coarse product, eliminating protecting group with hydrochloric acid solution of six-epoxy ring, washing, filtering to obtain Phe-Cys-Cys-Phe tetrapeptide, and storing at low temperature; preferably L-cystineThe molar ratio of the organic solvent to BOC-Phe-OSU is 1 (2.1-2.25). The structural formula of the Phe-Cys-Cys-Phe tetrapeptide is as follows:

the reaction formula of the preparation process of the modified hyaluronic acid is as follows:

preferably, the molecular weight of the hyaluronic acid in the step (1) is between 100 and 150 million.

The addition amount of the methacrylic anhydride in the step (1) is preferably 10 to 15% by mass of the hyaluronic acid.

Preferably, the alkaline conditions described in step (1) are such that the pH of the reaction is maintained between 8 and 12.

Preferably, the organic solvent in step (1) is absolute ethyl alcohol, acetone or dimethyl sulfoxide.

Preferably, the Phe-Cys-Cys-Phe tetrapeptide used in the step (2) is 0.1-1% of the mass of the primary modified hyaluronic acid.

Preferably, the reducing agent in the step (2) is dithiothreitol, 2-mercaptoethanol or tris (2-carbonylethyl) phosphate; the addition amount of the reducing agent is 10-15% of the mass of the first modified hyaluronic acid.

The reaction time in step (2) is preferably 3 to 4 hours.

Preferably, the addition amount of cucurbituril in the step (3) is 0.1-0.5% of the mass of the final modified hyaluronic acid.

Preferably, the photoinitiator in the step (3) is one of azobisisobutyramidine hydrochloride, azobisisobutyrimidazoline hydrochloride and 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropiophenone; the addition amount of the photoinitiator is 0.25-1% of the mass of the final modified hyaluronic acid.

Preferably, the mass concentration of the finally modified hyaluronic acid after the finally modified hyaluronic acid is dissolved in water in the step (3) is 1-5%.

Preferably, the parameters of the electrostatic spinning method in the step (4) are as follows: the voltage is 15-20 kv, the flow rate of the spinning solution is 0.5-2ml/h, and the receiving distance is 15-20 cm.

Preferably, the wavelength of the ultraviolet light is 365-; the irradiation time is 1-3 min.

The invention adjusts the spinning time according to the thickness of the required nanofiber membrane, and the transparent supermolecule nanofiber membrane can be obtained after being generally controlled to be 5-6 h.

Has the advantages that:

the invention adopts hyaluronic acid as raw material, and prepares the nano-fiber by modifying hyaluronic acid and combining with an electrostatic spinning technology. The simple hyaluronic acid has the problems of low mechanical strength, high hydrolysis speed and the like in practical application, and the requirement on materials in clinical application is difficult to meet. In addition, for the fields such as nerve repair and the like, the required materials have micro-nanofiber structures, and the conventional hyaluronic acid gel cannot meet the requirements, so the combination of the electrostatic spinning technology is particularly important. Therefore, through the modification of hyaluronic acid, more ideal biomedical materials can be prepared on hyaluronic acid macromolecules, and the modified hyaluronic acid is prepared into a nanofiber membrane. The obtained covalent bond and non-covalent bond-producing product has better mechanical strength and is more beneficial to cell adhesion and cell growth promotion, thereby improving the application range of the hyaluronic acid in the biomedical field.

Drawings

FIG. 1 is a schematic view of an electrospinning apparatus of the present invention;

FIG. 2 is a schematic representation of the first modified hyaluronic acid, the final modified hyaluronic acid and pure hyaluronic acid prepared in example 1;

fig. 3 is an SEM image of supramolecular nanofibers prepared by the electrospinning technique in example 1;

fig. 4 is an SEM image of supramolecular nanofibers prepared by the electrospinning technique in example 2;

fig. 5 is an SEM image of supramolecular nanofibers prepared by the electrospinning technique in example 3;

fig. 6 is a graph of the degradation rate of supramolecular hyaluronic acid and pure hyaluronic acid prepared in examples 1-3 under hyaluronidase.

Detailed Description

The present invention will be described below with reference to specific examples, but the present invention is not limited to these examples. The electrostatic spinning equipment of the invention is schematically shown in figure 1.