阅读说明：本技术 促进神经再生的活性多肽在材料表面共价接枝的方法及其应用 (Method for covalently grafting active polypeptide for promoting nerve regeneration on material surface and application thereof ) 是由 张鲁中 杨宇民 李贵才 于 2020-04-16 设计创作，主要内容包括：本发明公开了一种促进神经再生的活性多肽及其在材料表面共价接枝的方法,将生物相容性的天然多糖或蛋白质溶解,冷冻干燥,经酸碱或乙醇变性处理得到天然生物材料支架,天然生物材料支架的羟基与3-(甲基丙烯酰氧)丙基三甲氧基硅烷发生反应得到甲基丙烯酰基改性的生物材料；促进神经再生的多肽CRSYIG,CRSYIG-Biotin,CGQRKDP,CGQRKDP-Biotin,CFKIDKK,CFKIDKK-Biotin等通过硫醇-烯反应接枝到甲基丙烯酰基改性的生物材料。本发明的生物活性研究表明,多肽改性后的生物材料能够协同促进神经再生。(The invention discloses an active polypeptide for promoting nerve regeneration and a covalent grafting method on the surface of a material, which is characterized in that biocompatible natural polysaccharide or protein is dissolved, freeze-dried and subjected to acid-base or ethanol denaturation treatment to obtain a natural biomaterial scaffold, and hydroxyl of the natural biomaterial scaffold reacts with 3- (methacryloyloxy) propyl trimethoxy silane to obtain a methacryloyl modified biomaterial; the polypeptides CRSYIG, CRSYIG-Biotin, CGQRKDP, CGQRKDP-Biotin, CFKIDKK, CFKIDKK-Biotin and the like for promoting nerve regeneration are grafted to the methacryloyl modified biological material through thiol-ene reaction. The biological activity research of the invention shows that the biological material modified by the polypeptide can synergistically promote nerve regeneration.)

1. A method for covalently grafting an active polypeptide for promoting nerve regeneration on the surface of a material, which is a system for covalently grafting the polypeptide for promoting nerve regeneration on the surface of a biological material, comprises modifying a natural biological material scaffold, and covalently connecting the polypeptide for promoting nerve regeneration and the polypeptide to the biological material scaffold, and is characterized in that: the method is a polypeptide grafting method through mercaptan-alkene reaction, and comprises the following specific operations:

step one, preparing a natural biological material scaffold: dissolving natural polysaccharide or protein with biocompatibility, freeze-drying, and performing acid-base or ethanol denaturation treatment to obtain a natural biomaterial scaffold;

step two, preparation of the methacryloyl modified biomaterial: hydroxyl of the natural biomaterial scaffold reacts with 3- (methacryloyloxy) propyl trimethoxy silane to obtain a methacryloyl modified biomaterial;

step three, synthesizing bioactive polypeptide: firstly, screening a series of polypeptides through computer simulation, and synthesizing the polypeptides according to a polypeptide solid phase synthesis method: taking 2-chlorotrityl chloride resin and amino acid protected by Fmoc as starting raw materials, carrying out condensation of amido bonds between the amino acids by using HBTU, cutting off polypeptides from the resin by trifluoroacetic acid, removing protecting groups, and purifying by high performance liquid chromatography to obtain the bioactive polypeptides;

step four, preparing the polypeptide modified biological material: adding the aqueous solution containing the bioactive polypeptide into the solution of the methacryloyl modified biological material, and carrying out thiol-ene reaction on the thiol in the polypeptide and the methacryloyl on the surface of the biological material to obtain the polypeptide modified biological material which is prepared quantitatively and efficiently.

2. The method of claim 1, wherein the active polypeptide for promoting nerve regeneration is covalently grafted to the surface of the material, wherein: the natural polysaccharide is chitosan or alginic acid; the protein is silk fibroin or collagen; the acid, alkali or organic alcohol is hydrochloric acid, sulfuric acid, sodium hydroxide, potassium hydroxide or ethanol.

3. The method of claim 2, wherein the active polypeptide for promoting nerve regeneration is covalently grafted to the surface of the material, wherein: the chitosan is dissolved in an aqueous solution of acetic acid, is frozen and dried, is treated by sodium hydroxide or potassium hydroxide, and reacts with 3- (methacryloyloxy) propyltrimethoxysilane to obtain the methacryl modified chitosan.

4. The method of claim 2, wherein the active polypeptide for promoting nerve regeneration is covalently grafted to the surface of the material, wherein: the alginic acid is dissolved in aqueous solution of sodium hydroxide, is frozen and dried, is treated by hydrochloric acid or sulfuric acid, and reacts with 3- (methacryloyloxy) propyltrimethoxysilane to obtain the methacryl modified alginic acid.

5. The method of claim 2, wherein the active polypeptide for promoting nerve regeneration is covalently grafted to the surface of the material, wherein: dissolving the collagen in an aqueous solution of acetic acid, freeze-drying, neutralizing by a weak sodium bicarbonate aqueous solution, and reacting with 3- (methacryloyloxy) propyltrimethoxysilane to obtain the methacryloyl modified collagen.

6. The method of claim 2, wherein the active polypeptide for promoting nerve regeneration is covalently grafted to the surface of the material, wherein: the silk fibroin is dissolved in an aqueous solution of lithium bromide or calcium chloride/ethanol, is frozen and dried, is neutralized by a weak sodium bicarbonate aqueous solution, and reacts with 3- (methacryloyloxy) propyl trimethoxy silane to obtain the methacryl modified silk fibroin.

7. The method of claim 1, wherein the active polypeptide for promoting nerve regeneration is covalently grafted to the surface of the material, wherein: the concentration of the 3- (methacryloyloxy) propyl trimethoxy silane is 0.001-50% of one or two of methanol or ethanol solution.

8. The method of claim 1, wherein the active polypeptide for promoting nerve regeneration is covalently grafted to the surface of the material, wherein: the bioactive polypeptide is one or more of polypeptide CRSYIG, CRSYIG-Biotin, CGQRKDP, CGQRKDP-Biotin, CFKIDKK and CFKIDKK-Biotin which can promote nerve regeneration.

9. The method of claim 1, wherein the active polypeptide for promoting nerve regeneration is covalently grafted to the surface of the material, wherein: the aqueous solution of the biologically active polypeptide may be one or more of an aqueous solution, a phosphate buffer solution, a cell culture solution, or physiological saline.

10. Use of the method according to claim 1 for covalently grafting an active polypeptide for promoting nerve regeneration to a material surface, wherein the method comprises: the method for modifying the polypeptide can be used in the fields of cardiovascular and cerebrovascular, bone tissue engineering, nerve tissue engineering and skin tissue engineering.

Technical Field

The invention particularly relates to a method for covalently grafting active polypeptide for promoting nerve regeneration on the surface of a material and application thereof.

Background

The modification of the biological material is to endow the biological material with new functions on the premise of not changing the performance of the material body, and has wide application prospect in the field of biomedical materials. The most common methods for chemical modification of biological materials are physical adsorption, covalent grafting and the like. The physical adsorption is mainly determined by the properties of the material surface, for example, the material surface with positive charges can adsorb macromolecules, bioactive polypeptides and proteins with negative charges; by utilizing the specific combination of biotin and streptavidin, the material with biotin can adsorb the streptavidin, thereby realizing the specific adsorption. Although the physical adsorption method is simple and convenient to operate, the bioactive molecules adsorbed by the physical adsorption method are easy to leave or exchange with other proteins, so that the modified material surface loses the original functions. Covalent grafting is a method of covalently bonding a material to a biologically active substance, which is attached to the material at one end, and covalently grafting biologically active molecules that exhibit the same or even higher activity than soluble proteins relative to physical adsorption. For example, silanization is an effective method for achieving covalently grafted chitosan.

In recent years, orthogonal reactions are a class of chemical reactions that can be performed under mild conditions, even under physiological conditions in cells or animals. The reactions can have specific selectivity under complex physiological conditions, and the reaction process is not influenced by the complex physiological conditions and has no side effect. Orthogonal reactions are considered to be the most efficient method for surface modification because of their advantages of simplicity, high efficiency, and high specificity. The thiol-ene reaction is not affected by water and oxygen, quantitatively produces the target product, and is more valued because it does not require contamination by heavy metal catalysts. Through the orthogonal chemical reaction of thiol-ene, the biological activity of biological macromolecules, polypeptides or proteins can be evaluated more conveniently, and the specific labeling of biological active substances is realized under the condition of culturing cells.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the existing lack of effective bioactive molecules for promoting nerve regeneration, the invention aims to develop a novel polypeptide for promoting nerve regeneration and a method for covalently grafting the polypeptide to the surface of a modified biological material for surface modification of the biological material.

The technical scheme is as follows: a method for covalently grafting an active polypeptide for promoting nerve regeneration on the surface of a material, which is a system for covalently grafting the polypeptide for promoting nerve regeneration on the surface of a biomaterial and comprises a modified natural biomaterial scaffold, and a polypeptide for promoting nerve regeneration and a polypeptide are covalently connected to the biomaterial scaffold, wherein the method is a polypeptide grafting method through a thiol-ene reaction, and comprises the following specific operations:

step one, preparing a natural biological material scaffold: dissolving natural polysaccharide or protein with biocompatibility, freeze-drying, and performing acid-base or ethanol denaturation treatment to obtain a natural biomaterial scaffold;

step two, preparation of the methacryloyl modified biomaterial: hydroxyl of the natural biomaterial scaffold reacts with 3- (methacryloyloxy) propyl trimethoxy silane to obtain a methacryloyl modified biomaterial;

step three, synthesizing bioactive polypeptide: firstly, screening a series of polypeptides through computer simulation, and synthesizing the polypeptides according to a polypeptide solid phase synthesis method: taking 2-chlorotrityl chloride resin and amino acid protected by Fmoc as starting raw materials, carrying out condensation of amido bonds between the amino acids by using HBTU, cutting off polypeptides from the resin by trifluoroacetic acid, removing protecting groups, and purifying by high performance liquid chromatography to obtain the bioactive polypeptides;

step four, preparing the polypeptide modified biological material: adding the aqueous solution containing the bioactive polypeptide into the solution of the methacryloyl modified biological material, and carrying out thiol-ene reaction on the thiol in the polypeptide and the methacryloyl on the surface of the biological material to obtain the polypeptide modified biological material which is prepared quantitatively and efficiently.

As an optimization: the natural polysaccharide is chitosan or alginic acid; the protein is silk fibroin or collagen; the acid, alkali or organic alcohol is hydrochloric acid, sulfuric acid, sodium hydroxide, potassium hydroxide or ethanol.

As an optimization: the chitosan is dissolved in an aqueous solution of acetic acid, is frozen and dried, is treated by sodium hydroxide or potassium hydroxide, and reacts with 3- (methacryloyloxy) propyltrimethoxysilane to obtain the methacryl modified chitosan.

As an optimization: the alginic acid is dissolved in aqueous solution of sodium hydroxide, is frozen and dried, is treated by hydrochloric acid or sulfuric acid, and reacts with 3- (methacryloyloxy) propyltrimethoxysilane to obtain the methacryl modified alginic acid.

As an optimization: dissolving the collagen in an aqueous solution of acetic acid, freeze-drying, neutralizing by a weak sodium bicarbonate aqueous solution, and reacting with 3- (methacryloyloxy) propyltrimethoxysilane to obtain the methacryloyl modified collagen.

As an optimization: the silk fibroin is dissolved in an aqueous solution of lithium bromide or calcium chloride/ethanol, is frozen and dried, is neutralized by a weak sodium bicarbonate aqueous solution, and reacts with 3- (methacryloyloxy) propyl trimethoxy silane to obtain the methacryl modified silk fibroin.

As an optimization: the concentration of the 3- (methacryloyloxy) propyl trimethoxy silane is 0.001-50% of one or two of methanol or ethanol solution. The modification of the 3- (methacryloyloxy) propyl trimethoxy silane is that methoxy silane reacts with hydroxyl on polysaccharide or protein.

As an optimization: the bioactive polypeptide is one or more of polypeptide CRSYIG, CRSYIG-Biotin, CGQRKDP, CGQRKDP-Biotin, CFKIDKK and CFKIDKK-Biotin which can promote nerve regeneration. The polypeptide has the function of promoting nerve regeneration, and can promote the proliferation, migration and survival time of Schwann cells through coculture with Schwann cells and dorsal root neuronal cells. The polypeptide containing Biotin was able to increase significantly.

As an optimization: the aqueous solution of the polypeptide may be one or more of an aqueous solution, a phosphate buffer solution, a cell culture solution, or physiological saline.

According to the application of the method for covalently grafting the active polypeptide for promoting nerve regeneration on the surface of a material, the method for modifying the polypeptide can be used in the fields of cardiovascular and cerebrovascular diseases, bone tissue engineering, nerve tissue engineering and skin tissue engineering.

Has the advantages that: the specific advantages of the invention are as follows:

(1) the invention obtains the acryloyl modified biomaterial by reacting 3- (methacryloyloxy) propyl trimethoxy silane with hydroxyl on the biomaterial. The surface modification of the biological material can be efficiently and simply realized through thiol-ene reaction.

(2) The bioactive polypeptides CRSYIG, CGQRKDP and CFKIDKK in the invention can promote the proliferation and migration of Schwann cells, prolong the survival time of neurons and promote the secretion of neurotrophic factors. More importantly, compared with the Biotin group, the polypeptide group and the Biotin/polypeptide mixed group, the polypeptide coupled with the Biotin is more beneficial to the proliferation of Schwann cells and the survival of neurons, and the Biotin and the polypeptide have synergistic action. Compared with a polypeptide solution system, the effect of the polypeptide surface modified biomaterial for synergistically promoting nerve regeneration is more obvious.

Compared with the Biotin group, the polypeptide group and the Biotin/polypeptide mixed group, the covalently connected Biotin-polypeptide can greatly promote the polypeptide to promote the proliferation of schwann cells and prolong the survival time of neurons, and is a result of the dual action of a cell surface receptor-dependent multivitamin transporter of the Biotin and a laminin receptor of a YIGSR cell surface receptor.

The polypeptide surface modification method prepared by the invention is simple and convenient, and the preparation efficiency is high. Compared with the Biotin-polypeptide in the solution, the biological material modified with the Biotin-polypeptide on the surface is more beneficial to the proliferation of Schwann cells and the survival of neurons.

Drawings

FIG. 1 is an infrared spectrum of 3- (methacryloyloxy) propyltrimethoxysilane modified chitosan according to the invention; (a) chitosan, 2% (b), 5% (c), 10% (d), 20% (e) methacryl-modified chitosan;

FIG. 2 is a graph of the double bond content of 2%, 5%, 10%, 20% methacryl-modified chitosan of the present invention;

FIG. 3 is an amino group content of 2%, 5%, 10%, 20% methacryl-modified chitosan of the present invention;

FIG. 4 is a scanning electron microscope image of the surface and cross section of chitosan of the invention, 2%, 5%, 10%, 20% methacryl modified chitosan.

Detailed Description

The present invention will be further illustrated with reference to the following examples, which, however, do not limit the scope of the invention.