阅读说明：本技术 一种肝素化透明质酸水凝胶及其制备方法和应用 (Heparinized hyaluronic acid hydrogel and preparation method and application thereof ) 是由 鲁道欢 于珊 曾志文 国翠平 耿志杰 裴大婷 于 2021-09-13 设计创作，主要内容包括：本发明提供一种肝素化透明质酸水凝胶及其制备方法和应用。所述肝素化透明质酸水凝胶通过肝素改性透明质酸水凝胶,能改善细胞对透明质酸水凝胶的粘附,提高细胞活性。本发明的制备方法简单,不需要额外添加功能多肽或蛋白,所制得的肝素化透明质酸水凝胶对细胞粘附性好,提高了细胞活性,有望成为细胞治疗和各种组织工程应用的载体材料,具有广阔应用前景。(The invention provides a heparinized hyaluronic acid hydrogel and a preparation method and application thereof. The heparinized hyaluronic acid hydrogel can improve the adhesion of cells to the hyaluronic acid hydrogel and improve the activity of the cells by modifying the hyaluronic acid hydrogel with heparin. The preparation method is simple, functional polypeptide or protein does not need to be added additionally, the prepared heparinized hyaluronic acid hydrogel has good cell adhesion, improves cell activity, is expected to become a carrier material for cell therapy and various tissue engineering applications, and has wide application prospect.)

1. A heparinized hyaluronic acid hydrogel, comprising: the preparation raw materials comprise: the modified hyaluronic acid is formed by crosslinking with alkenyl modified heparin; the modified hyaluronic acid comprises at least one of alkenyl modified hyaluronic acid and thiol modified hyaluronic acid.

2. The heparinized hyaluronic acid hydrogel of claim 1, characterized in that: the modified hyaluronic acid is selected from at least one of methacrylic anhydride modified hyaluronic acid, acryloyl chloride modified hyaluronic acid, glycerol methacrylate modified hyaluronic acid and cysteamine modified hyaluronic acid; preferably, the alkenyl modified heparin is at least one selected from the group consisting of methacrylic anhydride modified heparin, acrylic anhydride modified heparin, and maleic anhydride modified heparin.

3. The heparinized hyaluronic acid hydrogel of claim 1, characterized in that: the mass ratio of the modified hyaluronic acid to the alkenyl modified heparin is (1-40): (1-10).

4. The heparinized hyaluronic acid hydrogel of claim 1, characterized in that: the alkenyl modified heparin is prepared by esterification reaction of hydroxyl in heparin and olefine acid anhydride group in an alkenyl modifying reagent, and the molar ratio of the hydroxyl in the heparin to the olefine acid anhydride group in the alkenyl modifying reagent is preferably 1: (5-20).

5. A method for preparing the heparinized hyaluronic acid hydrogel according to any one of claims 1 to 4, wherein: the method comprises the following steps:

s1: mixing the modified hyaluronic acid and the alkenyl modified heparin with a photoinitiator and water to obtain a hydrogel precursor solution;

s2: and (3) carrying out illumination crosslinking reaction on the hydrogel precursor solution prepared by the S1 to obtain the heparinized hyaluronic acid hydrogel.

6. The method for preparing a heparinized hyaluronic acid hydrogel according to claim 5, characterized in that: in S1, the concentration of the modified hyaluronic acid is 1-4 wt%; preferably, the concentration of the alkenyl modified heparin is 0.05 wt% to 10 wt%; more preferably, the concentration of the photoinitiator is 0.005 wt% to 0.1 wt%.

7. The method for preparing a heparinized hyaluronic acid hydrogel according to claim 5, characterized in that: in S1, the preparation method of the alkenyl modified heparin comprises the following steps: and (3) adjusting the heparin sodium solution to be alkaline, dropwise adding an alkenyl modified reagent under ice bath, and reacting to obtain the alkenyl modified heparin.

8. The method for preparing a heparinized hyaluronic acid hydrogel according to claim 7, characterized in that: the preparation method of the alkenyl modified heparin further comprises the step of purifying the solution after reaction to obtain the alkenyl modified heparin; preferably, the purification comprises precipitation, dialysis, and lyophilization.

9. The method for preparing a heparinized hyaluronic acid hydrogel according to claim 5, characterized in that: the alkenyl modifying agent is at least one selected from methacrylic anhydride, acrylic anhydride and maleic anhydride.

10. Use of the heparinized hyaluronic acid hydrogel of any of claims 1-4 in the preparation of a cell carrier, a drug carrier or a tissue repair material.

Technical Field

The invention belongs to the technical field of biological materials, and particularly relates to a heparinized hyaluronic acid hydrogel and a preparation method and application thereof.

Background

Hyaluronic Acid (HA) is a natural mucopolysaccharide, an important component in natural extracellular matrix, HAs unique viscoelasticity, excellent biological compatibility and degradability, and is an ideal tissue engineering material. The hyaluronic acid hydrogel has good application prospect in tissue engineering of cartilage, nerve, blood vessel, skin, throat and the like, and has attracted much attention in the biomedical field in recent years. However, the hyaluronic acid hydrogel itself has no adhesiveness to cells and proteins, and this property greatly limits its application in tissue engineering. The adhesion properties of the cells to the material greatly influence further cell behaviour, such as: proliferation, migration and differentiation, the simple hyaluronic acid hydrogel has poor adhesion to cells, and is not enough to maintain the activity of the cells for a long time, thereby influencing the proliferation of the cells.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a heparinized hyaluronic acid hydrogel, which can improve the adhesion of cells to the hyaluronic acid hydrogel and improve the activity of the cells by modifying the hyaluronic acid hydrogel with heparin.

The second aspect of the present invention provides a method for preparing the above-mentioned heparinized hyaluronic acid hydrogel.

The third aspect of the invention provides an application of the heparinized hyaluronic acid hydrogel.

According to the first aspect of the invention, the heparinized hyaluronic acid hydrogel is provided, and the preparation raw materials comprise modified hyaluronic acid and alkenyl modified heparin which are formed by crosslinking; the modified hyaluronic acid comprises at least one of alkenyl modified hyaluronic acid and thiol modified hyaluronic acid.

Generally, the adhesion of the hyaluronic acid hydrogel to cells is poor, and in the invention, the hyaluronic acid hydrogel is heparinized by utilizing the specific affinity of heparin to extracellular matrix proteins and cells, so that the cell adhesion environment is improved, cell adhesion is induced, an effective binding site is provided for the cell adhesion, the cell activity is improved, and the cell proliferation is promoted without any modification.

In some embodiments of the present invention, the modified hyaluronic acid is selected from at least one of methacrylic anhydride modified hyaluronic acid, acryloyl chloride modified hyaluronic acid, glycerol methacrylate modified hyaluronic acid, cysteamine modified hyaluronic acid.

In some preferred embodiments of the present invention, the modified heparin is selected from at least one of methacrylic anhydride modified heparin, acrylic anhydride modified heparin, maleic anhydride modified heparin.

In some more preferred embodiments of the present invention, the mass ratio of the modified hyaluronic acid to the alkenyl modified heparin is (1-40): (1-10).

In some more preferred embodiments of the present invention, the alkenyl modified heparin is prepared by esterification of hydroxyl group in heparin with an olefinic anhydride group in an alkenyl modifying reagent.

In some more preferred embodiments of the invention, the molar ratio of hydroxyl groups in the heparin to the olefinic anhydride groups in the alkenyl-modifying reagent is 1: (5-20).

According to a second aspect of the present invention, there is provided a method for preparing the above-mentioned heparinized hyaluronic acid hydrogel, comprising the steps of:

s1: mixing the modified hyaluronic acid and the alkenyl modified heparin with a photoinitiator and water to obtain a hydrogel precursor solution;

s2: and (3) carrying out illumination crosslinking reaction on the hydrogel precursor solution prepared by the S1 to obtain the heparinized hyaluronic acid hydrogel.

In some embodiments of the invention, the concentration of the modified hyaluronic acid in S1 is 1 wt% to 4 wt%; preferably, the concentration of the alkenyl modified heparin is 0.05 wt% to 10 wt%; more preferably, the concentration of the photoinitiator is 0.005 wt% to 0.1 wt%.

In some preferred embodiments of the present invention, in S1, the alkenyl modified heparin is prepared by the following steps: and (3) adjusting the heparin sodium solution to be alkaline, dropwise adding an alkenyl modified reagent under ice bath, and reacting to obtain the alkenyl modified heparin.

In some more preferred embodiments of the present invention, the alkalinity is a pH of 8 to 9.

In some more preferred embodiments of the present invention, the reaction time is 12h to 24 h.

In some more preferred embodiments of the present invention, the method for preparing alkenyl modified heparin further comprises purifying the reacted solution to obtain the alkenyl modified heparin; preferably, the purification comprises precipitation, dialysis, lyophilization; still more preferably, the precipitation is a precipitation of the reacted solution in pre-cooled ethanol.

In some more preferred embodiments of the present invention, the alkenyl modifying agent is selected from at least one of methacrylic anhydride, acrylic anhydride, maleic anhydride.

In some more preferred embodiments of the present invention, the photoinitiator is selected from at least one of 2-hydroxy-4' - (2-hydroxyethoxy) -2-methylpropiophenone (CAS number: 106797-53-9), 1-hydroxycyclohexyl-phenyl ketone (CAS number: 947-19-3), lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate (CAS number: 85073-19-4), 2-dimethoxy-phenyl ethanone (CAS number: 38002-92-5), eosin Y (CAS number: 15086-94-9).

In some more preferred embodiments of the present invention, in S2, the light crosslinking is performed by green light or ultraviolet light; preferably, the green light wavelength is 515nm-535nm, and the ultraviolet light wavelength is 350nm-380 nm.

In some more preferred embodiments of the present invention, in S2, the time for the crosslinking reaction is 5S to 120S.

According to a third aspect of the invention, the application of the heparinized hyaluronic acid hydrogel in preparing a cell carrier, a drug carrier or a tissue repair material is provided.

The invention has the beneficial effects that:

1. aiming at the problem of poor cell adhesion of the hyaluronic acid hydrogel, the heparan is modified by adopting natural mucopolysaccharide heparin, and the heparan and the modified hyaluronic acid are subjected to photocrosslinking to form gel, so that the heparinized hyaluronic acid hydrogel can be prepared, the preparation method is simple, and no functional polypeptide or protein is required to be additionally added.

2. The heparinized hyaluronic acid hydrogel improves the adhesion of cells to the hyaluronic acid hydrogel, improves the activity of the cells, is expected to become a carrier material for cell therapy and various tissue engineering applications, and has wide application prospect.

Drawings

The invention is further described with reference to the following figures and examples, in which:

FIG. 1 shows NMR spectra of methacrylic anhydride modified heparin and pre-modified heparin in example 1 of the present invention.

FIG. 2 is a graph showing the effect of the hydrogel prepared in example 1 of the present invention and the hydrogel prepared in comparative example 1 on cell adhesion.

FIG. 3 is a graph showing the effect of the hydrogel prepared in example 2 of the present invention and the hydrogel prepared in comparative example 1 on the activity of cells.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.

Example 1

The embodiment prepares a heparinized hyaluronic acid hydrogel, and the specific process comprises the following steps:

s1: preparing methacrylic anhydride modified heparin: preparing a 1 wt% heparin sodium solution, adjusting the pH of the solution to 8-9 with NaOH under an ice bath condition, slowly dropwise adding 3.2mL of methacrylic anhydride, and reacting for 12 hours under an ice bath condition. Pouring the reacted solution into precooled ethanol for precipitation, collecting the precipitate, filling the precipitate into a dialysis bag with molecular cut-off of 3500Da, dialyzing for 4 days, and freeze-drying to obtain methacrylic anhydride modified heparin;

s2: preparing a hydrogel precursor solution: dissolving methacrylic anhydride modified hyaluronic acid and methacrylic anhydride modified heparin in water to respectively form solutions with the mass fractions of 2% and 1%, adding 0.05% of photoinitiator lithium phenyl-2, 4, 6-trimethylbenzoyl phosphinate, and fully and uniformly mixing to obtain a hydrogel precursor solution;

s3: injecting the hydrogel precursor solution into a mold, and crosslinking for 10s by 365nm ultraviolet light to obtain the heparinized hyaluronic acid (HepHA) hydrogel.

Example 2

The embodiment prepares a heparinized hyaluronic acid hydrogel, and the specific process comprises the following steps:

s1: preparing methacrylic anhydride modified heparin: the preparation method of methacrylic anhydride modified heparin in this example is the same as that of example 1;

s2: preparing a hydrogel precursor solution: dissolving methacrylic anhydride modified hyaluronic acid and methacrylic anhydride modified heparin in water to respectively form solutions with the mass fractions of 2% and 0.1%, adding 0.05 wt% of photoinitiator lithium phenyl-2, 4, 6-trimethylbenzoyl phosphinate, and fully and uniformly mixing to obtain a hydrogel precursor solution;

s3: and injecting the hydrogel precursor solution into a mold, and crosslinking for 20s by 365nm ultraviolet light to obtain the HepHA hydrogel.

Comparative example 1

The comparative example prepared a hyaluronic acid hydrogel, which is different from example 1 in that the hydrogel does not contain modified heparin, and the specific process was as follows:

s1: dissolving methacrylic anhydride modified hyaluronic acid in water to form a solution with the mass fraction of 2%, adding 0.05 wt% of photoinitiator phenyl-2, 4, 6-trimethyl benzoyl lithium phosphinate, and fully and uniformly mixing to obtain a hydrogel precursor solution;

s2: injecting the hydrogel precursor solution into a mold, and crosslinking for 10s by 365nm ultraviolet light to form the hyaluronic acid (HAMA) hydrogel.

Test example 1

The results of nuclear magnetic characterization of the methacrylic anhydride-modified heparin obtained in S1 in example 1 and the heparin before modification are shown in fig. 1.

As can be seen from FIG. 1, comparing the NMR spectra of heparin and methacrylic anhydride modified heparin, the NMR spectra of methacrylic anhydride modified heparin have characteristic double bond peaks at 5.64ppm and 6.05ppm, indicating that methacrylic anhydride was successfully grafted on heparin.

Test example 2: adhesion assay of Human Umbilical Vein Endothelial Cells (HUVEC) to different hydrogels

The hydrogels prepared in example 1 and comparative example 1 were sterilized, placed in 48-well plates, and seeded with HUVEC at about 2X 10 per well⁴Individual cells, 5% CO at 37 ℃₂Culturing under the conditions of (1). After culturing for 6 and 78 hours respectively, the cells are marked by live cell fluorescent dye fluorescein diacetate, and the adhesion condition of the cells on the water is observed by an inverted fluorescence microscope. As shown in fig. 2. As can be seen from FIG. 2, the number of cells adhered on the HepHA hydrogel was greater than that of the HAMA hydrogel.

Test example 3: effect of different hydrogels on Human Umbilical Vein Endothelial Cell (HUVEC) Activity

The hydrogels prepared in example 2 and comparative example 1 were sterilized, placed in 48-well plates, and seeded with HUVEC at about 2X 10 per well⁴Individual cells, 5% CO at 37 ℃₂Culturing under the conditions of (1). Culturing for 1, 4 and 7 days, respectively, removing culture medium solution, adding culture medium solution containing 10% CCK-8 into each well, culturing at 37 deg.C with 5% CO₂After incubation for 2h, 100. mu.L of the supernatant was taken out of each well and put into a 96-well plate, and absorbance at 450nm was measured with a microplate reader, and the results are shown in FIG. 3. As can be seen from fig. 3, the HepHA hydrogel significantly improved the cell activity compared to the pure HAMA hydrogel, the cells still maintained higher activity at day 7, while the cells in the HAMA hydrogel decreased their activity at day 7.

The embodiments of the present invention have been described in detail, but the present invention is not limited to the embodiments, and various changes can be made without departing from the gist of the present invention within the knowledge of those skilled in the art. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.