1. An amphiphilic high-bioactivity modified hyaluronic acid is characterized in that hyaluronic acid HA is used as a main chain, active short peptide is used as a bioactive grafting group, and a lipophilic group is used as a transdermal promoting grafting group, and the chemical structural formula of the amphiphilic high-bioactivity modified hyaluronic acid is shown as a formula I:

wherein R in the formula I₁Is active short peptide, R₂Is a lipophilic group, or R in formula I₁Is a lipophilic group, R₂The peptide is an active short peptide, and n is 1-7000.

2. The amphiphilic biologically active modified hyaluronic acid of claim 1, wherein the active short peptide comprises a short peptide having the amino acid sequence RXDX, wherein X is E, G, V or S.

3. The amphiphilic biologically active modified hyaluronic acid according to claim 1, wherein the lipophilic group comprises any one or a combination of a carbonyl group of C4-C20, a polyoxypropylene group, a long-chain perfluoroalkyl group, a polysiloxane group, and a hydrocarbon group containing an aryl group, an ester group, an ether group, an amine group, an amide group, a cycloalkyl group, or a double bond.

4. The amphiphilic highly bioactive modified hyaluronic acid according to claim 1, wherein the molecular weight of the HA backbone of hyaluronic acid is between 1K and 2500 kDa.

5. The amphiphilic high-bioactivity modified hyaluronic acid of claim 1, wherein the hydrophilicity and hydrophobicity and the bioactivity of the modified hyaluronic acid shown in formula I can be adjusted by changing the grafting ratio of the active short peptide to the lipophilic group, and the ratio of the grafting ratio of the active short peptide to the lipophilic group is 1000: 1-1: 1000.

6. the amphiphilic biologically active modified hyaluronic acid of claim 1, wherein the active short peptide is directly covalently bonded to the HA backbone of hyaluronic acid or bonded thereto by a short peptide linker.

7. The amphiphilic highly bioactive modified hyaluronic acid according to claim 6, wherein said short peptide linker is WGRGDSP, GWGGLGPAGK or CCRR.

Background

Hyaluronic Acid (HA), also called Hyaluronic acid, is an important natural polysaccharide polymer biomedical material, the basic structure of which is a polysaccharide polymer composed of disaccharide units D-glucuronic acid and N-acetylglucosamine, and HAs the characteristics of excellent water retention, lubricity, viscoelasticity, biocompatibility and the like, so that HA HAs no natural substitute in the fields of medicines, cosmetics and functional foods, particularly when used as a tissue filler, a joint lubricant and a functional moisture retention component. Challenges currently faced in the use of conventional HAs include: (1) the half-life period is short, and the HA in the organism is easily degraded by enzyme; (2) single function, lack of cell adhesion and proliferation sites. Although the enzymolysis resistance of HA can be improved and the functionality can be endowed by means of crosslinking, blending modification (mixing and crosslinking with growth factors, collagen, chitosan and the like), and the like, the methods are easy to cause the problems of immunological rejection, difficult transdermal absorption and the like.

HA is widely distributed in the extracellular matrix of mammalian bone marrow cells, ocular vitreous body and soft tissues, and plays an important role in cellular responses such as proliferation, mutation, migration, adhesion and gene expression. HA with high relative molecular mass can ensure the water content of extracellular matrix and the integrity of cells; low molecular weight HA can induce receptor-mediated endocytosis. High-activity Short Peptides (BSP) are small molecular peptide chains which can simulate the physiological functions of in vivo active proteins and have no immunogenicity, and can realize high expression of effective components in unit density, such as antibiosis, tissue regeneration promotion, immunoregulation and the like. The BSP has clear synthetic route, has the advantages of rich active sites, stable conformation, easy synthesis, convenient storage and the like, and is a research and industrial hotspot in the field of current international functional biomaterials. At present, no report is found about the relevant technology of obtaining a novel HA material by modifying HA with a material with a bioactive function.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: how to modify HA by utilizing a bioactive functional material BSP so as to obtain the HA material with multiple functions.

In order to solve the technical problems, the invention provides an amphiphilic high-bioactivity modified hyaluronic acid, which takes hyaluronic acid HA as a main chain, takes active short peptide as a bioactive grafting group and takes a lipophilic group as a transdermal-promoting grafting group, and the chemical structural formula of the modified hyaluronic acid is shown as a formula I:

wherein R in the formula I₁Is active short peptide, R₂Is a lipophilic group, or R in formula I₁Is a lipophilic group, R₂The peptide is an active short peptide, and n is 1-7000.

Preferably, the active short peptide comprises a short peptide with an amino acid sequence of RXDX, wherein X is E, G, V or S.

Preferably, the lipophilic group comprises any one or a combination of a carbonyl group of C4-C20, a polyoxypropylene group, a long-chain perfluoroalkyl group, a polysiloxane group and a hydrocarbon group containing an aryl group, an ester group, an ether group, an amine group, an amide group, a cycloalkyl group or a double bond.

Preferably, the molecular weight of the hyaluronic acid HA backbone is between 1K and 2500 KDa.

Preferably, the hydrophilicity and hydrophobicity and the bioactivity of the modified hyaluronic acid shown in the formula I can be adjusted by changing the grafting ratio of the active short peptide to the lipophilic group, wherein the ratio of the grafting ratio of the active short peptide to the lipophilic group is 1000: 1-1: 1000. .

Preferably, the active short peptide is directly and covalently bonded with the HA backbone of hyaluronic acid or bonded with a short peptide linker.

More preferably, the short peptide linker is WGRGDSP, GWGLGPAGK or CCRR.

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

1. the amphiphilic high-bioactivity modified hyaluronic acid is grafted to the surface of HA by BSP with bioactivity function, so that the HA is endowed with bioactivity functions of promoting tissue regeneration and the like, lipophilic hydrophobic groups are grafted at the same time, the transdermal absorption effect is enhanced, and functional diversity of the modified hyaluronic acid can be obtained by adjusting the grafting ratio of the active short peptides to the lipophilic groups;

2. according to the invention, after the hyaluronic acid main chains with different molecular weights are modified, the modified hyaluronic acid with small molecular weight can be used as a functional raw material of cosmetics, and the modified hyaluronic acid with small molecular weight can be absorbed by smearing and penetrating skin, so that lipophilic groups of the modified hyaluronic acid can greatly promote skin absorption; the modified hyaluronic acid with large molecular weight can be used for medical and aesthetic materials or tissue regeneration or joint cavity lubrication by injection, has good biocompatibility and the function of promoting cell regeneration, and overcomes the defect of single physical filling function of a hyaluronic acid material.

Drawings

FIG. 1 shows the result of culturing fibroblasts with modified hyaluronic acid and ordinary hyaluronic acid HA according to example 1; wherein A is the culture result of ordinary hyaluronic acid HA on fibroblasts, B: the result of culturing fibroblasts with the modified hyaluronic acid of example 1;

FIG. 2 is a comparison graph of the transdermal absorption of the low molecular weight modified hyaluronic acid of example 2 and ordinary hyaluronic acid HA after being smeared; wherein (A): the condition of percutaneous absorption after smearing the common hyaluronic acid HA; (B) the method comprises the following steps The low molecular weight modified hyaluronic acid of example 2 was transdermally absorbed after being applied.

Detailed Description

In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings.

Example 1

A preparation method of amphiphilic high-bioactivity modified hyaluronic acid comprises the following steps:

high molecular weight HA greater than 800KDa (n 6500, or n 2500) was heated to 80 ℃ in a water bath and maintained at this temperature for 1 hour. Polypeptide RXDX (where X may be E or G, V and S, in this example polypeptide REDG) was grafted onto the carboxyl group of HA by wgrgdsp (linker) in dimethyl sulfoxide (DMSO) containing carbodiimide and 1-hydroxybenzotriazole (HOBt) at pH 6.8 for 1 hour at 37 ℃. Cetyl trimethyl ammonium bromide (CTA) and 6-primary alcohol hydroxyl of HA are reacted in dimethyl sulfoxide (DMSO) for 1 hour at 37 ℃ to generate HA-CTA, then the HA-CTA and di (p-nitrophenyl) carbonate (4-NPBC) are reacted for 1 hour in a phosphate-DMSO buffer system with the pH value of 8.0 to form activated ester, then the activated ester and aniline are reacted for 24 hours at 65 ℃, after the reaction is finished, ether is used for precipitation and washing the product, and the modified hyaluronic acid with the high molecular weight and the function of promoting the tissue repair bioactivity is obtained and is a compound 1-2.

In the obtained compound 1, n ═ 6500, R₁＝WGRGDSPREDG，R₂＝C₆H₅NH；

In the obtained compound 2, n is 2000, R₁＝WGRGDSPREDG，R₂＝C₆H₅NH。

The bioactivity of compound 1 or 2 in the above modified hyaluronic acid having the function of promoting tissue repair bioactivity was tested: taking common hyaluronic acid HA as a control group, adding fibroblasts into a culture dish containing hyaluronic acid, culturing for 24h, and observing the adhesion and proliferation conditions of the fibroblasts, as shown in fig. 1, it can be known from fig. 1 that the adhesion between the modified hyaluronic acid and the fibroblasts is good, the proliferation of the fibroblasts is effectively promoted, and the modified hyaluronic acid HAs good biocompatibility and high bioactivity.

Example 2

A preparation method of amphiphilic high-bioactivity modified hyaluronic acid comprises the following steps:

HA with a molecular weight of around 2.5KDa (n ═ 8) was heated to 80 ℃ in a water bath and stirred at this temperature for 1 hour in phosphate-DMSO, a polar solvent with pH 4.6. Polypeptide RXDX (where X may be E or G, V and S, in this example polypeptide REDG) was grafted to the carboxy derivative of HA by reaction in dimethyl sulfoxide (DMSO) containing carbodiimide and 1-hydroxybenzotriazole (HOBt) at pH 6.8 for 1 hour at 37 ℃. The primary hydroxyl group at the 6-position of the HA backbone was modified (with higher activity than the other sites) and butyric anhydride was esterified by stirring in DMSO at pH 8.0 for 1 hour to introduce the corresponding butyrate. The obtained modified hyaluronic acid with low molecular weight and the function of promoting the tissue repair bioactivity is compound 3.

In the obtained compound 3, n ═ 8, R₁＝REDG，R₂＝CH₃CH₂CH₂C＝O。

Wherein, the compound 3 in the low molecular weight modified hyaluronic acid can be absorbed transdermally after being smeared, can be completely absorbed within 3 seconds after being smeared, and can not be completely absorbed within 3 minutes after being smeared by the common hyaluronic acid HA, as shown in figure 2.

Example 3

A preparation method of amphiphilic high-bioactivity modified hyaluronic acid comprises the following steps:

HA with a molecular weight of around 80kDa (n-250) was heated to 80 ℃ in a water bath and maintained at this temperature for 1 hour. The polypeptide RXDX (where X may be E or G, V and S, in this example the polypeptide REDG) was grafted GWGLGPAGK(linker) to the carboxyl group of HA by reaction in dimethyl sulfoxide (DMSO) containing carbodiimide and 1-hydroxybenzotriazole (HOBt) at pH 6.8 for 1 hour at 37 ℃. The modified HA is dissolved in phosphate-DMSO environment containing palmitic acid and having pH of 6.0, and stirred at room temperature for 24 hours to obtain a medium molecular weight modified hyaluronic acid with tissue repair bioactivity promoting function, namely compound 4.

In the obtained compound 4, n is 250, R₁＝GWGLGPAGKREDG，R₂＝CH₃(CH₂)₁₄C＝O。

Example 4

A preparation method of amphiphilic high-bioactivity modified hyaluronic acid comprises the following steps:

HA with a molecular weight of around 10kDa (n-30) was heated to 80 ℃ in a water bath and maintained at this temperature for 1 hour. The polypeptide RXDX (where X can be E or G, V and S, and the polypeptide in this example is REDG) is grafted onto the carboxyl group of the prepared HA by CCRR (linker) (grafting method as in example 3). Specifically, palmitic acid and OH on HA molecules are stirred in DMSO with the pH of 8.0 for 1 hour to perform esterification reaction to generate ester, so that lipophilic groups are introduced, and the modified hyaluronic acid with the function of promoting the biological activity of tissue repair is obtained, namely the compound 5.

In the obtained compound 5, n ═ 30, R₁＝CCRRREDG，R₂＝CH₃(CH₂)₁₄C＝O。

The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way and substantially, it should be noted that those skilled in the art may make several modifications and additions without departing from the scope of the present invention, which should also be construed as a protection scope of the present invention.