CN111388741B

CN111388741B - Injectable self-repairing antibacterial hydrogel dressing preloaded with polypeptide and preparation method thereof

Info

Publication number: CN111388741B
Application number: CN202010250210.1A
Authority: CN
Inventors: 吴德群; 叶婷婷; 刘畅; 王学利; 李发学; 俞建勇
Original assignee: Donghua University
Current assignee: Donghua University
Priority date: 2020-04-01
Filing date: 2020-04-01
Publication date: 2021-09-07
Anticipated expiration: 2040-04-01
Also published as: CN111388741A

Abstract

The invention discloses an injectable self-repairing antibacterial hydrogel dressing with preloaded polypeptide and a preparation method thereof. The hydrogel dressing comprises a hydrogel bound to a fabric, the hydrogel having polypeptides distributed on and within its surface that promote migration of fibroblasts. The preparation method comprises the following steps: selectively oxidizing glucans with different molecular weights into aldehyde group glucan by an oxidant; reacting aldehyde dextran with fatty amine to obtain partially alkylated dextran; then preparing polypeptide in solid phase; the preparation method comprises the steps of respectively dissolving aldehyde dextran, adipic dihydrazide and partially alkylated dextran in a PBS buffer solution, adding the polypeptide into the aldehyde dextran solution, preloading the polypeptide synthesized by a solid phase, mixing the three solutions according to a certain mass ratio, preparing the injectable self-repairing antibacterial hydrogel of preloaded polypeptide, and finally preparing the dressing. The product prepared by the invention has good antibacterial performance, can reduce the infection chance of wounds, has dynamic self-repairing performance, can aim at irregular wounds, can keep the wound surfaces moist and accelerate the wound healing.

Description

Injectable self-repairing antibacterial hydrogel dressing preloaded with polypeptide and preparation method thereof

Technical Field

The invention relates to an injectable dynamic self-repairing antibacterial hydrogel dressing preloaded with polypeptides and a preparation method thereof, and belongs to the technical field of medical materials and preparation thereof.

Background

Wound healing is a complex and coordinated process involving hemostasis, inflammation, cell proliferation and extracellular matrix remodeling. Currently, bacterial infection and poor wound healing rates remain major challenges in wound therapy. Bacterial infection often results in the formation of exudates, which greatly impedes wound healing. In addition, chronic wounds and deep wounds are often difficult to vascularize and self-repair due to reduced activity of endogenous growth factors. To overcome these problems, many wound dressing materials such as films, hydrogels, foams and hydrocolloids have been developed to meet the requirements of preventing infection and accelerating wound closure. Among these materials, injectable hydrogels are a potential dressing due to their excellent properties, including encapsulation of cells or drugs, filling of irregular wound sites, and adequate adhesion to wounds.

Conventional injectable hydrogels are vulnerable to external forces during clinical treatment, especially for irregular and deep wound sites, due to their lack of self-healing capabilities, and are thus not conducive to wound repair. As an improvement, the self-repairing hydrogel can automatically repair the broken hydrogel in an active state by generating dynamic covalent bonds and non-covalent bonds, thereby prolonging the service time of the hydrogel, reducing the times of dressing change and being beneficial to the healing of irregular wounds. Dynamic covalent bonds, such as disulfide bonds, imine bonds (schiff bases), phenylboronate, acylhydrazone bonds and Diels-Alder reactions, exhibit unique advantages in the construction of self-healing hydrogels due to their excellent reversibility and stability. Thus, for irregular wound sites, self-healing hydrogels not only facilitate wound healing and tissue repair, but also can encapsulate different drugs to control delivery.

Polypeptide-based wound dressings have great potential in meeting medical needs due to their high specificity, effectiveness, and safety. The present invention utilizes solid phase synthesis to prepare novel polypeptides. The preloaded polypeptide contains KGRT sequence, is derived from the functional region of erythroid differentiation regulating factor 1(Erdr1), shares three amino acids with erythroid differentiation regulating factor 1(Erdr1), and can regulate stress related reaction. The preloaded novel polypeptides can stimulate wound healing by promoting fibroblast, keratinocyte and vascular endothelial cell migration and tissue infiltration. As a material of biological origin, glucan has been proved to have excellent biocompatibility and biodegradability and can be used as a self-repairing biomaterial for promoting wound healing.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: how to improve the tissue adhesion capability of glucan by modifying glucan.

In order to solve the technical problems, the invention provides an injectable self-repairing antibacterial hydrogel dressing preloaded with polypeptides, which is characterized by comprising a hydrogel combined with a fabric, wherein polypeptides for promoting migration of fiber cells are distributed on the surface and inside the hydrogel; the hydrogel is prepared by respectively dissolving aldehyde dextran, dihydrazide and partially alkylated dextran in PBS solution, mixing and crosslinking.

Preferably, the polypeptide contains a KGRT sequence, is derived from erythroid differentiation regulating factor 1, namely a functional region of Erdr1, and shares three amino acids with Erdr1, wherein Erdr1 can regulate stress-related reactions.

The invention also provides a preparation method of the injectable self-repairing antibacterial hydrogel dressing preloaded with the polypeptide, which is characterized by comprising the following steps:

step 1): selectively oxidizing glucans with different molecular weights into aldehyde group glucan by an oxidant;

step 2): reacting the aldehyde dextran with fatty amine to obtain partially alkylated dextran;

step 3): solid phase preparation of polypeptide:

step 3.1): adding dichlorotrityl chloride resin into a solvent for soaking, fully swelling the resin, and then discharging the solvent;

step 3.2): dissolving amino acid with solvent, transferring the solution into treated dichloro trityl chloride resin, adding catalyst, and allowing the amino acid and resin to interact at room temperature to fix on the resin;

step 3.3): washing the resin with a solvent, adding a piperidine solution into the resin for reaction, and removing a protecting group;

step 3.4): the resin was washed with solvent and checked for complete protection with ninhydrin;

step 3.5): dissolving amino acid, HBTU and HOBt in a solvent, transferring the solution into the resin obtained in the step 3.4), adding a catalyst, and reacting at room temperature; washing the resin with a solvent, checking whether the amino group is completely reacted with ninhydrin, and carrying out the next step if the amino group is colorless; if the color is blue, repeating the step until the color is detected to be colorless, and synthesizing the polypeptide;

step 3.6): washing the resin with dichloromethane, fully washing to dry, and then drying;

step 3.7): reacting trifluoroacetic acid with the product obtained in the step 3.6) to cut off polypeptide;

step 3.8): collecting filtrate and washing liquid, performing rotary evaporation and concentration, precipitating with anhydrous ether, and standing; after the precipitation is sufficient, carrying out suction filtration, washing and drying;

step 4): dissolving the aldehyde dextran obtained in the step 1), adipic acid dihydrazide and the partially alkylated dextran obtained in the step 2) in a PBS buffer solution respectively, adding the polypeptide prepared in the step 3) into the aldehyde dextran solution, preloading the polypeptide synthesized in a solid phase, and then mixing the three solutions to prepare the injectable self-repairing antibacterial hydrogel preloaded with the polypeptide;

step 5): the injectable self-repairing antibacterial hydrogel preloaded with the polypeptide is combined with the fabric to prepare the dressing.

Preferably, the number average molecular weight of the glucan in the step 1) is 5000-100000; the oxidant is KMnO₄，NaIO₄Or KIO₄。

Preferably, the number of carbon atoms of the aliphatic amine in the step 2) is 6-15.

Preferably, the step 2) is specifically: dissolving aldehyde dextran in deionized water at room temperature, dissolving fatty amine in ethanol solution, mixing the two solutions, and reacting for 1-4 hours; adding excessive sodium cyanoborohydride into the mixed solution, stirring at room temperature for 12-24 hours, and precipitating part of alkylated glucan by using ethanol; and dialyzing, freeze-drying and storing the reaction product.

More preferably, the molar ratio of sodium cyanoborohydride to aldehydized dextran is 3: 1.

preferably, the solvent in step 3) is DMF, and the solvent in the piperidine solution is also DMF; the catalyst is Diisopropylethylamine (DIEA).

Preferably, the mass ratio of the aldehyde dextran, the adipic acid dihydrazide and the partially alkylated dextran solution in the step 4) is (2-4):1: 1.

Preferably, the ratio of the polypeptide to the aldehyde dextran solution in step 4) is 1 μ g: 200 μ L.

The partially alkylated dextran is synthesized by reacting the aldehyde dextran with fatty amine. The polypeptides that promote fibroblast migration are synthesized by solid phase synthesis (polypeptides vary depending on the kind and number of amino acids and the order of arrangement). The aldehyde dextran, the dihydrazide and the alkyl modified dextran are respectively dissolved in a PBS solution, mixed according to a certain mass ratio, and preloaded with the novel polypeptide synthesized by the solid phase, so as to prepare the injectable dynamic self-repairing antibacterial hydrogel dressing preloaded with the polypeptide. The aldehyde groups of the partially alkylated dextran react with the amino groups of the dihydrazide, increasing the degree of crosslinking of the hydrogel. The novel polypeptide can promote the migration of fiber cells to stimulate wound healing and provide wound healing promotion performance for the hydrogel dressing. Preloaded solid-phase synthesized polypeptides, belonging to the preloaded type, are convenient to release.

The product obtained by the reaction of the aldehyde dextran and the fatty amine can improve the tissue adhesion performance of the hydrogel.

The strength of the self-repairing hydrogel is controlled by regulating the content ratio of the aldehyde dextran, the partially alkylated dextran and the bisamide.

The invention provides an injectable dynamic self-repairing antibacterial hydrogel dressing preloaded with polypeptides, which has good antibacterial performance, can reduce the infection chance of wounds, has dynamic self-repairing performance, can aim at irregular wounds, can keep the wound surfaces moist and accelerate the wound healing.

Compared with the existing hydrogel dressing, the hydrogel dressing has the beneficial effects that:

(1) the macromolecular monomer adopted by the invention is glucan which is proved to have excellent biocompatibility and biodegradability and can be used as a self-repairing biological material for promoting wound healing. The glucan can activate macrophages, thereby stimulating the increase of epidermal growth factors and improving the regeneration capacity of wound skin cells.

(2) According to the invention, through the acylhydrazone bond crosslinking reaction, redundant initiators are not required to be introduced, so that the toxicity of the reagent is reduced, and the safety of the prepared product is improved.

(3) The product obtained by the invention has dynamic self-repairing performance, the acylhydrazone bond is generated to quickly form gel in an injection mode, and the fractured hydrogel is automatically and dynamically repaired at the same time, so that the service time of the hydrogel is prolonged, the times of dressing change are reduced, and the healing of irregular wounds is facilitated.

(4) The alkyl chain of the partially alkylated glucan adopted by the invention is inserted into and anchored in the lipid bilayer of the cell membrane, so that the hydrogel dressing has a tissue adhesion function.

(5) The novel polypeptide prepared by the invention has good biocompatibility, and can accelerate wound healing by promoting the migration of fibroblasts, keratinocytes and vascular endothelial cells.

(6) The product prepared by the invention has good antibacterial performance, can reduce the infection chance of wounds, has dynamic self-repairing performance, can aim at irregular wounds, can keep the wound surfaces moist and accelerate the wound healing.

Drawings

FIG. 1 is a schematic of the synthesis of an aldehyde-based dextran;

FIG. 2 is a schematic of the synthesis of modified glucan;

fig. 3 is a flow chart of a method for preparing the dynamic self-repairing hydrogel dressing.

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 an injectable self-repairing antibacterial hydrogel dressing preloaded with polypeptides comprises the following steps:

a) preparation of aldehyde dextran:

dextran (formula 1 in figure 1) (6g) with number average molecular weight of 10000 is dissolved in 100mL deionized water at room temperature, sodium periodate (2g) is dissolved in 5mL deionized water, sodium periodate solution is added into the dextran solution drop by drop, reaction is carried out for 6 hours under the condition of keeping out of the sun, glycol (2g) is added for reaction for 1 hour at room temperature, the obtained product (formula 2 in figure 1) is dialyzed for one week, freeze-dried and stored at low temperature.

b) Preparation of modified glucan:

dissolving aldehyde dextran (1g) in deionized water at room temperature, dissolving dodecylamine (0.1g) in ethanol solution, adding dodecylamine (structure formula 3 in figure 2) solution into aldehyde dextran (structure formula 2 in figure 1) solution, reacting for 2 hours, adding excessive sodium cyanoborohydride (0.1g) into the mixture, stirring for 18 hours at room temperature, precipitating partially alkylated dextran (structure formula 4 in figure 2) with ethanol, dialyzing, lyophilizing, and storing at low temperature.

c) Solid phase preparation of polypeptide:

2g of dichlorotrityl chloride resin is taken to a polypeptide synthesis device, 20mL of dry DMF is added for soaking for half an hour to fully swell the resin, and finally the solvent DMF is discharged.

0.25g of amino acid was weighed and dissolved in DMF, and the solution was transferred to the above apparatus for peptide synthesis containing the treated resin, 2mL of Diisopropylethylamine (DIEA) as a catalyst was added, and the amino acid and the resin were allowed to interact for 1.5 hours at room temperature to sufficiently immobilize the amino acid on the resin.

The resin was washed with DMF.

20mL of 20 wt% piperidine/DMF solution was added to the resin in the previous step and reacted for half an hour to remove the protecting group.

The resin was washed with DMF and checked for complete protection with ninhydrin.

1.43g amino acid, 2.37g HBTU, 0.84g HOBt were weighed and dissolved in DMF, and the solution was transferred to the above apparatus for peptide synthesis containing treated resin, and 2mL of DIEA catalyst was added and reacted at room temperature for 1.5 hours.

The resin was washed with DMF. The completion of the amino group reaction was checked with ninhydrin. If the product is colorless, the condensation reaction can be carried out for the next step; if blue color is developed, the reaction solution is condensed to colorless, and then the next operation can be carried out.

The above steps are repeated until the desired polypeptide is synthesized.

The resin was washed with dichloromethane, thoroughly dried and dried in a vacuum oven for 24 hours.

The dried product was reacted with trifluoroacetic acid (20mL) for 2 hours to cleave the polypeptide.

Collecting filtrate and washing liquid, rotary evaporating for concentration, precipitating with anhydrous ether, and standing for a period of time. And after the precipitation is sufficient, carrying out suction filtration, washing and drying.

d) Preparation of injectable dynamic self-repairing antibacterial hydrogel dressing preloaded with polypeptide:

dissolving aldehyde dextran (structural formula 2 in figure 1), adipic acid dihydrazide and partially alkylated dextran (structural formula 4 in figure 2) in PBS buffer solution respectively, mixing according to the mass ratio of 3:1:1, preloading a trace amount of polypeptide synthesized by a solid phase (the ratio of the polypeptide to the aldehyde dextran solution is 1 mug: 200 muL), and preparing injectable dynamic self-repairing antibacterial hydrogel preloaded with the polypeptide;

e) the injectable self-repairing antibacterial hydrogel preloaded with the polypeptide is combined with non-woven fabrics or cotton fabrics to prepare the dressing.

Example 2

The preparation method of the injectable self-repairing antibacterial hydrogel dressing preloaded with the polypeptide is different from that of the example 1 in that:

the step a) is as follows:

a) preparation of aldehyde dextran:

dissolving dextran (formula 1 in figure 1) (6g) with number average molecular weight of 50000 in 100mL deionized water at room temperature, dissolving sodium periodate (2g) in 5mL deionized water, dropwise adding sodium periodate solution into dextran solution, reacting for 6 hours under dark condition, adding ethylene glycol (2g) to react for 1 hour at room temperature, dialyzing the obtained product (formula 2 in figure 1) for one week, freeze-drying, and storing at low temperature.

b) Preparation of modified glucan:

dissolving aldehyde dextran (1g) in deionized water at room temperature, dissolving dodecylamine (0.1g) in ethanol solution, adding dodecylamine (structure formula 3 in figure 2) solution into aldehyde dextran (structure formula 2 in figure 1) solution, reacting for 2 hours, adding excessive sodium cyanoborohydride (0.1g) into the mixed solution, stirring for 18 hours at room temperature, precipitating part of alkylated dextran (structure formula 4 in figure 2) with ethanol, dialyzing the reaction product, freeze-drying, and storing at low temperature.

Example 3

the step a) is as follows:

a) preparation of aldehyde dextran:

dissolving dextran (formula 1 in figure 1) (6g) with number average molecular weight of 100000 in 100mL deionized water at room temperature, dissolving sodium periodate (2g) in 5mL deionized water, adding sodium periodate solution into dextran solution dropwise, reacting for 6 hours under dark condition, adding ethylene glycol (2g) to react for 1 hour at room temperature, dialyzing the obtained product (formula 2 in figure 1) for one week, freeze-drying, and storing at low temperature.

b) Preparation of modified glucan:

Example 4

the step a) is as follows:

a) preparation of aldehyde dextran:

dissolving dextran (formula 1 in figure 1) (6g) with number average molecular weight of 50000 in 100mL deionized water at room temperature, dissolving potassium periodate (2g) in 5mL deionized water, adding potassium periodate solution into dextran solution dropwise, reacting for 6 hours under dark condition, adding ethylene glycol (2g) to react for 1 hour at room temperature, dialyzing the obtained product (formula 2 in figure 1) for one week, freeze-drying, and storing at low temperature.

b) Preparation of modified glucan:

Example 5

the step a) is as follows:

a) preparation of aldehyde dextran:

dissolving dextran (formula 1 in figure 1) (6g) with number average molecular weight of 50000 in 100mL deionized water at room temperature, dissolving potassium permanganate (1g) in 5mL deionized water, dropwise adding potassium permanganate solution into dextran solution, reacting for 6 hours under dark condition, adding ethylene glycol (2g) to react for 1 hour at room temperature, dialyzing the obtained product (formula 2 in figure 1) for 3 days, freeze-drying, and storing at low temperature.

b) Preparation of modified glucan:

Example 6

the step a) is as follows:

a) preparation of aldehyde dextran:

b) Preparation of modified glucan:

dissolving aldehyde dextran (1g) in deionized water at room temperature, dissolving tetradecylamine (0.1g) in ethanol solution, adding tetradecylamine solution into aldehyde dextran (formula 2 in figure 1), reacting for 2 hr, adding excessive sodium cyanoborohydride (0.1g) into the mixture, stirring at room temperature for 18 hr, precipitating partially alkylated dextran with ethanol, dialyzing the reaction product, lyophilizing, and storing at low temperature.

Example 7

the step a) is as follows:

a) preparation of aldehyde dextran:

b) Preparation of modified glucan:

dissolving aldehyde dextran (1g) in deionized water at room temperature, dissolving pentadecamine (0.1g) in ethanol solution, adding pentadecamine solution into aldehyde dextran (formula 2 in figure 1), reacting for 2 hr, adding excessive sodium cyanoborohydride (0.1g) into the mixture, stirring at room temperature for 18 hr, precipitating part of alkylated dextran with ethanol, dialyzing the reaction product, freeze drying, and storing at low temperature.

Example 8

the step a) is as follows:

a) preparation of aldehyde dextran:

dissolving dextran (formula 1 in figure 1) (6g) with number average molecular weight of 50000 in 100mL deionized water at room temperature, dissolving sodium periodate (4g) in 5mL deionized water, adding sodium periodate solution into dextran solution dropwise, reacting for 6 hours under dark condition, adding ethylene glycol (2g) to react for 1 hour at room temperature, dialyzing the obtained product (formula 2 in figure 1) for 3 days, freeze-drying, and storing at low temperature.

b) Preparation of modified glucan:

Example 9

the step a) is as follows:

a) preparation of aldehyde dextran:

b) Preparation of modified glucan:

dissolving aldehyde dextran (1g) in deionized water at room temperature, dissolving dodecylamine (1g) in ethanol solution, adding dodecylamine (structure formula 3 in figure 2) solution into aldehyde dextran (structure formula 2 in figure 1) solution, reacting for 2 hours, adding excessive sodium cyanoborohydride (0.1g) into the mixed solution, stirring for 18 hours at room temperature, precipitating partially alkylated dextran (structure formula 4 in figure 2) with ethanol, dialyzing the reaction product, freeze-drying, and storing at low temperature.

Example 10

the step a) is as follows:

a) preparation of aldehyde dextran:

b) Preparation of modified glucan:

dissolving aldehyde dextran (1g) in deionized water at room temperature, dissolving dodecylamine (1g) in ethanol solution, adding dodecylamine (structure formula 3 in figure 2) solution into aldehyde dextran (structure formula 2 in figure 1) solution, reacting for 2 hours, adding excessive sodium cyanoborohydride (1g) into the mixed solution, stirring for 18 hours at room temperature, precipitating part of alkylated dextran (structure formula 4 in figure 2) with ethanol, dialyzing the reaction product, freeze-drying, and storing at low temperature.

Claims

1. The injectable self-repairing antibacterial hydrogel dressing preloaded with the polypeptides is characterized by comprising a hydrogel combined with a fabric, wherein the surface and the inside of the hydrogel are distributed with the polypeptides capable of promoting migration of fiber cells; the hydrogel is prepared by respectively dissolving aldehyde dextran, dihydrazide and partially alkylated dextran in PBS solution, mixing and crosslinking; the partially alkylated glucan is prepared by the following method: dissolving aldehyde dextran in deionized water at room temperature, dissolving fatty amine in ethanol solution, mixing the two solutions, and reacting for 1-4 hours; adding excessive sodium cyanoborohydride into the mixed solution, stirring at room temperature for 12-24 hours, and precipitating part of alkylated glucan by using ethanol; and dialyzing, freeze-drying and storing the reaction product.

2. The injectable self-healing antimicrobial hydrogel dressing preloaded with polypeptides of claim 1, wherein said polypeptides contain a KGRT sequence derived from erythroid differentiation regulating factor 1, a functional region of Erdr1, sharing three amino acids with Erdr1, wherein Erdr1 modulates stress-related responses.

3. The injectable self-healing antimicrobial hydrogel dressing preloaded with polypeptides of claim 1, wherein the molar ratio of sodium cyanoborohydride to aldehydized dextran is 3: 1.

4. the method for preparing the injectable self-repairing antibacterial hydrogel dressing preloaded with the polypeptides in the claim 1 or 2, is characterized by comprising the following steps:

step 3): solid phase preparation of polypeptide:

5. The method for preparing the injectable self-repairing antibacterial hydrogel dressing preloaded with the polypeptides as claimed in claim 4, wherein the number average molecular weight of the glucan in the step 1) is 5000-100000; the oxidant is KMnO₄，NaIO₄Or KIO₄。

6. The method for preparing the injectable self-repairing antibacterial hydrogel dressing preloaded with the polypeptides in claim 4, wherein the number of carbon atoms of the aliphatic amine in the step 2) is 6-15.

7. The method for preparing the injectable self-repairing antibacterial hydrogel dressing preloaded with the polypeptides as claimed in claim 4, wherein a solvent in the step 3) is DMF, and a solvent in a piperidine solution is also DMF; the catalyst is diisopropylethylamine.

8. The method for preparing the injectable self-repairing antibacterial hydrogel dressing preloaded with the polypeptides as claimed in claim 4, wherein the mass ratio of the aldehyde dextran, the adipic acid dihydrazide and the partially alkylated dextran solution in the step 4) is (2-4) to 1: 1.

9. The method for preparing the injectable self-repairing antibacterial hydrogel dressing preloaded with the polypeptides according to claim 4, wherein the ratio of the polypeptides to the aldehyde dextran solution in the step 4) is 1 μ g: 200 μ L.