CN110947027A

CN110947027A - Bacteriostatic self-healing hydrogel dressing for promoting wound repair

Info

Publication number: CN110947027A
Application number: CN201911184089.0A
Authority: CN
Inventors: 陈敬华; 周娟; 周钰婷; 宿丹丹
Original assignee: Jiangnan University
Current assignee: Shanghai Zechong Biotechnology Co ltd
Priority date: 2019-11-27
Filing date: 2019-11-27
Publication date: 2020-04-03
Anticipated expiration: 2039-11-27
Also published as: CN110947027B

Abstract

The invention discloses a bacteriostatic self-healing hydrogel dressing for promoting wound repair, belonging to the field of medical biomaterials. The bacteriostatic self-healing hydrogel dressing for promoting wound repair provided by the invention takes collagen and benzaldehyde-terminated polyethylene glycol as hydrogel matrixes, and polydopamine is modified in an in-situ polymerization manner. The dressing has good mechanical property, self-healing property, adhesion property and biocompatibility, can prolong the service time of the dressing, reduce secondary damage caused by replacing the wound dressing, and has good adhesion property to cover the wound, especially the wound at the joint part. The polymerized dopamine in the dressing can play a role in bacteriostasis, the bacteriostasis rate to escherichia coli and staphylococcus epidermidis is close to 100% in a short time, and the wound healing experiment of the whole-skin excision also proves that the dressing has the potential of wound repair.

Description

Bacteriostatic self-healing hydrogel dressing for promoting wound repair

Technical Field

The invention relates to a bacteriostatic self-healing hydrogel dressing for promoting wound repair, belonging to the field of medical biomaterials.

Background

Due to surgery, burns and chronic ulcers, acute and chronic skin wounds occur worldwide, with over 8000 thousands of people requiring clinical care and over 200 billion dollars in the worldwide wound care market. Especially, chronic wounds usually require frequent dressing changes and long-term hospitalization to limit the body of patients, which imposes economic burden on patients and seriously reduces their life quality. In addition, this also places a heavy burden on the medical system.

Hydrogel-based wound dressings are one of the most promising materials in wound care, fulfilling important requirements for dressings, including: keeping the wound moist while absorbing large amounts of exudate, relieving pain by cooling, gently covering sensitive underlying tissues, and possibly promoting wound healing. Although there are already many hydrogel-based dressings on the market, there is an urgent need for new wound care treatment protocols to address the more and more serious acute and chronic wound problems in today's aging society. The complex preparation process, the safety problems of the materials and the lack of mechanical stability typical of hydrogels, hamper the clinical implementation of many new methods. To meet clinical needs, it would be desirable to develop new hydrogel wound dressings that overcome these disadvantages.

Collagen is a natural bioactive molecule, has the performances of biodegradability, strong biocompatibility, enhancement of cell adhesion proliferation, promotion of wound repair and the like, and is widely applied to wound repair at present. Because the mechanical properties of collagen are not ideal, glutaraldehyde, genipin, carbodiimide and other substances can be used for crosslinking the collagen so as to improve the mechanical properties of the collagen, but the common crosslinking agents have certain toxicity to influence the subsequent application of materials.

To avoid wound infection, the wound dressing should have good antimicrobial activity. In particular, the widespread use of antibiotics has led to increasingly severe bacterial resistance phenomena. Researchers have developed antimicrobial wound dressings by incorporating antimicrobial agents into the dressing or directly using materials with inherent antimicrobial activity. A dressing with inherent antimicrobial activity may have sustained antimicrobial activity and reduced tissue cytotoxicity compared to a dressing with a releasable antimicrobial agent. The polydopamine has inherent antibacterial activity and has the advantages of improving the adhesion performance of materials and the like in the aspect of developing hydrogel wound dressings.

Disclosure of Invention

The invention aims to solve the defects that the existing hydrogel dressing has poor mechanical property, poor adhesion property, no antibacterial property and can not promote wound healing, and the like, and provides the antibacterial self-healing hydrogel dressing for promoting wound healing and the preparation method thereof.

The first purpose of the invention is to provide a method for preparing bacteriostatic self-healing hydrogel dressing for promoting wound repair, which comprises the following steps:

(1) dissolving collagen in water to obtain a collagen solution, and dissolving benzaldehyde-terminated polyethylene glycol (APG) in the collagen solution to form a collagen/APG mixed solution; wherein the molar mass ratio of amino groups of the collagen to polyethylene glycol aldehyde groups is (0.4-3.5): 1;

(2) covering 8-15 mM dopamine solution on the mixed solution prepared in the step (1), standing for a certain time, and then removing the dopamine solution in the mold;

(3) and (3) rinsing the hydrogel treated in the step (2) for 2-3 times by adopting ultrapure water, thus preparing the hydrogel dressing with the antibacterial function.

In one embodiment, the molar ratio between the collagen amino group and the aldehyde group of the modified polyethylene glycol is 0.4-3.5.

In one embodiment, the molar ratio between the collagen amino groups and the aldehyde groups of the modified polyethylene glycol is 0.4, 1.3 or 3.5.

In one embodiment, the concentration of the polyethylene glycol modified by aldehyde groups is 0.3-2.9%.

In one embodiment, the concentration of the collagen solution is 2-4%.

In one embodiment, the polyethylene glycol is a mixture of polyethylene glycols of different molecular weights.

In one embodiment, the polyethylene glycol is polyethylene glycol 2000, polyethylene glycol 4000 and polyethylene glycol 6000 in a mass ratio of (23-34): (37-55): (80-120).

In one embodiment, the collagen of step (1) is mixed with 0.23-0.34% polyethylene glycol 2000, 0.37-0.55% polyethylene glycol 4000, and 0.8-1.2% polyethylene glycol 6000 at a concentration of 2-4% (m/v), and then allowed to stand at pH 7.0-7.8 for 5-30 min.

In one embodiment, the benzaldehyde-terminated polyethylene glycol is subjected to hydroformylation modification at both ends of the polyethylene glycol; the aldehyde modification step comprises: (1) polyethylene glycol and p-aldehyde benzoic acid are mixed according to a molar ratio of (0.5-1.5): (2-6) dissolving in anhydrous tetrahydrofuran, and adding 4-dimethyl aminopyridine into the solution; (2) dissolving N, N-dicyclohexylcarbodiimide in anhydrous tetrahydrofuran, dropwise adding the mixed solution prepared in the step (1), and stirring at room temperature under the protection of nitrogen; (3) stirring and reacting for 18-24 h, filtering, taking a clear solution, and slowly dripping the clear solution into a large amount of anhydrous ether to precipitate a product; (4) and (4) filtering and collecting the precipitate obtained in the step (3), dissolving the solid precipitate in anhydrous tetrahydrofuran, dripping into anhydrous ether for settling, repeating for three times, and purifying to obtain the aldehyde modified polyethylene glycol.

In one embodiment, the method comprises the steps of:

(1) aldehyde modification of polyethylene glycol:

dissolving polyethylene glycol, p-aldehyde benzoic acid and 4-dimethylamine pyridine in anhydrous tetrahydrofuran, wherein the molar ratio of the polyethylene glycol to the p-aldehyde benzoic acid is (0.5-1.5): (2-6); and then, mixing the mixture with polyethylene glycol according to the mass ratio of (0.5-1.5): and (2.5-7.5) dissolving N, N-dicyclohexyl carbodiimide in anhydrous tetrahydrofuran, dropwise adding the mixed solution, and stirring at room temperature under the protection of nitrogen. Stirring and reacting for 18-24 h, filtering, collecting a clear solution, and slowly dripping the clear solution into a large amount of anhydrous ether to precipitate a product. Filtering, collecting precipitate, dissolving the solid precipitate in anhydrous tetrahydrofuran, dripping into anhydrous ether for precipitation, repeating the process for three times, and purifying to obtain aldehyde modified polyethylene glycol.

(2) Preparation of Col/APG hydrogel: dissolving collagen in ultrapure water to form a uniform Col solution of 2-4% (m/v) at room temperature, dissolving required amount of aldehyde group modified polyethylene glycol in the collagen solution to form a Col/APG mixed solution with APG concentration of 0.3-21.2% (m/v), transferring the mixed solution into a mold, adjusting the pH value of the gel to 7.0-7.8, standing for a certain time, and rinsing with ultrapure water for three times to obtain the Col/APG hydrogel dressing.

(3) Preparation of PDA/Col/APG hydrogel: covering 0.1-20 mg/mL of fresh dopamine solution prepared by 10mM Tris-HCl (pH8.5) on the surface of the Col/APG hydrogel dressing prepared in the step (2), standing for 6-24 h, then discarding the dopamine solution in the mould, and rinsing with ultrapure water for three times to prepare the PDA/Col/APG hydrogel dressing.

The invention also claims a hydrogel dressing prepared by applying the method.

The invention also claims the application of the method in preparing wound repair materials in the fields of medicine and daily chemicals.

Has the advantages that: the bacteriostatic self-healing hydrogel dressing for promoting wound repair provided by the invention takes collagen and benzaldehyde-terminated polyethylene glycol as hydrogel matrixes, and polydopamine is modified in an in-situ polymerization manner. The dressing has good mechanical property, self-healing property, adhesion property and biocompatibility, gel bacteriostasis is endowed by the polydopamine property, the bacteriostasis rate to escherichia coli and staphylococcus epidermidis is close to 100% in a short time, and a wound healing experiment of full-skin excision also proves that the dressing has the potential of wound repair.

Drawings

Fig. 1 is a schematic view of the preparation process of the medical wound dressing of the present invention.

FIG. 2 shows the storage modulus and loss modulus of Col/APG, where (a) is the storage modulus and (b) is the loss modulus.

FIG. 3 shows the storage modulus and loss modulus of Col/APG combinations of APG of different molecular weights, where (a) is the storage modulus and (b) is the loss modulus.

FIG. 4 is a macroscopic self-healing behavior observation of 3% Col-APG 6000-1.2.

FIG. 5 shows the critical strain values for Col/APG.

FIG. 6 is an alternate stress sweep test of Col/APG.

FIG. 7 shows the storage modulus and loss modulus of PDA/Col/APG, where (a) is the storage modulus and (b) is the loss modulus.

FIG. 8 is the apparent viscosity of PDA/Col/APG.

FIG. 9 shows the critical strain values (a) and the alternative stress sweep test (b) for PDA/Col/APG.

FIG. 10 is a graph showing the adhesive strength (a) and adhesion at the joint of a hydrogel (b).

FIG. 11 shows the swelling ratio of the hydrogel.

FIG. 12 shows the total number of bacteria (Log values) on the hydrogel samples, where (a) is E.coli and (b) is Staphylococcus epidermidis.

FIG. 13 shows the viability of HFF-1 cells on hydrogel samples.

Figure 14 is an image of wound healing in a model full-thickness skin defect of mice on different days.

Detailed Description

The invention will be better understood by reference to the following description of a specific embodiment with reference to fig. 1. Unless otherwise specified, the concentrations (m/v) referred to in the examples refer to mass-to-volume concentrations, i.e., the compounds are contained in mass (g) per 100mL of solution, e.g., 2% (m/v) means 2g/100 mL.

Example 1

Polyethylene glycol 2000(1.63g, 0.815mmol), p-aldehyde benzoic acid (0.49g, 3.26mmol) and 4-dimethylamine pyridine (0.025g, 0.205mmol) were dissolved in 80mL of anhydrous tetrahydrofuran. Subsequently, N-dicyclohexylcarbodiimide (0.84g, 4.075mmol) was dissolved in 20mL of anhydrous tetrahydrofuran, and the mixture was added dropwise with stirring under nitrogen. Then, the whole system was reacted at 25 ℃ for 20 hours. Filtering, slowly dripping the clear solution into a large amount of anhydrous ether to precipitate the product, dissolving the solid in anhydrous tetrahydrofuran after filtering, dripping the solid into the anhydrous ether to precipitate, repeating the process for three times, and purifying the product to obtain the aldehyde modified polyethylene glycol 2000.

Polyethylene glycol 4000 and polyethylene glycol 6000 are subjected to hydroformylation modification respectively according to a method for preparing hydroformylation modified polyethylene glycol 2000, wherein polyethylene glycol 2000(1.63g, 0.815mmol) is replaced by polyethylene glycol 4000(3.26g, 0.815mmol) and polyethylene glycol 6000(4.89g, 0.815mmol), and products are named as APG2K, APG4K and APG6K respectively.

Example 2

Collagen was dissolved in ultrapure water to form a 2% (m/v) homogeneous Col solution at room temperature. Dissolving the APG2K prepared in example 1 in a collagen solution to form a Col/APG mixed solution with APG2K concentration of 2.9% (M/v), 1.0% (M/v) and 0.3% (M/v) to ensure that the molar mass of collagen amino and APG aldehyde groups is 0.4, 1.2 and 3.5 respectively, transferring the mixed solution into a mold, adjusting the pH of the gel to 7.0-7.8 by using 0.5M NaOH solution, standing, forming the gel within 5-120 s, standing for 30min, rinsing with ultrapure water for three times to prepare the hydrogel dressing, and naming the gel as 2% Col-APG2K-0.4, 2% Col-APG2K-1.2 and 2% Col-APG 2K-3.5.

TABLE 12% collagen gels with different APG2K concentrations

Example 3

Collagen was dissolved in ultrapure water to form a 2% (m/v) homogeneous Col solution at room temperature. Dissolving the APG4K prepared in example 1 in a collagen solution to form a Col/APG mixed solution with APG4K concentrations of 4.8%, 2.4% and 0.5% (M/v), wherein the collagen amino and APG aldehyde groups are 0.4, 1.2 and 3.5 mol respectively, then transferring the mixed solution into a mold, adjusting the pH of the gel to 7.0-7.8 by using 0.5M NaOH solution, standing, forming the gel within 5-120 s, rinsing with ultrapure water for three times after standing for 30min to prepare the hydrogel dressing, and the gel is named as 2% Col-APG4K-0.4, 2% Col-APG4K-1.2 and 2% Col-APG4K-3.5 hydrogel.

TABLE 22% collagen gels with various APG4K concentrations

Example 4

Collagen was dissolved in ultrapure water to form a 2% (m/v) homogeneous Col solution at room temperature. Dissolving the APG6K prepared in the example 1 in a collagen solution to form a Col/APG mixed solution with APG6K concentrations of 10.6%, 3.2% and 1.2% (M/v), wherein the collagen amino and APG aldehyde groups are 0.4, 1.2 and 3.5 in mol, respectively, transferring the mixed solution to a mold, adjusting the pH of the gel to 7.0-7.8 by using 0.5M NaOH solution, standing, forming the gel within 5-120 s, rinsing with ultrapure water for three times after standing for 30min to prepare the hydrogel dressing, and the gel is named as 2% Col-APG6K-0.4, 2% Col-APG6K-1.2 and 2% Col-APG 6K-3.5.

TABLE 32% collagen gels with various APG6K concentrations

Example 5

Collagen was dissolved in ultrapure water to form a 3% (m/v) homogeneous Col solution at room temperature. Meanwhile, dissolving the required amount of APG2K in a collagen solution to form a Col/APG mixed solution with APG2K concentrations of 4.3%, 1.4% and 0.5% (M/v), wherein the molar ratio of collagen amino groups to APG aldehyde groups is 0.4, 1.2 and 3.5 respectively, then transferring the mixed solution into a mold, adjusting the pH of the gel to 7.0-7.8 by using 0.5M NaOH solution, standing, forming the gel within 5-120 s, rinsing with ultrapure water for three times after standing for 30min to obtain the hydrogel dressing, and naming the gel as 3% Col-APG2K-0.4, 3% Col-APG2K-1.2 and 3% Col-APG 2K-3.5.

TABLE 43% collagen gels with different APG2K concentrations

Example 6

Collagen was dissolved in ultrapure water to form a 3% (m/v) homogeneous Col solution at room temperature. Meanwhile, dissolving the required amount of APG4K in a collagen solution to form a Col/APG mixed solution with APG4K concentrations of 7.2%, 2.4% and 0.8% (M/v), wherein the molar ratio of collagen amino groups to APG aldehyde groups is 0.4, 1.3 and 3.5 respectively, then transferring the mixed solution into a mold, adjusting the pH of the gel to 7.0-7.8 by using 0.5M NaOH solution, standing, forming the gel within 5-120 s, rinsing with ultrapure water for three times after standing for 30min to obtain the hydrogel dressing, and naming the gel as 3% Col-APG4K-0.4, 3% Col-APG4K-1.2 and 3% Col-APG 4K-3.5.

TABLE 53% collagen gels with various APG4K concentrations

Example 7

Collagen was dissolved in ultrapure water to form a 3% (m/v) homogeneous Col solution at room temperature. Meanwhile, dissolving the required amount of APG6K in a collagen solution to form a Col/APG mixed solution with APG6K concentrations of 15.9%, 5.3% and 1.8% (M/v), wherein the molar ratio of collagen amino groups to APG aldehyde groups is 0.4, 1.2 and 3.5 respectively, then transferring the mixed solution into a mold, adjusting the pH of the gel to 7.0-7.8 by using 0.5M NaOH solution, standing, forming the gel within 5-120 s, rinsing with ultrapure water for three times after standing for 30min to obtain the hydrogel dressing, and naming the gel as 3% Col-APG6K-0.4, 3% Col-APG6K-1.2 and 3% Col-APG 6K-3.5.

TABLE 63% collagen gels with various APG6K concentrations

Example 8

Collagen was dissolved in ultrapure water to form a 4% (m/v) homogeneous Col solution at room temperature. Meanwhile, dissolving the required amount of APG2K in a collagen solution to form a Col/APG mixed solution with APG2K concentrations of 5.7%, 1.9% and 0.7% (M/v), wherein the molar ratio of collagen amino groups to APG aldehyde groups is 0.4, 1.2 and 3.5 respectively, then transferring the mixed solution into a mold, adjusting the pH of the gel to 7.0-7.8 by using 0.5M NaOH solution, standing, forming the gel within 5-120 s, rinsing with ultrapure water for three times after standing for 30min to obtain the hydrogel dressing, and naming the gel as 4% Col-APG2K-0.4, 4% Col-APG2K-1.2 and 4% Col-APG 2K-3.5.

TABLE 74% collagen gels with various APG2K concentrations

Example 9

Collagen was dissolved in ultrapure water to form a 4% (m/v) homogeneous Col solution at room temperature. Meanwhile, dissolving the required amount of APG4K in a collagen solution to form a Col/APG mixed solution with APG4K concentrations of 9.6%, 3.2% and 1.1% (M/v), wherein the molar ratio of collagen amino groups to APG aldehyde groups is 0.4, 1.3 and 3.5 respectively, then transferring the mixed solution into a mold, adjusting the pH of the gel to 7.0-7.8 by using 0.5M NaOH solution, standing, forming the gel in 5-120 s, rinsing with ultrapure water for three times after standing for 30min to obtain the hydrogel dressing, and naming the gel as 4% Col-APG4K-0.4, 4% Col-APG4K-1.2 and 4% Col-APG 4K-3.5.

Gel of collagen of table 84% concentration and different APG4K concentration ratios

Example 10

Collagen was dissolved in ultrapure water to form a 4% (m/v) homogeneous Col solution at room temperature. Meanwhile, dissolving the required amount of APG6K in a collagen solution to form a Col/APG mixed solution with APG6K concentrations of 21.2%, 7.0% and 2.4% (M/v), wherein the molar ratio of collagen amino groups to APG aldehyde groups is 0.4, 1.3 and 3.5 respectively, then transferring the mixed solution into a mold, adjusting the pH of the gel to 7.0-7.8 by using 0.5M NaOH solution, standing, forming the gel within 5-120 s, rinsing with ultrapure water for three times after standing for 30min to obtain the hydrogel dressing, and naming the gel as 4% Col-APG6K-0.4, 4% Col-APG6K-1.2 and 4% Col-APG 6K-3.5.

Gel of collagen at 94% concentration in table with different APG6K concentrations

Example 11

The mechanical properties of the Col/APG hydrogels were tested. The storage modulus is an index for characterizing the rebound of a material after deformation. The storage modulus and loss modulus of the mesogels prepared in examples 2-10 were tested. The samples were all uniform cylinders. The rheology test was performed by rheometer (Discover DHR-2). The diameter of the parallel plate is 25mm, the distance is 1mm, the fixed strain gamma is 1%, and the scanning angular frequency range omega is 0.1-100 rad/s. The test results are shown in FIG. 2.

The mechanical property of the Col/APG hydrogel is crucial to the application of the Col/APG hydrogel in a wound dressing, and the test result is shown in figure 2, the storage modulus of the hydrogel with the collagen concentration of 2% is smaller and is lower than 500Pa, and the mechanical property of the sample with the molar ratio of 0.4 of the amino group and the aldehyde group of APG6K of the collagen is the highest and is about 300 Pa; for the 4% collagen gel, the overall mechanical property level was higher, and the mechanical properties of the samples with a molar ratio of collagen amino groups to APG2K aldehyde groups of 3.5 were up to about 1300 Pa. In the hydrogel sample with the collagen concentration of 3%, 3% Col-APG6K-1.2, 3% Col-APG6K-3.5 have the best mechanical property, and the highest mechanical property is 2000Pa, which is presumed to be because the space distance between the long chains of the collagen and APG6K is proper, and under the condition that the molar ratio of amino groups to aldehyde groups is 1.2 to 3.5, the gel cannot be formed due to too low concentration, and the internal structure of the gel cannot be unstable due to too high steric hindrance between the molecular chains due to too high concentration.

Example 12

Collagen was dissolved in ultrapure water to form a 2% (m/v) homogeneous Col solution at room temperature. Meanwhile, dissolving required amounts of APG2K and APG4K in a collagen solution to form a Col/APG mixed solution with APG2K concentration of 0.15% (M/v) and APG4K concentration of 0.25%, transferring the mixed solution into a mold, adjusting the pH of the gel to 7.0-7.8 by using 0.5M NaOH solution, standing, forming the gel within 5-120 s, rinsing with ultrapure water for three times after standing for 30min to obtain the hydrogel dressing, and naming the gel as 2% Col-APG 2K-4K.

Example 13

Collagen was dissolved in ultrapure water to form a 2% (m/v) homogeneous Col solution at room temperature. Meanwhile, dissolving required amounts of APG4K and APG6K in a collagen solution to form a Col/APG mixed solution with APG4K concentration of 0.25% (M/v) and APG6K concentration of 0.6%, transferring the mixed solution into a mold, adjusting the pH of the gel to 7.0-7.8 by using 0.5M NaOH solution, standing, forming the gel within 5-120 s, rinsing with ultrapure water for three times after standing for 30min to obtain the hydrogel dressing, and naming the gel as 2% Col-APG 4K-6K.

Example 14

Collagen was dissolved in ultrapure water to form a 2% (m/v) homogeneous Col solution at room temperature. Meanwhile, dissolving required amounts of APG2K and APG6K in a collagen solution to form a Col/APG mixed solution with APG2K concentration of 0.15% (M/v) and APG6K concentration of 0.6%, transferring the mixed solution into a mold, adjusting the pH of the gel to 7.0-7.8 by using 0.5M NaOH solution, standing, forming the gel within 5-120 s, rinsing with ultrapure water for three times after standing for 30min to obtain the hydrogel dressing, and naming the gel as 2% Col-APG 2K-6K.

Example 15

Collagen was dissolved in ultrapure water to form a 2% (m/v) homogeneous Col solution at room temperature. Meanwhile, dissolving required amounts of APG2K, APG4K and APG6K in a collagen solution to form a Col/APG mixed solution with the concentration of APG2K being 0.10% (M/v), the concentration of APG4K being 0.17% and the concentration of APG6K being 0.20%, transferring the mixed solution into a mold, adjusting the pH of the gel to 7.0-7.8 by using 0.5M NaOH solution, standing, forming the gel within 5-120 s, rinsing for three times by using ultrapure water after standing for 30min to prepare the hydrogel dressing, and naming the gel as 2% Col-APG 2K-4K-6K.

TABLE 102% concentration of collagen gels in combination with APG of different molecular weights

Example 16

Collagen was dissolved in ultrapure water to form a 3% (m/v) homogeneous Col solution at room temperature. Meanwhile, dissolving required amounts of APG2K and APG4K in a collagen solution to form a Col/APG mixed solution with APG2K concentration of 0.25% (M/v) and APG4K concentration of 0.40%, transferring the mixed solution into a mold, adjusting the pH of the gel to 7.0-7.8 by using 0.5M NaOH solution, standing, forming the gel within 5-120 s, rinsing with ultrapure water for three times after standing for 30min to obtain the hydrogel dressing, and naming the gel as 3% Col-APG 2K-4K.

Example 17

Collagen was dissolved in ultrapure water to form a 3% (m/v) homogeneous Col solution at room temperature. Meanwhile, dissolving required amounts of APG4K and APG6K in a collagen solution to form a Col/APG mixed solution with APG4K concentration of 0.40% (M/v) and APG6K concentration of 0.90%, transferring the mixed solution into a mold, adjusting the pH of the gel to 7.0-7.8 by using 0.5M NaOH solution, standing, forming the gel within 5-120 s, rinsing with ultrapure water for three times after standing for 30min to obtain the hydrogel dressing, and naming the gel as 3% Col-APG 4K-6K.

Example 18

Collagen was dissolved in ultrapure water to form a 3% (m/v) homogeneous Col solution at room temperature. Meanwhile, dissolving required amounts of APG2K and APG6K in a collagen solution to form a Col/APG mixed solution with APG2K concentration of 0.25% (M/v) and APG6K concentration of 0.90%, transferring the mixed solution into a mold, adjusting the pH of the gel to 7.0-7.8 by using 0.5M NaOH solution, standing, forming the gel within 5-120 s, rinsing with ultrapure water for three times after standing for 30min to obtain the hydrogel dressing, and naming the gel as 3% Col-APG 2K-6K.

Example 19

Collagen was dissolved in ultrapure water to form a 3% (m/v) homogeneous Col solution at room temperature. Meanwhile, dissolving required amounts of APG2K, APG4K and APG6K in a collagen solution to form a Col/APG mixed solution with the concentration of APG2K being 0.17% (M/v), the concentration of APG4K being 0.27% and the concentration of APG6K being 0.30%, transferring the mixed solution into a mold, adjusting the pH of the gel to 7.0-7.8 by using 0.5M NaOH solution, standing, forming the gel within 5-120 s, rinsing for three times by using ultrapure water after standing for 30min to prepare the hydrogel dressing, and naming the gel as 3% Col-APG 2K-4K-6K.

TABLE 113% concentration of collagen gels in combination with APG of different molecular weights

Example 20

Collagen was dissolved in ultrapure water to form a 4% (m/v) homogeneous Col solution at room temperature. Meanwhile, dissolving required amounts of APG2K and APG4K in a collagen solution to form a Col/APG mixed solution with APG2K concentration of 0.34% (M/v) and APG4K concentration of 0.55%, transferring the mixed solution into a mold, adjusting the pH of the gel to 7.0-7.8 by using 0.5M NaOH solution, standing, forming the gel within 5-120 s, rinsing with ultrapure water for three times after standing for 30min to obtain the hydrogel dressing, and naming the gel as 4% Col-APG 2K-4K.

Example 21

Collagen was dissolved in ultrapure water to form a 4% (m/v) homogeneous Col solution at room temperature. And meanwhile, dissolving APG4K and APG6K in a collagen solution to form a Col/APG mixed solution with APG4K concentration of 0.55% (M/v) and APG6K concentration of 1.20%, transferring the mixed solution into a mold, adjusting the pH of the gel to 7.0-7.8 by using 0.5M NaOH solution, standing, forming the gel within 5-120 s, rinsing with ultrapure water for three times after standing for 30min to obtain the hydrogel dressing, and naming the gel as 4% Col-APG 4K-6K.

Example 22

Collagen was dissolved in ultrapure water to form a 4% (m/v) homogeneous Col solution at room temperature. Meanwhile, dissolving required amounts of APG2K and APG6K in a collagen solution to form a Col/APG mixed solution with APG2K concentration of 0.34% (M/v) and APG6K concentration of 1.20%, transferring the mixed solution into a mold, adjusting the pH of the gel to 7.0-7.8 by using 0.5M NaOH solution, standing, forming the gel within 5-120 s, rinsing with ultrapure water for three times after standing for 30min to obtain the hydrogel dressing, and naming the gel as 4% Col-APG 2K-6K.

Example 23

Collagen was dissolved in ultrapure water to form a 4% (m/v) homogeneous Col solution at room temperature. Meanwhile, APG2K, APG4K and APG6K are dissolved in a collagen solution to form a Col/APG mixed solution with the concentration of APG2K being 0.23% (m/v), the concentration of APG4K being 0.37% and the concentration of APG6K being 0.80%, then the mixed solution is transferred into a mold, the pH of the gel is adjusted to 7.0-7.8 by 0.5M NaOH solution, standing is carried out, the gel is formed in 5-120 s, after standing for 30min, rinsing with ultrapure water for three times to prepare the hydrogel dressing, and the gel is named as 4% Col-APG 2K-4K-6K.

TABLE 124% concentration of collagen gels in combination with APG of different molecular weights

Example 24

The mechanical properties of the Col/APG hydrogel combined with APG with different molecular weights are measured, specifically the storage modulus and the loss modulus of the gel prepared in examples 12-23. The samples were all uniform cylinders. The rheology test was performed by rheometer (Discover DHR-2). The diameter of the parallel plate is 25mm, the distance is 1mm, the fixed strain gamma is 1%, and the scanning angular frequency range omega is 0.1-100 rad/s. The test results are shown in FIG. 3.

Based on the molar ratio of collagen amino groups to APG aldehyde groups of 3.5, hydrogels with different molecular weights for APG combination were prepared, and hydrogels with 12 formulations of examples 12 to 23 were obtained by adjusting the concentration of collagen and the combination of APG with different molecular weights, respectively. The combination of the three kinds of molecular weight polyethylene glycols has obviously improved mechanical properties on collagen hydrogel with the concentration of 3% and 4%, the storage modulus respectively reaches 1291.29Pa and 1116.87Pa, and the storage modulus of the collagen hydrogel prepared by the combination of the two kinds of polyethylene glycols is 126-412 Pa.

Example 24

In order to test the self-healing performance of Col/APG, 3% Col-APG6K-1.2 gel was cut up and cut off with a razor blade, the treated hydrogels were gathered together and left at room temperature for half an hour, and the self-healing behavior of the gels was recorded by photography. In order to observe the self-healing phenomenon of the gel more intuitively, the cut gel was placed on a glass plate, the cross sections were spliced, and the self-healing behavior at different times was recorded with an optical microscope. The test results are shown in FIG. 4.

After the gel is spliced again for 1 minute, 10 minutes and 30 minutes, the self-healing behavior of the gel can be obviously seen in fig. 4, and the areas of black shadows are gradually reduced, which indicates that the gel cuts are gradually reduced.

Example 25

Critical strain values of 4% Col-APG4K-1.2, 4% Col-APG2K-3.5, 4% Col-APG2K-4K-6K, 3% Col-APG6K-0.4, 3% Col-APG6K-1.2, 3% Col-APG2K-4K-6K were recorded by strain amplitude scanning, gamma ranged from 1% to 800%, fixed angular frequency of 1rad/s, using parallel plates with a diameter of 25mm, set at a pitch of 500 nm. The test results are shown in FIG. 5.

The critical strain value of the Col/APG hydrogel is crucial to the self-healing performance of the Col/APG hydrogel. The critical strain value of the hydrogel is evaluated by adopting a rheological test method. As shown in FIG. 5, the critical strain values for the six gel samples were all greater than 100%, with the 4% Col-APG4K-1.2 sample having a value of 122%; the critical strain values of the 3% Col-APGK-0.4, 4% Col-APG2K-3.5 and 4% Col-APG2K-4K-6K samples are all between 150% and 200%; the critical strain values of the 3% Col-APG6000-1.2 and 3% Col-APG2K-4K-6K samples are the highest and reach 208% and 247% respectively, which shows that the two gels can bear higher external strain force.

Example 26

The self-healing behavior of 4% Col-APG4K-1.2, 4% Col-APG2K-3.5, 4% Col-APG2K-4K-6K, 3% Col-APG6K-0.4, 3% Col-APG6K-1.2, 3% Col-APG2K-4K-6K was tested by an alternating strain-sweep test at a fixed angular frequency (1 rad. s-1). Amplitude oscillation strain goes from small to large: respectively, from γ ═ 1% to γ ═ 200%, γ ═ 1% to γ ═ 400%, γ ═ 1% to γ ═ 800%, at intervals of 60 seconds. Parallel plates with a diameter of 25mm were used, with a pitch of 500 nm. The test results are shown in FIG. 6.

The results of the alternating stress sweep test are shown in FIG. 6, and the storage modulus of the 3% Col-APG6K-1.2 sample is not much different from the initial modulus after different stress injuries, which indicates that the mechanical strength of the gel can still be restored to the initial level after the gel is subjected to strong tensile injuries. In contrast, the other five samples all had half of the mechanical properties damaged and had almost no storage modulus under 800% tension.

Example 27

The PDA/Col/APG-6 hydrogel dressing was prepared by covering the 3% Col-APG6K-1.2 with fresh dopamine solutions of 0.1mg/mL, 0.5mg/mL, 1.0mg/mL, 5.0mg/mL, 10.0mg/mL and 20.0mg/mL in Tris-HCl (10mM, pH8.5), respectively, leaving the mixture to stand for 6 hours, discarding the dopamine solution in the mold, and rinsing the mixture with ultrapure water three times.

Example 28

The PDA/Col/APG-12 hydrogel dressing was prepared by covering the 3% Col-APG6K-1.2 with fresh dopamine solutions of 0.1mg/mL, 0.5mg/mL, 1.0mg/mL, 5.0mg/mL, 10.0mg/mL and 20.0mg/mL each prepared from Tris-HCl (10mM, pH8.5), leaving the mixture to stand for 12 hours, discarding the dopamine solution in the mold, and rinsing the mixture with ultrapure water three times.

Example 29

The PDA/Col/APG-24 hydrogel dressing is prepared by covering the 3% Col-APG6K-1.2 with fresh dopamine solutions of 0.1mg/mL, 0.5mg/mL, 1.0mg/mL, 5.0mg/mL, 10.0mg/mL and 20.0mg/mL prepared by Tris-HCl (10mM, pH8.5), standing for 24h, discarding the dopamine solution in the mold, and rinsing with ultrapure water for three times.

Example 30

The storage modulus and loss modulus of the PDA/Col/APG hydrogels prepared in examples 27-29 were tested. The samples were all uniform cylinders. The rheology test was performed by rheometer (Discover DHR-2). The diameter of the parallel plate is 25mm, the distance is 1mm, the fixed strain gamma is 1%, and the scanning angular frequency range omega is 0.1-100 rad/s. The test results are shown in FIG. 7.

The rheological properties of PDA/Col/APG are shown in FIG. 7, where the storage modulus of all gels as a whole is less than 1000Pa, which is reduced compared to Col/APG, but the storage modulus at polydopamine concentrations of 0.5mg/mL and 1mg/mL is still optimistic. Therefore, the polydopamine solution can bear the external pressure of 600Pa and has relatively good mechanical property when the concentration is 0.5mg/mL and 1 mg/mL.

Example 31

The apparent viscosity of the PDA/Col/APG hydrogel prepared in the example 27-29 is measured by a rheometer, and the shear rate is in the range of 0.01-100s^-1. The test results are shown in FIG. 8.

As can be seen from FIG. 8, in 3% Col-APG6K-1.2 after immersion for various periods of time at all concentrations of polydopamine, the apparent viscosity decreased with increasing shear rate, but reached 0.016s at shear rate^-1When this is the case, the apparent viscosity of the gel is the highest. Wherein, the PDA/Col/APG apparent viscosity of the soaked polydopamine solution with the concentration of 1mg/mL is the highest and can reach 105 pas. From the visual results in FIG. 8, compareIn other concentrations, the apparent viscosity of the PDA/Col/APG is increased more obviously by the poly-dopamine solution with the concentration of 1mg/mL, and the apparent viscosity of the poly-dopamine solution with the concentration reaches 10271.70, 8983.66 and 5895.66 respectively at the polymerization time of 6h, 12h and 24h, which are all higher than other concentrations. Therefore, the experimental result shows that the PDA/Col/APG is between 0.01 and 100s^-1Has good shear thinning properties in the shear rate range of (a).

Example 31

The critical strain values of the three gel samples PDA/Col/APG-6, PDA/Col/APG-12 and PDA/Col/APG-24 prepared in examples 27-29 were recorded by strain amplitude scanning, with a gamma range of 1% to 800%, a fixed angular frequency of 1rad/s, the use of parallel plates with a diameter of 25mm and a set spacing of 500 nm. The test results are shown in FIG. 9.

Fixed angular frequency (1rad · s) by alternating strain sweep test^-1) Three gel samples, PDA/Col/APG-6, PDA/Col/APG-12, PDA/Col/APG-24, prepared in example 30 were tested for self-healing behavior. Amplitude oscillation strain goes from small to large: respectively, from γ ═ 1% to γ ═ 200%, γ ═ 1% to γ ═ 400%, γ ═ 1% to γ ═ 800%, at intervals of 60 seconds. Parallel plates with a diameter of 25mm were used, with a pitch of 500 nm. The test results are shown in FIG. 9.

And evaluating the critical strain value of the PDA/Col/APG by adopting a rheological test method to explore the self-healing performance of the gel. As shown in FIG. 9, the critical strain values of PDA/Col/APG at different polymerization times increased with time to 138%, 127%, 292%, respectively, so that the polymerization time of polydopamine was decreased within twelve hours for the gel, but the external strain that the gel could endure after twenty-four hours of polymerization was slightly increased. The results of the alternating stress scanning test show that after the 800% stress test, the storage modulus of three kinds of PDA/Col/APG can still approximately recover the initial level, but the storage modulus of the PDA/Col/APG-24 gel is lower and only about 500Pa, and the other two gels can reach 1000Pa, which is almost different from the modulus before polymerization of polydopamine.

Example 32

The bonding strength of the three gel samples PDA/Col/APG-6, PDA/Col/APG-12, PDA/Col/APG-24 prepared in example 30 were evaluated using the lap shear test. The ability of the hydrogel to adhere to the host tissue is performed by using pig skin. The method comprises the following specific steps: skin tissue was cut into 10mm x 40mm rectangles and then immersed in PBS prior to use. 100 μ L of hydrogel was applied to the surface of the pigskin and another skin was placed on the hydrogel solution. The bonding area was 10mm × 10 mm. Subsequently, the pigskin was left at room temperature for 3 hours. The adhesion performance was tested using a lap shear test at a rate of 5mm/min on an Instron materials testing system (mtscririon 43, MTS Criterion) equipped with a 50N load cell. Each group was tested in triplicate. The test results are shown in figure 10(a), and the adhesion strength of PDA/Col/APG-6 reaches 6.16kPa, which is 0.38kPa higher than that of the hydrogel not covered with dopamine solution. FIG. 10(b) is a schematic representation of hydrogel adhesion at the joint.

After application, the hydrogel dressing must adhere and seal the wound site completely to prevent bacterial proliferation and fluid seepage. The adhesive strength of the hydrogels was evaluated using the lap shear test. The hydrogels of this series all exhibited the desired adhesive strength and there was no significant difference between the groups, remaining with fibrin adhesive

(about 5kPa) equivalent strength.

Example 33

Three gel samples, PDA/Col/APG-6, PDA/Col/APG-12, PDA/Col/APG-24, prepared in example 30 were tested for their swelling capacity in PBS, and a 3% Col-APG6K-1.2 gel sample of unpolymerized dopamine was used as a control and designated as Col/APG. Approximately 5mg of the gel sample was weighed into an EP tube and the sample mass (W) was recorded₀). Then, 2ml of the solution of LPBS was added to the EP tube to immerse the sample, the temperature was maintained at 37 ℃, the surface of the sample was carefully wiped off after removal at various time points, the mass at each time point was accurately weighed using an analytical balance, and the wet weight (W. epsilon.) of the sample was recorded. Swelling capacity of the sample

Each group was tested in triplicate. MeasuringThe test results are shown in FIG. 11.

In general, hydrogel dressings can maintain a moist wound environment and can absorb excess exudate from tissue. In fig. 12, all hydrogels were allowed to swell dramatically, but the unpolymerized dopamine hydrogel group had a lower swelling ratio than the other three hydrogel groups.

Example 34

The three gel samples PDA/Col/APG-6, PDA/Col/APG-12 and PDA/Col/APG-24 prepared in examples 27-29 were tested for their bacteriostatic effects on Escherichia coli (ATCC 25922) and Staphylococcus epidermidis (ATCC 12228) by colony counting method, and a 3% Col-APG6K-1.2 gel sample of unpolymerized dopamine was used as a control and marked as Col/APG. Under sterile conditions, 200 μ L of hydrogel was prepared in each well of a 24-well plate. 20. mu.L of the bacterial suspension (cell concentration: 10)⁵CFU mL^-150% LB medium) was added to the surface of the hydrogel dish. Next, the 24-well plate was incubated in an incubator at 37 ℃ under a relatively humid atmosphere. After 2 hours, 2mL of sterilized was added to each well to resuspend all viable bacteria. As a negative control, 20. mu.L of bacterial suspension (10)⁵CFU mL^-150% LB medium) was added to 2mL of PBS to obtain a homogeneous solution. After 100-fold dilution of the bacterial suspension, 100. mu.L of the suspension was uniformly spread on the surface of a solidified nutrient agar medium plate, and after incubation at 37 ℃ for 12 hours, Colony Forming Units (CFU) on the petri dish were counted. Each group was tested in triplicate. The test results are shown in FIG. 12.

Total colony count (Log value) of sample Log (colony forming unit on the resulting culture dish × 2 × 10³)。

As can be seen from FIGS. 12(a) and (b), the PDA/Col/APG hydrogel dressing of the present invention has a good bacteriostatic effect on Escherichia coli and Staphylococcus epidermidis. Compared with Col/APG hydrogel, the PDA/Col/APG hydrogel dressing polymerized with dopamine in situ has obviously improved antibacterial effect, the polymerization time of dopamine is 12 hours, the effect is optimal, the antibacterial rate is close to 100% within 2 hours, and the antibacterial rate is 99.5% and 99.7% respectively after the polymerization time is 6 hours.

Example 35

The cytotoxicity of the three gel samples PDA/Col/APG-6, PDA/Col/APG-12 and PDA/Col/APG-24 prepared in examples 27-29 was measured by using the viability of NIH 3T3 cells, and the biocompatibility of the hydrogel was evaluated. A3% Col-APG6K-1.2 gel sample of unpolymerized dopamine was used as a control and was designated Col/APG. NIH 3T3 cells were first seeded at 5000 cells/well in 96-well cell culture plates and cultured for 24 h. The lyophilized gel was then soaked in DMEM medium containing 10% FBS, 100 μ L of sample soak was added to the wells of the plate, and cell control wells without added sample were left in the plate, and then cultured in the incubator for 1/2/3 days, respectively. After the incubation was completed, the solution in the plate was carefully removed, 100. mu.L of a phosphate buffer solution of MTT (0.5mg/ml) was added, the plate was incubated at 37 ℃ in a humid environment for 4 hours, MTT in the wells was discarded, 100. mu.L of DMSO was added each and mixed well by shaking at room temperature for 1min, and then the absorbance at 570nm was recorded with a microplate reader. Each group was tested in duplicate 6 times. The test results are shown in FIG. 13.

Cell relative survival (%) — absorbance measured with sample added/absorbance measured without sample added × 100%.

FIG. 13 shows that the hydrogel dressing provided by the invention has no obvious cytotoxicity on HFF-1 cells, and the hydrogel samples PDA/Col/APG-6 and PDA/Col/APG-12 have certain effects of promoting the proliferation of the HFF-1 cells, and can respectively improve the cell survival rate to 111-115% and 225-237% on the 1 st day and the 2 nd day.

Example 36

In vivo wound healing experiments were performed by full-thickness skin defect model to test the ability of the gel to promote wound healing, using a 3% Col-APG6K-1.2 gel sample of unpolymerized dopamine as control, denoted Col/APG. Male Kunming mice (30-40g, 5-6 weeks old) were used in this study. First, all mice were randomly divided into 3 groups. These three groups were Control, Col/APG hydrogel and PDA/Col/APG-12 hydrogel, each containing 3 mice. All mice were acclimated for 1 week prior to surgery. A full thickness skin circular wound of about 0.8cm diameter was created by needle biopsy. Then, 100 μ L PBS was added to the wound in the control group. For the hydrogel group, 100. mu.L of Col/APG hydrogel and PDA/Col/APG-12 hydrogel were applied to the wound, respectively. All wound area pictures were taken on day 0, day 3, day 5, day 9, day 13. The test results are shown in FIG. 14.

On day 3, some reduction in wound area occurred in each group, while the PDA/Col/APG-12 hydrogel exhibited the largest wound contraction area, indicating a relatively high promotion of wound healing. On day 9, both hydrogel groups showed better therapeutic effect than the control group. On day 13, the hydrogel group still had a larger wound contraction area than the blank control group, although all groups showed a smaller wound residual area. Therefore, the results of the PDA/Col/APG-12 hydrogel showed better wound healing effect than the control group by tracing the wound contraction area, due to the combined effect of the intrinsic antibacterial properties of polydopamine and the ideal promotion of wound healing by collagen, as well as the moist wound environment provided by the hydrogel dressing.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A method for preparing bacteriostatic self-healing hydrogel dressing for promoting wound repair is characterized by comprising the following steps of: (0.3-21.2) preparing a hydrogel matrix from the collagen and the aldehyde modified polyethylene glycol, and modifying with polydopamine in an in-situ polymerization manner to form the hydrogel dressing.

2. The method of claim 1, comprising the steps of:

(1) dissolving collagen in water to obtain a collagen solution, and dissolving benzaldehyde-terminated polyethylene glycol in the collagen solution to form a collagen-polyethylene glycol mixed solution; wherein the molar ratio of amino groups to polyethylene glycol aldehyde groups of the collagen is (0.4-3.5): 1;

(2) covering 8-15 mM dopamine solution on the mixed solution prepared in the step (1), standing for a certain time, and then removing the dopamine solution;

(3) and (3) rinsing the hydrogel treated in the step (2) for 2-3 times to obtain the hydrogel dressing with the antibacterial function.

3. The method according to claim 1 or 2, wherein the molar ratio between the amino groups of the collagen and the aldehyde groups of the modified polyethylene glycol is 0.4 to 3.5.

4. The method according to any one of claims 1 to 3, wherein the polyethylene glycol is one of polyethylene glycol 2000, polyethylene glycol 4000 and polyethylene glycol 6000, or a mixture of two or more thereof.

5. The method according to claim 4, wherein the polyethylene glycol is polyethylene glycol 2000, polyethylene glycol 4000 and polyethylene glycol 6000 in a mass ratio of (23-34): (37-55): (80-120).

6. The method according to claim 2, wherein the collagen of step (1) is mixed with 0.23-0.34% polyethylene glycol 2000, 0.37-0.55% polyethylene glycol 4000, and 0.8-1.2% polyethylene glycol 6000 at a concentration of 2-4%, and the mixture is allowed to stand at pH 7.0-7.8 for 5-30 min.

7. The method of claim 2, wherein the benzaldehyde-terminated polyethylene glycol is subjected to hydroformylation modification at both ends of the polyethylene glycol; the aldehyde modification step comprises:

(1) dissolving polyethylene glycol, p-aldehyde benzoic acid and 4-dimethylamine pyridine in anhydrous tetrahydrofuran, wherein the molar ratio of the polyethylene glycol to the p-aldehyde benzoic acid is (0.5-1.5): (2-6);

(2) the molar ratio of the compound to polyethylene glycol is (0.5-1.5): dissolving N, N-dicyclohexyl carbodiimide (2.5-7.5) in anhydrous tetrahydrofuran, and dropwise adding the mixed solution prepared in the step (1);

(3) stirring and reacting for 18-24 h, filtering, taking a clear solution, and slowly dripping the clear solution into a large amount of anhydrous ether to precipitate a product;

(4) and (4) filtering and collecting the precipitate obtained in the step (3), dissolving the solid precipitate in anhydrous tetrahydrofuran, dripping into anhydrous ether for settling, repeating for three times, and purifying to obtain the aldehyde modified polyethylene glycol.

8. The method of claim 2, wherein the dopamine solution is a dopamine-containing Tris-HCl solution; the concentration of the Tris-HCl solution is 8-15 mM, and the pH value is 8.0-8.5.

9. A hydrogel dressing prepared by the method of any one of claims 1 to 8.

10. The method of any one of claims 1 to 8, which is applied to the preparation of wound repair materials in the fields of medicine and daily chemicals.