CN116966335A - Hydrogel attached with antibacterial coating and application thereof - Google Patents

Abstract

The invention belongs to the field of biological medicine, and in particular relates to a hydrogel attached with an antibacterial coating and application thereof. The invention aims to solve the technical problems that the existing burn wound dressing has single function and cannot be applied to the whole burn healing process. The technical scheme of the invention is that the hydrogel with the antibacterial coating comprises macroporous polysaccharide hydrogel prepared from polysaccharide, and catechol compounds and antibacterial peptides which are sequentially attached to the surface of the macroporous polysaccharide hydrogel. The invention provides hydrogel which is used for bacterial infection after burn treatment and is used as a local drug delivery carrier, and the hydrogel can simultaneously meet the requirements for healing scalded wounds and reduce the formation of scar tissues.

Description

Hydrogel attached with antibacterial coating and application thereof

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to a hydrogel attached with an antibacterial coating and application thereof.

Background

Patients with severe burns will undergo a long-term healing process, and if not infected, II-degree and small-area III-degree burns will heal within 3-5 weeks, but may leave scars. In the case of extensive III degree burns, the burn will not heal by itself unless the patient is undergoing a skin grafting procedure. Burns can also lead to several difficult systemic pathological problems, such as fatal infections, pain, skin deformities, and hypertrophic scars. Realizing rapid healing of scalds and reducing scar tissue proliferation has great significance for burn patients. Autologous skin grafting can accelerate wound healing and reduce mortality, but it is limited by insufficient donor skin and unavoidable scar contractures.

To promote the ideal burn healing progression, complex factors in the healing progression need to be considered. Natural wound healing consists of four phases that overlap each other: hemostasis, inflammation, proliferation, remodeling. The hemostatic phase occurs after skin breakdown and then rapidly shifts to the inflammatory phase. The inflammatory phase is the rate limiting step in wound healing and moderate inflammatory response can clear necrotic cells and pathogens that invade the skin. However, tissue necrosis caused by thermal injury can release a large number of injury-associated molecular patterns (DAMPs), and exogenous bacterial infections can be recognized by pattern recognition receptors as pathogen-associated molecular pattern molecules (PAMPs), the excessive presence of which can exacerbate the inflammatory response at the wound site. The wound starts regeneration and repair after inflammation is eliminated, and enters a proliferation stage and a remodelling stage, wherein the two stages not only need proliferation and differentiation of cells, but also need to terminate the regeneration process at proper time, so that the proliferation of scar tissues is prevented.

Traditional wound dressings include vaseline gauze, silicone sheets, paraffin dressings and the like, but these may cause secondary damage to the wound when removed, while being single in function, failing to meet the complications in wound healing. This greatly limits their use. It is therefore necessary to develop new dressings that are more fully functional. In burn treatment, the hydrogel dressing can be used for absorbing wound exudates, can not generate adhesion when being attached to and removed from a wound, can cool the wound to relieve pain of the wound, and has the characteristics of low cost and high biocompatibility. In addition, the hydrogel dressing can be used as a tissue engineering scaffold to improve wound healing by carrying cells or cytokines. However, the conventional hydrogel dressing simply provides antibacterial and anti-inflammatory functions or adds components for promoting cell proliferation and angiogenesis. The design thought can only meet the requirements of healing in the inflammatory phase and the proliferation phase of the wound, and is a key for reducing scar tissue proliferation for the growth regulation of wound tissues in the remodelling phase. There are fewer hydrogel dressings currently available that have the ability to regulate the overall burn healing process.

Disclosure of Invention

The invention aims to solve the technical problems that the existing burn wound dressing has single function and cannot be applied to the whole burn healing process.

The technical scheme of the invention is that the hydrogel with the antibacterial coating comprises macroporous polysaccharide hydrogel prepared from polysaccharide, and polymers and antibacterial peptides of catechol compounds which are sequentially attached to the surface of the macroporous polysaccharide hydrogel.

Specifically, the polysaccharide is at least one of hyaluronic acid, carboxymethyl cellulose, carboxymethyl chitosan, sodium alginate or pectin.

In particular, the catechol compound is at least one of tannic acid, dopamine hydrochloride, gallic acid or gallate.

Wherein the antibacterial peptide is polypeptide DP7, or a modified product of polypeptide DP7, or a derivative of polypeptide DP 7.

Specifically, the amino acid sequence of the polypeptide DP7 is shown as SEQ ID No.1 (VQWRIRVAVIRK).

In particular, the modification is one of the following ways:

a. amidation or sulfation modification is carried out at the C terminal;

b. cholesterol or fatty acid modification is carried out at the N terminal;

c. intermediate residues are subject to glycosylation or phosphorylation modifications.

Specifically, the structure of the polypeptide DP7 is as follows:

Further, the hydrogel is loaded with a bioactive substance.

Specifically, the bioactive substance is at least one of a cell, a vesicle, a polypeptide, a protein or a drug.

Wherein the cells are at least one of mesenchymal stem cells, embryonic stem cells, multifunctional stem cells, skeletal muscle myoblasts, bone marrow derived monocytes, fibroblasts, endothelial cells or endothelial progenitor cells.

Further, the vesicle is at least one of an exosome, an apoptotic body, an autophagosome, a bacterial extracellular vesicle or a cell membrane vesicle.

Further, the polypeptide or protein is at least one of a cell growth factor or an immune cell chemokine.

In particular, the cell growth factor is VEGF or TGF-beta.

Further, the preparation method of the hydrogel comprises the following steps:

step s1: dissolving polysaccharide, catalyst and cross-linking agent adipic dihydrazide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide in a solvent, standing the mixed solution at-80-0 ℃ for 8-24 hours to obtain macroporous polysaccharide hydrogel; the catalyst is 1-ethyl- (3-dimethylaminopropyl) carbodiimide; the cross-linking agent is adipic dihydrazide;

Step s2: catechol compounds are dissolved in a solvent with pH of 7.0 to 8.5; the macroporous polysaccharide hydrogel obtained in the step s1 is restored to the room temperature, and is incubated in catechol compound solution for 1 to 8 hours at the room temperature; washing with pure water to obtain hydrogel attached with catechol compounds;

step s3: and (3) dissolving the antibacterial peptide in a solvent, soaking the hydrogel obtained in the step (s 2) in the antibacterial peptide solution, incubating for 1-8 hours at room temperature, and cleaning with pure water to obtain the hydrogel attached with the antibacterial coating.

Specifically, in step s1, the solvent is at least one of ultrapure water, phosphate buffer solution or physiological saline.

In step s2, the solvent is phosphate buffer, citrate buffer, carbonate buffer or Tris buffer.

In particular, in step s3, the solvent is at least one of ultrapure water, phosphate buffer solution or physiological saline.

Preferably, the preparation method further comprises one of the following steps:

step s4: freeze-drying the hydrogel obtained in the step s3 for later use;

step s5: step s5: preparing bioactive substances into a solution, dripping the solution onto the hydrogel obtained in the step s3, or soaking the hydrogel in the solution, and incubating.

Further, in step s2, the concentration of the catechol compound solution is 0.1 to 2mg/mL.

Preferably, in step s2, the concentration of catechol compound is 0.5mg/mL.

Further, in step s3, the concentration of the antibacterial peptide solution is 0.01 to 1mg/mL.

Preferably, in step s3, the concentration of the antimicrobial peptide solution is 150. Mu.g/mL.

Preferably, the hydrogel treated in step s4 is dissolved in pure water during use.

Specifically, in step s5, the bioactive substance is a cell, and the cell is adjusted to 6×10 ⁶ Density per mL, drop onto the hydrogel obtained in step s3, at 37℃with 5% CO ₂ Is incubated for 30 minutes in the incubator to allow the cells to penetrate into the gel.

Specifically, in step s5, the bioactive substance is vesicle, the vesicle is diluted to 100 mug/mL, the hydrogel obtained in step s3 is dehydrated, and the diluted cell vesicle solution is dripped on the dehydrated hydrogel and is placed at 4 ℃ for co-incubation for 4 hours; the unloaded vesicles were washed with PBS.

The invention also provides the application of the hydrogel in preparing products for treating burns, acute and chronic traumatic infection or skin infection; further, the product is a dressing.

The invention has the beneficial effects that: the present invention provides a macroporous polysaccharide hydrogel which can be used for the treatment of bacterial infections or skin infections, in particular bacterial infections after burns, and as a topical drug delivery vehicle. The hydrogel can simultaneously meet the requirements of scald and wound healing, and can reduce the formation of scar tissues. The macroporous polysaccharide hydrogel is prepared based on polysaccharide, catechol compounds, antibacterial coatings, loaded medicines and bioactive components are further sequentially attached, and different therapeutic components can be secreted at different stages of wound healing so as to play different functions. The hydrogel provided by the invention has good capability of inhibiting common pathogenic bacteria, and can promote mesenchymal stem cells to secrete cytokines related to wound healing, help fibroblast migration at a wound, generate new blood vessels and inhibit inflammatory response; the therapeutic components carried by the hydrogel can release the therapeutic factors such as antibacterial peptide, VEGF, TGF-beta and the like according to different characteristics of an inflammation stage, a proliferation stage and a remodelling stage in wound healing, so that the wound healing speed is accelerated, and the effect of no-scar repair is achieved, thereby realizing the whole-course regulation and control of the burn healing process. In addition, the materials used for preparing the hydrogel have good biocompatibility, simple preparation process and good stability, and have good industrial production and clinical application prospects.

Drawings

Fig. 1, frozen hydrogel (CG), dopamine-coated frozen hydrogel (DACG) and antimicrobial peptide-coated hydrogel (DA 7 CG) scanning electron micrographs, scale bars = 25 microns, 5 microns, 250 microns, respectively.

Fig. 2, scale = 100 microns, staining of stem cells loaded in hydrogels using Calicein AM/PI cell death and alive staining kit.

FIG. 3 shows staining of cytoskeleton loaded in hydrogel. A. The mesenchymal stem cell cytoskeleton loaded in the hydrogel was stained with phalloidin, scale = 100 microns. B. The hydrogel scaffold was labeled with FITC and the mesenchymal stem cell scaffold loaded within the hydrogel was stained with phalloidin, scale = 100 microns.

FIG. 4, fourier transform infrared spectroscopy detection of chemical bond differences between hydrogel lyophilized powder and hydrogel materials, hydrogel lyophilized powder at 1750cm ^-1 The appearance of characteristic peaks of amide bonds indicates that the hyaluronic acid is crosslinked into hydrogel through chemical reaction.

Fig. 5, rheometer data results for frozen hydrogels (CG).

FIG. 6, the release rate of the antimicrobial peptide DP7 from the surface of DA7CG was measured in a release medium having pH values of 5.0 and 7.4, respectively.

Fig. 7, comparing the effect of dopamine-frozen gel (DACG) loaded with DP7 on the paracrine capacity of mesenchymal stem cell (PMSC), it can be seen from the results that PMSC seeded inside the (DA 7 CG) hydrogel after DP7 addition had higher cytokine secretion levels, and mainly vascular growth factor (VEGF-Sub>A), epidermal Growth Factor (EGF) and Angiogenin (ANGPT) were detected.

FIG. 8, in vitro bacteriostasis test results. A. The inhibition effect of DA7CG on pseudomonas aeruginosa (PAO 1) escherichia coli (K12) and staphylococcus aureus (25923) is detected by a bacteriostasis circle experiment. B. The morphology changes of Pseudomonas aeruginosa (PAO 1) E.coli (K12) and Staphylococcus aureus (25923) after contact with DA7CG were observed by scanning electron microscopy. C. The inhibition effect of DA7CG on pseudomonas aeruginosa (PAO 1) escherichia coli (K12) and staphylococcus aureus (25923) is detected by a microdilution method, and the absorbance can reflect the quantity of bacteria.

FIG. 9, CCK-8 method for detecting cell viability, absorbance results were measured using a microplate reader.

FIG. 10, HUVEC cell in vitro tube experiments. A. Cell tube formation after treatment with different hydrogels, scale = 100 microns. B. The results were counted for the number of branches of the tube in each experimental group. C. Tube length statistics were performed for each experimental group.

FIG. 11 shows the results of cell migration experiments. Nih 3T3 cell scratch assay results, bar=100 microns. B. And counting scratch areas in the scratch experiment and drawing into a histogram.

FIG. 12, results of in vitro anti-inflammatory experiments. A. TNF-alpha expression level in THP-1 cells after treatment with different hydrogels was varied. B. IL-1 beta expression level in THP-1 cells is changed after different hydrogel treatments. C. IL-8 expression level in THP-1 cells was varied after treatment with different hydrogels.

FIGS. 13 and DP7 show experimental results of the ability of PMSC cells to promote secretion of cytokines. A. The change in the expression levels of VEGF, TGF-beta 1 and TGF-beta 3 after PMSC cells treated with different concentrations of DP7 were examined by the WB method. B. VEGF expression changes after PMSC treatment at different concentrations of DP7 were detected by q-PCR. C. The variation of TGF-beta 1 and TGF-beta 3 expression levels of PMSC cells treated with DP7 at different concentrations was detected by q-PCR.

Fig. 14, in vivo bacteriostasis test results. A. Colony growth after wound skin homogenate plating. B. Statistical results of colonies were counted.

Fig. 15, wound healing. A. Wound photographs at days 0,3,7, 14, 17 after molding and administration of treatment, scale = 0.5cm. B. Trend of wound shape change at day 0,3,7, 14, 17 after molding and administration of treatment. C. Wound area statistics.

Fig. 16, wound histopathological section staining results, CD31 immunohistochemical staining, sirius scarlet staining, M2 macrophage marker CD206 staining, nucleus staining, M1 macrophage marker INOS staining, scale = 100 microns, respectively, from right.

FIG. 17 shows the results of detection of changes in the expression level of VEGF and TGF-. Beta.3 in wound tissue at various time points after administration of treatment.

Fig. 18, scanning electron microscope images of DA7CG hydrogel loaded with human umbilical cord-derived mesenchymal stem cell vesicles (UCMSCV), and a spherical exosome in the DA7CG hydrogel can be seen, which indicates that the DA7CG hydrogel can successfully load cell vesicles.

Fig. 19, DACG, DA7CG, dacg@v and dacg@v hydrogel in vitro wound healing promoting effect. A. Cell scratch test results, scale = 50 microns. Transwell test results, scale = 100 microns. C. In vitro, umbilical vein endothelial cell (HUVEC cell) tube test results, scale = 200 microns. The hydrogel loaded with the mesenchymal stem cell vesicles from the human umbilical cord has good in-vitro effect of promoting migration of NIH 3T3 fibroblasts and catheterization of endothelial cells of the human umbilical vein.

FIG. 20 shows the results of in vitro proliferation-promoting fibroblasts (NIH 3T 3), in which more EdU dye fluorescence was observed in the DACG@V group and DA7CG@V group loaded with extracellular vesicles, indicating that the addition of extracellular vesicles promoted proliferation of NIH 3T3 cells.

Fig. 21 shows the experimental results of healing wounds of scalded infection in mice, and the highest healing speed of wounds on the backs of the mice in the DA7CG@V group, which shows that the DA7CG hydrogel can successfully load mesenchymal stem cell vesicles derived from human umbilical cords and successfully release the mesenchymal stem cell vesicles to play a role in promoting the healing of scalds.

Fig. 22 shows that the frozen gel prepared by sodium alginate and loaded with mesenchymal stem cells PMSC is incubated with NIH 3T3 cells, and the cell migration condition is observed by photographing at 0, 12 and 24 hours (h) after administration, and the result shows that the frozen gel prepared by sodium alginate as a material has the same capacity of promoting cell migration after loading PMSC cells.

Fig. 23, frozen hydrogel (ACG) prepared using sodium alginate was tested for inhibition ability of 3 bacteria, pseudomonas aeruginosa (PAO 1), escherichia coli (K12) and staphylococcus aureus (25923) after loading dopamine (ADACG) and DP7 (ADA 7 CG), and the results indicate that alginic acid also has inhibition ability after loading DP7 according to the prepared hydrogel.

Detailed Description

Polysaccharides are common ingredients that can be used to prepare wound dressings, such as hyaluronic acid, carboxymethyl cellulose, carboxymethyl chitosan, sodium alginate, pectin, and the like. Hyaluronic acid or sodium alginate is selected as one of the embodiments of the present invention. Hyaluronic Acid (HA) is a biocompatible polysaccharide that is widely present in mammalian tissues and is an integral component of the extracellular matrix. Therefore, HA-based hydrogels may be an ideal wound dressing. The reaction principle is that amino groups at two ends of the cross-linking agent can be subjected to amidation reaction with polysaccharide containing carboxyl groups under the action of a catalyst to crosslink the cross-linking agent into hydrogel. HA and adipic dihydrazide (AAD) a frozen macroporous hydrogel (Cryogel) prepared by amide reaction under freezing conditions. During gel freezing, large pores are formed inside the hydrogel scaffold due to ice crystal formation. The inventors have experimentally verified that these voids can be used to load drugs or cells, for example, mesenchymal Stem Cells (MSCs) can be loaded in the voids. Mesenchymal stem cells may help burn wounds heal rapidly, probably because they secrete tissue repair-related cytokines such as VEGF and TGF-beta.

In order to reduce bacterial infection during wound healing, the inventors considered whether the antibacterial properties of the gel material could be increased, and according to the characteristics of the hydrogel that formed large pores, the addition of a coating of antibacterial substances on the surface of the hydrogel was considered. The variety of antimicrobial substances is varied, and finally the inventors have chosen to attach antimicrobial peptides to hydrogels and hopefully to form a coating. The usual antimicrobial peptides can be used, and in one embodiment of the present invention the antimicrobial peptide DP7 (SEQ ID No.1: VQWRIRVAVIRK) is used. DP7 hydrogels have been shown to have bactericidal and scar-free wound healing effects. It was also found in earlier studies that Vascular Endothelial Growth Factor (VEGF) and transforming growth factor-beta (TGF-beta) secreted by placental-derived mesenchymal stem cells (PMSCs) were upregulated by PMSC cells after co-incubation with DP 7. VEGF can induce angiogenesis in wound tissue during the proliferative phase, while TGF-beta 3 can reduce scar tissue proliferation at the end of the healing phase.

In order to enhance the attachment of cells to the pore surface of the hydrogel, the inventors have found in research that catechol compounds act as an intermediate layer between the hydrogel and the cells. The catechol compound can not only effectively improve the attachment efficiency of cells, but also combine the cationic antibacterial peptide DP7 through the Schiff base and the electrostatic action, so that the hydrogel has antibacterial capability. Through experiments, the inventor reasonably configures the functions of hydrogel, catechol compounds and antibacterial peptides, so that the compounded hydrogel has better antibacterial and inflammation inhibiting effects. In addition, the adhesion function of catechol compounds is also beneficial for subsequent loading of vesicles, drugs, etc. (DP 7 is adsorbed on the dopamine layer, but not a continuous tight layer, the loading of DP7 does not consume all catechol groups, catechol has the remaining power to bind cell vesicles, etc.). The inventors have also found through experiments that in the process of sequentially attaching catechol compounds and antibacterial peptides, the concentrations of the catechol compounds and the antibacterial peptides have a certain influence on the attaching effect. Specifically, the hydrogel with the antibacterial coating having a good adhesion effect can be obtained by using the concentration of the catechol compound solution of 0.1-2 mg/mL and the concentration of the antibacterial peptide solution of 0.01-1 mg/mL. In a further optimization process, the effect is especially optimal with the concentration of catechol compound solution being 0.5mg/mL and the concentration of the antibacterial peptide solution being 150 mug/mL.

In the present invention, common catechol compounds such as tannic acid, dopamine hydrochloride, gallic acid, and gallate can be used. In one embodiment, dopamine hydrochloride (DA) is selected. Dopamine hydrochloride undergoes oxidation reaction under alkaline conditions to form Polydopamine (PDA) which is attached to the surface of the gel. PDA is a tissue-adhesive biomaterial that is synthesized by the oxidation of dopamine under alkaline conditions. PDA can react with amino-rich compounds such as peptides and cells via a schiff base reaction. This is also part of the mechanism by which PDA coatings can provide cell adhesion and hydrogel functionalization, as well as providing conditions for attachment of antimicrobial peptides. The antibacterial peptide DP7 is attached to the PDA through electrostatic adsorption and Schiff base reaction with the PDA coating, so that the antibacterial coating modification of the hydrogel is realized. Finally, the invention also proves that the PDA/DP7 coating and the frozen macroporous hydrogel (DA7CG@C) loaded with PMSCs are used as wound dressing, so that the inflammatory reaction of a secretory group and a wound part of the PMSCs can be regulated to regulate the wound healing process, and the scar-free repair is realized. The PDA/DP7 coating and the frozen macroporous hydrogel (DA7CG@V) loaded with UCMSCV are used as wound dressing, so that the inflammation of the wound site can be effectively inhibited.

Materials and reagents used in the examples below

Antimicrobial peptide DP7 (VQWRIRVAVIRK, synthesized by Shanghai peptide Biotechnology Co., ltd.), hyaluronic acid (HA, molecular weight 20-40 kDa, purchased from Beijing Kyoto Xihong BioCo., ltd.), sodium Alginate (Alginate, viscosity 200.+ -.20 mpa.s, purchased from Shanghai A Ding Shenghua Co.), dopamine hydrochloride (DA, purchased from Shanghai A Ding Shenghua Co.), adipic dihydrazide (AAD, purchased from Shanghai A Ding Shenghua Co.), 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC, purchased from Shanghai A Ding Shenghua Co.), pseudomonas aeruginosa PAO1 (Pseudomonas aeruginosa, laboratory seed, staphylococcus aureus ATCC 25923 (Staphylococcus aureus, laboratory seed), escherichia coli K12 (Escherichia coli, laboratory seed), human placenta-derived mesenchymal stem cells (PMSC, laboratory seed), human-derived mesenchymal stem cell vesicles (UCMSCV, laboratory preparation).

EXAMPLE 1 preparation of PMSC-loaded hydrogels

Preparation of frozen macroporous hydrogels:

hyaluronic acid 10mg,AAD 1.25mg,EDC 10mg was weighed and dissolved in 1mL of ultrapure water. The above mixed solution was put into a glass mold having a thickness of 1mm or directly put into a-20℃refrigerator to be frozen for 24 hours. The frozen hydrogel was taken out and left at room temperature, after returning to room temperature, the gel was cut into a round shape with a diameter of 1cm, and the remaining reagent was washed with purified water. The final product was the frozen macroporous hydrogel (CG).

Preparation of macroporous hydrogels containing antimicrobial peptide coatings:

DA was weighed and dissolved in Tris-HCl buffer at pH 8.0 to a concentration of 0.5mg/mL. The circular CG was immersed in DA solution and incubated on a shaker (40 rpm) at room temperature for 4 hours to form a dopamine coating modified cryogel (DACG). Excess dopamine molecules are then removed using purified water and buffered saline. DP7 was weighed and dissolved in PBS to a concentration of 150. Mu.g/mL. And soaking DACG in the DP7 solution, incubating the mixture for 2 hours at room temperature by using a shaking table (40 rpm), and then washing off excessive DP7 by using PBS to obtain the macroporous hydrogel (DA 7 CG) containing the antibacterial peptide coating. The prepared hydrogels (DACG, DA7CG, DACG@V) were subjected to ethanol gradient dehydration (50%, 70%, 85%, 95%, 100%), each concentration being soaked for 10min. Immediately after dehydration of 100% ethanol, the mixture was placed in a supercritical carbon dioxide dryer. Finally, the macroporous gel morphology (fig. 1) was observed using a Scanning Electron Microscope (SEM), and three graphs A, B, C in fig. 1 respectively show the hydrogel lattice morphology at different magnifications. It can be seen from fig. 1A that the hydrogel grids of the unmodified polydopamine and DP7 antimicrobial coating were smoother, particulate agglomerates appeared on the hydrogel grids of fig. 1B and C, and some shrinkage appeared on the grid morphology of fig. 1C due to the interaction of the positively charged DP7 with the negatively charged hydrogel grids and polydopamine coating. These results indicate that successful modification of polydopamine and DP7 brings about a certain change to the hydrogel lattice. However, the modification of polydopamine and DP7 did not affect the mesh size of the hydrogel much, both being around 25 μm.

Cell loading

DA7CG was sterilized by soaking in 75% ethanol for 40 minutes in a 24-well plate, followed by three washes with PBS. Placenta-derived mesenchymal stem cells were digested and counted at 6×10 ⁶ Density of/mL cells were resuspended and 50. Mu.L of cell suspension was added dropwise toGel, put at 37℃and 5% CO ₂ The cells were allowed to permeate into the gel by incubation for 30 minutes in the incubator of (C) to give DA7CG@C. Finally, 1mL of DMEM complete medium is added to each well, and the culture is carried out in an incubator. After culturing for 24, 48, 72h, cells were stained with Calcein-AM/PI stain and observed for cell growth using confocal microscopy (FIG. 2). It can be seen from confocal microscopy that none of the cells loaded into the hydrogel survived (green fluorescence) and no dead cells were observed (red), while the number of cells increased with increasing incubation time. This suggests that stem cells can be seeded within hydrogels without the hydrogels affecting the viability and proliferation capacity of the stem cells within.

Hydrogel inner cytoskeletal staining

100. Mu.L of a 1% FITC solution was added to each 1mL of the hydrogel solution at the time of preparation of the CG frozen gel to obtain FITC-labeled hyaluronic acid gel (FITC-CG). FITC-labeled DACG and DA7CG (FITC-DACG, FITC-DA7 CG) were prepared separately using FITC-CG. And FITC-CG, FITC-DACG and FITC-DA7CG were loaded with PMSC cells and cultured for 7 days as described previously, followed by staining of the cytoskeleton with phalloidin and observation with confocal microscopy (FIG. 3). The confocal microscope photograph shows that cells in the hydrogel are attached to the gel skeleton, cells are mostly in a bulk state in the FITC-CG group, and cell morphology is more regular in a shuttle state in the FITC-DACG group and the FITC-DA7CG group, which indicates that the dopamine and the dopamine-DP 7 are modified so that the cells are attached to the surface of the hydrogel more easily. The main reason is that the benzoquinone structure rich in the dopamine can react with the amino on the cell surface, and the residual positive charge carried on the surface of the DP7 after being adsorbed with the dopamine can not kill the cells, and is beneficial to cell adhesion carrying negative charges.

Fourier transform infrared spectrum detection

The hyaluronic acid macroporous frozen gel sample is freeze-dried in a freeze dryer and then crushed, and 50mg freeze-dried powder is taken. Then respectively weighing 50mg of sodium hyaluronate, AAD and EDC powder, preparing a detection sample by a potassium bromide tabletting method, and freeze-drying by Fourier transform infrared spectroscopy analysisThe chemical bonds in the hydrogel powder and the raw material powder are recorded in 4000-400 cm ^-1 Within the range. Fourier transform infrared spectrum detection of chemical bond difference between hydrogel freeze-dried powder and hydrogel raw material, wherein the hydrogel freeze-dried powder is 1750 cm ^-1 The appearance of characteristic peaks of amide bonds indicates that the hyaluronic acid has undergone chemical reaction to crosslink into hydrogel (fig. 4). The results demonstrate that hydrogels prepared using the freezing method have sufficient strength as wound dressings.

Rheometer detection

CG was prepared. The gel samples were placed on the sample stage of the rheometer and the time-dependent properties of the hydrogels were evaluated at 37 ℃ and 1 Hz (fig. 5). It can be seen that within 10 minutes of the test, the CG hydrogel has a storage modulus (G') greater than the loss modulus (G "), which indicates that the hydrogel is less fluid, mainly in solid form, demonstrating successful hydrogel preparation.

DP7 release rate determination

1) Determination of DP7 concentration standard curve: 1 mg of DP7 powder was dissolved in 1 mL purified water to give 1 mg/mL of DP7 solution. Subsequently, 500. Mu.L of 1. 1 mg/mL of the DP7 solution was pipetted into 500. Mu.L of pure water, diluted to 500. Mu.g/mL of the DP7 solution, followed by 2-fold gradient dilution in sequence. The ABS value (absorbance value) of the DP7 solution in a280 mode, i.e. 280 nm, was determined using an ultra-micro spectrophotometer Nanodrop 2000.

2) Gel surface DP7 release assay: 1 ml of PBS solution at pH 5.0 and 7.4, respectively, was added to a 24-well plate containing the frozen gel, and the gel was immersed. The well plate was placed on a shaker to achieve an even distribution of the DP7 concentration in the well at each section. Three parallel replicate wells were set up for each group and 2 μl of soak solution was taken at 15 min, 30 min, 1 h, 2 h, 3 h, 6 h, 12 h, 24 h, 48 h and 96 h, respectively, and the concentration of antimicrobial peptide DP7 released in the solution was determined using Nanodrop 2000. From the results of the release profile (fig. 6), it can be seen that the release rate of DP7 in the acidic medium is higher, since DP7 and dopamine are cross-linked by pH sensitive schiff base, and the oxidation product polydopamine of dopamine is more easily dissolved in the acidic medium. The pH sensitive property enables DA7CG to release antibacterial peptide according to acidic microenvironment generated by bacterial metabolism at a wound, achieves a controlled release effect, and is beneficial to reducing toxic and side effects.

DP7 demonstration of PMSC secretion growth factor secretion ability

In vitro experiments were performed to investigate the ability of dopamine-frozen gels (DACG) to modulate paracrine secretion of placenta-derived mesenchymal stem cell (PMSC) cytokines after DP7 addition. PMSCs were used at 3X 10 ⁵ The density of individual cells/wells was plated in 6-well plates. Meanwhile, the cells were treated by adding DP7 solutions at concentrations of 5. Mu.g/mL and 10. Mu.g/mL, respectively. The blank was treated with no DP7 solution. After 48 hours of co-incubation, total RNA and total protein were extracted from PMSCs. Expression levels of vascular endothelial growth factor-A (VEGF-A), transforming growth factor- β1 (TGF-. Beta.1) and transforming growth factor- β3 (TGF-. Beta.3) were detected using q-PCR and Western blot, respectively. From the results (FIG. 7), it can be seen that PMSC cells treated with DP7 have higher levels of growth factor secretion, and that these growth factors have a promoting effect on wound repair. PMSC inoculated inside the hydrogel after DP7 addition (DA 7 CG) had higher cytokine secretion levels, mainly examined for vascular growth factor (VEGF-Sub>A), epidermal Growth Factor (EGF) and Angiogenin (ANGPT).

In vitro bacteriostasis experiment

Frozen pseudomonas aeruginosa, escherichia coli and staphylococcus aureus were picked up and cultured overnight on a bacterial shaker at 37 ℃ in 3 mL of LB medium, respectively. The next day, the concentration of the resuscitated bacterial liquid is diluted to 10 ⁵ CFU/mL. Firstly, evaluating the bacteriostatic effect of DA7CG by using a bacteriostasis ring method, firstly, uniformly coating 3 diluted bacterial liquids on 3 LB plates, and respectively adding CG, DACG, DA CG three gels on each plate. The plates were placed upside down into a bacterial incubator at 37℃and incubated overnight. The next day, the size of the zone of inhibition around the three gels on the three bacterial plates was observed and photographed for recording (fig. 8A). The zone size was measured using Imagej software. Then evaluating the bacteriostatic effect of DA7CG by micro dilution, respectively adding 400 mu L of diluted bacterial solution (P.aeromonas, E.coll and S.aureus) into 24-well plate, respectively adding three different gels(CG, DACG, DA7 CG). The control group is bacterial liquid without gel treatment. After overnight incubation in an incubator at 37 ℃, 100ul of bacterial fluid per well was transferred to a 96-well plate. The viability of the bacteria was measured by reading the OD value of the bacterial liquid at 600nm (fig. 8C). Finally, we observed the morphological changes of the bacteria after contact with DA7CG hydrogel by scanning electron microscopy, three large pore size hydrogels (CG, DACG, DA CG) were placed in 24 well plates at a concentration of 1X 10 ⁸ CFU/mL of 100. Mu.L bacterial solution was co-cultured in a 37℃cell incubator for 4 hours, followed by fixation with 4% paraformaldehyde for 30min-1h. Ethanol gradient dehydration (50%, 70%, 85%, 95%, 100%) was then performed, with each concentration being soaked for 10min. Immediately after dehydration of 100% ethanol, the mixture was placed in a supercritical carbon dioxide dryer. Bacterial control preparation steps were co-incubated with the samples. And drying by supercritical carbon dioxide method. Finally, the bacterial morphology changes within the macroporous gel were observed using a Scanning Electron Microscope (SEM) (fig. 8B).

The DA7CG can be seen to effectively inhibit bacterial growth through a bacteriostasis ring and a micro dilution method, meanwhile, the structure of the bacterial outer membrane is changed after the DA7CG is incubated together according to SEM (scanning electron microscope) photographs, and the results show that the antibacterial coating formed by the DP7 can effectively inhibit bacterial growth through destroying the structure of the bacterial outer membrane.

Security detection

Fibroblasts (NIH 3T3, purchased from ATCC cell bank) were cultured in DMEM medium containing fetal bovine serum (10%) and penicillin-streptomycin (1%). After culturing to log phase, 3T3 cells were plated in 96-well plates at a density of 5X 10 ³ Well, culture for 24 hours.

At the same time, the inside of the container is 3 multiplied by 10 ⁶ CG, DACG, DA7CG samples of PMSC cells were soaked in DMEM for 24 hours.

The culture medium (100. Mu.L per well) in the 3T3 cell well plate was replaced with the leachate of the cell-loaded gels (CG@C, DACG@C, DA7CG@C) and the culture was continued for 24, 72 and 120 hours. Blank medium was incubated with 3T3 as a control. Subsequently 10. Mu.L of CCK-8 was added to each well followed by incubation at 37℃for 2 hours. The absorbance (OD value) of the solution in the wells at 450nm was measured using an enzyme-labeled instrument. Through comparison with the control group, it was found that none of the 3 stem cell-loaded hydrogel extracts had an effect on the activity of NIH 3T3 cells (fig. 9).

In vitro angiogenesis experiments

Human Umbilical Vein Endothelial Cells (HUVECs) were combined with a culture medium containing 3X 10 cells in a 12-well plate ⁶ Hydrogel dressing for PMSC cells (CG, DACG, DA7 CG) and blank hydrogel without cells (DA 7 CG) were co-cultured for 24h. Matrigel (Matrigel, available from corning company, usa) was thawed overnight at 4 ℃, and the vials were rotated after thawing to ensure Matrigel mixing. In addition, the gun tip used in the experiment and the EP tube for dispensing matrigel are pre-cooled in advance. To avoid repeated freeze thawing, matrigel was dispensed into 1.5mL ep tubes. And added to 96-well plates, 100 μl/well. The 96-well plate was placed at 37℃with 5% CO ₂ The matrigel was allowed to solidify for 40min in the incubator. Co-cultured HUVECs were counted after digestion and plated onto 96-well plates coated with matrix gel (1.5X10) ⁴ Individual cells/well). The group without gel treatment served as a control. Incubation was continued for 6 hours at 37 ℃. Blood vessel images in 96-well plates were acquired after 6 hours using an inverted microscope. Branch point and capillary length were calculated using ImageJ software. According to the statistical results (fig. 10), the PMSC-loaded hydrogel was seen to have a significant in vitro pro-angiogenic effect on HUVEC cells, wherein the DP 7-loaded DA7CG hydrogel group had a greater number of vascular branches and greater vascular lengths in each field than the control group (the difference was statistically significant), indicating that the DP 7-loaded hydrogel had some pro-angiogenic capacity. The hydrogel groups (CG@C, DACG@C and DA7CG@C) loaded with PMSC cells have the advantages that the number of blood vessel branches and the length of blood vessels in each visual field are larger than those of the control group and the DA7CG group, so that the hydrogel loaded with PMSC cells has stronger angiogenesis promoting capability.

Cell migration experiments

Cg@c, dacg@c (3×10 inoculated in each gel ⁵ Individual cells) and DA7CG were immersed in 1mL of serum-free DMEM medium for 24 hours. At the same time, fibroblast (NIH 3T 3) was cultured at 8X 10 ³ Density of wells/wells were seeded in 96-well plates and incubated in a 37 ℃ cell incubator for 24 hours. After washing the cells with PBS, a streaker device was used in a 96-well plateA uniform scratch was scraped into each well. And 100. Mu.L of the above hydrogel sample soak solution was added to each well. The control group was added with 100. Mu.L of serum-free medium alone. Scratch wounds after 0, 6, 12 and 24 hours of treatment were photographed using a Livecyte tracer (fig. 11A). Scratch areas were measured with imageJ software (fig. 11B), from which it can be seen that hydrogel groups loaded with PMSC cells had a higher degree of scratch healing than control groups and that the differences were statistically significant when the experiment was run to 24 hours. Wherein the score healing was approximately 100% in group CG@C, DACG@C, DA7CG@C.

In vitro anti-inflammatory test

Human monocytic leukemia cells (THP-1, purchased from ATCC cell bank) were cultured in 1640 medium containing fetal bovine serum (10%) and penicillin-streptomycin (1%). Culturing to a certain amount, spreading THP-1 cells into 6-well plate with density of 8X10 ³ Per well, 100ng/mL PMA was added for 24 hours to induce conversion of THP-1 cells to M0 macrophages. After 24 hours, 100ng/mL LPS and 20ng/mL IFN-. Gamma.were added to the 6-well plate and M0 macrophages were induced as M1 macrophages. After 24h a transwell was added and the upper chamber was filled with three cell-loaded cryogels (cg@c, dacg@c, da7cg@c) and non-cell-loaded DA7CG and co-cultured with cells for 24 h. The blank is the treatment of cells without LPS/IFN-gamma and cryogel. A control group stimulated with LPS+INF-gamma alone but not gel treated was also provided. mRNA was extracted after the completion of the treatment and the expression levels of inflammatory factors (TNF-. Alpha., IL-1β and IL-8) were measured by q-PCR. From the results (fig. 12), it can be seen that the frozen hydrogel loaded with stem cells has the ability to reduce the expression level of inflammatory factors, and the reduction of inflammatory levels facilitates the rapid entry of the wound into the regenerative phase through the inflammatory phase, thereby accelerating the wound healing process.

DP7 demonstration of PMSC secretion growth factor secretion ability

PMSCs were used at 3X 10 ⁵ The density of individual cells/wells was plated in 6-well plates. Meanwhile, the cells were treated by adding DP7 solutions at concentrations of 5. Mu.g/mL and 10. Mu.g/mL, respectively. The blank was treated with no DP7 solution. After 48 hours of co-incubation, total RNA and total protein were extracted from PMSCs. Respectively using q-PCR FIGS. 13B, 13C) and Western blo (FIG. 13A) t detect expression levels of vascular endothelial growth factor-A (VEGF-A), transforming growth factor- β1 (TGF- β1) and transforming growth factor- β3 (TGF- β3). From the results, it can be seen that PMSC cells treated with DP7 have higher secretion levels of growth factors, which promote wound repair.

Burn infection model establishment

Frozen PAO-1 bacteria were picked up, added to sterile BD tubes containing 3mL LB medium, and incubated on a bacterial shaker at 37℃for 12 hours to recover the bacteria. Subsequently, the bacterial liquid is diluted to 1X 10 in gradient ⁵ CFU/mL. Mice were intraperitoneally injected with 80 μl of 10% aqueous chloral solution, after anesthesia, the backs were shaved and dehaired, and the back dehaired areas were sterilized with alcohol cotton balls. A cylindrical iron of 10mm diameter was sterilized by wiping with an alcohol cotton ball, followed by heating to 200℃with a heat gun. Subsequently, the soldering iron was placed on the skin on the right side of the back spine of the mouse, without applying additional gravity, and left for 10s. Finally 20 mu L of 10 ⁵ The PAO-1 bacteria of CFU/mL are dripped into the scalding part, and after the liquid is stopped for 30 seconds, different preparations are given for treatment, and the indwelling needle film is used for wrapping the wound.

Evaluation of in vivo antibacterial Effect

The 18C 57BL/6 mice were randomly divided into 6 groups, namely a control group, a CG group, a DACG group, a DA7CG group, a PMSC suspension group and a DA7CG@C group. Three mice were used for each group. After molding according to the above method, the different preparations were placed on the back wound, and the PMSC group had a concentration of 3X 10 at 100. Mu.L ⁷ The PMSC cell suspension of cells/mL was injected subcutaneously around the wound. Then the catheter was covered with a patch of indwelling needle and fixed with 3M tape. After 3 days of treatment, the back wound skin of three mice was cut off from each group and placed in a 2mL homogenization tube for homogenization treatment. The homogenate was then diluted and spread on LB plates for cultivation. After 18 hours of incubation, the plates were removed, photographed (FIG. 14A) and colonies were counted using Imagej software (FIG. 14B). From the results, the DA7CG and DA7CG@C hydrogel show good in-vivo antibacterial effect, and bacteria attached to a wound can be effectively killed.

Evaluation of in vivo wound healing Effect

42C 57BL/6 mice were randomly divided into 6 groups, namely a control group, a CG group, a DACG group, a DA7CG group, a PMSC suspension group and a DA7CG@C group. Molding and administration of different formulations for treatment were performed as described above. The drug was changed every 3 days and on days 0, 3, 7, 14 and 17 of the treatment, a record of the wound healing was taken (fig. 15A) and the progress of wound healing was measured with Imagej software.

On days 3, 7, and 14 of treatment, one mouse was sacrificed for each group, skin at the wound was obtained, paraffin-embedded treatment was performed after fixation with 4% paraformaldehyde and sections were performed, and finally pathological forms of wound tissues were evaluated by HE, masson, sirius scarlet, immunohistochemistry, and immunofluorescence staining.

From the wound area changes (fig. 15B, 15C), it can be seen that the back wound healing rate was faster in the da7cg@c treated mice. It can be seen from HE and Masson staining (fig. 16) that the da7cg@c treated mice regenerated wound epithelium faster in collagen attachment. Based on CD31 immunohistochemical staining, more neovascularization was found in wound skin following da7cg@c treatment. According to the result of immunofluorescence staining of M2/M1 macrophages, the proportion of M2 macrophages in wound skin after DA7CG@C treatment is increased, and meanwhile, the proportion of type III collagen is also increased according to the Tianlangerhans scarlet staining, which shows that the wound has the potential of scar-free repair after DA7CG@C treatment.

Detection of cytokine expression levels in wound tissue

60 healthy C57BL/6 mice were randomly divided into five groups. The control group, CG group, DACG group, DA7CG group, PMSC suspension group and DA7CG@C group are respectively adopted. The method is used for molding and administering different preparations for treatment, wherein PMSC group is subcutaneously administered, and hydrogel group is externally applied. The drug was changed every 3 days and mice were sacrificed on days 0, 3, 7, 14 and 17 of treatment, followed by removal of wound tissue for milling to extract RNA. After each set of RNA concentrations was determined, reverse transcription was performed and the cDNA obtained by reverse transcription was subjected to qPCR reaction to verify the expression levels of VEGF-A and TGF-betSub>A 3 at various stages of the wound healing process. According to q-PCR results (FIG. 17), it can be seen that the expression level of VEGF in wound tissue is improved in mice treated with the hydrogel at the time of wound healing to the middle stage, wherein the PMSC group is the highest in elevation, which indicates that PMSC cells have strong angiogenesis promoting capacity, and the expression level of VEGF is also improved in the hydrogel group loaded with PMSC cells compared with the control group, which indicates that the hydrogel loaded with PMSC cells is helpful for the wound tissue in regeneration stage to generate new blood vessels. And the expression level of TGF-beta 3 in the tissues is improved when the wound heals to 14 days, wherein the rising amplitude of the hydrogel group loaded with PMSC is maximum, which is higher than that of the control group, the DACG group, the DA7CG group and the PMSC group. The increased expression of TGF-beta 3 helps the wound reduce scar tissue formation during remodeling.

Example 2 preparation of hydrogel da7cg@v

DA7CG was prepared by the method described in example 1. The UCMSC cell-derived cell vesicles were diluted to 100. Mu.g/mL. Subsequently, DA7CG hydrogel was prepared and placed on sterilized absorbent paper for dehydration. The diluted cell vesicle solution was added dropwise to 40. Mu.L of the dehydrated hydrogel, and incubated at 4℃for 4 hours. The unloaded cell vesicles were washed with PBS, followed by ethanol gradient dehydration (50%, 70%, 85%, 95%, 100%) of DA7CG (da7cg@v) after cell vesicle loading, each concentration soaked for 10min. Immediately after dehydration of 100% ethanol, the gel was placed in a supercritical carbon dioxide dryer, and the exosome form inside the macroporous gel was observed using a Scanning Electron Microscope (SEM) (fig. 18). The spherical exosomes within the DA7CG hydrogel can be seen, demonstrating that the DA7CG hydrogel can successfully load cell vesicles.

Da7cg@v contributes to fibroblast migration

DACG, DA7CG, DACG@V (loading method is the same as DA7CG@V) and DA7CG@V were immersed in 1mL serum-free DMEM medium for 24 hours. At the same time, fibroblast (NIH 3T 3) was cultured at 8X 10 ³ Density of wells/wells were seeded in 96-well plates and incubated in a 37 ℃ cell incubator for 24 hours. After washing the cells with PBS, uniform scratches were scraped in each well using a 96-well plate scratcher. And 100. Mu.L of the above hydrogel sample soak solution was added to each well. The control group was added with 100. Mu.L of serum-free medium alone. Using Livecyte single cells The tracer photographs the scored wounds after 0, 6, 12 and 24 hours of treatment (fig. 19A). From the change in scratch area in the photographs taken, it can be seen that the exosome-loaded hydrogels release exosomes into the medium and promote fibroblast migration.

In addition to the cell scratch experiments, cell migration capacity was also verified using Transwell, DACG, DA7CG, dacg@v and da7cg@v were immersed in 1mL serum-free DMEM medium for 24 hours. At the same time, fibroblast (NIH 3T 3) was cultured at 10 ⁵ Density of wells/Wells were inoculated in a 6-well plate Transwell upper chamber, cultured in a 37℃cell incubator for 16 hours, and 100. Mu.L of the above-mentioned hydrogel sample soak solution and 2mL of serum-free DMEM medium were added to the lower chamber. After further culturing for 6 hours, cells in the upper chamber are wiped off by using cotton balls, cells in the lower chamber are fixed, crystal violet staining is carried out, and finally, photographing is carried out by using an optical microscope. The results showed (fig. 19B) that more cells migrated to the back of the Transwell in the UCMSCV-containing hydrogel-dosed group, demonstrating their ability to promote fibroblast migration.

Da7cg@v in vitro angiogenesis

HUVECs were co-cultured with UCMSCV containing hydrogels (DACG@V, DA7CG@V) and non-cell loaded blank hydrogels (DA 7CG and DACG) in 12 well plates for 24h. The matrigel was thawed overnight at 4 ℃ and the vials were rotated after thawing to ensure uniform matrigel mixing. In addition, the gun tip used in the experiment and the EP tube for dispensing matrigel are pre-cooled in advance. To avoid repeated freeze thawing, matrigel was dispensed into 1.5mL ep tubes. And added to 96-well plates, 100 μl/well. The 96-well plate was placed at 37℃with 5% CO ₂ The matrigel was allowed to solidify for 40min in the incubator. Co-cultured HUVECs were counted after digestion and plated onto 96-well plates coated with matrix gel (1.5X10) ⁴ Individual cells/well). The group without gel treatment served as a control. Incubation was continued for 6 hours at 37 ℃. Blood vessel images in 96-well plates were acquired after 6 hours using an inverted microscope. Branch point and capillary length were calculated using ImageJ software. From the statistics (fig. 19C) it was seen that there was more vascular grid generation in the UCMSCV-loaded hydrogel group (dacg@v, da7cg@v), where the number of angiogenesis in the da7cg@v group was greater, without UCMSCV loadingThere was almost no angiogenesis in the hydrogel group (DACG, DA7 CG), similar to the case of the control group. This demonstrates that the UCMSCV-loaded hydrogels have a significant in vitro pro-angiogenic effect on HUVEC cells.

Da7cg@v in vitro contributes to fibroblast proliferation

DACG, DA7CG, DACG@V and DA7CG@V were immersed in 1mL serum-free DMEM medium for 24 hours. At the same time, fibroblast (NIH 3T 3) was cultured at 8X 10 ³ Density of wells/wells were seeded in 96-well plates and incubated in a 37 ℃ cell incubator for 24 hours. Subsequently, 100. Mu.L of the above hydrogel sample soak solution was added to each well. The control group was added with 100. Mu.L of serum-free medium alone. Cells were stained with EdU dye and DAPI after 24h incubation, indicating that the cell proliferation capacity was stronger when nuclei were stained red with EdU dye. Changes in cell proliferation potency were demonstrated by the degree of co-localization of EdU fluorescence and DAPI fluorescence in the photographs taken (FIG. 20). It can be seen that there was more EdU fluorescence in the UCMSCV loaded hydrogel group, while the EdU fluorescence was less in the non-UCMSCV loaded hydrogel group (DACG, DA7 CG), similar to the control group. This suggests that the UCMSCV-loaded hydrogel has a significant pro-proliferative effect on fibroblasts.

Burn infection model establishment

Frozen PAO-1 bacteria were picked up, added to sterile BD tubes containing 3mL LB medium, and incubated on a bacterial shaker at 37℃for 12 hours to recover the bacteria. Subsequently, the bacterial liquid is diluted to 1X 10 in gradient ⁵ CFU/mL. Mice were intraperitoneally injected with 80 μl of 10% aqueous chloral solution, after anesthesia, the backs were shaved and dehaired, and the back dehaired areas were sterilized with alcohol cotton balls. A cylindrical iron of 10mm diameter was sterilized by wiping with an alcohol cotton ball, followed by heating to 200℃with a heat gun. Subsequently, the soldering iron was placed on the skin on the right side of the back spine of the mouse, without applying additional gravity, and left for 10s. Finally 20 mu L of 10 ⁵ The PAO-1 bacteria of CFU/mL are dripped into the scalding part, and after the liquid is stopped for 30 seconds, different preparations are given for treatment, and the indwelling needle film is used for wrapping the wound.

Evaluation of in vivo wound healing Effect

30C 57BL/6 mice were randomly divided into 6 groups, namely a control group, a DACG group, a DA7CG group, a DACG@V group and a DA7CG@V group. Molding and administration of different formulations for treatment were performed as described above. The medications were changed every 3 days and photographs were taken of wound healing at days 0, 3, 7, 10, 14 and 17 of treatment. According to the change of the wound area (figure 21), the back wound healing speed of the mice treated by the DA7CG@V is faster, which shows that the DA7CG hydrogel can treat wound tissues through loading mesenchymal stem cells and also can treat wound tissues through loading exosomes, and is a delivery carrier compatible with cell therapy and cell vesicle therapy.

EXAMPLE 3 preparation of hydrogel ADA7CG@C

Preparation of frozen macroporous hydrogels

Sodium alginate 10mg,AAD 50mg,EDC 1g was weighed separately and dissolved in 1mL of ultrapure water each. 1.5mL of alginic acid was mixed with 1. Mu.L of AAD and 6. Mu.L of EDC solution, and then placed in a glass mold having a thickness of 1mm and frozen in a refrigerator at-20℃for 24 hours. Taking out the frozen hydrogel, placing at room temperature, waiting until the temperature is restored to the room temperature, cutting the gel into a round shape with the diameter of 1cm, and cleaning the residual reagent by using purified water to obtain the product, namely the sodium alginate frozen macroporous hydrogel (ACG).

Preparation of macroporous hydrogels containing antimicrobial peptide coatings

DA was weighed and dissolved in Tris-HCl buffer at pH 8.0 to a concentration of 0.5mg/mL. Circular ACG was immersed in DA solution and incubated on a shaker (40 rpm) at room temperature for 4 hours to form dopamine coating modified cryogel (ADACG). Excess dopamine molecules are then removed using purified water and buffered saline. DP7 was weighed and dissolved in PBS to a concentration of 150. Mu.g/mL. DAACG is soaked in DP7 solution, and after shaking table (40 rpm) incubation is carried out for 2 hours at room temperature, excess DP7 is washed off by PBS, thus obtaining the macroporous hydrogel (ADA 7 CG) containing the antibacterial peptide coating.

Cell loading

ADA7CG was sterilized by soaking in 75% ethanol for 40 min in 24-well plates, followed by three washes with PBS. Placenta-derived mesenchymal stem cells were digested and counted at 6×10 ⁶ Density of/mL cells were resuspended, 50. Mu.L of cell suspension was drop-added to the gel and placed at 37℃in 5% CO ₂ Is incubated for 30 minutes in the incubator to allow the cells to penetrate into the gel. Finally, 1mL of DMEM complete medium is added to each well, and the culture is carried out in an incubator.

Cell migration experiments

Preparing sodium alginate frozen macroporous hydrogel, loading dopamine and dopamine antibacterial peptide coated sodium alginate frozen macroporous hydrogel according to the steps, loading stem cells completely, and inoculating the obtained hydrogel ACG@C, ADACG@C and ADACG@C (each gel is inoculated with 3×10) ⁵ Individual cells) and ADA7CG were immersed in 1mL of serum-free DMEM medium for 24 hours. At the same time, fibroblast (NIH 3T 3) was cultured at 8X 10 ³ Density of wells/wells were seeded in 96-well plates and incubated in a 37 ℃ cell incubator for 24 hours. After washing the cells with PBS, uniform scratches were scraped in each well using a 96-well plate scratcher. And 100. Mu.L of the above hydrogel sample soak solution was added to each well. The control group was added with 100. Mu.L of serum-free medium alone. Scratch wounds after 0, 6, 12 and 24 hours of treatment were photographed using a Livecyte tracer (fig. 22). Scratch area was measured with imageJ software. The photographed photo shows that the residual scratch area of the hydrogel group loaded with the mesenchymal stem cells is smaller, which indicates that the sodium alginate hydrogels (ACG@C, ADACG@C and ADACG@C) after being loaded with the mesenchymal stem cells can promote the migration of the fibroblasts.

In vitro bacteriostasis experiment

Frozen pseudomonas aeruginosa, escherichia coli and staphylococcus aureus were picked up and cultured overnight on a bacterial shaker at 37 ℃ in 3mL of LB medium, respectively. The bacteriostatic effect of ADA7CG was then evaluated by microdilution, and after addition of 400 μl of diluted bacterial solutions (p.aeromonas, e.coll and s.aureus) to 24 well plates, three different gels (ACG, ADA CG and ADA7 CG) were added, respectively. The control group is bacterial liquid without gel treatment. After overnight incubation in an incubator at 37 ℃, 100ul of bacterial fluid per well was transferred to a 96-well plate. The viability of the bacteria was measured by reading the OD value of the bacterial liquid at 600nm (fig. 23). The bacterial growth in the hydrogel group without DP7 (ACG, ADACG) was observed to be similar to that in the control group by the microdilution method, while the bacterial activity was significantly reduced in the ADA7CG group with DP7, indicating that ADA7CG was effective in inhibiting bacterial growth.

Claims (14)

1. A hydrogel having an antimicrobial coating attached thereto, characterized by: comprises a macroporous polysaccharide hydrogel prepared from polysaccharide, a polymer of catechol compounds and antibacterial peptide, wherein the polymer and the antibacterial peptide are sequentially attached to the surface of the macroporous polysaccharide hydrogel.

2. The hydrogel of claim 1, characterized in that at least one of the following is satisfied:

1) The polysaccharide is at least one of hyaluronic acid, carboxymethyl cellulose, carboxymethyl chitosan, sodium alginate or pectin;

2) The catechol compound is at least one of tannic acid, dopamine hydrochloride, gallic acid or gallate;

3) The hydrogel of claim 1, wherein: the antibacterial peptide is polypeptide DP7, or a modifier of the polypeptide DP7, or a derivative of the polypeptide DP 7.

3. The hydrogel of claim 2, characterized in that at least one of the following is satisfied:

1) The amino acid sequence of the polypeptide DP7 is shown as SEQ ID No.1

2) The modification mode of the peptide DP7 is at least one of the following:

a. amidation or sulfation modification is carried out on the C-terminal;

b. cholesterol or fatty acid modification is carried out at the N-terminal;

c. intermediate residues are subject to glycosylation or phosphorylation modifications.

4. The hydrogel of claim 3, wherein: the structure of the polypeptide DP7 is as follows:

5. the hydrogel of claim 1, wherein: the hydrogel is also loaded with bioactive substances; further, the bioactive substance is at least one of a cell, a vesicle, a polypeptide, a protein, or a drug.

6. The hydrogel of claim 5, characterized in that at least one of the following is satisfied:

1) The cells are at least one of mesenchymal stem cells, embryonic stem cells, multifunctional stem cells, skeletal muscle myoblasts, bone marrow derived monocytes, fibroblasts, endothelial cells or endothelial progenitor cells;

2) The vesicle is at least one of exosomes, apoptotic bodies, autophagosomes, bacterial extracellular vesicles or cell membrane vesicles;

3) The polypeptide or protein is at least one of a cell growth factor or an immune cell chemokine.

7. The hydrogel of claim 6, wherein: the cell growth factor is VEGF or TGF-beta.

8. The hydrogel of claim 1, wherein: the preparation method of the hydrogel comprises the following steps:

step s1: dissolving polysaccharide, catalyst and cross-linking agent adipic dihydrazide and 1-ethyl- (3-dimethylaminopropyl) carbodiimide in a solvent, standing the mixed solution at-80-0 ℃ for 8-24 hours to obtain macroporous polysaccharide hydrogel; the catalyst is 1-ethyl- (3-dimethylaminopropyl) carbodiimide; the cross-linking agent is adipic dihydrazide;

Step s2: catechol compounds are dissolved in a solvent with pH of 7.0 to 8.5; the macroporous polysaccharide hydrogel obtained in the step s1 is restored to the room temperature, and is incubated in catechol compound solution for 1 to 8 hours at the room temperature; washing with pure water to obtain hydrogel attached with catechol compounds;

step s3: and (3) dissolving the antibacterial peptide in a solvent, soaking the hydrogel obtained in the step (s 2) in the antibacterial peptide solution, incubating for 1-8 hours at room temperature, and cleaning with pure water to obtain the hydrogel attached with the antibacterial coating.

9. The hydrogel of claim 14, characterized in that at least one of the following is satisfied:

1) In step s1, the solvent is at least one of ultrapure water, phosphate buffer solution or physiological saline;

2) In step s2, the solvent is phosphate buffer, citrate buffer, carbonate buffer or Tris buffer;

3) In step s3, the solvent is at least one of ultrapure water, phosphate buffer solution or physiological saline.

10. The hydrogel of claim 8, wherein: in the step s2, the concentration of the catechol compound solution is 0.1-2 mg/mL; further, in step s3, the concentration of the antibacterial peptide solution is 0.01 to 1mg/mL.

11. The hydrogel of claim 8, wherein: the preparation method further comprises one of the following steps:

step s4: freeze-drying the hydrogel obtained in the step s3 for later use;

alternatively, step s5: preparing bioactive substances into a solution, dripping the solution onto the hydrogel obtained in the step s3, or soaking the hydrogel in the solution, and incubating.

12. The hydrogel of claim 11, wherein: the hydrogel treated in the step s4 is dissolved by adding pure water when in use.

13. The hydrogel of claim 11, wherein: in step s5, when the bioactive substance is a cell, the cell is adjusted to 6×10 ⁶ Density per mL, drop onto the hydrogel obtained in step s3, at 37 ℃,5% co ₂ Is incubated in the incubator for 30 minutes to allow the cells to permeate into the gel;

or, in step s5, when the bioactive substance is a vesicle, diluting the vesicle to 100 μg/mL, dehydrating the hydrogel obtained in step s3, dripping the diluted cell vesicle solution on the dehydrated hydrogel, and incubating at 4 ℃ for 4 hours; the unloaded vesicles were washed with PBS.

14. Use of the hydrogel according to any one of claims 1 to 13 for the preparation of a product for the treatment of burns, acute and chronic traumatic infections or skin infections; further, the product is a dressing.