CN112933290B

CN112933290B - Hydrogel and application thereof in preparation of product for treating wound

Info

Publication number: CN112933290B
Application number: CN202110195508.1A
Authority: CN
Inventors: 李速明; 王元斗; 宿烽; 孔韶文; 许青良; 张萍; 邓金辉; 陈星宇
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-02-22
Filing date: 2021-02-22
Publication date: 2022-05-06
Anticipated expiration: 2041-02-22
Also published as: CN112933290A

Abstract

The invention provides a hydrogel which is prepared by combining aldehyde-group hyaluronic acid and carboxymethyl chitosan. The hydrogel prepared by the invention can be used for preparing a dressing for treating or assisting in treating wounds; the dressing is prepared by loading a therapeutic factor for treating or assisting in treating the wound on hydrogel; contains cells, active substances, growth factors, and medicines or nutrients for wound healing. The hydrogel dressing is also loaded with antibacterial peptide, and a specific amino acid sequence of the antibacterial peptide is SEQ ID NO. 1. The hydrogel dressing provided by the invention can be filled or smeared on an affected part for wound treatment. The hydrogel system has the functions of ventilation, moisture retention, wound permeation absorption and the like, and the prepared dressing for treating or assisting wound treatment can promote angiogenesis, improve and recover the anoxic and ischemic conditions of an affected part, regenerate epidermis and promote the healing of wounds and ulcers.

Description

Hydrogel and application thereof in preparation of product for treating wound

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to hydrogel and application thereof in preparation of a product for treating wounds.

Background

Wound healing is an important research content for wound medicine. The healing of any wound can generate non-healing, difficult healing or scar healing and the like due to the difference of individual organisms, and further intervention treatment is needed to promote the healing of the wound or prevent the occurrence of wound healing complications.

With the development of aging degree, bedsores caused by the elderly people lying in bed for a long time become a nursing institution and a clinical treatment difficulty. Due to slow metabolism of the old, poor blood circulation at wound parts, hypoxia, cell factor repair and the like, most of bedsores become chronic wounds which are difficult to heal.

Every year, about 3000 thousands of operations are performed in China, and wound surfaces of patients with surgical incisions, burns and scalds and the like need to be provided with a 'barrier' for continuously absorbing tissue exudates, keeping the wounds dry and preventing wound infection. Depending on individual differences of patients, certain wounds that are not or are difficult to heal must be healed by intervention of growth-promoting cells or factors. The healing sequelae are mostly seen in the population with scar constitution, and the currently used ray therapy cannot fundamentally solve the key problem of the lack of growth factors and regenerative cells.

The above problems of wound healing have become a clinical problem with long disease course, long time of occupying medical resources, high cost, pain of patients and influence the quality of life of patients to different degrees.

For example, one of the most common complications in the development of diabetes is diabetic ulcer, which is a disease of local skin tissue ulceration and necrosis caused by the dryness and ulceration of the skin of a patient due to the increase of sugar content in the blood of the patient, and the disease is mostly generated in a part with poor blood circulation environment, and is common to the feet of the patient, so that the term "diabetic foot" is born. When the disease is serious, the whole foot is necrotized and the amputation is needed, thereby severely limiting the life of the patient and bringing double pains to the spirit and the body of the patient. If effective nursing and treatment can be given, the treatment effect of the patient can be improved, the clinical symptoms of the patient can be relieved, and the life quality of the patient can be improved.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a hydrogel which is applied to preparation of a product for treating wounds. The hydrogel dressing can meet various requirements of wound care and treatment, and has the functions of ventilation, moisture retention and wound penetrating fluid absorption. The hydrogel dressing can retain the loaded components to the maximum extent when loading wound repair cells, bioactive substances such as growth promotion factors and various nutrient substances which can meet the requirements of wound treatment medicines or improve the wound healing rate.

The invention firstly provides a hydrogel, wherein the hydrogel is prepared by combining aldehyde-group hyaluronic acid and carboxymethyl chitosan;

the molar ratio of the carboxymethyl chitosan to the aldehyde-group hyaluronic acid meets the requirement of forming hydrogel through crosslinking;

specifically, the molar ratio of the carboxymethyl chitosan to the aldehyde-modified hyaluronic acid is 2:1 to 8: 1;

further, the aldehyde hyaluronic acid is prepared by adding a hyaluronic acid solution into a sodium periodate solution to react under a dark condition.

The hydrogel prepared by the invention can be used for preparing a dressing for treating or assisting in treating wounds;

in another aspect, the invention also provides a dressing for treating or assisting wound treatment, which is prepared by loading a treatment factor for treating or assisting wound treatment on the hydrogel;

the treatment factor comprises various cells (stem cells or tissue cells), active substances, growth factors and medicaments or nutrient substances (proteins, polypeptides, amino acids and the like) for wound treatment, which are required by wound repair.

As a concrete description of the examples, the repair cells are various stem cells, umbilical cord blood mononuclear cells or tissue cells;

more specifically, the hydrogel dressing is prepared by mixing aldehyde hyaluronic acid with a suspension of umbilical cord blood mononuclear cells and then mixing the mixture with carboxymethyl chitosan at room temperature.

Furthermore, the hydrogel dressing is also loaded with antibacterial peptide,

as a specific description of the examples, one specific amino acid sequence of the antimicrobial peptide is RKRKRFSRGAKLHLIKLAKKALSRGARAK (SEQ ID NO: 1);

the wound is specifically described as "diabetic foot" as an example.

The hydrogel dressing provided by the invention can be filled or smeared on an affected part for wound treatment. The hydrogel system has the functions of ventilation, moisture retention, wound permeation absorption and the like. The prepared composite auxiliary material loaded with the umbilical cord blood mononuclear cells is rich in a plurality of stem cells such as hematopoietic stem cells, neural stem cells, mesenchymal stem cells, endothelial cells and the like, can promote the regeneration of blood vessels, improve and recover the blood flow of affected limbs, treat the ischemia of the affected limbs and regenerate epidermis, thereby promoting the healing of ulcer surfaces.

Drawings

FIG. 1: 1HNMR detection photo picture of aldehyde group of aldehyde hyaluronic acid;

FIG. 2: a photograph of a freeze-dried aldehyde-modified hyaluronic acid;

FIG. 3: a photographic image of a freeze-dried hydrogel of the invention;

FIG. 4: a rheological performance diagram at a NOCC/A-HA volume ratio of 2: 1;

FIG. 5: a rheological performance diagram at a NOCC/A-HA volume ratio of 3: 1;

FIG. 6: a rheological performance diagram at a NOCC/A-HA volume ratio of 4: 1;

FIG. 7: a rheological performance diagram at a NOCC/A-HA volume ratio of 6: 1;

FIG. 8: a rheological performance diagram at a NOCC/A-HA volume ratio of 8: 1;

FIG. 9: figure of injectable hydrogel of the invention:

FIG. 10: the hydrogel self-healing picture of the invention:

FIG. 11: photo picture of activity detection of single nucleus cells of umbilical cord blood in hydrogel dressing.

Detailed Description

In the research of the applicant, the hydrogel prepared by aldehydized hyaluronic acid (A-HA) and carboxymethyl chitosan NOCC HAs self-healing property and injectability, can be used for loading the hydrogel of factors for treating wounds, HAs a three-dimensional network structure, can continuously absorb exudates, and keeps the moist environment of the wounds; the physical barrier function can ensure that the wound is not infected, and the effects of cleaning the wound and promoting healing are achieved. Meanwhile, the material characteristic of the extracellular matrix is imitated, so that the material can load various bioactive substances such as repairing cells, growth promotion factors and the like, various medicines capable of meeting the requirement of wound treatment and various nutrient substances capable of improving the wound healing rate.

The invention provides a dressing for treating or assisting wound treatment, which is characterized in that a treatment factor for treating or assisting wound treatment is loaded on the hydrogel; the treatment factor comprises wound repair cells, active substances, growth factors and medicaments or nutrient substances for wound treatment.

The classification of growth factors is as follows:

in tissue culture, a substance that promotes cell growth in place of a serum high-molecular substance in a medium may be used in addition to normal components such as amino acids, vitamins, glucose, and inorganic salts. The term "proliferation factor" is synonymous with development factor and proliferation factor, but is defined as a specific proliferation factor since it is a direct purpose of cell proliferation in tissue culture.

The growth factors are generally peptide hormones in a broad sense, and include insulin, Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), platelet-derived growth factor (PDGF), growth hormone release inhibitory factor (SRIH), and the like.

The generalized growth factor comprises basic group, porphyrin and derivatives thereof, sterol, amine, branched or straight chain fatty acid of C4-C6 besides vitamin medical | education net finishing; whereas growth factors in the narrow sense are generally only vitamins.

In addition to peptide hormones, cortisol and thyroxine (T3) and the like are also growth factors. Among these substances, substances other than the known hormones have been found as growth factors for cultured cells, but are also presumed to be growth factors for living bodies. Therefore, tissue culture is considered to be a good experimental means for searching for a factor for developing a living body.

② the growth factor is a cytokine with activity of stimulating cell growth.

The wound repair cells include adult stem cells, and generally include bone marrow stem cells, umbilical cord blood stem cells, and peripheral blood stem cells. They exist in tissues specific to an adult, and have the ability to form progenitor cells from stem progenitor cells and differentiate into cells having specific functions. For example: bone marrow stem cells, hematopoietic stem cells, neural stem cells.

The cord blood mononuclear cells used in the embodiment of the invention are a cell population containing multiple cell subsets after the cord blood is centrifuged to remove erythrocytes and granulocytes, are rich in hematopoietic stem cells, neural stem cells, mesenchymal stem cells, endothelial cells and other multiple stem cells, can promote the regeneration of blood vessels, improve and recover the blood flow of affected limbs, treat ischemia of the affected limbs and regenerate epidermis, thereby promoting the healing of ulcer surfaces and avoiding the pain of amputation of diabetic foot patients.

The hydrogel medical dressing loaded with umbilical cord blood mononuclear cells and suitable for treating diabetic foot comprises hydrogel and umbilical cord blood mononuclear cells, wherein the dressing is prepared by modifying natural high-molecular polysaccharide material hyaluronic acid to generate aldehyde-based hyaluronic acid, dissolving the aldehyde-based hyaluronic acid and carboxymethyl chitosan in normal saline, and mixing the aldehyde-based hyaluronic acid solution and umbilical cord blood mononuclear cell suspension at room temperature.

Further, the aldehyde-treated hyaluronic acid is prepared by dissolving hyaluronic acid in distilled water at a ratio of 1: adding sodium periodate solution with a molar ratio of 1, reacting for 24 hours at room temperature in the dark, adding ethylene glycol to quench the unreacted sodium periodate, reacting for 1 hour at room temperature, subjecting the obtained solution to complete dialysis (MWCO 10000) by distilled water for 3 days to purify the solution, and freeze-drying to obtain a dried product.

The present invention will be described in detail below with reference to examples and the accompanying drawings.

Example 1: synthesis of aldehyde-based hyaluronic acid (A-HA)

Sodium hyaluronate (1.0g,2.5mmol) was dissolved in 100mL of double distilled water at a concentration of 10 mg/mL. When the HA was completely dissolved, aqueous sodium periodate (2.5mmol,5mL) was added and the reaction was allowed to react for 24h at room temperature in the dark. 1mL of ethylene glycol was added to quench the unreacted sodium periodate. The reaction was stirred at room temperature for 1 hour, and the resulting solution was subjected to complete dialysis (MWCO 10000) with distilled water for 3 days to purify the solution, with water being changed at least three times a day during dialysis. The dried product was obtained by freeze-drying. 1HNMR experiments were used to confirm the formation of aldehyde groups-new chemical shifts at 4.90, 5.00 and 5.09ppm indicated the presence of aldehyde groups. The results in FIG. 1 show that an aldehyde-based hyaluronic acid has been made. FIG. 2 is a photograph of a freeze-dried aldehyde-modified hyaluronic acid.

Example 2: preparation of hydrogel for Loading cord blood mononuclear cells

Carboxymethyl chitosan NOCC and aldehyde-modified hyaluronic acid a-HA were dissolved in Phosphate Buffered Saline (PBS) at a concentration of 30mg/mL, respectively. NOCC has excellent hydrophilicity, and is prevented from aggregating into clusters in the powder dissolving process to influence the overall concentration of the solution, and bubbles are prevented from being generated in the dissolving process as much as possible. By mixing the NOCC and A-HA solutions in the molar ratios of 2:1, 3:1, 4:1, 6:1 and 8:1, the generation of bubbles is avoided during stirring, and the compactness of the hydrogel is influenced. Slow stirring promotes its in situ crosslinking to form a hydrogel. FIG. 3 is a photograph of a lyophilized hydrogel.

The rheological properties of hydrogels are an important property of interest for hydrogels as biomedical materials. The gelation time of the NOCC/a-HA hydrogel was monitored using a rheometer at room temperature, and the storage modulus (G') and loss modulus (G ") were measured as a function of time by mixing the NOCC and a-HA aqueous solutions in situ on the rheometer plate. For all samples, at the beginning of the experiment, the storage modulus was lower than the loss modulus (G ' < G "), indicating that the starting mixture was liquid, and that both G ' and G" began to increase after the sensing time, with G ' increasing much faster than G ". The cross-over point between G' and G ", i.e. the gel point, was detected, indicating a solution-gel transition. As shown in fig. 4. The Gel time decreased from 98s for Gel2-1 to 58s for Gel4-1 and then increased to 177s for Ge 8-1. This occurs because gelation occurs through cross-linking via imine bond formation and therefore depends on the ratio between the amino groups of the NOCC and the aldehyde groups of the a-HA. Initially, crosslinking is incomplete and thus gelation is slow. The gelling was gradually improved with increasing proportion of N0 CC. However, as the NOCC/a-HA ratio was further increased to 6:1 to 8:1, gelation became longer due to fewer aldehyde groups available for imine bond formation. As shown in fig. 4-8.

The prepared hydrogel has the advantages of transparent color, certain viscosity, high fluidity, good permeability, stable state, injectability and self-healing property, and is not easy to degrade in water. The hydrogel prepared was continuously injected through a 26G needle and maintained its integrity after injection (fig. 9).

In order to evaluate its self-healing performance. 2 samples of NOCC/A-HA hydrogel of different colors were cut into two semicircular pieces. After contacting for 3-5 minutes at room temperature, the two pieces of hydrogel can be fused into a whole again; the integrated hydrogel was removed and could support its own weight (fig. 10).

Example 4 preparation of hydrogel dressing for loading umbilical cord blood mononuclear cells

And centrifuging the cord blood sample by a gradient density method within 8h after collection to obtain mononuclear cells for trypan blue counting.

Adding cord blood mononuclear cells into PBS containing 1% bovine serum albumin, adding PE-labeled mouse anti-human CD34 monoclonal antibody, labeling at 4 deg.C for 20min, washing with PBS for 1 time, detecting with cytomics (TM) FC500 flow cytometer, and diluting until the concentration of cord blood mononuclear cells is 1 × 10⁷-1×10⁸one/mL.

Mixing the diluted cord blood mononuclear cell suspension with 10-40mg/mL carboxymethyl chitosan solution to make the concentration of cord blood mononuclear cells in the mixed solution be 1X 10⁶-1×10⁷And adding aldehyde hyaluronic acid into the mixture per mL, and mixing and gelling the mixture at room temperature. Wherein the molar ratio of the carboxymethyl chitosan to the aldehyde-based hyaluronic acid is 2: 1-8: 1.

The three-dimensional network structure of the hydrogel can provide a large surface area for drug loading and the like, a three-dimensional space structure for the cell scaffold material, and a large pore size can provide a channel for transporting nutrient and metabolic waste for cells.

The survival rate of the cell load in the hydrogel is observed by using a live-dead cell staining method, and the result shows that the survival rate of the umbilical cord blood mononuclear cells is not obviously reduced after 24 hours after the prepared hydrogel dressing is prepared. Therefore, the hydrogel dressing prepared by the invention can be used for treating the wound, the hydrogel dressing is placed on the wound, and the wound is treated by umbilical cord blood mononuclear cells. The use effect shows that the hydrogel dressing loaded with the umbilical cord blood mononuclear cells, prepared by the invention, can effectively treat chronic wounds which are difficult to heal, such as 'diabetic feet' and the like.

The prepared hydrogel can also be used for loading antibacterial peptides, so that the prepared hydrogel loaded with the antibacterial peptides has an antibacterial function.

One of the antibacterial peptides is isolated from fish egg mucin, has an amino acid sequence of RKRKRFSRGAKLHLIKLAKKALSRGARAK (SEQ ID NO:1) and is named AP 22.

The antibacterial peptide AP22 with the amino acid sequence of SEQ ID NO. 1 is obtained by screening from amphioxus, and the polypeptide is synthesized by commercial companies, the purity of the synthesized polypeptide is more than 95 percent, and the polypeptide can be used for detecting the biological activity.

Detecting the antibacterial property of the AP22 polypeptide by using a sterilized MH broth culture medium containing TTC (triphenyltetrazolium chloride) and having a pH of 7.0, wherein the detected bacteria comprise Escherichia coli O1 strain, Escherichia coli O2 strain and Edwardsiella tarda of gram-negative bacteria; staphylococcus aureus, a gram-positive bacterium.

The result shows that according to the growth characteristics of the bacteria in the detection hole and the control hole, comparison and judgment are carried out, and the lowest antibacterial peptide concentration without visible bacteria growth and the concentration of penicillin or streptomycin are used as the MIC of the test bacteria. In order to clearly show the result, 5 mu l of 0.5 percent triphenyltetrazolium chloride (TTC) can be added into each hole, and the mixture is incubated for 1-3h at 37 ℃, so that the red bacteria grower can be in red color, the color of the bacteria grower can not be changed, and the judgment of the test result is facilitated.

Wherein the MIC determination criteria are as follows:

the highest dilution that significantly inhibited bacterial growth compared to the control well was the end point, and the content of the antibacterial agent in this well was the MIC of the antibacterial agent against a certain bacterium.

In the case of bacterial production, TTC will appear red; if no bacteria are growing, TTC does not develop color. The results of determining the Minimum Inhibitory Concentration (MIC) of the AP22 polypeptide for the strains are shown in table 1.

Table 1: minimum inhibitory concentration value MIC (μ g/mL) of AP22 polypeptide

The results show that the AP22 polypeptide screened by the invention has bacteriostatic effect on gram-negative bacteria and gram-positive bacteria, belongs to a broad-spectrum antibacterial peptide, and has an optimal pH value of 5.6-7.4.

Scanning electron microscopy of escherichia coli treated by the antimicrobial peptide AP22 showed that the morphological structure of the bacteria was changed, and the surface of the bacteria was rough, wrinkled and severely deformed (fig. 2). The result shows that the antibacterial peptide AP22 can damage the cell wall of the Escherichia coli, thin the cell wall and even damage the cell wall, and lose the integrity.

The survival rate of cells under the action of the antibacterial peptide AP22 is measured by an MTT colorimetric method by using a RAW264.7 mouse macrophage cell line as a research object, so that whether the antibacterial peptide AP22 has a toxic effect or not is detected, and an experimental result shows that the antibacterial peptide AP22 has no obvious toxic and damage effects on RAW264.7 mouse macrophages under the condition that the concentration is 5-10 mu g/ml (table 2), and shows that the antibacterial peptide AP22 has no obvious toxicity on normal tissue cells of mammals.

Table 2: table showing the effect of different concentrations of the antimicrobial peptide AP22 on the survival rate of macrophages of RAW264.7 mice

The hemolytic effect of the antimicrobial peptide AP22 on mammalian cells was examined using human red blood cells as a subject. The experimental results show that the antimicrobial peptide AP22 still does not have significant hemolytic effect at concentrations as high as 25 μ M. Thus, the antimicrobial peptide AP22 was loaded in combination with cord blood mononuclear cells onto the hydrogel prepared in example 2 to prepare a complex for treatment of wounds. So that the prepared compound has the effect of treating wounds and can effectively inhibit bacterial infection.

It will be obvious to those skilled in the art that many simple derivations or substitutions can be made without inventive effort without departing from the inventive concept. Therefore, simple modifications to the present invention by those skilled in the art according to the present disclosure should be within the scope of the present invention. The above examples are preferred embodiments of the present invention, and all similar processes and equivalent variations to those of the present invention are intended to fall within the scope of the present invention.

Sequence listing

<110> honeycomb type honeycomb briquet

<120> hydrogel and application thereof in preparation of wound treatment product

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 29

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Arg Lys Arg Lys Arg Phe Ser Arg Gly Ala Lys Leu His Leu Ile Lys

1 5 10 15

Leu Ala Lys Lys Ala Leu Ser Arg Gly Ala Arg Ala Lys

20 25

Claims

1. A dressing for treating or assisting in treating a wound, wherein the dressing is formed by loading a treatment factor for treating or assisting in treating the wound on a hydrogel; the dressing is also loaded with antibacterial peptide, and the amino acid sequence of the antibacterial peptide is SEQ ID NO. 1; the hydrogel is prepared by combining aldehyde-group hyaluronic acid and carboxymethyl chitosan.

2. The dressing of claim 1, wherein the molar ratio of carboxymethyl chitosan to aldehyde-modified hyaluronic acid is 2:1 to 8: 1.

3. The dressing of claim 1 wherein said therapeutic factor comprises cells, active substances, wound treatment drugs or nutrients required for wound repair.

4. A dressing for use in the treatment or adjunct treatment of a wound according to claim 3, wherein said cells required for wound repair are various stem cells, cord blood mononuclear cells and tissue cells.