CN114015078B

CN114015078B - PH self-adjusting double-response hydrogel and synthetic method and application thereof

Info

Publication number: CN114015078B
Application number: CN202111498977.7A
Authority: CN
Inventors: 赵玲玲; 张佳颖; 崔明占; 陈凤娇; 戴范佳; 张聃; 梁洪泽
Original assignee: Ningbo University
Current assignee: Ningbo University
Priority date: 2021-12-09
Filing date: 2021-12-09
Publication date: 2023-05-12
Anticipated expiration: 2041-12-09
Also published as: CN114015078A

Abstract

The invention discloses a pH self-adjusting double-response hydrogel and a synthesis method and application thereof, and belongs to the technical field of preparation of diabetes wound dressing. The pH self-adjusting double-response hydrogel comprises the following raw materials: the preparation method comprises the following steps of high polymer materials containing amino and hydroxyl functional groups, high polymer materials containing aldehyde groups and hydroxyl functional groups, nano materials with hydroxyl functional groups on the surfaces, glucose oxidase and medicines. The pH self-regulating double-response hydrogel prepared by the invention has the pH self-regulating capability, can reduce the environmental pH from physiological pH (7.4) and maintain the environmental pH in a weak acid range with antibacterial activity, reduces blood glucose level and ROS (reactive oxygen species) concentration, and promotes the healing of chronic wounds; meanwhile, the medicine and cells are encapsulated, the medicine is continuously and controllably released, and the cell growth state is good, so that the hydrogel is an ideal target hydrogel.

Description

PH self-adjusting double-response hydrogel and synthetic method and application thereof

Technical Field

The invention relates to the field of biomedical materials, in particular to a pH self-adjusting double-response hydrogel, and a synthesis method and application thereof.

Background

Type 1 and type 2 diabetic ulcers are one of the most troublesome complications in clinic, and have become a medical, social and economic problem to be solved worldwide. Normally, wound healing can be generally divided into several partially overlapping phases, including hemostasis, inflammation, hyperplasia, and tissue remodeling. Acute wounds typically regularly undergo these several phases, eventually healing within weeks. The healing process of diabetic ulcers is more complex. Because of the high blood sugar in the patient, the nervous lesion and peripheral vascular disease can be caused, the healing process is blocked at a certain stage, and a chronic wound is formed. As tissue wounds persist, a large number of inflammatory cells rush into the wound, increasing Reactive Oxygen Species (ROS) and protease concentrations at the wound site, destroying the extracellular matrix (ECM) and degrading growth factors, and proteolysis of the ECM in turn recruits more inflammatory cells to the wound site, forming a vicious circle, making healing of the wound very difficult. Once a diabetic ulcer is formed, the risk of exacerbation of the wound increases and eventually leads to amputation, with lower survival rates due to its susceptibility to recurrent attacks. The data show that over 25% of diabetics will eventually receive amputation, with over 70% of diabetic amputees dying within 5 years.

Wound dressings or skin grafts are commonly used clinically to intervene in the healing of chronic wounds such as diabetic ulcers, which is also the most successful strategy at present. The wound dressing can provide temporary artificial matrix at the wound surface, can also load medicines, growth factors or cells and the like, acts on different stages of wound healing and promotes the wound healing. Researchers have been working on developing a wide variety of wound dressings for many years, with hydrogels being considered a promising and competitive wound dressing due to their nature similar to the natural extracellular matrix (ECM). Currently, the strategy commonly adopted in the literature is to combine various active substances (including growth factors, drugs, cells, bioactive materials, etc.) with hydrogels to form functional dressings. The research points are generally focused on the migration and proliferation of active substances, the regeneration of blood vessels and tissues and the influence of the active substances on wound healing, and the regulation of dressing on the microenvironment of the wound and the influence of dressing on the wound healing are rarely reported.

Research shows that parameters such as humidity, pH value, ROS level, biomolecule concentration, oxygen tension and the like of a wound microenvironment directly influence cell proliferation and tissue repair in the wound healing process, and are critical to wound healing. The pH has a significant impact on many physiological processes such as inflammatory response, collagen formation and angiogenesis. The pH value of the acute wound is generally about 4-6, and the acute wound has neutrophil activity to prevent bacterial colonization, while the pH value of the chronic wound is alkaline and is between 7 and 9, and the acute wound is easy to be infected by bacteria and is hindered from closing. Therefore, maintaining good physiological microenvironment of the wound, such as pH value, blood sugar level, ROS concentration and the like, has very important promotion effect on the healing of the diabetic wounds.

Disclosure of Invention

The invention aims to provide a pH self-adjusting double-response hydrogel, a synthesis method and application thereof, and the hydrogel with excellent performance is prepared by taking a polymer material containing amino and hydroxyl functional groups, a polymer material containing aldehyde and hydroxyl functional groups, a nano material with hydroxyl functional groups on the surface, glucose oxidase and a drug as raw materials.

In order to achieve the above object, the present invention provides the following solutions:

one of the technical schemes of the invention is as follows: a pH self-adjusting dual-response hydrogel comprising the following raw materials: a polymer material containing amino and hydroxyl functional groups, a polymer material containing aldehyde groups and hydroxyl functional groups, a nanomaterial with hydroxyl functional groups on the surface, glucose Oxidase (GOX) and a drug.

Still further, the feedstock also includes L929 cells.

Further, the polymer material containing amino and hydroxyl functional groups is a natural or synthetic polymer material; the polymer material containing aldehyde group and hydroxyl functional group is natural or synthetic polymer material; the nano material with the hydroxyl functional groups on the surface is an inorganic or organic nano material; the inorganic or organic nanomaterial is a nanofiber or nanotube.

Further, the polymer material containing amino and hydroxyl functional groups comprises any one of polyamino acid, chitosan (CS) and derivatives thereof.

Further, the polymer material containing aldehyde groups and hydroxyl functional groups comprises any one of oxidized dextran and Oligomeric Procyanidins (OPC).

The oligomeric procyanidine can be used as a cross-linking agent and an antioxidant at the same time, when the pH of the environment is reduced, schiff base with a chemical cross-linking effect breaks, the hydrogel structure is changed, the encapsulated medicine insulin is released, the oligomeric procyanidine used as the cross-linking agent is also released, and ROS and hydrogen peroxide generated when glucose oxidase oxidizes glucose at the wound surface can be removed.

Still further, the drug in the drug solution comprises one of a pro-inflammatory factor, a hypoglycemic agent, an antioxidant, an anti-inflammatory agent, or a cell growth factor.

Furthermore, the pH self-adjusting double-response hydrogel takes a macromolecule containing amino groups, aldehyde groups and hydroxyl groups and nano fibers/tubes as basic raw materials, glucose oxidase is introduced into the system, schiff base and hydrogen bonds are utilized for in-situ crosslinking to form the pH/glucose double-response hydrogel, and simultaneously medicines (such as hypoglycemic medicine insulin or antioxidant and the like) and cells can be respectively loaded in the network structures of the nano fibers/nano tubes and the hydrogel. The molecular weight of the polymer, the proportion of functional groups (amino, aldehyde and hydroxyl) in the polymer and the content of the nanofiber/tube can be regulated, so that hydrogels with different three-dimensional structures and performances can be prepared. The structure of the pH self-adjusting double-response hydrogel is shown as a formula (1):

wherein: a is a polymer material containing amino and hydroxyl functional groups; b is a polymer material containing aldehyde groups and hydroxyl groups; c is a nano material with hydroxyl functional groups on the surface, and can load medicines; d is an imine bond (also called Schiff base) generated by the reaction of amino and aldehyde groups, and has pH responsiveness; e is glucose oxidase, can be connected to a or b through chemical bonds (such as disulfide bonds, ester bonds or amide bonds, and the like), and can also be directly coated in a hydrogel network; f is hydrogen bond, which is formed by hydroxyl on the surface of the nanofiber/nanotube and amino or hydroxyl on the polymer.

The pH self-adjusting double-response hydrogel has a continuous through micropore structure, and the pore diameter and the modulus of the hydrogel are adjustable; the injection has the advantages of pH self-regulating capability, injectability and self-healing capability, can encapsulate drug molecules and living cells at the same time, and can continuously and controllably release drug molecules.

The second technical scheme of the invention is as follows: the preparation method of the pH self-adjusting double-response hydrogel comprises the following steps: and uniformly mixing the high polymer material containing amino and hydroxyl functional groups, the high polymer material containing aldehyde and hydroxyl functional groups, the nano material with hydroxyl functional groups on the surface, glucose oxidase and the medicine, and standing to obtain the pH self-regulating double-response hydrogel.

Further, the standing time is 1 to 30 minutes.

Further, the preparation method of the pH self-regulating double-response hydrogel further comprises the step of adding L929 cells in the preparation process.

Further, the mixing includes any one of the following modes:

before mixing, adding solvents into the polymer material containing amino and hydroxyl functional groups, the polymer material containing aldehyde and hydroxyl functional groups, the nano material with hydroxyl functional groups on the surface and the drug respectively to prepare the nano material: the preparation method comprises the steps of mixing a polymer material solution containing amino and hydroxyl functional groups, a polymer material solution containing aldehyde and hydroxyl functional groups, a nanomaterial solution with hydroxyl functional groups on the surface and a drug solution; the pH value of the medicine solution is 7-8;

(1) Loading the drug on the nano material with the hydroxyl functional group on the surface to obtain a drug-loaded nano material; dissolving glucose oxidase in a polymer material solution containing amino and hydroxyl functional groups to obtain a solution A, dispersing a drug-loaded nano material in the solution A to obtain a solution B, and uniformly mixing the solution B and the polymer material solution containing aldehyde and hydroxyl functional groups;

(2) Loading the drug on the nano material with the hydroxyl functional group on the surface to obtain a drug-loaded nano material; dissolving glucose oxidase in a polymer material solution containing amino and hydroxyl functional groups to obtain a solution A, dispersing a drug-loaded nano material in a polymer material solution containing aldehyde and hydroxyl functional groups to obtain a solution C, and uniformly mixing the solution A and the solution C;

(3) Loading the drug on the nano material with the hydroxyl functional group on the surface to obtain a drug-loaded nano material; dissolving glucose oxidase in a polymer material solution containing aldehyde groups and hydroxyl functional groups to obtain a solution D, dispersing a drug-loaded nano material in the solution D to obtain a solution E, and uniformly mixing the solution E and the polymer material solution containing amino groups and hydroxyl functional groups;

(4) Loading the drug on the nano material with the hydroxyl functional group on the surface to obtain a drug-loaded nano material; dissolving glucose oxidase in a polymer material solution containing aldehyde groups and hydroxyl functional groups to obtain a solution D, dispersing a drug-loaded nano material in the polymer material solution containing amino groups and hydroxyl functional groups to obtain a solution F, and uniformly mixing the solution D and the solution F;

(5) Grafting glucose oxidase on a polymer material containing amino and hydroxyl functional groups to obtain a substance I, dissolving the substance I, adding a drug solution, uniformly mixing to obtain a solution G, dispersing a nano material with the surface hydroxyl functional groups in a polymer material solution H containing aldehyde groups and hydroxyl functional groups, and uniformly mixing the solution G and the solution H.

Further, the preparation method of the pH self-adjusting double-response hydrogel further comprises the step of modifying the nano material with the surface having the hydroxyl functional groups.

The modification specifically comprises: adding chitosan or polyethylene glycol (PEG) solution into nano material dispersion liquid with hydroxyl functional groups on the surface, and obtaining polymer modified nano material (substance II) after reaction and separation;

still further, the preparation of the pH self-adjusting dual-response hydrogel specifically comprises: dispersing a substance II in a solvent, adding a drug solution, uniformly mixing to obtain a substance III, and uniformly mixing the substance III, glucose oxidase, a polymer material containing amino and hydroxyl functional groups and a polymer material containing aldehyde and hydroxyl functional groups; the concentration of the chitosan solution or the polyethylene glycol (PEG) solution is 0.1-20 mg/mL; further, the concentration of the solution is 1-5 mg/mL.

Further, the preparation of the substance II specifically comprises: adding chitosan or PEG solution into the nano material dispersion liquid with hydroxyl functional groups on the surface, stirring for 12-48 h at 20-60 ℃, centrifuging, and freeze-drying to obtain a substance II. Dispersing the substance II in a solvent, then adding a medicinal solution, vacuumizing and stirring or oscillating for 2-48 h at 20-60 ℃, centrifuging, and freeze-drying to obtain the substance III.

Further, the preparation of the drug-loaded nanomaterial specifically comprises: adding a drug solution into the nano material dispersion liquid with the surface having hydroxyl functional groups, vacuumizing and stirring or oscillating for 2-48 hours at 20-60 ℃, centrifuging, and freeze-drying to obtain the drug-loaded nano material.

Still further, the mixing specifically includes: adding a drug and glucose oxidase into a polymer material solution containing amino and hydroxyl functional groups to obtain a mixed solution M; dispersing the nano material with the surface having the hydroxyl functional group in a polymer material solution containing aldehyde groups and the hydroxyl functional groups, then adding L929 cells to obtain a mixed solution N, and uniformly mixing the mixed solution M and the mixed solution N.

Further, the pH of the polymer material solution containing amino and hydroxyl functional groups and the pH of the polymer solution containing aldehyde and hydroxyl functional groups are 7.4.

Further, the concentration of the polymer material solution containing amino and hydroxyl functional groups is 5-50 mg/mL; the concentration of the polymer material solution containing aldehyde groups and hydroxyl functional groups is 5-50 mg/mL.

Further, the concentration of the polymer material solution containing amino and hydroxyl functional groups is 16-30 mg/mL; the concentration of the polymer material solution containing aldehyde groups and hydroxyl functional groups is 16-30 mg/mL.

Further, the concentration of the nanomaterial dispersion liquid with hydroxyl functional groups on the surface is 1-50 mg/mL.

Further, the concentration of the nanomaterial dispersion liquid with hydroxyl functional groups on the surface is 2-20 mg/mL.

Further, the concentration of the drug solution is 0.1-10 mg/mL.

Further, the concentration of the drug solution is 2-5 mg/mL.

Further, the vacuum pressure of the vacuuming is 1-20 mm Hg.

Further, the vacuum pressure of the vacuuming is 5-10 mm Hg.

Still further, the solvent may include any one of water, PBS solution, ethanol, methanol, acetone, or DMSO.

Further, the molar ratio of the polymer material containing amino and hydroxyl functional groups to the polymer material containing aldehyde and hydroxyl functional groups is 10:1-1:10.

Further, the molar ratio of the polymer material containing amino and hydroxyl functional groups to the polymer material containing aldehyde and hydroxyl functional groups is 5:1-1:1.

Further, the Glucose Oxidase (GOX) is contained in the hydrogel in an amount of 0.5 to 2wt%.

GOX can be fixed in the hydrogel through chemical bonds so as to avoid enzyme loss in the use process of the hydrogel; the chemical bond is an ester bond, an amide bond, an imine bond or a disulfide bond.

Still further, the chemical bond is a disulfide bond.

Further, the preparation of the substance I specifically comprises: glucose Oxidase (GOX) was grafted onto a polymer material containing amino and hydroxyl functionalities via disulfide bonds using SPDP (N-hydroxysuccinimide 3- (2-pyridinedimercapto) propionate).

The third technical scheme of the invention: an application of the pH self-adjusting double-response hydrogel in preparing a diabetic wound repair product.

Under the condition of high blood sugar level, glucose oxidase oxidizes glucose to generate gluconic acid, which can reduce the glucose concentration at the wound surface and maintain the pH value at the wound in a weak acid range with antibacterial activity; meanwhile, the change of pH value can cause the structural change of hydrogel, trigger the controlled release of drug molecules at the wound surface, reduce blood glucose level and ROS (reactive oxygen species) concentration, promote the healing of chronic wounds; the double-network structure between the polymer and between the polymer and the nanofiber/tube can improve the mechanical strength of the hydrogel and meet the mechanical property requirement of the dressing; dynamic bond in situ crosslinking can impart injectability and self-healing properties to the hydrogel, facilitate filling of irregular tissue wounds, and maintain structural stability of the dressing during wound healing.

The invention discloses the following technical effects:

(1) The pH self-adjusting double-response hydrogel has the pH self-adjusting capability, and can reduce the environmental pH from the physiological pH (7.4) and maintain the environmental pH in a weak acid range (pH 6) with antibacterial activity.

(2) The pH self-regulating double-response hydrogel can simultaneously encapsulate drugs and cells, continuously and controllably release the drugs, and has good cell growth state.

(3) The pH self-adjusting double-response hydrogel can be used as an active dressing for repairing diabetic wounds. On one hand, the pH value of the wound can be maintained in a weak acid range with antibacterial activity, and on the other hand, the controlled release of drug molecules at the wound surface can be triggered, so that the blood glucose level and the ROS (reactive oxygen species) concentration are reduced, and the healing of chronic wounds is promoted.

(4) The pH self-adjusting double-response hydrogel has injectability and self-repairing property.

(5) The preparation method of the pH self-adjusting double-response hydrogel is simple and easy to implement, has mild conditions, and can be used for encapsulating living cells; the microporous structure, modulus, pH responsiveness, regulatory capacity, drug release rate and the like of the hydrogel can be regulated by changing the concentration, proportion and the like of each component, thereby obtaining the required ideal target hydrogel.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a graph showing the procedure from solution to gel for the hydrogel prepared in example 1 of the present invention;

FIG. 2 is a scanning electron microscope image of hydrogels prepared in example 3 and example 4 of the present invention, (A) is example 3, and (B) is example 4;

FIG. 3 is a time-scanning flow chart of hydrogels prepared in examples 3 to 5 of the present invention;

FIG. 4 is a graph showing drug release under various conditions of the hydrogel prepared in example 3 according to the present invention;

FIG. 5 shows the surrounding environment of the hydrogel prepared in example 4 of the present invention (PBS release, pH7.4, initial glucose concentrations of 100 and 400mg dL, respectively) ^-1 ) A graph of glucose concentration versus pH, (a) glucose concentration and (b) pH;

FIG. 6 is a graph showing the growth of L929 cells in the hydrogel prepared in example 2, wherein (A) is 1 day and (B) is 3 days;

FIG. 7 is a graph showing the repair of a wound on the back of a diabetic rat using the hydrogel prepared in example 5 as a wound dressing.

Detailed Description

Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.

In the following examples, HCl or NaOH solutions with a concentration of 2M were used to adjust the pH.

Example 1

Preparation of a glucose oxidase grafted chitosan/oligomeric procyanidine/halloysite nanotube (CS-GOX/OPC/HNTs) composite hydrogel:

(1) Preparation of CS-GOX

Weighing 320mg of CS, dissolving in 320mL of PBS (pH 6.0), stirring at room temperature for dissolving, then dropwise adding 0.19mg of SPDP solution (the solvent is DMSO), and stirring for 1-3 h to obtain a CS solution; 40mg of GOX was dissolved in 40mL of PBS (pH 6.0) to give a GOX solution; adding GOX solution into CS solution, stirring at room temperature for 24h, transferring the reaction solution into a dialysis bag with molecular weight cut-off of 14kDa, dialyzing with deionized water for 3d, changing deionized water every 4h, collecting the reserved solution, and freeze-drying to obtain light yellow product glucose oxidase grafted chitosan (CS-GOX).

(2) Weighing a certain amount of CS-GOX, adding deionized water to prepare CS-GOX solution with the concentration of 35mg/mL, adjusting the pH value to 7.4, adding a certain amount of insulin (the final concentration of insulin is 6 mg/mL), and stirring to fully dissolve the insulin to obtain CS-GOX/insulin solution; weighing a certain amount of OPC, adding deionized water to prepare an OPC solution with the concentration of 40mg/mL, adjusting the pH value to 7.4, then adding a certain amount of halloysite nanotubes (the final concentration of the nanotubes is 10 mg/mL), and stirring to uniformly disperse the halloysite nanotubes to obtain an OPC/halloysite nanotube dispersion; mixing CS-GOX/insulin solution and OPC/halloysite nanotube dispersion liquid in a volume ratio of 1:1, rapidly stirring to fully and uniformly mix the mixture, and standing for about 1min to obtain CS-GOX/OPC/HNTs composite carrier hydrogel (pH self-adjusting double-response hydrogel); the process diagram of the hydrogel changing from solution to gel is shown in fig. 1.

Example 2

Preparation of Chitosan/oxidized dextran/halloysite nanotube (CS/Oxd/HNTs) double-loaded hydrogel

(1) Dissolving 20mg of CS in 1mL of deionized water, stirring at room temperature for dissolution, adjusting the pH value to 7.4 to obtain a CS solution with the concentration of 20mg/mL, adding a certain amount of insulin (the final concentration of insulin is 6 mg/mL) and GOX (the final concentration of GOX is 6 mg/mL) into the CS solution, and stirring to fully dissolve the insulin and the GOX to obtain a mixed solution A.

(2) A certain amount of oxidized dextran (Oxd) is weighed, sterile deionized water is added to prepare Oxd solution with the concentration of 15mg/mL, and the pH value is adjusted to 8.

(3) Weighing a certain amount of halloysite nanotubes, adding the halloysite nanotubes into Oxd solution, stirring to uniformly disperse the halloysite nanotubes, and obtaining mixed solution B, wherein the concentration of the halloysite nanotubes is 10mg/mL.

(4) Collecting L929 cells with good growth state, dispersing with mixed solution B to obtain cell suspension with cell density of 1×10 ⁵ Each mL gave Oxd/halloysite nanotube/L929 cell dispersion.

(5) Mixing the mixed solution A with Oxd/halloysite nanotube/L929 cell dispersion liquid according to the volume ratio of 1:1, rapidly stirring to fully and uniformly mix the mixed solution A and the halloysite nanotube/L929 cell dispersion liquid, and standing for about 5min to form CS/Oxd/HNTs double-loaded hydrogel loaded with the medicine and living cells simultaneously, wherein the growth condition diagram of the L929 cells in the hydrogel is shown in figure 6.

Example 3

(1) 40mg of CS is dissolved in 1mL of deionized water, stirred and dissolved at room temperature, the pH value is adjusted to 7.4, a CS solution with the concentration of 40mg/mL is obtained, and then a certain amount of insulin (the final concentration of insulin is 6 mg/mL) and GOX (the final concentration of GOX is 4 mg/mL) are added into the CS solution, and the CS solution is stirred to be fully dissolved, so that a mixed solution A is obtained.

(2) A certain amount of oxidized dextran (Oxd) is weighed, sterile deionized water is added to prepare Oxd solution with the concentration of 40mg/mL, and the pH value is adjusted to 8.

(3) Weighing a certain amount of halloysite nanotubes, adding the halloysite nanotubes into Oxd solution, stirring to uniformly disperse the halloysite nanotubes, and obtaining a mixed solution B, wherein the concentration of the halloysite nanotubes is 0.4 mg/mL.

(4) Mixing the mixed solution A and the mixed solution B in a volume ratio of 1:1, rapidly stirring to fully and uniformly mix the mixed solution A and the mixed solution B, standing for about 5min to form hydrogel, wherein a scanning electron microscope diagram of the prepared hydrogel is shown in fig. 2, a time scanning rheological diagram is shown in fig. 3, and a drug release diagram under different conditions is shown in fig. 4.

Example 4

(1) 50mg of CS is dissolved in 1mL of deionized water, stirred and dissolved at room temperature, the pH value is adjusted to 7.4, a CS solution with the concentration of 50mg/mL is obtained, and then a certain amount of insulin (the final concentration of insulin is 6 mg/mL) and GOX (the final concentration of GOX is 4 mg/mL) are added into the CS solution, and the CS solution is stirred to be fully dissolved, so that a mixed solution A is obtained.

(4) Mixing the mixed solution A and the mixed solution B in a volume ratio of 1:1, rapidly stirring to fully and uniformly mix the mixed solution A and the mixed solution B, standing for about 5min to form hydrogel, wherein a scanning electron microscope image of the prepared hydrogel is shown in fig. 2, a time scanning rheological image is shown in fig. 3, and a surrounding environment change image is shown in fig. 5.

Example 5

(1) 25mg of CS is dissolved in 1mL of deionized water, stirred and dissolved at room temperature, the pH value is adjusted to 7.4, a CS solution with the concentration of 25mg/mL is obtained, and then a certain amount of insulin (the final concentration of insulin is 6 mg/mL) and GOX (the final concentration of GOX is 4 mg/mL) are added into the CS solution, and the CS solution is stirred to be fully dissolved, so that a mixed solution A is obtained.

(4) Mixing the mixed solution A and the mixed solution B in a volume ratio of 1:1, rapidly stirring to fully and uniformly mix the mixed solution A and the mixed solution B, standing for about 5min to form hydrogel, wherein a time scanning rheogram of the prepared hydrogel is shown in figure 3, and a repairing condition of the hydrogel serving as a wound dressing on back wounds of diabetic rats is shown in figure 7.

Example 6

(1) Dispersing halloysite nanotubes in PBS (pH 6.0) to obtain a halloysite nanotube dispersion with a concentration of 10 mg/mL; insulin was dissolved in PBS (pH 6.0) to give an insulin solution at a concentration of 6 mg/mL; mixing insulin solution and halloysite nanotube dispersion, vacuumizing at 30 ℃ for 10 hours (the vacuum pressure is 5mm Hg), centrifuging the reaction mixture, washing with deionized water, and freeze-drying to obtain the drug-loaded nano material.

(2) 20mg of CS was dissolved in 1mL of PBS (pH 6.0), and the solution was stirred at room temperature to adjust the pH to 7.4, thereby obtaining a CS solution, and 4mg of GOX was added to the CS solution and stirred to dissolve the CS solution, thereby obtaining a mixed solution.

(3) Dispersing the drug-loaded nano-materials in the mixed solution prepared in the step (2) to obtain a drug-loaded nano-material mixed dispersion liquid with the concentration of 2wt%.

(4) A certain amount of Oxd is weighed, deionized water is added to prepare Oxd solution with the concentration of 15mg/mL, and the pH value is adjusted to 7.4, so as to obtain Oxd solution.

(5) Mixing the drug-loaded nano material mixed dispersion liquid and Oxd solution according to the volume ratio of 1:1, rapidly stirring to fully and uniformly mix the materials, and standing for about 10min to obtain the pH self-adjusting double-response hydrogel.

Example 7

(2) 20mg of CS was dissolved in 1mL of PBS (pH 6.0), and the solution was stirred at room temperature to adjust the pH to 7.4, thereby obtaining a CS solution, and 4mg of GOX was added to the CS solution, and the solution was stirred and dissolved, thereby obtaining a mixed solution A.

(3) A certain amount of Oxd is weighed, deionized water is added to prepare Oxd solution with the concentration of 10mg/mL, the pH value is regulated to 7.4, oxd solution is obtained, and then the drug-carrying nano material is added into Oxd solution, so that the concentration of the drug-carrying nano material is 2wt%, and the mixed solution B is obtained.

(4) Mixing the mixed solution A and the mixed solution B according to the volume ratio of 1:1, rapidly stirring to fully and uniformly mix the mixed solution A and the mixed solution B, and standing for about 10min to obtain the pH self-adjusting double-response hydrogel.

Example 8

(2) Weighing a certain amount of Oxd, adding 1mL of deionized water to prepare an OPC solution with the concentration of 20mg/mL, adjusting the pH value to 7.4 to obtain the OPC solution, adding 4mg of GOX into the CS solution, and stirring for dissolution to obtain a mixed solution.

(4) 20mg of CS was dissolved in 1mL of PBS (pH 6.0), and the solution was stirred at room temperature to adjust the pH to 7.4, thereby obtaining a CS solution.

(5) And mixing the drug-loaded nano material mixed dispersion liquid and the CS solution in a volume ratio of 1:1, rapidly stirring to fully and uniformly mix the materials, and standing for about 10min to obtain the pH self-adjusting double-response hydrogel.

Example 9

(2) Weighing a certain amount of OPC, adding 1mL of deionized water to prepare an OPC solution with the concentration of 20mg/mL, adjusting the pH value to 7.4 to obtain the OPC solution, adding 4mg of GOX into the CS solution, and stirring for dissolution to obtain a mixed solution A.

(3) Dissolving 20mg of CS in 1mL of PBS (pH 6.0), stirring at room temperature for dissolution, adjusting the pH value to 7.4 to obtain CS solution, and adding the drug-loaded nanomaterial into the OPC solution to make the concentration of the drug-loaded nanomaterial be 2wt% to obtain a mixed solution B.

(4) Mixing the mixed solution A and the mixed solution B according to the volume ratio of 1:1, rapidly stirring to fully and uniformly mix the mixed solution A and the mixed solution B, and standing for about 1min to obtain the pH self-adjusting double-response hydrogel.

Example 10

(1) 100mgCS was dissolved in 100mL of deionized water, and the solution was stirred at room temperature to obtain a CS solution.

(2) 100mg of halloysite nanotubes were dispersed in 100mL of deionized water to give a halloysite nanotube dispersion with a concentration of 1 mg/mL.

(3) Adding CS solution into halloysite nanotube dispersion liquid, stirring at 30 ℃ for 24 hours, performing ultrafiltration and centrifugal separation on the mixture, removing supernatant, alternately washing with water and ethanol, finally dispersing in water, and performing freeze-drying to obtain CS modified halloysite nanotubes.

(4) 100mg of CS-modified halloysite nanotubes were dispersed in 10ml of PBS (pH 6.0), and the dispersion was homogenized by sonication to obtain a CS-modified halloysite nanotube dispersion having a concentration of 10mg/ml.

(5) 6mg of insulin was dissolved in 1mL of PBS (pH 6.0) to give an insulin solution having a concentration of 6 mg/mL.

(6) Adding insulin solution into halloysite nanotube dispersion liquid, vacuumizing and stirring for 10 hours at 30 ℃, centrifuging the reaction mixture, washing with deionized water, and freeze-drying to obtain CS modified halloysite nanotubes carrying medicine; CS-modified drug-loaded halloysite nanotubes were dispersed in PBS (pH 6.0) to give a CS-modified drug-loaded halloysite nanotube dispersion at a concentration of 10mg/mL.

(7) Weighing a certain amount of Oxd, adding deionized water to prepare Oxd solution with the concentration of 20mg/mL, and adjusting the pH value to 7.4 to obtain Oxd solution; 4mg of GOX was dissolved in 1mL of PBS (pH 6.0) to give a GOX solution; a certain amount of chitosan is weighed and dissolved in 1mL of PBS (pH 6.0), and the solution is stirred and dissolved at room temperature, and the pH value is regulated to 7.4, so as to obtain CS solution.

(8) And mixing CS modified drug-loaded halloysite nanotube dispersion liquid and Oxd solution in an equal volume ratio, rapidly stirring to fully and uniformly mix the CS solution and the GOX solution, and standing for about 10min to obtain the pH self-regulating double-response hydrogel.

Example 11

(1) 150mg of PEG was dissolved in 100mL of deionized water and stirred at room temperature to give a PEG solution.

(3) Adding PEG solution into halloysite nanotube dispersion, stirring at 30deg.C for 24 hr, ultrafiltering, centrifuging, removing supernatant, alternately washing with water and ethanol, dispersing in water, and lyophilizing to obtain PEG-modified halloysite nanotube.

(4) 100mg of PEG-modified halloysite nanotubes were dispersed in 10ml of PBS (pH 6.0) and sonicated to homogenize the dispersion, resulting in a PEG-modified halloysite nanotube dispersion at a concentration of 10mg/ml.

(6) Adding insulin solution into halloysite nanotube dispersion liquid, vacuumizing and stirring for 10 hours at 30 ℃, centrifuging a reaction mixture, washing with deionized water, and freeze-drying to obtain a drug-loaded PEG modified halloysite nanotube; PEG-modified drug-loaded halloysite nanotubes were dispersed in PBS (pH 6.0) to give a PEG-modified drug-loaded halloysite nanotube dispersion at a concentration of 10mg/mL.

(8) And mixing the PEG modified drug-loaded halloysite nanotube dispersion liquid, oxd solution, CS solution and GOX solution in an equal volume ratio, rapidly stirring to fully and uniformly mix the two solutions, and standing for about 10min to obtain the pH self-regulating double-response hydrogel.

The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.

Claims

1. A pH self-adjusting double-response hydrogel, comprising the following materials: a polymer material containing amino and hydroxyl functional groups, a polymer material containing aldehyde groups and hydroxyl functional groups, a nano material with hydroxyl functional groups on the surface, glucose oxidase and a drug;

the high polymer material containing amino and hydroxyl functional groups comprises any one of polyamino acid, chitosan and derivatives thereof;

the high polymer material containing aldehyde groups and hydroxyl functional groups comprises any one of oxidized dextran and oligomeric procyanidins;

the nano material with the hydroxyl functional groups on the surface is nanofiber or nanotube.

2. A method of preparing a pH self-adjusting dual-response hydrogel according to claim 1, comprising the steps of: and uniformly mixing the high polymer material containing amino and hydroxyl functional groups, the high polymer material containing aldehyde and hydroxyl functional groups, the nano material with hydroxyl functional groups on the surface, glucose oxidase and the medicine, and standing to obtain the pH self-regulating double-response hydrogel.

3. The method of preparing a pH self-adjusting dual response hydrogel according to claim 2, further comprising adding L929 cells during the preparation.

4. The method of preparing a pH self-adjusting dual response hydrogel according to claim 2, wherein the mixing comprises any of the following:

before mixing, adding solvents into the polymer material containing amino and hydroxyl functional groups, the polymer material containing aldehyde and hydroxyl functional groups, the nano material with hydroxyl functional groups on the surface and the drug respectively to prepare the nano material: the preparation method comprises the steps of mixing a polymer material solution containing amino and hydroxyl functional groups, a polymer material solution containing aldehyde and hydroxyl functional groups, a nanomaterial solution with hydroxyl functional groups on the surface and a drug solution;

5. The method for preparing a pH self-adjusting double-response hydrogel according to claim 4, further comprising modifying the nanomaterial with hydroxyl functional groups on the surface.

6. The method for preparing a pH self-adjusting double-response hydrogel according to claim 4, wherein the pH of the polymer material solution containing amino and hydroxyl functional groups and the pH of the polymer solution containing aldehyde and hydroxyl functional groups are each 7.4; the concentration of the polymer material solution containing the amino and hydroxyl functional groups is 5-50 mg/mL; the concentration of the polymer material solution containing aldehyde groups and hydroxyl functional groups is 5-50 mg/mL.

7. The method for preparing the pH self-adjusting double-response hydrogel according to claim 4, wherein the concentration of the drug solution is 0.1-10 mg/mL.

8. Use of the pH self-adjusting dual response hydrogel of claim 1 in the preparation of a diabetic wound repair product.