CN108452370B

CN108452370B - Composite supermolecule hydrogel and preparation method thereof

Info

Publication number: CN108452370B
Application number: CN201810421517.6A
Authority: CN
Inventors: 张翼; 宋茹茹; 王杨; 郑俊; 刘依林
Original assignee: Central South University
Current assignee: Central South University
Priority date: 2018-05-04
Filing date: 2018-05-04
Publication date: 2020-09-11
Anticipated expiration: 2038-05-04
Also published as: CN108452370A

Abstract

The invention discloses a composite type supermolecule hydrogel and a preparation method thereof, belonging to the technical field of supermolecule hydrogels. Chitosan is used as a gel factor, and a freezing-thawing method is adopted to form milky composite supramolecular hydrogel with traditional Chinese medicine monomer cordycepin in a PBS buffer solution. The components of the hydrogel are natural products with good biocompatibility, and the antibacterial performance of the hydrogel is enhanced by the synergistic effect of chitosan and cordycepin. The composite supermolecule hydrogel disclosed by the invention has the performances of self-healing, smearing and the like, and the two components of the composite supermolecule hydrogel not only improve the antibacterial performance of the composite material through synergistic action, but also can reduce the formation of scars of skin wounds, and has a wide application prospect in the fields of antibacterial materials, wound dressings and the like.

Description

Composite supermolecule hydrogel and preparation method thereof

Technical Field

The invention relates to a composite type supermolecule hydrogel and a preparation method thereof, belonging to the technical field of supermolecule hydrogels.

Background

To date, various wound healing materials have been commercialized; however, clinical practice has shown that there is still a new need as they serve only as temporary barriers to infection and fluid loss. Inadequate wound healing is prone to the formation of chronic wounds or to life-threatening complications. Wound healing is a complex and dynamic process divided into predictable phases: blood clotting (hemostasis), inflammation, tissue growth (proliferation) and tissue remodeling (maturation). At each stage, a series of cytokines and growth factors secreted by inflammatory cells play an important role in restoring damaged cellular structures and tissue layers. In this regard, attention has been focused on the use of growth factor-functionalized and/or cell-based skin grafts. Among the cells involved in the wound healing process, macrophages may be responsible for infection, tissue repair and resolution of inflammation. As a host defense, macrophages recognize and destroy foreign organisms, clear dead and damaged tissue components (classical activation), and produce cytokines that regulate tissue growth and remodeling, growth factors, and angiogenic factor activation). Therefore, there is a great need in the art to develop hydrogel wound dressings that are well biocompatible and have no side effects.

Hydrogels (hydrogels) are gels that use water as the dispersion medium. It has good biocompatibility and biodegradability, and is often used in the fields of tissue engineering scaffolds, wound repair, drug delivery and the like. An increasing number of researchers are currently concerned with their use as wound dressings. The hydrogel used as wound dressing has effects of absorbing wound exudate, sterilizing, moisturizing, and promoting wound healing. The design of the supermolecule hydrogel composite material with ideal antibacterial property and biocompatibility has very important significance for developing novel wound dressings.

Disclosure of Invention

In order to solve the technical problems, the invention provides the composite type supermolecule hydrogel with good antibacterial property and the preparation method thereof.

In order to achieve the purpose, the technical scheme of the invention is as follows: a composite supramolecular hydrogel comprises a cordycepin solution, a chitosan solution and a PBS buffer solution.

As a preferable scheme of the invention, the concentration of the cordycepin solution is 100 mg/mL.

In a preferable embodiment of the present invention, the solvent of the chitosan solution is 1% HAc, and the concentration of the chitosan solution is 20-40 mg/L.

In a preferable embodiment of the present invention, the solvent of the PBS buffer solution is ultrapure water, the PH of the PBS buffer solution is 8.3 to 8.5, and the concentration of each phosphate used is 0.1 mol/L.

As a preferred embodiment of the present invention, the composite supramolecular hydrogel is applied to an antibacterial agent or/and a wound dressing.

A preparation method of a composite type supermolecule hydrogel comprises the following steps:

step 1, preparing a cordycepin solution; dissolving the cordycepin solid powder with dimethyl sulfoxide, and performing ultrasonic dispersion to obtain a cordycepin solution;

step 2, preparing a chitosan solution; dissolving chitosan with 1% HAc, and magnetically stirring to accelerate the dissolution of chitosan to prepare a chitosan solution;

step 3, preparing a PBS buffer solution; with Na₂HPO₄And NaH₂PO₄The solution is prepared into PBS buffer solutions with different Ph;

and 4, uniformly mixing the cordycepin solution, the chitosan solution and the PBS buffer solution, putting the mixture into a refrigerator at the temperature of 20 ℃ below zero, freezing the mixture till the mixture is completely frozen, and then putting the frozen mixture at room temperature for thawing to form the composite supramolecular hydrogel.

As an optimized scheme for the preparation method, in the step 1, the time of ultrasonic dispersion of the solid cordycepin powder dissolved in dimethyl sulfoxide is 5-10 min, the concentration of the obtained cordycepin solution is 100mg/mL, and light-shielding treatment is carried out in the process of preparing the cordycepin solution.

As an optimized scheme for the preparation method, in the step 2, the concentration of the chitosan solution is 20-40 mg/ml; in the step 3, the Ph of the PBS buffer solution is 8.3-8.5, and the concentrations of Na2HPO4 and NaH2PO4 in the PBS buffer solution are both 0.1 mol/L.

Taking 80-100 mu L of the cordycepin solution, adding the cordycepin solution into a cleaned and dried 2.0mL light-shielding treated screw bottle, quickly adding 400-420 mu L of the PBS buffer solution, quickly shaking up, adding 500 mu L of the chitosan solution, preparing 1.0mL mixed solution, and carrying out the preparation process of the mixed solution under magnetic stirring; and after the mixed solution is uniformly mixed, putting the screw bottle into a refrigerator at the temperature of-20 ℃ for freezing until the mixed solution is completely frozen, and then putting the bottle at room temperature for thawing to form the composite supramolecular hydrogel.

Through the technical scheme, the technical scheme of the invention has the beneficial effects that: the hydrogel factor used in the invention is chitosan, which is obtained by non-covalent bond self-assembly, and the chitosan hydrogel is in a spongy structure.

Cordycepin is the first nucleoside antibiotic separated from fungus and has molecular formula of C₁₀H₁₃N₅O₃. The antibacterial has an important function in the wound healing process, and the cordycepin has a broad-spectrum antibacterial effect and can inhibit the growth of pathogenic bacteria such as bacteria, fungi and the like. In addition, cordycepin also has broad-spectrum biological activities of resisting virus, regulating immunity, eliminating free radicals, resisting tumor, resisting inflammation and the like.

Chitosan (Chitosan) is a hydrophilic natural polymer. The chitosan has good broad-spectrum antibacterial activity and can inhibit the growth and reproduction of fungi, bacteria and viruses. Meanwhile, chitosan has the function of promoting blood coagulation and can be used as a hemostatic. Can also be used for wound healing, has the effects of reducing wound bacterial growth, absorbing wound exudate, being not easy to dehydrate and shrink, and the like, and is an excellent wound healing dressing component. In addition, chitosan has the function of a thickening agent, and can enhance the mechanical strength of the composite material. Chitosan may be one of the functional biomaterials responsible for wound healing. The chitosan has great superiority as a drug sustained-release matrix, can combine the antibacterial advantage of cordycepin, and has synergistic effect in the antibacterial and anti-inflammatory fields.

1) The composite natural product supermolecule hydrogel prepared by the invention has good biocompatibility, is easy to degrade and has no cytotoxicity.

2) The composite natural product supermolecule hydrogel prepared by the invention has proper hardness, elasticity and viscosity, is very suitable for being applied to skin and is self-healing gel.

3) The preparation process of the invention does not add any reducing agent and dispersant, and is green and environment-friendly.

4) The chitosan added in the invention can be cooperated with cordycepin, so that the antibacterial property of the hydrogel is enhanced, the hydrogel is easier to be smeared, and the hydrogel is beneficial to skin absorption and is more suitable for being used as wound dressing.

5) The preparation method is simple, low in cost, capable of being commercialized and suitable for large-scale production.

6) The composite supermolecule hydrogel prepared by the invention has good antibacterial property, can effectively promote wound healing, and has good application prospect in the fields of antibacterial agents, wound dressings and the like.

Drawings

FIG. 1 is a Scanning Electron Microscope (SEM) image of a chitosan hydrogel;

FIG. 2 is a Scanning Electron Microscope (SEM) image of the composite supramolecular hydrogel in example 1 of the invention;

FIG. 3 is a digital photograph of the composite supramolecular hydrogel of example 1 according to the present invention taken before it is formed into a hydrogel;

FIG. 4 is a digital photograph of the composite supramolecular hydrogel of example 1 after being hydrated into hydrogel

FIG. 5 is a digital photograph showing the growth of bacteria after the bacterial liquid in the liquid culture medium No. 1-3 of example 1 is inoculated on a solid culture medium for 12 hours, with No. 1-3 arranged from left to right;

fig. 6 and 7 are graphs showing the contrast effect. FIG. 6 is a digital photograph of wounds from SD rats of similar body weight. Fig. 7 is a digital photograph of the wound healing condition after 15 days, the left column in fig. 7 is a digital photograph of the wound without any treatment, the middle column is a digital photograph of the chitosan-only hydrogel applied thereto, and the right column is a digital photograph of the composite type supramolecular hydrogel applied thereto in example 2 of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

Weighing 50.0mg of cordycepin solid powder, dissolving in 500 μ L of dimethyl sulfoxide, and ultrasonically dispersing for 5-10 min to obtain 100mg/mL cordycepin solution (stored in dark place); 400mg of chitosan is weighed, 10mL of glacial acetic acid with the volume ratio of 1% is added to promote the dissolution of the chitosan under the action of magnetic stirring, and a chitosan solution with the concentration of 40mg/mL is prepared.

Adding 100 μ L of cordycepin solution into a 2.0mL screw-top bottle cleaned, dried and protected from light, rapidly adding 400 μ L of PBS buffer solution, rapidly shaking, adding 500 μ L of chitosan solution, rapidly shaking, and magnetically stirring to mix well. With reference to fig. 3 and 4, the screw bottle is frozen in a refrigerator at-20 ℃ and then thawed at room temperature (when the temperature is overhigh in summer, the bottle is thawed under an air conditioner) to obtain the composite supramolecular hydrogel. Scanning the composite supramolecular hydrogel to obtain a Scanning Electron Microscope (SEM) image of the composite supramolecular hydrogel shown in fig. 2. Compared with fig. 1, the composite supramolecular hydrogel has a sponge-like structure with a smoother inner part and different pore diameters.

The antibacterial experiment steps are as follows: weighing 10.0g of tryptone, 5.0g of yeast extract and 10.0g of sodium chloride in a beaker, adding ultrapure water into the beaker to dissolve, stirring with a glass rod to promote dissolution, adjusting the pH of the mixed solution to 7.4 with sodium hydroxide, and then adding ultrapure water to a 1L conical flask to prepare a liquid culture medium.

Weighing 10.0g of tryptone, 5.0g of yeast extract, 10.0g of sodium chloride and 15.0g of agar, adding ultrapure water into a beaker for dissolution, stirring with a glass rod to promote dissolution, adjusting the pH of the mixed solution to 7.4 with sodium hydroxide, then preparing a liquid culture medium in a conical flask with the constant volume of ultrapure water to 1L, plugging the bottle mouth with cotton, tightly wrapping with gauze, wrapping with tin foil paper, and tightly tying with cotton threads. Sealing, and autoclaving at 110 deg.C for 30 min. 3 10mL centrifuge tubes are numbered 1-3 respectively and represent blank groups (without any treatment); a chitosan hydrogel group; a chitosan/cordycepin hydrogel group; taking out the Escherichia coli (gram negative bacteria) liquid frozen in a refrigerator at 4 ℃, respectively sucking 100 mu L of the liquid in a sterile operating platform, and adding 3 centrifugal tubes. 6mL of liquid culture medium is added into the No. 1 centrifuge tube; adding 1.0mL of chitosan hydrogel into a No. 2 centrifuge tube, and then adding 5mL of liquid culture medium; adding 1.0mL of the composite supramolecular hydrogel in the example 1 into a No. 3 centrifuge tube, and then adding 5mL of liquid culture medium; after shaking up, the mixture is placed in a constant temperature air shaking incubator at 37 ℃ for culture for 4 hours. Taking 3 sterilized bacteria culture dishes, numbering 1-3 respectively, heating and melting the solid culture medium, shaking uniformly, and pouring into 3 culture dishes before cooling and not solidifying, wherein each dish is poured by 20 mL. And (3) taking 10 mu L of bacterial liquid from the centrifugal tube, inoculating the bacterial liquid to a solid culture medium, turning over after the substrate is solidified, placing the bacterial liquid in a constant-temperature air shaking incubator at 37 ℃ for 12 hours to form bacterial colonies, observing, taking out and taking a picture to obtain a picture shown in figure 5.

Example 2

100 mu L of the cordycepin solution obtained in the above example 1 was taken and added into a 2.0mL screw bottle which was cleaned, dried and protected from light, 400 mu L of the PBS buffer solution prepared in the same manner as in the example 1 was rapidly added, shaking was rapidly carried out, 500 mu L of the 40mg/mL chitosan solution prepared in the same manner as in the example 1 was added, and the mixture was uniformly mixed with a magnetic stirrer. The screw bottle is put into a refrigerator at the temperature of 20 ℃ below zero for freezing, and the composite natural product supermolecule hydrogel can be obtained after the bottle is unfrozen at the room temperature.

Animal experiment steps: healthy male Sprague-Dawley rats (180 to 220g)

A rat full-thickness skin wound model is manufactured to study the effect of the composite natural product supramolecular hydrogel on wound healing. The whole animal experiment process is completed in the animal school department of the New school of Hunan ya medical college of the university of China. Rats were weighed, anesthetized by intraperitoneal injection using pentobarbital (2% sodium pentobarbital, 2ml/kg), the anesthetized rat dorsal hairs were shaved off with an electric shaver, and the skin was disinfected and cleaned with a complex iodine solution before surgery. Three circles with a diameter of 1cm are symmetrically drawn on the back of a rat by using a mold, and a circular wound with a diameter of 1cm is cut by using surgical scissors with the assistance of tweezers, as shown in fig. 6. Three wounds per rat corresponded to three groups: group I was a blank group (no treatment of wounds), group II was a chitosan hydrogel group (control group), group III was a composite supramolecular hydrogel group, and 5 rats per group. After treatment, the rats were individually housed in cages, fed with equal amounts of food and water, photographed for recording, continuously kept for 15 days, and the rats were photographed for recording the wound healing conditions by changing bedding, water and food every day. After 15 days, the photographs shown in FIG. 7 were obtained. The chitosan can act synergistically with cordycepin, can be applied to antibacterial agents and wound dressings, and can achieve the purposes of enhancing the bactericidal performance and promoting wound healing.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The composite supramolecular hydrogel is characterized by comprising a cordycepin solution, a chitosan solution and a PBS buffer solution; the concentration of the cordycepin solution is 100 mg/mL; the solvent of the chitosan solution is 1% HAc, and the concentration of the chitosan solution is 20-40 mg/L.

2. The composite supramolecular hydrogel as claimed in claim 1, wherein solvent of PBS buffer solution is ultrapure water, pH of PBS buffer solution is 8.3-8.5, and each phosphate concentration is 0.1 mol/L.

3. Composite supramolecular hydrogel according to any of claims 1 to 2, characterized in that it is applied in antimicrobial agents or/and wound dressings.

4. A method for preparing a composite supramolecular hydrogel according to any one of claims 1 to 2, characterized in that it comprises the following steps:

step 3, preparing a PBS buffer solution; with Na₂HPO₄And NaH₂PO₄Solution preparationPBS buffer solutions with different Ph;

5. The preparation method of the composite supramolecular hydrogel as claimed in claim 4, wherein in the step 1, the time for dissolving the cordycepin solid powder in the dimethyl sulfoxide is 5-10 min through ultrasonic dispersion, the concentration of the obtained cordycepin solution is 100mg/mL, and light-shielding treatment is performed in the process of preparing the cordycepin solution.

6. The method for preparing the composite supramolecular hydrogel as claimed in claim 5, wherein in the step 2, the concentration of the chitosan solution is 20-40 mg/ml; in the step 3, the Ph of the PBS buffer solution is 8.3-8.5, and the concentrations of Na2HPO4 and NaH2PO4 in the PBS buffer solution are both 0.1 mol/L.

7. The preparation method of the composite supramolecular hydrogel as claimed in claim 6, wherein, in step 4, 80 μ L to 100 μ L of the cordycepin solution is added into a cleaned and dried 2.0mL of a screw bottle which is processed in a dark place, 400 μ L to 420 μ L of the PBS buffer solution is rapidly added, the mixture is rapidly shaken up, 500 μ L of the chitosan solution is added to prepare a 1.0mL mixed solution, and the preparation process of the mixed solution is carried out under magnetic stirring; and after the mixed solution is uniformly mixed, putting the screw bottle into a refrigerator at the temperature of-20 ℃ for freezing until the mixed solution is completely frozen, and then putting the bottle at room temperature for thawing to form the composite supramolecular hydrogel.