CN114796597A

CN114796597A - Sphingosine-based hydrogel and preparation method and application thereof

Info

Publication number: CN114796597A
Application number: CN202210459745.9A
Authority: CN
Inventors: 朱虎; 常爱平; 叶泽立
Original assignee: Fujian Normal University
Current assignee: Fujian Normal University
Priority date: 2022-04-24
Filing date: 2022-04-24
Publication date: 2022-07-29
Anticipated expiration: 2042-04-24
Also published as: CN114796597B

Abstract

The invention discloses a sphingosine-based hydrogel and a preparation method and application thereof. The preparation raw materials of the sphingosine-based hydrogel comprise sphingosine WL gel, citric acid, graphene oxide and the like. The hydrogel prepared by the raw materials and the preparation method has good stability in aqueous solution, is not easy to hydrolyze, has high porosity, can effectively absorb wound exudate when being used as a dressing, and can reduce the risk of impregnation of the wound and the healthy tissues around the wound. After the ciprofloxacin is loaded, escherichia coli and staphylococcus aureus can be inhibited for a long time, and the escherichia coli and the staphylococcus aureus are common and representative pathogenic bacteria in wound healing, so that the medicine carrying gel disclosed by the invention has a good treatment effect on skin wounds.

Description

Sphingosine-based hydrogel and preparation method and application thereof

Technical Field

The invention belongs to the technical field of hydrogel, and particularly relates to sphingosine-based hydrogel and a preparation method and application thereof.

Background

Hydrogels are polymeric materials possessing a 3D network structure that absorbs large amounts of water into their porous polymer network by hydration and capillary forces. Due to the polymer cross-linked network, the hydrogel exhibits the characteristics of an elastic solid, can be deformed under the action of an external force, and recovers when the external force is removed. Also, hydrogels can absorb large amounts of water, have liquid-like properties, such as flowability, permeability to chemical or biological molecules, and the like. In addition, hydrogels have other unique properties, such as swellability and the ability to respond to external stimuli.

In recent decades, as researchers at home and abroad continue to make intensive studies on hydrogels, the types of hydrogels have been increasing, and synthetic polymers formed by chemical reactions of small organic monomers have been widely used for the construction of hydrogels. Although hydrogels based on synthetic polymers have strong water absorption and excellent mechanical properties, their poor biodegradability and potential toxicity greatly limit their application in the biomedical field.

The skin is the largest organ of the human body and is the first immune barrier against external damage and invasion. It is therefore also one of the most commonly injured organs of the human body. Wound healing is a complex and continuous process, affected by a number of factors, requiring appropriate circumstances to accelerate healing. Wound dressings are vital to wound care and not only provide a physical barrier between the wound and the external environment, preventing further injury or infection, but also promote the wound healing process. Therefore, there is a need to develop an adjuvant that can promote the healing of skin wounds.

Disclosure of Invention

Aiming at the prior art, the invention provides a sphingosine-based hydrogel as well as a preparation method and application thereof, so as to prepare the hydrogel which is beneficial to creating a mild and moist microenvironment for wound healing.

In order to achieve the purpose, the invention adopts the technical scheme that: the preparation method of the sphingosine-based hydrogel comprises the following steps of: 15-25 parts of sphingosine WL glue and 1-4 parts of citric acid.

On the basis of the technical scheme, the invention can be further improved as follows.

Further, the preparation raw materials of the hydrogel comprise the following components in parts by mass: 20 parts of sphingosine WL glue and 2 parts of citric acid.

Further, the raw materials for preparing the hydrogel also comprise graphene oxide, and the mass of the graphene oxide is 0.1-10% of that of the sphingosine WL gel.

Further, the sphingosine WL glue is prepared by the following steps:

s1: adding the activated sphingomonas bacterium liquid into a seed culture medium according to the volume ratio of 1:100, and culturing for 16h at constant temperature to obtain a first-stage seed liquid; the sphingomonas is sphingomonas WG, and the preservation number is CCTCC No. M2013161;

s2: adding the primary seed liquid into a seed culture medium according to the volume ratio of 1:20, and culturing for 16h at constant temperature to obtain a secondary seed liquid;

s3: adding the secondary seed liquid into a fermentation medium according to the volume ratio of 1:10, and culturing for 96 hours at constant temperature to obtain fermentation liquid;

s4: mixing the fermentation liquor with 95% alcohol according to the volume ratio of 1:4, standing until the precipitate is not increased any more, performing solid-liquid separation, dialyzing the precipitate for 3 days by using a dialysis bag with the molecular weight cutoff of 8000-14000 Da, and freeze-drying to obtain a crude product of sphingosine WL gel;

s5: dissolving the crude product of sphingosine WL glue in water, mixing the obtained solution with isopropanol containing 5 wt% of sodium chloride according to the volume ratio of 1:4, standing until the precipitate does not increase any more, separating the precipitate, washing with an isopropanol aqueous solution with gradient concentration, dialyzing, and drying to obtain the final product;

the seed culture medium is prepared by the following steps: dissolving 1g of yeast powder, 5g of peptone, 11g of glucose, 2g of potassium dihydrogen phosphate and 0.205g of magnesium sulfate heptahydrate in 1000mL of ultrapure water, and sterilizing at 121 ℃ for 25min to obtain the yeast extract;

the fermentation medium is prepared by the following steps: dissolving 73.8g glucose, 0.205g magnesium sulfate heptahydrate, 2g potassium dihydrogen phosphate, 3g yeast powder and 4g dipotassium hydrogen phosphate in 1000mL ultrapure water, and sterilizing at 121 deg.C for 25 min.

Further, the graphene oxide is prepared by the following steps:

s1: dissolving graphite powder and sodium nitrate into concentrated sulfuric acid, cooling the mixture to 0 ℃, adding potassium permanganate, reacting for 1 hour, heating to 35 ℃, continuing to react for 3 hours, adding ultrapure water, heating to 95 ℃, preserving heat, reacting for 0.5 hour, sequentially washing with 30% hydrochloric acid and ultrapure water until the solution becomes colloidal, performing ultrasonic treatment for 80min, centrifuging, and taking upper layer liquid; the material ratio of the graphite powder, the sodium nitrate and the concentrated sulfuric acid is 2g:1g:46mL, and the mass ratio of the added potassium permanganate to the graphite powder is 3: 1;

s2: and (4) sequentially dialyzing and freeze-drying the supernatant to obtain the finished product.

The invention also discloses a preparation method of the sphingosine-based hydrogel, which comprises the following steps:

(1) dispersing the graphene oxide with the formula amount in ultrapure water to obtain a graphene oxide dispersion liquid; dispersing sphingosine WL glue and citric acid in the formula amount in ultrapure water to obtain a base solution;

(2) adding the graphene oxide dispersion liquid into the base liquid, uniformly mixing, and removing bubbles in the mixed solution to obtain a hydrogel pre-solution;

(3) and flatly spreading the hydrogel pre-solution on a mold, drying water at 30 ℃, and heating to 80 ℃ for reaction for 24 hours to obtain the hydrogel.

The hydrogel prepared by the invention has excellent performance and can be used for preparing wound dressings. The wound dressing of the present invention includes a sphingosine-based hydrogel substrate and an antimicrobial drug supported on the substrate.

Further, the antibacterial agent is ciprofloxacin.

Further, the wound dressing is prepared by the following steps:

s1: dissolving ciprofloxacin in a hydrochloric acid solution to form a drug solution with the concentration of 20mg/mL, and adjusting the pH value of the drug solution to be 3;

s2: immersing the sphingosine-based hydrogel substrate into the medicinal solution, performing rotary culture at 37 ℃ for 24h, taking out the sphingosine-based hydrogel substrate, and drying at 30 ℃ to obtain the sphingosine-based hydrogel.

The invention has the beneficial effects that:

1. the sphingosine WL glue and CA are mixed, the sphingosine WL glue and CA are subjected to chemical crosslinking reaction to form a hydrogel film, and the thermal stability and the mechanical strength of the hydrogel film are improved through crosslinking between the sphingosine WL glue and CA, so that the obtained hydrogel has better mechanical properties.

2. The hydrogel obtained by the invention has good stability in aqueous solution and is not easy to hydrolyze; and has high porosity, and can be used as dressing to effectively absorb wound exudate, and reduce the risk of impregnation of wound and the surrounding healthy tissue.

3. After the ciprofloxacin is loaded on the hydrogel, escherichia coli and staphylococcus aureus can be inhibited for a long time, and the escherichia coli and the staphylococcus aureus are common and representative pathogenic bacteria in wound healing, so that the medicine-carrying gel has a good treatment effect on skin wounds.

4. The hydrogel disclosed by the invention has good biocompatibility and is suitable for biomedical application.

Drawings

FIG. 1 shows cell viability of 3T3 cells, HUVEC cells, and 293E cells after 24 hours of culture with hydrogel conditioned medium;

FIG. 2 is an ATR-FTIR spectrum of a WL-CA film;

FIG. 3 is the evolution of the hydroxyl vibration band associated with the CA crosslinking reaction: -ratio of OH/β -1-4 glycosidic linkages;

FIG. 4 is a TG curve of CA and WL-CA films;

FIG. 5 is a DTG curve for CA and WL-CA films;

FIG. 6 is a photograph of a WL-CA hydrogel swollen at different pH values, the initial film being a 4.5mm circular disc;

fig. 7 is a plot of swelling rate of WL-CA hydrogel at pH 9.5(a), pH 7.4(B), and pH 5.5(C) over time; FIG. 7D is a graph of the swelling ratio of WL-CA hydrogels with different concentrations of citric acid over 2 hours;

fig. 8 is a graph showing the swelling rate of WL-CA hydrogel at pH 9.5(a), pH 7.4(B) and pH 5.5(C) over 87 hours;

FIG. 9 is an SEM cross-section of a WL-CA hydrogel swollen at different pH;

FIG. 10 is a photograph of swollen WL-CA10 hydrogel adhered to a frequently moving joint and hand of a human body;

FIG. 11 is the rheological behavior of a swollen WL-CA hydrogel, with a set strain of 1%;

FIG. 12 is the in vitro cumulative release of CIP in WL-CA-CIP hydrogel films at different pH values;

FIG. 13 is an in vitro antimicrobial activity of a hydrogel; a is a photograph of an inhibition zone of escherichia coli after 24 hours of culture, B is a photograph of an inhibition zone of staphylococcus aureus after 24 hours of culture, C is a bacterial survival rate of escherichia coli, and D is a bacterial survival rate of staphylococcus aureus;

FIG. 14 is a photograph of colonies after co-culturing E.coli with WL-CA0, WL-CA10 and WL-CA-CIP hydrogel;

FIG. 15 is a photograph of colonies of Staphylococcus aureus co-cultured with WL-CA0, WL-CA10 and WL-CA-CIP hydrogel;

FIG. 16 is the results of twisting and stretching of WL-CA-GO hydrogel films;

fig. 17 and 18 show the rheological behavior of the sphingosine WL glue gel.

Detailed Description

The following examples are provided to illustrate specific embodiments of the present invention.

Example 1: preparation of citric acid crosslinked sphingosine WL glue gel

Preparation of sphingosine WL glue

The fermentation steps of the sphingosine WL glue are as follows: the bacterial extracellular polysaccharide sphingosine WL glue is prepared by fermenting a Sphingomonas WG (Sphingomonas sp. WG, the preservation number of CCTCC No. M2013161). The culture of the fermentation process is carried out in a conical flask, the whole process is carried out in a vertical constant temperature oscillator, the temperature is set to be 28 ℃, and the rotating speed is set to be 150 rpm; the strain transfer process is carried out in a sterile workbench, the sterile workbench is sterilized for 30min before operation, and 75% alcohol is sprayed before entering the sterile workbench.

First, Sphingomonas sp.WG strain frozen at-80 ℃ was taken out, transferred to a 250mL Erlenmeyer flask containing 50mL Luria-Bertina medium, activated, and cultured for 20h in a vertical homothermal shaker. And adding 1mL of activated Sphingomonas sp.WG bacterial liquid into a 250mL conical flask with the content of 100mL of seed culture medium, and culturing for 16h to obtain a primary seed liquid. And adding 5mL of the primary seed solution into a 250mL conical flask with the content of 100mL of seed culture medium, and culturing for 16h to obtain an orange-yellow secondary seed solution. Then 20mL of the secondary seed culture was transferred to a 500mL Erlenmeyer flask containing 200mL of fermentation medium and cultured for 96h to obtain a thick yellow-orange fermentation broth. Seed culture medium: 1g of yeast powder, 5g of peptone, 11g of glucose, 2g of monopotassium phosphate and 0.205g of magnesium sulfate heptahydrate are dissolved in 1000mL of ultrapure water; setting the sterilization temperature at 121 ℃, and sterilizing for 25 min. Fermentation medium: 73.8g of glucose, 0.205g of magnesium sulfate heptahydrate, 2g of potassium dihydrogen phosphate, 3g of yeast powder and 4g of dipotassium hydrogen phosphate are dissolved in 1000mL of ultrapure water. Setting the sterilization temperature at 121 ℃, and sterilizing for 25 min.

The alcohol precipitation step of the sphingosine WL glue crude product is as follows: the fermentation broth was first mixed with 95% alcohol (4: 1 alcohol to broth by volume) to precipitate the polysaccharide. And (3) carrying out solid-liquid separation on the precipitated polysaccharide through a vacuum filtration device, dialyzing the polysaccharide for 3 days by using a dialysis bag (the cut-off molecular weight is 8000-14000 Da), and freeze-drying to obtain a sphingosine WL gum crude product.

The crude product was re-dissolved in water and stirred for three days to give a crude solution of sphingosine WL gum. Adding an isopropanol solution containing 5% of sodium chloride into the crude solution, wherein the volume ratio of the isopropanol to the crude solution is 4:1, mixing and stirring for 2h, and re-precipitating overnight. The precipitate was filtered and washed with a gradient concentration of aqueous isopropanol (isopropanol to water volume ratio of 10:0, 9:1, 8:2 and 7:3) to remove impurities. Finally, dialysis was performed for 3 days. Pure sphingosine gel was obtained after freeze drying and it was confirmed by uv testing that nucleic acids and proteins were removed.

Secondly, preparing citric acid cross-linked sphingosine WL glue gel

Dissolving Citric Acid (CA) in water to prepare high-concentration citric acid solutions, and adding the citric acid solutions with different volumes into 1 wt% of sphinganine WL glue (WL) solutions respectively. Finally, in 20mL of ultrapure water, 0.2g of sphinganine WL gel and 0g, 0.01g, 0.02g or 0.04g of citric acid were stirred for 3 days using a mechanical stirrer to uniformly mix the solution. And (3) carrying out ultrasonic treatment on the uniform viscous solution for 30min, vacuumizing until the solution is free of bubbles, pouring the solution into a polytetrafluoroethylene mold with the length of 10cm, the width of 3cm and the height of 0.5cm, and scraping. Subsequently, the sample was placed in a 30 ℃ oven to remove moisture and held in an 80 ℃ oven for 24 h. Finally, the WL-CA film was immersed in pure water to pH 7 to remove unreacted citric acid, and then dried in an oven at 30 ℃. The resulting hydrogel film was stored in a drying dish. The mass ratios of CA to WL were 0, 0.05, 0.10 and 0.20. The corresponding WL-CA films were named WL-CA0, WL-CA5, WL-CA10, WL-CA 20.

Example 2: preparation of graphene oxide modified citric acid cross-linked sphingosine WL glue gel

Firstly, preparing graphene oxide

Sequentially adding 2g of graphite powder, 1g of sodium nitrate and 46mL of concentrated sulfuric acid into a 500mL beaker, and carrying out ice-water bath at the temperature of 0 ℃ for 1 h; then slowly adding about 0.2g of potassium permanganate into the mixture in an amount of about 6g each time under an ice bath condition, finishing the addition within about 1 hour, and continuing to react for 1 hour; then the mixed solution is heated to 35 ℃ while being stirred, the reaction is continued for 3h, 92mL of ultrapure water is dripped and heated to 95 ℃, and the reaction is carried out for 0.5h at 95 ℃. After dilution with 184mL of ultrapure water, unreacted potassium permanganate was reduced with 30% hydrogen peroxide until the slurry became yellowish brown. After cooling in air, the mixture was washed several times and centrifuged (10000rpm, 10min) at room temperature, washed three times with 30% hydrochloric acid to remove unreacted potassium permanganate, and then washed with ultrapure water until the solution became gel. Carrying out ultrasonic treatment on the washed Graphene Oxide (GO) aqueous solution for 80min, and stripping unreacted scale graphite and GO with low oxidation degree; centrifuging (3500rpm, 10min) to obtain upper layer liquid, and obtaining GO dispersion liquid.

100mL of GO dispersion is dialyzed in 1000mL of ultrapure water for three days, water is changed every 2h in the first day, and water is changed every 4h in the last two days. Freeze drying yielded a dark brown freeze dried sample of GO. And dissolving the freeze-dried graphene oxide in water at the concentration of 5mg/mL, and performing ultrasonic treatment for 1h to keep the stable state for more than 20 days.

Secondly, preparing graphene oxide modified citric acid cross-linked sphingosine WL glue gel

0.02g of freeze-dried graphene oxide is added into 1mL of ultrapure water, stirred for 30min and then subjected to ultrasonic treatment for 10min to obtain a uniform GO dispersion solution.

Sphingosine WL gum and citric acid were added to 60mL of ultrapure water, and the solution was mixed uniformly by stirring at 500rpm for 3 days using mechanical stirring. Wherein the mass fraction of the sphingosine WL gel is 1 wt%, and the mass of the citric acid is 10% of that of the sphingosine WL gel. The mixed solution was divided into 5 parts on average, transferred to 50mL centrifuge tubes, and GO dispersion solutions of different volumes were added to the four centrifuge tubes so that the mass ratios of graphene oxide to sphingosine WL gel were 0, 0.001, 0.01, 0.05 and 0.1, respectively. The hydrogel pre-solution was mixed for 30min using a vortex apparatus and then sonicated for 30 min. The mixed pre-solution was vacuumed to remove air bubbles, and then the mixed liquid containing no air bubbles was spread on a polytetrafluoroethylene mold (mold size: length × width × height ═ 3cm × 2cm × 0.5 cm). And (3) placing the hydrogel pre-solution in a drying oven, drying at 30 ℃, and then heating to 80 ℃ for reaction for 24 hours to obtain the WG-CA-GO hydrogel membrane. According to the content change of graphene oxide, the composite hydrogel films are named as WL-CA10, WL-CA-GO0.1, WL-CA-GO1, WL-CA-GO5 and WL-CA-GO10 respectively. In addition, no citric acid is added, no graphene oxide is added, and a hydrogel film named WL is obtained; adding graphene oxide with the mass ratio of 0.01 to sphingosine WL glue without adding citric acid to obtain a hydrogel film named WL-GO 1.

Example 3: loading ciprofloxacin on hydrogel

Ciprofloxacin (CIP) was dissolved in hydrochloric acid solution to form CIP solution (pH 3). The hydrogel lyophilized films (about 3.5mg) prepared in examples 1 and 2 were immersed in 3mL of CIP solution having a concentration of 20mg/mL, incubated at 37 ℃ and 70rpm for 24 hours, and the samples were taken out and dried at 30 ℃ until use.

Analysis of results

1. In vitro cytotoxicity assay

To evaluate the potential of the WL-CA-based hydrogel as a wound dressing, the cytotoxicity of WL-CA0, WL-CA10 and WL-CA-CIP hydrogels was evaluated by using mouse NIH 3T3 fibroblasts, Human Umbilical Vein Endothelial Cells (HUVECs) and human embryonic kidney cells (293E), and the results are shown in fig. 1. The hydrogel soaking solution method is a more common method, after the cells are co-cultured by using WL-CA0, WL-CA10 and WL-CA-CIP hydrogel extraction media for 24 hours, the cell activity of mouse NIH 3T3 and HUVECs is higher than 90 percent and the cell activity of 293E cells is higher than 80 percent through MTT method detection. ISO 10993-5 states that this material is considered cytotoxic in the case of viability drops by more than 30%, and therefore these hydrogels are excellent in biocompatibility and suitable for biomedical applications.

2. Structural characterization of hydrogel films

The chemical structure of the WL-CA film was analyzed by ATR-FTIR. As shown in FIG. 2, the IR spectra of WL-CA5, WL-CA10 and WL-CA20 are very similar to that of WL-CA0, and all have the characteristic absorption peaks of sphingosine WL gel. There was no new infrared absorption peak after the crosslinking reaction, since 1724cm ^-1 The absorbance of (A1724) corresponds not only to the new ester bond formed in the esterification cross-linking but also to the O-acetyl group of WL. For WL-CA0, WL-CA5, WL-CA10 and WL-CA20, A1724 and 896cm ^-1 The ratios of the beta-1-4 glycosidic bond reference bands (A896) were 3.4, 3.8, 4.3, and 4.7, respectively (FIG. 3), indicating that as CA increased, more ester bonds were formed. Furthermore, 3300cm ^-1 The absorbance of (A3300) corresponds mainly to-OH of WL, which can react with-COOH of CA. For WL-CA0, WL-CA5, WL-CA10 and WL-CA20, the ratios of A3300 and A896 were 2.1, 1.7, 1.5 and 1.2 respectively (FIG. 3), which means that more hydroxyl groups were reacted as CA increased. These results indicate that as the citric acid content increases, more esterification reactions between-OH of WL and-COOH of CA occur, thereby increasing the crosslink density of the film.

As shown in fig. 4 and 5, the TG curve and the first Derivative of TG (DTG) curve laterally confirmed the presence of chemical reactions and confirmed the different thermal stability of the films for different citric acid contents. All films showed a three-stage weight loss. In the first stage, there was a small mass loss of the WL-CA film, probably 3%, in addition to the citric acid, due to the evaporation of free and bound water. The temperature range over which mass loss occurs for WL-CA films containing citric acid is smaller compared to the physical hydrogel WL-CA 0. The reason for this is the reduced consumption of hydroxyl groups when WL cross-links with CA, and the reduced hydrophilic groups that can interact with water molecules. The second and third stages correspond to the decomposition of the polysaccharide chains and the vaporization and elimination of volatile products. The hydroxyl groups on the gel are cross-linked with citric acid by an esterification reaction, which reduces the hydroxyl content in the WG. The second stage of WL-CA0 started at 200 ℃ higher than those films where chemical crosslinking was present (175 ℃ for WL-CA5, 193 ℃ for WL-CA10, 165 ℃ for WL-CA 20). One possible explanation is that the crosslinking reaction can reduce the number of remaining hydrogen bonds in the WL-CA film, reducing the energy required for degradation. In contrast to WL-CA20, the mass loss rate for WL-CA10 is lower, with the highest point at which the maximum mass loss rate is at (269 ℃ C.), which indicates that the crosslinks formed between WL and CA improve the thermal stability of the film, but that excessive amounts of CA (e.g., WL-CA20) reduce the stability. In fig. 5, the crosslinked film exhibited an additional shoulder around the temperature of 240 ℃, which may be due to excessive CA. The above results confirmed the formation of chemical crosslinks in WL-CA5, WL-CA10 and WL-CA 20.

3. Analysis of pH-responsive swelling behavior

The swelling behavior of WL-CA films in PBS at pH 9.5, 7.4 and 5.5 was investigated. It was observed that after 2 hours of swelling in PBS, there was no significant change in the diameter of any of WL-CA0, WL-CA5, WL-CA10, or WL-CA 20. However, the thickness varied greatly, increasing by about 50-fold (2mm/40 μm) (FIG. 6). And the film changed from translucent to white and opaque.

The water absorption capacity of the WL-CA film was further quantitatively evaluated by a weighing method. As shown in FIG. 7, all of the films WL-CA0, WL-CA5, WL-CA10 and WL-CA20 showed a rapid swelling capacity in the first 0.5 hours, and all of the hydrogel films reached a swelling equilibrium in 2 hours. Whereas the swelling degree of the hydrogel decreases with increasing amount of the crosslinking agent. The reason is that the increase of the crosslinking agent increases the crosslinking density of the hydrogel, and the crosslinking density prevents the diffusion of water, limiting the swelling of the hydrogel. For WL-CA films, the water absorption capacity increases with pH due to deprotonation of the carboxyl groups, resulting in charge repulsion and increased hydrophilicity. The maximum swelling ratios of WL-CA0, WL-CA5, WL-CA10 and WL-CA20 were 38g/g, 29g/g, 27g/g and 21g/g, respectively. The WL-CA hydrogel film has high water absorption and is suitable for use in wound dressing to absorb wound exudate. To test the stability of WL-CA hydrogels in water, the swelling ratio was hardly changed after swelling WL-CA0, WL-CA5, WL-CA10, and WL-CA20 films in PBS at pH 9.5, pH 7.4, pH 9.5, and pH 5.5 for 87 hours. This shows that there is no tendency for the weight of all WL-CA hydrogels to decrease after reaching swelling equilibrium (fig. 8), i.e. these hydrogel structures have a certain stability in aqueous solution and are not easily hydrolyzed, and when used as a dressing, they can absorb wound exudate effectively and reduce the risk of maceration of the wound and the healthy tissue surrounding the wound.

4. Analysis of porous morphology and porosity

The ability of a hydrogel to absorb and retain water is closely related to the porosity of the hydrogel. First, the morphological characteristics of the swollen hydrogel were observed by SEM. In FIG. 9, all WL-CA hydrogels showed a three-dimensional porous network structure. The porous structure of the physically crosslinked hydrogel WL-CA0 is obviously destroyed after swelling, and after soaking at pH 9.5, the WL-CA0 hydrogel hardly has the porous appearance of the hydrogel; the other covalently linked hydrogels did not appear to be characteristic of the WL-CA0 hydrogel, indicating that chemical crosslinking effectively preserved the microscopic morphology of the hydrogel.

Analysis by Image J software revealed that the pore diameters (pH 5.5) of the swollen WL-CA5, WL-CA10 and WL-CA20 were 102.9 μm, 39.6 μm and 9.4 μm, respectively, the pore diameters (pH 7.4) of WL-CA5, WL-CA10 and WL-CA20 were 242.1 μm, 54.9 μm and 17.3 μm, respectively, and the pore diameters (pH 9.5) of WL-CA5, WL-CA10 and WL-CA20 were 246.9 μm, 71.1 μm and 20.0 μm, respectively. These results indicate that as the crosslinking agent is increased, the crosslinking density increases and the pore size of the hydrogel becomes smaller; and with the increase of the pH value, the carboxyl is deprotonated, electrostatic repulsion occurs between groups, the molecular chain distance is increased, the pore diameter of the hydrogel is enlarged, and more water enters the hydrogel. This is consistent with the tendency of the swelling ratio to change.

5. Skin adhesion and rheological Properties analysis

The WL-CA10 hydrogel was applied to the skin, including the skin of the elbow or interphalangeal joint, during the course of moving the joint and was found to be free of any retraction or rupture (fig. 10). When subjected to a tearing test with the skin-adherent WL-CA hydrogel, the hydrogel was easily separated from the skin tissue without any residue and the tearing process did not produce pain. The WL-CA hydrogel was shown to have moderate tissue adhesion and may not be prone to secondary wound injury when the hydrogel was replaced.

The rheological properties of the hydrogel at a strain of 1% and a modulus varying with frequency were tested by a rotational rheometer. As shown in fig. 11, the storage modulus (G') was much higher than the loss modulus (G ") for all hydrogels over the frequency range tested, which indicates that the hydrogels have good solid properties. The G 'and G' of the hydrogel WL-CA0 in the physically crosslinked state increased significantly with increasing frequency, indicating the presence of physical crosslinking. As the citric acid content increased, the storage modulus value of the covalently linked hydrogels increased, decreasing in frequency dependence, indicating that hydrogels with higher chemical crosslink density had relatively better stability and a stiffer network. The modulus of WL-CA20 is particularly weakly frequency dependent and exhibits a solid-like behavior. The WL-CA5 had a G ' of about 0.6kPa, the WL-CA10 had a G ' of about 1.2kPa, and the WL-CA20 had a G ' of about 2kPa, indicating that the WL-CA hydrogel had some mechanical strength.

6. Drug release profile

Based on the comparison of the pH response performance, the porosity and the porous morphology of the WL-CA hydrogel and the comparison of the rheological properties of the hydrogel, WL-CA10 showed the best overall performance in potential wound dressing applications, and the WL-CA10 hydrogel film was selected for drug loading and release studies.

The CIP loading on WL-CA-CIP, calculated by spectrophotometry, was 24.5%.

Drug release experiments were performed using the conditions of the swelling experiments and PBS solutions of three pH values were used as release media, and the results are shown in fig. 12. As can be seen from the figure, burst release of the drug occurred within 10 minutes, 25% at pH 9.5, 52% at pH 7.4, 62% at pH 5.5, followed by sustained release. The explosive release may be caused by desorption of ciprofloxacin adsorbed on the surface of the hydrogel and the rapid gel swelling described above. The burst release results in a high exposure of the antimicrobial drug at the wound site, providing immediate and effective bacterial infection prevention. In the first 6 hours, the cumulative CIP release decreased with increasing pH, mainly because ciprofloxacin's water solubility decreased with increasing pH. Then, due to the swelling of the hydrogel, the cumulative release of ciprofloxacin at pH 9.5 gradually exceeded the release at pH 7.4. After 12 hours, approximately 90% of the loaded CIP was released into the surrounding PBS at pH 9.5 and pH 5.5, while only 75% was released at pH 7.4.

7. Analysis of antibacterial Activity

Gram-negative escherichia coli and gram-positive staphylococcus aureus are typical pathogenic bacteria common in wound healing. Gram-positive bacteria such as staphylococcus aureus and streptococcus pyogenes appear early in wound formation; after a period of time following wound formation, gram-negative bacteria such as E.coli and P.aeruginosa may be present in the wound.

First, the antibacterial activity of the WL-CA0, WL-CA10 and WL-CA-CIP hydrogels against Escherichia coli and Staphylococcus aureus in vitro was investigated by the zone of inhibition test, and as shown in FIG. 13A, after 24 hours of culture, the inhibition zones of WL-CA0, WL-CA10 and WL-CA-CIP against Escherichia coli were 0cm, 1.3 cm and 4.9 cm, respectively. For Staphylococcus aureus, only WL-CA-CIP showed a significant zone of inhibition, with a zone of inhibition of 3.5 cm (FIG. 13B). These results indicate that WL-CA has a slight inhibitory effect on E.coli due to unreacted residual CA. In contrast, WL-CA-CIP has strong inhibiting and killing effects on escherichia coli and staphylococcus aureus, and the antibacterial source of the hydrogel is mainly antibiotic ciprofloxacin.

In addition, to test for long-term antimicrobial activity, fresh PBS solution and freshly activated S.aureus or E.coli suspension were co-cultured daily with WL-CA0, WL-CA10 and WL-CA-CIP and surviving colonies were characterized using plate coating and photographical recording. As can be seen from fig. 13C & D, fig. 14, and fig. 15, WL-CA10 killed escherichia coli within the first three days, without antimicrobial activity against staphylococcus aureus; WL-CA-CIP inhibits growth of Escherichia coli for 10 days and Staphylococcus aureus for 7 days. The long-term antimicrobial properties of the hydrogel may be related to the incomplete release of the drug present in the WL-CA10 hydrogel. All antibacterial test results show that the WL-CA-CIP has good long-term antibacterial action.

8. Mechanical property analysis of graphene oxide modified hydrogel

In order to research the influence of graphene oxide on the performance of the WL-CA-GO hydrogel film, the mechanical behavior of WL-CA-GO layered hybrid films with different graphene oxide contents is researched through room-temperature manual twisting and tensile tests. The mechanical properties of the films are shown in fig. 16, which shows the flexibility of WL-CA-GO hydrogel films with different GO contents, which can be arbitrarily distorted. It can be seen that the addition of graphene oxide significantly improves the tensile properties of the WL-CA-GO hydrogel film. WL-CA-GO1 has better stretching behavior, and the stretching length is one third of the original length. The content of graphene oxide of WL-CA-GO0.1 is too low, the hardness of the hydrogel film is low, and the hydrogel film does not have stretching behavior. The content of graphene oxide in the hydrogel of WL-CA-GO5 and WL-CA-GO10 is too high, so that the hydrogel film has too high hardness and is easy to break.

The storage modulus (G') and loss modulus (G ") of each sample were evaluated using oscillatory rheological measurements. When the oscillating shear strain was fixed at 1% and the angular frequency increased from 0.1rad/s to 100rad/s, G' was significantly higher than G "for each group, with no crossing traces (FIG. 17), indicating that the WL-CA10 hydrogel was predominantly elastic in nature. More importantly, with increasing GO concentration, G 'of GO-reinforced hydrogels gradually increased, up to 7kPa and with increasing GO concentration, G' was significantly higher than that of no GO hydrogel, indicating that more stable, stiffer network systems existed in GO composite hydrogels.

In addition, to explore the effect of the crosslinker Citric Acid (CA) on the WL-CA-GO system, the oscillatory rheological behavior of WL, WL-CA10, WL-GO1 and WL-CA-GO1 were compared. As is evident from fig. 18, the elastic behavior of the hydrogel films with citric acid added is significantly better than that of the hydrogels without citric acid, and the elastic modulus ordering of these four hydrogels is from small to large: WL, WL-GO1, WL-CA10 and WL-CA-GO 1. The presence of the chemically cross-linked network was shown to enhance the rigidity and stability of the hydrogel network, and the greater the resistance of the hydrogel film to external denaturation relative to a physically cross-linked hydrogel (WL, WL-GO1) incorporating citric acid.

While the present invention has been described in detail with reference to the embodiments, it should not be construed as limited to the scope of the patent. Various modifications and changes may be made by those skilled in the art without inventive step within the scope of the appended claims.

Claims

1. The sphingosine-based hydrogel is characterized in that raw materials for preparing the hydrogel comprise the following components in parts by mass: 15-25 parts of sphingosine WL glue and 1-4 parts of citric acid.

2. The sphingosine-based hydrogel according to claim 1, wherein the hydrogel is prepared from the following components in parts by mass: 20 parts of sphingosine WL glue and 2 parts of citric acid.

3. The sphingosine-based hydrogel according to claim 1 or 2, wherein: the hydrogel is prepared from graphene oxide, wherein the mass of the graphene oxide is 0.1-10% of that of the sphingosine WL gel.

4. The sphingosine-based hydrogel according to claim 3, wherein said sphingosine WL gel is prepared by the following steps:

5. The sphingosine-based hydrogel according to claim 3, wherein said graphene oxide is prepared by the following steps:

6. The method of making a sphingosine-based hydrogel according to claim 3 comprising the steps of:

7. Use of a sphingosine-based hydrogel according to any of claims 1 to 5 for the preparation of a wound dressing.

8. Use according to claim 7, characterized in that: the wound dressing includes a sphingosine-based hydrogel substrate and an antimicrobial drug supported on the substrate.

9. Use according to claim 8, characterized in that: the antibacterial agent is ciprofloxacin.

10. The use of claim 9, wherein the wound dressing is prepared by: