CN114796597B - Sphingosine-based hydrogel and preparation method and application thereof - Google Patents

Abstract

The invention discloses a sphingosine-based hydrogel and a preparation method and application thereof. The raw materials for preparing the sphingosine-based hydrogel comprise sphingosine WL glue, 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 higher porosity, can be used as dressing, can effectively absorb wound exudates, and can reduce the impregnation risk of wounds and healthy tissues around the wounds. After ciprofloxacin is loaded, escherichia coli and staphylococcus aureus which are common and representative pathogenic bacteria in wound healing can be inhibited for a long time, so that the medicine-carrying gel has 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 hydrogels, and particularly relates to a sphingosine-based hydrogel, and a preparation method and application thereof.

Background

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

In recent decades, as scholars at home and abroad continuously research hydrogels, the types of hydrogels are increasing, and synthetic polymers formed by chemical reaction of small organic monomers have been widely used for constructing hydrogels. Although hydrogels based on synthetic polymers have very strong water absorption and excellent mechanical properties, their poor biodegradability and potential toxicity greatly limit their use in the biomedical field.

The skin is the largest organ of the human body and is the first immune barrier against external injury 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 that is affected by a number of factors, requiring a proper environment to accelerate healing. Wound dressings are critical to wound care, and can not only provide a physical barrier between the wound and the external environment, preventing further injury or infection, but can also promote the wound healing process. Therefore, it is necessary to develop an adjuvant capable of promoting the healing of skin wounds.

Disclosure of Invention

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

In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: the preparation raw materials of the sphingosine-based hydrogel comprise the following components in parts by mass: 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 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 preparation raw materials of the hydrogel further comprise graphene oxide, and the mass of the graphene oxide is 0.1-10% of the mass of the sphingosine WL glue.

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

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

s2: adding the first-stage seed liquid into a seed culture medium according to the volume ratio of 1:20, and culturing for 16 hours at constant temperature to obtain a second-stage seed liquid;

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

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

s5: dissolving a sphingosine WL glue crude product in water, mixing the obtained solution with isopropanol containing 5wt% of sodium chloride according to a volume ratio of 1:4, standing until precipitation is not increased, separating the precipitation, washing with isopropanol water solution with gradient concentration, dialyzing, and drying to obtain the sphingosine WL glue;

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 product;

the fermentation medium is prepared by the following steps: 73.8g of glucose, 0.205g of magnesium sulfate heptahydrate, 2g of monopotassium phosphate, 3g of yeast powder and 4g of dipotassium phosphate are dissolved in 1000mL of ultrapure water, and sterilized at 121 ℃ for 25min to obtain the compound.

Further, graphene oxide is prepared through the following steps:

s1: the preparation method comprises the steps of co-dissolving graphite powder and sodium nitrate in concentrated sulfuric acid, reducing the temperature of a mixture to 0 ℃, adding potassium permanganate, reacting for 1h, heating to 35 ℃, continuing to react for 3h, adding ultrapure water, heating to 95 ℃, preserving heat, reacting for 0.5h, washing with 30% hydrochloric acid and ultrapure water in sequence until the solution becomes gelatinous, performing ultrasonic treatment for 80min, and centrifuging to obtain upper-layer liquid; the material ratio of the graphite powder to the sodium nitrate to the concentrated sulfuric acid is 2g to 1g to 46mL, and the mass ratio of the added potassium permanganate to the graphite powder is 3 to 1;

s2: and sequentially dialyzing and freeze-drying the supernatant to obtain the 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 graphene oxide dispersion liquid; dispersing the sphingosine WL glue and citric acid in the formula amount into ultrapure water to obtain a base solution;

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

(3) Spreading the hydrogel pre-solution on a die, drying the 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 dressing. The wound dressing of the present invention comprises 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: ciprofloxacin is dissolved in hydrochloric acid solution to form a drug solution with the concentration of 20mg/mL, and the pH=3 of the drug solution is adjusted;

s2: immersing the sphingosine-based hydrogel substrate into a medicinal solution, rotating and culturing for 24 hours at 37 ℃, taking out the sphingosine-based hydrogel substrate, and drying at 30 ℃ to obtain the sphingosine-based hydrogel.

The beneficial effects of the invention are as follows:

1. the sphingosine WL glue and the CA are mixed, the sphingosine WL glue and the CA are subjected to chemical crosslinking reaction to form the hydrogel film, and the crosslinking between the sphingosine WL glue and the CA improves the thermal stability and the mechanical strength of the hydrogel film, 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 higher porosity, and can effectively absorb wound exudates when being used as dressing, thereby reducing the impregnation risk of wounds and healthy tissues around the wounds.

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

4. The hydrogel provided 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 incubation with hydrogel conditioned medium;

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

FIG. 3 shows the evolution of the hydroxyl vibrational bands associated with the CA cross-linking reaction: -OH/beta-1-4 glycosidic bond ratio;

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

FIG. 5 is a DTG plot of CA and WL-CA films;

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

fig. 7 is a plot of swelling ratio versus time for WL-CA hydrogels at ph=9.5 (a), ph=7.4 (B) and ph=5.5 (C); FIG. 7D is the swelling rate of WL-CA hydrogels containing different concentrations of citric acid within 2 hours;

fig. 8 is a graph of the swelling ratio of WL-CA hydrogel over time at ph=9.5 (a), ph=7.4 (B) and ph=5.5 (C) for 87 hours;

FIG. 9 is a SEM cross-sectional view of a WL-CA hydrogel swelled at different pH's;

FIG. 10 is a photograph of swollen WL-CA10 hydrogel adhering 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 fixation strain of 1%;

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

FIG. 13 is an in vitro antimicrobial activity of hydrogels; a is a photo of a inhibition zone of escherichia coli after 24 hours of culture, B is a photo of a 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 hydrogels;

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

FIG. 16 is a graph showing the twist and stretch results of WL-CA-GO hydrogel films;

fig. 17 and 18 are rheological behavior of sphingosine WL glue gels.

Detailed Description

The following describes the present invention in detail with reference to examples.

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

1. Preparation of sphingosine WL glue

The fermentation steps of sphingosine WL gum are as follows: bacterial exopolysaccharide sphingosine WL gum was prepared by fermentation of a Sphingomonas WG (sphingamonas sp.wg, accession number cctccc No. m 2013161) strain. Culturing in a conical flask during fermentation, wherein the whole process is cultured in a vertical constant temperature oscillator, the temperature is set to 28 ℃, and the rotating speed is set to 150rpm; the strain transferring process is carried out in an aseptic workbench, the aseptic workbench is sterilized for 30min before operation, and 75% alcohol is sprayed before entering the aseptic workbench.

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

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

The crude product was redissolved in water and stirred for three days to give a crude solution of sphingosine WL gum. Adding an isopropanol solution containing 5% sodium chloride into the crude solution, wherein the volume ratio of isopropanol to the crude solution is 4:1, mixing and stirring for 2h, and reprecipitating overnight. The precipitate was filtered and washed with graded concentrations of aqueous isopropanol (isopropanol to water volume ratios of 10:0,9:1,8:2 and 7:3) to remove impurities. Finally, dialysis was performed for 3 days. The pure sphingosine gel product is obtained after freeze drying, and the removal of nucleic acid and protein is confirmed by ultraviolet test.

2. Preparation of citric acid crosslinked sphingosine WL glue gel

Citric Acid (CA) is dissolved in water to prepare a high-concentration citric acid solution, and different volumes of citric acid aqueous solutions are respectively added into 1wt% of a sphingosine WL glue (WL) solution. Finally, in 20mL of ultrapure water, 0.2g of the sphingosine WL gum and 0g, 0.01g, 0.02g or 0.04g of citric acid were mixed uniformly by stirring for 3 days using a mechanical stirrer. And carrying out ultrasonic treatment on the uniform viscous solution for 30min, vacuumizing until the solution is bubble-free, pouring the solution into a polytetrafluoroethylene mould with the length of 10cm, the width of 3cm and the height of 0.5cm, and strickling. Subsequently, the sample was placed in a 30 ℃ oven to remove moisture and held at 80 ℃ oven for 24 hours. Finally, the WL-CA film was immersed in pure water to ph=7 to wash out unreacted citric acid, and then dried in an oven at 30 ℃. The resulting hydrogel film was stored in a dry dish. The mass ratio of CA to WL was 0, 0.05, 0.10 and 0.20. The corresponding WL-CA films are designated WL-CA0, WL-CA5, WL-CA10, WL-CA20.

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

1. Preparation of graphene oxide

2g of graphite powder, 1g of sodium nitrate and 46mL of concentrated sulfuric acid are sequentially added into a 500mL beaker, and the mixture is subjected to ice water bath at 0 ℃ for 1h; slowly adding 6g of potassium permanganate in total amount of about 0.2g each time under ice bath condition, and continuing to react for 1h after about 1h; the mixture was then heated to 35℃with stirring, the reaction was continued for 3 hours, 92mL of ultrapure water was added dropwise and heated to 95℃and reacted at 95℃for 0.5 hour. After dilution with 184mL of ultrapure water, unreacted potassium permanganate was reduced again with 30% hydrogen peroxide until the slurry became earthy yellow. After cooling in air, the mixture was centrifuged (10000 rpm,10 min) at room temperature by washing with 30% hydrochloric acid three times, unreacted potassium permanganate was removed, and then washed with ultrapure water until the solution became gel. Ultrasonic treatment is carried out on the washed Graphene Oxide (GO) aqueous solution for 80 minutes, and unreacted crystalline flake graphite and GO with low oxidation degree are stripped; centrifuging (3500 rpm,10 min) to obtain the upper liquid, and collecting GO dispersion.

100mL of GO dispersion was dialyzed in 1000mL of ultra pure water for three days, with water changed every 2 hours on the first day and every 4 hours on the second day. Freeze-drying to obtain dark brown GO freeze-dried sample. The freeze-dried graphene oxide is dissolved in water, the concentration is 5mg/mL, and the ultrasonic treatment is carried out for 1h, so that the stable state can be maintained for more than 20 days.

2. Preparation of 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, so that a uniform GO dispersion solution is obtained.

Sphingosine WL gum and citric acid were added to 60mL of ultrapure water, and the solution was stirred at 500rpm for 3 days using mechanical stirring to mix the solution uniformly. Wherein the mass fraction of the sphingosine WL glue is 1wt%, and the mass of the citric acid is 10% of the sphingosine WL glue. The mixed solution is divided into 5 parts averagely, transferred into 50mL centrifuge tubes, and GO dispersion solutions with different volumes are added into the four centrifuge tubes, so that the mass ratio of graphene oxide to sphingosine WL glue is 0,0.001,0.01,0.05 and 0.1 respectively. The hydrogel pre-solution was mixed for 30min using a vortex machine, followed by sonication for 30min. The mixed pre-solution was evacuated of bubbles, and then the mixed liquid without bubbles was spread on a polytetrafluoroethylene mold (mold size: length×width×height=3 cm×2cm×0.5 cm). And (3) placing the hydrogel pre-solution in an oven, drying at 30 ℃, and then heating to 80 ℃ for reaction for 24 hours to obtain the WG-CA-GO hydrogel film. 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 and no graphene oxide were added to obtain a hydrogel film, designated WL; and adding graphene oxide with the mass ratio of 0.01 to the sphingosine WL glue without adding citric acid to obtain a hydrogel film named WL-GO1.

Example 3: supported ciprofloxacin on hydrogel

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

Analysis of results

1. In vitro cytotoxicity test

To evaluate the potential of WL-CA based hydrogels as wound dressing, 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 method is a more common method, and after the WL-CA0, WL-CA10 and WL-CA-CIP hydrogel extraction media are used for being cultured together with cells for 24 hours, the cell viability of the mice NIH 3T3 and HUVECs is higher than 90% and the cell activity of 293E cells is higher than 80% as detected by the MTT method. ISO 10993-5 states that the material is considered cytotoxic in the event of a drop in viability of more than 30%, so that these hydrogels have excellent biocompatibility and are 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 infrared spectra of WL-CA5, WL-CA10 and WL-CA20 are very similar to WL-CA0, and all possess characteristic absorption peaks of sphingosine WL glue. There was no new infrared absorption peak after the crosslinking reaction because of 1724cm ^-1 The absorbance (A1724) of (A) corresponds not only to the new ester bond formed in the esterification crosslinking but also to the O-acetyl group of WL. For WL-CA0, WL-CA5, WL-CA10 and WL-CA20, A1724 and 896cm ^-1 The ratio of β -1-4 glycosidic bond reference band (A896) was 3.4, 3.8, 4.3 and 4.7, respectively (FIG. 3), indicating that more ester bonds formed with increasing CA. Furthermore, 3300cm ^-1 The absorbance (A3300) of (C) corresponds mainly to the-OH of WL, which can react with the-COOH of CA. The ratios of A3300 and A896 were 2.1, 1.7, 1.5 and 1.2 for WL-CA0, WL-CA5, WL-CA10 and WL-CA20, respectively (FIG. 3), which means that more hydroxyl groups reacted as CA increased. These results indicate that as the citric acid content increases, more esterification reactions occur between the-OH of WL and the-COOH of CA, 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 verify the presence of chemical reactions from the side and confirm the difference in thermal stability of films of different citric acid content. All films showed a weight loss in three stages. In the first stage, there is a small mass loss of WL-CA film, approximately 3%, except for citric acid, due to evaporation of free and bound water. The temperature range at which the quality loss of the WL-CA film containing citric acid occurs is smaller than that of the physical hydrogel WL-CA 0. The reason for this is the reduced consumption of hydroxyl groups when WL and CA cross-link, 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 cross-link with citric acid by esterification, which reduces the hydroxyl content in WG. The second stage of WL-CA0 starts at 200deg.C, above those films where chemical cross-linking is present (175 deg.C for WL-CA5, 193 deg.C for WL-CA10, 165 deg.C for WL-CA 20). One possible explanation is that the crosslinking reaction can reduce the number of residual hydrogen bonds in the WL-CA film, reducing the energy required for degradation. In contrast to WL-CA20, WL-CA10 has a lower rate of mass loss and its maximum rate of mass loss is at the highest temperature point (269 ℃ C.), which indicates that the cross-linking formed between WL and CA improves the thermal stability of the film, but that excessive CA (e.g., WL-CA 20) reduces the stability. In fig. 5, the crosslinked film showed an additional shoulder around the temperature reached 240 ℃, which may be due to excessive CA. The above results confirm the formation of chemical crosslinks in WL-CA5, WL-CA10 and WL-CA20.

3. Analysis of pH responsive swelling behavior

Swelling behavior of WL-CA films in PBS at pH 9.5, 7.4 and 5.5 was studied. It was found by observation that after swelling in PBS for 2 hours, there was no significant change in the diameters of WL-CA0, WL-CA5, WL-CA10 and WL-CA20. However, the thickness was greatly varied, increasing by about 50 times (2 mm/40 μm) (FIG. 6). And the film changed from translucent to white opaque.

The water absorption capacity of the WL-CA film was further quantitatively evaluated using a weighing method. As shown in FIG. 7, all of the WL-CA0, WL-CA5, WL-CA10 and WL-CA20 films exhibited rapid swelling capacity within the first 0.5 hour, and all of the hydrogel films reached swelling equilibrium within 2 hours. While the swelling degree of the hydrogel decreases with the increase of the crosslinking agent. The reason is that the increase of the crosslinking agent increases the crosslinking density of the hydrogel, which prevents the diffusion of water and limits the swelling of the hydrogel. For WL-CA films, the water uptake capacity increases with pH due to deprotonation of the carboxyl groups, leading to 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 super absorbent nature of WL-CA hydrogel films is suitable for use in wound dressings, absorbing wound exudates. To test the stability of WL-CA hydrogels in water, the swelling rate was hardly changed after swelling WL-CA0, WL-CA5, WL-CA10 and WL-CA20 films in PBS at ph=9.5, ph=7.4 and ph=9.5, ph=5.5 for 87 hours. This shows that after the swelling equilibrium is reached, the weight of all WL-CA hydrogels does not decrease (fig. 8), i.e. the hydrogel structures have a certain stability in aqueous solution and are not easily hydrolyzed, and as a dressing, wound exudates can be effectively absorbed, and the risk of maceration of the wound and healthy tissue around the wound can be reduced.

4. Porous morphology and porosity analysis

The ability of hydrogels to absorb and retain moisture is closely related to the porosity of the hydrogels. First, morphological properties of the swollen hydrogel were observed by SEM. In FIG. 9, all WL-CA hydrogels showed three-dimensional porous network structures. 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 sees the porous morphology of the hydrogel; other covalently linked hydrogels did not exhibit the characteristics of WL-CA0 hydrogels, indicating that chemical cross-linking effectively protected the microscopic morphology of the hydrogels.

From Image J software analysis, the pore diameters of the swollen WL-CA5, WL-CA10 and WL-CA20 (pH=5.5) were-102.9 μm, -39.6 μm and-9.4 μm, respectively, the pore diameters of WL-CA5, WL-CA10 and WL-CA20 (pH=7.4) were-242.1 μm, -54.9 μm and-17.3 μm, respectively, and the pore diameters of WL-CA5, WL-CA10 and WL-CA20 (pH=9.5) were-246.9 μm, -71.1 μm and-20.0 μm, respectively. These results indicate that as the crosslinking agent increases, 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 is generated 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. Analysis of skin adhesion and rheological Properties

WL-CA10 hydrogel was applied to the skin, including the elbow or interphalangeal joint skin, during articulation and found to be free of any retraction or rupture (fig. 10). The WL-CA hydrogel adhered to the skin was subjected to a tearing test, the hydrogel was easily separated from the skin tissue, no residue was left, and the tearing process did not produce pain. The WL-CA hydrogels were shown to have moderate tissue adhesion and may not be prone to secondary injury from wounds when the hydrogels were replaced.

The rheological properties of the hydrogels were measured by a rotarheometer at a strain of 1% and a modulus as a function of frequency. 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 indicated that the hydrogels had good solid properties. The G 'and G' of the hydrogels WL-CA0 in the physically crosslinked state increased significantly with increasing frequency, indicating the presence of physical crosslinking. As the citric acid content increases, the storage modulus value of the covalently linked hydrogels increases and the dependence on frequency decreases, indicating that hydrogels with higher chemical crosslink densities have relatively better stability and stiffer networks. The modulus value of WL-CA20 is particularly weakly dependent on frequency and exhibits solids-like behavior. The WL-CA5 has a G ' of about 0.6kPa, the WL-CA10 has a G ' of about 1.2kPa, and the WL-CA20 has a G ' of about 2kPa, indicating that the WL-CA hydrogel has a certain mechanical strength.

6. Drug release profile

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

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

Drug release experiments were performed using swelling experimental conditions and three pH PBS solutions were used as release media, the results are shown in fig. 12. As can be seen, the explosive 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. Burst release may be caused by desorption of ciprofloxacin adsorbed on the surface layer of the hydrogel and rapid gel swelling as described above. Burst release results in a high exposure of the antimicrobial drug to the wound site, providing immediate and effective prevention of bacterial infection. In the first 6 hours, the CIP cumulative release decreased with increasing pH, mainly because ciprofloxacin 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, at ph=9.5 and ph=5.5, approximately 90% of the loaded CIP was released into the surrounding PBS, while at ph=7.4 only 75% was released.

7. Analysis of antibacterial Activity

Gram-negative E.coli and gram-positive Staphylococcus aureus are common, representative pathogens in wound healing. Gram positive bacteria including staphylococcus aureus and streptococcus pyogenes appear in early stages of wound formation; after a period of time has elapsed from the formation of the wound, gram-negative bacteria such as E.coli and P.aeruginosa may appear in the wound.

First, antibacterial activities of WL-CA0, WL-CA10 and WL-CA-CIP hydrogels against E.coli and Staphylococcus aureus in vitro were studied by a zone of inhibition test, and the results are shown in FIG. 13A, in which inhibition zones of WL-CA0, WL-CA10 and WL-CA-CIP against E.coli were 0cm, 1.3 cm and 4.9 cm, respectively, after 24 hours of cultivation. In contrast, only WL-CA-CIP showed a distinct inhibition zone for Staphylococcus aureus, with a zone of inhibition up to 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 the ciprofloxacin.

In addition, to test for long-term antimicrobial activity, fresh PBS solution and freshly activated Staphylococcus aureus or Escherichia coli suspension were used daily, co-cultured with WL-CA0, WL-CA10 and WL-CA-CIP, and surviving colonies were characterized using plate coating and photographic recording. From FIGS. 13C & D, 14, and 15, WL-CA10 kills E.coli within the first three days, with no antibacterial ability against Staphylococcus aureus; WL-CA-CIP inhibited escherichia coli growth for 10 days and staphylococcus aureus for 7 days. The long-term antimicrobial properties of hydrogels may be related to the incomplete release of drug present in WL-CA10 hydrogels. All antibacterial test results show that the WL-CA-CIP has good long-term antibacterial effect.

8. Mechanical property analysis of graphene oxide modified hydrogels

In order to study the influence of graphene oxide on the performance of the WL-CA-GO hydrogel film, the mechanical behaviors of WL-CA-GO layered hybrid films with different graphene oxide contents are studied 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, and can be distorted arbitrarily. It can be seen that the addition of graphene oxide significantly improved 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 WL-CA-GO0.1 has too little graphene oxide content, the hydrogel film has lower hardness and does not have stretching behavior. The graphene oxide content in the WL-CA-GO5 and WL-CA-GO10 hydrogels is too high, so that the hydrogel films are too high in hardness and are easy to break.

The storage modulus (G ') and loss modulus (G') of each sample were evaluated using an oscillatory rheology measurement method. When the oscillatory shear strain was set at 1% and the angular frequency increased from 0.1rad/s to 100rad/s, each group had a G' significantly higher than G "and no intersecting traces (FIG. 17), indicating that WL-CA10 hydrogels were predominantly elastic in nature. More importantly, as the GO concentration increases, the G 'of the GO-enhanced hydrogels increases gradually, up to 7kPa, and as the GO concentration increases, G' is significantly higher than the no GO hydrogels, indicating the presence of a more stable, stiffer network system in the GO composite hydrogels.

Furthermore, to investigate 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 was compared. As is evident from fig. 18, the elastic behavior of the hydrogel film with added citric acid is significantly better than that of the hydrogel without citric acid, and the elastic modulus of the four hydrogels are ordered from small to large: WL, WL-GO1, WL-CA10 and WL-CA-GO1. It is demonstrated that the presence of a chemically crosslinked network can enhance the hardness and stability of the hydrogel network, and that the hydrogel film incorporating citric acid is more resistant to external denaturation than the physically crosslinked hydrogel (WL, WL-GO 1).

While specific embodiments of the invention have been described in detail in connection with the examples, it should not be construed as limiting the scope of protection of the patent. Various modifications and variations which may be made by those skilled in the art without the creative effort are within the scope of the patent described in the claims.

Claims (7)

1. The sphingosine-based hydrogel is characterized by comprising the following raw materials in parts by mass: 20 parts of sphingosine WL glue and 2 parts of citric acid; the preparation method comprises the steps of preparing a sphingosine WL glue, and further comprises graphene oxide, wherein the mass of the graphene oxide is 0.1-10% of the mass of the sphingosine WL glue;

the sphingosine WL glue is prepared through the following steps:

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

s2: adding the first-stage seed liquid into a seed culture medium according to the volume ratio of 1:20, and culturing for 16 hours at constant temperature to obtain a second-stage seed liquid;

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

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

s5: dissolving a sphingosine WL glue crude product in water, mixing the obtained solution with isopropanol containing 5wt% of sodium chloride according to a volume ratio of 1:4, standing until precipitation is not increased, separating the precipitation, washing with isopropanol water solution with gradient concentration, dialyzing, and drying to obtain the sphingosine WL glue;

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 product;

the fermentation medium is prepared by the following steps: 73.8g of glucose, 0.205g of magnesium sulfate heptahydrate, 2g of monopotassium phosphate, 3g of yeast powder and 4g of dipotassium phosphate are dissolved in 1000mL of ultrapure water, and sterilized at 121 ℃ for 25min to obtain the compound.

2. The sphingosine-based hydrogel according to claim 1, wherein the graphene oxide is prepared by the steps of:

s1: the preparation method comprises the steps of co-dissolving graphite powder and sodium nitrate in concentrated sulfuric acid, reducing the temperature of a mixture to 0 ℃, adding potassium permanganate, reacting for 1h, heating to 35 ℃, continuing to react for 3h, adding ultrapure water, heating to 95 ℃, preserving heat, reacting for 0.5h, washing with 30% hydrochloric acid and ultrapure water in sequence until the solution becomes gelatinous, performing ultrasonic treatment for 80min, and centrifuging to obtain upper-layer liquid; the material ratio of the graphite powder to the sodium nitrate to the concentrated sulfuric acid is 2g to 1g to 46mL, and the mass ratio of the added potassium permanganate to the graphite powder is 3 to 1;

s2: and sequentially dialyzing and freeze-drying the supernatant to obtain the product.

3. The method for preparing a sphingosine-based hydrogel according to claim 1, comprising the steps of:

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

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

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

4. Use of the sphingosine-based hydrogel according to any of claims 1 to 2 for the preparation of a wound dressing.

5. The use according to claim 4, characterized in that: the wound dressing includes a sphingosine-based hydrogel substrate and an antimicrobial drug supported on the substrate.

6. The use according to claim 5, characterized in that: the antibacterial drug is ciprofloxacin.

7. The use according to claim 6, wherein the wound dressing is prepared by the steps of:

s1: ciprofloxacin is dissolved in hydrochloric acid solution to form a drug solution with the concentration of 20mg/mL, and the pH=3 of the drug solution is adjusted;

s2: immersing the sphingosine-based hydrogel substrate into a medicinal solution, rotating and culturing for 24 hours at 37 ℃, taking out the sphingosine-based hydrogel substrate, and drying at 30 ℃ to obtain the sphingosine-based hydrogel.