A sorangium / hyaluronic acid antibacterial hydrogel and a preparation method and application thereof

Abstract

The application discloses a preparation method of a solagel / hyaluronic acid hydrogel, and the method comprises the following steps: solagel is grafted with adipic acid dihydrazide through an amide reaction to obtain adipic acid dihydrazide solagel; sodium periodate is used to oxidize hyaluronic acid to obtain oxidized hyaluronic acid; and the adipic acid dihydrazide solagel and the oxidized hyaluronic acid are reacted through a Schiff base reaction to obtain the solagel / hyaluronic acid hydrogel. The adipic acid dihydrazide solagel, the oxidized hyaluronic acid and PHMB are blended to prepare the PHMB / Sal-ADH / OHA hydrogel, the hydrogel has good self-healing characteristics, biocompatibility, antibacterial performance and wound healing promotion performance, is an ideal biomaterial, and provides a new idea for development of a hydrogel wound dressing.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medical devices, and particularly relates to a sorghum gum / hyaluronic acid antibacterial hydrogel and a preparation method and application thereof. BACKGROUND

[0002] The information disclosed in this Background section is only for the purpose of increasing the understanding of the background of the present application and should not be taken as an acknowledgement or any form of suggestion that this information forms prior art that is already commonly known to a person of ordinary skill in the art.

[0003] As a protective barrier against harsh environments, human skin is often in conflict and inevitably suffers from trauma. When the skin is damaged, the damaged area is prone to bacterial infection, which hinders wound healing and even causes serious complications. Scientists have devoted to developing wound dressings for clinical practice. Traditional wound dressings such as gauze and cotton cloth have good moisture absorption and can absorb wound exudates, but their promotion of wound healing is limited. In order to promote wound healing, researchers have improved gauze and cotton cloth by introducing antibacterial drugs into cotton cloth and gauze, such as loading antibacterial polymers to prevent bacterial infection of wounds. However, the problems of gauze and cotton cloth cannot be changed, and cotton cloth and gauze will adhere to the newly growing granulation tissue and cause pain and secondary damage when removed. Compared with dry dressings, wet dressings not only prevent damage to the wound but also increase the speed of epithelialization. Hydrogels have good flexibility and biocompatibility, and hydrogels with antibacterial activity are considered to be good candidates for wound wet dressings. However, the internal network structure of traditional hydrogels is irreversible, and when damaged by external force, the hydrogel will crack and lead to bacterial invasion. Therefore, it is of great significance to prepare a hydrogel dressing with good antibacterial performance and self-repairing ability in wound repair. SUMMARY

[0004] In order to overcome the above problems, the present application provides a sorghum gum / hyaluronic acid antibacterial hydrogel and a preparation method and application thereof. The present application uses adipic acid dihydrazide to react with sorghum gum to obtain sorghum gum with hydrazide groups at the end, and oxidizes hyaluronic acid with sodium periodate to obtain hyaluronic acid with aldehyde groups. Based on the dynamic acylhydrazone bond, a hydrogel with self-repairing performance is constructed using the two as raw materials, and an antibacterial agent polyhexamethylene biguanide (PHMB) is introduced to improve the antibacterial ability of the hydrogel.

[0005] To achieve the above technical purposes, the present application adopts the following technical solutions:

[0006] In a first aspect of the present application, a preparation method of a sorghum gum / hyaluronic acid hydrogel is provided, the method comprising:

[0007] Salecan (Sal) is grafted with adipic acid dihydrazide (ADH) by an amidation reaction to obtain adipic acid dihydrazide-modified Salecan (Sal-ADH); hyaluronic acid (HA) is oxidized by sodium periodate to obtain oxidized hyaluronic acid (OHA); and Salecan is grafted with adipic acid dihydrazide to obtain adipic acid dihydrazide-modified Salecan (Sal-ADH), and the Salecan grafted with adipic acid dihydrazide is reacted with oxidized hyaluronic acid by a Schiff base reaction to obtain a Salecan / hyaluronic acid (Sal-ADH / OHA) hydrogel.

[0008] In a second aspect of the present application, the Salecan / hyaluronic acid hydrogel prepared by the above preparation method is provided.

[0009] In a third aspect of the present application, a preparation method of a Salecan / hyaluronic acid antibacterial hydrogel is provided, and the method comprises the following steps:

[0010] Polyhexamethylene biguanide hydrochloride (PHMB) is added to the above Salecan / hyaluronic acid hydrogel, and after sufficient stirring, a Salecan / hyaluronic acid antibacterial (PHMB / Sal-ADH / OHA) hydrogel is obtained.

[0011] In a fourth aspect of the present application, the Salecan / hyaluronic acid antibacterial hydrogel prepared by the above method is provided.

[0012] In a fifth aspect of the present application, a wound dressing is provided, and the wound dressing comprises the above Salecan / hyaluronic acid antibacterial hydrogel.

[0013] In a sixth aspect of the present application, the above Salecan / hyaluronic acid antibacterial hydrogel is applied to skin repair, wound suture or wearable devices.

[0014] The present application has the following beneficial effects:

[0015] (1) The present application prepares a PHMB / Sal-ADH / OHA hydrogel by blending adipic acid dihydrazide-modified Salecan with oxidized hyaluronic acid and PHMB, and the hydrogel has good self-healing properties, biocompatibility, antibacterial performance and wound healing promotion performance, and is an ideal biomaterial, which provides a new idea for the development of hydrogel wound dressings.

[0016] (2) The Salecan / hyaluronic acid hydrogel prepared by the present application exhibits good swelling behavior in a liquid; and the macroscopic healing experiment and the continuous strain and recovery scanning test results of the hydrogel show that the hydrogel has self-healing properties.

[0017] (3) The cell survival rate of the sorangium cellulosum / hyaluronic acid antibacterial hydrogel prepared in the application was determined by MTT experiment, and the cell survival rate of all hydrogel groups was more than 80% after the hydrogel extract was co-incubated with L929 cells for 24 hours and 48 hours. In the live / dead cell staining experiment, most of the L929 cells were green live cells with spindle shape and uniform distribution, and there were basically no red cells and dead cells. In addition, the hemolysis rate of the hydrogel was within 5% in the hemolysis experiment, which proved that the hydrogel had good biocompatibility.

[0018] (4) Further experiments by animal wound experiment showed that the sorangium cellulosum / hyaluronic acid antibacterial hydrogel prepared in the application could promote the healing process of wound tissue. BRIEF DESCRIPTION OF DRAWINGS

[0019] The drawings accompanying the specification of the application form a part of the application and serve to further understand the application. The illustrative embodiments of the application and their description serve to explain the application without constituting an improper limitation of the application.

[0020] Figure 1 is a scanning electron microscope image of the sorangium cellulosum / hyaluronic acid hydrogel prepared in Example 1;

[0021] Figure 2 is a sorangium cellulosum / hyaluronic acid hydrogel swelling property graph prepared in Example 1, wherein a is a hydrogel swelling rate graph, and b is an equilibrium swelling rate graph;

[0022] Figure 3 is a sorangium cellulosum / hyaluronic acid hydrogel water vapor permeability graph prepared in Example 1;

[0023] Figure 4 is a sorangium cellulosum / hyaluronic acid hydrogel self-healing graph prepared in Example 1;

[0024] Figure 5 is a sorangium cellulosum / hyaluronic acid hydrogel mechanical property graph prepared in Example 1, wherein a is a compression strain curve, and b is a compression strength;

[0025] Figure 6 is a sorangium cellulosum / hyaluronic acid hydrogel continuous strain recovery scanning test graph prepared in Example 1, wherein a is Gel-0.6, b is Gel-0.7, c is Gel-0.8, d is Gel-0.9, and e is Gel-1.0;

[0026] Figure 7 is a scanning electron microscope image of the sorangium cellulosum / hyaluronic acid antibacterial hydrogel prepared in Example 3

[0027] Figure 8Figure 3 is a drug release profile of the solagene / hyaluronic acid antibacterial hydrogel prepared in Example 3, wherein a is the drug release profile of different antibacterial gels, b is the drug release profile of SOP-0.10 in PBS solution at different pH values (pH = 5, 7.4);

[0028] Figure 9 Figure 4 is an injectability analysis profile of the solagene / hyaluronic acid antibacterial hydrogel prepared in Example 3, wherein a is a macroscopic injection profile, b is a profile of the viscosity of the hydrogel as a function of shear rate.

[0029] Figure 10 Figure 5 is an antibacterial performance profile of the solagene / hyaluronic acid antibacterial hydrogel prepared in Example 3, wherein a is a bacterial inhibition zone profile of the hydrogel, b is an OD value of the antibacterial hydrogel.

[0030] Figure 11 Figure 6 is a cell survival rate profile of L929 cells co-incubated with the extract of the solagene / hyaluronic acid antibacterial hydrogel prepared in Example 3 for different times, wherein a is 24 h, b is 48 h.

[0031] Figure 12 Figure 7 is a live / dead staining profile of L929 cells cultured with the extract of the hydrogel for 1 day.

[0032] Figure 13 Figure 8 is a hemolysis profile of the solagene / hyaluronic acid antibacterial hydrogel prepared in Example 3, wherein a is a hydrogel hemolysis effect profile, b is a hydrogel hemolysis rate profile.

[0033] Figure 14 Figure 9 is a mouse wound healing profile, wherein a is a wound healing change, b is a wound healing rate. DETAILED DESCRIPTION

[0034] It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0035] Salopolysaccharide-based hydrogels are usually prepared with toxic cross-linking agents or monomers for copolymerization to enhance the mechanical properties of the hydrogel, which has a large safety hazard and limits its use in clinical applications. Hyaluronic acid lacks active groups for rapid gelation, so it is necessary to modify solagene and hyaluronic acid to broaden their application prospects in wound dressings. The first typical embodiment of the present application provides a preparation method of a solagene / hyaluronic acid hydrogel, which comprises:

[0036] The solagum is grafted with adipic acid dihydrazide by an amide reaction to obtain adipic acid dihydrazide solagum; the hyaluronic acid is oxidized by sodium periodate to obtain oxidized hyaluronic acid; and the adipic acid dihydrazide solagum and the oxidized hyaluronic acid are reacted by a Schiff base reaction to obtain the solagum / hyaluronic acid hydrogel.

[0037] In one or more embodiments, the specific synthesis method of the solagum grafted with adipic acid dihydrazide by an amide reaction to obtain adipic acid dihydrazide solagum is as follows: the solagum and adipic acid dihydrazide are added to water, and then stirred at room temperature for 25-40 min; a hydrochloric acid solution is used to adjust the pH to 4-5; 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) is added, and then a hydrochloric acid solution is used to adjust the pH to 4-5, and the stirring is continued for 3-5 h; a NaOH solution is used to adjust the pH to 7 to terminate the reaction; the reaction solution is placed in a dialysis bag for dialysis, and the solution after dialysis is freeze-dried for 30-50 h to obtain the adipic acid dihydrazide solagum.

[0038] Preferably, the mass ratio of the solagum, adipic acid dihydrazide, 1-ethyl-(3-dimethylaminopropyl) carbodiimide, and water is 0.2-0.5:1.8-2.2:0.5-0.6:18-22 0, and more preferably 0.3:2.08:0.57:200.

[0039] Preferably, the solagum and adipic acid dihydrazide are added to water, and the stirring time at room temperature is 30 min.

[0040] Preferably, the concentration of the hydrochloric acid solution is 0.05-0.15 mol / L, and more preferably 0.1 mol / L.

[0041] Preferably, the concentration of the NaOH solution is 0.05-0.15 mol / L, and more preferably 0.1 mol / L.

[0042] Preferably, the hydrochloric acid solution is used to adjust the pH to 4.75.

[0043] Preferably, the molecular weight cut off of the dialysis bag is 7000 Da.

[0044] Preferably, the specific dialysis method is as follows: the dialysis is performed with a 0.1 mol / L NaCl aqueous solution for 24 h, then with 25% ethanol for 24 h, and finally with pure water for 4 days, and the dialysis solution is changed at least three times a day during the dialysis to remove unreacted substances.

[0045] Preferably, the freeze-drying time is 48 h.

[0046] In one or more embodiments, the specific synthesis method of oxidized hyaluronic acid obtained by sodium periodate oxidizing hyaluronic acid is as follows: NaIO4 and HA are dissolved in water respectively to obtain a NaIO4 solution and an HA solution, the NaIO4 solution is added dropwise into the HA solution, and the solution is stirred in the dark for 5-7 hours, then ethylene glycol is added to terminate the reaction, and the stirring is continued for 0.5-2 hours, the reaction solution is placed in a dialysis bag for dialysis, and the solution after dialysis is freeze-dried for 30-50 hours to obtain the oxidized hyaluronic acid.

[0047] Preferably, the mass ratio of NaIO4 to HA is 1.5-2.5:1.1-1.3, and preferably 2.0:1.13; the concentration of the NaIO4 solution is 0.4-6 mol / L, and preferably 0.5 mol / L; and the concentration of the HA solution is 8-12 mg / mL, and preferably 10 mg / mL.

[0048] Preferably, the stirring in the dark is performed for 6 hours.

[0049] Preferably, the mass ratio of NaIO4 to ethylene glycol is 1.5-2.5:0.5-0.6, and preferably 2.0:1.1.

[0050] Preferably, the stirring is continued for 1 hour.

[0051] Preferably, the specific method of dialysis is as follows: the dialysis is performed with pure water for 3 days, and the dialysate is changed at least three times a day during the dialysis to remove unreacted substances through dialysis.

[0052] Preferably, the freeze-drying is performed for 48 hours.

[0053] In one or more embodiments, the specific method of obtaining the solagel / hyaluronic acid hydrogel by Schiff base reaction of adipic acid dihydrazide solagel and oxidized hyaluronic acid is as follows: the adipic acid dihydrazide solagel is dissolved in water, heated in a water bath at 70-90°C for 15-25 minutes, then the oxidized hyaluronic acid is added, and the solagel / hyaluronic acid hydrogel is obtained after stirring for 20-40 seconds and cooling and standing.

[0054] Preferably, the temperature of the water bath is 80°C; and the heating is performed for 20 minutes.

[0055] Preferably, the stirring is performed for 30 seconds.

[0056] Preferably, the mass ratio of adipic acid dihydrazide solagel to oxidized hyaluronic acid is 0.6-1.0:0.2, and further preferably 0.8:0.2.

[0057] Preferably, the concentration of adipic acid dihydrazide solagel is 0.6-1.0 wt%, preferably 0.8 wt%; and the concentration of oxidized hyaluronic acid is 0.2 wt%.

[0058] In a second exemplary embodiment of the present application, a solagel / hyaluronic acid hydrogel prepared by the above method is provided.

[0059] In a third exemplary embodiment of the present application, a method for preparing a solagel / hyaluronic acid antibacterial hydrogel is provided, which comprises:

[0060] Polyhexamethylene biguanide hydrochloride (PHMB) is added to the solagel / hyaluronic acid hydrogel, and after sufficient stirring, a solagel / hyaluronic acid antibacterial (PHMB / Sal-ADH / OHA) hydrogel is obtained.

[0061] In one or more embodiments, the solagel / hyaluronic acid hydrogel needs to be prepared on demand.

[0062] In one or more embodiments, the concentration of polyhexamethylene biguanide hydrochloride is 0.05 wt%-0.20 wt%, preferably 0.05 wt%, 0.10 wt%, 0.15 wt%, or 0.20 wt%.

[0063] In one or more embodiments, the stirring time is 20-40 s, preferably 30 s.

[0064] In a fourth exemplary embodiment of the present application, a solagel / hyaluronic acid antibacterial hydrogel prepared by the above method is provided.

[0065] In a fifth exemplary embodiment of the present application, a wound dressing is provided, which comprises the above solagel / hyaluronic acid hydrogel and / or the above solagel / hyaluronic acid antibacterial hydrogel.

[0066] In a sixth exemplary embodiment of the present application, the above solagel / hyaluronic acid antibacterial hydrogel and / or the above solagel / hyaluronic acid antibacterial hydrogel is used in skin repair, wound suture, or wearable devices.

[0067] In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with specific examples.

[0068] Example 1: Preparation of solagel / hyaluronic acid hydrogel

[0069] Sal-ADH / OHA hydrogel was prepared as follows: 0.3 g Sal was dissolved in 200 mL pure water, 2.08 g ADH powder was added into the solution, stirred at room temperature for 30 min, the pH was adjusted to 4.75 with 0.1 mol / L HCl solution, then 0.57 g EDC powder was added and dissolved, the pH was adjusted to 4.75 again with 0.1 mol / L HCl solution, the reaction was continued for 4 hours, then the pH was adjusted to 7 with 0.1 mol / L NaOH solution to terminate the reaction. The obtained reaction solution was added into a dialysis bag (molecular weight cut-off 7000 Da), first dialyzed against 0.1 mol / L NaCl aqueous solution for 24 h, then against 25% ethanol for 24 h, and finally against pure water for 4 days, and the dialysis solution was changed at least three times a day to remove unreacted substances by dialysis. The dialyzed solution was freeze-dried for 48 h to obtain Sal-ADH, which was stored in a desiccator for later use.

[0070] 2.0 g HA powder was weighed and dissolved in 200 mL pure water to prepare a solution with a concentration of 10 mg / mL. 1.13 g NaIO4 was dissolved in 10.55 mL pure water (concentration 0.5 mol / L), and then the prepared NaIO4 solution was added dropwise into the HA solution, stirred in the dark for 6 h, then 1 mL of ethylene glycol was added to terminate the reaction, and the reaction was continued for 1 h. The reacted solution was placed in a dialysis bag (molecular weight cut-off 7000 Da) and dialyzed against pure water for 3 days. The dialyzed solution was freeze-dried for 48 h to obtain OHA.

[0071] A certain amount of Sal-ADH powder was dissolved in pure water, heated in a water bath at 80°C for 20 min, and then mixed with OHA at different ratios. After stirring for 30 s, the rotor was removed and the gel was allowed to cool and stand. The ratios of Sal-ADH and OHA in each gel are shown in Table 1.

[0072] Table 1 Composition of Sal-ADH / OHA hydrogel

[0073]

[0074] Example 2

[0075] In this example, the Sal gel / hyaluronic acid hydrogel prepared in Example 1 was characterized.

[0076] As Figure 1As shown, the microstructure of the hydrogels was observed by scanning electron microscopy. The hydrogels showed a porous structure, and the hydrogel network gradually became denser as the concentration of Sal-ADH increased, and the average pore size showed a decreasing trend. This is because the hydrazine groups on Sal-ADH provide active sites for OHA crosslinking agents to form acylhydrazone bonds. The higher the concentration of Sal-ADH, the higher the crosslinking density, and the more compact the microstructure of the hydrogel. This structure is conducive to quickly absorbing a large amount of water and facilitating the timely transport of cell nutrients and metabolic waste, and conducive to cell adhesion, proliferation and secretion of cytokines.

[0077] An ideal wound dressing should absorb exudates from the wound site, and the water absorption or swelling behavior of the hydrogel in water allows the penetration of nutrients and bioactive molecules. As shown in Figure 2 As shown in Figure a, the Sal-ADH / OHA hydrogels all showed strong water absorption capacity within the first 10 hours. When the concentration of Sal-ADH was 0.6wt%, the gel reached equilibrium swelling at 15 hours, and then the swelling degree showed a downward trend and then stabilized, because the hydrogel network structure was loose at this time, and Sal-ADH and OHA contained a large number of hydroxyl groups, resulting in the phenomenon of gel disintegration due to excessive swelling. As the concentration of Sal-ADH increased, the hydrogel tended to be stable after reaching equilibrium swelling, because the internal structure of the gel gradually became denser, and the structure of the hydrogel was more stable. As the concentration of Sal-ADH increased from 0.6% to 1.0%, the equilibrium swelling of the hydrogel decreased from 3133.5% to 2290.9%, because the higher the concentration of Sal-ADH, the higher the crosslinking density of the hydrogel. Therefore, due to the high crosslinking density, it is difficult for the hydrogel to swell, which increases the stability and structure of the hydrogel network, resulting in a decrease in the ability of the hydrogel to absorb liquid and a decrease in the swelling rate. The swelling degree of all hydrogels is above 2000%, confirming that the hydrogel shows good swelling behavior in liquid, indicating the potential of Sal-ADH / OHA hydrogel in wound dressing.

[0078] Water vapor transmission rate is a parameter for testing the applicability of materials for external applications such as wound dressings. For wound dressing materials, the moisture permeability should neither be too high, which leads to extreme water loss, nor too low, which can cause exudate accumulation. As shown in Figure 3 The water vapor transmission rate of different hydrogels is 3300-3050 g / m 2 / day, and the surface hydrogel has moderate permeability.

[0079] Hydrogel wound dressings may be subjected to mechanical forces and deformed, worn out or even damaged during use. Hydrogels with the ability of automatic self-healing can recover from injury, thereby effectively prolonging their service life. As shown in Figure 4As shown, two hydrogel films, one stained with methyl orange and the other unstained, were bonded together. After one hour, the hydrogels spontaneously healed into a single, intact piece. This macroscopically demonstrates the self-healing properties of the hydrogel. This property may be due to the dynamic chemical bonds of the acylhydrazone bonds within the hydrogel. These bonds can spontaneously form at room temperature through a Schiff base reaction, ultimately exhibiting the self-healing characteristics of the hydrogel.

[0080] like Figure 5 As shown, with the Sal-ADH concentration increasing from 0.6% to 0.9%, the compressive stress rose from 17.18 kPa to 74.77 kPa. This is because the increased crosslinking density of the hydrogel led to a significant improvement in its mechanical properties. However, when the Sal-ADH concentration was 1.0 wt%, the hydrogel fractured at approximately 35% compressive strain, while it fractured at 50%. This indicates that excessively high crosslinking density leads to brittleness and a decrease in compressive stress. The compressive modulus of the hydrogel also showed the same trend as the pressure-strain curve.

[0081] The mechanical and self-healing properties of the hydrogel were assessed using rheological recovery testing. Figure 6 As shown in Figure b, after the strain increases from 1% to 400%, the storage modulus (G') of the hydrogel decreases and becomes less than the loss modulus (G”), indicating a sol state. When the strain decreases from 400% to 1%, the storage modulus G' of the hydrogel rapidly rises back to its original value, at which point the hydrogel recovers its gel structure. This demonstrates that these hydrogels have better recovery capabilities. These results indicate that the prepared Sal-ADH / OHA hydrogels possess self-healing properties, which may be because the groups forming the hydrogel are acylhydrazone bonds, which are dynamic chemical bonds. Acylhydrazone groups break when the gel structure is disrupted, but can re-form acylhydrazone bonds through a Schiff base reaction at low strain, thus forming a hydrogel and restoring the storage modulus G' value to its original value. Furthermore, the equilibrium creep compliance (Jo) was determined, defined as the ratio of G' to G” calculated from the end creep measurement region. Gel-0.8 was the highest among all composite hydrogels, at 96.31%, while the recovery rates of other hydrogels ranged from 44.9% to 75.9%. The results show that although different hydrogels can achieve partial structural recovery, they still lose some integrity under strain. Among the Sal-ADH / OHA hydrogels with different ratios, Gel-0.8 showed a greater recovery rate and exhibited better self-healing properties.

[0082] Example 3 (Preparation of a Sola Gel / Hyaluronic Acid Antibacterial Hydrogel)

[0083] A certain amount of Sal-ADH powder was dissolved in pure water, and after heating at 80°C for 20 min, it was mixed with OHA at different ratios, and different concentrations of PHMB were added. After stirring for 30 s, the rotor was removed, cooled, and left to form a gel. The concentrations of Sal-ADH and OHA in the antibacterial gel were 0.8 wt% and 0.2 wt%, and the concentrations of PHMB were 0.05 wt%, 0.10 wt%, 0.15 wt%, and 0.20 wt%. The antibacterial gels were named SOP-0.05, SOP-0.10, SOP-0.15, and SOP-0.20, respectively.

[0084] The morphology of the SOP hydrogel was examined by SEM in a freeze-dried state. As shown in Figure 7 , the SOP hydrogel has a porous and dense network structure with a pore size ranging from 200 to 100 μm. The addition of PHMB enhances the network structure of the gel, making the pore size of the hydrogel smaller and enhancing the cross-linking density of the hydrogel.

[0085] Example 4

[0086] The antibacterial gels SOP-0.05, SOP-0.10, SOP-0.15, and SOP-0.20 prepared in Example 3 were subjected to drug release analysis.

[0087] The prepared antibacterial hydrogels SOP-0.05, SOP-0.10, SOP-0.15, and SOP-0.20 were placed in centrifuge tubes containing 10 mL of PBS solution, and were placed in a constant temperature shaker at 37°C and 100 rpm. Samples were taken at different time intervals, and after each 2 mL of solution was taken, an equal volume of PBS buffer was added to the centrifuge tube until the end of the set time node. The absorbance value of the solution was measured at 235 nm using a UV spectrophotometer, and a release curve was made to determine the concentration of PHMB in the sample, as shown in Figure 8 .

[0088] As can be seen from Figure 8 , within the first 8 hours, the drug shows rapid release, and there is a certain burst release behavior. In the later stage, PHMB is released in a sustained and stable manner. The dense network of the composite material also helps to delay the diffusion of PHMB as a physical barrier for fixing PHMB. When the PHMB content is high, the electrostatic interaction between the negative charges of Sal-ADH and hyaluronic acid and PHMB is strong, and the PHMB loaded inside the gel is difficult to overcome the restraint of Sal-ADH and hyaluronic acid and be fully released. When the PHMB content is low, the negative charges of Sal-ADH and hyaluronic acid are neutralized by PHMB, and the restraint on PHMB is weakened, making the release amount of loaded PHMB significantly increased.

[0089] As can be seen from Figure 8As shown in Figure b, initially, due to the large concentration difference, the drug release rate of the PBS solution was rapid. Gradually, the drug release rates of the two PBS solutions showed differences. The hydrogel exhibited obvious pH-sensitive drug release behavior, and showed the fastest drug release rate in the PBS solution at pH=5. This is because the acylhydrazone bonds in the hydrogel network broke in the acidic microenvironment, releasing PHMB more quickly. Subsequently, the hydrogel exhibited a stable, long-term, but slower release.

[0090] Example 5

[0091] Injectability analysis was performed on the antibacterial gels SOP-0.05, SOP-0.10, SOP-0.15, and SOP-0.20 prepared in Example 3.

[0092] like Figure 9 As shown in Figure a, the hydrogel precursor solution was injected into a syringe, and then the hydrogel was squeezed into deionized water through the syringe needle. The hydrogel still maintained the characteristics of a gel and did not break, indicating that the hydrogel has injectable properties.

[0093] like Figure 9 As shown in Figure b, the viscosity of the hydrogel decreases with increasing shear rate, indicating that the hydrogel has good shear-thinning properties. From a clinical application perspective, this property is very advantageous in wound treatment because this hydrogel can be evenly applied to the wound and fill irregular defects.

[0094] Example 5

[0095] The in vitro antibacterial properties of the antibacterial gels SOP-0.05, SOP-0.10, SOP-0.15, and SOP-0.20 prepared in Example 3 were evaluated using Escherichia coli and Staphylococcus aureus.

[0096] Inhibition zone method: First, prepare solid and liquid culture media. The solid culture medium is prepared as follows: Take 25g LB broth and 15g agar powder, add 1000mL of deionized water, dispense into Erlenmeyer flasks, and autoclave at 121℃ for 20min. Then, take 15mL-20mL of the culture medium and pour it into sterile disposable plastic dishes. After cooling, invert and store at 4℃ in a sealed container. Activate and culture *Escherichia coli* and *Staphylococcus aureus* at 37℃ and 140rpm for 12h. Dilute the obtained bacterial suspension to the required concentration before use.

[0097] The hydrogel needs to be pretreated. Using a mold, the hydrogel is prepared into a shape with a diameter of 15 mm and a height of 10 mm. After formation, it is irradiated under ultraviolet light for 12 hours to prepare a sterile hydrogel. Take approximately 100 μL of the diluted bacterial suspension (10... 6CFU / mL), the bacterial suspension was evenly coated on the solid medium with a coating rod, the hydrogel was placed on the solid medium coated with the bacterial solution, and the culture was inverted and placed in a bacterial incubator at 37°C for 24 h. Then, the antibacterial activity was compared according to the inhibition zone. Figure 10 As shown in FIG. 8a, the size of the inhibition zone gradually increased with the increase of the PHMB concentration. The antibacterial ability of PHMB was derived from the interaction between the positively charged PHMB and the negatively charged phospholipids in the bacterial cell membrane when PHMB contacted with the bacteria, which destroyed the lipid bilayer and the integrity of the cell membrane, then penetrated into the cytoplasm, resulting in ion leakage and loss of function of the bacterial cells, and finally leading to cell death. Due to the strong antibacterial activity of PHMB, the SOP hydrogel showed strong antibacterial activity.

[0098] OD value method: 1 mL of the hydrogel was added to a 24-well plate, and the plate was irradiated under ultraviolet light for 12 h. Then, 1 mL of the bacterial suspension (about 10 8 CFU / mL) was added to the plate, and the plate was cultured in an incubator for 24 h. Then, the OD value at 600 nm was measured by an enzyme-labeled instrument. The smaller the OD value, the better the antibacterial effect.

[0099] As shown in FIG. 8b, the OD value of the SOP-treated bacterial suspension was much lower than that of the control group, indicating that the SOP had good antibacterial activity. Figure 10

[0100] Example 6

[0101] Cell compatibility of the antibacterial gels SOP-0.05, SOP-0.10, SOP-0.15, and SOP-0.20 prepared in Example 3.

[0102] MTT method: The four groups of SOP hydrogels were prepared into discs with a diameter of 20 mm and a thickness of 4 mm. Before the experiment, the hydrogels needed to be pretreated, and the sample treatment method was as follows:

[0103] Firstly, the four groups of weighed hydrogels were soaked in 75% ethanol for 4 h, which was to sterilize the hydrogels. Then, the hydrogels were washed with sterile PBS for 3 times, which was to remove the alcohol in the hydrogels. Then, the hydrogels were placed in DEME culture medium (according to the ratio of hydrogel mass to culture medium volume, 1 g: 10 mL), and incubated at 37°C on a shaker (100 rpm) for 24 h. Then, the hydrogels were taken out of the culture medium, and the remaining solution was filtered to obtain the hydrogel extract, and the extract concentration was 1, 5, 25, and 50 mg / mL, respectively. MTT solution was prepared by weighing 50 mg of MTT powder and dissolving it in 10 mL of PBS solution to obtain a stock solution with a final concentration of 5 mg / mL. After filtration with a sterile filter head of 0.22 μm, the solution was stored at -20°C in the dark. ​

[0104] Second, after reviving mouse fibroblasts (L929), the cells were suspended in DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody. L929 cells in the logarithmic growth phase were digested with trypsin containing EDTA, and then diluted with fresh medium to obtain a final volume of 5 × 10⁶ cells. 5 Cell suspension at 100 μL / mL. Add 100 μL of cell suspension to a 96-well cell culture plate to achieve a seeding density of 5 × 10⁶ cells / mL. 4 One cell per well was incubated in a 37°C cell culture incubator for 24 hours.

[0105] Third, after the cells reached confluence, remove the culture medium solution from the 96-well plates. Add 100 μL of hydrogel extraction buffer and 100 μL of fresh culture medium to the experimental and control groups, and continue incubation at 37°C for 24 h and 48 h, respectively. After 24 h and 48 h, remove the culture medium, add 150 μL of MTT solution to each well, and continue incubation at 37°C for 4 h. Then remove the MTT solution and add 150 μL of dimethyl sulfoxide (DMSO) to each well containing cells in the 96-well plates. Incubate in the dark on a shaker at 37°C for 10 min. Finally, measure the absorbance at 570 nm using a microplate reader and calculate the cell viability using the following formula:

[0106]

[0107] Among them, A t The absorbance values ​​are: A0 for the wells containing hydrogel extract, Ae for the wells containing sterile water, and Ae for the wells containing DMEM medium.

[0108] Live / dead cell staining assay: Take 1 mL of cells at a density of 5 × 10⁻⁶ 5 L929 cells per mL were seeded into 24-well plates. After incubation for 1 day, the culture medium was removed. 1 mL of hydrogel extract and 1 mL of fresh culture medium were added to the experimental and control groups, respectively, and then added to the 24-well plates. The plates were incubated at 37°C for another day. The hydrogel extract and culture medium were removed, and the cells were washed with PBS. 250 μL of AM / PI mixture was added, and the plates were incubated at room temperature in the dark for 30 min. The cell state was carefully observed and photographed using an inverted fluorescence microscope.

[0109] like Figure 11As shown in Figure 6, the cell viability of L929 cells incubated with SOP hydrogels for 24 and 48 hours was over 100% for all four groups, indicating that the alginate and hyaluronic acid as part of the material itself can promote the proliferation of L929 cells, because these two polysaccharides provide nutrients for cell proliferation and differentiation. The decrease in cell viability of SOP-0.20 hydrogel may be related to the release of high concentrations of PHMB, but all cell viabilities were over 80%, indicating that the PHMB-loaded hydrogel has good cell compatibility for L929 cells.

[0110] As shown in Figure 6, the cell viability of L929 cells incubated with SOP hydrogels for 24 and 48 hours was over 100% for all four groups, indicating that the alginate and hyaluronic acid as part of the material itself can promote the proliferation of L929 cells, because these two polysaccharides provide nutrients for cell proliferation and differentiation. The decrease in cell viability of SOP-0.20 hydrogel may be related to the release of high concentrations of PHMB, but all cell viabilities were over 80%, indicating that the PHMB-loaded hydrogel has good cell compatibility for L929 cells. Figure 12

[0111] Example 7

[0112] Hemolytic activity of the antibacterial gels SOP-0.05, SOP-0.10, SOP-0.15, and SOP-0.20 prepared in Example 3.

[0113] Hemolysis can occur when red blood cells are ruptured after contact with the material. By measuring the absorbance value, the blood compatibility of the material can be determined.

[0114] Rat blood was centrifuged at 1000 rpm for 10 min to obtain red blood cells, which were washed 3 times with PBS and diluted to a concentration of 5% (v / v). Then, different hydrogel samples (0.3 mL) and red blood cells (0.7 mL) were mixed and placed on a shaker at 37°C and 100 rpm for 1 h. After centrifugation (1000 rpm, 10 min), 0.3 mL of distilled water and 0.3 mL of PBS were mixed with 0.7 mL of red blood cell suspension (0.7 mL) as positive and negative controls, respectively. The absorbance at 540 nm was recorded using a microplate reader, and the hemolysis rate was calculated:

[0115]

[0116] where A s , A n and A p are the absorbance values of the sample, negative control and positive control.

[0117] Figure 13 ​The hemolysis chart of the SOP hydrogel, the PBS solution treatment is the blank control group, and the deionized water is the positive control group. The supernatant of the four groups of hydrogels and the blank control group shows light yellow, and the positive control group shows red, indicating that the SOP hydrogel does not produce hemolysis phenomenon, and the hemolysis rate of the four kinds of hydrogels is 0.37, 1.17, 1.83, and 2.34%, respectively. When the hemolysis rate of the material is less than 5%, the material has good blood compatibility. It shows that the four kinds of hydrogels have good blood compatibility and will not produce toxic reactions on the blood.

[0118] Example 8

[0119] The antibacterial gels SOP-0.05, SOP-0.10, SOP-0.15, and SOP-0.20 prepared in Example 3 were subjected to a wound healing promotion experiment.

[0120] A rat dorsal skin full-thickness defect model was established to test the function of the hydrogel as a wound dressing to promote wound healing. SD rats weighing between 180-220 g were used in the experiment. In order to adapt the rats to the environment, they were fed for one week before the experiment began. Before the experiment, the mice were divided into three groups: (1) blank group; (2) commercial dressing 3M hydrogel dressing group; (3) hydrogel group. 10% chloral hydrate was injected intraperitoneally into the SD rats to anesthetize them, and then a hair removal machine was used to remove the hair on the back of the SD male rats. Subsequently, a full-thickness skin wound with a diameter of 1.5 cm was created on the back of the rats using scissors, and 100 μL of bacterial suspension (10 8 CFU / mL) was injected into the wound of each mouse to create a bacterial environment, and then the wounds of the rats were treated with 3M dressing and hydrogel dressing, respectively. The wound healing was observed by taking photos on day 1, day 7, and day 14. Image J software was used to process and calculate the wound area and wound contraction rate (%). The formula for calculating the wound contraction rate (%) is:

[0121]

[0122] A0is the area of the wound on day 0, and Atis the area of the wound on day t t

[0123] As shown in Figure 14 Figure 1, on day 0, all the wounds were similar. By day 7, differences in wound size began to appear between the blank group and the hydrogel group and the 3M dressing group. Significant wound closure was found in the hydrogel group, and the wound areas of the rats in the commercial group and the gel group were reduced to 65.17% and 68.70%, respectively, which was higher than that of the control group. On day 14, the wound closure area of the blank group and the commercial dressing group increased, and the wound healing rates were 90.01% and 95.55%, respectively, while the wound closure of the gel group was basically complete, and new hair began to cover the wound surface. It shows that the hydrogel has the best wound healing effect on the rat wounds.​

Claims

1. A method for preparing a Sola gel / hyaluronic acid hydrogel, characterized in that, The method includes: Sola gel was grafted with adipic dihydrazide via an amide reaction to obtain adipic dihydrazide-modified sola gel; sodium periodate was used to oxidize hyaluronic acid to obtain oxidized hyaluronic acid; adipic dihydrazide-modified sola gel and oxidized hyaluronic acid were reacted with a Schiff base to obtain a sola gel / hyaluronic acid hydrogel. The specific synthesis method of obtaining adipic dihydrazide-modified solar via amide reaction of solar is as follows: Solar and adipic dihydrazide are added to water, and then stirred at room temperature for 25-40 min. The pH is adjusted to 4-5 with hydrochloric acid solution. 1-Ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) is added, and the pH is adjusted to 4-5 again with hydrochloric acid solution. Stirring is continued for 3-5 h. NaOH solution is added to adjust the pH to 7 to terminate the reaction. The reaction solution is placed in a dialysis bag for dialysis. The dialysis solution is freeze-dried for 30-50 h to obtain adipic dihydrazide-modified solar. The concentration of the adipic acid dihydrazide-based solara gel is 0.6~1.0 wt%; the concentration of oxidized hyaluronic acid is 0.2 wt%.

2. The preparation method according to claim 1, characterized in that, The specific synthesis method for obtaining oxidized hyaluronic acid by oxidizing hyaluronic acid with sodium periodate is as follows: NaIO4 and HA are dissolved in water to obtain NaIO4 solution and HA solution respectively. The NaIO4 solution is added dropwise to the HA solution and stirred in the dark for 5-7 h. Then, ethylene glycol is added to terminate the reaction and stirring is continued for 0.5-2 h. The reaction solution is placed in a dialysis bag for dialysis. After dialysis, the solution is freeze-dried for 30-50 h to obtain oxidized hyaluronic acid.

3. The preparation method according to claim 1, characterized in that, The specific method for obtaining Sola gel / hyaluronic acid hydrogel by reacting adipic acid dihydrazide-modified Sola gel with oxidized hyaluronic acid through Schiff base reaction is as follows: dissolve adipic acid dihydrazide-modified Sola gel in water, heat in a water bath at 70~90℃ for 15~25 min, then add oxidized hyaluronic acid, stir for 20~40 s, and then cool and let stand to obtain Sola gel / hyaluronic acid hydrogel.

4. The solara gel / hyaluronic acid hydrogel prepared by the preparation method according to any one of claims 1 to 3.

5. A method for preparing a Sola gel / hyaluronic acid antibacterial hydrogel, characterized in that, The method includes: Polyhexamethylene biguanide hydrochloride was added to the Sola gel / hyaluronic acid hydrogel according to claim 4, and after thorough stirring, a Sola gel / hyaluronic acid antibacterial hydrogel was obtained.

6. The Solar agar / hyaluronic acid antibacterial hydrogel prepared by the preparation method of claim 5.

7. A wound dressing, characterized in that, The wound dressing comprises the Sola gel / hyaluronic acid hydrogel of claim 4 and / or the Sola gel / hyaluronic acid antibacterial hydrogel of claim 6.

Images