A thermosensitive hydrogel capable of promoting wound healing, and its preparation method and application

Abstract

The present invention is applicable to the field of thermosensitive hydrogel technology, and provides a thermosensitive hydrogel and its preparation method and application that can promote wound healing, including the following raw materials: hydroxybutyl chitosan, oxidized bletilla striata polysaccharide, shikonin cerium complex and injection aqueous solution; wherein, the concentration of hydroxybutyl chitosan in the hydrogel is 1 200 mg / mL, the concentration of oxidized bletilla striata polysaccharide is 1 200 mg / mL, and the concentration of shikonin cerium complex is 1 50 mg / mL. The present invention can play a stronger wound healing effect by regulating the wound microenvironment, inhibiting bacteria, removing reactive oxygen species and other synergistic multiple effects. At the same time, it also has thermosensitive properties, has better conformability and closure to the wound, can be widely used in the preparation of drugs that promote wound healing, and is conducive to promoting the reduction of wound area and promoting the recovery of skin function at the wound.

Description

Technical Field

[0001] The present invention belongs to the technical field of thermosensitive hydrogels, and in particular relates to a thermosensitive hydrogel capable of promoting wound healing, and a preparation method and application thereof. Background Art

[0002] Chitosan, a product of the natural polysaccharide chitin with some acetyl groups removed, has excellent biodegradability and biocompatibility. It also promotes skin repair, including moisturizing, promoting wound healing, reducing scarring, inhibiting melanin production, and providing antibacterial and anti-inflammatory benefits. Consequently, it is widely used in a wide range of fields, including food additives, textiles, agriculture, environmental protection, beauty and health care, cosmetics, antimicrobial agents, medical fibers, medical dressings, artificial tissue materials, sustained-release drug materials, gene transduction vectors, biomedical applications, medical absorbable materials, tissue engineering carrier materials, medical treatment, and drug development, as well as in other daily chemical industries. However, chitosan's poor solubility in water significantly limits its use.

[0003] Hydroxybutyl chitosan is a chitosan derivative modified from chitosan, which has good water solubility and thermosensitive properties, thus expanding the application of chitosan; Bletilla striata polysaccharide is a polysaccharide compound extracted from the plant Bletilla striata, which has skin repairing effects such as hemostasis, detumescence, tissue regeneration, wound healing, anti-oxidation, improvement of skin texture, lightening of spots, and whitening; Shikonin is a quinone compound extracted from the plant Lithospermum officinale, which has anti-inflammatory, anti-allergic, wound healing, whitening, anti-oxidation and other skin repairing effects, and is widely used in eczema, especially childhood eczema.

[0004] The prior art does not disclose the use of the three raw materials in combination in a hydrogel. Summary of the Invention

[0005] The purpose of the embodiments of the present invention is to provide a thermosensitive hydrogel that can promote wound healing, aiming to solve the problems raised in the above background technology.

[0006] The embodiment of the present invention is achieved as follows: a thermosensitive hydrogel that can promote wound healing includes the following raw materials: hydroxybutyl chitosan, oxidized bletilla striata polysaccharide, a shikonin-cerium complex, and an injectable aqueous solution; wherein the concentration of hydroxybutyl chitosan in the hydrogel is 1-200 mg / mL, the concentration of oxidized bletilla striata polysaccharide is 1-200 mg / mL, and the concentration of the shikonin-cerium complex is 1-50 mg / mL.

[0007] Another object of the present invention is to provide a method for preparing a thermosensitive hydrogel that can promote wound healing, comprising the following steps:

[0008] Preparation of shikonin-cerium complex: dissolve water-soluble cerium salt in water, add to alcohol solution of shikonin, stir at room temperature for reaction, then add water and centrifuge to precipitate to obtain shikonin-cerium complex;

[0009] Preparation of hydroxybutyl chitosan: chitosan powder is dispersed in a NaOH aqueous solution, stirred at room temperature, and then filtered. The filter cake is added to an isopropanol / water solution, stirred at room temperature, and then 1,2-butylene oxide is added. The mixture is stirred at room temperature and then heated and stirred. The pH is adjusted, filtered, the filtrate is dialyzed, and then freeze-dried to obtain hydroxybutyl chitosan.

[0010] Preparation of oxidized Bletilla striata polysaccharide: dissolving Bletilla striata polysaccharide and NaIO4 in water, stirring at room temperature for reaction, then adding ethylene glycol, continuing stirring to terminate the reaction, dialyzing the reaction solution, and freeze-drying to obtain oxidized Bletilla striata polysaccharide;

[0011] Preparation of thermosensitive hydrogel: adding hydroxybutyl chitosan, oxidized bletilla striata polysaccharide and shikonin cerium complex into injection aqueous solution and stirring to prepare thermosensitive hydrogel.

[0012] Preferably, in the step of preparing the shikonin cerium complex, the water-soluble cerium salt is one of cerium nitrate, cerium chloride, cerium sulfate and cerium acetate.

[0013] Preferably, in the step of preparing the shikonin-cerium complex, the alcohol solution of shikonin is one of a methanol solution of shikonin, an ethanol solution of shikonin, and a glycerol solution of shikonin.

[0014] Another object of an embodiment of the present invention is to provide a use of a thermosensitive hydrogel that can promote wound healing in the preparation of a medicament that promotes wound healing.

[0015] Preferably, the wound is one or more of trauma, bacterial infection, burn, surgery and diabetic wound.

[0016] Another object of the embodiments of the present invention is to provide a use of a thermosensitive hydrogel that can promote wound healing in the preparation of antibacterial drugs.

[0017] The embodiment of the present invention provides a thermosensitive hydrogel that can promote wound healing, which is added with hydroxybutyl chitosan, oxidized bletilla striata polysaccharide, and shikonin cerium complex. The amino groups in hydroxybutyl chitosan and the carbonyl groups in oxidized bletilla striata polysaccharide can form a hydrogel by forming a Schiff base self-assembly, which has high strength. The hydrogel is loaded with the shikonin cerium complex formed by shikonin and cerium ions. The cerium ions Ce 4+ and Ce 3+The conversion in the organism can expel ROS free radicals and enhance its wound healing effect. The hydrogel provided by the embodiment of the present invention can exert a stronger wound healing effect through synergistic multiple effects such as regulating the wound microenvironment, inhibiting bacteria, and removing reactive oxygen species. At the same time, it also has a temperature-sensitive property. It is liquid at 20°C and forms a gel at body temperature (37°C). Compared with traditional hydrogels, this intelligent temperature-sensitive feature has better adhesion and sealing properties to wounds, and can be widely used in the preparation of drugs that promote wound healing, which is beneficial to promoting the reduction of wound area and promoting the recovery of skin function at the wound. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a SEM schematic diagram of the hydrogel prepared in Example 1 of the present invention;

[0019] Figure 2 Schematic diagram of the transition between the liquid state and the gel state at different temperatures of the hydrogel prepared in Example 1 of the present invention;

[0020] Figure 3 The cellular ROS scavenging activity results of different samples provided in Example 5 of the present invention;

[0021] Figure 4 The DPPH scavenging activity results of different samples provided in Example 6 of the present invention;

[0022] Figure 5 The antibacterial activity results of different samples provided in Example 7 of the present invention (a is a photo of each group of culture dishes, b is the antibacterial rate results of different samples against Escherichia coli, and c is the antibacterial rate results of different samples against Staphylococcus aureus);

[0023] Figure 6 The wound healing activity results of mice using different samples provided in Example 8 of the present invention;

[0024] Figure 7 This is a fitting diagram of the wound areas of mice using different samples provided in Example 8 of the present invention. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0026] The specific implementation of the present invention is described in detail below with reference to specific embodiments.

[0027] Example 1: A thermosensitive hydrogel capable of promoting wound healing, wherein the preparation method comprises the following steps:

[0028] Step 1, preparation of shikonin cerium complex: 100 mg of cerium nitrate was dissolved in 1 mL of water, and 10 mg of shikonin was dissolved in 1 mL of ethanol to obtain a shikonin ethanol solution. The cerium nitrate solution was added to the above shikonin ethanol solution, stirred at room temperature for 24 h, 100 mL of water was added, and centrifuged at 5000 r / min for 10 min. The precipitate was separated, washed with water, and dried under reduced pressure at 70°C to obtain 11 mg of shikonin cerium complex;

[0029] Step 2, preparation of hydroxybutyl chitosan: 1 g of chitosan was dispersed in 10 mL of 50% by mass NaOH water, stirred at room temperature for 24 h, filtered to obtain a filter cake, and the excess NaOH solution in the filter cake was squeezed out with filter paper, 10 mL of water and 10 mL of isopropanol were added, and stirred at room temperature for 24 h, and then 20 mL of 1,2-butylene oxide was added, stirred at room temperature for 1 h, and then heated and stirred at 60 ° C for 36 h, and the pH was adjusted to 6-7 with concentrated hydrochloric acid, filtered, and the filtrate was dialyzed with a dialysis membrane (MW = 3500) for three days, and freeze-dried to obtain 0.8 g of hydroxybutyl chitosan;

[0030] Step 3. Preparation of oxidized Bletilla striata polysaccharide: 1 g of Bletilla striata polysaccharide was added to 50 mL of water, and 1 g of NaIO4 was added, and the mixture was stirred at room temperature for 24 h. 5 mL of ethylene glycol was added, and the mixture was stirred for 0.5 h to terminate the reaction. The reaction solution was dialyzed using a dialysis bag (MW = 3500) for three days and freeze-dried to obtain 0.6 g of oxidized Bletilla striata polysaccharide.

[0031] Step 4. Preparation of thermosensitive hydrogel: Dissolve 10 mg of oxidized Bletilla striata polysaccharide in 1 mL of injection water, add 1 mg of cerium shikonin and disperse evenly, then add 30 mg of hydroxybutyl chitosan and stir until dissolved to prepare a hydrogel.

[0032] Example 2: A thermosensitive hydrogel capable of promoting wound healing, wherein the preparation method comprises the following steps:

[0033] Step 1, preparation of shikonin cerium complex: 50 mg of cerium chloride was dissolved in 1 mL of water, and 10 mg of shikonin was dissolved in 1 mL of methanol to obtain a shikonin methanol solution. The cerium chloride solution was added to the above shikonin methanol solution, stirred at room temperature for 24 h, 100 mL of water was added, and the mixture was centrifuged at 5000 r / min for 10 min. The precipitate was separated, washed with water, and dried under reduced pressure at 70°C to obtain 10 mg of shikonin cerium complex;

[0034] Step 2 and step 3 are the same as in Example 1;

[0035] Step 4: Preparation of thermosensitive hydrogel: Dissolve 30 mg of oxidized Bletilla striata polysaccharide in 1 mL of injection water, add 2 mg of cerium shikonin and disperse evenly, then add 50 mg of hydroxybutyl chitosan and stir until dissolved to prepare a hydrogel.

[0036] Example 3: A thermosensitive hydrogel capable of promoting wound healing, wherein the preparation method comprises the following steps:

[0037] Step 1, preparation of shikonin cerium complex: 50 mg of cerium sulfate was dissolved in 1 mL of water, and 10 mg of shikonin was dissolved in 1 mL of glycerol to obtain a shikonin glycerol solution. The cerium chloride solution was added to the above shikonin glycerol solution, stirred at room temperature for 24 h, 100 mL of water was added, and the mixture was centrifuged at 5000 r / min for 10 min. The precipitate was separated, washed with water, and dried under reduced pressure at 70°C to obtain 10 mg of shikonin cerium complex;

[0038] Step 2 and step 3 are the same as in Example 1;

[0039] Step 4: Preparation of thermosensitive hydrogel: Dissolve 1 mg of oxidized Bletilla striata polysaccharide in 1 mL of injection water, add 1 mg of cerium shikonin and disperse evenly, then add 1 mg of hydroxybutyl chitosan and stir until dissolved to prepare a hydrogel.

[0040] Example 4: A thermosensitive hydrogel capable of promoting wound healing, the preparation method of which comprises the following steps:

[0041] Step 1, preparation of shikonin cerium complex: 50 mg of cerium acetate was dissolved in 1 mL of water, and 10 mg of shikonin was dissolved in 1 mL of methanol to obtain a shikonin methanol solution. The cerium chloride solution was added to the above shikonin methanol solution, stirred at room temperature for 24 h, 100 mL of water was added, and the mixture was centrifuged at 5000 r / min for 10 min. The precipitate was separated, washed with water, and dried under reduced pressure at 70°C to obtain 10 mg of shikonin cerium complex;

[0042] Step 2 and step 3 are the same as in Example 1;

[0043] Step 4: Preparation of thermosensitive hydrogel: Dissolve 200 mg of oxidized Bletilla striata polysaccharide in 1 mL of injection water, add 50 mg of cerium shikonin and disperse evenly, then add 200 mg of hydroxybutyl chitosan and stir until dissolved to prepare a hydrogel.

[0044] The hydrogel prepared in Example 1 was freeze-dried and then subjected to SEM scanning, and the results were as follows: Figure 1 As shown, the hydrogel presents a three-dimensional network structure; its morphological transformation at different temperatures is as follows Figure 2 As shown in the figure, the hydrogel is in liquid state below 10℃ and in gel state above 25℃, which proves that the hydrogel has good temperature sensitivity.

[0045] Example 5, ROS scavenging experiment:

[0046] The fluorescent probe DCFH-DA was used to evaluate the ROS level in L929 cells (American type culture collection, ATCC). 1×10 L929 cells were cultured. 6 The cells were incubated at 37°C for 5 h with the samples of each group [Control: blank control group; HBC: hydroxybutyl chitosan hydrogel group; HBC+ORBSP: chitosan+oxidized bletilla striata polysaccharide hydrogel group; Shik-Ce(Ⅲ): shikonin cerium complex group; HBC+ORBSP / Shik-Ce(Ⅲ): chitosan+oxidized bletilla striata polysaccharide / shikonin cerium complex hydrogel group (hydrogel prepared in Example 1)] for 48 h, and the culture medium was aspirated and replaced with complete culture medium containing 100 μM H2O2. The cells were incubated at 37°C for 5 h, and then the culture medium was aspirated and washed three times with PBS. 10 μM fluorescent probe DCFH-DA was added and incubated with the cells for 15 min, and then washed three times with PBS. The liquid in the wells was aspirated, and the well plate was placed under an inverted fluorescence microscope to observe the fluorescence intensity of the cells. The fluorescence intensity of the control group was set as 100%, and the fluorescence intensity of each group was calculated. The results are shown in FIG. Figure 3 As shown in the figure, compared with the control group, the fluorescence intensity of the HBC+ORBSP / Shik-Ce(Ⅲ) hydrogel group was the lowest, proving that it had good cellular ROS scavenging activity and the ROS scavenging rate was greater than 60%.

[0047] Example 6, DPPH free radical scavenging experiment:

[0048] The free radical scavenging performance of the hydrogel in vitro was evaluated by DPPH free radical scavenging experiment. 0.1 g of each sample [Control: blank control group; HBC: hydroxybutyl chitosan hydrogel group; HBC+ORBSP: chitosan + oxidized bletilla striata polysaccharide hydrogel group; Shik-Ce(Ⅲ): shikonin cerium complex group; HBC+ORBSP / Shik-Ce(Ⅲ): chitosan + oxidized bletilla striata polysaccharide / shikonin cerium complex hydrogel group (hydrogel prepared in Example 1)] was added to 1 mL of 0.1 mM DPPH ethanol solution, and the mixture was incubated in a shaker at 37 °C in the dark for 30 min. The ultraviolet absorbance of the supernatant at 517 nm was measured using a microplate reader. The absorbance of DPPH solution was A d The sample absorbance is A h ; The scavenging efficiency of DPPH free radicals was calculated according to the following formula: DPPH scavenging rate = (A d -A h ) / A d ×100%;

[0049] The results are as follows Figure 4 As shown in the results, compared with the control group, the HBC+ORBSP / Shik-Ce(Ⅲ) hydrogel group had good DPPH free radical scavenging activity, and the DPPH free radical scavenging rate was greater than 80%.

[0050] Example 7, hydrogel antibacterial experiment:

[0051] The antibacterial activity of the hydrogel was determined by the plate coating method. The blank culture medium was used as the control, and Gram-positive bacteria (Staphylococcus aureus: Beijing Putian Tongchuang Biotechnology Co., Ltd.) and Gram-negative bacteria (Escherichia coli: Beijing Putian Tongchuang Biotechnology Co., Ltd.) were used as the target bacteria. 6 The bacterial suspension with a CFU / ml bacterial liquid concentration was incubated with each group of samples [Control: blank control group; HBC: hydroxybutyl chitosan hydrogel group; HBC+ORBSP: chitosan+oxidized bletilla striata polysaccharide hydrogel group; Shik: shikonin group; Shik-Ce(Ⅲ): shikonin cerium complex group; HBC+ORBSP / Shik-Ce(Ⅲ): chitosan+oxidized bletilla striata polysaccharide / shikonin cerium complex hydrogel group (hydrogel prepared in Example 1)] for 12 h. After the incubation, the bacterial suspension was diluted 5 times and inoculated onto the surface of the solid nutrient culture medium. It was cultured at 37°C for 24 h, the culture dishes were taken out, photographed and counted, and the plate count method was used to calculate the inhibition rate. The results are as follows: Figure 5 As shown in the figure, it can be seen that the HBC+ORBSP / Shik-Ce(Ⅲ) group has the strongest antibacterial effect, and the antibacterial rates against Staphylococcus aureus and Escherichia coli are both greater than 95%.

[0052] Example 8, hydrogel wound healing experiment:

[0053] Kunming mice (source: Liaoning Changsheng Biotechnology Co., Ltd.) were anesthetized with isoflurane gas, and the hair on their backs was shaved to create circular wounds with a diameter of 10 mm. Samples from each group [Control: blank control group; HBC: hydroxybutyl chitosan hydrogel group; HBC+ORBSP: chitosan+oxidized bletilla striata polysaccharide hydrogel group; Shik-Ce(Ⅲ): shikonin cerium complex group; HBC+ORBSP / Shik-Ce(Ⅲ): chitosan+oxidized bletilla striata polysaccharide / shikonin cerium complex hydrogel group (hydrogel prepared in Example 1)] were taken to treat the wounds, and the wound healing was monitored on days 7 and 14 after surgery. Figure 6 As shown, Image J software was used to analyze the wound area. Figure 7 As shown in the figure, it can be seen that the HBC+ORBSP / Shik-Ce(Ⅲ) group has the strongest wound healing activity compared with other groups.

[0054] In summary, the hydrogel prepared in the embodiment of the present invention has high strength and temperature sensitivity, can eliminate ROS free radicals, has good antibacterial and wound healing effects, and can be used in the preparation of wound healing drugs and antibacterial drugs.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A thermosensitive hydrogel capable of promoting wound healing, characterized in that: The invention comprises the following raw materials: hydroxybutyl chitosan, oxidized bletilla striata polysaccharide, shikonin cerium complex and injection aqueous solution; wherein the concentration of hydroxybutyl chitosan in the hydrogel is 1-200 mg / mL, the concentration of oxidized bletilla striata polysaccharide is 1-200 mg / mL, and the concentration of shikonin cerium complex is 1-50 mg / mL.

2. A method for preparing the thermosensitive hydrogel capable of promoting wound healing according to claim 1, characterized in that: The following steps are involved: Preparation of shikonin-cerium complex: dissolve water-soluble cerium salt in water, add to alcohol solution of shikonin, stir at room temperature for reaction, then add water and centrifuge to precipitate to obtain shikonin-cerium complex; Preparation of hydroxybutyl chitosan: chitosan powder is dispersed in a NaOH aqueous solution, stirred at room temperature, and then filtered. The filter cake is added to an isopropanol / water solution, stirred at room temperature, and then 1,2-butylene oxide is added. The mixture is stirred at room temperature and then heated and stirred. The pH is adjusted, filtered, the filtrate is dialyzed, and then freeze-dried to obtain hydroxybutyl chitosan. Preparation of oxidized Bletilla striata polysaccharide: dissolving Bletilla striata polysaccharide and NaIO4 in water, stirring at room temperature for reaction, then adding ethylene glycol, continuing stirring to terminate the reaction, dialyzing the reaction solution, and freeze-drying to obtain oxidized Bletilla striata polysaccharide; Preparation of thermosensitive hydrogel: adding hydroxybutyl chitosan, oxidized bletilla striata polysaccharide and shikonin cerium complex into injection aqueous solution and stirring to prepare thermosensitive hydrogel.

3. The method for preparing the thermosensitive hydrogel capable of promoting wound healing according to claim 2, wherein: In the step of preparing the shikonin-cerium complex, the water-soluble cerium salt is one of cerium nitrate, cerium chloride, cerium sulfate and cerium acetate.

4. The method for preparing the thermosensitive hydrogel capable of promoting wound healing according to claim 2, wherein: In the step of preparing the shikonin-cerium complex, the alcohol solution of shikonin is one of a methanol solution of shikonin, an ethanol solution of shikonin, and a glycerol solution of shikonin.

5. Use of the thermosensitive hydrogel capable of promoting wound healing as claimed in claim 1 in the preparation of a medicament for promoting wound healing.

6. Use of the thermosensitive hydrogel capable of promoting wound healing as claimed in claim 1 in the preparation of antibacterial drugs.

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