Temperature-sensitive hydrogel capable of promoting wound healing as well as preparation method and application of temperature-sensitive hydrogel

Abstract

The invention is suitable for the technical field of temperature-sensitive hydrogel, and provides a temperature-sensitive hydrogel capable of promoting wound healing and a preparation method and application thereof, the temperature-sensitive hydrogel comprises the following raw materials: hydroxybutyl chitosan, oxidized bletilla striata polysaccharide, an alkannin cerium complex and an injection aqueous solution; wherein in the hydrogel, the concentration of the hydroxybutyl chitosan is 1 to 200 mg / mL, the concentration of the oxidized bletilla striata polysaccharide is 1 to 200 mg / mL, and the concentration of the alkannin cerium complex is 1 to 50 mg / mL. According to the present invention, the strong wound healing promoting effect can be provided through the synergistic multiple effects of wound microenvironment adjustment, bacteriostasis, active oxygen removal and the like, and the temperature-sensitive wound healing promoting agent further has characteristics of temperature sensitivity and good wound fitting and sealing property, can be widely used in the preparation of the wound healing promoting drug, and further has advantages of wide application prospect and wide application prospect. The wound area is reduced, and the skin function recovery at the wound is promoted.

Description

Technical Field

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

[0002] Chitosan is a product obtained by removing partial acetyl groups from natural polysaccharide chitin. It has good biodegradability and biocompatibility, and at the same time has functions such as moisturizing, promoting wound healing, reducing scar formation, inhibiting melanin production, and antibacterial and anti-inflammatory effects for promoting skin repair. Therefore, it is widely used in many fields such as food additives, textiles, agriculture, environmental protection, beauty care, cosmetics, antibacterial agents, medical fibers, medical dressings, artificial tissue materials, drug sustained-release materials, gene transduction vectors, biomedical fields, medical absorbable materials, tissue engineering carrier materials, medical treatment, and drug development, as well as other daily chemical industries. However, chitosan has poor solubility in water, so it is greatly limited in use.

[0003] Hydroxybutyl chitosan is a chitosan derivative modified from chitosan, which has good water solubility and at the same time has thermosensitive properties, thus expanding the application of chitosan; Bletilla striata polysaccharide is a polysaccharide compound extracted from the plant Bletilla striata, which has functions such as hemostasis, detumescence, granulation promotion, wound healing promotion, antioxidant, skin texture improvement, age spot fading, and whitening for skin repair; Shikonin is a quinone compound extracted from the plant Lithospermum erythrorhizon, which has functions such as anti-inflammatory, anti-allergic, wound healing promotion, whitening, and antioxidant for promoting skin repair, and is widely used in eczema, especially pediatric eczema.

[0004] The prior art does not disclose the content of combining the three raw materials and applying them in a hydrogel. Summary of the Invention

[0005] The purpose of the embodiment of the present invention is to provide a thermosensitive hydrogel capable of promoting wound healing, aiming to solve the problems proposed in the above background art.

[0006] The embodiment of the present invention is implemented as follows. A thermosensitive hydrogel capable of promoting wound healing includes the following raw materials: hydroxybutyl chitosan, oxidized Bletilla striata polysaccharide, shikonin cerium complex, and an 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.

[0007] Another purpose of the embodiment of the present invention is to provide a preparation method of a thermosensitive hydrogel capable of promoting wound healing, including the following steps: Preparation of shikonin cerium complex: Dissolve water-soluble cerium salt in water, then add it to the alcoholic solution of shikonin, stir and react at room temperature, and then add water and centrifuge to separate the precipitate to obtain shikonin cerium complex; Preparation of hydroxybutyl chitosan: Disperse chitosan powder in an aqueous NaOH solution, stir at room temperature and then filter. Add the filter cake to an isopropanol / water solution, stir at room temperature, add 1,2-epoxybutane, stir at room temperature and then heat and stir, adjust the pH, filter, dialyze the filtrate, and then freeze-dry to obtain hydroxybutyl chitosan; Preparation of oxidized Bletilla striata polysaccharide: Dissolve Bletilla striata polysaccharide and NaIO4 in water, stir and react at room temperature, then add ethylene glycol and continue to stir to terminate the reaction. Dialyze the reaction solution and freeze-dry to obtain oxidized Bletilla striata polysaccharide; Preparation of thermosensitive hydrogel: Add hydroxybutyl chitosan, oxidized Bletilla striata polysaccharide and shikonin cerium complex to an injection aqueous solution and stir to obtain a thermosensitive hydrogel.

[0008] Preferably, in the step of preparing shikonin cerium complex, the water-soluble cerium salt is one of cerium nitrate, cerium chloride, cerium sulfate, and cerium acetate.

[0009] Preferably, in the step of preparing shikonin cerium complex, the alcoholic solution of shikonin is one of the methanol solution of shikonin, the ethanol solution of shikonin, and the glycerol solution of shikonin.

[0010] Another object of the embodiments of the present invention is to provide an application of a thermosensitive hydrogel capable of promoting wound healing in the preparation of a drug for promoting wound healing.

[0011] Preferably, the wound is one or more of trauma, bacterial infection, scald, surgery, and diabetic wound.

[0012] Another object of the embodiments of the present invention is to provide an application of a thermosensitive hydrogel capable of promoting wound healing in the preparation of an antibacterial drug.

[0013] A thermosensitive hydrogel capable of promoting wound healing provided by the embodiments of the present invention is added with hydroxybutyl chitosan, oxidized Bletilla striata polysaccharide, and shikonin cerium complex. The amino group in hydroxybutyl chitosan and the carbonyl group in oxidized Bletilla striata polysaccharide can form a Schiff base and self-assemble to form a hydrogel with high strength, and is loaded with the shikonin cerium complex formed by shikonin and cerium ions. Cerium ions Ce 4+ and Ce 3+The conversion in vivo can expel ROS free radicals and enhance its wound healing promotion effect. The hydrogel provided by the embodiments of the present invention can play a stronger wound healing promotion effect through synergistic multiple effects such as regulating the wound microenvironment, antibacterial, and scavenging reactive oxygen species. At the same time, it also has thermosensitive properties, being liquid at 20°C and forming a gel under body temperature (37°C). This intelligent thermosensitive feature has better conformability and sealing property to the wound compared with traditional hydrogels, and can be widely used in the preparation of drugs for promoting wound healing, which is beneficial to promoting the reduction of wound area and the recovery of skin function at the wound site. Brief Description of the Drawings

[0014] Figure 1 SEM schematic diagram of the hydrogel prepared in Example 1 of the present invention; Figure 2 Schematic diagram of different temperature transitions of the hydrogel prepared in Example 1 of the present invention in liquid state and gel state; Figure 3 Cell ROS scavenging activity results of different samples provided in Example 5 of the present invention; Figure 4 DPPH scavenging activity results of different samples provided in Example 6 of the present invention; Figure 5 Antibacterial activity results of different samples provided in Example 7 of the present invention (a is the photo of each culture dish, b is the antibacterial rate result of different samples against Escherichia coli, and c is the antibacterial rate result of different samples against Staphylococcus aureus); Figure 6 Wound healing activity results of mice using different samples provided in Example 8 of the present invention; Figure 7 Wound area fitting diagram of mice using different samples provided in Example 8 of the present invention. Detailed Embodiments

[0015] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, 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 used to limit the present invention.

[0016] The following describes the specific implementation of the present invention in detail with reference to specific embodiments.

[0017] Example 1. A thermosensitive hydrogel capable of promoting wound healing, and its preparation method includes the following steps: Step 1. Preparation of shikonin cerium complex: Dissolve 100 mg of cerium nitrate in 1 mL of water, and dissolve 10 mg of shikonin in 1 mL of ethanol to obtain a shikonin ethanol solution. Add the cerium nitrate solution to the above shikonin ethanol solution, stir at room temperature for 24 h, add 100 mL of water, centrifuge at 5000 r / min for 10 min, separate the precipitate, wash with water, and dry under reduced pressure at 70 °C to obtain 11 mg of shikonin cerium complex; Step 2. Preparation of hydroxybutyl chitosan: Disperse 1 g of chitosan in 10 mL of 50% (mass fraction) NaOH aqueous solution, stir at room temperature for 24 h, filter to obtain a filter cake, and squeeze out the excess NaOH solution in the filter cake with filter paper. Add 10 mL of water and 10 mL of isopropanol, stir at room temperature for 24 h, then add 20 mL of 1,2-epoxybutane, stir at room temperature for 1 h, then heat and stir at 60 °C for 36 h, adjust the pH to 6 - 7 with concentrated hydrochloric acid, filter, dialyze the filtrate through a dialysis membrane (MW = 3500) for three days, and freeze-dry to obtain 0.8 g of hydroxybutyl chitosan; Step 3. Preparation of oxidized Bletilla striata polysaccharide: Add 1 g of Bletilla striata polysaccharide to 50 mL of water, add 1 g of NaIO4, stir and react at room temperature for 24 h, add 5 mL of ethylene glycol, stir for 0.5 h to terminate the reaction, dialyze the reaction solution through a dialysis bag (MW = 3500) for three days, and freeze-dry to obtain 0.6 g of oxidized Bletilla striata polysaccharide; Step 4. Preparation of thermosensitive hydrogel: Dissolve 10 mg of oxidized Bletilla striata polysaccharide in 1 mL of injection water, add 1 mg of shikonin cerium and disperse evenly, then add 30 mg of hydroxybutyl chitosan and stir until dissolved to prepare the hydrogel.

[0018] Example 2. A thermosensitive hydrogel that can promote wound healing, and its preparation method includes the following steps: Step 1. Preparation of shikonin cerium complex: Dissolve 50 mg of cerium chloride in 1 mL of water, and dissolve 10 mg of shikonin in 1 mL of methanol to obtain a shikonin methanol solution. Add the cerium chloride solution to the above shikonin methanol solution, stir at room temperature for 24 h, add 100 mL of water, centrifuge at 5000 r / min for 10 min, separate the precipitate, wash with water, and dry under reduced pressure at 70 °C to obtain 10 mg of shikonin cerium complex; Steps 2 and 3 are the same as those in Example 1; Step 4. Preparation of thermosensitive hydrogel: Dissolve 30 mg of oxidized Bletilla striata polysaccharide in 1 mL of injection water, add 2 mg of shikonin cerium and disperse evenly, then add 50 mg of hydroxybutyl chitosan and stir until dissolved to prepare the hydrogel.

[0019] Example 3. A thermosensitive hydrogel that can promote wound healing, and its preparation method includes the following steps: Step 1. Preparation of shikonin cerium complex: Dissolve 50 mg of cerium sulfate in 1 mL of water, and dissolve 10 mg of shikonin in 1 mL of glycerol to obtain a shikonin glycerol solution. Add the cerium chloride solution to the above shikonin glycerol solution, stir at room temperature for 24 h, add 100 mL of water, centrifuge at 5000 r / min for 10 min, separate the precipitate, wash with water, and dry under reduced pressure at 70 °C to obtain 10 mg of shikonin cerium complex; Steps 2 and 3 are the same as those in Example 1; Step 4. Preparation of thermosensitive hydrogel: Dissolve 1 mg of oxidized bletilla striata polysaccharide in 1 mL of injection water, add 1 mg of shikonin cerium and disperse evenly, then add 1 mg of hydroxybutyl chitosan and stir until dissolved to prepare a hydrogel.

[0020] Example 4. A thermosensitive hydrogel that can promote wound healing, and its preparation method includes the following steps: Step 1. Preparation of shikonin cerium complex: Dissolve 50 mg of cerium acetate in 1 mL of water, and dissolve 10 mg of shikonin in 1 mL of methanol to obtain a shikonin methanol solution. Add the cerium chloride solution to the above shikonin methanol solution, stir at room temperature for 24 h, add 100 mL of water, centrifuge at 5000 r / min for 10 min, separate the precipitate, wash with water, and dry under reduced pressure at 70 °C to obtain 10 mg of shikonin cerium complex; Steps 2 and 3 are the same as those in Example 1; Step 4. Preparation of thermosensitive hydrogel: Dissolve 200 mg of oxidized bletilla striata polysaccharide in 1 mL of injection water, add 50 mg of shikonin cerium and disperse evenly, then add 200 mg of hydroxybutyl chitosan and stir until dissolved to prepare a hydrogel.

[0021] The hydrogel prepared in Example 1 was freeze-dried and then scanned by SEM, and the results are as Figure 1 shown. The hydrogel has a three-dimensional network structure; its morphological transformation at different temperatures is as Figure 2 shown. The hydrogel is in a liquid state below 10 °C and in a gel state above 25 °C, indicating that the hydrogel has good thermosensitivity.

[0022] Example 5. ROS scavenging experiment: The ROS level in L929 cells (American type culture collection, ATCC) was evaluated using the fluorescent probe DCFH-DA. 1×10 6At different densities, they were cultured with each group of samples [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 (the hydrogel prepared in Example 1)] for 48 h. Then, the culture medium was aspirated and replaced with a complete medium containing 100 μM H2O2. After incubating at 37 °C for 5 h, the culture medium was aspirated, and the cells were washed 3 times with PBS; 10 μM of the fluorescent probe DCFH-DA was added, and after co-incubating with the cells for 15 min, the cells were washed 3 times with PBS; the liquid in the wells was aspirated, and the 96-well plate was placed under an inverted fluorescence microscope to observe the fluorescence intensity of the cells; taking the fluorescence intensity of the Control group as 100%, the fluorescence intensity of each group was calculated. The results are as Figure 3 shown. Compared with the control group, the HBC+ORBSP / Shik-Ce(Ⅲ) hydrogel group had the lowest fluorescence intensity, indicating good cellular ROS scavenging activity, and the ROS scavenging rate was greater than 60%. Example 6. DPPH free radical scavenging experiment: The free radical scavenging performance of the hydrogels in vitro was evaluated by the DPPH free radical scavenging experiment. 0.1 g of each group of samples [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 (the hydrogel prepared in Example 1)] was added to 1 mL of 0.1 mM DPPH ethanol solution respectively, and cultured in the dark on a shaker at 37 °C for 30 min. The ultraviolet absorbance of the supernatant at 517 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader; the absorbance of the DPPH solution was A d , and the absorbance of the sample was 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%; The results are as Figure 4 shown. 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%.

[0023] Example 7. Antibacterial experiment of the hydrogel: The antibacterial activity of the hydrogel was determined by the plate coating method, with a blank medium as the control. 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 strains. A bacterial suspension with a concentration of 10 6 CFU / ml 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 (the hydrogel prepared in Example 1)] for 12 h. After the incubation, the bacterial suspension was diluted 5-fold and inoculated onto the surface of a solid nutrient medium, and cultured at 37°C for 24 h. The culture dish was taken out, photographed and counted. The plate counting method was used to calculate the antibacterial rate, and the results are as Figure 5 shown. 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%.

[0024] Example 8. Hydrogel wound healing experiment: Kunming mice (source: Liaoning Changsheng Biotechnology Co., Ltd.) were anesthetized with isoflurane gas, and the hair on the back of the mice was shaved off to create a circular wound with a diameter of 10 mm. Each group of samples [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 (the hydrogel prepared in Example 1)] were used to treat the wounds, and the wound healing conditions were monitored on the 7th and 14th days after the operation, as Figure 6 shown. The wound area was analyzed by Image J software, as Figure 7 shown. It can be seen that the HBC+ORBSP / Shik-Ce(Ⅲ) group has the strongest activity in promoting wound healing compared with other groups.

[0025] In summary, the hydrogel prepared in the examples of the present invention has high strength, is thermosensitive, can scavenge ROS free radicals, has good antibacterial and wound healing promoting effects, and can be applied to the preparation of wound healing drugs and antibacterial drugs.

[0026] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A temperature-sensitive hydrogel that can promote wound healing, characterized in that, It includes the following raw materials: hydroxybutyl chitosan, oxidized bletilla striata polysaccharide, shikonin cerium complex, and an 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 a temperature-sensitive hydrogel capable of promoting wound healing as described in claim 1, characterized in that, It includes the following steps: Preparing shikonin cerium complex: Dissolve the water-soluble cerium salt in water, then add it to the alcoholic solution of shikonin, stir and react at room temperature, and then add water to centrifuge and separate the precipitate to obtain shikonin cerium complex; Preparing hydroxybutyl chitosan: Disperse the chitosan powder in an aqueous NaOH solution, stir at room temperature and then filter. Add the filter cake to an isopropanol / aqueous solution, stir at room temperature, then add 1,2-epoxybutane, stir at room temperature and then heat and stir, adjust the pH, filter, dialyze the filtrate, and then freeze-dry to obtain hydroxybutyl chitosan; Preparing oxidized bletilla striata polysaccharide: Dissolve bletilla striata polysaccharide and NaIO4 in water, stir and react at room temperature, then add ethylene glycol and continue to stir to terminate the reaction. Dialyze the reaction solution and freeze-dry to obtain oxidized bletilla striata polysaccharide; Preparing a thermosensitive hydrogel: Add hydroxybutyl chitosan, oxidized bletilla striata polysaccharide, and shikonin cerium complex to the injection aqueous solution and stir to obtain a thermosensitive hydrogel.

3. The preparation method of the temperature-sensitive hydrogel capable of promoting wound healing according to claim 2, characterized in that, In the step of preparing shikonin cerium complex, the water-soluble cerium salt is one of cerium nitrate, cerium chloride, cerium sulfate, and cerium acetate.

4. The preparation method of the temperature-sensitive hydrogel capable of promoting wound healing according to claim 2, wherein In the step of preparing shikonin cerium complex, the alcoholic solution of shikonin is one of the methanol solution of shikonin, the ethanol solution of shikonin, and the glycerol solution of shikonin.

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

6. The application according to claim 5, wherein The wound is one or more of traumatic wounds, bacterial infections, burns, surgeries, and diabetic wounds.

7. Use of a thermosensitive hydrogel as claimed in claim 1 for promoting wound healing in the preparation of an antibacterial drug.

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