Preparation method and application of drug-loaded hydrogel

Abstract

The invention discloses a preparation method and application of drug-loaded hydrogel, and relates to the technical field of biological medicines.The preparation method comprises the following steps that HA and glycerin are mixed and stirred, and the PH is adjusted to 7; adding the Poloxamer407 and the Poloxamer188, and carrying out standing until the Poloxamer407 and the Poloxamer188 are completely dissolved in the system, so as to obtain the hyaluronic acid poloxamer407 and the Poloxamer188, wherein the Poloxamer407 and the Poloxamer188 are completely dissolved in the system; and adding DHM, physically stirring until the medicine has no precipitate, centrifuging and discharging bubbles to obtain the drug-loaded hydrogel. According to the technical scheme, the plant active substance DHM is adopted to replace existing antibiotics, the HA hydrogel is doped to endow the injection with the antibacterial property, and the HA hydrogel is endowed with thermosensitivity by adding the heat sensitizer Poloxamer, so that the injection forms a gel state in a living body after being injected, the slow release effect on the medicine is realized, the bioavailability of the DHM is improved, the problem that the DHM is difficult to dissolve is solved, and the preparation method is simple and convenient. The disinfection and killing on the drug-resistant strains are realized. The antibacterial wound healing promoting temperature-sensitive hydrogel is simple to prepare and high in biocompatibility, and can be widely applied to the field of biological medicines.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a preparation method and application of a drug-loaded hydrogel. Background Art

[0002] As the largest defense organ of the body, the skin is easily susceptible to external stimuli, including strong impact, severe friction and sharp cuts, which often lead to skin trauma. Although minor skin injuries can heal themselves, severe skin trauma, if not treated promptly and effectively, can lead to wound infection and even tissue necrosis due to microbial invasion and inflammatory infiltration.

[0003] At present, the treatment of bacterial infections mainly uses antibiotics. Since the advent of antibiotics in the 20th century, countless lives have been saved, which is an important turning point in the long struggle between humans and pathogenic bacteria. However, due to the excessive abuse of antibiotics, bacterial resistance has become a major global public health threat. Studies have shown that the mortality rate of people infected with drug-resistant bacteria is 0.7 to 12.0 times that of those infected with sensitive bacteria. This means that skin infections caused by drug-resistant bacteria may lead to more serious consequences or even death. With the increase of drug resistance, there are fewer and fewer effective antibiotics, which limits the options for doctors to treat infectious diseases. At the same time, drug-resistant bacteria increase the socioeconomic burden, resulting in rising medical costs, which not only puts economic pressure on families, but also burdens the social medical security system. Therefore, public health experts and medical workers around the world are working hard to find new ways to fight drug-resistant bacteria, improve existing antibiotic use strategies, and raise public awareness of the rational use of antibiotics. Plant-derived substances have always attracted much attention, and as natural antibacterial compounds, they are expected to become an alternative strategy to antibiotics. Dihydromyricetin (DHM) is an extract of the grape plant rattan tea and is the main active flavonoid compound in rattan tea. It is well-known for its multifaceted pharmacological properties, including antibacterial, anti-inflammatory, anti-cancer, antioxidant and anti-diabetic properties, and has good biosafety. However, the bioavailability of dihydromyricetin is extremely low and its solubility is extremely poor, which is an urgent problem that needs to be solved.

[0004] Hyaluronic acid (HA), also known as hyaluronic acid, is a high molecular weight acidic mucopolysaccharide. It is a disaccharide unit glycosaminoglycan composed of D-glucuronic acid and N-acetylglucosamine. These disaccharide units can be repeated thousands of times to form long-chain polymers. Hyaluronic acid is widely present in the human body, such as connective tissue, vitreous body, synovial fluid, etc., and plays an important physiological function. Hyaluronic acid hydrogel (HA hydrogel) is a biomaterial with hyaluronic acid as the main component. Due to its unique physical and chemical properties, it has been widely studied and applied in the biomedical field, including antibacterial, anti-inflammatory, wound healing, cancer treatment, regenerative medicine, etc. These applications utilize the biocompatibility and biodegradability of hyaluronic acid, as well as its ability to interact well with cells and tissues. Studies have shown that the use of hyaluronic acid monomers with different molecular weights can affect the biophysical properties of hydrogels, such as viscosity, gel time, shear storage modulus, etc., and thus affect its application effect in biomedicine.

[0005] Poloxamer is a triblock copolymer composed of polyethylene oxide (PEO) and polypropylene oxide (PPO) with unique thermoresponsive properties. In the preparation of hydrogels, poloxamer, as a thermoplastic polymer, can remain liquid at room temperature and transform into a gel state at body temperature, which makes it very useful in drug delivery systems, especially in applications where hydrogels need to be formed in situ. The thermopolymerization properties of poloxamer hydrogels are mainly reflected in their unique reverse thermogel properties, that is, they are liquid at lower temperatures (such as 4~10℃) and transform into a gel state when the temperature rises to body temperature. This temperature sensitivity makes poloxamer hydrogels have broad application potential in biomedical fields such as tissue engineering, drug / gene delivery, therapeutic diagnostic devices, and the design and application of 3D printed bio-inks. The preparation of poloxamer hydrogels usually involves mixing poloxamer with water to form a solution, and then inducing the formation of a gel by increasing temperature or other triggering factors (such as pH changes, ionic strength changes, etc.). This hydrogel can be used as a drug carrier to improve drug release and bioavailability, and can also be used as a scaffold material for tissue engineering to promote cell growth and tissue regeneration. The biocompatibility and biodegradability of poloxamer hydrogels make them less toxic and have good safety when used in vivo. In addition, the amphiphilic nature of poloxamer and its potential to self-assemble into micelles make it a promising drug carrier. In general, as a hydrogel thermopolymer, poloxamer has a wide range of application prospects in the biomedical field due to its unique thermal responsiveness and biocompatibility. Summary of the invention

[0006] The technical solution of the present invention to solve the above technical problem is to provide a method for preparing a drug-loaded hydrogel, comprising the following steps: Mix HA and glycerol and adjust the pH to 7; Poloxamer 407 and Poloxamer 188 were added, and the mixture was allowed to stand until Poloxamer 407 and Poloxamer 188 were completely dissolved in the system to obtain hyaluronic acid poloxamer hydrogel; DHM was added and physically stirred until no drug precipitation occurred. The drug-loaded hydrogel was obtained after centrifugation to remove bubbles.

[0007] Furthermore, the step of mixing HA and glycerol and adjusting the pH to 7 comprises: HA was placed in distilled water and magnetically stirred at room temperature; After the powder is dissolved, add glycerin and continue stirring; Sodium hydroxide was added dropwise while stirring to adjust the pH to 7.

[0008] Furthermore, in the step of placing HA in distilled water and stirring it magnetically at room temperature, The mass of HA in the HA solution is 0.14 g, and the molecular mass is 600 kDa; the solution system is 5 mL, and the magnetic stirring time is 4-6 hours.

[0009] Furthermore, in the step of adding Poloxamer 407 and Poloxamer 188 and standing the mixture until Poloxamer 407 and Poloxamer 188 are completely dissolved in the system to obtain the hyaluronic acid poloxamer hydrogel, The ratio of Poloxamer 407 to Poloxamer 188 is 1.8:0.2.

[0010] Furthermore, in the step of adding Poloxamer 407 and Poloxamer 188 and standing the mixture until Poloxamer 407 and Poloxamer 188 are completely dissolved in the system to obtain the hyaluronic acid poloxamer hydrogel, After adding Poloxamer 407 and Poloxamer 188, place in a refrigerator at 4°C for 36h-40h; The system was allowed to stand until Poloxamer 407 and Poloxamer 188 were completely dissolved in the system to obtain hyaluronic acid poloxamer hydrogel.

[0011] Furthermore, in the step of adding DHM, physically stirring until there is no precipitation of the drug, and centrifuging to remove bubbles to obtain the drug-loaded hydrogel, The mass of DHM is 40 mg; the mass of hyaluronic acid poloxamer hydrogel is 1 mL; the physical stirring time is 3-5 min; the centrifuge speed is 1200-1500 r / min, and the time is 10 min.

[0012] In order to solve the above technical problems, the present invention also proposes an application of a thermosensitive hydrogel in promoting the healing of wounds infected with drug-resistant bacteria. The thermosensitive hydrogel is prepared by the preparation method of the drug-loaded hydrogel described above. The present invention uses plant active substance DHM to replace existing antibiotics, adds HA hydrogel to give it antibacterial properties, and gives HA hydrogel temperature sensitivity by adding thermosensitizer Poloxamer, so that it becomes a gel in the body after injection, realizes the sustained release of drugs, thereby improving the bioavailability of DHM, solving its insoluble problem, and achieving the elimination of drug-resistant strains. The antibacterial and wound healing-promoting thermosensitive hydrogel is simple to prepare and has high biocompatibility, and can be widely used in the field of biomedicine.

[0013] Compared with the previous technology, this application has the following beneficial effects: 1. Rapid preparation: The preparation method of the hydrogel in the present invention may significantly shorten the preparation time and improve the production efficiency compared with the traditional method.

[0014] 2. Improved biocompatibility: The present invention improves the biocompatibility of the thermosensitive hydrogel and reduces immune response or other side effects.

[0015] 3. Environmentally friendly: The present invention is more environmentally friendly, reduces waste generation, and the materials used in preparing the thermosensitive hydrogel pose no risk of environmental pollution.

[0016] 4. Easy operation: The present invention simplifies the preparation process of the thermosensitive hydrogel, making the preparation process easier and greatly shortening the preparation time.

[0017] 5. Adjustable performance parameters: The present invention has a wide range of adjustment space in terms of performance parameters, and exhibits different physical and chemical capabilities by selecting appropriate ratios.

[0018] 6. Clinical transformation potential: The present invention increases the clinical transformation potential of the hydrogel and gives the hydrogel a wider application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0020] Figure 1 This is the SEM image of the HPP@DHM hydrogel of the present invention; Figure 2 The fluidity and injectability images of the HPP@DHM hydrogel of the present invention; Figure 3 This is the degradation image of the HPP@DHM hydrogel of the present invention; Figure 4 This is the drug release image of the HPP@DHM hydrogel of the present invention; Figure 5 This is the in vitro antibacterial image of the HPP@DHM hydrogel of the present invention; Figure 6 This is the wound healing image of the mouse of the present invention; Figure 7 The images of the inhibition zones of the injectable drug-loaded chitosan poloxamer thermosensitive hydrogel of the present invention at different concentrations are shown; Figure 8 It is the Masson staining image of the present invention; Fig. 9 This is a Masson staining image of mice after 9 days of treatment with the present invention; Fig.10 The present invention is a flowchart of the steps of the method for preparing the drug-loaded hydrogel. DETAILED DESCRIPTION

[0021] The present invention provides a preparation method and application of a drug-loaded hydrogel, aiming to design a preparation method of an injectable thermosensitive hydrogel for promoting wound healing caused by drug-resistant bacteria infection.

[0022] The preparation method of the drug-loaded hydrogel proposed by the present invention will be described in the following specific examples: In the technical solution of this embodiment, a method for preparing a drug-loaded hydrogel is as follows: Fig.10 As shown, the following steps are included: Step 1: Mix HA and glycerol and adjust the pH to 7; Step 2: Add Poloxamer 407 and Poloxamer 188, and let stand until Poloxamer 407 and Poloxamer 188 are completely dissolved in the system to obtain hyaluronic acid poloxamer hydrogel; Step 3: Add DHM and physically stir until there is no drug precipitation. Centrifuge to remove bubbles to obtain the drug-loaded hydrogel.

[0023] Furthermore, the step of mixing HA and glycerol and adjusting the pH to 7 comprises: HA was placed in distilled water and magnetically stirred at room temperature; After the powder is dissolved, add glycerin and continue stirring; Sodium hydroxide was added dropwise while stirring to adjust the pH to 7.

[0024] Furthermore, in the step of placing HA in distilled water and stirring it magnetically at room temperature, The mass of HA in the HA solution is 0.14 g, and the molecular mass is 600 kDa; the solution system is 5 mL, and the magnetic stirring time is 4-6 hours.

[0025] Furthermore, in the step of adding Poloxamer 407 and Poloxamer 188 and standing the mixture until Poloxamer 407 and Poloxamer 188 are completely dissolved in the system to obtain the hyaluronic acid poloxamer hydrogel, The ratio of Poloxamer 407 to Poloxamer 188 is 1.8:0.2.

[0026] Furthermore, in the step of adding Poloxamer 407 and Poloxamer 188 and standing the mixture until Poloxamer 407 and Poloxamer 188 are completely dissolved in the system to obtain the hyaluronic acid poloxamer hydrogel, After adding Poloxamer 407 and Poloxamer 188, place in a refrigerator at 4°C for 36h-40h; The system was allowed to stand until Poloxamer 407 and Poloxamer 188 were completely dissolved in the system to obtain hyaluronic acid poloxamer hydrogel.

[0027] Furthermore, in the step of adding DHM, physically stirring until there is no precipitation of the drug, and centrifuging to remove bubbles to obtain the drug-loaded hydrogel, The mass of DHM is 40 mg; the mass of hyaluronic acid poloxamer hydrogel is 1 mL; the physical stirring time is 3-5 min; the centrifuge speed is 1200-1500 r / min, and the time is 10 min.

[0028] In order to solve the above technical problems, the present invention also proposes an application of a thermosensitive hydrogel in promoting the healing of wounds infected with drug-resistant bacteria. The thermosensitive hydrogel is prepared by the preparation method of the drug-loaded hydrogel described above. The technical solution of this application can reduce the preparation time: The preparation of the hydrogel in the present invention uses a physical crosslinking method. The formation of the physically crosslinked hydrogel is usually completed within a few minutes to a few hours, while the chemical crosslinking process may take several hours to several days, especially in the curing or post-treatment steps.

[0029] Applying technical solutions can reduce immune response: During the treatment, tissues around the wound were collected and the content of anti-inflammatory factor interleukin 10 (IL-10) was checked. The results showed that the IL-10 content of the drug-loaded hydrogel group was much higher than that of the blank control group, indicating that the drug-loaded hydrogel can treat inflammation and reduce the body's immune response.

[0030] The difficulty to be overcome in injectable thermosensitive hydrogels is that the hydrogels must have good fluidity to be injected through a syringe, and must also be able to gel in the mouse body to achieve a sustained release effect of the drug. Therefore, the present application designs thermosensitive hydrogels.

[0031] The present application requires only three raw materials, does not require any catalyst, and the entire production process is a physical cross-linking method.

[0032] This application adopts the control variable method, the design scheme, fixes the ratio of two materials and changes the ratio of another material to achieve the preset effect. The later drug loading will also affect the fluidity of the hydrogel. The appropriate amount interval is continuously determined between 10-60mg of the drug, and finally the drug loading that can achieve the maximum antibacterial effect without affecting the fluidity of the hydrogel is determined. The experimental time of each batch needs to be based on the degradation time of the hydrogel, and the whole process takes more than 6 months. Example 1: A method for preparing an injectable drug-loaded chitosan thermosensitive hydrogel, comprising the following steps: Weigh 0.14 g of HA powder in 5 mL of distilled water and stir magnetically at room temperature for 4 h.

[0033] After the HA powder is completely dissolved, add glycerin and continue stirring.

[0034] Sodium hydroxide (NaOH) solution was added dropwise while stirring to adjust the pH to 7.

[0035] Then Poloxamer 407 and Poloxamer 188 were added in a ratio of 1.8:0.2.

[0036] The mixture was placed in a 4°C refrigerator and allowed to stand until Poloxamer 407 and Poloxamer 188 were completely dissolved in the system to obtain hyaluronic acid poloxamer hydrogel (HPP).

[0037] Weigh DHM into a 5 mL centrifuge tube, inject 1 mL of hyaluronic acid poloxamer hydrogel using a syringe, physically stir until there is no drug precipitation (physical stirring time is 3-5 min), and centrifuge to remove bubbles to obtain the drug-loaded hydrogel (HPP@DHM) (centrifuge speed is 1200-1500 r / min, time is 10 min). SEM image of HPP hydrogel prepared in Example 1 The microstructure of the hydrogel was observed by SEM, such as Figure 1As shown, the HPP hydrogel presents an interconnected three-dimensional network without forming aggregates. The highly porous network structure can increase the uniform distribution and structural content of DHM in the hydrogel, enabling DHM to enter the wound site and be released. The appropriate macroporous structure helps to increase the specific surface area and enhance the interaction with water molecules.

[0038] Temperature sensitivity and fluidity images of HPP hydrogels, such as Figure 2 As shown; the gel is in a flowing sol state at room temperature and can be freely drawn with a syringe, indicating that it has good fluidity, and turns into a gel state at around 34°C, indicating that the hydrogel is thermosensitive.

[0039] Degradation test of HPP hydrogel: The Cy5 dye was prepared into a 0.1 mg / mL solution, 10 uL was taken and added to 1 mL of HPP hydrogel, and the Cy5 solution was evenly distributed in the HPP hydrogel using physical stirring to obtain HPP@Cy5. The free Cy5 solution and HPP@Cy5 were injected into depilated Kunming mice (6-8 week old female Kunming mice (KM mice) provided by Yisi Experimental Animal Technology Co., Ltd.) using a syringe, and the degradation of the hydrogel was obtained by observing the subcutaneous fluorescence intensity of the mice using a small animal in vivo imager.

[0040] The degradation of HPP hydrogel under mouse skin, such as Figure 3 As shown in the figure, in the mouse body, the HPP gel formed a regular gel state and gradually degraded until the 9th day when the gel was almost completely degraded. The fluorescence intensity of the free Cy5 fluorescent dye group decreased from 100% to 54.34% after 1 day. After the 4th day, no fluorescence was detected in the free Cy5 group, while the degradation time of the HPP@Cy5 group reached 9 days, indicating that the HPP hydrogel can be degraded and can play a role in sustained drug release.

[0041] Drug release test of HPP@DHM hydrogel: 1 mL of HPP@DHM hydrogel was injected into a 50 mL centrifuge tube, placed in a 34 °C constant temperature water bath to allow the hydrogel to gel, then 30 mL of PBS was slowly injected, and then placed in a 37 °C 100 r / min constant temperature shaker. At a specific time point, 3 mL of the solution was taken out and the absorbance was measured at 290 nm by a UV spectrophotometer, and the drug release amount was calculated to draw a drug release curve.

[0042] like Figure 4As shown in the figure, the release of DHM from HPP@DHM hydrogel increased significantly within eight hours, with the cumulative release of dihydromyricetin being approximately 62.4%. The release reached 65.5% at 11.5 h. This indicates that the dihydromyricetin encapsulated in the hydrogel was released once the gel was immersed in the medium.

[0043] In vitro antibacterial test of HPP@DHM hydrogel: The experiment was divided into three groups, namely blank control group, HPP hydrogel group and HPP@DHM hydrogel group. For the blank group, 100 μL of methicillin-resistant Staphylococcus aureus (MRSA) solution (108 CFU / mL) was evenly spread on the agar plate. For the HPP hydrogel group and HPP@DHM hydrogel group, 500 uL of HPP hydrogel and HPP@DHM hydrogel were taken respectively using a one-ml syringe on the basis of the blank group, and the hydrogel was evenly spread on the plate. The three groups were cultured at 37°C at the same time for 16 hours, and the number of colonies was observed.

[0044] like Figure 5 As shown in the figure, the antibacterial activity of HPP@DHM hydrogel was studied using the hydrogel to study the antibacterial activity of MRSA. Compared with the control group, the number of colonies in the HPP hydrogel group was partially reduced, indicating that the HPP hydrogel itself has a weak antibacterial effect; the HPP@DHM hydrogel group has a significant antibacterial effect compared with the other two groups, indicating that the HPP@DHM hydrogel has a strong inhibitory effect on MRSA.

[0045] HPP@DHM hydrogel promotes wound healing test: The mice were randomly divided into 3 groups, 5 in each group. After depilation and anesthesia of the back of each group of mice, a circular full-thickness wound with a diameter of 10 mm was cut on the back of the mice using surgical forceps and surgical scissors. 50uL of MRSA bacteria droplets with a concentration of 108CFU were applied to the wound to establish a MRSA infection model. After 48 hours of infection, 100uL of PBS solution, blank hydrogel, and drug-loaded hydrogel were injected around the wound. Subsequently, the wound changes of the mice on different days after treatment were recorded using a mobile phone on days 1, 5, 9, and 13, and the size of the wound was quantified.

[0046] Wound healing images of mice in each group on days 1, 5, 9, and 13. Figure 6 As shown, during the entire wound healing process, the drug-loaded hydrogel group had better therapeutic effects on each infection model than the other groups, and the infected wounds were almost completely healed after 13 days of treatment.

[0047] The wound healing rate of mice in each group was Figure 7 As shown, the back wounds of mice in the HPP@DHM group were almost completely healed after thirteen days of treatment, while the remaining healing area in the blank control group was 32.17%.

[0048] HPP@DHM hydrogel promotes collagen deposition at the wound site: Masson staining was used to confirm collagen deposition during wound healing, because the gradual increase in the amount of collagen fiber deposition indicated the main signs of external matrix deposition, tissue remodeling and maturation during wound healing. Skin tissues in each treatment group were collected on the 9th day after treatment and Masson staining was used to observe collagen deposition in the wound site. Sample tissues were dewaxed to water and soaked in xylene I for 5 minutes, xylene II for 5 minutes, xylene III for 5 minutes, anhydrous ethanol for 1 minute, 95% ethanol for 1 minute, 75% ethanol for 1 minute, and rinsed with tap water for a few seconds; stained with the prepared Weigert iron hematoxylin staining solution for 8 minutes; differentiated with acidic ethanol differentiation solution for 15 seconds, washed with water; and returned to blue with Masson blueing solution for 5 minutes, washed with water. Wash with distilled water for 1 minute; stain with Ponceau fuchsin staining solution for 5 minutes; wash with weak acid working solution for 1 minute; wash with phosphomolybdic acid solution for 1 minute, and wash with weak acid working solution for 1 minute; stain with aniline blue staining solution for 2 minutes, and wash with weak acid for 1 minute; dehydrate and make transparent: rapid dehydration with 95% ethanol for 2-3 seconds, dehydration with anhydrous ethanol 3 times, 5-10 seconds each time, and xylene make transparent 3 times, 1-2 minutes each time, and observe the results under an optical microscope after sealing with neutral gum.

[0049] Masson staining images, such as Figure 8 As shown in the figure, the collagen deposition area of ​​HPP@DHM hydrogel is 86.08%, while the collagen deposition area of ​​PBS control group is 31.02%. This result is consistent with the previous experimental results, indicating that HPP@DHM hydrogel has a good promoting effect on collagen deposition in MRSA-infected wounds.

[0050] HPP@DHM hydrogel safety testing: After 9 days of treatment, the main organs of mice (heart, liver, spleen, lung, and kidney) were taken and rinsed in pre-cooled PBS. After absorbing the water with clean filter paper, they were fixed in 4% paraformaldehyde for more than 24 hours, and then dehydrated by gradient: 70% ethanol solution for 30 minutes, 80% ethanol solution for 30 minutes, 90% ethanol solution for 30 minutes, 95% ethanol solution for 30 minutes, anhydrous ethanol for 60 minutes, anhydrous ethanol for 60 minutes, xylene for 120 minutes, and paraffin for 180 minutes; after dehydration, the main organs were embedded in paraffin. Use a slicer to cut into 5um thick tissues. Dewaxing steps for staining: xylene for 20 minutes, anhydrous ethanol for 10 minutes, 95% ethanol for 5 minutes, 85% ethanol for 5 minutes, 70% ethanol for 5 minutes, and pure water for 5 minutes. Staining: hematoxylin for 4 minutes, tap water for 10 minutes, hydrochloric acid ethanol differentiation for 3 seconds, tap water for 10 minutes, eosin for 25 seconds to 1 minute. Dehydration: anhydrous ethanol for 15 minutes. Permeabilization: xylene for 30 minutes. Sealing: After sealing with neutral resin, wait for it to dry naturally and then place it under a microscope for photography and observation.

[0051] like Fig. 9 After 9 days of treatment, the main organs of the mice were collected for H&E staining. Compared with the HE results of normal mice, no obvious pathological abnormalities were found, indicating that HPP@DHM hydrogel has excellent safety. The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed by the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for preparing a drug-loaded hydrogel, characterized in that: The following steps are involved: Mix HA and glycerol and adjust the pH to 7; Poloxamer 407 and Poloxamer 188 were added, and the mixture was allowed to stand until Poloxamer 407 and Poloxamer 188 were completely dissolved in the system to obtain hyaluronic acid poloxamer hydrogel; DHM was added and physically stirred until no drug precipitation occurred. The drug-loaded hydrogel was obtained after centrifugation to remove bubbles.

2. The method for preparing the drug-loaded hydrogel according to claim 1, characterized in that: The step of mixing HA and glycerol and adjusting the pH to 7 comprises: HA was placed in distilled water and magnetically stirred at room temperature; After the powder is dissolved, add glycerin and continue stirring; Sodium hydroxide was added dropwise while stirring to adjust the pH to 7.

3. The method for preparing the drug-loaded hydrogel according to claim 2, characterized in that: In the step of placing HA in distilled water and stirring it magnetically at room temperature, The mass of HA in the HA solution is 0.14 g, and the molecular mass is 600 kDa; the solution system is 5 mL, and the magnetic stirring time is 4-6 hours.

4. The method for preparing the drug-loaded hydrogel according to claim 1, characterized in that: In the step of adding Poloxamer 407 and Poloxamer 188 and standing until Poloxamer 407 and Poloxamer 188 are completely dissolved in the system to obtain hyaluronic acid poloxamer hydrogel, The ratio of Poloxamer 407 to Poloxamer 188 is 1.8:0.

2.

5. The method for preparing the drug-loaded hydrogel according to claim 1, characterized in that: In the step of adding Poloxamer 407 and Poloxamer 188 and standing until Poloxamer 407 and Poloxamer 188 are completely dissolved in the system to obtain hyaluronic acid poloxamer hydrogel, After adding Poloxamer 407 and Poloxamer 188, place in a refrigerator at 4°C for 36h-40h; The system was allowed to stand until Poloxamer 407 and Poloxamer 188 were completely dissolved in the system to obtain hyaluronic acid poloxamer hydrogel.

6. The method for preparing the drug-loaded hydrogel according to claim 1, characterized in that: In the step of adding DHM, physically stirring until there is no precipitation of the drug, and centrifuging to remove bubbles to obtain the drug-loaded hydrogel, The mass of DHM is 40 mg; the mass of hyaluronic acid poloxamer hydrogel is 1 mL; the physical stirring time is 3-5 min; the centrifuge speed is 1200-1500 r / min, and the time is 10 min.

7. Application of a thermosensitive hydrogel in promoting wound healing caused by drug-resistant bacterial infection, characterized in that: The thermosensitive hydrogel is prepared by the method for preparing the drug-loaded hydrogel according to any one of claims 1 to 6.

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