Multifunctional temperature-sensitive slow-release hydrogel, preparation method thereof and application of multifunctional temperature-sensitive slow-release hydrogel in preparation of itching-relieving and pain-easing preparation

Abstract

The invention discloses multifunctional temperature-sensitive slow-release hydrogel, a preparation method thereof and application of the multifunctional temperature-sensitive slow-release hydrogel in preparation of an itching-relieving and pain-easing preparation. The preparation method comprises the following steps: sequentially mixing microspheres formed by compounding hyaluronic acid and quaternary ammonium salt chitosan with a medicine, a substance which is dissolved by a solubilizer and has a cool smell and poloxamer 407 by adopting a simpler magnetic stirring method, and preparing the multifunctional temperature-sensitive slow-release hydrogel on the premise of not using a thickening agent. The hydrogel prepared by the preparation method disclosed by the invention has relatively strong cool smell and slow release effect, and has good biocompatibility; when the used medicine is a substance with the itching-relieving and pain-relieving effect, such as oxymatrine, the hydrogel also has the effects of treating non-histamine-dependent pruritus and easing pain.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a multifunctional thermosensitive sustained-release hydrogel and a preparation method thereof, and application of the hydrogel in the preparation of an antipruritic and analgesic preparation. Background Art

[0002] Hydrogel is a polymer system composed of a three-dimensional viscoelastic network. It can absorb a large amount of water and swell in a physiological environment, while physical or chemical cross-linking can enhance its structure and performance. It has good hydrophilicity, softness and biocompatibility, and is widely used in the field of biomedicine. Hydrogel can control drug release through two ways: self-swelling and external stimulation of its network structure, so it can be used as a drug carrier to assist in achieving the purpose of controlled drug release. According to the cross-linking method of gel formation, hydrogels can be divided into physically cross-linked hydrogels, chemically cross-linked hydrogels and radiation cross-linked hydrogels; according to their functions and uses, they can be divided into drug carrier hydrogels and tissue scaffold hydrogels, etc.

[0003] According to the function of hydrogel, hydrogel can be divided into thermosensitive hydrogel, sustained-release hydrogel and antipruritic and analgesic hydrogel. Hydrogel with thermosensitive function has a certain proportion of hydrophilic groups (such as carboxyl, amide, hydroxyl, etc.) and hydrophobic groups (such as methyl, ethyl, propyl and other alkyl groups) on its macromolecular chain. Changes in the external environment temperature will affect the interaction between the hydrophilic and hydrophobic groups and the hydrogen bonding of the macromolecular chain, thereby causing it to gel at a certain temperature. Different hydrogel synthesis conditions and material composition will directly affect the release rate of drugs in hydrogels. Hydrogels with high cross-linking degree usually show a slower release rate, thereby achieving a sustained release effect; hydrogels with low cross-linking degree show a higher release dynamic rate. Hydrogels can be used as carriers of drugs to assist the dispersion of drugs in the system, and the drugs can play their effects normally. Depending on the drugs carried, hydrogels can embody different functions: hydrogels containing antipruritic drugs such as calamine have a certain antipruritic effect. When the drugs contained are analgesics such as ropivacaine, the hydrogels can play an analgesic effect.

[0004] Nevertheless, hydrogels still have certain defects:

[0005] (1) The mechanical properties are low, and subsequent processing and sterilization are difficult. Existing hydrogels for antipruritic and analgesic purposes are generally in the form of patches;

[0006] (2) The environmental response speed is slow, and the thermosensitive response performance of the thermosensitive hydrogel with a single structure is poor;

[0007] (3) Low drug loading and poor controlled drug release limit clinical application;

[0008] (4) The improvement conditions of different functions of hydrogels interfere with or even contradict each other, making it difficult to achieve overall functional improvement;

[0009] (5) The hydrogel improvement process is relatively complicated and the improvement cost is high, which increases the actual use cost.

[0010] Commonly used antipruritic and analgesic topical preparations currently mainly include ointments, films, suppositories and effervescent tablets, and each dosage form has certain defects: Ointment preparations mix the drug with an appropriate amount of matrix, apply it directly to the affected skin, and slowly absorb through penetration, but its composition is relatively fixed and its modifiability is poor. Film preparations can quickly form a film to cover the affected area and isolate the impact of external factors on the affected area, but the film is thin, the drug loading is small, and the therapeutic effect is limited. Suppositories are inserted into the cavity and in close contact with the mucosa, quickly softening and dissolving to release the drug, but they have more matrix and less active ingredient content, requiring frequent dressing changes, and requiring high storage conditions. Effervescent tablets produce a large amount of carbon dioxide through effervescent reactions, which can help carry drugs to lesions that are difficult to administer with ordinary preparations. The production process is prone to sticking and splitting, and the production requirements are high.

[0011] In view of the above problems, the present invention has the advantages of simple manufacturing process and low production cost, and at the same time has good temperature-sensitive function, good drug sustained-release effect and obvious antipruritic and analgesic effects. Summary of the invention

[0012] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and to provide a method for preparing a multifunctional temperature-sensitive sustained-release hydrogel.

[0013] Another object of the present invention is to provide a multifunctional thermosensitive sustained-release hydrogel obtained by the above preparation method, wherein the hydrogel has the characteristics of thermosensitive and sustained-release.

[0014] Another object of the present invention is to provide an application of the multifunctional temperature-sensitive sustained-release hydrogel.

[0015] The purpose of the present invention is achieved through the following technical solutions:

[0016] A method for preparing a multifunctional thermosensitive sustained-release hydrogel comprises the following steps:

[0017] (1) mixing the quaternary ammonium salt chitosan aqueous solution and the hyaluronic acid aqueous solution uniformly, centrifuging and collecting the supernatant, and rotary evaporating and concentrating to obtain a quaternary ammonium salt chitosan / hyaluronic acid microsphere aqueous solution;

[0018] (2) mixing the drug with the quaternary ammonium salt chitosan / hyaluronic acid microsphere aqueous solution obtained in step (1) to obtain a drug-loaded microsphere solution;

[0019] (3) mixing the substance having a refreshing smell with a solubilizing agent, and dissolving them in water to obtain a solution having a refreshing smell;

[0020] (4) mixing the drug-loaded microsphere solution obtained in step (2) with the solution with a refreshing smell obtained in step (3) to obtain a drug-loaded microsphere solution with a refreshing smell;

[0021] (5) Poloxamer 407 (also known as segmented polyether F-127) is added to the solution obtained in step (4), water is added to mix, and the mixture is allowed to stand to obtain a multifunctional thermosensitive sustained-release hydrogel.

[0022] The method of uniformly mixing the quaternary ammonium salt chitosan aqueous solution and the hyaluronic acid aqueous solution in step (1) is preferably: injecting the hyaluronic acid aqueous solution into the quaternary ammonium salt chitosan aqueous solution under stirring.

[0023] The injection speed of the quaternary ammonium salt chitosan aqueous solution into the hyaluronic acid aqueous solution is 0.1 to 10.0 mL / s, preferably 1.3 to 1.5 mL / s.

[0024] The stirring condition is preferably 800-1000 rpm for 1-6 hours, and more preferably 900 rpm for 4 hours.

[0025] The molecular weight of the quaternary ammonium salt chitosan described in step (1) is preferably 5.0×10 4 ~40.0×10 4 Dalton; more preferably 20.0×10 4 Dalton.

[0026] The molecular weight of the hyaluronic acid in step (1) is preferably 5.0×10 4 ~40.0×10 4 Dalton; more preferably 20.0×10 4 Dalton.

[0027] The concentration of the quaternary ammonium chitosan aqueous solution described in step (1) is preferably 0.1-2.0 mg / mL; more preferably 1 mg / mL.

[0028] The concentration of the hyaluronic acid aqueous solution in step (1) is preferably 0.1 to 2.0 mg / mL; more preferably 1 mg / mL.

[0029] The volume ratio of the quaternary ammonium salt chitosan aqueous solution to the hyaluronic acid aqueous solution in step (1) is preferably 2:1 to 1:2; more preferably 1:1.

[0030] The centrifugation conditions in step (1) are preferably centrifuged at a speed of 2000-18000 rpm for 5-30 minutes; more preferably centrifuged at 7000 rpm for 15 minutes.

[0031] The conditions for rotary evaporation concentration described in step (1) are preferably as follows: a rotation speed of 20 to 200 rpm, a pressure of 0.01 to 0.1 MPa, a temperature of 25 to 65° C., and a time of 2 to 10 h; more preferably as follows: a rotation speed of 60 rpm, a pressure of 0.095 MPa, a temperature of 50° C., and a time of 3.5 to 4.5 h.

[0032] The concentration of the microspheres in the quaternary ammonium chitosan / hyaluronic acid microsphere aqueous solution in step (1) is 0.10-2.00% (w / w); preferably 1.2-1.3% (w / w); more preferably 1.25% (w / w).

[0033] The particle size of the quaternary ammonium salt chitosan / hyaluronic acid microspheres described in step (1) is preferably 100 to 1000 nm; more preferably 450 to 600 nm.

[0034] The drug described in step (2) is preferably a water-soluble drug; more preferably at least one of an alkaloid and a flavonoid component; most preferably at least one of an alkaloid and a flavonoid component having the effect of treating non-histamine-dependent pruritus and analgesia.

[0035] The alkaloid is preferably at least one of oxymatrine, sophocarpine and oxymatrine; more preferably oxymatrine.

[0036] The flavonoid component is preferably at least one of calycosin and sophora flavanoids.

[0037] The drug and the quaternary ammonium salt chitosan / hyaluronic acid microspheres in step (2) are preferably calculated at a mass ratio of 240 to 1:1; more preferably, they are calculated at a mass ratio of 80:9.

[0038] The mixing method in step (2) is preferably stirring; more preferably magnetic stirring.

[0039] The stirring condition is preferably 100-200 rpm for 1-72 h, more preferably 100 rpm for 48 h.

[0040] The substance with a refreshing smell described in step (3) is preferably at least one of menthol, natural borneol (borneol), artificial borneol and camphor; more preferably at least one of menthol and natural borneol (borneol).

[0041] The content of the substance with a refreshing smell in the refreshing smell solution in step (3) is 0.1-10.0% (w / w); more preferably 5% (w / w).

[0042] The solubilizing agent described in step (3) is preferably PEG-DA258, PEG660CSA or PEG40COH; more preferably PEG40COH.

[0043] The amount of the solubilizer used in step (3) is preferably calculated based on a mass ratio of the substance with a cool smell to the solubilizer of 1:1 to 18; more preferably, the mass ratio of the substance with a cool smell to the solubilizer is 1:4.

[0044] The drug-loaded microsphere solution described in step (4) and the solution having a refreshing smell are preferably mixed in a volume ratio of 1:1 to 10:1; more preferably, they are mixed in a volume ratio of 5:2 (v / v).

[0045] The mixing method in step (4) is preferably stirring.

[0046] The stirring condition is preferably room temperature and stirring at 100 rpm for 10 min.

[0047] The room temperature is 25±1°C.

[0048] The content of poloxamer 407 in the multifunctional thermosensitive sustained-release hydrogel system in step (5) is preferably 10.0% to 16.0% (w / w); more preferably 12 to 13% (w / w); and most preferably 12 to 12.5%.

[0049] The amount of water used in step (5) is preferably calculated in a ratio of 140-200 μL:100-160 mg to poloxamer 407; more preferably, in a ratio of 170-180 μL:120-130 mg to poloxamer 407; and most preferably, in a ratio of 175-180 μL:120-125 mg to poloxamer 407.

[0050] The mixing method in step (5) is preferably stirring.

[0051] The stirring conditions are preferably -20 to 40°C, 50 to 150 rpm, and 0.2 to 10 h; more preferably 4 to 6°C, 100 rpm, and 4 to 6 h.

[0052] When the standing temperature in step (5) is not less than the phase transition temperature, the multifunctional thermosensitive sustained-release hydrogel is in a semisolid form. When used, it is placed in an environment below the phase transition temperature and converted into a solution state, which can be used by spraying or applying. When the standing temperature is less than the phase transition temperature, the multifunctional thermosensitive sustained-release hydrogel is in a solution form, which can be used by spraying or applying.

[0053] The product is named as multifunctional sustained-release hydrogel in the present invention according to its form when used on the human body.

[0054] The standing in step (5) is preferably performed at -6 to -20°C for 12 to 24 hours.

[0055] A multifunctional thermosensitive sustained-release hydrogel is prepared by the above preparation method.

[0056] The multifunctional thermosensitive sustained-release hydrogel is a multifunctional thermosensitive sustained-release hydrogel with a refreshing smell; when the drug therein is a drug with the effects of treating non-histamine-dependent pruritus and relieving pain, the multifunctional thermosensitive sustained-release hydrogel has the effects of treating non-histamine-dependent pruritus and relieving pain.

[0057] The multifunctional thermosensitive sustained-release hydrogel is used in the preparation of an antipruritic and analgesic preparation.

[0058] The antipruritic and analgesic effect refers to the effect of treating non-histamine dependent pruritus and relieving pain.

[0059] The antipruritic and analgesic preparations include but are not limited to the following dosage forms: external solutions, lotions, liniments, ointments, pastes, patches, emulsions, sprays; films; soft capsules, emulsions, suspensions; microsphere preparations, microcapsule preparations, nanocapsule preparations; and local injections.

[0060] Example results and conclusions:

[0061] After successfully preparing a multifunctional thermosensitive sustained-release hydrogel with a refreshing smell, the present invention found through temperature scanning that the phase transition temperature of the gel was 21-30°C, and the gel smelled minty and refreshing. The Franz transdermal cell was used at 32±0.5°C and physiological saline was used as the receiving liquid to evaluate the transdermal release behavior of the active ingredients in the prepared multifunctional thermosensitive sustained-release hydrogel within 24 hours, and the cumulative drug permeability and the cumulative permeation per unit area were calculated and the corresponding curves were drawn. The results showed that compared with the solution containing only the drug, the multifunctional thermosensitive sustained-release hydrogel had a longer drug release time. The results of the biocompatibility and hemolysis experiments showed that the sustained-release hydrogel had better biosafety.

[0062] Compared with the prior art, the present invention has the following advantages and effects:

[0063] 1. In the embodiment of the present invention, microspheres formed by the composite of hyaluronic acid and quaternary ammonium salt chitosan are mixed with oxymatrine, menthol solution solubilized with PEG40COH, and poloxamer 407 in sequence by a simpler magnetic stirring method to prepare a multifunctional thermosensitive sustained-release hydrogel solution without using a thickener.

[0064] 2. The microspheres formed by physical crosslinking between hyaluronic acid and quaternary ammonium salt chitosan through electrostatic action in the present invention can "encircle" oxymatrine therein, inhibiting the excessive release of oxymatrine. The added poloxamer 407 can convert the gel solution from a liquid state to a gel state when the temperature rises, and can also restore the gel solution from a gel state to a liquid state when the temperature drops, adding the sprayable property of non-contact administration and the non-contact cleaning property that can be easily washed with cold water to the gel solution, providing a convenient administration and dressing change method for patients with skin lesions, wounds or hypersensitivity on the affected part, while avoiding secondary infection of skin lesions or wounds caused by direct contact with the affected part.

[0065] 3. The present invention does not involve the separation and purification of drug-loaded microspheres, and the administered drugs can be 100% utilized, thus reducing the cost of drug use.

[0066] 4. Compared with most composite materials using hyaluronic acid-quaternary ammonium salt chitosan, the present invention adds menthol, which can bring a local cool smell to the user and relieve discomfort such as redness, swelling, heat, pain, itching, etc.

[0067] 5. The multifunctional thermosensitive sustained-release hydrogel provided by the present invention has a cumulative transdermal permeability and cumulative permeation per unit area reduced by about 50% compared with the aqueous solution after 24 hours of use, and has a strong sustained-release effect.

[0068] 6. In animal experiments, it was found that the multifunctional thermosensitive sustained-release hydrogel provided by the present invention has an antipruritic effect that can last for at least 3 days after a single use, and has a strong and ultra-long antipruritic effect.

[0069] 7. The multifunctional thermosensitive sustained-release hydrogel involved in the present invention has relatively good biocompatibility, will not produce hemolytic effect, and will not cause harm to the human body when applied externally.

[0070] 8. The multifunctional thermosensitive sustained-release hydrogel provided by the present invention has good antipruritic and analgesic effects and has no effect on the immune organs of experimental animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 The figures are transmission electron micrographs of quaternary ammonium chitosan / hyaluronic acid microspheres before and after drug loading; wherein, (A) is quaternary ammonium chitosan / hyaluronic acid microspheres without drug loading (15000×), (B) is a crystallization image of oxymatrine (50000×), and (C) is a quaternary ammonium chitosan / hyaluronic acid microspheres loaded with oxymatrine (10000×).

[0072] Figure 2The figures are temperature scanning results of the multifunctional thermosensitive sustained-release hydrogel when the proportion of F-127 is 12-13%; wherein, (A) is the temperature scanning result of the multifunctional thermosensitive sustained-release hydrogel when the proportion of F-127 is 13%, (B) is the temperature scanning result of the multifunctional thermosensitive sustained-release hydrogel when the proportion of F-127 is 12.5%, and (C) is the temperature scanning result of the multifunctional thermosensitive sustained-release hydrogel when the proportion of F-127 is 12%.

[0073] Figure 3 This is a graph showing the cytotoxicity of different concentrations of multifunctional thermosensitive sustained-release hydrogel extracts on L929.

[0074] Figure 4 The figures are the results of hemolysis of multifunctional thermosensitive sustained-release hydrogels of different concentrations; wherein, (A) is a statistical bar graph of the hemolysis rate of multifunctional thermosensitive sustained-release hydrogels of different concentrations compared with the positive control 1% Triton-X100 solution, and (B) is a schematic diagram of the hemolysis of multifunctional thermosensitive sustained-release hydrogels of different concentrations compared with the positive control 1% Triton-X100 solution.

[0075] Figure 5 The figures are the results of in vitro transdermal test of drug aqueous solution and multifunctional thermosensitive sustained-release hydrogel; wherein, (A) is the statistical graph of transdermal permeability of drug aqueous solution and multifunctional thermosensitive sustained-release hydrogel over time; (B) is the statistical graph of transdermal permeability of drug aqueous solution and multifunctional thermosensitive sustained-release hydrogel over time.

[0076] Figure 6 It is a statistical graph showing the effect of the sustained-release properties of the multifunctional thermosensitive sustained-release hydrogel on the antipruritic efficacy of oxymatrine over time; wherein, (A) is a statistical graph showing the antipruritic efficacy of the drug aqueous solution over time; (B) is a statistical graph showing the antipruritic efficacy of the multifunctional thermosensitive sustained-release hydrogel over time.

[0077] Figure 7 The figures are statistical diagrams showing the antipruritic and analgesic efficacy of the multifunctional thermosensitive sustained-release hydrogel, and its effects on the immune-related thymus and spleen organs; wherein, (A) is a statistical diagram showing the antipruritic efficacy of the multifunctional thermosensitive sustained-release hydrogel; (B) is a statistical diagram showing the analgesic efficacy of the multifunctional thermosensitive sustained-release hydrogel; (C) is a statistical diagram showing the effect of the multifunctional thermosensitive sustained-release hydrogel on the thymus; and (D) is a statistical diagram showing the effect of the multifunctional thermosensitive sustained-release hydrogel on the spleen. DETAILED DESCRIPTION

[0078] The present invention is described in further detail below in conjunction with Examples and accompanying drawings, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents described in the present invention are all commercially available.

[0079] Example 1

[0080] (1) Weigh 50 mg of hyaluronic acid (20.0 × 10 4 Da, the same below) and 50 mg quaternary ammonium salt chitosan (20.0×10 4 Da, the same below), were placed in 50 ml centrifuge tubes, and after adding water, they were shaken on a shaker for 4 hours to dissolve the solids evenly, and a 1 mg / ml concentration solution was prepared;

[0081] (2) The quaternary ammonium salt chitosan solution was poured into a beaker and stirred at 900 rpm at room temperature until the speed was stable. Then, the hyaluronic acid solution was added to the quaternary ammonium salt chitosan solution using a separatory funnel (speed 1.3-1.5 ml / s). After continuous stirring for 4 hours, the solution was centrifuged at 7000 rpm for 15 minutes and the supernatant was collected. Under a transmission electron microscope, it was found that the diameter of the solution was at the micro-nano scale (e.g. Figure 1 As shown), rotary evaporation and concentration for 3.5 hours to a solution with a microsphere mass fraction of 1.25%;

[0082] (3) Weigh 50 mg of oxymatrine and add 450 μl of concentrated microsphere solution, stir at 100 rpm for 48 hours at room temperature to prepare a microsphere solution containing oxymatrine, wherein the mass ratio of oxymatrine to microsphere is 80:9. Under transmission electron microscopy, oxymatrine crystals (such as Figure 1 shown);

[0083] (4) Weigh 10 mg of menthol and 40 mg of PEG40COH (Nantong Jiuze Chemical Co., Ltd.; Product No.: 2023031315), add 150 μL of water to dissolve, add the oxymatrine drug-loaded microsphere solution prepared in step (3), and stir at 100 rpm for 10 min at room temperature to prepare a menthol and oxymatrine drug-loaded microsphere solution;

[0084] (5) Weigh 125 mg of poloxamer 407 (F-127) and add it to the menthol and oxymatrine drug-loaded microsphere solution prepared in step (4), add 175 μL of water, stir at 100 rpm for 4 to 6 hours at room temperature, and then place in a refrigerator overnight to complete the preparation of the multifunctional thermosensitive sustained-release hydrogel;

[0085] (6) The obtained multifunctional thermosensitive sustained-release hydrogel was subjected to temperature scanning using a rotational rheometer, and the gel phase transition temperature was measured to be approximately 26.5°C ( Figure 2 The multifunctional thermosensitive sustained-release hydrogel is in liquid state at 4°C and in slightly viscous sprayable liquid state at room temperature (24°C). It can become gel after being sprayed on the skin for 1-3 seconds and can be washed off in clean water at about 18°C ​​for 5 seconds.

[0086] (7) The multifunctional thermosensitive sustained-release hydrogel extract was prepared according to the soluble materials section in GB16886.12-2017, and its cytotoxicity study was carried out according to GB / T16886-5-2017. The gel had no potential cytotoxicity (such as Figure 3 shown);

[0087] (8) Whole blood was collected from the abdominal aorta of 8-12 week old rats (purchased from Zhuhai Baishitong Biotechnology Co., Ltd.), and the 4% red blood cell suspension obtained by diluting the anticoagulated purified saline was used for hemolytic study. The gel solution with a concentration range of 0.25 mg / mL to 0.1 g / mL in saline did not cause red blood cell rupture and hemolysis (such as Figure 4 shown);

[0088] (9) An in vitro transdermal test based on a Franz cell was conducted using the dorsal skin of 8- to 12-week-old Kunming mice (purchased from Zhuhai Baishitong Biotechnology Co., Ltd.). It was found that the 24-hour release rate of oxymatrine from the multifunctional thermosensitive sustained-release hydrogel was approximately 22.3%, and the sustained-release effect was superior to that of an aqueous solution of the drug with the same concentration as the sustained-release hydrogel (e.g. Figure 5 shown);

[0089] (10) The multifunctional thermosensitive sustained-release hydrogel and the drug aqueous solution were mixed at a rate of 100 μL / cm 2 When applied to the cheek skin of mice modeled with allergic contact dermatitis (ACD) using squaric acid dibutyl ester (SADBE) (modeling process reference "Zhang Z et al. (2019). Differences in itch and pain behaviors accompanying the irritant and allergic contact dermatitis produced by a contact allergen in mice. PAIN Reports, 4 (5), e781."), it was found that its antipruritic effect could last for at least 3 days, while the aqueous solution of the drug could only last for 1 day (e.g. Figure 6 The blank control group (Control) was a group without modeling, and the model group (Model) was a group using SADBE to build an ACD model. Both groups were treated with 100 μL / cm 2 Administered in a drug-free gel carrier.

[0090] (11) The multifunctional thermosensitive sustained-release hydrogel was heated at 100 μL / cm 2After applying it to the cheek skin of mice modeled with allergic contact dermatitis (ACD) using squaric acid dibutyl ester (SADBE) for 3 consecutive days (the modeling process refers to "Zhang Z et al. (2019). Differences in itch and pain behaviors accompanying the irritant and allergic contact dermatitis produced by a contact allergenin mice. PAIN Reports, 4 (5), e781."), it was found that it had comparable antipruritic (reduced the number of times mice scratched their cheeks) and analgesic (reduced the number of times mice scratched and rubbed) effects as compound dexamethasone cream, and had no significant effect on immune-related organs such as the spleen and thymus (such as Figure 7 shown).

[0091] Example 2

[0092] (1) Weigh 50 mg of hyaluronic acid and 50 mg of quaternary ammonium salt chitosan, place them in 50 ml centrifuge tubes respectively, add water and shake on a shaker for 4 hours to dissolve the solids evenly, and prepare a 1 mg / ml concentration solution;

[0093] (2) Pour the quaternary ammonium salt chitosan solution into a beaker and stir at 900 rpm at room temperature until the speed is stable. Then use a separatory funnel to add the hyaluronic acid solution into the polyammonium salt chitosan solution (speed 1.3-1.5 ml per second). After continuing to stir for 4 hours, centrifuge at 7000 rpm for 15 minutes and take the supernatant. Under a transmission electron microscope, it can be seen that the diameter of the particles is at the micro-nano scale (such as Figure 1 As shown), rotary evaporation and concentration for 3.5 hours to a solution with a microsphere mass fraction of 1.25%;

[0094] (3) Weigh 50 mg of oxymatrine and add 450 μl of concentrated microsphere solution, stir at 100 rpm for 48 hours at room temperature to prepare a microsphere solution containing oxymatrine. Under transmission electron microscopy, oxymatrine crystals (such as Figure 1 shown);

[0095] (4) Weigh 10 mg of menthol and 40 mg of PEG40COH, add 150 μL of water to dissolve, add the oxymatrine drug-loaded microsphere solution prepared in step (3), and stir at room temperature and 100 rpm for 10 min to prepare a menthol and oxymatrine drug-loaded microsphere solution;

[0096] (5) Weigh 120 mg of poloxamer 407 (F-127) and add it to the menthol and oxymatrine drug-loaded microsphere solution prepared in step (4), add 180 μL of water, stir at room temperature and 100 rpm for 4 to 6 hours, and then put it in a refrigerator overnight to complete the preparation of the multifunctional thermosensitive sustained-release hydrogel;

[0097] (6) The obtained multifunctional thermosensitive sustained-release hydrogel was subjected to temperature scanning using a rotational rheometer, and the gel phase transition temperature was measured to be approximately 30°C ( Figure 2 The multifunctional thermosensitive sustained-release hydrogel is in liquid form at room temperature, can become gel in 6-7 seconds after being sprayed on the skin, and can be washed off in 5 seconds with room temperature water.

[0098] Example 3

[0099] (1) Weigh 50 mg of hyaluronic acid and 50 mg of quaternary ammonium salt chitosan, place them in 50 ml centrifuge tubes respectively, add water and shake on a shaker for 4 hours to dissolve the solids evenly, and prepare a 1 mg / ml concentration solution;

[0100] (2) Pour the quaternary ammonium salt chitosan solution into a beaker and stir at 900 rpm at room temperature until the speed is stable. Then use a separatory funnel to add the hyaluronic acid solution into the polyammonium salt chitosan solution (speed 1.3-1.5 ml per second). After continuing to stir for 4 hours, centrifuge at 7000 rpm for 15 minutes and take the supernatant. Under a transmission electron microscope, it can be seen that the diameter of the particles is at the micro-nano scale (such as Figure 1 As shown), rotary evaporation and concentration for 3.5 hours to a solution with a microsphere mass fraction of 1.25%;

[0101] (3) Weigh 50 mg of oxymatrine and add 450 μl of concentrated microsphere solution, stir at 100 rpm for 48 hours at room temperature to prepare a microsphere solution containing oxymatrine. Under transmission electron microscopy, oxymatrine crystals (such as Figure 1 shown);

[0102] (4) Weigh 10 mg of menthol and 40 mg of PEG40COH, add 150 μL of water to dissolve, add the oxymatrine drug-loaded microsphere solution prepared in step (3), and stir at room temperature and 100 rpm for 10 min to prepare a menthol and oxymatrine drug-loaded microsphere solution;

[0103] (5) Weigh 130 mg of poloxamer 407 (F-127) and add it to the menthol and oxymatrine drug-loaded microsphere solution prepared in step (4), add 170 μL of water, stir at room temperature and 100 rpm for 4 to 6 hours, and then put it in a refrigerator overnight to complete the preparation of the multifunctional thermosensitive sustained-release hydrogel;

[0104] (6) The obtained multifunctional thermosensitive sustained-release hydrogel was subjected to temperature scanning using a rotational rheometer, and the gel phase transition temperature was measured to be approximately 21°C ( Figure 2 The multifunctional thermosensitive sustained-release hydrogel is in a gel state at room temperature. It can form a film in 1-2 seconds after being taken out of a refrigerator at 4°C and sprayed on the skin, and can be washed off in 5 seconds using cold water at about 4°C.

[0105] Comparative Example 1

[0106] The difference between the method for preparing the aqueous solution of chitosan / hyaluronic acid microspheres containing quaternary ammonium salt in Example 1 is that the time of rotary evaporation concentration is different, as follows:

[0107] (1) Weigh 50 mg of hyaluronic acid and 50 mg of quaternary ammonium salt chitosan, place them in 50 ml centrifuge tubes respectively, add water and shake on a shaker for 4 hours to dissolve the solids evenly, and prepare a 1 mg / ml concentration solution;

[0108] (2) The quaternary ammonium salt chitosan solution was poured into a beaker, stirred at 900 rpm at room temperature until the speed was stable, and then the hyaluronic acid solution was added to the quaternary ammonium salt chitosan solution (speed 1.3-1.5 ml per second) using a separatory funnel. After stirring for 4 hours, the supernatant was taken after centrifugation at 7000 rpm for 15 minutes, and the microspheres were concentrated by rotary evaporation for 10 hours. The microspheres were agglomerated and could not be redissolved by adding any volume of water. It can be seen that the rotary evaporation concentration time was too long and high-concentration and uniformly distributed microspheres could not be obtained.

[0109] Comparative Example 2

[0110] The difference between the method for preparing the aqueous solution of chitosan / hyaluronic acid microspheres containing quaternary ammonium salt in Example 1 is that the time of rotary evaporation concentration is different, as follows:

[0111] (1) Weigh 50 mg of hyaluronic acid and 50 mg of quaternary ammonium salt chitosan, place them in 50 ml centrifuge tubes respectively, add water and shake on a shaker for 4 hours to dissolve the solids evenly, and prepare a 1 mg / ml concentration solution;

[0112] (2) The quaternary ammonium salt chitosan solution was poured into a beaker, stirred at 900 rpm at room temperature until the speed was stable, and then the hyaluronic acid solution was added to the quaternary ammonium salt chitosan solution (speed 1.3-1.5 ml per second) using a separatory funnel. After continuing to stir for 4 hours, the supernatant was taken after centrifugation at 7000 rpm for 15 minutes, and the mass fraction was detected to be about 0.078% after rotary evaporation concentration for 2 hours, which did not meet the concentration requirements. Therefore, if the rotary evaporation concentration time is too short, the microsphere concentration will be too low, which will affect the formation of gel during the gel preparation process.

[0113] Comparative Example 3

[0114] The difference between the preparation of the solution with a refreshing smell in Example 1 is that the solubilizer and the amount used are different, as follows:

[0115] 10 mg of menthol and 190 mg of PEG-DA258 (CAS No. 26570-48-9, PEG-DA258 is liquid at room temperature) were weighed and mixed to obtain solution A; solution A was applied to the skin of the test animal and immediately induced local irritant contact dermatitis. It can be seen that PEG-DA258, as a solubilizer for menthol, requires a large amount to dissolve menthol, and the obtained mixed solution has a certain irritation and is not suitable for preparing the multifunctional sustained-release gel of the present invention.

[0116] Comparative Example 4

[0117] The difference between the preparation of the solution with a refreshing smell in Example 1 is that the solubilizer and the amount used are different, as follows:

[0118] (1) Weigh 50 mg of hyaluronic acid and 50 mg of quaternary ammonium salt chitosan, place them in 50 ml centrifuge tubes respectively, add water and shake on a shaker for 4 hours to dissolve the solids evenly, and prepare a 1 mg / ml concentration solution;

[0119] (2) The quaternary ammonium salt chitosan solution was poured into a beaker and stirred at 900 rpm at room temperature until the speed was stable. Then, the hyaluronic acid solution was added to the quaternary ammonium salt chitosan solution using a separatory funnel (speed 1.3-1.5 ml / s). After continuous stirring for 4 hours, the solution was centrifuged at 7000 rpm for 15 minutes and the supernatant was collected. Under a transmission electron microscope, it was found that the diameter of the solution was at the micro-nano scale (e.g. Figure 1 As shown), rotary evaporation and concentration for 3.5 hours to a solution with a microsphere mass fraction of 1.25%;

[0120] (3) Weigh 50 mg of oxymatrine and add 450 μL of concentrated microsphere solution, stir at 100 rpm at room temperature for 48 hours to prepare a solution of oxymatrine-loaded microspheres;

[0121] (4) After 10 mg of menthol and an appropriate weight of PEG660CSA were weighed and dissolved in a corresponding volume of water, ① white flocculent floating matter and ② poor fluidity at room temperature appeared (see Table 1 below for details). Therefore, when PEG660CSA is selected as a solubilizer, most proportions are not suitable for preparing the multifunctional sustained-release gel of the present invention.

[0122] (5) Based on Table 1, 10 mg of menthol and 60-70 mg of PEG660CSA were weighed, and 420-430 μL of water was added to prepare a menthol solution, and after mixing with the oxymatrine drug-loaded microsphere solution, the resulting volume was close to the final volume of 1 mL. After adding the corresponding amount of F-127 to make up the volume to 1 mL, F-127 could be dissolved at room temperature, but could not form gel at room temperature (24° C.) and 32.5° C. (see Table 2 below for details). Therefore, the menthol solution prepared using PEG660CSA is not suitable for preparing the multifunctional sustained-release gel of the present invention.

[0123] Table 1 Experimental results of using PEG660CSA to solubilize 10 mg menthol

[0124]

[0125] Table 2 Gelation Experimental Conditions

[0126]

[0127] Comparative Example 5

[0128] The difference between the preparation of the solution with a refreshing smell in Example 1 is that the amount of the solubilizer is different. The specific steps are as follows:

[0129] Weigh 10 mg of menthol and 10 mg of PEG40COH, add 180 μl of water to dissolve, and white floccules will appear. Therefore, when the amount of PEG40COH is insufficient, menthol will not dissolve after mixing with water, but will form white flocs floating on the liquid surface.

[0130] Comparative Example 6

[0131] The difference between the drug-loaded microsphere solution with a refreshing smell prepared in Example 1 is that the amount of the solubilizer is different. The specific steps are as follows:

[0132] Weigh 10 mg of menthol and 190 mg of PEG40COH, add to the oxymatrine drug-loaded microsphere solution, the solution is in the form of an ointment. Therefore, if the amount of PEG40COH is increased and water is not added to mix and dissolve menthol, then after mixing it with the drug-loaded microspheres, a liquid drug-loaded microsphere solution with a refreshing smell cannot be obtained.

[0133] Comparative Example 7

[0134] The difference between the multifunctional sustained-release gel prepared in Example 1 is that the amount of poloxamer 407 is different. The specific steps are as follows:

[0135] (1) Weigh 50 mg of hyaluronic acid and 50 mg of quaternary ammonium salt chitosan, place them in 50 ml centrifuge tubes respectively, add water and shake on a shaker for 4 hours to dissolve the solids evenly, and prepare a 1 mg / ml concentration solution;

[0136] (2) Pour the quaternary ammonium salt chitosan solution into a beaker, stir at 900 rpm at room temperature until the speed is stable, use a separatory funnel to add the hyaluronic acid solution to the quaternary ammonium salt chitosan solution (speed 1.3-1.5 ml per second), continue stirring for 4 hours, centrifuge at 7000 rpm for 15 minutes, take the supernatant, and concentrate it by rotary evaporation to a solution with a microsphere mass fraction of 1.25%;

[0137] (3) Weigh 50 mg of oxymatrine and add 450 μL of concentrated microsphere solution, and stir at room temperature (100 rpm) for 48 hours to prepare a solution of oxymatrine-loaded microspheres;

[0138] (4) Weigh 10 mg of menthol and 40 mg of PEG40COH, add 150 μL of water to dissolve them, and then add the oxymatrine drug-loaded microsphere solution to prepare a menthol and oxymatrine drug-loaded microsphere solution;

[0139] (5) Weigh 100 mg of poloxamer 407 and add it to the drug-loaded microsphere solution containing menthol and oxymatrine, add 200 μL of water, stir at room temperature and 100 rpm for 4 to 6 hours, and then put it in a refrigerator overnight to complete the preparation of the multifunctional thermosensitive sustained-release hydrogel;

[0140] (6) The obtained multifunctional thermosensitive sustained-release hydrogel was subjected to viscosity scanning using a rotational rheometer, and it was found that there was no significant viscosity change at any temperature, and the gel phase transition temperature could not be measured using temperature scanning. Therefore, changing the ratio of poloxamer 407 to water, that is, reducing the amount of poloxamer 407 and increasing the amount of water, could not produce a temperature-sensitive hydrogel.

[0141] Comparative Example 8

[0142] The difference between the multifunctional sustained-release gel prepared in Example 1 is that the amount of poloxamer 407 is different. The specific steps are as follows:

[0143] (1) Weigh 50 mg of hyaluronic acid and 50 mg of quaternary ammonium salt chitosan, place them in 50 ml centrifuge tubes respectively, add water and shake on a shaker for 4 hours to dissolve the solids evenly, and prepare a 1 mg / ml concentration solution;

[0144] (2) Pour the quaternary ammonium salt chitosan solution into a beaker, stir at 900 rpm at room temperature until the speed is stable, use a separatory funnel to add the hyaluronic acid solution to the quaternary ammonium salt chitosan solution (speed 1.3-1.5 ml per second), continue stirring for 4 hours, centrifuge at 7000 rpm for 15 minutes, take the supernatant, and concentrate it by rotary evaporation to a solution with a microsphere mass fraction of 1.25%;

[0145] (3) Weigh 50 mg of oxymatrine and add 450 μL of concentrated microsphere solution, stir at 100 rpm at room temperature for 48 hours to prepare a solution of oxymatrine-loaded microspheres;

[0146] (4) Weigh 10 mg of menthol and 40 mg of PEG40COH, add 150 μL of water to dissolve them, and then add the oxymatrine drug-loaded microsphere solution to prepare a menthol and oxymatrine drug-loaded microsphere solution;

[0147] (5) Weigh 160 mg of poloxamer 407 and add it to the drug-loaded microsphere solution containing menthol and oxymatrine, add 140 μL of water, stir at room temperature and 100 rpm for 4 to 6 hours, but fail to mix successfully. Stir at -20°C and 100 rpm for 4 to 6 hours, and mix successfully. After being placed in a refrigerator overnight, the solution changes from liquid to solid, and can only be melted at -20°C. Therefore, changing the ratio of poloxamer 407 to water, that is, increasing the amount of poloxamer 407 and reducing the amount of water beyond a certain range; the multifunctional sustained-release gel can only be successfully prepared under low temperature conditions, and the phase transition temperature is too low to be convenient to use.

[0148] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a multifunctional thermosensitive sustained-release hydrogel, characterized in that: The steps include: (1) mixing the quaternary ammonium salt chitosan aqueous solution and the hyaluronic acid aqueous solution uniformly, centrifuging and collecting the supernatant, and rotary evaporating and concentrating to obtain a quaternary ammonium salt chitosan / hyaluronic acid microsphere aqueous solution; (2) mixing the drug with the quaternary ammonium salt chitosan / hyaluronic acid microsphere aqueous solution obtained in step (1) to obtain a drug-loaded microsphere solution; (3) mixing the substance having a refreshing smell with a solubilizing agent, and dissolving them in water to obtain a solution having a refreshing smell; (4) mixing the drug-loaded microsphere solution obtained in step (2) with the solution with a refreshing smell obtained in step (3) to obtain a drug-loaded microsphere solution with a refreshing smell; (5) Poloxamer 407 is added to the solution obtained in step (4), water is added to mix, and the mixture is allowed to stand to obtain a multifunctional thermosensitive sustained-release hydrogel.

2. The method for preparing the multifunctional thermosensitive sustained-release hydrogel according to claim 1, characterized in that: The method for uniformly mixing the quaternary ammonium salt chitosan aqueous solution and the hyaluronic acid aqueous solution in step (1) is: injecting the hyaluronic acid aqueous solution into the quaternary ammonium salt chitosan aqueous solution under stirring.

3. The method for preparing the multifunctional thermosensitive sustained-release hydrogel according to claim 2, characterized in that: The speed of injecting the quaternary ammonium salt chitosan aqueous solution into the hyaluronic acid aqueous solution is 0.1 to 10.0 mL / s; The stirring condition is 800-1000 rpm for 1-6 hours.

4. The method for preparing the multifunctional thermosensitive sustained-release hydrogel according to claim 1, characterized in that: The drug described in step (2) is a water-soluble drug; The substance with a refreshing smell in step (3) is at least one of menthol, natural borneol, artificial borneol and camphor; The solubilizing agent described in step (3) is PEG-DA258, PEG660CSA or PEG40COH.

5. The method for preparing the multifunctional thermosensitive sustained-release hydrogel according to claim 1, characterized in that: The molecular weight of the quaternary ammonium salt chitosan described in step (1) is 5.0×10 4 ~40.0×10 4 Dalton; The molecular weight of the hyaluronic acid described in step (1) is 5.0×10 4 ~40.0×10 4 Dalton.

6. The method for preparing the multifunctional thermosensitive sustained-release hydrogel according to claim 1, characterized in that: The concentration of the quaternary ammonium salt chitosan aqueous solution described in step (1) is 0.1-2.0 mg / mL; The concentration of the hyaluronic acid aqueous solution in step (1) is 0.1 to 2.0 mg / mL; The volume ratio of the quaternary ammonium salt chitosan aqueous solution to the hyaluronic acid aqueous solution described in step (1) is 2:1 to 1:2; The mass fraction of microspheres in the quaternary ammonium chitosan / hyaluronic acid microsphere aqueous solution in step (1) is 0.10-2.00% (w / w); The drug described in step (2) and the quaternary ammonium salt chitosan / hyaluronic acid microspheres are calculated in a mass ratio of 240 to 1:1; The content of the substance with a refreshing smell in the refreshing smell solution in step (3) is 0.1 to 10.0% (w / w); The amount of the solubilizer in step (3) is calculated based on a mass ratio of the substance having a refreshing odor to the solubilizer of 1:1 to 18; The drug-loaded microsphere solution described in step (4) is mixed with the solution having a refreshing smell in a volume ratio of 1:1 to 10:1; The content of poloxamer 407 in the sustained-release hydrogel system in step (5) is 10.0% to 16.0% (w / w); The amount of water used in step (5) is calculated based on the ratio of water to poloxamer 407 of 170-180 μL:120-130 mg.

7. The method for preparing the multifunctional thermosensitive sustained-release hydrogel according to claim 1, characterized in that: The conditions of the rotary evaporation concentration in step (1) are as follows: a rotation speed of 20 to 200 rpm, a pressure of 0.01 to 0.1 MPa, a temperature of 25 to 65° C., and a time of 2 to 10 h; The mixing method in step (2) is stirring; The mixing method in step (4) is stirring; The mixing method in step (5) is stirring; The standing in step (5) is standing at -6 to -20°C for 12 to 24 hours.

8. The method for preparing the multifunctional thermosensitive sustained-release hydrogel according to claim 7, characterized in that: The conditions for rotary evaporation concentration in step (1) are as follows: rotation speed 120 rpm, pressure 0.095 MPa, temperature 50° C., and time 3.5 to 4.5 h.

9. A multifunctional thermosensitive sustained-release hydrogel, characterized in that: The method is obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the multifunctional thermosensitive sustained-release hydrogel according to claim 9 in the preparation of antipruritic and analgesic preparations.

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