A multifunctional thermosensitive sustained-release hydrogel and its preparation method and application in the preparation of antipruritic and analgesic preparations

Abstract

The present invention discloses a multifunctional thermosensitive sustained-release hydrogel, its preparation method, and its use in the preparation of antipruritic and analgesic preparations. The present invention utilizes microspheres formed by a composite of hyaluronic acid and quaternary ammonium salt chitosan. These microspheres are sequentially mixed with a drug, a solubilizing agent-dissolved cooling substance, and poloxamer 407 using a simpler magnetic stirring method. This multifunctional thermosensitive sustained-release hydrogel is prepared without the use of a thickener. The hydrogel prepared by the present invention exhibits a strong cooling odor and sustained-release effect, along with good biocompatibility. When the drug used is an antipruritic and analgesic substance, such as oxymatrine, the hydrogel also has therapeutic and analgesic effects for non-histamine-dependent pruritus.

Description

Technical Field

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

[0002] Hydrogels are polymer systems composed of three-dimensional viscoelastic networks. They can absorb large amounts of water and swell in physiological environments. Physical or chemical crosslinking enhances their structure and properties, resulting in excellent hydrophilicity, flexibility, and biocompatibility. Hydrogels are widely used in the biomedical field. Hydrogels can control drug release through both self-swelling and external stimulation of their network structure, thus serving as drug carriers to achieve controlled drug release. Based on the crosslinking method used to form the gel, hydrogels can be categorized as physically crosslinked, chemically crosslinked, and radiation-crosslinked. Based on their function and application, they can be further divided into drug carrier hydrogels, tissue scaffold hydrogels, and other types.

[0003] Hydrogels can be categorized by their function as thermosensitive, sustained-release, and antipruritic and analgesic hydrogels. Thermosensitive hydrogels possess a certain ratio of hydrophilic groups (such as carboxyl, amide, and hydroxyl groups) and hydrophobic groups (such as methyl, ethyl, and propyl alkyl groups) on their macromolecular chains. Changes in ambient temperature influence the interactions between these groups and the hydrogen bonding within the macromolecular chains, leading to gelation at a certain temperature. Different hydrogel synthesis conditions and material composition directly affect the release rate of drugs from the hydrogel. Hydrogels with a high degree of cross-linking typically exhibit a slower release rate, achieving a sustained-release effect; hydrogels with a low degree of cross-linking exhibit a higher release kinetic rate. Hydrogels can serve as drug carriers, facilitating drug dispersion within the system while allowing the drug to exert its effect. Depending on the drug carried, hydrogels can exhibit different functions: hydrogels containing antipruritic drugs such as calamine exhibit a moderate antipruritic effect, while hydrogels containing analgesics such as ropivacaine can also exert an analgesic effect.

[0004] Despite this, hydrogels still have certain defects:

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

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

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

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

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

[0010] Commonly used topical antipruritic and analgesic preparations currently include ointments, films, suppositories, and effervescent tablets. Each of these dosage forms has certain drawbacks: Ointments mix the drug with an appropriate amount of matrix and apply it directly to the affected skin, where it is slowly absorbed through osmosis. However, their composition is relatively fixed and their modifiability is poor. Films can quickly form a film that covers the affected area, isolating it from external factors, but the film is thin, the drug loading capacity is low, and the therapeutic effect is limited. Suppositories are inserted into the cavity and come into close contact with the mucosa, rapidly softening and dissolving to release the drug. However, they contain more matrix and less active ingredients, requiring frequent dressing changes and requiring strict storage conditions. Effervescent tablets produce large amounts of carbon dioxide through effervescent reactions, which can help carry drugs to lesions that are difficult to administer with ordinary preparations. However, the production process is prone to sticking and flakes, and the production requirements are high.

[0011] In response to the above problems, the present invention has the advantages of simple production 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 thermosensitive 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, which has the characteristics of thermosensitivity and sustained release.

[0014] Another object of the present invention is to provide applications 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) uniformly mixing the quaternary ammonium salt chitosan aqueous solution and the hyaluronic acid aqueous solution, centrifuging and concentrating the supernatant by rotary evaporation 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 the mixture 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 having a refreshing smell obtained in step (3) to obtain a drug-loaded microsphere solution having a refreshing smell;

[0021] (5) Poloxamer 407 (also known as segmented polyether F-127) is added to the solution obtained in step (4), mixed with water, and 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 Daltons; 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 Daltons; more preferably 20.0×10 4 Dalton.

[0027] The concentration of the quaternary ammonium salt chitosan aqueous solution described in step (1) is preferably 0.1 to 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 centrifugal at a speed of 2000 to 18000 rpm for 5 to 30 minutes; more preferably centrifugal at 7000 rpm for 15 minutes.

[0031] The conditions for the rotary evaporation concentration in step (1) are preferably as follows: a 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 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 salt 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 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, sophoracarpine and oxymatrine; more preferably oxymatrine.

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

[0037] The drug described in step (2) and the quaternary ammonium salt chitosan / hyaluronic acid microspheres 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 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 in step (3) is preferably PEG-DA258, PEG660CSA or PEG40COH; more preferably PEG40COH.

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

[0044] The drug-loaded microsphere solution 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, in a volume ratio of 5:2 (v / v).

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

[0046] The stirring conditions are preferably room temperature and 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 based on a ratio of 140-200 μL:100-160 mg of water to poloxamer 407; more preferably, a ratio of 170-180 μL:120-130 mg of water to poloxamer 407; and most preferably, a ratio of 175-180 μL:120-125 mg of water 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 obtained is in a semisolid form. When used, it is placed in an environment below the phase transition temperature, and after it is converted into a solution state, it can be used by spraying or applying. When the standing temperature is less than the phase transition temperature, the multifunctional thermosensitive sustained-release hydrogel obtained 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 based on 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 effect of treating non-histamine-dependent pruritus and analgesia, the multifunctional thermosensitive sustained-release hydrogel has the effect of treating non-histamine-dependent pruritus and analgesia.

[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 analgesia.

[0059] The antipruritic and analgesic preparations include but are not limited to the following dosage forms: external use 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 scent, the present invention discovered through temperature scanning that the gel's phase transition temperature is 21-30°C, and the gel has a minty, refreshing odor. Using a Franz transdermal cell at 32±0.5°C and physiological saline as the receiving fluid, the transdermal release behavior of the active ingredient in the prepared multifunctional, thermosensitive, sustained-release hydrogel over 24 hours was evaluated. The cumulative drug permeation rate and cumulative permeation per unit area were calculated, and corresponding curves were plotted. The results showed that compared with a solution containing only the drug, the multifunctional, thermosensitive, sustained-release hydrogel had a longer drug release time. Biocompatibility and hemolysis test results demonstrated that the sustained-release hydrogel has improved biosafety.

[0062] The present invention has the following advantages and effects compared to the prior art:

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

[0064] 2. The microspheres formed by electrostatically physically crosslinking hyaluronic acid and quaternary ammonium salt chitosan in the present invention can "encircle" oxymatrine, inhibiting its rapid release. The added poloxamer 407 allows the gel solution to transform from a liquid state to a gel state when heated and to return from a gel state to a liquid state when cooled. This enhances the gel solution's sprayability for contactless administration and its contactless cleansing properties, allowing for easy cleansing with cold water. This provides convenient administration and dressing changes for patients with lesions, wounds, or hypersensitivity, while also preventing secondary infections of lesions or wounds caused by direct contact with the affected area.

[0065] 3. The present invention does not involve the separation and purification of drug-loaded microspheres, and the administered drugs can be 100% utilized, thereby 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 transdermal cumulative 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, does 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; (A) is the quaternary ammonium chitosan / hyaluronic acid microspheres without drug loading (15000×), (B) is the crystallization image of oxymatrine (50000×), and (C) is the quaternary ammonium chitosan / hyaluronic acid microspheres loaded with oxymatrine (10000×).

[0072] Figure 2These 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 to L929.

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

[0075] Figure 5 These are the results of in vitro transdermal experiments on drug aqueous solutions and multifunctional thermosensitive sustained-release hydrogels; among them, (A) is a statistical graph showing the changes in transdermal permeability of drug aqueous solutions and multifunctional thermosensitive sustained-release hydrogels over time; (B) is a statistical graph showing the changes in transdermal permeation of drug aqueous solutions and multifunctional thermosensitive sustained-release hydrogels over time.

[0076] Figure 6 This 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 These are statistical graphs showing the antipruritic and analgesic efficacy of the multifunctional thermosensitive sustained-release hydrogel, and its effects on the immune-related thymus and spleen organs; among them, (A) is a statistical graph showing the antipruritic efficacy of the multifunctional thermosensitive sustained-release hydrogel; (B) is a statistical graph showing the analgesic efficacy of the multifunctional thermosensitive sustained-release hydrogel; (C) is a statistical graph showing the effect of the multifunctional thermosensitive sustained-release hydrogel on the thymus; and (D) is a statistical graph showing the effect of the multifunctional thermosensitive sustained-release hydrogel on the spleen. DETAILED DESCRIPTION

[0078] Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but 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, 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, shaken on a shaker for 4 hours to dissolve the solid evenly, to prepare a 1 mg / ml concentration solution;

[0081] (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 quaternary ammonium salt chitosan solution (speed 1.3-1.5 ml per second). After continuous stirring for 4 hours, centrifuge at 7000 rpm for 15 minutes and take the supernatant. Under transmission electron microscopy, it can be seen that the diameter of the hyaluronic acid solution is at the micro-nano scale level (such as Figure 1 As shown), the solution was concentrated by rotary evaporation for 3.5 hours to 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 solution of drug-loaded microspheres containing oxymatrine. The mass ratio of oxymatrine to microspheres 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 number: 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-loaded microsphere solution prepared in step (4), add 175 μL of water, stir at 100 rpm at room temperature for 4 to 6 hours, 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 (e.g. Figure 2 The multifunctional thermosensitive sustained-release hydrogel is in liquid form at 4°C and in a slightly viscous, sprayable liquid form at room temperature (24°C). It becomes a gel within 1-3 seconds after being sprayed on the skin and can be rinsed off within 5 seconds using clean water at about 18°C.

[0086] (7) The multifunctional thermosensitive sustained-release hydrogel extract was prepared according to the soluble material part of 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 hemolysis 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 lead to hemolysis (such as Figure 4 shown);

[0088] (9) In vitro transdermal experiments based on Franz sample cells were conducted on the dorsal skin of 8-12 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 about 22.3%, and the sustained-release effect was better than that of aqueous solutions of drugs with the same concentration as the sustained-release hydrogel (such as Figure 5 shown);

[0089] (10) The multifunctional thermosensitive sustained-release hydrogel and the drug aqueous solution were mixed at a concentration 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) (reference for the modeling process: "Zhang Z et al. (2019). Differences in itch and pain behaviors accompanying the irritant and allergic contact dermatitis produced by acontact 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 with ACD modeling using SADBE. Both groups were treated with 100 μL / cm 2 Administer a drug-free gel carrier.

[0090] (11) The multifunctional thermosensitive sustained-release hydrogel was placed at a flow rate of 100 μL / cm 2After being applied 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 is referenced in "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 chitosan, place them in 50 ml centrifuge tubes, add water, and shake on a shaker for 4 hours to dissolve the solids evenly, to prepare a 1 mg / ml 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). Continue stirring for 4 hours, centrifuge at 7000 rpm for 15 minutes, and take the supernatant. Under the transmission electron microscope, it can be seen that the diameter of the quaternary ammonium salt chitosan solution is at the micro-nano scale level (such as Figure 1 As shown), the solution was concentrated by rotary evaporation for 3.5 hours to 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 solution of oxymatrine-loaded microspheres. Under a transmission electron microscope, oxymatrine crystals (e.g., 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-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 place 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 (e.g. Figure 2 The multifunctional thermosensitive sustained-release hydrogel is in liquid form at room temperature and can be turned into gel within 6-7 seconds after being sprayed on the skin and can be rinsed off within 5 seconds using room temperature water.

[0098] Example 3

[0099] (1) Weigh 50 mg of hyaluronic acid and 50 mg of quaternary ammonium chitosan, place them in 50 ml centrifuge tubes, add water, and shake on a shaker for 4 hours to dissolve the solids evenly, to prepare a 1 mg / ml 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). Continue stirring for 4 hours, centrifuge at 7000 rpm for 15 minutes, and take the supernatant. Under the transmission electron microscope, it can be seen that the diameter of the quaternary ammonium salt chitosan solution is at the micro-nano scale level (such as Figure 1 As shown), the solution was concentrated by rotary evaporation for 3.5 hours to 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 solution of oxymatrine-loaded microspheres. Under a transmission electron microscope, oxymatrine crystals (e.g., 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-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 place 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 (e.g. 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 rinsed 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 salts and Example 1 is that the time for rotary evaporation and concentration is different, as follows:

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

[0108] (2) The quaternary ammonium salt chitosan solution was poured into a beaker and stirred at 900 rpm at room temperature until the speed stabilized. The hyaluronic acid solution was then added to the quaternary ammonium salt chitosan solution using a separatory funnel (at a speed of 1.3 to 1.5 ml per second). Stirring was continued for 4 hours, and the supernatant was collected after centrifugation at 7000 rpm for 15 minutes. The supernatant was 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, 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 salts and Example 1 is that the time for rotary evaporation and concentration is different, as follows:

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

[0112] (2) The quaternary ammonium salt chitosan solution was poured into a beaker and stirred at 900 rpm at room temperature until the speed stabilized. Then, a separatory funnel was used to add the hyaluronic acid solution to the quaternary ammonium salt chitosan solution (at a speed of 1.3 to 1.5 milliliters per second). After stirring for 4 hours, the supernatant was centrifuged at 7000 rpm for 15 minutes and the supernatant was collected. After rotary evaporation and concentration for 2 hours, the mass fraction thereof was approximately 0.078%, which did not meet the concentration requirement. Therefore, if the rotary evaporation concentration time is too short, the microsphere concentration will be too low, which will affect the formation of the gel during the gel preparation process.

[0113] Comparative Example 3

[0114] The difference between the preparation of the solution with a refreshing smell and Example 1 lies in the difference in the solubilizer and the amount used, 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 mixed and dissolved to obtain Solution A. When Solution A was applied to the skin of test animals, it immediately induced local irritant contact dermatitis. This indicates that PEG-DA258, as a solubilizer for menthol, requires a relatively large amount to dissolve it. Furthermore, the resulting mixed solution is somewhat irritating and unsuitable for preparing the multifunctional sustained-release gel described herein.

[0116] Comparative Example 4

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

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

[0119] (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 quaternary ammonium salt chitosan solution (speed 1.3-1.5 ml per second). After continuous stirring for 4 hours, centrifuge at 7000 rpm for 15 minutes and take the supernatant. Under transmission electron microscopy, it can be seen that the diameter of the hyaluronic acid solution is at the micro-nano scale level (such as Figure 1 As shown), the solution was concentrated by rotary evaporation for 3.5 hours to 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 dissolving 10 mg of menthol and an appropriate weight of PEG660CSA in a corresponding volume of water, problems such as 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 solubilizing agent, 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 added to 420-430 μL of water to prepare a menthol solution. After mixing with the oxymatrine-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, the F-127 was dissolved at room temperature. However, no gel was formed at room temperature (24°C) or 32.5°C (see Table 2 below for details). Therefore, the menthol solution prepared with 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 of menthol

[0124]

[0125] Table 2 colloid formation experiment results

[0126]

[0127] Comparative Example 5

[0128] The difference between the preparation of the solution with a refreshing smell and 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 and dissolve them in 180 μL of water. A white, floating flocculent substance appears. Therefore, when the amount of PEG40COH is insufficient, the menthol will not dissolve after mixing with water, but will form a white flocculent substance 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 the amount of solubilizer used. The specific steps are as follows:

[0132] When 10 mg of menthol and 190 mg of PEG40COH were added to a solution of oxymatrine-loaded drug-loaded microspheres, the solution became an ointment. Therefore, if the amount of PEG40COH was increased without adding water to dissolve the menthol, the menthol-loaded microsphere solution with the drug-loaded microspheres would not produce a liquid solution with a refreshing smell.

[0133] Comparative Example 7

[0134] The difference between the multifunctional sustained-release gel prepared in Example 1 and that 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 chitosan, place them in 50 ml centrifuge tubes, add water, and shake on a shaker for 4 hours to dissolve the solids evenly, to prepare a 1 mg / ml solution;

[0136] (2) Pour the quaternary ammonium salt chitosan solution into a beaker and stir at 900 rpm at room temperature until the speed stabilizes. Then, use a separatory funnel to add the hyaluronic acid solution to the quaternary ammonium salt chitosan solution (at a speed of 1.3 to 1.5 ml per second). Continue stirring for 4 hours, centrifuge at 7000 rpm for 15 minutes, collect the supernatant, and concentrate 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, 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 a solution of drug-loaded microspheres containing menthol and oxymatrine, add 200 μL of water, stir at room temperature and 100 rpm for 4-6 hours, and then place in a refrigerator overnight to complete the preparation of a multifunctional thermosensitive sustained-release hydrogel;

[0140] (6) The obtained multifunctional thermosensitive sustained-release hydrogel was subjected to viscosity scanning using a rotational rheometer. 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, did not produce a temperature-sensitive hydrogel.

[0141] Comparative Example 8

[0142] The difference between the multifunctional sustained-release gel prepared in Example 1 and that 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 chitosan, place them in 50 ml centrifuge tubes, add water, and shake on a shaker for 4 hours to dissolve the solids evenly, to prepare a 1 mg / ml solution;

[0144] (2) Pour the quaternary ammonium salt chitosan solution into a beaker and stir at 900 rpm at room temperature until the speed stabilizes. Then, use a separatory funnel to add the hyaluronic acid solution to the quaternary ammonium salt chitosan solution (at a speed of 1.3 to 1.5 ml per second). Continue stirring for 4 hours, centrifuge at 7000 rpm for 15 minutes, collect the supernatant, and concentrate 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, 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 a solution of drug-loaded microspheres containing menthol and oxymatrine. Then add 140 μL of water. After stirring at room temperature and 100 rpm for 4 to 6 hours, the mixture cannot be successfully mixed. However, after stirring at -20°C and 100 rpm for 4 to 6 hours, the mixture is successfully mixed. 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, requires low temperature conditions to successfully prepare the multifunctional sustained-release gel, and the phase transition temperature is too low to be used.

[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 considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for preparing a multifunctional thermosensitive sustained-release hydrogel, characterized in that: The steps include: (1) The quaternary ammonium salt chitosan aqueous solution and the hyaluronic acid aqueous solution are uniformly mixed, and after centrifugation, the supernatant is concentrated by rotary evaporation 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 odor with a solubilizer, and dissolving the mixture in water to obtain a solution having a refreshing odor; (4) mixing the drug-loaded microsphere solution obtained in step (2) with the solution having a refreshing smell obtained in step (3) to obtain a drug-loaded microsphere solution having a refreshing smell; (5) Poloxamer 407 is added to the solution obtained in step (4), water is added and mixed, and the mixture is allowed to stand to obtain a multifunctional thermosensitive sustained-release hydrogel; The method of uniformly mixing the quaternary ammonium salt chitosan aqueous solution and the hyaluronic acid aqueous solution in step (1) is as follows: injecting the hyaluronic acid aqueous solution into the quaternary ammonium salt chitosan aqueous solution under stirring; 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 in step (1) is 5.0×10 4 ~40.0×10 4 Dalton; The concentration of the quaternary ammonium 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 in step (1) is 2:1 to 1:2; The conditions for the rotary evaporation concentration described 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 3.5 to 4.5 hours; The drug described in step (2) is a water-soluble drug; The substance with a refreshing smell in step (3) is menthol; The solubilizing agent in step (3) is PEG40COH; 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 1:4 to 18; 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.

2. The method for preparing the multifunctional thermosensitive sustained-release hydrogel according to claim 1, 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.

3. The method for preparing the multifunctional thermosensitive sustained-release hydrogel according to claim 1, wherein: The water-soluble drug is oxymatrine.

4. The method for preparing the multifunctional thermosensitive sustained-release hydrogel according to claim 1, characterized in that: The mass fraction of microspheres in the quaternary ammonium salt 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 refreshing smell substance in the refreshing smell solution in step (3) is 0.1 to 10.0% (w / w); 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).

5. The method for preparing the multifunctional thermosensitive sustained-release hydrogel according to claim 1, characterized in that: 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.

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

7. 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 6.

8. Use of the multifunctional thermosensitive sustained-release hydrogel according to claim 7 in the preparation of an antipruritic and analgesic preparation.

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