Human epidermal growth factor temperature-sensitive sustained-release gel and preparation method and application thereof

Abstract

The invention discloses a human epidermal growth factor temperature-sensitive sustained-release gel as well as a preparation method and application thereof, and belongs to the technical field of biological pharmacy. The preparation method of the human epidermal growth factor temperature-sensitive sustained-release gel comprises the following steps: S1, adding poloxamer and a thickening agent into purified water, and uniformly mixing to form a gel matrix; s2, respectively preparing a human epidermal growth factor solution and a chitosan-sodium tripolyphosphate colloid, and adding the human epidermal growth factor solution into the chitosan-sodium tripolyphosphate colloid for adsorption to obtain the chitosan-sodium tripolyphosphate colloid loaded with the human epidermal growth factor; s3, adding the chitosan-sodium tripolyphosphate colloid loaded with the human epidermal growth factors, a humectant and a stabilizer into the gel matrix, and adjusting the pH value. The gel can realize stable loading and controllable slow release of the human epidermal growth factor, so that the administration frequency and repeated administration operation are reduced, and the gel has important application value in treatment of refractory wounds.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biopharmaceuticals, in particular to a human epidermal growth factor temperature-sensitive sustained-release gel, a preparation method and uses thereof. BACKGROUND

[0002] Human epidermal growth factor (hEGF) is a small molecule polypeptide composed of 53 amino acids, with a molecular weight of about 6000 daltons, containing three pairs of disulfide bonds, and having acid and alkali resistance and high temperature characteristics. hEGF mainly activates downstream multiple signaling pathways by binding to cell surface receptors (EGFR) to exert effects, mainly showing significant proliferation, migration and other effects on various cells. In the field of tissue regeneration and skin wound repair, hEGF as an effective healing factor has important value for the treatment of ulcers, burns and surgical wounds, and shows a more extensive application prospect in the preparation of functional biomaterials. However, hEGF has a short half-life in the body (about 1 hour) and is easily degraded by proteases, and conventional dosage forms (such as solutions, sprays) need to be frequently administered, resulting in large fluctuations in drug concentration and low patient compliance. SUMMARY

[0003] Therefore, the present application aims to solve the technical problems of existing dosage forms that need to be frequently used or repeatedly administered, have large fluctuations in drug concentration, and have low compliance, and provides a human epidermal growth factor temperature-sensitive sustained-release gel, a preparation method and uses thereof. The human epidermal growth factor temperature-sensitive sustained-release gel can achieve slow and long-acting release of human epidermal growth factor, has good repair effect, avoids frequent use or repeated administration, and can naturally flow flat and closely adhere to the wound surface without the need for application, thereby improving patient medication compliance.

[0004] To this end, the present application provides the following technical solutions: In a first aspect, the present application provides a preparation method of a human epidermal growth factor temperature-sensitive sustained-release gel, comprising the following steps: S1. Poloxamer and thickening agent are added to purified water and mixed uniformly to form a gel matrix; S2. A human epidermal growth factor solution and a chitosan-sodium tripolyphosphate colloid are prepared respectively, the human epidermal growth factor solution is added to the chitosan-sodium tripolyphosphate colloid for adsorption, and a chitosan-sodium tripolyphosphate colloid loaded with human epidermal growth factor is obtained; S3. The chitosan-sodium tripolyphosphate colloid loaded with human epidermal growth factor, a humectant and a stabilizer are added to the gel matrix, and the pH is adjusted.

[0005] In an alternative embodiment, in step S1, the content of poloxamer in the gel matrix is 16.65wt%-24.10wt%; and the content of the thickening agent is 0.08wt%-0.46wt%.

[0006] In one optional embodiment, the human epidermal growth factor includes naturally extracted human epidermal growth factor or recombinant human epidermal growth factor. In one alternative implementation, the poloxamer includes poloxamer 407; In one alternative embodiment, the thickener includes at least one of polycarbofil, sodium hyaluronate, and carbomer.

[0007] In one optional implementation, step S2 includes: S21. Preparation of human epidermal growth factor solution: Human epidermal growth factor was dissolved in a solvent consisting of 15-30 mmol / L PBS buffer solution (pH 5.0-7.5) and 30%-60% glycerol in a ratio of (2-4):(3-5) (v / v). S22. Preparation of chitosan-sodium tripolyphosphate colloid: Chitosan was dissolved in a 0.5wt%~1.5wt% glacial acetic acid solution to obtain a chitosan-glacial acetic acid solution; Sodium tripolyphosphate was dissolved in 15-25 mmol / L PB buffer solution with pH 6.5-7.0 to obtain a sodium tripolyphosphate solution. The chitosan-glacial acetic acid solution was stirred at 20-30℃ and 150-600 rpm, and sodium tripolyphosphate solution was added dropwise to obtain chitosan-sodium tripolyphosphate colloid. The pH was then adjusted to 5.0-6.5. S23. Adsorption: Human epidermal growth factor solution was added to chitosan-sodium tripolyphosphate colloid and adsorbed at 15-25℃ for 8-12 hours.

[0008] In one optional embodiment, the concentration of the human epidermal growth factor solution is 0.5~2 mg / mL; In one optional embodiment, the concentration of chitosan in the chitosan-glacial acetic acid solution is 0.25~1.0 g / mL; In one optional embodiment, the concentration of sodium tripolyphosphate in the sodium tripolyphosphate solution is 1~3 g / mL; In one optional embodiment, the mass ratio of chitosan to sodium tripolyphosphate in the chitosan-sodium tripolyphosphate colloid is (3~15):4; In one optional embodiment, the volume ratio of the human epidermal growth factor solution to the chitosan-sodium tripolyphosphate colloid is (1~4):(10~23).

[0009] In an optional embodiment, in step S3, the ratio of the gel matrix to the chitosan-sodium tripolyphosphate colloid loaded with human epidermal growth factor is (600.5~663):(58~120) (m:v).

[0010] In an optional embodiment, in step S3, the moisturizer is any one of glycerin, dextropanthenol, and propylene glycol, and its content in the human epidermal growth factor thermosensitive sustained-release gel is 10wt%~15wt%.

[0011] In one optional embodiment, the stabilizer is at least one of disodium edetate, human serum albumin, and Tween 80, and its content in the human epidermal growth factor thermosensitive sustained-release gel is 0.01wt% to 0.1wt%.

[0012] In one optional embodiment, the pH adjuster is at least one selected from sodium hydroxide, citric acid, triethanolamine, hydrochloric acid, and acetic acid. In one alternative implementation, the pH is adjusted to 6.0-7.0.

[0013] Secondly, the present invention provides a human epidermal growth factor thermosensitive sustained-release gel prepared by the above preparation method. In the human epidermal growth factor thermosensitive sustained-release gel, the amino acid cations in chitosan and the negatively charged phosphate ions in sodium tripolyphosphate attract each other, adsorb and polymerize, and form a cross-linked network structure, thereby providing a structural basis for the adsorption and release of human epidermal growth factor. In one optional embodiment, in the human epidermal growth factor thermosensitive sustained-release gel, the thickener serves to reduce the fluidity of the sample, preventing the sample from flowing away from the target area quickly during use. In one optional embodiment, the humectant in the human epidermal growth factor thermosensitive sustained-release gel serves to maintain the sample's moisture level after use and reduce wound discomfort. In one optional embodiment, in the human epidermal growth factor thermosensitive sustained-release gel, poloxamer is in a solution when it is not cross-linked, and forms a network structure when the temperature is raised. The other excipients are distributed in it. After application, the poloxamer network structure gradually collapses, and the growth factors that are not adsorbed by chitosan-sodium tripolyphosphate in the solution are released first. The adsorbed growth factors are gradually released as the structure changes, thereby prolonging the release cycle of the growth factors.

[0014] In one optional embodiment, the optimal gelation temperature range of the human epidermal growth factor thermosensitive sustained-release gel is 32~37℃. In one optional embodiment, the human epidermal growth factor content in the thermosensitive sustained-release gel is 0.001% to 0.002%.

[0015] Thirdly, the present invention provides the use of a human epidermal growth factor thermosensitive sustained-release gel prepared by the above preparation method, or the human epidermal growth factor thermosensitive sustained-release gel prepared by the above preparation method, in the preparation of a medicament for treating refractory wounds and / or in the preparation of cosmetic skin care products.

[0016] The technical solution of this invention has the following advantages: 1. This invention provides a method for preparing a thermosensitive sustained-release gel of human epidermal growth factor (HGF), comprising the following steps: S1. Adding poloxamer and a thickener to purified water and mixing evenly to form a gel matrix; S2. Preparing a HGF solution and a chitosan-sodium tripolyphosphate colloid, respectively, and adding the HGF solution to the chitosan-sodium tripolyphosphate colloid for adsorption to obtain a chitosan-sodium tripolyphosphate colloid loaded with HGF; S3. Adding the chitosan-sodium tripolyphosphate colloid loaded with HGF, a humectant, and a stabilizer to the gel matrix, and adjusting the pH. In the above scheme, the sodium tripolyphosphate molecule contains multiple negatively charged phosphate ions, while the chitosan molecule contains multiple amino cations and hydroxyl groups. When they encounter certain conditions (such as under appropriate pH conditions), the phosphate ions and amino cations will undergo electrostatic interactions to generate a structure in which one phosphate ion and two or three amino cations are cross-linked, forming multiple cross-linked network structures of sodium tripolyphosphate and chitosan. Then, after mixing the chitosan-sodium tripolyphosphate colloid with human epidermal growth factor (HEP), due to the cross-linked network structure of the chitosan-sodium tripolyphosphate colloid, HEP can be adsorbed on the surface and inside of the chitosan-sodium tripolyphosphate colloid. This allows the prepared gel to be suitable for skin contact surfaces or skin wounds. It ensures that even in relatively dry conditions with little moisture or flow, the HEP in the gel can still be slowly and effectively released to the contact surface or wound to achieve the therapeutic purpose.Furthermore, chitosan-sodium tripolyphosphate colloid loaded with human epidermal growth factor was added to the gel matrix, along with humectants and stabilizers. These components work together to form a network structure that can form stable micelles with increasing temperature. Poloxamer undergoes a phase transition to form a stable framework structure, with the chitosan-sodium tripolyphosphate colloid and humectant embedded within it. Upon increasing ambient temperature, poloxamer cross-links to form a stable colloid. Below skin temperature, the temperature-sensitive gel system is in solution, and the sodium tripolyphosphate-chitosan system, after adsorbing growth factors, is suspended in the solution. Above skin temperature, poloxamer forms a stable colloid, and the adsorbed growth factors are distributed within the colloid. A thickener ensures that the system is in contact with the skin before the colloidal structure has fully formed. The reduced fluidity of the gel allows the sample to remain on the skin for a period of time upon contact, achieving the following combined effects: Firstly, it ensures the slow and long-lasting release of human epidermal growth factor (HGF) onto the skin or wound surface when moisture is lacking. Secondly, it regulates the gelation temperature, allowing it to solidify using skin temperature. This not only controls the gel thickness but also synergistically enhances the sustained-release properties, achieving a slow and long-lasting release of HGF with stable drug concentration and good repair effects. Furthermore, it avoids the need for application, naturally leveling on the skin or wound surface and adhering closely to it, preventing pain caused by application during medication and improving patient compliance. In summary, the aforementioned gel achieves a long-lasting, slow release of growth factors, providing excellent repair effects, avoiding frequent or repeated administration, and naturally leveling and adhering closely to the wound surface without application, improving patient compliance. It also adheres smoothly to the wound after application, thus achieving a longer release time for growth factors.

[0017] Meanwhile, the addition of a humectant to the gel matrix of this invention enhances its moisturizing effect on wounds, maintains wound hydration, and promotes wound healing. Attached Figure Description

[0018] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 This is the result of the gelation state of the example in Test Example 2 at 37°C; Figure 2 This is the gelation state result of the comparative example in test example 2 at 37°C; Figure 3The leveling results in the culture dishes of the examples and comparative examples in Test Example 2 at 37°C; Figure 4 This is the result of the human epidermal growth factor release rate test in test case 3. Detailed Implementation

[0020] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0021] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0022] In the following examples and comparative examples, recombinant human epidermal growth factor was purchased from Shenzhen Huashengyuan Gene Engineering Development Co., Ltd.; poloxamer 407 was purchased from Xi'an Tianzheng Pharmaceutical Excipients Co., Ltd.; chitosan was purchased from Hunan Xinlvfang Pharmaceutical Co., Ltd.; sodium tripolyphosphate was purchased from Sinopharm Chemical Reagent Co., Ltd.; polycarbofil was purchased from Lubrizol Specialty Chemicals (Shanghai) Co., Ltd.; sodium hyaluronate was purchased from Shandong Zhongshan Biotechnology Co., Ltd.; carbomer was purchased from Guizhou Xinzihong Pharmaceutical Excipients Co., Ltd.; glycerol was purchased from Hunan Jiudian Hongyang Pharmaceutical Co., Ltd.; and dextropanthenol was purchased from Shanghai Maclean's. The following products were purchased from the following pharmaceutical companies: Chemical Technology Co., Ltd.; Propylene glycol from Hunan Ercon Pharmaceutical Co., Ltd.; Disodium edetate from Hebei Huachen Pharmaceutical Group Co., Ltd.; Human serum albumin from Chengdu Rongsheng Pharmaceutical Co., Ltd.; Tween 80 from Hunan Ercon Pharmaceutical Co., Ltd.; Sodium hydroxide from Hunan Ercon Pharmaceutical Co., Ltd.; Citric acid from Chengdu Huayi Pharmaceutical Excipients Manufacturing Co., Ltd.; Triethanolamine from Chengdu Huayi Pharmaceutical Excipients Manufacturing Co., Ltd.; Hydrochloric acid from Sinopharm Chemical Reagent Co., Ltd.; Acetic acid from Chengdu Huayi Pharmaceutical Excipients Manufacturing Co., Ltd.

[0023] Example 1 This embodiment provides a method for preparing a thermosensitive sustained-release gel of human epidermal growth factor, including: (1) Gel matrix preparation: Dissolve 150g of poloxamer 407 in 500g of ultrapure water to form a poloxamer 407 solution; add 1g of polycarbamide to the solution and stir until dissolved; (2) Preparation of sustained-release colloid: 0.25 g chitosan was added to 100 mL of glacial acetic acid solution (concentration of 1%) and stirred to dissolve to obtain chitosan solution; 1 g sodium tripolyphosphate was dissolved in 100 mL of 20 mmol / L pH 6.5 PB solution to obtain sodium tripolyphosphate solution; 25 mL of sodium tripolyphosphate solution was slowly added dropwise to 75 mL of chitosan solution to obtain chitosan-sodium tripolyphosphate colloid. The pH of the colloid was adjusted to 5.5 with sodium hydroxide solution, and then 20 mL of recombinant human epidermal growth factor solution (concentration of 0.5 mg / mL, solvent of 20 mmol / L pH 5.0 PBS buffer and 40% glycerol, volume ratio of 2:3) was added to allow the colloid to adsorb. The adsorption conditions were 25℃ and the adsorption time was 10 h. (3) Mixing of sustained-release colloid and gel matrix: Chitosan-sodium tripolyphosphate sustained-release colloid was added to poloxamer gel matrix, 100g of glycerol and 1g of disodium edetate were added, the pH of the colloid was adjusted to 6.5 with sodium hydroxide solution, and ultrapure water was added to make up the system weight to 1000g to obtain human epidermal growth factor thermosensitive gel.

[0024] Example 2 This embodiment provides a method for preparing a thermosensitive sustained-release gel of human epidermal growth factor, including: (1) Gel matrix preparation: Dissolve 140g of poloxamer 407 in 500g of ultrapure water to form a poloxamer 407 solution; add 3g of sodium hyaluronate to the solution and stir until dissolved; (2) Preparation of sustained-release colloid: 0.50 g chitosan was added to 100 mL of glacial acetic acid solution (concentration of 1%) and stirred to dissolve to obtain chitosan solution; 1 g sodium tripolyphosphate was dissolved in 100 mL of 15 mmol / L pH 6.5 PB solution to obtain sodium tripolyphosphate solution; 40 mL of sodium tripolyphosphate solution was slowly added dropwise to 75 mL of chitosan solution to obtain chitosan-sodium tripolyphosphate colloid. The pH of the colloid was adjusted to 5.5 with sodium hydroxide solution, and 5 mL of recombinant human epidermal growth factor solution (concentration of 2 mg / mL, solvent of 20 mmol / L pH 7.5 PBS buffer and 40% glycerol, volume ratio of 3:4) was added to allow the colloid to adsorb. The adsorption conditions were 23℃ and the adsorption time was 8 h. (3) Mixing of sustained-release colloid and gel matrix: Chitosan-sodium tripolyphosphate sustained-release colloid was added to poloxamer gel matrix to obtain a mixture. 150g of glycerol, 1g of disodium edetate and 20mg of human serum albumin were added to the mixture. The pH of the colloid was adjusted to 7.0 with citric acid. Ultrapure water was added to make up the system weight to 1000g to obtain human epidermal growth factor thermosensitive gel.

[0025] Example 3 This embodiment provides a method for preparing a thermosensitive sustained-release gel of human epidermal growth factor, including: (1) Gel matrix preparation: Dissolve 160g of poloxamer 407 in 500g of ultrapure water to form a poloxamer 407 solution; add 3g of carbomer to the solution and stir until dissolved; (2) Preparation of sustained-release colloid: 1 g of chitosan was added to 100 mL of glacial acetic acid solution (concentration of 0.5 wt%) and stirred to dissolve to obtain chitosan solution; 1 g of sodium tripolyphosphate was dissolved in 100 mL of 20 mmol / L pH 7.0 PB solution to obtain sodium tripolyphosphate solution; 20 mL of sodium tripolyphosphate solution was slowly added dropwise to 75 mL of chitosan solution to obtain chitosan-sodium tripolyphosphate colloid. The pH of the colloid was adjusted to 5.5 with 0.1 mol / L sodium hydroxide solution, and then 12.5 mL of recombinant human epidermal growth factor solution (concentration of 1.6 mg / mL, solvent of 20 mmol / L pH 5.0 PBS buffer and 40% glycerol, volume ratio of 4:5) was added to allow the colloid to adsorb. The adsorption conditions were 20℃ and the adsorption time was 12 h. (3) Mixing of sustained-release colloid and gel matrix: Chitosan-sodium tripolyphosphate sustained-release colloid was added to poloxamer gel matrix to obtain a mixture. 150g of propylene glycol, 1g of Tween 80, and 10mg of human serum albumin were added to the mixture. The pH of the colloid was adjusted to 6.7 using triethanolamine. Ultrapure water was added to make up the system weight to 1000g to obtain human epidermal growth factor thermosensitive gel.

[0026] Example 4 This embodiment provides a method for preparing a thermosensitive sustained-release gel of human epidermal growth factor, including: (1) Gel matrix preparation: Dissolve 130g of poloxamer 407 in 500g of ultrapure water to form a poloxamer 407 solution; add 1g of carbomer and 1g of polycarbofil to the solution and stir until dissolved; (2) Preparation of sustained-release colloid: 1 g of chitosan was added to 100 mL of glacial acetic acid solution (concentration of 1.5%) and stirred to dissolve to obtain chitosan solution; 1 g of sodium tripolyphosphate was dissolved in 100 mL of 25 mmol / L pH 6.5 PB solution to obtain sodium tripolyphosphate solution; 40 mL of sodium tripolyphosphate solution was mixed with 40 mL of chitosan solution to obtain chitosan-sodium tripolyphosphate colloid. The pH of the colloid was adjusted to 5.5 with 0.1 mol / L sodium hydroxide solution, and then 16 mL of recombinant human epidermal growth factor solution (concentration of 1.25 mg / mL, solvent of 20 mmol / L pH 5.0 PBS buffer and 40% glycerol, volume ratio of 3:3) was added to allow the colloid to adsorb. The adsorption conditions were 20℃ and the adsorption time was 9 h. (3) Mixing of sustained-release colloid and gel matrix: Chitosan-sodium tripolyphosphate sustained-release colloid was added to poloxamer gel matrix to obtain a mixture. 100g of dextropanthenol and 1g of disodium edetate were added to the mixture. The pH of the colloid was adjusted to 6.0 with hydrochloric acid. Ultrapure water was added to make up the weight of the system to 1000g to obtain human epidermal growth factor thermosensitive gel.

[0027] Example 5 This embodiment provides a method for preparing a thermosensitive sustained-release gel of human epidermal growth factor, including: (1) Gel matrix preparation: Dissolve 145g of poloxamer 407 in 500g of ultrapure water to form a poloxamer 407 solution; add 3g of sodium hyaluronate to the solution and stir until dissolved; (2) Preparation of sustained-release colloid: 1 g of chitosan was added to 100 mL of glacial acetic acid solution (concentration of 1%) and stirred to dissolve to obtain chitosan solution; 0.75 g of sodium tripolyphosphate was dissolved in 50 mL of 15 mmol / L pH 6.5 PB solution to obtain sodium tripolyphosphate solution; 20 mL of sodium tripolyphosphate solution was mixed with 30 mL of chitosan solution to obtain chitosan-sodium tripolyphosphate colloid. The pH of the colloid was adjusted to 5.0 with 0.1 mol / L sodium hydroxide solution, and 8 mL of recombinant human epidermal growth factor solution (concentration of 1.25 mg / mL, solvent of 30 mmol / L pH 5.0 PBS buffer and 30% glycerol, volume ratio of 4:3) was added to allow the colloid to adsorb. The adsorption conditions were 20℃ and the adsorption time was 9 h. (3) Mixing of sustained-release colloid and gel matrix: Chitosan-sodium tripolyphosphate sustained-release colloid was added to poloxamer gel matrix to obtain a mixture. 100g of glycerol and 1g of disodium edetate were added to the mixture. The pH of the colloid was adjusted to 6.7 with acetic acid. Ultrapure water was added to make up the weight of the system to 1000g to obtain human epidermal growth factor thermosensitive gel.

[0028] Example 6 This embodiment provides a method for preparing a thermosensitive sustained-release gel of human epidermal growth factor, including: (1) Gel matrix preparation: Dissolve 100g of poloxamer 407 in 500g of ultrapure water to form a poloxamer 407 solution; add 0.5g of carbomer to the solution and stir until dissolved; (2) Preparation of sustained-release colloid: 0.8 g of chitosan was added to 100 mL of glacial acetic acid solution (concentration of 0.5%) and stirred to dissolve to obtain chitosan solution; 3 g of sodium tripolyphosphate was dissolved in 100 mL of 20 mmol / L pH 7.0 PB solution to obtain sodium tripolyphosphate solution; 10 mL of sodium tripolyphosphate solution was mixed with 50 mL of chitosan solution to obtain chitosan-sodium tripolyphosphate colloid. The pH of the colloid was adjusted to 6.5 with 0.1 mol / L sodium hydroxide solution, and then 13.3 mL of recombinant human epidermal growth factor solution (concentration of 1.50 mg / mL, solvent of 15 mmol / L pH 7.5 PBS buffer and 60% glycerol, volume ratio of 2:5) was added to allow the colloid to adsorb. The adsorption conditions were 20℃ and the adsorption time was 11 h. (3) Mixing of sustained-release colloid and gel matrix: Chitosan-sodium tripolyphosphate sustained-release colloid was added to poloxamer gel matrix to obtain a mixture. 100g of glycerol and 1g of disodium edetate were added to the mixture. The pH of the colloid was adjusted to 6.0 using sodium hydroxide and acetic acid. Ultrapure water was added to make up the system weight to 1000g to obtain human epidermal growth factor thermosensitive gel.

[0029] Comparative Example 1 This comparative example provides a method for preparing a thermosensitive sustained-release gel of human epidermal growth factor, including: (1) Dissolve 145g of poloxamer 407 in 500g of ultrapure water to form a poloxamer 407 solution; add 3g of sodium hyaluronate to the solution and stir until dissolved; (2) Dissolve 0.5g of sodium tripolyphosphate in 50mL of 20mmol / L pH 7.0 PB solution to obtain sodium tripolyphosphate solution; add 1g of chitosan to 100mL of glacial acetic acid solution and stir to dissolve to obtain chitosan solution; take 20mL of sodium tripolyphosphate solution and 30mL of chitosan solution and add them to poloxamer and thickener solution; adjust the pH of the colloid to 5.5 with 0.1mol / L sodium hydroxide solution, add 8mL of recombinant human epidermal growth factor solution (concentration of 1.25mg / mL, solvent of 20mmol pH 5.0 PBS buffer + 40% glycerol), and stir the colloid to allow it to adsorb; (3) Add 100g of glycerol and 1g of disodium edetate to the gel matrix, stir to dissolve, adjust the pH of the colloid to 6.7 with 0.1mol / L sodium hydroxide solution, add ultrapure water to make up the weight of the system to 1000g, and obtain human epidermal growth factor thermosensitive gel.

[0030] Comparative Example 2 This comparative example provides a method for preparing a thermosensitive sustained-release gel of human epidermal growth factor, including: 10.8 g of sodium β-glycerophosphate was dissolved in 100 mL of 20 mmol / L pH 7.0 PB solution to obtain a sodium β-glycerophosphate solution; 0.25 g of chitosan was added to 100 mL of 1% glacial acetic acid solution and stirred to dissolve to obtain a chitosan solution; 40 mL of sodium β-glycerophosphate solution was slowly added dropwise to 120 mL of chitosan solution to obtain a chitosan-sodium β-glycerophosphate colloid. The pH of the colloid was adjusted to 5.5 using 0.1 mol / L sodium hydroxide solution, and then recombinant human epidermal growth factor solution was added to allow the colloid to adsorb.

[0031] Comparative Example 3 This comparative example provides a method for preparing a thermosensitive sustained-release gel of human epidermal growth factor, including: 10.8 g of sodium β-glycerophosphate was dissolved in 100 mL of 20 mmol / L pH 7.0 PB solution to obtain a sodium β-glycerophosphate solution; 0.5 g of chitosan was added to 100 mL of 1% glacial acetic acid solution and stirred to dissolve to obtain a chitosan solution; 45 mL of sodium β-glycerophosphate solution was slowly added dropwise to 50 mL of chitosan solution to obtain a chitosan-sodium β-glycerophosphate colloid. The pH of the colloid was adjusted to 5.5 using 0.1 mol / L sodium hydroxide solution, and then recombinant human epidermal growth factor solution was added to allow the colloid to adsorb.

[0032] Comparative Example 4 This comparative example provides a method for preparing a thermosensitive sustained-release gel of human epidermal growth factor, including: 10.8 g of sodium β-glycerophosphate was dissolved in 100 mL of 20 mmol / L pH 7.0 PB solution to obtain a sodium β-glycerophosphate solution; 2.0 g of chitosan was added to 200 mL of 1% glacial acetic acid solution and stirred to dissolve to obtain a chitosan solution; 20 mL of sodium β-glycerophosphate solution was slowly added dropwise to 140 mL of chitosan solution to obtain a chitosan-sodium β-glycerophosphate colloid. The pH of the colloid was adjusted to 5.5 using 0.1 mol / L sodium hydroxide solution, and then recombinant human epidermal growth factor solution was added to allow the colloid to adsorb.

[0033] Comparative Example 5 This comparative example provides a method for preparing a thermosensitive sustained-release gel of human epidermal growth factor, including: Dissolve 1.08 g of sodium hyaluronate in 100 mL of 20 mmol / L pH 7.0 PB solution to obtain a sodium hyaluronate solution; add 0.25 g of chitosan to 100 mL of 1% glacial acetic acid solution and stir to dissolve to obtain a chitosan solution; slowly add 20 mL of sodium hyaluronate solution to 60 mL of chitosan solution to obtain a chitosan-sodium hyaluronate colloid; adjust the pH of the colloid to 5.5 using 0.1 mol / L sodium hydroxide solution, and then add recombinant human epidermal growth factor solution to allow the colloid to adsorb.

[0034] Comparative Example 6 This comparative example provides a method for preparing a thermosensitive sustained-release gel of human epidermal growth factor, including: Dissolve 1.08 g of sodium hyaluronate in 100 mL of 20 mmol / L pH 7.0 PB solution to obtain a sodium hyaluronate solution; add 0.50 g of chitosan to 100 mL of 1% glacial acetic acid solution and stir to dissolve to obtain a chitosan solution; slowly add 20 mL of sodium hyaluronate solution to 75 mL of chitosan solution to obtain a chitosan-sodium hyaluronate colloid; adjust the pH of the colloid to 5.5 using 0.1 mol / L sodium hydroxide solution, and then add recombinant human epidermal growth factor solution to allow the colloid to adsorb.

[0035] Comparative Example 7 This comparative example provides a method for preparing a thermosensitive sustained-release gel of human epidermal growth factor, including: Dissolve 1.08 g of sodium hyaluronate in 100 mL of 20 mmol / L pH 7.0 PB solution to obtain a sodium hyaluronate solution; add 1.00 g of chitosan to 100 mL of 1% glacial acetic acid solution and stir to dissolve to obtain a chitosan solution; slowly add 20 mL of sodium hyaluronate solution to 75 mL of chitosan solution to obtain a chitosan-sodium hyaluronate colloid; adjust the pH of the colloid to 5.5 using 0.1 mol / L sodium hydroxide solution, and then add recombinant human epidermal growth factor solution to allow the colloid to adsorb.

[0036] Comparative Example 8 This comparative example provides a method for preparing a thermosensitive sustained-release gel of human epidermal growth factor, including: Dissolve 1.00 g of sodium alginate in 100 mL of 20 mmol / L pH 6.5 PB solution to obtain a sodium alginate solution; add 0.25 g of chitosan to 100 mL of 1% glacial acetic acid solution and stir to dissolve to obtain a chitosan solution; slowly add 20 mL of sodium alginate solution to 60 mL of chitosan solution to obtain a chitosan-sodium alginate colloid; adjust the pH of the colloid to 5.5 with 0.1 mol / L sodium hydroxide solution, and then add recombinant human epidermal growth factor solution to allow the colloid to adsorb.

[0037] Comparative Example 9 This comparative example provides a method for preparing a thermosensitive sustained-release gel of human epidermal growth factor, including: Dissolve 1.00 g of sodium alginate in 100 mL of 20 mmol / L pH 6.5 PB solution to obtain a sodium alginate solution; add 0.50 g of chitosan to 100 mL of 1% glacial acetic acid solution and stir to dissolve to obtain a chitosan solution; slowly add 20 mL of sodium alginate solution to 75 mL of chitosan solution to obtain a chitosan-sodium alginate colloid. Adjust the pH of the colloid to 5.5 with 0.1 mol / L sodium hydroxide solution, and then add recombinant human epidermal growth factor solution to allow the colloid to adsorb.

[0038] Comparative Example 10 This comparative example provides a method for preparing a thermosensitive sustained-release gel of human epidermal growth factor, including: Dissolve 1.00 g of sodium alginate in 100 mL of 20 mmol / L pH 6.5 PB solution to obtain a sodium alginate solution; add 1.00 g of chitosan to 100 mL of 1% glacial acetic acid solution and stir to dissolve to obtain a chitosan solution; slowly add 20 mL of sodium alginate solution to 75 mL of chitosan solution to obtain a chitosan-sodium alginate colloid; adjust the pH of the colloid to 5.5 with 0.1 mol / L sodium hydroxide solution, and then add recombinant human epidermal growth factor solution to allow the colloid to adsorb.

[0039] Comparative Example 11 This comparative example provides a method for preparing a thermosensitive sustained-release gel of human epidermal growth factor, including: The difference between this comparative example and Example 3 is that (2) in the preparation of the sustained-release colloid: take 20 mL of sodium tripolyphosphate solution, then add 25 μg of recombinant human epidermal growth factor to the sodium tripolyphosphate solution, stir evenly, and then slowly drop it into 75 mL of chitosan solution to obtain hEGF-chitosan-sodium tripolyphosphate colloid.

[0040] Comparative Example 12 The difference between this comparative example and Example 1 is that the thickener is replaced with polyethylene glycol 2000, and the amount used is 50g.

[0041] Comparative Example 13 The difference between this comparative example and Example 1 is that the thickener in the formulation has been removed.

[0042] Comparative Example 14 The difference between this comparative example and Example 1 is that the humectant is replaced with butylene glycol, and the amount used is 100g.

[0043] Comparative Example 15 The difference between this comparative example and Example 1 is that the moisturizer in the formulation has been removed.

[0044] Test Example 1: Protein Adsorption and Desorption Test Protein adsorption and desorption tests were performed on the sustained-release colloids in Examples 1-6 and Comparative Examples 2-11. The test process is as follows.

[0045] (1) Protein adsorption test: Take 20 mL of the sustained-release gel without adsorbed recombinant human epidermal growth factor, add 2 mL of recombinant human epidermal growth factor solution (protein concentration of 60 ug / mL), and add an appropriate amount of NaCl to prepare an isotonic solution of 280-320 mOsm / L. Stir at a constant speed of 100 rpm for 12 h at room temperature to allow recombinant human epidermal growth factor to adsorb on the colloid. After filtering an appropriate amount of liquid, detect the concentration of recombinant human epidermal growth factor and calculate the adsorption rate of recombinant human epidermal growth factor in the colloid. The calculation formula is as follows:

[0046] For Comparative Example 11, the only difference in the protein adsorption test was: take 20 mL of sustained-release gel adsorbed with recombinant human epidermal growth factor, add 2 mL of solution without recombinant human epidermal growth factor (protein concentration of 0 μg / mL), and add an appropriate amount of NaCl to prepare an isotonic solution.

[0047] (2) Protein desorption test: The pH of the sample after adsorption was adjusted to 9.0, and the sample was stirred at a constant speed of 100 rpm for 12 h at room temperature to desorb the recombinant human epidermal growth factor adsorbed on the colloid. After filtering an appropriate amount of liquid, the concentration of recombinant human epidermal growth factor was measured, and the desorption rate of recombinant human epidermal growth factor in the colloid was calculated. The formula for calculating the desorption rate is as follows:

[0048] The adsorption and desorption rates of Examples 1-6 and Comparative Examples 2-11 are shown in Table 1.

[0049] Table 1 Adsorption and desorption results of the sustained-release colloid

[0050] The results showed that the adsorption and desorption of recombinant human epidermal growth factor (rGF) by adsorbent colloids prepared with different excipients varied greatly. Specifically, sodium β-glycerophosphate-chitosan colloids (Comparative Examples 2, 3, and 4) exhibited poor adsorption of rGF and failed to adsorb it; sodium hyaluronate-chitosan colloids (Comparative Examples 5, 6, and 7) showed strong adsorption of rGF but poor desorption; sodium alginate-chitosan colloids (Comparative Examples 8, 9, and 10) showed strong adsorption of rGF but low desorption, thus having little significance for the sustained-release effect of the formulation; while sodium tripolyphosphate-chitosan colloids showed a certain adsorption effect on rGF, and the rGF not adsorbed in the colloid could take effect at the initial stage of administration, and its desorption capacity was significantly better than that of the comparative examples.

[0051] Test Example 2: Basic Properties of Gel The basic physical properties of Examples 1-6 and the control samples were determined, including gelation temperature, viscosity, and post-coating state. The test results are shown in Table 2. Figure 1 , Figure 2 The solidification states of the examples and comparative examples at 37°C are shown respectively.

[0052] Gelation temperature: Take 2 mL of sample into a vial, seal it, and keep it warm in different water baths. The sample gelation temperature is the one that does not fall within 10 seconds after the vial is inverted. Viscosity: The viscosity data of the sample was measured at 25°C and 100 rpm using an IKA ROTAISC me-vi viscometer.

[0053] After natural rheological changes: Add 2 mL of sample to a petri dish or glass plate placed in a 37°C oven and observe the state of the sample after drying. Figure 3 The natural leveling state of the examples and comparative examples at 37°C.

[0054] Table 2 Results of physical property determination of gel samples

[0055] As can be seen from the table above, Comparative Examples 1 and 11 have similar gel properties to the examples; however, Comparative Example 12 uses polyethylene glycol 2000, a commonly used thickener for gels, but due to its strong hydrophilicity, its addition disrupts the stable structure of poloxamer 407, preventing it from gelling; Comparative Example 13 has a gelling temperature of 38°C, which is higher than the normal human body temperature, making it difficult to gel when applied to the skin; Comparative Examples 14 and 15 are... Figure 3 It can be seen that the gel exhibits obvious shrinkage, which indicates poor structural stability of the gel and may affect the effect due to poor adhesion to the skin during application.

[0056] In summary, by adjusting the amounts of poloxamer 407, thickener, and humectant in the formulation, the gelation temperature of the gel will change accordingly, thereby altering the thickness of the gel after application to the skin. This results in the formation of gel structures of varying thicknesses on wounds or skin surfaces, while simultaneously providing a moisturizing environment for wound healing. Adjustments to the thickener and humectant will have a significant impact on the gel's performance.

[0057] Test Example 3: Human Epidermal Growth Factor Release Characteristics of Gel Samples The diffusion cell method (Chinese Pharmacopoeia 2025 Edition, Part IV, page 196, 0931, Dissolution and Release Determination, Method 9, Diffusion Cell Method) was used to test the samples of Examples 1-6 and Comparative Examples 1 and 11 (Comparative Example 12 could not gel and could not be tested; Comparative Example 13 had no thickener and was highly fluid; Comparative Examples 14-15 had poor condition after application). The receiving solution was 20 mmol / L PBS buffer at pH 7.5. The stirring speed was set to 100 rpm, the test temperature was 37°C, and the device was used for 48 hours. 1 mL of the receiving solution was taken at 2h, 4h, 6h, 8h, 12h, 24h, 36h, and 48h, and fresh receiving solution was added. The concentration of recombinant human epidermal growth factor in the receiving solution was measured, and the release rate was calculated using the formula shown below. The release results of recombinant human epidermal growth factor from the examples and comparative examples are as follows. Figure 4 As shown in Table 3.

[0058]

[0059] according to Figure 4 As shown in Table 3, the chitosan-sodium tripolyphosphate colloid in Comparative Example 1 showed poor adsorption of recombinant human epidermal growth factor, with a rapid release rate, reaching a plateau in about 6 hours. Comparative Example 11 achieved a release rate of 20.5% in 2 hours, reaching 80% release in about 8 hours, exhibiting a significant burst release and rapid release rate. Examples 1-6 showed release rates ranging from 5.0% to 13.0% within 2 hours, requiring more than 12 hours to reach over 80% release, with Example 3 even reaching 80% release in 48 hours. The concentration of recombinant human epidermal growth factor (rGF) in samples varied from over 80% in Examples 1 to 6 due to differences in the adsorption effect of chitosan-sodium tripolyphosphate colloid on recombinant human epidermal growth factor (rGF). This difference in rGF concentration affected the release detection. Samples 1, 2, and 6 reached a plateau at 24 hours, while samples 3, 4, and 5 reached a plateau at 36 hours. This resulted in the sustained release of rGF and maintained a stable concentration of rGF protein at the application site.

[0060] Table 3 Release rate results of the examples and comparative examples

[0061] In summary, although Comparative Examples 1 and 11 showed similar effects to the Examples after application, their drug release behavior differed significantly from the Examples: Comparative Examples 1 and 11 reached a release plateau in approximately 8 hours, while Examples 1-6 reached a sustained-release plateau after 24 hours. In contrast, the release saturation time of the Examples was significantly later than that of the Comparative Examples, indicating a longer sustained-release time. Furthermore, the release rates differed among the different Examples, providing a basis for the sustained release of human epidermal growth factor.

[0062] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing a thermosensitive sustained-release gel of human epidermal growth factor, characterized in that, Includes the following steps: S1. Add poloxamer and thickener to purified water and mix well to form a gel matrix; S2. Prepare human epidermal growth factor solution and chitosan-sodium tripolyphosphate colloid respectively. Add human epidermal growth factor solution to chitosan-sodium tripolyphosphate colloid for adsorption to obtain chitosan-sodium tripolyphosphate colloid loaded with human epidermal growth factor. S3. Add chitosan-sodium tripolyphosphate colloid loaded with human epidermal growth factor, humectant, and stabilizer to the gel matrix, and adjust the pH.

2. The method for preparing the human epidermal growth factor thermosensitive sustained-release gel according to claim 1, characterized in that, In step S1, the content of poloxamer in the gel matrix is ​​16.65wt%~24.10wt%; the content of thickener is 0.08wt%~0.46wt%.

3. The method for preparing the human epidermal growth factor thermosensitive sustained-release gel according to claim 1 or 2, characterized in that, The human epidermal growth factor includes naturally extracted human epidermal growth factor or recombinant human epidermal growth factor; The poloxamer includes poloxamer 407; The thickener includes at least one of polycarbofil, sodium hyaluronate, and carbomer.

4. The method for preparing the human epidermal growth factor thermosensitive sustained-release gel according to claim 1, characterized in that, Step S2 includes: S21. Preparation of human epidermal growth factor solution: Human epidermal growth factor was dissolved in a solvent consisting of 15-30 mmol / L PBS buffer solution (pH 5.0-7.5) and 30%-60% glycerol in a ratio of (2-4):(3-5) (v / v). S22. Preparation of chitosan-sodium tripolyphosphate colloid: Chitosan was dissolved in a 0.5wt%~1.5wt% glacial acetic acid solution to obtain a chitosan-glacial acetic acid solution; Sodium tripolyphosphate was dissolved in 15-25 mmol / L PB buffer solution with pH 6.5-7.0 to obtain a sodium tripolyphosphate solution. The chitosan-glacial acetic acid solution was stirred at 20-30℃ and 150-600 rpm, and sodium tripolyphosphate solution was added dropwise to obtain chitosan-sodium tripolyphosphate colloid. The pH was then adjusted to 5.0-6.

5. S23. Adsorption: Human epidermal growth factor solution was added to chitosan-sodium tripolyphosphate colloid and adsorbed at 15-25℃ for 8-12 hours.

5. The method for preparing the human epidermal growth factor thermosensitive sustained-release gel according to claim 4, characterized in that, The concentration of the human epidermal growth factor solution is 0.5~2 mg / mL; In the chitosan-glacial acetic acid solution, the concentration of chitosan is 0.25~1.0 g / mL; The sodium tripolyphosphate solution has a concentration of 1~3 g / mL. In the chitosan-sodium tripolyphosphate colloid, the mass ratio of chitosan to sodium tripolyphosphate is (3~15):4; The volume ratio of the human epidermal growth factor solution to the chitosan-sodium tripolyphosphate colloid is (1~4):(10~23).

6. The method for preparing the human epidermal growth factor thermosensitive sustained-release gel according to claim 1, characterized in that, In step S3, The ratio of the gel matrix to the chitosan-sodium tripolyphosphate colloid loaded with human epidermal growth factor is (600.5~663):(58~120) (m:v).

7. The method for preparing the human epidermal growth factor thermosensitive sustained-release gel according to claim 1, characterized in that, In step S3, the moisturizer is any one of glycerin, dextropanthenol, and propylene glycol, and its content in the human epidermal growth factor thermosensitive sustained-release gel is 10wt%~15wt%. The stabilizer is at least one of disodium edetate, human serum albumin, and Tween 80, and its content in the human epidermal growth factor thermosensitive sustained-release gel is 0.01wt%~0.1wt%. The pH adjuster is at least one of sodium hydroxide, citric acid, triethanolamine, hydrochloric acid, and acetic acid. Optionally, adjust the pH to 6.0~7.

0.

8. A thermosensitive sustained-release gel of human epidermal growth factor prepared by the preparation method according to any one of claims 1-7, characterized in that, In the human epidermal growth factor thermosensitive sustained-release gel, the amino acid cations in chitosan and the negatively charged phosphate ions in sodium tripolyphosphate attract each other, undergo adsorption and polymerization, and form a cross-linked network structure, thereby providing a structural basis for the adsorption and release of human epidermal growth factor. In the human epidermal growth factor thermosensitive sustained-release gel, the thickener serves to reduce the fluidity of the sample, preventing it from flowing away from the target area quickly during use. In the human epidermal growth factor thermosensitive sustained-release gel, the function of the moisturizer is to maintain the moist state of the sample after use and reduce wound discomfort; In the human epidermal growth factor thermosensitive sustained-release gel, poloxamer is in solution when it is not cross-linked. When the temperature is raised, it forms a network structure in which the other excipients are distributed. After application, the poloxamer network structure gradually collapses, and the growth factors that are not adsorbed by chitosan-sodium tripolyphosphate in the solution are released first. The adsorbed growth factors are gradually released as the structure changes, thereby prolonging the release cycle of the growth factors.

9. The human epidermal growth factor thermosensitive sustained-release gel prepared by the preparation method according to claim 8, characterized in that, The optimal gelation temperature range for the human epidermal growth factor thermosensitive sustained-release gel is 32~37℃. The human epidermal growth factor content in the thermosensitive sustained-release gel is 0.001%~0.002%.

10. Use of a human epidermal growth factor thermosensitive sustained-release gel prepared by any one of claims 1-7 or the human epidermal growth factor thermosensitive sustained-release gel prepared by claim 8 or 9 in the preparation of a medicament for treating refractory wounds and / or in the preparation of cosmetic skin care products.

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