Composite hydrogel as well as preparation method and application thereof

Abstract

The invention discloses composite hydrogel as well as a preparation method and application thereof. The curcumin-EGCG composite hydrogel is prepared by forming a complex through hydrogen-bond interaction between gelatin and epigallocatechin gallate and carrying out chemical crosslinking through Schiff base reaction of curcumin and gelatin. The composite hydrogel disclosed by the invention has a self-healing characteristic or viscosity and a shear thinning characteristic, and is beneficial to local application. The composite hydrogel is loose and porous in structure, the drug loading rate can be increased, the drug can be slowly and controllably released, the treatment effect of the composite hydrogel in androgenetic alopecia is promoted, meanwhile, the solubility of a drug monomer can be remarkably improved, and the problem that the drug solubility is low is solved. The composite hydrogel provided by the invention provides an effective transdermal drug delivery preparation for treatment of skin diseases, and has good clinical application prospects and development values.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical nanomaterials, and in particular to a composite hydrogel and a preparation method and application thereof. Background Art

[0002] Androgenetic alopecia (AGA), also known as seborrheic alopecia, is the most common type of hair loss in the world. It is a progressive hair follicle miniaturization disease that begins in adolescence or late adolescence. AGA is often clinically manifested as progressive hair thinning and receding hairline on the top of the head in men, and diffuse hair thinning in the central scalp in women, but the hairline is preserved. The pathological characteristics of AGA are mainly androgen overreaction, resulting in an inhibitory effect on hair follicles. Its pathogenesis is directly related to the androgen metabolic pathway, including the increase in testosterone (TS), dihydrotestosterone (DHT) and androgen receptor (AR) concentrations. 5α-reductase II, an important enzyme for the conversion of TS to DHT, is also commonly found in AGA patients. Therefore, inhibition of 5α-reductase II may be a good target for the treatment of AGA.

[0003] Currently, the treatments approved by the U.S. Food and Drug Administration (FDA) include topical minoxidil (MXD) and oral finasteride, but these two treatments often exhibit certain side effects. In addition, other therapies such as low-laser therapy, autologous platelet-rich plasma therapy, and hair transplantation technology have limitations such as poor patient compliance and convenience as well as high costs. As a result, more and more researchers have turned their focus to the study of the topical use of traditional Chinese medicine and its active ingredients to treat AGA. In addition, a variety of traditional Chinese medicine monomers have been shown to have a good effect in promoting hair regeneration, and significant progress has been made in the research on the treatment of AGA.

[0004] Curcumin (cur), an active ingredient of traditional Chinese medicine, can inhibit the expression of AR, promote the regeneration of hair follicles, and achieve hair regeneration treatment; Epigallocatechin gallate (EGCG), another active ingredient of traditional Chinese medicine, is a 5α-reductase inhibitor, which can inhibit the production of DHT and reduce the binding to AR receptors, thereby improving hair follicle cell apoptosis and promoting hair regeneration; therefore, these two active ingredients of traditional Chinese medicine can be used as effective AGA treatment drugs. However, curcumin has the defects of low solubility, poor stability and intestinal permeability, as well as rapid metabolism in the gastrointestinal tract and liver, which limit its application; and EGCG has poor stability, low bioavailability and limited efficacy, which also greatly limit its application. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for preparing a composite hydrogel.

[0006] Another object of the present invention is to provide a composite hydrogel.

[0007] Another object of the present invention is to provide applications of the composite hydrogel.

[0008] To achieve the above object, the technical solution adopted by the present invention is:

[0009] In a first aspect, the present invention provides a method for preparing a composite hydrogel, comprising the following steps:

[0010] S1, completely dissolving gelatin and EGCG in an organic solvent to obtain a hydrogel precursor solution;

[0011] S2. Slowly drop the curcumin solution into the hydrogel precursor solution obtained in step S1, stir, and allow to stand to form a gel, thereby obtaining a composite hydrogel loaded with curcumin and EGCG.

[0012] Hydrogel is a very hydrophilic three-dimensional network structure gel with good biocompatibility, similar to the structure of the extracellular matrix, adjustable physical and mechanical properties, protection of wounds from microbial invasion, and biodegradability, making it an ideal carrier widely used in technical fields such as wound dressings, drug sustained-release matrices, and human soft tissue filling. Its loose and porous structure can load hydrophobic drugs to help improve drug dissolution, improve drug stability and drug loading capacity to promote efficacy, and can release drugs slowly and controllably to reduce the frequency of drug administration; relying on the shear-thinning properties of hydrogels, it is also conducive to the local application of drugs and expands the application of drugs.

[0013] Gelatin (Gel) is the product of partial hydrolysis of collagen. It is a large molecular hydrophilic colloid with the characteristics of low immunogenicity, non-toxicity, low price, good film-forming property and biocompatibility. It also has many unique properties of collagen, such as high biocompatibility and low skin irritation. It is regarded as a substitute for collagen and is often used as a drug delivery carrier.

[0014] As a preferred embodiment of the method for preparing the composite hydrogel of the present invention, the organic solvent in step S1 is ethanol, and more preferably 50% ethanol. Ethanol or anhydrous ethanol is low in toxicity, environmentally friendly, and can achieve low-cost, large-scale production.

[0015] As a preferred embodiment of the method for preparing the composite hydrogel of the present invention, the ratio of gelatin, EGCG, and ethanol in step S1 is gelatin: EGCG: organic solvent = 80-120 mg: 3-7 mg: 1 ml, more preferably 100 mg: 5 mg: 1 ml. When the ratio is 100 mg: 5 mg: 1 ml, that is, the mass fraction of gelatin is 10%, the prepared hydrogel has shear thinning properties, can be used for local administration, and has good self-healing ability.

[0016] As a preferred embodiment of the method for preparing the hydrogel of the present invention, the curcumin solution is a 0.5-2 mg / mL curcumin ethanol solution; more preferably a 1 mg / mL curcumin ethanol solution. The preparation of the curcumin solution includes the following steps: weighing curcumin as needed under light-proof conditions, adding it to anhydrous ethanol, stirring and dissolving it, and temporarily storing it at 4°C; the curcumin solution should be prepared and used immediately.

[0017] As a preferred embodiment of the method for preparing the hydrogel of the present invention, the volume ratio of the curcumin solution to the hydrogel precursor solution is 1:8-12; more preferably 1:10.

[0018] As a preferred embodiment of the method for preparing the composite hydrogel of the present invention, the complete dissolution described in step S1 is achieved by heating at a temperature of 65±5°C for 24±5h; and the static gelation described in step S2 is achieved at a low temperature of 4±2°C.

[0019] As a preferred embodiment of the method for preparing the composite hydrogel of the present invention, the dropping in step S2 is dropping while stirring; the stirring is magnetic stirring, and the rotation speed is 1000±200 rpm / min.

[0020] As a preferred embodiment of the method for preparing the composite hydrogel of the present invention, the stirring time in step S2 is 2±1 h.

[0021] As a preferred embodiment of the method for preparing the composite hydrogel of the present invention, the hydrogel precursor solution in step S1 should be reddish brown to wine red, more preferably wine red; step S2 is carried out under light-proof conditions.

[0022] The composite hydrogel loaded with curcumin and EGCG needs to be stored at 4° C. in the dark.

[0023] In a second aspect, the present invention provides a composite hydrogel prepared by the above-mentioned composite hydrogel preparation method.

[0024] In a third aspect, the present invention provides use of the composite hydrogel in preparing a drug for treating skin diseases.

[0025] As a preferred embodiment of the use of the composite hydrogel of the present invention in the preparation of a drug for treating skin diseases, the skin disease is at least one of androgenic alopecia, infection, burns, mechanical trauma, and diabetic dermatosis.

[0026] The curcumin-EGCG composite hydrogel prepared by the present invention is expected to be used as a drug delivery strategy in the multi-directional and multi-dimensional nanobiomedicine field such as androgenic alopecia, infection, burns, diabetes, mechanical trauma and other diseases mainly with skin lesions.

[0027] As a preferred embodiment of the use of the composite hydrogel of the present invention in the preparation of a drug for treating skin diseases, the drug is an external dressing.

[0028] Compared with the prior art, the present invention has the following advantages and effects:

[0029] 1. The curcumin-EGCG nanocomposite hydrogel loaded with curcumin prepared by the present invention loads curcumin and EGCG in a three-dimensional network structure of the gel. The prepared composite hydrogel has dynamic chemical bonds, has viscosity or self-healing properties, and shear thinning properties. After topical administration, it adheres to the skin surface as a drug reservoir, and effectively penetrates the drug into the epidermis and dermis, releases the drug in a controlled manner, reduces the frequency of administration, and allows the drug to be continuously absorbed into the hair follicle microenvironment, and can be used as an excellent gel preparation for skin administration.

[0030] 2. The present invention utilizes the easily hydrolyzed Schiff base bond formed by the Schiff base reaction between the carbonyl group of Cur and the amino group of Gel, so that the hydrogel has good degradability, and the drug delivery system based on curcumin-EGCG is easily degraded into non-toxic substances. Therefore, the composite hydrogel loaded with curcumin-EGCG described in the present invention has excellent biocompatibility and is suitable for disease treatment.

[0031] 3. The EGCG described in the present invention prepares Cur-EGCG-Gel composite hydrogel by self-assembly through hydrogen bonding between its phenolic hydroxyl group and the carbonyl group of Cur and / or the amino group of Gel, thereby improving the stability and solubility of the drug, the self-healing and viscosity of the hydrogel, and being beneficial to the treatment of diseases.

[0032] 4. The preparation method of the curcumin-EGCG loaded composite hydrogel provided by the present invention has a relatively simple production process. The solvent involved in synthesizing the composite hydrogel is ethanol or anhydrous ethanol, which is non-toxic, environmentally friendly, and can achieve low-cost, large-scale production.

[0033] 5. The curcumin-EGCG loaded composite hydrogel provided by the present invention is expected to be used as a drug delivery strategy in the multi-directional and multi-dimensional nanobiomedicine field such as androgenic alopecia, infection, burns, diabetes, mechanical trauma and other diseases mainly with skin lesions. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the phase change of Cur-EGCG-Gel hydrogel prepared in the present invention.

[0035] Figure 2 Scanning electron microscope (SEM) characterization images of the Gel crystals and Cur-EGCG-Gel freeze-dried samples prepared in the present invention (scale: 10 μm); wherein a is the SEM image of Gel, and b is the SEM image of Cur-EGCG-Gel.

[0036] Figure 3 RXD spectra of Cur monomer powder, EGCG monomer, Gel crystal and Cur-EGCG-Gel freeze-dried sample prepared in the present invention.

[0037] Figure 4 The UV-visible spectra of the Cur solution, EGCG solution and Cur-EGCG-Gel solution prepared in the present invention.

[0038] Figure 5 FTIR spectra of EGCG, Cur monomer powder, Gel crystal and Cur-EGCG-Gel freeze-dried samples prepared in the present invention.

[0039] Figure 6 These are the rheological property result graphs of the Gel crystals and Cur-EGCG-Gel freeze-dried samples prepared in the present invention; wherein, a is the rheological property result graph of Cur-EGCG-Gel, and b is the rheological property result graph of Gel.

[0040] Figure 7 The mechanical properties of the Gel crystals and the freeze-dried samples of Cur-EGCG-Gel prepared by the present invention are shown in Figure 1; wherein a is the mechanical properties of Cur-EGCG-Gel, and b is the mechanical properties of Gel.

[0041] Figure 8 These are stability result diagrams of the Gel crystals and Cur-EGCG-Gel freeze-dried samples prepared in the present invention; wherein a is the stability result diagram of Cur-EGCG-Gel, and b is the stability result diagram of Gel.

[0042] Fig. 9 Schematic diagram of the change of viscosity of the Gel crystals and Cur-EGCG-Gel freeze-dried samples prepared in the present invention with shear rate.

[0043] Fig.10 This is a graph showing the self-healing performance of the Cur-EGCG-Gel hydrogel prepared in the present invention.

[0044] Fig.11 The concentration standard curves of the Cur solution and the EGCG solution prepared in the present invention at 430 nm and 276 nm are shown; wherein a is the concentration standard curve of Cur, and b is the concentration standard curve of EGCG.

[0045] Fig.12 The in vitro release results of Cur and EGCG prepared in the present invention in the Cur-EGCG-Gel hydrogel system and the Cur solution and the EGCG solution are shown in FIG. a. The release results of EGCG in Cur-EGCG-Gel and the EGCG solution are shown in FIG. b. The release results of Cur in Cur-EGCG-Gel and the Cur solution are shown in FIG. (n=3).

[0046] Fig.13 This is a graph showing the in vitro skin permeation results of EGCG in Cur-EGCG-Gel and EGCG solution prepared in the present invention.

[0047] Fig.14 This is a graph showing the intradermal retention results of Cur-EGCG-Gel and EGCG prepared in the present invention.

[0048] Fig.15 The cytotoxicity test results of Cur, EGCG and Cur-EGCG-Gel prepared by the present invention are shown in FIG. a, wherein a is the cytotoxicity test result of Cur, b is the cytotoxicity test result of EGCG, and c is the cytotoxicity test result of Cur-EGCG-Gel.

[0049] Fig.16 It is a time-dependent uptake analysis diagram of Cur-EGCG-Gel prepared in the present invention on human skin fibroblasts (HSF); wherein a is a cell uptake fluorescence image, b is a fluorescence quantitative analysis diagram, scale bar = 100 μm, n = 3.

[0050] Fig.17 This is a concentration-dependent uptake analysis diagram of Cur-EGCG-Gel prepared in the present invention on HSF cells; wherein a is a cell uptake fluorescence image, b is a fluorescence quantitative analysis diagram, scale bar = 100 μm, n = 3.

[0051] Fig.18 This is a diagram showing the uptake mechanism of Cur-EGCG-Gel prepared in the present invention on HSF cells.

[0052] Fig.19 The graph is a result of flow cytometry analysis of the uptake mechanism of Cur-EGCG-Gel prepared in the present invention on HSF cells; wherein, a is a flow cytometry analysis graph of cell uptake, and b is a quantitative analysis graph of cell uptake, ***p<0.001 vs Control, n=3.

[0053] Fig. 20 Schematic diagram of animal experimental administration of Cur-EGCG-Gel prepared in the present invention.

[0054] Fig.21 This is a diagram showing the experimental results of the effects of Cur, EGCG and Cur-EGCG-Gel prepared in the present invention on animal hair growth after administration.

[0055] Fig. 22 The figures are statistical results of the evaluation of the quality of new hair growth in animals after treatment with Cur, EGCG and Cur-EGCG-Gel prepared in the present invention; the left figure is a statistical figure of new hair coverage area (n=8), the middle figure is a statistical figure of new hair weight (n=8), and the right figure is a statistical figure of new hair length (n=5).

[0056] Fig.23 This is the SEM image of newly grown hair after treatment with Cur-EGCG-Gel prepared in the present invention (scale bar: 10 μm).

[0057] Fig.24 This is a HE staining result diagram showing the effect of Cur, EGCG and Cur-EGCG-Gel prepared in the present invention on the cycle changes of animal hair follicles after administration (scale scale is 200 μm).

[0058] Fig.25 This is a diagram showing the experimental results of Ki67 immunohistochemical staining on the proliferation of hair follicle cells of animals after administration of Cur, EGCG and Cur-EGCG-Gel prepared in the present invention (scale bar is 200 μm).

[0059] Fig.26 The results of immunohistochemical staining of type II 5α-reductase that affects the androgen level of animals after administration of Cur, EGCG and Cur-EGCG-Gel prepared by the present invention (scale bar is 200μm).

[0060] Fig. 27 This is a diagram showing the experimental results of AR receptor immunohistochemical staining that affects the androgen level in animals after administration of Cur, EGCG and Cur-EGCG-Gel prepared in the present invention (scale bar is 200 μm).

[0061] Fig.28 This is a graph showing the changes in animal body weight during treatment with Cur, EGCG and Cur-EGCG-Gel prepared in the present invention.

[0062] Fig.29 This is a graph showing the results of animal blood biochemical analysis after administration of Cur, EGCG and Cur-EGCG-Gel prepared by the present invention.

[0063] Fig.30This is a HE staining result of pathological detection of animal organs after administration of Cur, EGCG and Cur-EGCG-Gel prepared in the present invention (scale bar is 200 μm). DETAILED DESCRIPTION

[0064] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0065] Sources of materials in the following examples: Gelatin: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; Epigallocatechin gallate (EGCG): purchased from Nantong Feiyu Biotechnology Co., Ltd.; Curcumin: purchased from Shanghai Shaoyuan Reagent Co., Ltd.; Phosphate buffer solution (pH = 5.0): purchased from Guangjian (Guangzhou) Testing Technology Co., Ltd.; Anhydrous ethanol: purchased from Guangzhou Bell Biotechnology Co., Ltd.; Fetal bovine serum: purchased from Thermo Fisher Scientific, USA; pH 7.4 PBS: purchased from Thermo Fisher Scientific, USA; bFGF: purchased from Wuhan Pronosai Life Science Co., Ltd.; recombinant human insulin solution: purchased from Wuhan Pronosai Life Science Co., Ltd.; L-glutamine: purchased from Thermo Fisher Scientific, USA; sodium pyruvate: purchased from Thermo Fisher Scientific, USA; CCK8 kit: purchased from Tongren Chemical Research Institute; DMEM / F12: purchased from Thermo Fisher Scientific, USA; 4% paraformaldehyde: purchased from Beijing White Shark Easy Technology Co., Ltd.; hydrocortisone: purchased from Beijing Solebao Technology Co., Ltd.; serum-free cryopreservation solution: purchased from Thermo Fisher Scientific, USA; anti-fluorescence quenching sealing solution: purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; xylene: purchased from Guangzhou Bell Biotechnology Co., Ltd.; Ki67 antibody: purchased from Annolunsheng Co., Ltd.; AR antibody: purchased from Hangzhou Yikang Biotechnology Co., Ltd.; SRD5A2 antibody: purchased from Beijing Bioson Biotechnology Co., Ltd.; DAB colorimetric solution: purchased from Zhongshan Jinqiao Biotechnology Co., Ltd.; minoxidil: purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; testosterone: purchased from Beijing Solebow Technology Co., Ltd.; hematoxylin-eosin stain: purchased from Beijing White Shark Easy Technology Co., Ltd.; sodium citrate repair solution: purchased from Beijing White Shark Easy Technology Co., Ltd.; goat serum: purchased from Beijing Solebow Technology Co., Ltd.; Leica 819 blade: purchased from Ernst Leitz, Germany; histochemistry rabbit two-step kit: purchased from Zhongshan Jinqiao Biotechnology Co., Ltd.; primary antibody diluent: purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; neutral gum: purchased from Beijing White Shark Easy Technology Co., Ltd.

[0066] Embodiment 1:

[0067] 1. Preparation of composite hydrogel, including the following steps:

[0068] 1. Measure 5 ml of anhydrous ethanol and mix with 5 ml of pure water to obtain 50% ethanol.

[0069] 2. Weigh 100 mg of Gel (mass fraction 10%) and 5 mg of EGCG monomer and dissolve them in 1 ml of 50% ethanol. Place them in an oven at 65°C for 24 hours until the Gel is completely dissolved and the solution is wine red. This can be used as a hydrogel precursor solution.

[0070] 3. Under light-proof conditions, weigh 1 mg of curcumin monomer, add it to 1 ml of anhydrous ethanol, and stir until dissolved to obtain a curcumin solution (Cur solution) with a concentration of 1 mg / ml.

[0071] 4. Slowly add 100 μl of 1 mg / ml curcumin solution to the hydrogel precursor solution prepared in step 2, stirring while adding, and stirring at 1000 rpm / min at room temperature for 2 h, and let stand at low temperature (4°C) to gel, to obtain a composite hydrogel loaded with curcumin-EGCG, named Cur-EGCG@Gel.

[0072] 5. Part of Cur-EGCG@Gel was gradient freeze-dried and named as the freeze-dried sample of Cur-EGCG@Gel.

[0073] 2. Preparation of blank hydrogel Gel, including the following steps:

[0074] 1. Measure 5 ml of anhydrous ethanol and mix with 5 ml of pure water to obtain 50% ethanol.

[0075] 2. Weigh 100 mg of Gel (mass fraction is 10%), dissolve it in 1 ml of 50% ethanol, and place it in a 65°C oven for 24 hours until the Gel is completely dissolved to obtain a blank hydrogel solution, and place it at 4°C to gel to obtain a blank hydrogel Gel.

[0076] 3. Preparation of EGCG solution, including the following steps:

[0077] 1. Weigh 5 mg of EGCG monomer, add it to 1 ml of 50% ethanol, stir to dissolve, and obtain an EGCG solution with a concentration of 5 mg / ml.

[0078] Comparative Example 1:

[0079] The difference between preparing the composite hydrogel and Example 1 is that the EGCG and Gel in step 2 are placed in an oven at 55° C., 60° C., 65° C., and 70° C. for observation. The results show that the color of the hydrogel precursor solution at 65° C. is darker and the effect is better.

[0080] Comparative Example 2:

[0081] The composite hydrogel was prepared, and the difference from Example 1 was that the anhydrous ethanol in step 3 was replaced with a PBS buffer solution. It was found that when the drug monomer was dissolved in a PBS buffer solution, Cur could not be detected in the drug release experiment, which may be because Cur, as a fat-soluble drug, was difficult to dissolve in the PBS buffer solution. Therefore, after the medium was replaced with an organic solvent, EGCG and Cur could be dissolved at the same time.

[0082] Comparative Example 3:

[0083] In the preliminary experiment, when EGCG, Cur and Gel were physically mixed at 5mg, 1mg and 100mg, the drug release ratio of Cur to EGCG was 1:1 and 1:2, and the release of Cur was not completely detected, while the release of EGCG was almost complete after 44h. The analysis of Cur showed that the absorption peak was not detected because it could not pass through the dialysis bag due to covalent cross-linking with Gel and was retained in the bag. After three days, no Cur was detected in the internal and external fluids, and EGCG was completely released because it did not non-covalently bind to Gel. Therefore, according to the gelation ratio of EGCG to Gel of 1:20, the preparation conditions were changed to weigh EGCG and Gel and dissolve them to prepare the hydrogel precursor solution, and then add Cur to prepare the hydrogel carrier.

[0084] Effect Example 1

[0085] Characterization and performance testing of the product prepared in Example 1:

[0086] 1. The phase change test of Cur-EGCG@Gel was carried out. The results are as follows Figure 1 As shown, at high temperature (37°C), the hydrogel state changes from gel to sol, and at low temperature (20°C), it returns to the gel state.

[0087] 2. SEM characterization of Cur-EGCG@Gel freeze-dried samples and Gel crystals. The results are as follows Figure 2 As shown in the figure, compared with Gel, the structure of the freeze-dried sample of Cur-EGCG@Gel exhibits a typical 3D porous structure and high connectivity, and the mesoporous interconnection also provides sufficient space for the diffusion of drugs from the hydrogel, while the pore size of the Gel crystal structure is small, the number of pores is small, and the pores are not connected to each other. It is confirmed that the structure of Cur-EGCG@Gel is loose and porous, which is conducive to drug loading and release.

[0088] 3. X-ray powder diffraction (XRD) was performed on EGCG monomer, Cur monomer powder, Gel crystal and Cur-EGCG@Gel freeze-dried samples. The results are as follows: Figure 3 As shown in the figure, the sharp diffraction peak of Cur is its crystal diffraction peak, which proves that Cur has a higher crystal structure; the characteristic peak of EGCG also has a sharp peak, indicating that it is more crystalline; and the characteristic peak of Gel is a diffusely scattered bun peak, indicating that the structure is amorphous. In the freeze-dried sample of Cur-EGCG-Gel, the sharp characteristic peaks of Cur and EGCG disappeared, and only the diffraction peak of Gel was retained, proving that the drug exists in the composite hydrogel in an amorphous form.

[0089] 4. UV-visible absorption spectra of EGCG solution, Cur solution and hydrogel Cur-EGCG@Gel were tested. The results are as follows: Figure 4 As shown, the characteristic absorption peak of the EGCG spectrum is around 276nm, and the characteristic absorption peak of the Cur spectrum is at 430nm. The ultraviolet absorption of the prepared hydrogel Cur-EGCG-Gel detected the characteristic absorption of EGCG and Cur at 276nm and 430nm, confirming the successful construction of Cur-EGCG-Gel.

[0090] 5. EGCG monomer, Cur monomer powder, Gel crystal and Cur-EGCG@Gel freeze-dried samples were characterized by Fourier transform infrared (FT-IR). The results are as follows: Figure 5 As shown, the FT-IR of Cur is 3503 cm -1 、1626cm -1 and 1596cm -1 The absorption at 3473 cm in FT-IR of EGCG corresponds to the phenolic hydroxyl groups on the benzene ring. -1 ,3350cm -1 and 3295cm -1 The stretching vibration absorption peak of phenolic OH is 1694 cm -1 and 1608cm -1 The C=O stretching vibration absorption peak of the phenolic hydroxyl group in EGCG is at 3291 cm; the NH stretching vibration of the amide bond and the free -OH in the FT-IR of Gel are at a high wavelength of 3291 cm -1 There is a small broad peak at 1626cm -1 The peak at 1515 cm is the stretching vibration of C=O of the amide bond. -1 The peak at 1634 cm-1 is due to the -NH bending vibration of the amide bond; -1 The new peak represents the formation of -C=N-, with a high wavelength of 3291cm -1The -OH stretching vibration wave number and intensity decreased, indicating the formation of intermolecular hydrogen bonds; this confirmed the occurrence of Schiff base reaction and the formation of intermolecular hydrogen bonds in Cur-EGCG-Gel.

[0091] 6. Temperature scanning test was performed on the rheological test of Cur-EGCG@Gel freeze-dried samples and Gel crystals. The results are as follows: Figure 6 As shown in the figure, as the temperature gradually rises to 26°C, G' and G" intersect. Before this, G' is always greater than G", indicating that it is always in a gel state within this temperature range. After 26°C, G" begins to be greater than G', indicating that the gel state changes from a viscoelastic solid to a viscoelastic liquid to form a sol, confirming that 26°C is the critical temperature for gelation. The rheological test of Cur-EGCG@Gel freeze-dried samples and Gel crystals was carried out by oscillation scanning test, and the results are shown in Figure 7 As shown in the figure, as the stress gradually increases to 1000Pa, the molecular structure of the hydrogel is destroyed and changes from solid to liquid, showing a wide linear viscoelastic region, which proves that the composite hydrogel has low strength, is easy to spread, and is convenient for local application. The rheological test of Cur-EGCG@Gel freeze-dried samples and Gel crystals was carried out by frequency scanning test, and the results are shown in the figure. Figure 8 As shown in the figure, as the frequency changes, the storage modulus (G') is always greater than the loss modulus (G"), which proves that it is always in a gel state and is relatively stable during this period. The rheological test of Cur-EGCG@Gel and blank hydrogel Gel described in the example was carried out by dynamic viscosity test, and the results are as follows Fig. 9 As shown in the figure, with the increase of shear rate, the viscosity of the two hydrogel systems showed a downward trend, proving that both hydrogels have shear thinning properties. The rheological test of Cur-EGCG@Gel and Gel was carried out for self-healing test, and the results are shown in the figure. Fig.10 As shown in the figure, when high stress and low stress are cyclically applied to the Cur-EGCG-Gel hydrogel system, the modulus changes and returns to the original modulus value when entering the next cycle, confirming that the hydrogel has a certain degree of self-healing performance.

[0092] 7. Drug release test was performed on EGCG solution, Cur solution and Cur-EGCG@Gel. Fig.11 It can be seen that the standard curve of Cur at 430nm is y=0.1612x+0.0575, R 2 is 0.9995; the standard curve of EGCG at 276nm is y=0.0252x-0.0042, R 2 The drug release results were Fig.12It can be seen that EGCG is slowly released from Cur-EGCG-Gel hydrogel, and the overall release tends to be stable. The drug release in 48 hours is about 80%; while Cur experienced a burst release stage in the first 12 hours, and then the release began to tend to be slow. The cumulative release was better than that of Cur solution, which greatly improved the dissolution of Cur. The drug release in 48 hours was almost complete, confirming that Cur-EGCG-Gel has good drug sustained-release properties.

[0093] 8. In vitro transdermal tests were performed on EGCG solution and Cur-EGCG@Gel. The results are as follows: Fig.13 As shown in Figure 2, the cumulative percutaneous penetration of Cur-EGCG-Gel per unit area for 24 hours was 635.31±158.70μg / cm 2 The cumulative permeation of EGCG solution per unit area for 24 hours was 417.79±118.09μg / cm 2 .Depend on Fig.14 It can be seen that the intradermal retention amounts of Cur-EGCG-Gel and EGCG were 2.26±0.14 mg and 2.03±0.19 mg, respectively, indicating that in the in vitro transdermal process, Cur-EGCG-Gel had better skin penetration than EGCG solution, and the skin deposition was also slightly higher than that of EGCG solution.

[0094] Effect Example 2

[0095] The in vitro safety evaluation experiment of Cur-EGCG@Gel prepared in Example 1 included the safety evaluation of Cur-EGCG@Gel at the level of human skin fibroblasts (HSF).

[0096] HSF cells were cultured at 5 × 10 3 Cells were seeded at a density of 100 cells / well in a 96-well plate. After culturing for 24 hours, gradient concentrations of Cur solution, EGCG solution and Cur-EGCG@Gel were added and incubated together for another 24 hours. CCK-8 solution was then added and incubated for 4 hours. The absorbance at 562 nm was measured using an ELISA reader. Cell viability was expressed as the percentage of the drug-treated group relative to the blank control group at A562 nm. The results are shown in Fig.15 As shown, after incubation with HSF for 24 h, there was no significant change in cell viability within the concentration range of Cur in the range of 5-80 μg / ml, EGCG in the range of 0.625-10 μg / ml, and Cur-EGCG@Gel in the range of 6.25-100 μg / ml, indicating good biocompatibility.

[0097] Effect Example 3

[0098] The cellular uptake experiment of Cur-EGCG@Gel prepared in Example 1 includes the uptake behavior and uptake mechanism of Cur-EGCG@Gel at the level of human skin fibroblasts (HSF).

[0099] 1. HSF cells were cultured at 1×10 5 The cells were inoculated at a density of 100 μg / ml on a 12-well plate slide. After culturing for 24 hours, the culture medium was discarded, and 800 μl of 100 μg / ml Cur-EGCG-Gel solution was added and incubated in the dark for 0.5, 1, and 3 hours, and after adding gradient concentrations of Cur-EGCG-Gel solution and incubating in the dark for 3 hours, the culture medium was discarded, the cells were washed three times with PBS, DAPI was added and incubated in the dark for 15 minutes to stain the cell nucleus, DAPI was discarded, the cells were washed three times with PBS, 4% paraformaldehyde was added for fixation for 15 minutes, PBS was washed three times, and anti-fluorescence quencher was used to seal the slides. The uptake of Cur-EGCG-Gel by HSF cells over time and concentration was observed under a confocal fluorescence microscope. The results are shown in Fig.16 and Fig.17 As shown, the fluorescence intensity of Cur-EGCG-Gel gradually increased with the extension of incubation time, showing time dependence. In addition, as the concentration of Cur-EGCG-Gel (25-100 μg / ml) gradually increased, the fluorescence intensity of Cur-EGCG-Gel became stronger, showing concentration dependence. It was confirmed that Cur-EGCG-Gel was successfully taken up by HSF cells and had the characteristics of concentration and time dependence.

[0100] 2. Uptake mechanism of Cur-EGCG@Gel at the level of human skin fibroblasts (HSF): HSF cells were cultured at a rate of 1×10 5 The cells were inoculated at a density of 100 μg / ml on a 12-well plate. After 24 h of culture, the culture medium was discarded, the cells were washed with PBS, and then 500 μl of 100 μg / ml of Cur-EGCG-Gel solution (control group) and 250 μl DMEM / F12 medium containing methyl-β-cyclodextrin (MβCD) (5 mM), chlorpromazine (CPZ) (10 μg / ml) and 5-(ethyl-N-isopropyl) amiloride (EIPA) (10 μg / ml) uptake inhibitors, respectively, were incubated in the dark for 30 min, and then 250 μl Cur-EGCG-Gel solution was added to each group and incubated in the dark for 3 h. The medium was discarded, and the cells were washed three times with PBS. DAPI was added and incubated in the dark for 15 min to stain the cell nucleus. The cells were washed three times with PBS, fixed with 4% paraformaldehyde for 15 min, washed with PBS, and sealed with an anti-fluorescence quencher. The uptake of Cur-EGCG-Gel by HSF cells in different uptake inhibitor groups was observed under a confocal fluorescence microscope. The results are shown in Figure 2 Fig.18shown.

[0101] Flow cytometry was used to investigate the uptake mechanism of Cur-EGCG@Gel at the level of human skin fibroblasts (HSF): HSF cells were cultured at a rate of 1×10 6 The cells were inoculated at a density of 100 μg / ml / well on a six-well plate. After incubation for 24 h, the culture medium was discarded and the cells were washed with PBS. 2 ml of 100 μg / ml Cur-EGCG-Gel solution was added to the control group. 1 ml of DMEM / F12 culture medium containing MβCD (5 mM), CPZ (10 μg / ml) and EIPA (10 μg / ml) was added to the uptake inhibitor group. After incubation in the dark for 30 min, 1 ml of Cur-EGCG-Gel solution was added to a final concentration of 100 μg / ml. The cells were incubated in the dark for 3 h, and then all the cells in the wells were scraped off as much as possible with a cell scraper. The cells were centrifuged at 1500 rpm at room temperature for 5 min, and centrifuged three times. The cell uptake inhibition of different groups was observed by flow cytometry. The results are shown in the figure. Fig.19 shown.

[0102] Fig.18 and Fig.19 The results showed that compared with the control group, the fluorescence intensity of cell uptake was significantly reduced in the CPZ and MβCD groups after treatment, which reduced the internalization of Cur-EGCG-Gel by HSF cells; the uptake of EIPA cells was significantly increased, confirming that the uptake of Cur-EGCG-Gel by HSF cells mainly depended on caveolin- and clathrin-mediated endocytosis.

[0103] Effect Example 4

[0104] The pharmacodynamics of the topical gel preparation against androgenic alopecia in vivo was studied using Cur-EGCG-Gel prepared in Example 1:

[0105] Six-week-old male C57BL / 6 mice weighing 18-22 g were used. Before the experiment, the mice were kept in an SPF environment with a normal rhythm of 12 h light and 12 h dark, a temperature of 25 ± 2 °C, and a relative humidity of 55 ± 5%. After one week of adaptive feeding, animal experiments were conducted. Fig. 20As shown: First, remove the hair on the back of the mouse with a razor, evenly apply the depilatory cream on the depilatory area, and gently wipe off the depilatory cream with a wet cotton ball after 30 seconds. 24 hours after depilation, TS (TS was dissolved in 50% ethanol) was administered at a concentration of 3 mg / ml, and each mouse was administered 200 μl. The modeling lasted for seven days, and then the mice were randomly divided into 6 groups: Control group (no treatment after depilation), TS group (3 mg / ml, 200 μl), MXD group (250 mg / kg), Cur group (1 mg / ml, 100 μl), EGCG group (5 mg / ml, 200 μl), Cur-EGCG-Gel group (200 μl). Except for the Control group, all groups applied testosterone ethanol solution (3 mg / ml, 200 μl) to the depilatory area every day until the end of the experiment. The positive drug minoxidil group (MXD group) and Cur-EGCG-Gel group were topically administered once every other day until the end of the experiment; the Cur group and EGCG group were topically administered once a day until the end of the experiment;

[0106] After the administration, a new hair growth evaluation experiment was performed, which included a new hair growth quality evaluation experiment, a skin tissue HE staining experiment, a Ki67 immunohistochemical staining experiment, a type II 5α-reductase immunohistochemical staining experiment, and an androgen receptor immunohistochemical staining experiment.

[0107] New hair growth quality evaluation experiment: Take photos to record the skin color and hair growth of mice on the 1st, 10th, 24th, 36th and 45th days after the start of drug administration. After the end of the experiment, collect the new hair of the mice, weigh the new hair of each group with an analytical balance, measure the length of the new hair with a vernier caliper, and quantify the coverage of the new hair with ImageJ. In addition, select the new hair of each group on the 45th day to observe the hair structure under a scanning electron microscope. The results are as follows: Fig.21 , Fig. 22 , Fig.23 shown.

[0108] Skin tissue HE staining experiment: At the end of the experiment, the mice were killed, the skin of the hair removal area on the back was removed, the adhesion tissue was removed, and the skin was fixed in 4% paraformaldehyde for 48 hours. The fixed skin tissue was taken out, cut appropriately and placed in an embedding box, rinsed with running water for 24 hours, the paraformaldehyde on the surface was washed off, and then dehydrated in 70% ethanol (overnight), 80% ethanol (2h), 90% ethanol (2h), 95% ethanol I (overnight), 95% ethanol II (2h), and n-butanol (overnight). Then it was embedded, and the wax block was cut into 4μm thick paraffin specimens and baked overnight. Before staining, the paraffin specimens were baked at 62°C for 2h, placed in xylene I (20min) and xylene II (20min) in a fume hood for dewaxing, and then hydrated with gradient alcohol: 100% ethanol I (5min), 100% ethanol II (5min), 95% ethanol I (5min), 95% ethanol II (5min), 90% ethanol (5min), 80% ethanol (5min), 70% ethanol (5min), and then washed with PBS three times, each time for 5min. After drying PBS, appropriate amount of hematoxylin was added to cover the specimens, and after staining for 15s, the specimens were rinsed with running water, and then placed in PBS for 5min to return to blue, and then the slides were placed in a dyeing jar filled with eosin dye for 2min, and then the slides were placed in a dyeing jar filled with water to wash away the eosin dye, and the washing was repeated three times until clean, and then naturally dried in a fume hood, and then soaked in xylene for 20min to make it transparent, and finally sealed with neutral resin. The morphology of hair follicles in different groups was observed by pathological scanner the next day. The results are as follows Fig.24 shown.

[0109] Ki67 immunohistochemical staining experiment: After dewaxing and hydrating the paraffin sections according to the above method, wash them with PBS three times, 5 minutes each time, and perform antigen repair with sodium citrate in a pressure cooker. After boiling for 90 seconds, take out the specimens, cool them naturally to room temperature, and wash them with PBS three times, 5 minutes each time. Shake dry and wipe off the PBS around the tissue, then draw a hydrophobic line around the tissue with a tissue pen, add an appropriate amount of endogenous catalase blocker to each specimen to cover the specimen, place it in a humidified box, and incubate it in a 37℃ oven for 20 minutes, and then wash it with PBS three times, 5 minutes each time. After shaking off the PBS, each specimen is blocked by adding 10% goat serum consistent with the source of the secondary antibody, placed in a humidified box and then incubated in a 37℃ oven for 20 minutes, shake off the serum, and do not wash. Then add the primary antibody diluted in an appropriate proportion and incubate it overnight at 4℃. Note: anti-Ki67 (1:100, Affinity, AF0198) detects hair follicle cell proliferation. The next day, wash with PBS three times, each time for 10 minutes, add an appropriate amount of secondary antibody to the specimen (determine the source of the secondary antibody according to the source of the primary antibody), place it in a wet box and then incubate it in a 37°C oven for 20 minutes, and then wash with PBS three times, each time for 10 minutes. Next, add DAB staining under a microscope and observe its color development time. After the color development is completed, rinse with tap water to remove the DAB stain. Restain with hematoxylin for 6-10 seconds, rinse gently with running water, and then return to blue with PBS for 5 minutes. Finally, dehydrate with gradient alcohol, and place in 70% ethanol (5s), 80% ethanol (5s), 90% ethanol (5s), 95% ethanol I (5min), 95% ethanol II (5min), 100% ethanol I (5min), 100% ethanol II (5min), xylene transparent for 20 minutes, and finally seal with neutral resin. The sealed sections were placed under a pathology scanner to observe the expression levels of Ki67 in different groups. The results are as follows Fig.25 shown.

[0110] Type II 5α-reductase immunohistochemical staining experiment: Follow the above Ki67 immunohistochemical staining experimental steps, and dilute the anti-SRD5A2 with a certain proportion of the immunohistochemical primary antibody diluent. Fig.26 shown.

[0111] Androgen receptor immunohistochemical staining experiment: According to the above Ki67 immunohistochemical staining experimental steps, the primary antibody anti-AR was diluted to a certain ratio with the immunohistochemical primary antibody diluent. Fig. 27 shown.

[0112] The results of the new hair growth evaluation experiment showed that the new hair coverage area of ​​the mice in the Cur-EGCG@Gel group was significantly increased after drug treatment, and the hair weight was also significantly higher than that of the other groups. The length of the new hair was basically the same as that of the control group, but there were differences with other groups. The SEM images showed that compared with the TS group, the new hair in the Cur-EGCG-Gel group showed complete scales and thicker hair shafts. The results of the HE staining experiment showed that the hair bulbs in the Cur-EGCG-Gel group were larger in volume, with a large amount of melanin precipitation, and the same hair growth pattern. The results of the two groups were consistent; in the Ki67 immunohistochemical staining experiment, the positive expression of the Cur-EGCG-Gel group was more obvious; in the type II 5α-reductase immunohistochemical staining experiment, the Cur-EGCG-Gel group played the role of EGCG in inhibiting 5α-reductase; in the androgen receptor immunohistochemical staining experiment, weak positive expression appeared around the hair follicles of the TS group (the red arrow points to positive expression), while no obvious positive expression was found in the other three groups, suggesting that Cur, as an AR inhibitor, played an antagonistic role in AR in Cur-EGCG-Gel. These results show that the prepared transdermal gel preparation Cur-EGCG-Gel has the effect of improving the growth of new hair in AGA mice.

[0113] Effect Example 5

[0114] The in vivo safety evaluation experiment of Cur-EGCG-Gel prepared in Example 1 includes a statistical experiment on the weight change of mice after the administration of Cur-EGCG@Gel, a blood sample detection experiment and an organ HE staining experiment.

[0115] The weight changes of mice in each group during the experiment were statistically analyzed. Fig.28 As shown in the data, the weight of mice in the TS / MXD / Cur / group continued to increase and they were in good health. The weight of mice in the Cur-EGCG-Gel / EGCG / Control group fluctuated during the administration period, but then gradually returned to normal. At the end of the experiment, there was no significant difference in the weight of mice in each group, indicating that the dosage was relatively safe during the experiment and did not cause a significant impact on the health of the mice.

[0116] After the experiment, blood samples were collected from mice in each group for relevant tests. The blood sample analysis indicators included the concentration of red blood cells (RBC), white blood cells (WBC) and platelets (PLT). The results are as follows: Fig.29As shown, the relevant indicators of all drug-dosing groups were within a reasonable range and did not show significant differences. Further biochemical analysis was performed on the blood of mice, and the evaluation indicators included blood urea nitrogen (BUN) and creatinine (CREA) to evaluate renal function; aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin test (TBIL), and albumin (ALB) to evaluate liver function. The results showed that no abnormalities were found in the relevant parameters of all drug-dosing groups, indicating that each drug-dosing group had good biosafety and did not cause functional damage to the liver and kidneys of mice.

[0117] After the experiment, the main organs (heart, liver, spleen, lung, and kidney) of each group of mice were sliced ​​and stained with HE. Fig.30 As shown in the figure, the myocardial cells in each group were intact without obvious aggregation, the liver cells had normal morphology without inflammation and fibrosis, the lung cells had no obvious changes in morphology, and no pathological tissue was found in the kidneys, which confirmed the good biocompatibility of Gel and the low toxicity of the drug monomer.

[0118] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

[0119] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the described embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a composite hydrogel, characterized in that: The following steps are involved: S1, completely dissolving gelatin and EGCG in an organic solvent to obtain a hydrogel precursor solution; S2. Slowly drop the curcumin solution into the hydrogel precursor solution obtained in step S1, stir, and allow to stand to form a gel, thereby obtaining a composite hydrogel loaded with curcumin and EGCG.

2. The method for preparing the composite hydrogel according to claim 1, characterized in that: The organic solvent described in step S1 is ethanol or 50% ethanol.

3. The method for preparing the composite hydrogel according to claim 1, characterized in that: The ratio of gelatin, EGCG and organic solvent in step S1 is gelatin: EGCG: ethanol = 80-120 mg: 3-7 mg: 1 ml.

4. The method for preparing the composite hydrogel according to claim 1, characterized in that: The ratio of gelatin, EGCG and organic solvent in step S1 is gelatin: EGCG: organic solvent = 100 mg: 5 mg: 1 ml.

5. The method for preparing the composite hydrogel according to claim 1, characterized in that: The curcumin solution is a 0.5-2 mg / mL curcumin ethanol solution; The volume ratio of the curcumin solution to the hydrogel precursor solution is 1:8-12.

6. The method for preparing the composite hydrogel according to claim 1, characterized in that: The curcumin solution is a 1 mg / mL curcumin ethanol solution; The volume ratio of the curcumin solution to the hydrogel precursor solution is 1:

10.

7. The method for preparing the composite hydrogel according to any one of claims 1 to 6, characterized in that: The complete dissolution described in step S1 is achieved by heating at a temperature of 65±5°C for 24±5h; the standing gelation described in step S2 is achieved at a low temperature of 4±2°C; The dropping in step S2 is dropping while stirring; the stirring is magnetic stirring, and the rotation speed is 1000±200rpm / min; The stirring time in step S2 is 2±1 h.

8. A composite hydrogel, characterized in that: The method is obtained by the preparation method described in any one of claims 1 to 7.

9. Use of the composite hydrogel according to claim 8 in preparing medicines for treating skin diseases.

10. The use according to claim 9, characterized in that: The skin disease is at least one of androgenic alopecia, infection, burns, mechanical trauma, and diabetic dermatosis.

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