Morusin nanoparticles, pH-responsive hydrogel and application thereof

Abstract

The present application relates to a kind of mulberry pigment nanoparticles, pH-responsive hydrogel and application.The mulberry pigment nanoparticles provided in the present application are quickly degraded in alkaline environment, have certain stability in acidic environment, have the ability to release mulberry pigment in response to pH, can realize the precise control of mulberry pigment release according to the change of environmental pH.The present application further methyl acrylate treatment is carried out to hyaluronic acid, and MeHA is obtained.MeHA is more easily degraded in acidic environment, as hydrogel load C F M N P, can be effectively used to treat the wound caused by burn and frostbite.Further, based on pigment nanoparticle-basic fibroblast growth factor pH-responsive double-layer hydrogel, can realize the sequential release of effective components according to the pH change of burn or frostbite wound.Effective inhibition of inflammation, resistance to oxidative stress, promote the proliferation and migration of endothelial cells, promote the formation of new blood vessels, and ultimately accelerate wound healing.

Description

TECHNICAL FIELD

[0001] The present application relates to a mulberry pigment nanoparticle, a pH-responsive hydrogel and applications. BACKGROUND

[0002] Skin, as the largest organ of the human body, has crucial physical, chemical and microbial barrier functions. It has unique biological functions and wholeheartedly protects the host from external threats. However, the skin is extremely fragile. When the skin suffers physical damage such as burns, frostbite (B / F), its normal structure and function will be severely damaged. In the case of burns, high temperatures can cause protein denaturation and cell necrosis in skin tissue, thereby damaging the barrier function of the skin and increasing the risk of infection. In severe cases, it can cause sepsis, which is life-threatening. Frostbite is caused by low temperature, which causes blood vessels in the skin to constrict and blood flow to slow down, causing tissue ischemia and hypoxia. In addition, severe frostbite can cause skin tissue necrosis, causing local skin to appear red, blistering, ulceration and other symptoms, and also increasing the risk of infectious diseases. Burns and frostbite not only directly damage the skin, but also significantly hinder wound healing by causing inflammation, oxidative stress and vascularization difficulties. After burns and frostbite occur, the damaged tissue will initiate an inflammatory response. Persistent inflammation can cause tissue edema and pain, and immune cells will release a large amount of reactive oxygen species (ROS), which will exacerbate inflammation and hinder wound healing. At the same time, burns and frostbite can damage the vascular endothelium, causing vasospasm and thrombosis. As a result, the damaged tissue lacks nutrients, has decreased anti-infection ability, and has difficulty in expelling metabolic products, causing vascularization difficulties, delaying wound healing, and causing complications. Currently, the main treatment for burns and frostbite is comprehensive treatment, including early debridement, use of external drugs containing antibiotics and growth factors, and skin grafting surgery. However, there are still certain limitations in the overall effectiveness of existing treatment options. Therefore, there is an urgent need for effective strategies to regulate the inflammatory response, reduce oxidative stress, promote early regeneration of endothelial cells and vascular reconstruction to address the clinical problem of poor wound healing after burns and frostbite.

[0003] Mulberry pigment has excellent anti-inflammatory and antioxidant properties and shows broad application prospects in the medical field. Studies have shown that mulberry pigment has significant anti-inflammatory and antioxidant effects in various disease models such as ulcerative colitis, neurodegenerative diseases and diabetes. For example, in cardiovascular diseases, mulberry pigment can reduce oxidative stress and inflammatory response in vascular endothelial cells and improve vascular endothelial function. However, the hydrophobicity and low bioavailability of mulberry pigment limit its application. Specifically, there is currently no literature reporting that mulberry pigment can be applied to the skin surface to treat physical damage such as burns and frostbite (B / F) using its anti-inflammatory and antioxidant effects. SUMMARY

[0004] The present application aims to overcome the shortcomings and deficiencies of the prior art, and provides a mulberry pigment nanoparticle, a pH-responsive hydrogel and application

[0005] In a first aspect of the present application, a mulberry pigment nanoparticle is provided, which is formed by self-assembly of 4-hydroxyphenylboronic pinacol ester modified fucoidan, chitosan and mulberry pigment.

[0006] In a second aspect of the present application, a preparation method of the mulberry pigment nanoparticle is provided, comprising the following steps:

[0007] (1) preparing hydroxyl-modified fucoidan;

[0008] (2) reacting the hydroxyl-modified fucoidan with 4-hydroxyphenylboronic pinacol ester to obtain 4-hydroxyphenylboronic pinacol ester-modified fucoidan;

[0009] (3) self-assembling the 4-hydroxyphenylboronic pinacol ester-modified fucoidan, chitosan and mulberry pigment to obtain the mulberry pigment nanoparticle.

[0010] Preferably, in step (1), the fucoidan is mixed and reacted with a strong base and a hydroxymethylating agent, after the reaction is completed, the reaction mixture is neutralized by adding an acid, and then dialysis and freeze-drying are performed to obtain the freeze-dried product of the hydroxyl-modified fucoidan.

[0011] Preferably, in step (2), the hydroxyl-modified fucoidan is mixed and reacted with a carbodiimide activator under a nucleophilic catalyst to activate the carboxyl group, then 4-hydroxyphenylboronic pinacol ester is added, after the reaction is completed, dialysis and freeze-drying are performed to obtain the freeze-dried product of the 4-hydroxyphenylboronic pinacol ester-modified fucoidan.

[0012] Preferably, in step (3), the chitosan is dissolved in an acetic acid aqueous solution to obtain a chitosan solution, the 4-hydroxyphenylboronic pinacol ester-modified fucoidan is dissolved in water to obtain a 4-hydroxyphenylboronic pinacol ester-modified fucoidan aqueous solution, the mulberry pigment is dissolved in dimethyl sulfoxide to obtain a mulberry pigment solution, the chitosan solution, the fucoidan aqueous solution and the mulberry pigment solution are mixed to obtain a mixed solution, the mixed solution is treated under ultrasonic waves, then stirring reaction is performed, after the reaction is completed, purification is performed to obtain the mulberry pigment nanoparticle.

[0013] In a third aspect of the present application, a pH-responsive hydrogel loaded with the mulberry pigment nanoparticle is provided.

[0014] Preferably, the pH-responsive hydrogel loaded with the mulberry pigment nanoparticle is a methacrylated hyaluronic acid.

[0015] In a fourth aspect of the present application, a pH-responsive double-layer hydrogel is provided, comprising:

[0016] An upper layer hydrogel comprising methacrylated hyaluronic acid and basic fibroblast growth factor;

[0017] A lower layer hydrogel comprising methacrylated hyaluronic acid and mulberry pigment nanoparticles as described above;

[0018] The concentration of methacrylated hyaluronic acid in the upper layer hydrogel is greater than the concentration of methacrylated hyaluronic acid in the lower layer hydrogel.

[0019] Preferably, the concentration of methacrylated hyaluronic acid in the upper layer hydrogel is 2% mass / volume, and the concentration of methacrylated hyaluronic acid in the lower layer hydrogel is 0.8% mass / volume.

[0020] In a fifth aspect of the present application, the use of the pH-responsive double-layer hydrogel as described above for the preparation of a product for treating a wound surface caused by burns or frostbite is provided.

[0021] The beneficial effects of the present application are as follows:

[0022] (1) The present application uses 4-hydroxyphenylboronic acid pinacol ester (PAPE) to modify fucoidan (Fu), forming a pH-responsive material. Subsequently, through self-assembly, it is combined with mulberry pigment and chitosan (Cs) to form mulberry pigment-loaded nanoparticles (CFMNPs). The nanoparticles degrade rapidly in an alkaline environment and have a certain stability in an acidic environment, with the ability to release mulberry pigment in response to pH, and can achieve precise regulation of mulberry pigment release according to changes in environmental pH. This feature makes it a promising drug delivery carrier, especially suitable for treatment scenarios that require pH-triggered release.

[0023] (2) The present application further acrylates hyaluronic acid (HA) to obtain methacrylated hyaluronic acid (MeHA). Using the characteristic that MeHA is more easily degraded in an acidic environment, MeHA is used as a hydrogel to load CFMNPs. In the early acidic stage of burn and frostbite wounds, MeHA rapidly degrades and releases CFMNPs. As the healing process of the wound progresses, the wound gradually becomes alkaline, and CFMNPs further release mulberry pigment with anti-inflammatory and antioxidant effects, achieving the effect of promoting the healing of wounds caused by burns and frostbite.

[0024] (3) The pH-responsive double-layer hydrogel based on mulberry pigment nanoparticles-basic fibroblast growth factor (bFGF) provided by the application loads CFMNPs in the lower layer hydrogel with low concentration of MeHA, and loads bFGF in the upper layer hydrogel with high concentration of MeHA, and the two layers of hydrogel jointly constitute a double-layer hydrogel (bFGF-CFMN), and the design purpose is to adapt to the dynamic change of the pH value of the wound surface and realize the sequential release of the treatment effective components. In short, in terms of burns and frostbite, local tissue cells are damaged, leading to ischemia and hypoxia. The metabolic process in cells changes from aerobic metabolism to anaerobic metabolism, producing a large amount of lactic acid and other acidic metabolites, thereby reducing the pH value of the wound (pH 5.5-6.5). With the body starting the repair reaction to the damage, inflammatory cells infiltrate, and some alkaline substances are released by cells such as neutrophils, leading to an increase in the pH value of the wound surface (pH 7.4-8.0). When the wound enters the remodeling healing stage, the pH value of the wound will further approach the pH value of normal skin (pH 6.5-7.0). The double-layer hydrogel (bFGF-CFMN) of the application releases CFMNPs in the lower layer with low concentration of MeHA in the early acid stage of the burn and frostbite wound surface. With the advancement of the wound healing process, the wound gradually becomes alkaline, and CFMNPs further release mulberry pigment with anti-inflammatory and antioxidant effects. At the same time, the upper layer with high concentration of MeHA slowly releases bFGF in the alkaline stage of the wound surface in the later stage, continuously promoting wound healing.

[0025] The application proves that the bFGF-CFMN double-layer hydrogel can realize the sequential release of effective components according to the pH value change of the burn or frostbite wound surface through in vitro and in vivo experiments. It can effectively inhibit inflammation, resist oxidative stress, promote the proliferation and migration of endothelial cells, promote the formation of new blood vessels, and ultimately accelerate wound healing. BRIEF DESCRIPTION OF DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiment or prior art description. Obviously, the drawings in the following description only some embodiments of the application, and for those skilled in the art, according to these drawings, other drawings can be obtained without creative labor, which still belong to the scope of the application.

[0027] Figure 1Schematic diagram of bFGF-CFMN double-layer hydrogel for treating B / F wounds, (A) Schematic diagram of the synthesis process of bFGF-CFMN double-layer hydrogel; (B) Mechanism of bFGF-CFMN double-layer hydrogel for treating B / F wounds;

[0028] Figure 2 For the characterization of CFMNPs: (A) Photograph of CFMNPs powder; (B) Photograph of CFMNPs solution (left) and morin solution (right); (C) Transmission electron microscopy (TEM) image of CFMNPs, the scale bar is 100 nanometers; (D) Particle size distribution of CFMNPs; (E) Fourier transform infrared spectroscopy (FTIR) of CFMNPs; (F) Particle size change of CFMNPs in different pH environments in vitro; (G) Particle size of CFMNPs in different pH environments in vitro at 24 hours; (H) Release curve of CFMNPs in different pH environments in vitro;

[0029] Figure 3 For the characterization of bFGF-CFMN double-layer hydrogel: (A) Appearance of hydrogel, I represents the upper layer of bFGF hydrogel, II represents the lower layer of CFMN hydrogel, and III represents the bFGF-CFMN double-layer hydrogel; (B) Transmission electron microscopy (TEM) image of bFGF-CFMN double-layer hydrogel, I represents the lower layer of CFMN hydrogel, Ia is the enlarged view of the local area, and II represents the upper layer of bFGF hydrogel, the red triangle points to CFMNPs, the scale bar in the main figure is 200 μm, and the scale bar in the insert is 20 μm; (C) Fourier transform infrared spectroscopy (FTIR) of hydrogel; (D) Storage modulus (G') and loss modulus (G'') of hydrogel; (E) Shear viscosity of hydrogel; (F) In vitro degradation curve of hydrogel in different pH environments (n=3); (G) In vitro release curve of hydrogel in different pH environments (n=3);

[0030] Figure 4 For the in vitro cell experiment of bFGF-CFMN double-layer hydrogel: Live / dead staining results of human umbilical vein endothelial cells (HUVECs) after hydrogel treatment for 24 hours (A) and 72 hours (C); Survival rate of human umbilical vein endothelial cells after hydrogel treatment for 24 hours (B) and 72 hours (D); Scratch experiment of human umbilical vein endothelial cells treated by hydrogel, the scale bar is 100 μm; Scratch healing rate of human umbilical vein endothelial cells treated by hydrogel (n=3); Tube lumen formation experiment of human umbilical vein endothelial cells treated by hydrogel, the scale bar is 50 μm; Quantitative analysis of tube lumen formation in tube lumen formation experiment (n=3);

[0031] Figure 5In vitro anti-inflammatory, antioxidant and proliferation-promoting effects of bFGF-CFMN double-layer hydrogel: (A) RAW 264.7 cells were loaded with fluorescent probes and then treated with Rosup, followed by treatment with the hydrogel, and then the fluorescence was observed under a fluorescence microscope. The scale bar is 100 μm. (B) Quantitative analysis of the fluorescence intensity of RAW 264.7 cells (n = 3). After lipopolysaccharide (LPS) stimulation and hydrogel treatment, the levels of (C) tumor necrosis factor-α (TNF-α), (D) interleukin-1β (IL-1β), and (E) interleukin-6 (IL-6) in RAW 264.7 cells were detected by enzyme-linked immunosorbent assay (ELISA) (n = 3). (F) EdU staining results of human umbilical vein endothelial cells (HUVECs) after treatment with the hydrogel. The scale bar is 100 μm. (G) Quantitative analysis of the EdU staining results (n = 3);

[0032] Figure 6 In vivo degradation and release of bFGF-CFMN double-layer hydrogel: (A) Changes in the pH value of the burn and freeze wound surface over time (n = 5). (B) Changes in the lactic acid concentration of the burn and freeze wound surface over time (n = 3). (C) In vivo imaging was performed on days 1, 3, 5, and 14 after administration to monitor the degradation of rhodamine B-labeled CFMN hydrogel and FITC-labeled bFGF on the wound surface. (D) Quantitative analysis of in vivo fluorescence imaging of the burn wound (n = 3). (E) Quantitative analysis of in vivo fluorescence imaging of the freeze wound (n = 3). (F) In vivo drug release curve of bFGF-CFMN double-layer hydrogel on the burn wound (n = 3). (G) In vivo drug release curve of bFGF-CFMN double-layer hydrogel on the freeze wound (n = 3).

[0033] Figure 7 Timeline of animal experiments using bFGF-CFMN double-layer hydrogel to treat burn and freeze (B / F) wounds;

[0034] Figure 8Therapeutic effects of bFGF-CFMN double-layer hydrogel on burn wound: (A) Representative photographs of wound healing at 1, 3, 5, 10 and 14 days after the corresponding treatment in each group; (B) HE staining of the burn wound tissue at 5 and 14 days after different treatments to observe the changes in the tissue. The scale bar is 100 pm; (C) Masson staining of the burn wound tissue at 5 and 14 days after different treatments to observe the changes in the tissue. The scale bar is 100 pm; (D) Wound healing rate at 5 and 14 days in each treatment group (n = 3); (E) Quantitative analysis of inflammatory infiltration in the burn wound tissue in each group (n = 3); (F) Quantitative analysis of collagen deposition in the burn wound tissue in each group (n = 3); Levels of inflammatory cytokines in the burn wound tissue at different time points after different treatments: tumor necrosis factor-a (TNF-a) (G), interleukin-1b (IL-1b) (H) and interleukin-6 (IL-6) (I) (n = 3);

[0035] Figure 9 Therapeutic effects of bFGF-CFMN double-layer hydrogel on frostbite wound: (A) Representative photographs of wound healing at 1, 3, 5, 10 and 14 days after the corresponding treatment in each group; (B) HE staining of the frostbite wound tissue at 5 and 14 days after different treatments to observe the changes in the tissue. The scale bar is 100 pm; (C) Masson staining of the frostbite wound tissue at 5 and 14 days after different treatments to observe the changes in the tissue. The scale bar is 100 pm; (D) Wound healing rate at 5 and 14 days in each treatment group (n = 3); (E) Quantitative analysis of inflammatory infiltration in the frostbite wound tissue in each group (n = 3); (F) Quantitative analysis of collagen deposition in the frostbite wound tissue in each group (n = 3); Levels of inflammatory cytokines in the frostbite wound tissue at different time points after different treatments: tumor necrosis factor-a (TNF-a) (G), interleukin-1b (IL-1b) (H) and interleukin-6 (IL-6) (I) (n = 3);

[0036] Figure 10Antioxidant effect of bFGF-CFMN double-layer hydrogel on burn wound: (A) DHE fluorescence staining of burn wound on day 7 and day 14 after hydrogel treatment, scale bar 100 μm; (B) quantitative analysis of DHE fluorescence intensity in each group (n = 3); (C) DHE fluorescence staining of frostbite wound on day 7 and day 14 after hydrogel treatment, scale bar 100 μm; (D) quantitative analysis of DHE fluorescence intensity in each group (n = 3); Detection of antioxidant indexes in burn wound tissue on day 5 and day 14 after corresponding treatment: superoxide dismutase (SOD) (E), glutathione (GSH) (F) and malondialdehyde (MDA) (G) (n = 3); Detection of antioxidant indexes in frostbite wound tissue on day 5 and day 14 after corresponding treatment: superoxide dismutase (SOD) (H), glutathione (GSH) (I) and malondialdehyde (MDA) (J) (n = 3);

[0037] Figure 11 Pro-angiogenic effect of bFGF-CFMN double-layer hydrogel on burn wound: (A) CD31 immunofluorescence staining of burn wound tissue on day 5 and day 14 after corresponding treatment, scale bar 50 μm; (B) vascular endothelial growth factor (VEGF) immunohistochemical staining of burn wound tissue on day 5 and day 14 after corresponding treatment, scale bar 50 μm; (C) Quantitative analysis of CD31 fluorescence intensity in each group (n = 3); (D) Quantitative analysis of VEGF positive expression level in each group (n = 3); Detection of platelet-derived growth factor (PDGF) (E) and Ki67 (F) content in burn wound tissue by enzyme-linked immunosorbent assay (ELISA) on day 5 and day 14 after corresponding treatment (n = 3);

[0038] Figure 12 Pro-angiogenic effect of bFGF-CFMN double-layer hydrogel on frostbite wound: (A) CD31 immunofluorescence staining of frostbite wound tissue on day 5 and day 14 after corresponding treatment, scale bar 50 μm; (B) vascular endothelial growth factor (VEGF) immunohistochemical staining of frostbite wound tissue on day 5 and day 14 after corresponding treatment, scale bar 50 μm; (C) Quantitative analysis of CD31 fluorescence intensity in each group (n = 3); (D) Quantitative analysis of VEGF positive expression level in each group (n = 3); Detection of platelet-derived growth factor (PDGF) (E) and Ki67 (F) content in frostbite wound tissue by enzyme-linked immunosorbent assay (ELISA) on day 5 and day 14 after corresponding treatment (n = 3). DETAILED DESCRIPTION

[0039] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application will be further described in detail below with the accompanying drawings.

[0040] The raw materials used in the following examples were as follows:

[0041] Hyaluronic acid (HA, molecular weight: 150-250 kDa), N,N-dimethylformamide (DMF), 4-hydroxyphenylboronic acid pinacol ester (PAPE), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 4-dimethylaminopyridine (DMAP), and anhydrous formamide were purchased from Macklin (Shanghai, China). Fucoidan (Fu), chloroacetic acid, dihydroethidium (DHE), and sodium hydroxide were purchased from Aladdin (Shanghai, China). Morin, chitosan, methacrylic acid, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), fluorescein isothiocyanate (FITC), and rhodamine B were purchased from Sigma-Aldrich (St. Louis, MO, USA). Basic fibroblast growth factor (bFGF), dimethyl sulfoxide (DMSO), a live / dead cell activity assay kit, a reactive oxygen species assay kit, an EdU cell proliferation kit, 4',6-diamidino-2-phenylindole (DAPI) staining solution, a cell counting kit-8 (CCK-8), and a lactate assay kit were purchased from Biyun Tian (Shanghai, China). Lipopolysaccharide (LPS) and phosphate-buffered saline (PBS) were purchased from Solabio (Beijing, China). Mouse tumor necrosis factor-alpha enzyme-linked immunosorbent assay (ELISA) kit, mouse interleukin-1 beta ELISA kit, mouse interleukin-6 ELISA kit, mouse interleukin-10 ELISA kit, mouse vascular endothelial growth factor ELISA kit, mouse platelet-derived growth factor ELISA kit, and mouse transforming growth factor-beta ELISA kit were purchased from Hangzhou Link Biological Technology Co., Ltd. (Hangzhou, China). Mouse Ki67 ELISA kit was purchased from Jianglai Biological Technology Co., Ltd. (Shanghai, China). CD31 rabbit antibody (ab9498) and VEGF rabbit antibody (ab32152) were purchased from Abeam (Cambridge, UK). Superoxide dismutase (SOD) assay kit (WST-1 method), malondialdehyde (MDA) assay kit (TBA method), and reduced glutathione (GSH) assay kit were purchased from Nanjing Jiancheng Biological Engineering Institute (Nanjing, China). Human umbilical vein endothelial cells (HUVECs) and RAW 264.7 macrophages were purchased from Pnonyase Biotechnology Co., Ltd. (Wuhan, China).

[0042] Example 1. Preparation of morin-loaded nanoparticles (CFMNPs):

[0043] The present embodiment uses a self-assembly method to prepare C-FMNPs. Specifically, Fu is modified by PAPE to have pH-responsive ability. Subsequently, Cs, Fu-PAPE and morin are self-assembled by stirring and ultrasonic treatment to form nanoparticles, i.e. C-FMNPs (A). Figure 2 Due to intermolecular hydrogen bonding and π-π stacking, the solubility of morin in water is poor, often appearing as a turbid suspension and easily precipitating, which greatly limits its application in the biomedical field. However, when morin participates in the self-assembly process, Cs and Fu-PAPE bind to morin through hydrogen bonding and electrostatic interaction, breaking its aggregation force, effectively improving its solubility in water. It can be observed that the C-FMNPs solution is more uniform and transparent than the morin solution (B). The specific preparation steps are as follows: Figure 2

[0044] (1) First, 1.7 grams of fucoidan (Fu) was dissolved in 20 milliliters of ultrapure water. Then, 3.2 grams of sodium hydroxide (NaOH) and 3.7 grams of chloroacetic acid were added in sequence. After stirring at room temperature for 12 hours, the reaction mixture was neutralized with a 2 molar per liter hydrochloric acid (HCl) solution. Then, the mixture was dialyzed using a dialysis bag (molecular weight cut-off value 1000) for 3 days, with the external dialysis water being replaced 6 times a day. Finally, the dialysis product was collected and freeze-dried for 3 days to obtain the freeze-dried product Fu-COOH.

[0045] (2) 100 milligrams of Fu-COOH was added to a 50 milliliter round-bottom flask, followed by 20 milliliters of anhydrous formamide. Stirring for about 30 minutes ensured that Fu-COOH was completely dissolved. Then, 30 milligrams of DMAP was added to the above solution and stirred until completely dissolved. Subsequently, 50 milligrams of EDC was added and stirred for 15 minutes to ensure that it was fully dissolved and activated the carboxyl group. Finally, 50 milligrams of PAPE was added to the above mixture. The reaction flask was sealed and stirred at room temperature in the dark for 24 hours. After the reaction was completed, the reaction mixture was transferred to a pre-prepared dialysis bag (molecular weight cut-off value 1000) and dialyzed for 3 days, with the external dialysis water being replaced 8 times a day. After dialysis was completed, the final mixture was collected and freeze-dried to obtain Fu-PAPE.

[0046] ​(3) An equal volume of 5 mg / mL chitosan (Cs) solution (prepared by dissolving chitosan in 2% acetic acid aqueous solution) was mixed with 5 mg / mL Fu-PAPE aqueous solution. Then, 5 mg / mL morin solution (prepared by dissolving morin in dimethyl sulfoxide) was added. The mixture was sonicated for 1 hour to promote the self-assembly of morin with chitosan and Fu-PAPE, followed by stirring for another 1 hour. Next, the mixture was dialyzed for 6 hours using a dialysis bag (molecular weight cutoff 3000). After dialysis, the solution was collected and filtered through a 0.45 μm filter membrane. After filtration, the filtrate was centrifuged at 15000 rpm for 15 minutes, and the CFMNPs precipitate was collected. Finally, the precipitate was freeze-dried to obtain CFMNPs.

[0047] Subsequently, the prepared CFMNPs were further characterized to comprehensively explore their structural features and potential application properties. Transmission electron microscopy (TEM) was used to observe the microstructure of the CFMNPs, revealing that they possess a relatively uniform spherical structure. Figure 2 C). The particle size analysis results showed that the diameter of CFMNPs was approximately 154.63 ± 24.20 nm, and the polydispersity index (PDI) was 0.25 ± 0.09, indicating that their particle size distribution was uniform. Figure 2 D). The successful preparation of CFMNPs was verified by infrared spectroscopy analysis. Figure 2 (E) Fourier transform infrared spectroscopy (FTIR) showed that Fu exhibited a hydroxyl (OH) stretching vibration peak at approximately 3400 cm⁻¹. After introducing a carboxyl group to form Fu-COOH, while retaining the hydroxyl stretching vibration peak at the same position, a new carbon-oxygen double bond (C=O) stretching vibration peak appeared near 1700 cm⁻¹, indicating that a carboxyl group had been successfully introduced into the fucoidan molecule. Cs showed stretching vibration peaks of amino (NH₂) and hydroxyl (OH) groups at the same wavenumber. Morusin exhibited a carbon-carbon double bond (C=C) stretching vibration peak at 1450-1600 cm⁻¹ and significant absorption at 1000-1300 cm⁻¹. The spectrum of the CFMN composite material showed characteristic peaks of the three components, proving that the three components successfully combined and formed stable nanoparticles through interaction. In addition, high performance liquid chromatography (HPLC) determined that the encapsulation efficiency and drug loading rate of CFMNs for morulain were approximately 87.23% and 8.31%, respectively.

[0048] To further verify the pH-responsive ability of the Fu-COOH-grafted CFMNPs, degradation experiments of the CFMNPs under different pH conditions were first carried out. The experimental results showed that in the alkaline environment (pH 8), the diameter of the CFMNPs gradually decreased with time, reaching 14.77 ± 5.47 nm at 24 hours; in the neutral environment (pH 7), the diameter of the CFMNPs decreased to a relatively small extent; in the acidic environment (pH 6), the diameter of the CFMNPs hardly changed, still being 142.79 ± 20.90 nm at 24 hours Figure 2 F and Figure 2 G). This showed that the CFMNPs were significantly degraded in the alkaline environment, slightly degraded in the neutral environment (pH 7.0), and hardly degraded in the acidic environment. At the same time, it can be observed that the supernatant of the CFMNPs gradually changed from colorless to yellow in the alkaline environment with time, and the spherical morphology of the CFMNPs was significantly destroyed, while the supernatant of the CFMNPs had no obvious color change in the neutral and acidic environments, and the morphology of the CFMNPs remained relatively intact in the acidic environment. This fully explained that the CFMNPs could be rapidly degraded in the alkaline environment and had certain stability in the acidic environment. The pH responsiveness of the CFMNPs might be due to the fact that the pinacol borate bond in the PAPE was easily attacked by hydroxyl ions in the alkaline environment, which destroyed the structural stability of the nanoparticles; the amino group of Cs was proton dissociated under alkaline conditions, which reduced the solubility and changed the intermolecular interaction between the molecular chains, further affecting the stability of the nanoparticles; in addition, the intermolecular force between Fu and Cs was destroyed in the alkaline environment.

[0049] In addition, the ability of CFMNPs to release morin in response to pH was also verified. Quantitative analysis of the amount of morin released from CFMNPs at different time points by high-performance liquid chromatography (HPLC) showed that the cumulative release of morin exceeded 80% only after 12 hours. This rapid initial release stage was attributed to the rapid destruction of the nanoparticle structure in the alkaline environment, allowing morin to quickly pass through the loose nanoparticle shell into the surrounding environment. By 24 hours, the cumulative release of morin was as high as about 95%, meaning that most of the morin had been released from the CFMNPs. In sharp contrast, in a neutral environment, the rate of release of morin from CFMNPs was significantly slower, with a cumulative release of morin of only about 20% after 12 hours and about 24% after 24 hours. This phenomenon can be due to the relatively slow rate of nanoparticle degradation in a neutral environment, with limited structural damage, thereby hindering the diffusion of morin from the interior of the nanoparticles to the surrounding environment. In an acidic environment, CFMNPs released very little morin, with a release rate of only about 11% after 24 hours, fully demonstrating the stability of CFMNPs in an acidic environment. This can be because the structure of CFMNPs is almost not destroyed under acidic conditions, and morin is tightly wrapped inside and difficult to release Figure 2 H}.

[0050] In summary, CFMNPs have excellent pH-responsive morin release ability and can precisely control the release of morin according to changes in the environmental pH. This property makes them promising as drug delivery carriers, especially for therapeutic scenarios that require pH-triggered release.

[0051] Example 2 Preparation of double-layer hydrogel containing CFMNPs:

[0052] (1) Dissolve 1 gram of hyaluronic acid (HA) in a mixed solvent of 30 milliliters of N,N-dimethylformamide (DMF) and 60 milliliters of pure water, and stir until the HA is completely dissolved. Then, slowly add 3 milliliters of methacrylic acid under ice bath conditions, and continue to react for 24 hours. After the reaction is complete, adjust the pH of the solution to 8-9 with sodium hydroxide (NaOH). Next, add 3 times the volume of pre-cooled anhydrous ethanol to precipitate the product. After precipitation, discard the supernatant, centrifuge the precipitate at 5000 rpm for 10 minutes, discard the supernatant again, add an appropriate amount of pure water to dissolve the precipitate, and dialyze at 4°C for 3 days, changing the water 3 times a day. After dialysis, collect the aqueous solution and freeze-dry it to obtain a solid of methacrylated hyaluronic acid (MeHA).

[0053] (2) The methylacrylate hyaluronic acid (MeHA) was dissolved in pure water, and after complete dissolution, LAP photoinitiator (0.25%, w / v) was added to obtain MeHA solutions with concentrations of 0.8% (w / v) and 2% (w / v). The preparation of the double-layer hydrogel was as follows: first, 2% MeHA solution was added to the mold, and then basic fibroblast growth factor (bFGF, 100 μg per milliliter of hydrogel system) was added, and the solution was irradiated with a UV light source for 30 seconds; then, 0.8% MeHA solution and CFMNPs (10 mg per milliliter of hydrogel system) were added, and the solution was irradiated with a UV light source for 45 seconds to obtain bFGF-CFMN double-layer hydrogel.

[0054] Example 3 Preparation of bFGF-CFMN double-layer hydrogel

[0055] (1) One gram of hyaluronic acid (HA) was dissolved in 30 milliliters of N,N- dimethylformamide (DMF) and 60 milliliters of pure water mixed solvent, and stirred until the HA was completely dissolved. Then, 3 milliliters of methacrylic acid was slowly added dropwise under ice bath conditions, and the reaction was continued for 24 hours. After the reaction was completed, the pH value of the solution was adjusted to 8-9 with sodium hydroxide (NaOH). Next, 3 times the volume of pre-cooled anhydrous ethanol was added to precipitate the product. After precipitation, the supernatant was discarded, the precipitate was centrifuged at 5000 rpm for 10 minutes, the supernatant was again discarded, and the precipitate was dissolved with an appropriate amount of pure water and dialyzed at 4°C for 3 days, with water changed 3 times a day. After dialysis, the aqueous solution was collected and freeze-dried, and finally methylacrylate hyaluronic acid (MeHA) solid was obtained.

[0056] (2) The methylacrylate hyaluronic acid (MeHA) was dissolved in pure water, and after complete dissolution, LAP photoinitiator (0.25%, w / v) was added to obtain MeHA solutions with concentrations of 0.8% (w / v) and 2% (w / v). The preparation of the double-layer hydrogel was as follows: first, 2% MeHA solution was added to the mold, and then basic fibroblast growth factor (bFGF, 100 μg per milliliter of hydrogel system) was added, and the solution was irradiated with a UV light source for 30 seconds; then, 0.8% MeHA solution and CFMNPs (10 mg per milliliter of hydrogel system) were added, and the solution was irradiated with a UV light source for 45 seconds to obtain bFGF-CFMN double-layer hydrogel.

[0057] In this example, methylacrylate hyaluronic acid (MeHA) was used as the main material to construct a unique double-layer structure by taking advantage of the concentration-dependent properties of MeHA and its sensitivity to acidic environments. Specifically, the upper layer of the hydrogel was composed of 2% MeHA, which was loaded with basic fibroblast growth factor (bFGF); the lower layer of the hydrogel was composed of 0.8% MeHA, which was loaded with CFMNPs (CFMN); these two layers together formed a pH-responsive double-layer hydrogel (bFGF-CFMN) Figure 3A). The microstructure of bFGF-CFMN double-layer hydrogel was further observed by scanning electron microscopy (SEM) Figure 3 B). The results showed that the CFMN hydrogel structure was relatively loose, and the CFMNPs were uniformly distributed on the surface, which was conducive to its rapid degradation and release. In contrast, the bFGF hydrogel presented a more dense network structure, which could effectively protect bFGF and achieve its sustained release. Fourier transform infrared spectroscopy (FTIR) analysis showed that there was no significant difference in the spectral peaks between the upper hydrogel loaded with bFGF and the upper hydrogel without loading, and similarly, the main peaks of the lower hydrogel also did not change significantly whether loaded with CFMNPs or not Figure 3 C). These results showed that the introduction of bFGF and CFMNPs did not change the chemical structure of MeHA, and both substances existed in the hydrogel matrix in the form of non-covalent binding (physical embedding).

[0058] Rheological tests further elucidated the viscoelastic properties of bFGF-CFMN double-layer hydrogel. The storage modulus / loss modulus of bFGF hydrogel was greater than that of CFMN hydrogel Figure 3 D), indicating that the bFGF hydrogel had higher stiffness. Similarly, the viscosity of the bFGF hydrogel was also greater than that of the CFMN hydrogel Figure 3 E). This rheological difference enabled the CFMN hydrogel to more effectively conform to the wound surface and achieve controlled degradation, while the bFGF hydrogel with higher stiffness could maintain structural integrity and resist external mechanical interference.

[0059] Preparation of blank double-layer hydrogel without bFGF and CFMNPs (Comparative Example 1):

[0060] 2% MeHA was added to the mold, then irradiated with a UV light source for 30 seconds; then, 0.8% MeHA was added, and the UV light source was irradiated again for 45 seconds to obtain a blank double-layer hydrogel without bFGF and CFMNPs.

[0061] Preparation of double-layer hydrogel containing bFGF (Comparative Example 2):

[0062] 2% MeHA solution was added to the mold, then basic fibroblast growth factor (bFGF, 100 μg per milliliter of hydrogel system) was added, and irradiated with a UV light source for 30 seconds; then, 0.8% MeHA solution was added, and the UV light source was irradiated again for 45 seconds to obtain a bFGF hydrogel.

[0063] Test Example 1: In vitro experiment:

[0064] (1) pH-responsive degradation and release experiment of bFGF-CFMN double-layer hydrogel:

[0065] The bFGF-CFMN double-layer hydrogel was immersed in PBS with different pH values, i.e. pH 6, pH 7 and pH 8. The hydrogel was incubated at 37 °C. At predetermined time points, the hydrogel was taken out, freeze-dried and weighed to calculate the degradation rate of the hydrogel. Meanwhile, the supernatant after the hydrogel was immersed was taken out at predetermined time points. The contents of morin and bFGF were detected by high performance liquid chromatography (HPLC) and bFGF enzyme-linked immunosorbent assay (ELISA) kit, respectively, to calculate the release rate of morin and bFGF.

[0066] The results show that both the bFGF hydrogel and the CFMN hydrogel are significantly degraded under acidic conditions (pH 6), while the hydrogels remain stable in neutral (pH 7) and alkaline (pH 8) environments. Further degradation experiments confirm that the upper and lower hydrogels degrade faster in acidic environments, and the degradation rate of the lower hydrogel is higher than that of the upper hydrogel ( Figure 3 F). Drug release experiments show that the release rate of bFGF in the upper hydrogel is related to its degradation rate, while the release rate of morin in the lower hydrogel does not match its degradation rate. Specifically, the release rate of morin in alkaline environment is faster than in neutral or acidic conditions ( Figure 3 G), which may be due to the rapid degradation of CFMNP in alkaline environment. To verify this hypothesis, the release of CFMNP in the CFMN hydrogel was further studied. The results show that CFMNP is released fastest in acidic environment and slowest in alkaline conditions, which is consistent with the degradation behavior of the CFMN hydrogel. This indicates that the release of morin in the CFMN hydrogel is influenced by both the hydrogel itself and CFMNP. In summary, the double-layer hydrogel has unique pH-responsive properties, the release rate of bFGF in the bFGF hydrogel matches its degradation rate, while the release of morin in the CFMN hydrogel is regulated by both the hydrogel and CFMNP, highlighting the complexity and specificity of the drug release mechanism.

[0067] (2) Cell experiments:

[0068] Cell biocompatibility is a key indicator for the application of hydrogels in the biomedical field, directly determining their safety and effectiveness in vivo. To evaluate the biocompatibility of the bFGF-CFMN double-layer hydrogel, human umbilical vein endothelial cells (HUVECs) related to wound repair were selected for subsequent experiments. First, the effect of bFGF-CFMN double-layer hydrogel on cell viability was evaluated by live / dead staining ( Figure 4 A and Figure 4C). The results showed that the green fluorescence intensity of the CFMN group, bFGF group, and bFGF-CFMN group was higher than that of the control group at 24 and 72 hours of culture, indicating higher cell viability. Propidium iodide (PI) staining further confirmed that the red fluorescence level was lower in all treatment groups, indicating low cell mortality. This indicates that the bFGF-CFMN double-layer hydrogel has biocompatibility and does not cause significant cytotoxicity. To further verify these results, CCK-8 cell proliferation experiments were performed Figure 4 B and Figure 4 D). The results showed that there was no significant difference in cell viability between the CFMN group, bFGF group, bFGF-CFMN group, and control group at 24 and 72 hours of culture, further confirming the biocompatibility of the bFGF-CFMN double-layer hydrogel. The scratch test and tube formation experiment are standard methods for evaluating the wound repair process at the cellular level and can be used to explore cell migration and angiogenesis. The results of the scratch test showed that the wound closure rate of the CFMN group, bFGF group, and bFGF-CFMN group was significantly higher than that of the control group ( Figure 4 E), with the highest closure rate in the bFGF-CFMN group ( Figure 4 F). This indicates that the bFGF-CFMN hydrogel can effectively promote HUVECs migration and accelerate the wound healing process in vitro. In addition, the wound closure rate of the bFGF group and bFGF-CFMN group was similar and higher than that of the CFMN group, suggesting that bFGF is the main driving force for promoting HUVECs migration, while CFMN may play a synergistic role. Next, the tube formation experiment further verified the pro-angiogenic effect of the bFGF-CFMN hydrogel. Compared with the control group, the CFMN group, bFGF group, and bFGF-CFMN group induced the formation of more tubular structures ( Figure 4 G), with the highest number in the bFGF-CFMN group ( Figure 4 H), followed by the bFGF group. This indicates that bFGF plays a dominant role in angiogenesis, while CFMN provides auxiliary support. In summary, these results indicate that the bFGF-CFMN double-layer hydrogel not only has excellent biocompatibility, but also can significantly enhance cell migration and angiogenesis, supporting its potential in promoting wound repair.

[0069] (3) In vitro antioxidant and anti-inflammatory evaluation:

[0070] To further confirm the antioxidant and anti-inflammatory capabilities of the bFGF-CFMN hydrogel, the RAW 264.7 macrophage cell line, commonly used in studies of inflammatory responses and immunomodulation, was employed. First, an oxidative stress environment was constructed using a reactive oxygen species (ROS) inducer. After co-incubating the hydrogel with RAW 264.7 cells for a period of time, intracellular ROS levels were detected using the DCFH-DA fluorescent probe. The results showed that the positive control group exhibited significant green fluorescence compared to the negative control group, indicating increased ROS levels and successful induction of oxidative stress. Further observation revealed a significant decrease in green fluorescence intensity in the CFMN group, bFGF group, and bFGF-CFMN treatment group, indicating reduced intracellular ROS production and that the hydrogel possesses antioxidant activity. Figure 5 A). In addition, the blank hydrogel also showed some antioxidant activity. Quantitative fluorescence analysis showed that the bFGF-CFMN group had the lowest average fluorescence intensity, while the CFMN group was slightly lower than the bFGF group (A). Figure 5 B). This suggests that the bFGF-CFMN bilayer hydrogel has the strongest antioxidant effect, which may be attributed to CFMNPs. Its strong antioxidant capacity may originate from morin released by CFMNPs in the CFMN hydrogel. Regarding anti-inflammatory effects, an inflammatory environment was constructed by introducing lipopolysaccharide (LPS). After co-incubating the hydrogel with cells for a specific time, the cell supernatant was collected, and the levels of inflammatory cytokines (TNF-α, IL-1β, and IL-6) were detected using enzyme-linked immunosorbent assay (ELISA) to assess the anti-inflammatory capacity of the bFGF-CFMN bilayer hydrogel. The results showed that compared with the control group, the CFMN group, bFGF group, and bFGF-CFMN group all significantly reduced the concentration of these inflammatory cytokines, with the bFGF-CFMN group showing the most significant inhibitory effect. Similarly, the anti-inflammatory effect of the CFMN group was comparable to that of the bFGF-CFMN group. Figure 5 C-5E). This indicates that the bFGF-CFMN hydrogel possesses excellent anti-inflammatory efficacy, and that CFMN plays a crucial role in inflammation suppression. Simultaneously, bFGF may synergistically promote cell repair and regeneration, creating a favorable microenvironment for inflammation resolution and tissue repair. In summary, the bFGF-CFMN bilayer hydrogel not only alleviates oxidative stress but also effectively inhibits inflammatory responses, providing a stable and favorable microenvironment for wound healing. Furthermore, to evaluate the proliferation-promoting effect of the bFGF-CFMN bilayer hydrogel on human umbilical vein endothelial cells (HUVECs), an EdU cell proliferation assay was performed. Fluorescence results showed that the red fluorescence intensity of the CFMN group, bFGF group, and bFGF-CFMN group was higher than that of the control group, indicating enhanced cell proliferation activity (…). Figure 5 F). Quantitative fluorescence analysis further showed that the bFGF-CFMN group had the highest average fluorescence intensity, followed by the bFGF group (F). Figure 5G). These findings suggest that while CFMN plays a dominant role in antioxidant and anti-inflammatory activities, bFGF exhibits more significant advantages in promoting cell proliferation. In summary, bFGF-CFMN double-layer hydrogel not only has excellent antioxidant and anti-inflammatory capacity, but also can significantly promote endothelial cell proliferation. This multifunctionality makes it potential in biomedical applications, especially in the field of wound repair.

[0071] Test Example 2 Animal experiment:

[0072] Healthy male C57 BL / 6 mice aged 6-8 weeks were selected and randomly divided into 5 groups: ① control group, ② blank group (treated with blank double-layer hydrogel prepared in Comparative Example 1), ③ CFMN group (treated with CFMN hydrogel prepared in Example 2), ④ bFGF group (treated with bFGF hydrogel prepared in Comparative Example 2) and ⑤ bFGF-CFMN group (treated with bFGF-CFMN double-layer hydrogel prepared in Example 3), with 6 mice in each group. After anesthesia with 3% sodium pentobarbital (50 mg / kg), all mice were subjected to shaving and depilation treatment, and their skin was disinfected with povidone iodine. A 10 mm skin puncher was used to make a full-thickness circular skin wound with a diameter of 10 mm on the back of the mice. A heated metal rod and liquid nitrogen were used to make burn and frostbite wounds, respectively. Specifically, for burn wounds, the metal rod was heated to 100°C and then contacted with the wound for 5 seconds; for frostbite wounds, a coin was placed in liquid nitrogen for 2 minutes and then taken out to contact the wound for 5 seconds. The day of wound making was recorded as day 1. A circular hydrogel with a diameter of 10 mm was applied on the wound on the back of each mouse.

[0073] To further study its in vivo pH-responsive degradation and drug release, this example established a burn / frostbite (B / F) wound model in mice and monitored the pH value of the wound surface during the entire healing process using a pH meter. The results showed that in both burn / frostbite models, the wound pH value gradually acidified in the early stage of healing, became alkaline in the middle stage, and then returned to a weakly acidic state in the late stage (Fig. 4A). Figure 6 A). The initial acidification may be due to the release of acidic substances by damaged cells, metabolic acid produced by inflammatory cells, and hydrolysis of plasma proteins. As healing progresses, fibroblast proliferation, angiogenesis, and macrophage phenotype conversion are associated with the increase in pH value to alkaline. The return to a weakly acidic state in the late stage may be related to the production of metabolic acid by keratinocytes and the maintenance of skin barrier function. The dynamic changes in wound pH value may affect the in vivo degradation and drug release of bFGF-CFMN double-layer hydrogel. At the same time, the key factor affecting the pH value of the wound, lactic acid, was also measured. In both burn and frostbite models, the lactic acid level rapidly increased in the early stage of wound healing and gradually decreased to a stable level in the middle and late stages (Fig. 4B). Figure 6B). The initial increase in lactate levels can be attributed to enhanced anaerobic glycolysis due to tissue hypoxia, active metabolism of inflammatory cells, and accelerated glycogenolysis, which is consistent with the observed decrease in pH. The subsequent decrease in lactate levels can be due to improved blood circulation, changes in cellular metabolism, and systemic pH regulation, leading to an increase in wound pH. The pH-responsive behavior of bFGF-CFMN double-layer hydrogels was further investigated in vivo. The upper bFGF hydrogel was fluorescently labeled with FITC, and the lower CFMN hydrogel was fluorescently labeled with rhodamine B. In the burn / freeze injury model, the fluorescence signal intensity of the two-layer hydrogel was monitored over time using small-animal in vivo imaging technology. The results showed that in both models, by the 5th day, the fluorescence intensity of the bFGF hydrogel decreased very little, while that of the CFMN hydrogel decreased significantly. By the 14th day, the CFMN hydrogel was almost completely degraded, while the bFGF hydrogel still retained measurable fluorescence intensity. Figure 6 C). Quantitative analysis of fluorescence intensity showed that the CFMN hydrogel degraded rapidly in the early stage of wound healing, while the bFGF hydrogel degraded more slowly. Figure 6 D and Figure 6 E). This indicates that the CFMN hydrogel responds rapidly to the early acidic environment, while the bFGF hydrogel degrades slowly in the later stage. Combining the dynamic changes in wound pH and lactate levels, it can be inferred that the bFGF-CFMN double-layer hydrogel responds to the pH changes in burn / freeze injury wounds by adjusting its degradation rate, thereby controlling drug release to better adapt to different stages of wound healing. To further evaluate the drug release of bFGF-CFMN double-layer hydrogels in vivo, high-performance liquid chromatography (HPLC) and fluorescence labeling were used to monitor the levels of morin and bFGF in local tissues, respectively. The results showed that by the 5th day, the release rate of morin in burn and freeze injury wounds reached 80.33 ± 6.22% and 74.27 ± 4.74%, respectively, while the release rate of bFGF did not exceed 20% and 30%, respectively. Figure 6 F and Figure 7 G). The in vivo drug release curve is consistent with the degradation behavior. In the early inflammatory stage, the acidic environment of the wound triggers the rapid degradation of the CFMN hydrogel, releasing CFMNP to combat inflammation. As the wound enters the proliferation and remodeling stage, the alkaline environment slows down the degradation of the bFGF hydrogel, allowing bFGF to be released continuously to promote tissue repair. In summary, the bFGF-CFMN double-layer hydrogel can respond to the dynamic pH changes in burn / freeze injury wounds in vivo, precisely regulate its degradation rate and drug release, achieve stage-matched delivery, and highlight its potential as a new type of wound treatment material.

[0074] To comprehensively evaluate the potential of bFGF-CFMN double-layer hydrogels in promoting burn and freeze injury wound healing in vivo, this embodiment established a burn / freeze (B / F) wound model in mice to conduct in vivo experiments.Figure 8 This study investigated the therapeutic effects of bFGF-CFMN bilayer hydrogel on burn / frostbite wounds, applying it to burn and frostbite wounds respectively, and comparing the efficacy with a control group. Residual wound area was recorded and measured on days 1, 3, 5, 10, and 14. Figure 9 A and Figure 8 A). Compared with the control group and the blank group, the CFMN group, bFGF group, and bFGF-CFMN group all showed accelerated wound closure. Among them, the bFGF-CFMN group achieved near-complete closure on day 14, indicating that it had the best ability to promote wound healing. Further analysis of the wound healing rate of each group showed that the bFGF-CFMN group had the highest healing rate for burn wounds (95.92±1.22%) and frostbite wounds (94.47±3.63%), respectively, with the fastest healing speed. The wound healing rate of the CFMN group was slightly higher than that of the bFGF group ( Figure 9 D and Figure 8 D). This may be because morin released by CFMNPs in CFMN hydrogel has strong antioxidant and anti-inflammatory properties. In the early stages of burns / frostbite, morin scavenge free radicals and inhibit pro-inflammatory factors, creating a favorable wound microenvironment. Meanwhile, bFGF mainly promotes cell proliferation and migration in the mid-to-late stages. The CFMN group controlled inflammatory stress in the early stages, laying the foundation for repair, thus resulting in a slightly better healing process. Next, histological analysis was performed using HE and Masson staining to further confirm the therapeutic effect of the bFGF-CFMN group. For both burns and frostbite, on day 5, compared with the control group, the bFGF-CFMN group showed a significant reduction in inflammatory cell infiltration. By day 14, the overall tissue structure of the bFGF-CFMN group was relatively more regular, with orderly cell arrangement, reducing cell damage caused by excessive inflammatory stimulation. Figure 9 B and Figure 8 B). Further quantitative analysis of inflammatory cell infiltration showed that the bFGF-CFMN group had the lowest degree of inflammatory infiltration, followed by the CFMN group. The degree of inflammatory infiltration in the bFGF group was lower than that in the control group and the blank group, but slightly higher than that in the CFMN group. Figure 9 E and Figure 8 E). Furthermore, the study found that inflammatory damage in burn wounds was more severe than in frostbite wounds. This validates the significant efficacy of morin released from CFMNPs in CFMN hydrogels in controlling inflammation, and the synergistic advantages of bFGF-CFMN bilayer hydrogels in modulating the wound inflammatory microenvironment. Masson staining showed that in both burn / frostbite wounds, the bFGF-CFMN group had more collagen deposition, denser, larger, and more orderly arranged collagen fibers in the skin tissue, and more complete epithelial coverage. In contrast, the control and blank groups showed only a small amount of loose and disordered collagen fibers ( Figure 9 C and Figure 8C). Quantitative analysis of collagen area showed that the bFGF-CFMN group had the highest amount of collagen deposition. The bFGF group was similar to the bFGF-CFMN group, slightly higher than the CFMN group Figure 9 F and Figure 8 F). This may be due to the direct and significant role of bFGF in promoting collagen synthesis. Although the CFMN group laid the foundation for wound repair through the early anti-inflammatory effect of morin, its collagen deposition was slightly lower than the bFGF group and the bFGF-CFMN group due to the lack of direct stimulation of bFGF on cell proliferation and collagen synthesis. However, the collagen deposition of the CFMN group was still significantly higher than the control group and the blank group, further indicating the positive role of morin in regulating the wound microenvironment and indirectly promoting collagen synthesis. These results suggest that the bFGF-CFMN double-layer hydrogel shows excellent potential in promoting burn / freeze injury wound healing by regulating the inflammatory response, enhancing collagen synthesis, and epithelial coverage.

[0075] Excessive and persistent inflammation is a key factor that hinders burn / flash injury (B / F) wound repair, so the anti-inflammatory effect of bFGF-CFMN double-layer hydrogel is particularly important. After injury, the wound quickly enters a state of stress, activating the inflammatory signaling pathway and triggering the release of inflammatory cytokines. Excessive secretion of these cytokines not only directly damages surrounding cells, but also exacerbates inflammation, forming a vicious cycle that hinders the normal healing of the wound. Therefore, the anti-inflammatory effect of bFGF-CFMN double-layer hydrogel in burn / flash injury wounds was further studied. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of key inflammatory cytokines (TNF-a, IL-1b, and IL-6) in wound tissue to reflect the inflammatory state Figure 9 G-8I and Figure 10G-9I). In both burn and scald models, the control group had higher levels of TNF-a, IL-1b, and IL-6 on day 5, which is consistent with the early inflammatory response after injury. In contrast, these cytokine levels were lower in the bFGF-CFMN group, suggesting that it can have an anti-inflammatory effect. Moreover, the cytokine levels in the CFMN group were slightly lower than in the bFGF group, which is consistent with the case where morin plays a more significant role in anti-inflammatory activity. To further evaluate the anti-inflammatory ability of the bFGF-CFMN double-layer hydrogel, the levels of the anti-inflammatory cytokine IL-10 were also detected. IL-10 plays a key role in inhibiting macrophage and T cell activation and reducing the release of inflammatory mediators. The results showed that the IL-10 level in the bFGF-CFMN group was higher than in the control group, the CFMN group, and the bFGF group. Although the IL-10 level decreased by day 14, the IL-10 level in the bFGF-CFMN group was still the highest. This suggests that the bFGF-CFMN double-layer hydrogel can promote increased expression of IL-10, which can help limit the overactivation of inflammatory cells and reduce the release of pro-inflammatory cytokines, thereby creating a stable microenvironment for wound healing. In the early stages of wound healing, the CFMN hydrogel rapidly degrades in response to an acidic environment, releasing CFMNs. As the wound progresses, the pH becomes alkaline, triggering the degradation of CFMNs and the release of morin, which plays a key anti-inflammatory role. In the later stages, the alkaline environment inhibits the degradation of the bFGF hydrogel, allowing bFGF to be released continuously, which can further modulate inflammation. In summary, the bFGF-CFMN double-layer hydrogel achieves a potent anti-inflammatory effect through its pH-responsive degradation and drug release mechanism, providing effective and sustained anti-inflammatory support for burn and scald wound healing.

[0076] In addition to excessive inflammatory response, oxidative stress is also an important factor that hinders wound repair during burn and scald (B / F) wound healing. Numerous studies have shown that burns and frostbite can lead to excessive production of reactive oxygen species (ROS). The accumulation of ROS can cause cell and tissue damage, which in turn delays wound healing. Therefore, evaluating the antioxidant effect of the bFGF-CFMN double-layer hydrogel is crucial for a comprehensive understanding of its mechanism for promoting wound healing. To explore the antioxidant capacity of the bFGF-CFMN double-layer hydrogel, dihydroethidium (DHE) staining was performed on the mouse burn and scald wound tissues, and the degree of oxidative stress damage was evaluated by red fluorescence intensity Figure 10 A and Figure 10C). Fluorescence results showed that on days 5 and 14, the wound tissue in the control group exhibited strong red fluorescence, indicating a higher level of oxidative stress. The fluorescence intensity of the burn wound was higher than that of the frostbite wound, indicating that the oxidative stress in the burn wound was more severe. This may be because burns cause more extensive and severe tissue damage, leading to a stronger inflammatory response and more ROS production. In contrast, the red fluorescence intensity of the CFMN group and the bFGF group was reduced, but not as significantly as that of the bFGF-CFMN group. This indicates that both CFMN hydrogel and bFGF hydrogel alleviated oxidative damage to some extent, but the bFGF-CFMN bilayer hydrogel had a more significant inhibitory effect on oxidative stress. Quantitative analysis showed that on days 5 and 14, the red fluorescence intensity of the bFGF-CFMN group was significantly lower than that of the other groups ( Figure 10 B and Figure 10 D). These results indicate that the bFGF-CFMN bilayer hydrogel possesses strong antioxidant properties in burn wounds, effectively reducing oxidative stress levels and promoting wound healing. Furthermore, the red fluorescence intensity of the CFMN group was lower than that of the bFGF group, indicating that morin in the CFMN hydrogel played a more prominent role in the antioxidant process. Although bFGF can also alleviate oxidative stress to some extent, its effect is limited. Antioxidant and oxidative stress indicators include superoxide dismutase (SOD), the antioxidant glutathione (GSH), and the lipid peroxidation end product malondialdehyde (MDA), which play a crucial role in oxidative stress during wound healing, and their levels are important biomarkers reflecting the oxidative stress status of wound tissue. Therefore, we further examined SOD activity, GSH levels, and MDA levels in burn wound tissues. Compared with the control group, SOD activity in the wound tissues of both the CFMN and bFGF groups was increased ( Figure 10 E and Figure 10 H), GSH levels increased ( Figure 10 F and Figure 10 I) Decreased MDA levels ( Figure 10 G and Figure 11 However, these changes were not as pronounced as in the bFGF-CFMN group. These results are related to the bFGF-CFMN bilayer hydrogel, which showed higher SOD activity and GSH levels and lower MDA levels in the wound tissue. This is consistent with enhanced tissue antioxidant defense and reduced lipid peroxidation, thus confirming its antioxidant effect. In conclusion, the bFGF-CFMN bilayer hydrogel can enhance the antioxidant defense capacity of burn wound tissue.

[0077] Angiogenesis provides essential nutrients and oxygen to the wound site, promoting cell proliferation and tissue repair, and plays a crucial role in burn / wound healing. Therefore, it is of great significance to study the pro-angiogenic effect of bFGF-CFMN double-layer hydrogel. To evaluate the pro-angiogenic ability of bFGF-CFMN hydrogel, immunofluorescence (IF) staining was used to detect the expression of CD31 in burn / wound tissue. Compared with the control group, the bFGF-CFMN group had higher fluorescence intensity of CD31 Figure 12 A and Figure 11 A). Further quantitative analysis showed that the average CD31 fluorescence intensity of the bFGF-CFMN group was the highest on day 5, followed by the bFGF group, and then the CFMN group. By day 14, the difference in average fluorescence intensity between the bFGF-CFMN group and the control group further increased Figure 12 C and Figure 11 C). These results confirmed the pro-angiogenic effect of bFGF-CFMN hydrogel. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis, which can stimulate endothelial cell proliferation, migration, and vascular formation. Therefore, we used immunohistochemistry (IHC) to detect the expression of VEGF in wound tissue. The results showed that the expression level of VEGF in the bFGF-CFMN group was higher than that in the control group in the burn / wound model Figure 12 B and Figure 11 B). This indicates that bFGF-CFMN hydrogel can more effectively promote VEGF expression, which may contribute to angiogenesis. Quantitative analysis of VEGF immunohistochemistry results showed that the bFGF-CFMN group had the highest VEGF positive rate, followed by the bFGF group, which was slightly lower but still higher than the other groups. Although the VEGF positive rate of the bFGF-CFMN group decreased slightly by day 14, it remained the highest Figure 12 D and Figure 11D). These results indicate that bFGF hydrogel has the ability to promote angiogenesis, while C-FMN hydrogel may synergistically enhance this effect, together promoting angiogenesis and tissue repair in burn and scald wounds. Compared with single-layer hydrogel, bFGF-CFMN double-layer hydrogel can cope with the complex microenvironment of burn and scald wounds, and may create a more favorable intracellular and extracellular environment, thus more effectively promoting angiogenesis. In addition, the VEGF positive rate of the bFGF-CFMN group decreased slightly at day 14, which may reflect a physiological feedback mechanism that inhibits the overexpression of VEGF. This embodiment also detects the content of VEGF in burn and scald wounds, and the results are consistent with the immunohistochemical results. The VEGF level of the bFGF-CFMN group is the highest. This further confirms the excellent ability of the double-layer hydrogel to promote the synthesis and secretion of VEGF, thereby supporting wound vascularization. Platelet-derived growth factor (PDGF) and transforming growth factor-β1 (TGF-β1) also play an important role in angiogenesis, and they work synergistically with VEGF to promote burn and scald wound healing. PDGF stimulates the proliferation and migration of fibroblasts, smooth muscle cells and endothelial cells, and is involved in the construction and stabilization of blood vessel walls. TGF-β1 regulates the synthesis and degradation of extracellular matrix, and affects the remodeling and maturation of blood vessels. To further explore the effect of bFGF-CFMN double-layer hydrogel on these key growth factors, we detected the levels of PDGF and TGF-β1 in burn and scald wounds. At day 5, the PDGF content of the bFGF-CFMN group was higher than that of the control group, and similar to the level of the bFGF group. By day 14, although the PDGF content began to decrease and stabilize, the PDGF content of the bFGF-CFMN group was still higher than that of the control group (p < 0.05) Figure 12 E and Figure 11 E). Similarly, the differences and changes in the TGF-β1 content of each group are consistent with those of PDGF. Overall, bFGF-CFMN hydrogel has a positive regulatory effect on multiple key growth factors throughout the burn and scald wound healing process. The strong pro-angiogenic ability of bFGF lays the foundation, while the synergistic regulation of C-FMN hydrogel enhances this effect, together promoting angiogenesis and tissue repair in burn and scald wounds. Finally, to further verify the pro-proliferative effect of bFGF-CFMN double-layer hydrogel, we detected the expression of Ki67 in burn and scald wound tissues. Ki67 is a nuclear protein closely related to cell proliferation, and its expression level can reflect the proliferative activity of cells. The results show that at day 5 and day 14, the Ki67 level of the bFGF-CFMN group is higher than that of the control group and other experimental groups, and the highest Ki67 level is observed at day 5 (p < 0.05) Figure 12 F and ​F). This indicates that bFGF-CFMN double-layer hydrogel not only can effectively promote angiogenesis, but also can significantly enhance the cell proliferation capacity, ensuring the sustained growth and repair of new tissues. This sustained proliferative effect may be of great significance for the complete healing of burn and scald wounds. In the early stage of wound healing, the acidic environment, CFMN hydrogel degrades rapidly, releasing CFMNP. As the wound progresses, the pH gradually increases to alkaline, and CFMNP degrades and releases morin, which may create a favorable microenvironment for later wound healing and vascularization. As the wound healing process progresses, the pH value further increases, and the bFGF hydrogel slowly degrades in an alkaline environment, allowing bFGF to be released continuously, thereby up-regulating the expression of angiogenesis-related factors such as CD31, VEGF, PDGF, and TGF-β1, promoting wound cell proliferation, and ultimately contributing to the formation and maturation of new blood vessels. Based on the above results, bFGF-CFMN double-layer hydrogel, through the strong pro-angiogenic ability of bFGF and the synergistic effect of CFMN, together creates a microenvironment conducive to vascularization, cell proliferation, and tissue regeneration, thereby accelerating the healing process of burn and scald wounds.

[0078] The in vivo biocompatibility of bFGF-CFMN double-layer hydrogel is a key factor in evaluating its clinical application value. To comprehensively evaluate the biocompatibility of the hydrogel, we conducted a series of in vivo experiments. To evaluate the hemocompatibility of bFGF-CFMN double-layer hydrogel, we first conducted a hemolysis test. This test can directly determine whether the hydrogel will cause red blood cell hemolysis. The results showed that after the bFGF-CFMN hydrogel was in contact with blood, no significant lysis of red blood cells occurred, and the hemolysis rate was far below 5%, indicating that it had good hemocompatibility with red blood cells. In addition, we applied bFGF-CFMN double-layer hydrogel to the wounds of healthy mice, and after 14 days, we took their main organs (liver, kidney, heart, lung, and spleen) for HE staining. The staining results showed that compared with healthy mice, the main organ tissues of mice treated with bFGF-CFMN hydrogel were intact in structure and normal in cell morphology, and no obvious pathological damage or inflammatory infiltration occurred. These results collectively confirm that bFGF-CFMN double-layer hydrogel has good in vivo biocompatibility.

[0079] In summary, the present application provides a novel bFGF-CFMN double-layer hydrogel. The hydrogel regulates drug release by virtue of its unique pH-responsive degradation characteristics, thereby adapting to the dynamic physiological microenvironment of different stages of wound healing and exhibiting excellent therapeutic effect. The bFGF-CFMN double-layer hydrogel uses methacrylated hyaluronic acid (MeHA) as a substrate, utilizes the concentration dependence and pH responsiveness of MeHA, combines pH-responsive CFMNPs and the healing-promoting factor bFGF, and constructs a double-layer structure with clear functional division. The lower layer is composed of 0.8% MeHA loaded with CFMNPs, and the upper layer is 1.5% MeHA loaded with bFGF, which together form a double-layer hydrogel that can respond to the dynamic pH changes of the wound, and release morin and bFGF in a time-dependent manner by regulating the degradation process, to achieve precise delivery of therapeutic ingredients. Experimental results confirm that the bFGF-CFMN double-layer hydrogel has excellent pH responsiveness and mechanical properties. In vitro cell studies show that the double-layer hydrogel has good biocompatibility, can promote the migration, tube formation and proliferation of human umbilical vein endothelial cells (HUVECs), and preliminarily shows good antioxidant and anti-inflammatory ability. In animal burn / cold injury wound models, the bFGF-CFMN double-layer hydrogel accelerates wound healing by inhibiting inflammation, resisting oxidative stress and promoting angiogenesis. In addition, the double-layer hydrogel also has good biocompatibility. In summary, the bFGF-CFMN double-layer hydrogel realizes the precise delivery of antioxidant, anti-inflammatory and pro-angiogenic therapeutic ingredients through its unique pH-responsive degradation and time-dependent release mechanism, providing an efficient and intelligent solution for the treatment of burn / cold injury wounds, and has broad clinical application prospects.

[0080] The above disclosure is only the preferred embodiment of the present application, and of course cannot limit the scope of the rights of the present application, so the equivalent changes made according to the claims of the present application still fall within the scope covered by the present application.

Claims

1. A pH-responsive double-layer hydrogel, characterized in that, The application relates to a pH-responsive double-layer hydrogel and a preparation method thereof. The upper layer hydrogel comprises methacrylated hyaluronic acid and basic fibroblast growth factor; The lower layer hydrogel comprises methacrylated hyaluronic acid and mulberry pigment nanoparticles; The methacrylated hyaluronic acid concentration of the upper layer hydrogel is higher than that of the lower layer hydrogel; The mulberry pigment nanoparticles are formed by self-assembly of 4-hydroxyphenylboronic acid pinacol ester modified fucoidan, chitosan and mulberry pigment.

2. The pH-responsive double-layer hydrogel of claim 1, wherein, The application further discloses a preparation method of the pH-responsive double-layer hydrogel. The method comprises the following steps: (1) preparing hydroxyl-modified fucoidan; (2) reacting the hydroxyl-modified fucoidan with 4-hydroxyphenylboronic acid pinacol ester to obtain 4-hydroxyphenylboronic acid pinacol ester modified fucoidan; 3. The pH-responsive double-layer hydrogel of claim 2, wherein: (3) self-assembling the 4-hydroxyphenylboronic acid pinacol ester modified fucoidan, chitosan and mulberry pigment to obtain mulberry pigment nanoparticles.

4. The pH-responsive double-layer hydrogel of claim 2, wherein: In step (1), the fucoidan is mixed with a strong base and a hydroxymethylating agent for reaction, then an acid is added to neutralize the reaction mixture, and the reaction mixture is dialyzed and freeze-dried to obtain the freeze-dried product hydroxyl-modified fucoidan.

5. The pH-responsive double-layer hydrogel of claim 2, wherein: In step (2), the hydroxyl-modified fucoidan is mixed with a carbon diimide activator under the catalysis of a nucleophile to activate the carboxyl group, then 4-hydroxyphenylboronic acid pinacol ester is added, and the reaction mixture is dialyzed and freeze-dried to obtain the freeze-dried product 4-hydroxyphenylboronic acid pinacol ester modified fucoidan.

6. The pH-responsive double-layer hydrogel of claim 1, wherein: In step (3), the chitosan is dissolved in an acetic acid aqueous solution to obtain a chitosan solution, the 4-hydroxyphenylboronic acid pinacol ester modified fucoidan is dissolved in water to obtain a 4-hydroxyphenylboronic acid pinacol ester modified fucoidan aqueous solution, the mulberry pigment is dissolved in dimethyl sulfoxide to obtain a mulberry pigment solution, the chitosan solution, the fucoidan aqueous solution and the mulberry pigment solution are mixed to obtain a mixed solution, the mixed solution is ultrasonically treated and then stirred for reaction, and the mulberry pigment nanoparticles are obtained by filtering the dialyzed solution. The methacrylated hyaluronic acid concentration of the upper layer hydrogel is 2% (mass / volume), and the methacrylated hyaluronic acid concentration of the lower layer hydrogel is 0.8% (mass / volume).

7. The pH-responsive double-layer hydrogel according to claim 1 is used for preparing a drug for treating a burn or frostbite caused wound.

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