Morrin nano-particles, pH responsive hydrogel and application of morin nano-particles and pH responsive hydrogel

Abstract

The invention relates to morin nanoparticles, pH responsive hydrogel and application of the morin nanoparticles and the pH responsive hydrogel. The morin-loaded nanoparticles provided by the invention can be rapidly degraded in an alkaline environment, have certain stability in an acidic environment, have the capability of releasing morin in response to pH, and can realize accurate regulation and control of morin release according to the change of environmental pH. According to the invention, hyaluronic acid is further subjected to methacrylation treatment, such that MeHA is obtained. The characteristic that MeHA is easier to degrade in an acid environment is utilized, and MeHA is used as hydrogel to load CFMNPs and can be effectively used for treating wounds caused by burns and frostbite. Furthermore, the pH responsive double-layer hydrogel based on pigment nanoparticles-basic fibroblast growth factors can realize sequential release of the effective components according to the change of the pH value of the burn or frostbite wound. The composition can effectively inhibit inflammation, resist oxidative stress, promote proliferation and migration of endothelial cells, promote neovascularization and finally 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 In a first aspect of the present application, a mulberry pigment nanoparticle is provided, which is formed by self-assembly of 4-hydroxyphenylboronic acid pinacol ester modified fucoidan, chitosan and mulberry pigment.

[0005] In a second aspect of the present application, a preparation method of the mulberry pigment nanoparticle is provided, comprising 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) self-assembling 4-hydroxyphenylboronic acid pinacol ester modified fucoidan, chitosan and mulberry pigment to obtain the mulberry pigment nanoparticle.

[0006] 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 hydroxyl-modified fucoidan.

[0007] 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 acid pinacol ester is added, after the reaction is completed, dialysis and freeze-drying are performed to obtain the freeze-dried product 4-hydroxyphenylboronic acid pinacol ester modified fucoidan.

[0008] Preferably, 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 treated under ultrasonic waves, then stirring reaction is performed, after the reaction is completed, purification is performed to obtain the mulberry pigment nanoparticle.

[0009] In a third aspect of the present application, a pH-responsive hydrogel loaded with the mulberry pigment nanoparticle is provided.

[0010] Preferably, the pH-responsive hydrogel loaded with the mulberry pigment nanoparticle is methacrylated hyaluronic acid.

[0011] In a fourth aspect of the present application, a pH-responsive double-layer hydrogel is provided, comprising: an upper-layer hydrogel comprising methacrylated hyaluronic acid and basic fibroblast growth factor; The lower layer hydrogel comprises methacrylated hyaluronic acid and the morin nanoparticle as described above; The concentration of the methacrylated hyaluronic acid of the upper layer hydrogel is greater than the concentration of the methacrylated hyaluronic acid of the lower layer hydrogel.

[0012] Preferably, the concentration of the methacrylated hyaluronic acid of the upper layer hydrogel is 2% (mass / volume), and the concentration of the methacrylated hyaluronic acid of the lower layer hydrogel is 0.8% (mass / volume).

[0013] In a fifth aspect of the present application, the use of the pH-responsive double-layer hydrogel as described above for preparing a product for treating a wound surface caused by burns or frostbite is provided.

[0014] The beneficial effects of the present application are as follows: (1) The present application uses 4-hydroxyphenylboronic acid pinacol ester (PAPE) to modify fucoidan (Fu) to form a pH-responsive material. Subsequently, through self-assembly, it is combined with morin and chitosan (Cs) to form morin-loaded nanoparticles (CFMNPs). The nanoparticles degrade rapidly in an alkaline environment and have a certain stability in an acidic environment, have the ability to release morin in response to pH, and can achieve precise regulation of the release of morin according to the change of the environmental pH. This property makes it a promising drug delivery carrier, especially suitable for treatment scenarios that require pH-triggered release.

[0015] (2) The present application further acrylates hyaluronic acid (HA) to obtain methacrylated hyaluronic acid (MeHA). By taking advantage of 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 morin with anti-inflammatory and antioxidant effects, achieving the effect of promoting the healing of wounds caused by burns and frostbite.

[0016] (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 a wound surface and realize sequential release of a treatment effective component. In short, in terms of burns and frostbite, local tissue cells are damaged, leading to ischemia and hypoxia. The metabolic process in cells is changed from aerobic metabolism to anaerobic metabolism, a large amount of acidic metabolites such as lactic acid is produced, and thus the pH value of a wound is reduced (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.

[0017] The application proves that the bFGF-CFMN double-layer hydrogel can realize 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. The bFGF-CFMN double-layer hydrogel 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

[0018] 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 embodiments or prior art description. Obviously, the drawings in the following description only some embodiments of the application, and for those skilled in the art, other drawings can be obtained according to these drawings without creative labor.

[0019] 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; 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; 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); Figure 4 For the in vitro cell experiments 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); 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); Figure 6 In vivo degradation and release of bFGF-CFMN double-layer hydrogel: (A) Changes in the pH value of burn and frostbite wounds with healing time (n = 5). (B) Changes in the lactic acid concentration of burn and frostbite wounds with healing 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 burn wounds (n = 3). (E) Quantitative analysis of in vivo fluorescence imaging of frostbite wounds (n = 3). (F) In vivo drug release curve of bFGF-CFMN double-layer hydrogel on burn wounds (n = 3). (G) In vivo drug release curve of bFGF-CFMN double-layer hydrogel on frostbite wounds (n = 3). Figure 7 Timeline of animal experiments using bFGF-CFMN double-layer hydrogel to treat burn and frostbite (B / F) wounds; 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 respective treatments in each group; (B) HE staining of the burn wound tissue at 5 and 14 days after the 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 the different treatments to observe the changes in the tissue. The scale bar is 100 pm; (D) Wound healing rates 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 the different treatments: tumor necrosis factor-a (TNF-a) (G), interleukin-1b (IL-1b) (H) and interleukin-6 (IL-6) (I) (n = 3); 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 respective treatments in each group; (B) HE staining of the frostbite wound tissue at 5 and 14 days after the 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 the different treatments to observe the changes in the tissue. The scale bar is 100 pm; (D) Wound healing rates 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 the different treatments: tumor necrosis factor-a (TNF-a) (G), interleukin-1b (IL-1b) (H) and interleukin-6 (IL-6) (I) (n = 3); 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); 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 on day 5 and day 14 after corresponding treatment by enzyme-linked immunosorbent assay (ELISA) (n = 3); 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 on day 5 and day 14 after corresponding treatment by enzyme-linked immunosorbent assay (ELISA) (n = 3). DETAILED DESCRIPTION

[0020] 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 reference to the drawings.

[0021] In the following examples, the raw materials used are as follows: Hydroxyapatite (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), live / dead cell viability assay kit, reactive oxygen species assay kit, EdU cell proliferation kit, 4',6-diamidino-2-phenylindole (DAPI) staining solution, cell counting kit-8 (CCK-8), and lactic acid 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 Pnonye Biotech Co., Ltd. (Wuhan, China).

[0022] Example 1. Preparation of morin-loaded nanoparticles (CFMNPs): In this example, CFMNPs were prepared by self-assembly. Specifically, Fu was modified with PAPE to have pH-responsive ability. Subsequently, Cs, Fu-PAPE, and morin were self-assembled by stirring and ultrasonic treatment to form nanoparticles, i.e., CFMNPs (Fig. 1). Figure 2A). Due to the intermolecular hydrogen bonding and π-π stacking, the solubility of morin in water is poor, often appearing as a turbid suspension and easy to precipitate, which greatly limits its application in biomedical field. However, when morin participates in the self-assembly process, Cs and Fu-PAPE combine with morin through hydrogen bonding and electrostatic interaction, breaking its aggregation force, effectively improving its solubility in water. It can be observed that the solution of C F MNPs is more uniform and transparent than the solution of morin Figure 2 B). The specific preparation steps are as follows: (1) First, 1.7 grams of fucoidan (Fu) is dissolved in 20 milliliters of ultrapure water. Then, 3.2 grams of sodium hydroxide (NaOH) and 3.7 grams of chloroacetic acid are added in turn. After stirring at room temperature for 12 hours, the reaction mixture is neutralized with a 2 molar per liter hydrochloric acid (HC1) solution. Then, the mixture is dialyzed using a dialysis bag (molecular weight cut-off value 1000) for 3 days, changing the external dialysis water 6 times a day. Finally, the dialysis product is collected and freeze-dried for 3 days to obtain the freeze-dried product Fu-COOH.

[0023] (2) 100 milligrams of Fu-COOH is added to a 50 milliliter round-bottom flask, and 20 milliliters of anhydrous formamide is added. Stir for about 30 minutes until the Fu-COOH is completely dissolved. Then, 30 milligrams of DMAP is added to the above solution and stirred until completely dissolved. Then, 50 milligrams of EDC is added and stirred for 15 minutes to ensure that it is fully dissolved and activates the carboxyl group. Finally, 50 milligrams of PAPE is added to the above mixture. Seal the reaction flask and stir at room temperature in the dark for 24 hours. After the reaction is complete, the reaction mixture is transferred to a pre-prepared dialysis bag (molecular weight cut-off value 1000) and dialyzed for 3 days, changing the external dialysis water 8 times a day. After dialysis is complete, the final mixture is collected and freeze-dried to obtain Fu-PAPE.

[0024] (3) Equal volumes of 5 mg / mL chitosan (Cs) solution (chitosan is dissolved in 2% acetic acid aqueous solution to prepare) and 5 mg / mL Fu-PAPE aqueous solution are mixed. Then, 5 mg / mL morin solution (morin is prepared by dissolving in dimethyl sulfoxide in advance) is added. The mixture is ultrasonically treated for 1 hour to promote the self-assembly of morin with chitosan and Fu-PAPE, and then continues to stir for 1 hour. Then, the mixture is dialyzed using a dialysis bag (molecular weight cut-off value 3000) for 6 hours. After dialysis is complete, the solution is collected and filtered through a 0.45 μm filter membrane. After filtration, the filtrate is centrifuged at 15000 rpm for 15 minutes, and the C F MNPs precipitate is collected. Finally, the precipitate is freeze-dried to obtain C F MNPs.

[0025] Subsequently, the prepared CFMNPs were further characterized to fully explore their structural characteristics and potential application performance. The micromorphology of CFMNPs was observed using transmission electron microscopy (TEM), and the results showed that CFMNPs had a relatively uniform spherical structure ( Figure 2 C). The results of the nanoparticle size analyzer 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 its particle size distribution was uniform ( Figure 2 D). The successful preparation of CFMNPs was verified by infrared spectroscopy analysis ( Figure 2 Fourier transform infrared spectroscopy (FTIR) revealed that Fu exhibited a hydroxyl (OH) stretching vibration peak at approximately 3400 cm⁻¹. After the introduction of 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 the successful introduction of carboxyl groups into the fucoidan molecule. Cs exhibited amino (NH2) and hydroxyl (OH) stretching vibration peaks at the same wavenumbers. Morin 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 displayed characteristic peaks for the three components, demonstrating their successful association and interaction to form stable nanoparticles. Furthermore, high-performance liquid chromatography (HPLC) analysis revealed that the encapsulation efficiency and drug loading efficiency of CFMNPs for morin were approximately 87.23% and 8.31%, respectively.

[0026] To further verify the pH responsiveness of Fu-COOH-endowed CFMNPs, degradation experiments of CFMNPs under different pH conditions were first conducted. The experimental results showed that in an alkaline environment (pH 8), the diameter of CFMNPs gradually decreased over time, reaching 14.77±5.47 nm after 24 hours; in a neutral environment (pH 7), the diameter of CFMNPs decreased, but the decrease was relatively small; in an acidic environment (pH 6), the diameter of CFMNPs showed almost no significant change, remaining at 142.79±20.90 nm after 24 hours ( Figure 2 F and Figure 2G). This indicates that CFMNPs undergo significant degradation in basic environments, slight degradation in neutral environments (pH 7.0), and almost no degradation in acidic environments. It can also be observed that the supernatant of CFMNPs gradually changes from colorless to yellow over time in basic environments, and the spherical morphology of CFMNPs is significantly destroyed, while there is no obvious color change in the supernatant of CFMNPs in neutral and acidic environments, and the morphology of CFMNPs in acidic environments remains relatively intact. This fully demonstrates that CFMNPs can quickly degrade in basic environments and have certain stability in acidic environments. The pH responsiveness of CFMNPs may be due to the fact that the boronic acid pinacol ester bond in PAPE is easily attacked by hydroxyl ions in basic environments, destroying the structural stability of the nanoparticles; the amino group of Cs undergoes proton dissociation under basic conditions, reducing the solubility and changing the intermolecular interactions between the molecular chains, further affecting the stability of the nanoparticles; in addition, the intermolecular forces between Fu and Cs are destroyed in basic environments.

[0027] In addition, the ability of CFMNPs to release morin in response to pH was also verified. Quantitative analysis of the morin released by 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 phase is attributed to the rapid destruction of the nanoparticle structure in basic environments, allowing morin to quickly pass through the loose nanoparticle shell into the surrounding environment. By 24 hours, the cumulative release of morin was about 95%, meaning that most of the morin had been released from CFMNPs. In sharp contrast, in neutral environments, the rate of morin release from CFMNPs was significantly slower, with a cumulative release of only about 20% after 12 hours and about 24% after 24 hours. This phenomenon may be due to the relatively slow degradation rate of nanoparticles in neutral environments, with limited structural damage, thereby hindering the diffusion of morin from the interior of the nanoparticles to the surrounding environment. In acidic environments, CFMNPs released very little morin, with a release rate of only about 11% after 24 hours, fully demonstrating the stability of CFMNPs in acidic environments. This may be because the structure of CFMNPs is almost not destroyed in acidic conditions, and morin is tightly wrapped inside and difficult to release Figure 2 H}.

[0028] In summary, CFMNPs have excellent pH-responsive morin release ability and can precisely control the release of morin according to changes in environmental pH. This feature makes it a promising drug delivery carrier, especially for therapeutic scenarios that require pH-triggered release.

[0029] Example 2 Preparation of double-layer hydrogel containing CFMNPs: (1) 1 gram of hyaluronic acid (HA) was dissolved in 30 milliliters of a mixed solvent of N,N-dimethylformamide (DMF) and 60 milliliters of pure water, 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 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, an appropriate amount of pure water was added to dissolve the precipitate, and the solution was dialyzed at 4°C for 3 days, with water being changed 3 times per day. After dialysis, the aqueous solution was collected and freeze-dried, and finally, methacrylated hyaluronic acid (MeHA) solid was obtained.

[0030] (2) The methacrylated hyaluronic acid (MeHA) was dissolved in pure water, and after complete dissolution, the LAP photoinitiator (0.25%, w / v) was added to obtain MeHA solutions with concentrations of 0.8% (w / v) and 2% (w / v). The 2% MeHA solution was added to the mold, and irradiated with a UV light source for 30 seconds; then, the 0.8% MeHA solution and CFMNPs (10 mg per milliliter of hydrogel system) were added, and irradiated again with a UV light source for 45 seconds to obtain the CFMN hydrogel.

[0031] Example 3 Preparation of bFGF-CFMN double-layer hydrogel: (1) 1 gram of hyaluronic acid (HA) was dissolved in 30 milliliters of a mixed solvent of N,N-dimethylformamide (DMF) and 60 milliliters of pure water, 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 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, an appropriate amount of pure water was added to dissolve the precipitate, and the solution was dialyzed at 4°C for 3 days, with water being changed 3 times per day. After dialysis, the aqueous solution was collected and freeze-dried, and finally, methacrylated hyaluronic acid (MeHA) solid was obtained.

[0032] (2) Methacrylated hyaluronic acid (MeHA) was dissolved in pure water, after complete dissolution, LAP photoinitiator (0.25%, w / v) was added to obtain MeHA solution with concentrations of 0.8% (w / v) and 2% (w / v). The preparation steps of the double-layer hydrogel are as follows: first, 2% MeHA solution was added to the mold, then basic fibroblast growth factor (bFGF, 100 μg per milliliter of hydrogel system) was added, and it was irradiated with an ultraviolet light source for 30 seconds; then, 0.8% MeHA solution and CFMNPs (10 mg per milliliter of hydrogel system) were added, and it was irradiated with an ultraviolet light source for 45 seconds again to obtain bFGF-CFMN double-layer hydrogel.

[0033] This example uses methacrylated hyaluronic acid (MeHA) as the main material, and uses the concentration-dependent properties of MeHA and its sensitivity to acidic environment to construct a unique double-layer structure. Specifically, the upper layer hydrogel is composed of 2% MeHA, loaded with basic fibroblast growth factor (bFGF); the lower layer hydrogel is composed of 0.8% MeHA, loaded with CFMNPs (CFMN); the two layers together constitute a pH-responsive double-layer hydrogel (bFGF-CFMN) Figure 3 A). Scanning electron microscopy (SEM) was further used to observe the microstructure of the bFGF-CFMN double-layer hydrogel Figure 3 B). The results show that the CFMN hydrogel structure is relatively loose, and the CFMNPs are uniformly distributed on the surface, which is conducive to its rapid degradation and release. In contrast, the bFGF hydrogel presents a more dense network structure, which can effectively protect bFGF and achieve its sustained release. Fourier transform infrared spectroscopy (FTIR) analysis shows that there is no significant difference in the spectral peaks between the upper layer hydrogel loaded with bFGF and the upper layer hydrogel without loading Figure 3 C). These results show that the introduction of bFGF and CFMNPs does not change the chemical structure of MeHA, and both substances exist in the hydrogel matrix in the form of non-covalent binding (physical embedding).

[0034] Rheological tests further clarify the viscoelastic properties of the bFGF-CFMN double-layer hydrogel. The storage modulus / loss modulus of the bFGF hydrogel is greater than that of the CFMN hydrogel Figure 3 D), indicating that the bFGF hydrogel has higher stiffness. Similarly, the viscosity of the bFGF hydrogel is also greater than that of the CFMN hydrogel Figure 3 E). This rheological difference enables the CFMN hydrogel to more effectively conform to the wound surface and achieve controlled degradation, while the bFGF hydrogel with higher stiffness can maintain structural integrity and resist external mechanical interference.

[0035] Preparation of blank double-layer hydrogel without bFGF and C-FMNPs: 2% of MeHA was added into the mold, and then irradiated with a UV light source for 30 seconds; subsequently, 0.8% of MeHA was added, and irradiated with a UV light source for 45 seconds again, to obtain the blank double-layer hydrogel without bFGF and C-FMNPs.

[0036] Preparation of double-layer hydrogel with bFGF: 2% of MeHA solution was added into the mold, and 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; subsequently, 0.8% of MeHA solution was added, and irradiated with a UV light source for 45 seconds again, to obtain the bFGF hydrogel.

[0037] Test Example 1: In vitro experiment: (1) pH-responsive degradation and release experiment of bFGF-CFMN double-layer hydrogel: The bFGF-CFMN double-layer hydrogel was soaked in PBS solutions 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, at predetermined time points, the supernatant after soaking of the hydrogel was taken out. 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 rates of morin and bFGF.

[0038] The results show that under acidic conditions (pH 6), both the bFGF hydrogel and the C-FMN hydrogel are significantly degraded, while in neutral (pH 7) and alkaline (pH 8) environments, the hydrogels remain stable. 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 (Fig. F). Figure 3 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 is not consistent with its degradation rate. Specifically, the release rate of morin in alkaline environment is faster than that in neutral or acidic conditions (Fig. F). Figure 3G), which might be due to the fast degradation of CFMNPs in alkaline environment. To verify this hypothesis, the release of CFMNPs from CFMN hydrogel was further investigated. The results showed that CFMNPs were released fastest in acidic environment and slowest in alkaline condition, which was consistent with the degradation behavior of CFMN hydrogel. This indicated that the release of morin from CFMN hydrogel was influenced by both the hydrogel itself and CFMNPs. In summary, the dual-layer hydrogel had unique pH-responsive properties, the release rate of bFGF from bFGF hydrogel matched its degradation rate, while the release of morin from CFMN hydrogel was regulated by both the hydrogel and CFMNPs, which highlighted the complexity and specificity of drug release mechanism.

[0039] (2) Cell experiments: Cell biocompatibility is a key indicator for the application of hydrogels in biomedical field, which directly determines their safety and effectiveness in vivo. To evaluate the biocompatibility of bFGF-CFMN dual-layer hydrogel, human umbilical vein endothelial cells (HUVECs) related to wound repair were selected for subsequent experiments. First, the effect of bFGF-CFMN dual-layer hydrogel on cell viability was evaluated by live / dead staining ( Figure 4 A and Figure 4 C). The results showed that the green fluorescence intensity of CFMN group, bFGF group and bFGF-CFMN group was higher than that of the control group at 24 hours and 72 hours of culture, indicating higher cell viability. Propidium iodide (PI) staining further confirmed that the red fluorescence level of all treatment groups was low, indicating low cell death rate. This indicated that bFGF-CFMN dual-layer hydrogel was biocompatible and did 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 CFMN group, bFGF group, bFGF-CFMN group and control group at 24 hours and 72 hours of culture, further confirming the biocompatibility of bFGF-CFMN dual-layer hydrogel. Scratch test and tube formation test are standard methods for evaluating wound repair process at cellular level, which can be used to explore cell migration and angiogenesis. The results of scratch test showed that the wound closure rate of CFMN group, bFGF group and bFGF-CFMN group was significantly higher than that of the control group ( Figure 4 E), among which the closure rate of bFGF-CFMN group was the highest ( Figure 4F). This indicates that bFGF-CFMN hydrogel can effectively promote HUVECs migration in vitro and accelerate the wound healing process. In addition, the wound closure rates of the bFGF group and the bFGF-CFMN group were 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 angiogenic effect of bFGF-CFMN hydrogel. Compared with the control group, the CFMN group, the bFGF group, and the bFGF-CFMN group induced the formation of more tubular structures ( Figure 4 G), among which the bFGF-CFMN group had the largest number ( Figure 4 H), followed closely by the bFGF group. This suggests that bFGF plays a dominant role in angiogenesis, while CFMN provides auxiliary support. Taken together, these results demonstrate that the bFGF-CFMN bilayer hydrogel not only has excellent biocompatibility but also significantly enhances cell migration and angiogenesis, supporting its potential in promoting wound repair.

[0040] (3) In vitro antioxidant and anti-inflammatory evaluation: To further confirm the antioxidant and anti-inflammatory capabilities of bFGF-CFMN hydrogel, the study used RAW 264.7 cells, a macrophage cell line commonly used in inflammatory response and immunomodulation research. First, an oxidative stress environment was constructed using a reactive oxygen species (ROS) inducer. After the hydrogel was co-incubated with RAW 264.7 cells for a period of time, the intracellular ROS level was detected using a DCFH-DA fluorescent probe. The results showed that compared with the negative control group, the positive control group exhibited obvious green fluorescence, indicating that the ROS level was elevated and oxidative stress was successfully induced. Further observation revealed that the green fluorescence intensity of the CFMN group, bFGF group, and bFGF-CFMN-treated group was significantly reduced, indicating that the generation of ROS in the cells was reduced and the hydrogel had antioxidant activity ( Figure 5 A). In addition, the blank hydrogel also showed a certain antioxidant activity. Fluorescence quantitative analysis showed that the average fluorescence intensity of the bFGF-CFMN group was the lowest, and the CFMN group was slightly lower than the bFGF group ( Figure 5B). This suggests that the bFGF-CFMN double-layer hydrogel has the strongest antioxidant effect, which may be attributed to the CFMNPs. Its strong antioxidant capacity may be due to the release of morin from CFMNPs in the CFMN hydrogel. In terms of anti-inflammatory effects, by introducing lipopolysaccharide (LPS) to create an inflammatory environment, the hydrogel was co-incubated with cells for a certain period of time, and then the cell supernatant was collected. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of inflammatory cytokines (TNF-α, IL-1β and IL-6) to evaluate the anti-inflammatory ability of bFGF-CFMN double-layer hydrogel. The results showed that compared with the control group, the CFMN group, the bFGF group and the bFGF-CFMN group significantly reduced the concentration of these inflammatory cytokines, and the inhibition effect of the bFGF-CFMN group was the most significant. Similarly, the anti-inflammatory effect of the CFMN group was comparable to that of the bFGF-CFMN group (Figs. 6A-6C). Figure 5 C-5E). This indicates that the bFGF-CFMN hydrogel has excellent anti-inflammatory efficacy, and CFMN plays a key role in inflammation suppression. At the same time, bFGF may promote cell repair and regeneration through synergistic effects, creating a favorable microenvironment for inflammation resolution and tissue repair. In summary, the bFGF-CFMN double-layer hydrogel not only reduces oxidative stress, but also effectively suppresses inflammatory responses, providing a stable and favorable microenvironment for wound repair. In addition, to evaluate the proliferation-promoting effect of bFGF-CFMN double-layer hydrogel on human umbilical vein endothelial cells (HUVECs), an EdU cell proliferation experiment was conducted. The fluorescence results showed that the red fluorescence intensity of the CFMN group, the bFGF group and the bFGF-CFMN group was higher than that of the control group, indicating that the cell proliferation activity was enhanced (Figs. 7A-7C). Figure 5 F). Fluorescence quantitative analysis further showed that the average fluorescence intensity of the bFGF-CFMN group was the highest, followed by the bFGF group (Figs. 7D-7F). Figure 5 G). These findings suggest that although CFMN plays a dominant role in antioxidant and anti-inflammatory activity, bFGF shows more significant advantages in promoting cell proliferation. In summary, the bFGF-CFMN double-layer hydrogel not only has excellent antioxidant and anti-inflammatory ability, but also significantly promotes endothelial cell proliferation. This multifunctionality makes it potential in biomedical applications, especially in the field of wound repair.

[0041] Test Example 2 Animal Experiment: Healthy male C57 BL / 6 mice aged 6-8 weeks were randomly divided into five groups: ① control group, ② blank group (treated with the blank bilayer hydrogel prepared in Comparative Example 1), ③ CFMN group (treated with the CFMN hydrogel prepared in Example 2), ④ bFGF group (treated with the bFGF hydrogel prepared in Comparative Example 2), and ⑤ bFGF-CFMN group (treated with the bFGF-CFMN bilayer hydrogel prepared in Example 3), with 6 mice per group. After anesthesia with 3% sodium pentobarbital (50 mg / kg), all mice were shaved and depilated, and their skin was disinfected with povidone-iodine. A full-thickness circular wound with a diameter of 10 mm was created on the back of the mice using a 10 mm skin punch. Burn and frostbite wounds were created using a heated metal rod and liquid nitrogen, respectively. Specifically, for burn wounds, the metal rod was heated to 100°C and then in contact with the wound for 5 seconds. For frostbite wounds, a coin was placed in liquid nitrogen for 2 minutes, removed, and then in contact with the wound for 5 seconds. The day of wound creation was recorded as day 1. A circular hydrogel with a diameter of 10 mm was applied to the wound on the back of each mouse.

[0042] To further investigate its pH-responsive degradation and drug release in vivo, a burn / frostbite (B / F) wound model was established in mice. A pH meter was used to monitor the pH value of the wound surface throughout the healing process. The results showed that in both burn / frostbite models, the wound pH gradually acidified in the early healing phase, became alkaline in the middle phase, and then returned to a weakly acidic state in the late healing phase ( Figure 6 A). Initial acidification may be due to the release of acidic substances by damaged cells, the production of metabolic acids by inflammatory cells, and the hydrolysis of plasma proteins. As healing progresses, fibroblast proliferation, angiogenesis, and macrophage phenotypic conversion are associated with an increase in pH toward alkalinity. The later recovery to a weakly acidic state may be related to the production of metabolic acids by keratinocytes and the maintenance of skin barrier function. The dynamic changes in wound pH may affect the in vivo degradation and drug release of bFGF-CFMN bilayer hydrogels. At the same time, lactic acid, a key factor affecting wound pH, was also measured. In both burn and frostbite models, lactate levels increased rapidly in the early stages of wound healing and gradually decreased to a stable level in the middle and late stages ( 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 attributed to improved blood circulation, changes in cellular metabolism, and systemic pH regulation, resulting in 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 day 5, the fluorescence intensity of the bFGF hydrogel decreased very little, while the fluorescence intensity of the CFMN hydrogel decreased significantly. By day 14, 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 day 5, 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.

[0043] 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 The study investigated the efficacy of bFGF-CFMN bilayer hydrogel on burn / frostbite wounds. It was applied to burn and frostbite wounds and compared with the efficacy of the control group. The remaining 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, the bFGF group, and the bFGF-CFMN group all showed accelerated wound closure. Among them, the bFGF-CFMN group achieved nearly complete closure on the 14th day, indicating that it had the best ability to promote wound healing. Further analysis of the wound healing rates of each group showed that the bFGF-CFMN group had the fastest healing rates for burn and frostbite wounds, with healing rates of 95.92±1.22% and 94.47±3.63%, respectively. 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 the mulberry pigments released by CFMNPs in the CFMN hydrogel have strong antioxidant and anti-inflammatory properties. In the early stages of burns / frostbite, mulberry pigments scavenge free radicals and inhibit pro-inflammatory factors, creating a favorable wound microenvironment. At the same time, bFGF mainly promotes cell proliferation and migration in the middle and late stages. The CFMN group controls inflammatory stress in the early stage and lays the foundation for repair, so the healing process is slightly better. Next, histological analysis was performed by HE and Masson staining to further confirm the therapeutic effect of the bFGF-CFMN group. Regardless of burns or frostbite, on the 5th day, the inflammatory cell infiltration in the bFGF-CFMN group was significantly reduced compared with the control group. By the 14th day, the overall tissue structure of the bFGF-CFMN group was relatively more regular, and the cells were arranged in an orderly manner, 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). In addition, the study found that the inflammatory damage in burn wounds was more severe than that in frostbite wounds. This verifies the significant efficacy of mulberry pigment released by CFMNPs in CFMN hydrogel in controlling inflammation, as well as the synergistic advantage of bFGF-CFMN bilayer hydrogel in regulating the wound inflammatory microenvironment. Masson staining showed that in both burn / frostbite wounds, the bFGF-CFMN group had more collagen deposition, the collagen fibers in the skin tissue were denser, thicker, more neatly arranged, and the epithelial coverage was more complete. In contrast, the control group and the blank group only had 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.

[0044] 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-α, IL-1β, 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.

[0045] 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 the 5th and 14th days, the wound tissue of the control group showed strong red fluorescence, indicating a high level of oxidative stress. The fluorescence intensity of the burn wound was higher than that of the frostbite wound, indicating that the oxidative stress of the burn wound was more severe. This may be because the tissue damage caused by the burn is more extensive and more severe, which will trigger a stronger inflammatory response and more ROS production. In contrast, the red fluorescence intensity of the CFMN group and the bFGF group decreased, but not as significantly as the bFGF-CFMN group. This shows that the CFMN hydrogel and the bFGF hydrogel reduced oxidative damage to a certain extent, and the bFGF-CFMN double-layer hydrogel had a more significant inhibitory effect on oxidative stress. Quantitative analysis showed that on the 5th and 14th days, 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 has strong antioxidant properties in burn wounds, can effectively reduce the level of oxidative stress, and promote wound healing. In addition, the red fluorescence intensity of the CFMN group was lower than that of the bFGF group, indicating that the morin in the CFMN hydrogel played a more prominent role in the antioxidant process. Although bFGF can also alleviate oxidative stress to a certain extent, its effect is limited. Antioxidant and oxidative stress indicators include superoxide dismutase (SOD), antioxidant glutathione (GSH), and lipid peroxidation end product malondialdehyde (MDA), which play a key role in oxidative stress in the wound healing process, and their levels are important biomarkers reflecting the oxidative stress status of wound tissue. Therefore, we further detected the SOD activity, GSH level, and MDA level in burn wound tissue. Compared with the control group, the SOD activity in the wound tissue of the CFMN group and the bFGF group 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 J), but these changes were less pronounced than in the bFGF-CFMN group. These results correlate with the bFGF-CFMN bilayer hydrogel, which showed higher SOD activity and GSH levels and lower MDA levels in wound tissue. This is consistent with enhanced tissue antioxidant defenses and reduced lipid peroxidation, confirming its antioxidant effect. In summary, the bFGF-CFMN bilayer hydrogel can enhance the antioxidant defenses of burn wound tissue.

[0046] Angiogenesis provides essential nutrients and oxygen to the wound site, promotes cell proliferation and tissue repair, and plays a vital role in the healing process of burn and scald (B / F) wounds. Therefore, it is of great significance to study the angiogenesis-promoting effect of bFGF-CFMN bilayer hydrogel. To evaluate the angiogenesis-promoting ability of bFGF-CFMN hydrogel, immunofluorescence (IF) staining was used to detect the expression of CD31 in burn and scald wound tissue. Compared with the control group, the CD31 fluorescence intensity in the bFGF-CFMN group was higher ( Figure 12 A and Figure 11 A). Further quantitative analysis showed that on day 5, the average CD31 fluorescence intensity was highest in the bFGF-CFMN group, 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 confirm the angiogenic effect of bFGF-CFMN hydrogel. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis, stimulating endothelial cell proliferation, migration, and blood vessel formation. Therefore, we used immunohistochemistry (IHC) to detect VEGF expression in wound tissue. The results showed that in the burn wound model, the VEGF expression level in the bFGF-CFMN group was higher than that in the control group ( Figure 12 B and Figure 11 B). This suggests that bFGF-CFMN hydrogel can more effectively promote VEGF expression, which may in turn contribute to angiogenesis. Quantitative analysis of VEGF immunohistochemistry results showed that the bFGF-CFMN group had the highest VEGF positivity rate, which was slightly lower in the bFGF group but still higher than in the other groups. Although the VEGF positivity rate in the bFGF-CFMN group decreased slightly by day 14, it still 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.

[0047] 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.

[0048] 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.

[0049] 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, therefore the equivalent changes made according to the claims of the present application still belong to the scope covered by the present application.

Claims

1. A morin nanoparticle, characterized in that: It is formed by self-assembly of 4-hydroxyphenylboronic acid pinacol ester-modified fucoidan, chitosan and morin.

2. The method for preparing morin nanoparticles according to claim 1, wherein: The following steps are involved: (1) Preparation of 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) Fucoidan modified with 4-hydroxyphenylboronic acid pinacol ester, chitosan and morin were mixed and self-assembled to obtain morin nanoparticles.

3. The method for preparing morin nanoparticles according to claim 2, wherein: In step (1), fucoidan is mixed with a strong base and a hydroxymethylation agent for reaction. After the reaction is completed, an acid is added to neutralize the reaction mixture, dialyzed, and freeze-dried to obtain a freeze-dried product of hydroxyl-modified fucoidan.

4. The method for preparing morin nanoparticles according to claim 2, wherein: In step (2), the hydroxyl-modified fucoidan is mixed with a carbodiimide activator in the presence of a nucleophilic catalyst to activate the carboxyl group, and then 4-hydroxyphenylboronic acid pinacol ester is added. After the reaction is completed, the mixture is dialyzed and freeze-dried to obtain a freeze-dried product of 4-hydroxyphenylboronic acid pinacol ester-modified fucoidan.

5. The method for preparing morin nanoparticles according to claim 2, wherein: In step (3), chitosan is dissolved in an acetic acid aqueous solution to obtain a chitosan solution, 4-hydroxyphenylboronic acid pinacol ester-modified fucoidan is dissolved in water to obtain a 4-hydroxyphenylboronic acid pinacol ester-modified fucoidan aqueous solution, morin is dissolved in dimethyl sulfoxide to obtain a morin solution, the chitosan solution, the fucoidan aqueous solution, and the morin solution are mixed to obtain a mixed solution, the mixed solution is treated under ultrasound, and then stirred for reaction, and then purified to obtain morin nanoparticles.

6. A pH-responsive hydrogel, characterized in that: It is loaded with the mulberry pigment nanoparticles as claimed in claim 1.

7. The pH-responsive hydrogel according to claim 6, wherein: It uses methacryloyl hyaluronic acid to load morin nanoparticles.

8. The pH-responsive hydrogel according to claim 6, wherein include: an upper hydrogel layer comprising methacryloylated hyaluronic acid and basic fibroblast growth factor; a lower layer of hydrogel comprising methacryloylated hyaluronic acid and the morin nanoparticles as claimed in claim 1; The concentration of methacryloyl hyaluronic acid in the upper hydrogel layer is greater than that in the lower hydrogel layer.

9. The pH-responsive double-layer hydrogel according to claim 8, characterized in that: The concentration of methacryloyl hyaluronic acid in the upper hydrogel layer was 2% by weight by volume, and the concentration of methacryloyl hyaluronic acid in the lower hydrogel layer was 0.8% by weight by volume.

10. Use of the pH-responsive double-layer hydrogel according to any one of claims 6 to 9 for preparing a product for treating wounds caused by burns or frostbite.

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