A polyphenol-loaded biomimetic mussel photo-thermal hydrogel dressing and a preparation method and application thereof

Abstract

The application belongs to the technical field of diabetic wound healing, and discloses a polyphenol-loaded biomimetic mussel photothermal hydrogel dressing as well as a preparation method and application thereof.The hydrogel can simultaneously meet the treatment requirements of three pathological foundations of a diabetic wound.The hydrogel is based on gelatin, is modified through dopamine polymerization of a biomimetic mussel, and is loaded with active polyphenol, so that targeted treatment of the three pathological foundations of the diabetic wound can be realized.The preparation method is simple, and is convenient for large-scale preparation and application in home and clinical treatment of the diabetic wound.

Description

Technical Field

[0001] This invention belongs to the field of diabetic wound healing technology, specifically relating to a biomimetic mussel photothermal gel dressing loaded with polyphenols, its preparation method, and its application. Background Technology

[0002] Diabetic wounds, especially diabetic foot, are typical examples of slow-healing wounds. As one of the most common complications of diabetes, they not only affect the physical and mental health of patients but also impose a heavy economic burden on families and society. Diabetic foot is caused by peripheral neuropathy and peripheral vascular disease in diabetic patients due to hyperglycemia. Under excessive mechanical pressure, it causes damage and deformity of the soft tissues and musculoskeletal system of the foot. As diabetes worsens, it can also lead to distal lower limb neuropathy and foot ulcers, infections, and deep tissue damage associated with varying degrees of peripheral vascular disease.

[0003] Currently, neuropathy, vascular disease, and recurring bacterial infection around diabetic wounds are considered the three major pathological bases of diabetic wounds. The pathological basis of diabetic wounds is neuropathy and vascular disease, while bacterial infection further aggravates the condition. For example, damage to vascular cells in densely vascularized areas of the skin can lead to peripheral nerve necrosis and loss of sensation. Furthermore, wounds are susceptible to bacterial infection in a high-glucose environment. The infecting bacteria further disrupt the body's balance, and the damaged blood vessels prevent essential substances and cells from reaching the area in time, leading to the body's immune system being unable to resist further bacterial infection and a gradual loss of self-healing ability. These three pathological bases of diabetic wounds coexist and are mutually causal, complicating the assessment of the wound's course. Therefore, discussing the treatment of diabetic foot wounds from the perspective of only one symptom is incomplete. Researchers should comprehensively consider the multiple pathological features of diabetic wounds to simplify treatment and reduce patient suffering. Current research has confirmed the advantages of hydrogels as dressings for diabetic wound treatment, but few researchers have focused on developing hydrogels that can simultaneously meet the three major pathological conditions of diabetic wounds.

[0004] Treatment for the three major pathological bases of diabetic wounds primarily involves oral or injectable medications. However, achieving the required blood drug concentrations for the wound inevitably results in large dosages and significant side effects. Therefore, effective topical medications are needed to meet the treatment needs of diabetic wounds. However, the topical use of different medications still presents certain problems. For example, to avoid inducing bacterial resistance, guidelines for the treatment of diabetic wounds do not recommend the direct use of antibiotics or other drugs on the wound. Currently, many domestic and international guidelines for the treatment of diabetic wounds do not recommend typical and effective topical treatment ingredients.

[0005] Polyphenols are a class of versatile drugs with strong antioxidant and anti-inflammatory effects. In recent years, polyphenols have been considered potential neuroprotective substances against the blood-brain barrier in diabetes; polyphenolic compounds can serve as effective nutritional drugs for diabetic patients, while also possessing advantages such as antibacterial and anti-drug resistance properties. Furthermore, polydopamine (PDP) coatings, inspired by the high adhesion of mussels, exhibit good biocompatibility and can improve the physical / adhesive properties, photothermal activity, and impart certain physiological activities to materials.

[0006] The dressing of the present invention is a hydrogel dressing, which has good photothermal antibacterial effect under near-infrared light irradiation, and combined with the antibacterial effect of polyphenol loading, prevents bacterial infection of the wound; the hydrogel can simultaneously promote blood vessel and nerve regeneration at the wound site; in summary, the hydrogel can simultaneously meet the treatment needs of the three major pathological bases of diabetic wounds. Summary of the Invention

[0007] To overcome the shortcomings of existing hydrogels and their preparation techniques, the present invention aims to provide a biomimetic mussel photothermal hydrogel loaded with polyphenols. This hydrogel can simultaneously meet the three major pathological treatment needs of diabetic wounds. At the same time, the hydrogel formulation provided by the present invention has a simple preparation process and low cost.

[0008] To achieve the above objectives, the technical solution adopted by the present invention to overcome its shortcomings is as follows:

[0009] This invention provides a method for preparing a polyphenol-loaded biomimetic mussel photothermal gel that simultaneously targets the three major pathological bases of diabetic wounds. The method primarily employs in-situ polymerization, and the specific steps include:

[0010] S1: Dissolve dopamine hydrochloride in alkaline PBS buffer to obtain a polydopamine solution;

[0011] S2: Add gelatin to the polydopamine solution obtained in step S1, stir, heat and swell to obtain a melt;

[0012] S3: Add polyphenols to the melt described in step S2, stir and mix well, spread evenly, and let stand and refrigerate to obtain the biomimetic mussel photothermal hydrogel loaded with polyphenols; the polyphenols are caffeic acid or monocaffeoylglycerol esters.

[0013] Preferably, in step S1, the concentration of dopamine hydrochloride is 1–100 mM;

[0014] The alkaline PBS buffer in step S1 has a pH of 7-10 and a concentration of 5-100 mM;

[0015] During the dissolution process, the stirring speed is 200-1500 rpm and the stirring time is 0.1-36 h.

[0016] Preferably, in step S2, the concentration of the gelatin in the polydopamine solution is 5-50% w / w, the stirring speed is 200-1500 rpm, the swelling temperature is 35-60°C, and the swelling time is 20-60 min.

[0017] Preferably, in step S3, the concentration of the polyphenol is 0.1–100 mM, the stirring speed is 200–1500 rpm, the stirring temperature is 35–60°C, and the stirring time is 5–60 min; the refrigeration temperature is 4–10°C, and the refrigeration time is 6–24 hours.

[0018] Preferably, in step S3, the structural formula of the monocaffeoylglycerol is:

[0019]

[0020] Preferably, the monocaffeoylglycerol ester in step S3 is prepared by the following method:

[0021] (1) Add ethyl caffeate, glycerol and catalyst to the reaction vessel at the same time, and heat and stir to react;

[0022] (2) Dissolve the reactants thoroughly in water, terminate the reaction, and remove excess reactants from the system by vacuum filtration under reduced pressure.

[0023] (3) Extract the reaction solution 3 to 5 times, take the upper extract, evaporate by rotary evaporation, refrigerate to crystallize, filter, and dry to obtain the monocaffeoylglycerol.

[0024] More preferably, in step (1), the catalyst is 10% by volume of lysozyme 435; the molar ratio of ethyl caffeate to glycerol is 1:1 to 1:10; the reaction temperature is 65±10℃; and the reaction time is 24±12h.

[0025] More preferably, in step (3), the extraction step is to add dichloromethane of the same volume as the reaction solution for extraction; the rotary evaporation temperature is 50±5℃; the cold crystallization time is 6~16h; and the drying conditions are drying in a cold and low humidity environment for at least 48h.

[0026] The present invention also provides a biomimetic mussel photothermal gel loaded with polyphenols prepared by the above preparation method.

[0027] The present invention also provides the application of the above-mentioned polyphenol-loaded biomimetic mussel photothermal gel in wound healing.

[0028] The beneficial effects of this invention are:

[0029] 1. The biomimetic mussel photothermal hydrogel dressing loaded with polyphenols prepared above exhibits both collagenase and photothermal responses. The release of polyphenols and the degradation process of the hydrogel can be controlled by the collagenase level of the wound and the needs of the wound surface. In addition, this hydrogel has good biocompatibility and is safe for application on wounds.

[0030] 2. The aforementioned biomimetic mussel photothermal hydrogel dressing loaded with polyphenols can promote nerve repair by upregulating the expression of nerve repair factors; it achieves the antibacterial effect of the hydrogel by combining the antibacterial ability of polyphenols and the photothermal effect; the gelatin hydrolysate of the gelatin hydrogel further provides nutritional support for wound repair; and it promotes angiogenesis and blood circulation in the wound by combining the near-infrared "heat therapy" effect. This hydrogel can simultaneously target the three major pathological bases of diabetic wounds and promote the repair of diabetic wounds. Attached Figure Description

[0031] Figure 1 The chemical structure of the monocaffeoylglycerol prepared in Example 1 is shown.

[0032] Figure 2 Mass spectrometry characterization of the monocaffeoylglycerol ester prepared in Example 1.

[0033] Figure 3 The FTIR spectrum of the biomimetic photothermal gel prepared in Example 2 is shown.

[0034] Figure 4 The image shows the SEM pattern of the biomimetic photothermal gel prepared in Example 2.

[0035] Figure 5 Photothermal image of the biomimetic photothermal gel prepared in Example 2 under NIR irradiation.

[0036] Figure 6 This is a diagram showing the joint adhesion effect of the biomimetic photothermal gel prepared in Example 2.

[0037] Figure 7 The polyphenol release behavior of the biomimetic photothermal gel prepared in Example 2 under the presence of collagenase and NIR irradiation.

[0038] Figure 8 The biocompatibility of the biomimetic photothermal hydrogel prepared in Example 2 is shown in Figure A, which represents RAW263.7 macrophages, Figure B represents SH-SY5Y nerve cells, and Figure C represents HaCat epidermal cells. The horizontal axis represents extracts from different systems, where M represents 1-MCG-PDP hydrogel extract and C represents CA-PDP hydrogel extract.

[0039] Figure 9 The biomimetic photothermal gel prepared in Example 2 was used to promote wound healing in rats.

[0040] Figure 10 Immunohistochemical image of the blood vessels in rat wounds promoted by the biomimetic photothermal hydrogel prepared in Example 2.

[0041] Figure 11 The biomimetic photothermal gel prepared in Example 2 promotes the expression level of nerve repair factors in rat wounds. Detailed Implementation

[0042] Example 1

[0043] Example 1: Preparation and characterization of monocaffeoyl glycerol (1-MCG)

[0044] (1) Preparation of 1-MCG: In a 25 mL jacketed flask, a transesterification reaction was carried out between 3 mmol of ethyl caffeate (EC) and 30 mmol of glycerol catalyzed by 10% (w / w) Lysozyme 435. The reaction was carried out in a thermostat at 65 °C and 300 rpm for 24 h. After dissolving thoroughly in 500 mL of distilled water, the immobilized enzyme particles and undissolved ethyl caffeate were removed by vacuum filtration. Subsequently, the mixture was extracted 3–5 times with an equal volume of dichloromethane. The lower and middle layers in the separatory funnel were discarded, and the upper extract was retained. The extract was then rotary evaporated at 50 °C to remove excess dichloromethane. The reaction solution was then placed in a refrigerator overnight for crystallization. Subsequently, the liquid was filtered to obtain white crystalline 1-MCG, which was dried under refrigerated and low-humidity conditions for at least 48 h. The dried white powder was collected and stored at 4 °C in the dark.

[0045] (2) Mass spectrometric characterization of monocaffeoylglycerol (1-MCG): The molecular weight and molecular formula of the water-soluble caffeic acid structure oil were identified using an Agilent 1290 / maXis impact high-resolution mass spectrometer. Detection conditions: Ion source: ESI + and ESI - Capillary voltage: 3.5kV; cone voltage: 2kV; mass-to-charge ratio scanning range: 200~1000m / z.

[0046] The sample and mobile phase were prepared according to the above high-performance liquid chromatography method. After injection, the sample was separated by an SB-C18 reversed-phase column (RRHD 1.8μm 2.1×50mm). The substances in the sample entered the high-resolution mass spectrometer in order of decreasing polarity. After electrospray ionization, the charged particles entered the ion hydrazine mass analyzer. The charged particles were detected, and the molecular ion peak and fragment peak of monocaffeic acid glycerol were determined based on the liquid phase elution time.

[0047] Figure 1 and Figure 2 The figures show the chemical structure and mass spectrometry characterization of 1-MCG. The product molecular formula, as shown in the figure, is C1.12 H 14 O6 has a molar mass of 254 g / mol.

[0048] Example 2

[0049] This embodiment provides a method for preparing a biomimetic mussel photothermal hydrogel loaded with polyphenols, the preparation method comprising:

[0050] Add 32 mg of dopamine hydrochloride to 20 mL of PBS (pH 8.8, 10 mM) buffer solution and stir at 800 rpm for 20 min to oxidize. Add 4 g of gelatin (20% w / w) to the obtained polydopamine (PDP) solution, gradually heat the system to 45 °C, stir at 500 rpm and swell for 30 min. Add 18 mg of CA or 25.6 mg of 1-MCG to the swollen gelatin-PDP solution system, stir continuously for 10 min, and then inject the hydrogel into the corresponding container. Spread the hydrogel using the flow casting method, let it stand at room temperature for 30 min to initially form a gel, and then refrigerate at 4 °C overnight to form a gel. The hydrogels in each group are: Gel hydrogel: 20% w / w gelatin hydrogel; PDP hydrogel: PDP + 20% w / w gelatin hydrogel; CA-PDP hydrogel: CA + PDP + 20% w / w gelatin hydrogel; 1-MCG-PDP hydrogel: 1-MCG + PDP + 20% w / w gelatin hydrogel.

[0051] Example 3

[0052] Characterization of hydrogel dressings

[0053] FTIR characterization of hydrogels

[0054] Thin hydrogel films (2 mm thick) of different groups of hydrogels were prepared according to Example 1. After freeze-drying, they were characterized by Fourier transform infrared spectroscopy (FTIR). The FTIR-ATR mode of a Fourier transform infrared spectrometer (Tensor 37, Bruker) was used for detection. During testing, a small amount of freeze-dried hydrogel sample was cut with a fine blade and placed on the ATR (Attenuated Total Reflection) detection module for scanning, with a scan count of 64. Data analysis and processing were performed using OMNIC software, followed by plotting using Origin 8.

[0055] Depend on Figure 3 Compared to gel hydrogels, PDP hydrogels, CA-PDP hydrogels, and 1-MCG-PDP hydrogels exhibit better performance at 1280 cm⁻¹. -1A new peak appears, corresponding to the stretching vibration of CN. The appearance of the new peak may be due to the interaction between the amino groups in gelatin and the phenolic hydroxyl groups in PDP. In addition, the intensity of this peak is greater in CA-PDP hydrogel and 1-MCG-PDP hydrogel than in PDP hydrogel.

[0056] SEM characterization of hydrogels

[0057] The surface morphology of the hydrogel was observed using a scanning electron microscope (SEM). The general procedure was as follows: First, a thin hydrogel film (1 mm thick) was prepared according to Example 2. Then, the hydrogel film was freeze-dried. During the measurement, different freeze-dried hydrogels were cut into appropriate sizes, and the samples were attached to the sample stage with conductive adhesive. After sputtering gold for 30 min, the SEM images of the samples were obtained by SEM (Zeiss EVO18, Carl Zeiss).

[0058] Depend on Figure 4 It can be seen that the addition of PDP reduces the pore structure of the hydrogel and introduces more ridges and microfibers. The formation of these microfibers can be attributed to the π-π and hydrogen bond interactions between PDP and gelatin chains. Further addition of 1-MCG results in a more regular, dense, and ordered overall hydrogel structure with uniform pore size and no cracks. While a noticeable pore structure also appears in CA-PDP colloid, its structural order and pore uniformity are inferior to those of 1-MCG-PDP.

[0059] Photothermal effect of hydrogels

[0060] One mL of the hydrogel solution prepared according to Example 2 was placed in a 1.5 mL centrifuge tube and incubated overnight at 4°C to form a gel. Before testing, the centrifuge tube containing the hydrogel was placed at room temperature for 1 hour to allow the hydrogel temperature to reach room temperature. Subsequently, the hydrogel was irradiated with an 808 nm infrared laser module (2W), with the light source 20 cm away from the centrifuge tube. The highest temperature in the centrifuge tube was recorded every 30 seconds using an infrared thermal imager (Hikvision H10), and thermal images were taken every 2 minutes for a total of 10 minutes. Each group was repeated three times. The highest temperature of the hydrogel at different times was recorded.

[0061] Depend on Figure 5 It is known that gel hydrogels have almost no photothermal effect, while hydrogels containing PDP all have good photothermal conversion efficiency, with PDP hydrogels exhibiting the highest conversion efficiency. The addition of 1-MCG and CA reduces the thermal response of PDP hydrogels to some extent. This may be due to the antioxidant effect of 1-MCG and CA, which reduces the auto-oxidative polymerization of dopamine during gelatin swelling during the preparation process, thus resulting in a slower heating rate compared to PDP colloids.

[0062] Joint adhesion of hydrogels

[0063] Different hydrogels, each measuring 11cm × 3cm × 3mm, were applied to a horizontal human wrist joint. The wrist was then gradually moved to 45° and 90° before returning to a horizontal position. Photos were taken to observe the adhesion of the hydrogels during movement.

[0064] Depend on Figure 6 It was found that when gel hydrogel was applied to the wrist joint, as the wrist moved from 45° to 90°, the edges of the gel gradually lifted up. When the wrist returned to a horizontal position, the middle of the hydrogel arched and detached from the skin, indicating that the gel hydrogel could not adhere tightly to the skin of the joint. In contrast, PDP hydrogel, CA-PDP hydrogel, and 1-MCG-PDP hydrogel all showed better adhesion to the joint during movement: when the wrist moved, the hydrogel adhered tightly to the wrist without lifting or arching.

[0065] Polyphenol release behavior of photothermal hydrogels in the presence of collagenase and under NIR irradiation

[0066] To further investigate the release behavior of 1-MCG-PDP and CA-PDP hydrogels at wound sites, this study constructed three release systems: PBS, collagenase + PBS, and collagenase + NIR + PBS. The concentrations of 1-MCG and CA in these systems were quantified using HPLC-UV peak integration to explore the release characteristics of 1-MCG and CA under different conditions, aiming to match the drug administration concentration with the pathological requirements of the wound. Standard solutions of CA and 1-MCG (1000, 500, 250, 125, 62.5, 31.25, and 15.65 μM) were prepared using PBS (pH 6, 10 mM). After filtration through a 0.22 μm syringe filter, the solutions were placed in HPLC vials, with three replicates per group. Sample detection was performed using HPLC, with the following specific method: mobile phase A was ultrapure water; mobile phase B was methanol; gradient elution was used, specifically: from 0-6 min, phase B concentration increased from 35% to 75%; from 6-9 min, phase B concentration decreased from 75% to 35%; and from 9-12 min, phase B concentration was maintained at 35%. The UV detector wavelength was 325 nm, and the injection volume was 5 μL. The corresponding peaks were integrated. The integrated areas and corresponding concentrations were entered into Excel, compiled, and then imported into Prism 8 to plot a standard curve of CA and 1-MCG concentration versus peak area, and a linear equation was obtained through fitting.

[0067] Subsequently, hydrogel samples with a diameter of 4 cm and a thickness of 3 mm prepared according to Example 2 were taken and placed in 20 mL of PBS (pH 6, 10 mM) or PBS containing 5 U / mL collagenase (pH 6, 10 mM). They were incubated at 25 °C for 60 min; the NIR+collagenase group was simultaneously irradiated with NIR for 60 min. The reaction was terminated and samples were taken at 2, 4, 6, 8, 10, 15, 20, 25, 30, 40, 50, and 60 min, with three replicates for each group. 800 μL of the supernatant was taken and filtered using a 0.22 μm syringe filter. The content of CA or 1-MCG in the supernatant was determined using HPLC-UV detection at a wavelength of 325 nm with an injection volume of 5 μL. After obtaining the integrated area, the release amount of CA / 1-MCG was calculated using a standard curve of CA and 1-MCG. The data were then imported into Prism to plot the release of CA and 1-MCG in the hydrogel over time.

[0068] Depend on Figure 7 It can be seen that 1-MCG-PDP hydrogel and CA-PDP hydrogel both possess controlled release properties that respond to collagenase and NIR photothermal response.

[0069] Biocompatibility of hydrogels

[0070] Cell compatibility is crucial for hydrogel dressings. 1-MCG-PDP and CA-PDP hydrogel samples, prepared according to Example 2 (4 cm diameter, 3 mm thickness), were placed in three release systems: 20 mL of PBS (pH 6, 10 mM), PBS containing 5 U / mL collagenase, or PBS containing 5 U / mL collagenase combined with NIR irradiation. After 24 h, the supernatant was collected and vortexed. The mixed supernatant was filtered through a 0.22 μm aqueous injection needle filter. Next, 50 μL of the hydrogel extract was mixed with 50 μL of DMEM medium containing 10% FBS and co-cultured with RAW 264.7, SH-SY5Y, or HaCat cells in 96-well plates at 37°C and 5% CO2 for 24 h. Cell viability was assessed using CCK 8 assay.

[0071] Depend on Figure 8 It can be seen that the survival rate of RAW 264.7, SH-SY5Y and HaCat cells cultured with hydrogel extracts obtained by different methods is higher than 85%, indicating that the 1-MCG-PDP hydrogel prepared in this study has good biocompatibility with CA-PDP hydrogel extract.

[0072] Example 3

[0073] Hydrogel promotes the repair of diabetic wounds infected with S. aureus.

[0074] Thirty diabetic rats were randomly divided into 5 groups (n=6). The rats were anesthetized, shaved, and depilated with hair removal cream. Four circular skin defects, each 10 mm in diameter, were made on each side of the back of each rat, extending to the subcutaneous layer. 100 μL of Staphylococcus aureus suspension (10...) was then added... 8 Inoculate the wound with CFU / mL and seal the wound for 24 hours to create an infected wound model.

[0075] Rats with successful modeling were divided into five groups: a control group, a gel group, a PDP group, a CA-PDP group, and a 1-MCG-PDP group. The control group was covered with 3M adhesive, while the other groups received different hydrogels plus 3M adhesive. Wounds were cleaned and treated every 3 days: after cleaning, 150 μL of 10 mm diameter, 2 mm thick hydrogel was applied to the wounds of different groups, followed by irradiation with an 808 nm laser for 10 minutes. After photothermal treatment, the wounds were bandaged with 3M adhesive. On days 0, 3, 7, 10, and 14 of treatment, rats were anesthetized, and the wounds and surrounding areas were cleaned. A 10 mm diameter circular ruler was used to wrap the wounds, and the wounds of the infected diabetic rats after different hydrogel treatments were photographed at a certain distance using a camera (Z6Ⅱ, Nikon). The wound area was statistically analyzed using ImageJ. After the photo was taken, a new hydrogel dressing was applied, and NIR treatment was performed using an 808nm laser (LR-MFJ-808 / 5000mW, Changchun New Industries). An infrared imager (E85, USA) was used to control the treatment temperature below 45℃. After NIR irradiation for 10 minutes, the wound was wrapped with 3M adhesive.

[0076] Depend on Figure 9 It was found that in 1-MCG-PDP hydrogel and CA-PDP hydrogel, the photothermal effect of PDP hydrogel combined with the antibacterial effect of 1-MCG / CA led to an enhanced antibacterial ability of the hydrogel. The wound area of ​​diabetic rats treated with all hydrogels gradually decreased over time, and the order of wound recovery from fastest to slowest among the different groups was: 1-MCG-PDP group > CA-PDP group > PDP group > Gel group > Control group.

[0077] Immunohistochemical analysis of angiogenesis in wounds of diabetic rats

[0078] Immunohistochemical analysis was performed on wound tissue collected from diabetic rats on day 7 of treatment. Paraffin sections were dewaxed and washed. The washed tissue sections were placed in a retrieval chamber filled with EDTA antigen retrieval buffer (pH 9.0) and incubated in a microwave oven for antigen retrieval. After washing with PBS (pH 7.4) for 15 min, the sections were placed in 3% hydrogen peroxide solution and incubated in the dark for 25 min. They were then washed again with PBS (pH 7.4) for 15 min. After slightly drying the sections, circles were drawn around the tissue using a histochemical pen (GT1001, Genentech). Then, 3% BSA was added to the circle and incubated for 30 min. Subsequently, primary antibody (CD 31:PBS = 1:2000) was added to the section, and the section was incubated overnight at 4°C in a humidified chamber. The sections were washed again with PBS (pH 7.4). After drying the sections, HRP-labeled goat anti-rabbit secondary antibody (1:200, diluted with PBST) was added to the circle to cover the tissue, and the section was incubated in the dark at room temperature for 50 min. Then, the slides were washed in PBS (pH 7.4) with agitation for 15 min. After slightly drying the sections, DAB chromogenic agent was added to the inner circle. After complete development, the slides were rinsed with running water, counterstained with hematoxylin, differentiated with 1% hydrochloric acid ethanol, rinsed with running water, blued with ammonia, and rinsed with running water. Finally, the slides were dehydrated with anhydrous ethanol and mounted with xylene-based neutral resin. After staining, the slides were observed and images were acquired under a Nikon inverted fluorescence microscope.

[0079] Depend on Figure 10 It was found that after 7 days of treatment with different hydrogels, the vascular density of different groups, from highest to lowest, was: 1-MCG-PDP group > CA-PDP group > PDP group > Gel group > Control group. The number of blood vessels in the 1-MCG-PDP group and CA-PDP group was significantly higher than that in the other groups, and the vascular density of the 1-MCG-PDP group was the best.

[0080] Expression levels of nerve repair factors in wounds of diabetic rats

[0081] Immunofluorescence full scan analysis was performed on wound tissue from diabetic rats on day 14 of treatment. Paraffin sections were dewaxed and washed. The tissue sections were then placed in a retrieval chamber filled with citrate antigen retrieval buffer (pH 6.0) and subjected to antigen retrieval in a microwave oven, followed by washing with PBS for 15 min. After slightly drying the sections, circles were drawn around the tissue with a histochemical pen, and autofluorescence quencher was added to the circle for 5 min, followed by rinsing with running water for 10 min. 3% BSA was added to the circle and incubated for 30 min. After removing the blocking solution, primary antibody (GFAP:PBS = 1:200) was added to the section, and the section was incubated overnight at 4°C in a humidified chamber. The overnight slide was washed with PBS (pH 7.4) for 15 min. After slightly drying the sections, HRP-labeled goat anti-rabbit secondary antibody (1:400, diluted with PBST) was added to the circle to cover the tissue, and the section was incubated at room temperature in the dark for 50 min, followed by washing with PBS (pH 7.4). Add DAPI staining solution and incubate at room temperature in the dark for 10 min to counterstain cell nuclei. Wash and dry the sections, then mount them with anti-fluorescence quenching mounting medium. Observe and image the obtained sections under a Nikon fluorescence microscope.

[0082] Depend on Figure 11 It was found that, compared with the Control group, neither the Gel group nor the PDP group showed a significant increase in GDNF levels. However, the GDNF levels in both the CA-PDP hydrogel treatment group and the 1-MCG-PDP hydrogel treatment group were significantly increased, with the 1-MCG-PDP hydrogel treatment group showing the highest GDNF level. This result indicates that 1-MCG can promote the growth and survival of neurons at the wound site by regulating the expression level of GDNF in the wound.

[0083] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a biomimetic mussel photothermal hydrogel loaded with polyphenols, characterized in that, The specific steps include: S1: Dissolve dopamine hydrochloride in alkaline PBS buffer to obtain a polydopamine solution; S2: Add gelatin to the polydopamine solution obtained in step S1, stir, heat and swell to obtain a melt; S3: Add polyphenols to the melt described in step S2, stir and mix well, spread evenly, and let stand and refrigerate to obtain the biomimetic mussel photothermal hydrogel loaded with polyphenols; the polyphenols are monocaffeoylglycerol esters. In step S2, the concentration of the gelatin in the polydopamine solution is 5~50% w / w; In step S3, the structural formula of the monocaffeoylglycerol is: ； The concentration of the polyphenol is 0.1~100 mM.

2. The preparation method according to claim 1, characterized in that, The monocaffeoylglycerol ester was prepared by the following method: (1) Add ethyl caffeate, glycerol and catalyst to the reaction vessel at the same time, and heat and stir to react; (2) Dissolve the reactants completely in water, terminate the reaction, and then remove excess reactants from the system by vacuum filtration under reduced pressure. (3) Extract the reaction solution 3 to 5 times, take the upper extract, evaporate by rotary evaporation, refrigerate to crystallize, filter, and dry to obtain the monocaffeoylglycerol.

3. The preparation method according to claim 1, characterized in that, In step S1, the concentration of dopamine hydrochloride is 1~100 mM; The alkaline PBS buffer has a pH of 7-10 and a concentration of 5-100 mM. During the dissolution process, the stirring speed is 200~1500 rpm and the stirring time is 0.1~36 h.

4. The preparation method according to claim 1, characterized in that, In step S2, the stirring speed is 200~1500 rpm, the heating and swelling temperature is 35~60℃, and the swelling time is 20~60 min.

5. The preparation method according to claim 1, characterized in that, In step S3, the stirring speed is 200~1500 rpm, the stirring temperature is 35~60℃, and the stirring time is 5~60 min; the refrigeration temperature is 4~10℃, and the refrigeration time is 6~24 hours.

6. The preparation method according to claim 2, characterized in that, In step (1), the catalyst is 10% lipozyme 435 by volume; the molar ratio of ethyl caffeate to glycerol is 1:1 to 1:10; the reaction temperature is 65±10℃ and the reaction time is 24±12h.

7. The preparation method according to claim 2, characterized in that, In step (3), the extraction step is to add dichloromethane of the same volume as the reaction solution for extraction; the rotary evaporation temperature is 50±5℃; the cold crystallization time is 6~16h; and the drying conditions are drying in a cold and low humidity environment for at least 48h.

8. A biomimetic mussel photothermal gel loaded with polyphenols prepared by the method described in any one of claims 1 to 7.

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