Melanin-hydroxypropyl chitosan / hyaluronic acid-bisamino polyethylene glycol composite hydrogel and its preparation method and application

Abstract

The present invention discloses a melanin-hydroxypropyl chitosan / hyaluronic acid-bisamino polyethylene glycol composite hydrogel and its preparation method and application. The present invention first improves the properties of CS, HA and melanin, grafts melanin onto HPCS to prepare M-HPCS to improve its solubility, and then mixes M-HPCS and HA-PEG to form a composite hydrogel, and soluble metal ions can be added. The two composite hydrogels prepared by the present invention have a strong degree of internal network cross-linking and better toughness; and have good photothermal properties and can be used for photothermal therapy of wound repair; have strong antioxidant properties and good free radical scavenging ability. The hydrogel is non-cytotoxic and has good biocompatibility; has good ability to promote cell growth; and can promote the formation of new blood vessels. After actual verification, the composite hydrogels prepared by the present invention can promote wound healing by promoting the formation of new blood vessels.

Description

Technical Field

[0001] The invention belongs to the technical field of hydrogel preparation, and particularly relates to a melanin-hydroxypropyl chitosan / hyaluronic acid-bisamino polyethylene glycol composite hydrogel and a preparation method and application thereof. Background Art

[0002] A variety of dressings (including films, semipermeable foams, hydrocolloids, and hydrogels) have been studied for the treatment of chronic wounds. Hydrogels, due to their excellent structural and functional properties, hold broad application prospects in wound repair. In recent decades, natural polymers such as chitosan, hyaluronic acid, sodium alginate, and glucose have been widely used in the preparation of hydrogel dressings.

[0003] Currently, the research on hydrogel dressings is still in the extensive research stage. The present invention conducts research in order to design a hydrogel dressing for chronic wounds with good performance. Summary of the Invention

[0004] The present invention aims to provide a melanin-hydroxypropyl chitosan (M-HPCS) / hyaluronic acid-bisamino polyethylene glycol (HA-PEG) hydrogel, and also provides a preparation method and application of the composite hydrogel to overcome the deficiencies of the prior art.

[0005] To achieve the above object, the present invention is achieved through the following technical solutions:

[0006] A melanin-hydroxypropyl chitosan / hyaluronic acid-bisamino polyethylene glycol (M-HPCS / HA-PEG) composite hydrogel is prepared by mixing hydroxypropyl chitosan grafted with melanin and hyaluronic acid-bisamino polyethylene glycol.

[0007] Furthermore, the preparation method of the composite hydrogel comprises the following steps:

[0008] (1) Chitosan was hydrophilically modified by etherification reaction to obtain hydroxypropyl chitosan, and then melanin was grafted onto HPCS through amide reaction to prepare M-HPCS; HA-PEG was prepared through amide reaction;

[0009] (2) preparing an M-HPCS solution and a HA-PEG solution, and then evenly mixing the M-HPCS solution and the HA-PEG solution and placing them in a water bath to prepare a composite hydrogel.

[0010] Furthermore, in step (2), the prepared M-HPCS and HA-PEG are dissolved in deionized water to prepare solutions with concentrations of 7% and 4% (w / v), respectively; the ratio of the M-HPCS solution to the HA-PEG solution is 3-7:7-3 (v / v).

[0011] Furthermore, the M-HPCS solution: HA-PEG solution is 7:3 (v / v); the water bath temperature is 45° C.-60° C., preferably 60° C. for 30 minutes.

[0012] The invention discloses an M-HPCS / HA-PEG / soluble metal ion composite hydrogel. The composite hydrogel is prepared by stirring and mixing a hydroxypropyl chitosan solution grafted with melanin and a hyaluronic acid-bisamino polyethylene glycol solution, and adding a soluble metal ion solution at the same time.

[0013] The metal ion can be a magnesium ion, that is, a M-HPCS / HA-PEG+Mg 2+ The composite hydrogel is prepared by mixing a hydroxypropyl chitosan solution grafted with black pigment and a hyaluronic acid-bisamino polyethylene glycol solution while adding Mg 2+ Made of solution.

[0014] Furthermore, the preparation method of the M-HPCS / HA-PEG / soluble metal ion composite hydrogel comprises the following steps:

[0015] (1) Chitosan was hydrophilically modified by etherification reaction to obtain hydroxypropyl chitosan, and then melanin was grafted onto HPCS through amide reaction to prepare M-HPCS; HA-PEG was prepared through amide reaction;

[0016] (2) preparing an M-HPCS solution and a HA-PEG solution, then uniformly mixing the M-HPCS solution and the HA-PEG solution, adding a soluble metal ion solution dropwise while mixing, and placing the solution in a water bath for a certain period of time to obtain the composite hydrogel.

[0017] Furthermore, in step (2), the prepared M-HPCS and HA-PEG are dissolved in deionized water to prepare solutions with concentrations of 7% and 4% (w / v), respectively; the ratio of the M-HPCS solution to the HA-PEG solution is 3-7:7-3 (v / v).

[0018] Furthermore, the ratio of the M-HPCS solution to the HA-PEG solution is 3:7 (v / v); the concentration of the added soluble metal ion solution is 4% (w / v), and the added volume percentage is 5%.

[0019] The M-HPCS / HA-PEG composite hydrogel or the M-HPCS / HA-PEG / soluble metal ion composite hydrogel is used in the preparation of products that resist oxidation, promote cell growth, and promote new blood vessel formation.

[0020] Application of the M-HPCS / HA-PEG composite hydrogel or the M-HPCS / HA-PEG / soluble metal ion composite hydrogel in the preparation of photothermal performance products.

[0021] The M-HPCS / HA-PEG composite hydrogel or the M-HPCS / HA-PEG / soluble metal ion composite hydrogel is used in the preparation of skin wound healing products, especially in the preparation of skin wound healing products for diabetic patients.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention first improves the properties of CS, HA, and melanin. Chitosan is hydrophilically modified through an etherification reaction. Melanin is grafted onto HPCS through an amide reaction to prepare M-HPCS, improving its solubility. HA-PEG is prepared through an amide reaction to improve its mechanical properties and provide a large number of amino groups for the hydrogel network. M-HPCS and HA-PEG are then mixed to form a composite hydrogel, to which soluble metal ions can be added.

[0024] The two composite hydrogels prepared by the present invention have an adhesive force suitable for wound repair. They can not only stably adhere to the skin, but also avoid secondary damage to the wound and surrounding skin during the peeling process. The internal network cross-linking degree of the hydrogel is strong, and the toughness is better. It also has good photothermal properties and can be used for photothermal therapy of wound repair. It has strong antioxidant properties and good free radical scavenging ability. The hydrogel is non-cytotoxic and has good biocompatibility. It has good cell growth promotion ability and can promote the formation of new blood vessels. Experimental results show that the composite hydrogels prepared by the present invention can promote wound healing by promoting the formation of new blood vessels. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 (I) Infrared spectra of melanin (A), chitosan, hydroxypropyl chitosan, melanin-hydroxypropyl chitosan (B), hyaluronic acid and hyaluronic acid-bisamino polyethylene glycol (C); UV-visible spectrum of melanin (D); NMR spectra of hydroxypropyl chitosan, melanin-hydroxypropyl chitosan (E), hyaluronic acid and hyaluronic acid-bisamino polyethylene glycol (F); (II) Scanning electron microscopy (A) and transmission electron microscopy (B) images of melanin; (III) Scanning electron microscopy (A) and transmission electron microscopy (B) images of melanin-hydroxypropyl chitosan; (IV) Particle size distribution (A) and potential distribution (C) of melanin; Particle size distribution (B) and potential distribution (D) of melanin-hydroxypropyl chitosan.

[0026] Figure 2The gelation pictures of M-HPCS / HA-PEG hydrogel (A); M-HPCS / HA-PEG (B) and M-HPCS / HA-PEG+Mg 2+ (C) Schematic diagram of the hydrogel gelation network.

[0027] Figure 3 The scanning electron micrograph (A), porosity (B) and pore size (C) of M-HPCS / HA-PEG hydrogel, M-HPCS / HA-PEG+Mg 2+ Scanning electron microscopy images of the hydrogels (D, porosity (E) and pore size (F) (*p < 0.05, **p < 0.01, ***p < 0.001).

[0028] Figure 4 are photos of M-HPCS / HA-PEG hydrogels adhered to different substrates.

[0029] Figure 5 Schematic diagram of hydrogel adhesion (A); adhesion strength of M-HPCS / HA-PEG (B) and M-HPCS / HA-PEG+Mg2+ (C) (*p<0.05, **p<0.01, ***p<0.001).

[0030] Figure 6 M-HPCS / HA-PEG and M-HPCS / HA-PEG+Mg 2+ Temperature sweep of hydrogel modulus.

[0031] Figure 7 M-HPCS / HA-PEG and M-HPCS / HA-PEG+Mg 2+ Rheological properties of hydrogels. Frequency sweep (A) and strain sweep (B) of the modulus of M-HPCS / HA-PEG hydrogel; M-HPCS / HA-PEG+Mg 2+ Frequency sweep (C) and strain sweep (D) of hydrogel modulus.

[0032] Figure 8 M-HPCS / HA-PEG hydrogel (A) and M-HPCS / HA-PEG+Mg 2+ Swelling ratio of hydrogel (B); M-HPCS / HA-PEG and M-HPCS / HA-PEG+Mg 2+ Photothermal properties of hydrogels: Temperature rise curves of M-HPCS / HA-PEG hydrogels at different powers (C). M-HPCS / HA-PEG hydrogels at 1 W / cm 2 Temperature rise curve (D) and thermal imaging (E) under different powers; M-HPCS / HA-PEG+Mg under different powers 2+Heating curve of hydrogel (F), M-HPCS / HA-PEG+Mg 2+ Hydrogel at 1W / cm 2 Temperature rise curve (G) and thermal imaging (H) under power; M-HPCS / HA-PEG (I) and M-HPCS / HA-PEG+Mg 2+ (J) Photothermal stability of hydrogels.

[0033] Figure 9 DPPH scavenging ability (A) and ABTS scavenging ability (B) of M-HPCS / HA-PEG+Mg 2+ DPPH scavenging ability (C) and ABTS scavenging ability (D) of hydrogel.

[0034] Figure 10 The relative proliferation rates of L929 cells (A) and HUVECs cells (B) after treatment with different concentrations of M-HPCS7 / HA-PEG3 hydrogel extract at 24h and 48h.

[0035] Figure 11 The cell growth status of L929 cells and HUVECs cells after treatment with different concentrations of M-HPCS7 / HA-PEG3 hydrogel extract at 24h and 48h (200×; Bar=200μm).

[0036] Figure 12 Different concentrations of M-HPCS3 / HA-PEG7+Mg 2+ Relative proliferation rates of L929 cells (A) and HUVECs cells (B) after treatment with hydrogel extract at 24h and 48h.

[0037] Figure 13 Different concentrations of M-HPCS3 / HA-PEG7+Mg 2+ Cell growth status of L929 cells and HUVECs cells after treatment with hydrogel extract at 24h and 48h (Bar = 200μm)

[0038] Figure 14 This is the growth status of L929 cells after co-culture with different hydrogel extracts for 1, 3, 5, and 7 days.

[0039] Figure 15 Figure 3 HUVEC cell migration after co-culture with hydrogel extract for 0, 12, 24, and 36 hours. (n = 5, *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control group).

[0040] Figure 16Figure 3 shows the migration of L929 cells after co-culture with the hydrogel extract for 0, 12, 24, and 36 hours (n = 5, *p < 0.05, **p < 0.01, ***p < 0.001 compared with the control group).

[0041] Figure 17 Figure 1 shows the angiogenesis of HUVEC cells after co-culture with hydrogel extract for 2, 4, 6, and 8 hours. (A) Statistical analysis of angiogenesis at 8 hours (B) Statistical analysis of vascular nodes at 8 hours (C) Statistical analysis of tubular formation at 8 hours (*p < 0.05, **p < 0.01, ***p < 0.001).

[0042] Figure 18 The hydrogel's antioxidant effect at the cellular level. (A) Effects of different H2O2 concentrations on L929 cell viability; (B) Protective effect of the hydrogel on H2O2-stimulated L929 cells; (C) DCFH-DA fluorescence staining was used to assess the ROS-scavenging ability of the hydrogel (*p < 0.05, **p < 0.01, ***p < 0.001).

[0043] Figure 19 M-HPCS7 / HA-PEG3 hydrogel and M-HPCS3 / HA-PEG7+Mg 2+ Hydrogel used for wound healing of diabetic skin burns. Photos of wound healing in different treatment groups at 0, 7, 14, 21, and 28 days. Schematic diagram of diabetic skin wound treatment and photos of wound healing.

[0044] Figure 20 Wound healing rates of diabetic skin burns treated with M-HPCS7 / HA-PEG3 hydrogel and M-HPCS3 / HA-PEG7+Mg2+ hydrogel were analyzed. Wound healing rates were calculated for the different treatment groups at 0, 7, 14, 21, and 28 days (*p<0.05, **p<0.01, ***p<0.001 compared to the control group).

[0045] Figure 21 M-HPCS7 / HA-PEG3 hydrogel (A) and M-HPCS3 / HA-PEG7+Mg under different powers 2+ Photothermal heating of hydrogel (B) at the wound site of rats and at 1.5W / cm 2 Thermal image under power (C).

[0046] Figure 22 These are the hematoxylin-eosin (H&E) staining images of the wound sites of diabetic skin burn wounds in different treatment groups 0, 7, 14, 21, and 28 days after treatment.

[0047] Figure 23These are Masson staining images of the wound sites of diabetic skin burn wounds in different treatment groups 0, 7, 14, 21, and 28 days after treatment.

[0048] Figure 24 M-HPCS / HA-PEG hydrogel and M-HPCS / HA-PEG+Mg 2+ In vivo anti-inflammatory ability of hydrogels. Wound tissues were immunofluorescently stained for iNOS (red) and cell nuclei (DAPI, blue) on day 7 after treatment with diabetic skin wounds in different treatment groups.

[0049] Figure 25 M-HPCS / HA-PEG hydrogel and M-HPCS / HA-PEG+Mg 2+ In vivo anti-inflammatory ability of hydrogels. Wound tissues were immunofluorescently stained for CD206 (green) and cell nuclei (DAPI, blue) on day 7 after treatment with diabetic skin wounds in different treatment groups.

[0050] Figure 26 M-HPCS / HA-PEG hydrogel and M-HPCS / HA-PEG+Mg 2+ Immunofluorescence staining of α-SMA (green) and cell nuclei (DAPI, blue) was performed on the wound tissues of diabetic skin wounds treated with different treatment groups on day 7.

[0051] Figure 27 M-HPCS / HA-PEG hydrogel and M-HPCS / HA-PEG+Mg 2+ Immunofluorescence staining of CD31 (red) and cell nuclei (DAPI, blue) was performed on the wound tissues of diabetic skin wounds in different treatment groups on day 7. DETAILED DESCRIPTION

[0052] The technical solution of the present invention is further described below with reference to embodiments.

[0053] The following reagents were required for the experiment: melanin extracted from the needleless cuttlefish, chitosan (Mw: 40 kDa, DD: 95.43%), commercially available reagents: hyaluronic acid (HA), carbodiimide hydrochloride (EDC·HCL) and N-hydroxysuccinimide (NHS), isopropyl alcohol, propylene oxide, and magnesium chloride hexahydrate (MgCl2·6H2O), HUVEC human umbilical vein endothelial cells and L929 mouse fibroblasts, DMEM high-glucose medium, Calcein-AM / PI kit and DCFH-DA fluorescent dye, MTT, sodium pentobarbital, paraformaldehyde, potassium dihydrogen phosphate, sodium dihydrogen phosphate, citric acid, and sodium citrate, streptozotocin (STZ), hemostatic guaiac gauze (positive control), hematoxylin and eosin staining kit and modified Masson's trichrome staining kit, immunohistofluorescence kit, xylene, and neutral gum.

[0054] Example 1: In this example, melanin, hydroxypropyl chitosan (HPCS), melanin-hydroxypropyl chitosan (M-HPCS) and hyaluronic acid-bisamino polyethylene glycol (HA-PEG) were prepared.

[0055] 1. Preparation of melanin: Cuttlefish ink is taken and washed with anhydrous ethanol to obtain a primary extract of cuttlefish ink melanin; the primary extract is soaked in deionized water. After 24 hours, the precipitate is obtained by centrifugation, and the precipitate is placed in 50 times the mass of deionized water, the pH is adjusted to 10.5, 1.5wt% alkaline protease is added, and after inactivation of the enzyme, the precipitate is washed with deionized water until neutral, freeze-dried, and purified melanin is obtained, which is then stored in a refrigerator at 4°C.

[0056] 2. Preparation of HPCS: 3 g of chitosan was dissolved in 20 mL of NaOH solution with stirring, then frozen at -40°C overnight. After thawing, the solution was filtered to remove excess alkali. The chitosan was then added to 40 mL of isopropanol and stirred at room temperature for 30 min. Propylene oxide was then added dropwise and stirred in a 50°C water bath. The reaction was terminated after 24 h. The reaction product was filtered to remove excess reagents, washed several times with anhydrous ethanol, dissolved in deionized water, and adjusted to a neutral pH by the addition of 1 mol / L hydrochloric acid solution. HPCS was then dialyzed and freeze-dried to obtain HPCS.

[0057] 3. Preparation of M-HPCS: Accurately weigh a certain amount of hydroxypropyl chitosan and dissolve it in Tris buffer (0.01 g / mL) for later use. Add 0.66 g of melanin to 33 mL of Tris buffer (0.02 g / mL) and ultrasonically disperse for 20 minutes. Then, add 0.31 g of EDC and 0.23 g of NHS. Stir at room temperature for 2 hours. Then, add the fully dissolved hydroxypropyl chitosan solution dropwise and stir for another 12 hours. After the reaction, dialyze in deionized water for 48 hours to remove impurities and lyophilize.

[0058] 4. Preparation of HA-PEG: Dissolve HA in ultrapure water (0.01 g / mL). After dissolution, add 0.31 g EDC and 0.23 g NHS to activate the carboxyl group. After stirring at room temperature for 1 hour, add NH2-PEG-NH2 to the above solution and stir at room temperature for 12 hours. After the reaction is completed, dialyze in deionized water for 48 hours to remove impurities and freeze-dry.

[0059] The properties of the melanin, HPCS, M-HPCS and HA-PEG prepared above were characterized:

[0060] 1. The infrared absorption spectra of melanin, CS, HPCS, M-HPCS, HA and HA-PEG were collected using a Fourier transform infrared spectrometer. The hydrogen nuclear magnetic resonance spectra of HPCS, M-HPCS, HA and HA-PEG were obtained by nuclear magnetic resonance characterization, and the chemical structures of HPCS, M-HPCS and HA-PEG were studied.

[0061] The chemical structure of melanin was analyzed by measuring the absorbance of the melanin solution in the wavelength range of 200-600 nm using a UV-visible spectrophotometer.

[0062] Melanin and M-HPCS were dissolved in deionized water to prepare a solution. The particle size distribution, Zeta potential and polydispersity index (PDI) were measured using a Malvern laser particle size analyzer, and the morphology was observed using a scanning electron microscope and a transmission electron microscope.

[0063] Table 1

[0064]

[0065] The degree of substitution of propylene oxide and melanin in melanin-hydroxypropyl chitosan and the degree of substitution of PEG in HA-PEG were determined by elemental analysis. After freeze-drying CS, HPCS, M-HPCS, and HA-PEG, a certain amount was taken and tested in an elemental analyzer to determine the C, H, and N content of each substance. The degree of substitution of IPA, melanin, and NH2-PEG-NH2 was calculated based on the C / N ratio of the substance. The calculation formula is as follows:

[0066]

[0067]

[0068] Where (C / N) HPCS is the carbon-nitrogen ratio of HPCS, DA is the deacetylation degree of chitosan, DS IPA is the degree of substitution of propylene oxide, (C / N) M-HPCS is the carbon-nitrogen ratio of M-HPCS, DS MEis the degree of substitution of melanin, (C / N) HA-PEG is the carbon-nitrogen ratio of HA-PEG, DS PEG is the degree of substitution of PEG. By simplifying formulas 2-1, 2-2 and 2-3, the degrees of substitution can be obtained.

[0069] Result analysis:

[0070] 1. Preparation and characterization of melanin, HPCS, M-HPCS and HA-PEG

[0071] Figure 1 In the Fourier infrared spectrum of melanin, 1330cm -1 The absorption peak at 1585 cm is caused by the deformation of phenolic hydroxyl group OH and CO stretching; -1 The characteristic peak at 3408cm is attributed to the stretching vibration of C=C on the aromatic ring; -1 The strong and broad band at 1024cm in the infrared spectrum of chitosan may be attributed to the stretching vibration of OH and NH. These absorption peaks are representative characteristic peaks of melanin, and the results show that melanin extraction is successful. -1 ,1080cm -1 ,1155cm -1 and 891cm -1 The characteristic peak shows its polysaccharide structure, 1155cm -1 The characteristic peak at 1155cm is caused by the stretching vibration of CO at the C6 position of chitosan. -1 The characteristic peak at 2973 cm -1 The -CH3 stretching vibration peak appears at 1463cm -1 The -CH3 bending vibration peak appears at 1068cm -1 The characteristic peaks at 1595 cm also reflect the introduction of hydroxypropyl. -1 The absorption peak disappeared, indicating that a small amount of hydroxypropyl was introduced on -NH2, which shows that hydroxypropyl chitosan was successfully synthesized.

[0072] The 3417cm in the infrared spectrum of melanin-hydroxypropyl chitosan -1 ,1523cm -1 ,1646cm -1 The absorption peak at 1378 cm is considered to be the representative characteristic of melanin. -1 The absorption peak at 1523 cm is attributed to the OH deformation and CO stretching of the phenolic hydroxyl group; -1 The characteristic peak at 1646cm is caused by the stretching vibration of C=C on the aromatic ring; -1The stretching vibration of C=O on the amide bond is caused by 3417cm -1 The strong and broad bands were caused by the stretching vibrations of OH and NH, indicating that melanin was successfully grafted onto hydroxypropyl chitosan.

[0073] In the infrared spectrum of hyaluronic acid, 1323 cm -1 The characteristic peak at 1375 cm is caused by the bending vibration of -OH. -1 and 1406cm -1 The characteristic peak at 1563 cm is caused by CH bending vibration. -1 The characteristic peak at 1618 cm is caused by NH stretching vibration. -1 The characteristic peak at 2923cm is caused by C=O stretching vibration. -1 The characteristic peaks at 1738 cm are attributed to the stretching vibration peaks of -CH2 and -CH3. -1 The characteristic peak at 1480 cm is attributed to the stretching vibration peak of C=O in the amide bond. -1 The characteristic peak at is due to the -COC- vibration in NH2-PEG-NH2, which indicates that HA-PEG was successfully synthesized.

[0074] like Figure 1 -(Ⅱ), in the hydroxypropyl chitosan 1 In the H-NMR spectrum, the signal peaks at 0.97ppm and 1.08ppm represent the C9 proton absorption peaks, indicating the hydroxypropylation of chitosan; the signal peaks at 3.20ppm-3.84ppm represent the C3-C8 proton absorption peaks, the signal peak at 3.01ppm represents the C2 proton absorption peak, and the signal peak at 4.76ppm reflects the proton peak on C1. Compared with hydroxypropyl chitosan, 1 H-NMR spectrum of melanin-hydroxypropyl chitosan 1 In the H-NMR spectrum, H 3.49ppm, δ H A new chemical shift appeared at 2.48-2.69 ppm, which once again proved that HPCS and M-HPCS were successfully synthesized.

[0075] In HA and HA-PEG 1 In the H-NMR spectrum, the signal peak at a shift of approximately 8 ppm is the hydrogen on the NH of the amide bond, the proton peak b at a shift of approximately 1.6 ppm is the hydrogen ion on the NH2 of PEG, the peak e at approximately 3.5 ppm is the hydrogen carried by the methylene group adjacent to the oxygen in the PEG structure, and the proton peaks c and d between 2.5 and 3.0 ppm belong to the hydrogen carried by the methylene group connected to the amino group in the PEG structure. The results show that PEG was successfully grafted onto HA.

[0076] The UV-visible spectrum of melanin is shown in Figure 2. Figure 1 -(Ⅱ)-D shows that there is a maximum absorption peak at 200nm, which is attributed to the benzene ring, and the absorbance in the visible light region gradually decreases with the increase of wavelength, which is caused by the conjugated structure in the melanin structure.

[0077] Figure 1 -(III) is the scanning electron microscopy and transmission electron microscopy images of melanin. It can be clearly seen from the images that the melanin nanoparticles are uniform spheres or ellipsoids with clear edges, easy to agglomerate, and the particle size is about 200nm. Figure 1 -(IV) shows scanning electron microscopy (A) and transmission electron microscopy (B) images of M-HPCS. Figure A shows a uniform spherical or ellipsoidal shape, with an average particle size of approximately 200 nm and uniform distribution, consistent with the DLS analysis results. The results demonstrate that melanin grafted onto hydroxypropyl chitosan via an amide reaction can still self-assemble into nanospheres. The transmission electron microscopy image (Figure B) clearly demonstrates a distinct core-shell structure in M-HPCS, indicating successful melanin grafting onto hydroxypropyl chitosan.

[0078] like Figure 1 -(V) shows that the average particle size of melanin is 164±3.95nm, and PDI=0.064±0.007, indicating that the prepared melanin nanoparticles have relatively uniform particle size and good dispersibility. Figure 2-8 The zeta potential of melanin is -25.3 ± 0.36 mV. The average particle size of M-HPCS is 396 ± 0.24 nm, and the PDI is 0.18 ± 0.021, indicating that the prepared M-HPCS is nanoparticles with relatively uniform particle size and good dispersion. Figure 2-14 This is the Zeta potential detection result of M-HPCS. The Zeta potential of M-HPCS is +10.06±1.12 mV.

[0079] The C, N, and H element contents of CS, HPCS, M-HPCS, and HA-PEG were measured by elemental analysis and are shown in Table 1. The C / N ratio was calculated. According to formulas 2-1, 2-2, and 2-3 above, the deacetylation degree was 80.56%, the grafting degree of hydroxypropyl was 116%, and the melanin substitution was 87.54%, indicating that melanin was successfully grafted onto HPCS. The degree of substitution of PEG was 45.23%. The results showed that M-HPCS and HA-PEG were successfully synthesized with a high grafting rate.

[0080] Example 2: Preparation of M-HPCS / HA-PEG and M-HPCS / HA-PEG+Mg 2+ Composite hydrogel

[0081] 1. Preparation of M-HPCS / HA-PEG composite hydrogels: Determination of hydrogel gelation time using the vial inversion method. Specific method: Dissolve the prepared M-HPCS and HA-PEG in deionized water to prepare solutions with concentrations of 7% and 4% (w / v), respectively. After thorough stirring, mix the M-HPCS solution and HA-PEG solution in different ratios (1:9; 3:7; 1:1; 7:3; 9:1) in a glass vial. Immerse the vial in a water bath at different temperatures at the initial temperature. Test the gelation time of the hydrogel at each temperature. After a certain period of time in the water bath, remove the vial and invert it. If no solution flows down the wall within 1 minute, the hydrogel is considered to have formed, and the time is recorded.

[0082] 2. Preparation of M-HPCS / HA-PEG+Mg 2+ hydrogel

[0083] Weigh 0.4066 g of magnesium chloride and dissolve it in 10 mL of deionized water to prepare a 4% (w / v) magnesium chloride solution. Add 0.1 mL of the magnesium chloride solution dropwise to 2 mL of the mixed solution to prepare a hydrogel containing 10 mM magnesium ions.

[0084] Based on the above experiments, the prepared M-HPCS and HA-PEG were dissolved in deionized water to prepare solutions with concentrations of 7% and 4% (w / v), respectively. After being fully stirred, the M-HPCS solution and the HA-PEG solution were mixed in glass bottles according to different ratios (3:7; 1:1; 7:3). Mg was added dropwise while stirring. 2+ Solution (ratio: 5%, v / v), 2 mL of each ratio was prepared, and the mixed M-HPCS / HA-PEG+Mg 2+ The solution was immersed in water baths at different temperatures at the initial temperature, and the gelation time of the hydrogel was tested under each temperature condition. That is, the solution was placed in the water bath for a certain period of time, and then the glass bottle was inverted after being taken out. If no solution flowed down the wall within 1 minute, it was considered to have formed a hydrogel, and the time was recorded.

[0085] Since the melanin structural unit monomer has a large number of phenolic hydroxyl groups, it can produce a strong chelating effect with metal ions. Therefore, it is inferred from the mechanism of action that various metal ions (Fe 2+ 、Cu 2+ , Ca 2+ 、Fe 2+ etc.) can further cross-link with the melanin-based hydrogel (M-HPCS3 / HA-PEG7), so the technical protection of the method for constructing the complex metal ion hydrogel in the present invention is not limited to Mg 2+ , other metal ions should also be covered. In this embodiment, Mg 2+ Take this as an example to illustrate.

[0086] 1. M-HPCS / HA-PEG and M-HPCS / HA-PEG+Mg 2+ Microscopic morphology of hydrogel

[0087] Different ratios of M-HPCS / HA-PEG and M-HPCS / HA-PEG+Mg 2+ After the hydrogel is formed, it is placed in a -20℃ refrigerator and frozen overnight. The xerogel is obtained by vacuum freeze-drying. The xerogel is cut to obtain a cross section. After processing, the microstructure inside the hydrogel is observed under a scanning electron microscope. 2+ The hydrogel was measured from five different regions, with at least five pore sizes selected from each region.

[0088] 2. M-HPCS / HA-PEG and M-HPCS / HA-PEG+Mg 2+ Adhesion of hydrogels

[0089] The adhesion properties of the hydrogel were tested using a universal testing machine and a macroscopic adhesion test. The adhesion properties of the hydrogel were macroscopically studied using materials such as pig skin, polyethylene film, glass bottles, weights, and rubber through a tensile adhesion test. Fresh pig skin was soaked in alcohol to remove fat, and then soaked in PBS buffer for later use. The treated pig skin was cut into strips with a width of 2 cm and a length of 5 cm, and bonded to the hydrogel (adhesion area of ​​20 mm × 20 mm) and a certain weight was applied at room temperature to allow the hydrogel and pig skin to interact for 1 hour. Then, a lap shear test was performed using a universal testing machine at a constant speed of 2 mm / min (n≥5) in a tensile mode, and the maximum force N was recorded. Max The tensile adhesion strength of the hydrogel was determined by dividing the maximum force (N) by the adhesion area (m 2 ) were calculated, and each group of experiments was repeated 3 times.

[0090] 3. M-HPCS / HA-PEG and M-HPCS / HA-PEG+Mg 2+ Rheological properties of hydrogels

[0091] Based on the determination of hydrogel gelation time using the vial inversion method, 5 mL of each solution mixture was prepared for the selected gelation ratios. The hydrogel gelation temperature was determined using a rheometer. The following steps were performed: the prepared M-HPCS / HA-PEG solution was injected onto the rheometer's test platform with the plate spacing set to 1000 μm, the frequency to 1 Hz, the strain to 1%, and the temperature set to 25°C-70°C at a rate of 2°C / min. The gelation temperature was determined as the temperature corresponding to the intersection of the storage modulus and the loss modulus.

[0092] Oscillation frequency scanning was performed on hydrogels with different proportions to explore the structural stability of hydrogels with different proportions. The parameter settings of the rheometer were as follows: scanning frequency range 0.1-100 Hz, shear strain 1%, temperature 37°C, and the change of modulus with frequency was recorded after the measurement.

[0093] Oscillatory strain scanning was performed on hydrogels with different proportions to explore the reversible deformation and stability of hydrogels with different proportions. The parameters of the rheometer were set as follows: shear strain range of 1%-100%, frequency of 1Hz, temperature of 37°C. After the measurement, the change of modulus with shear strain was recorded.

[0094] 4. M-HPCS / HA-PEG and M-HPCS / HA-PEG+Mg 2+ Swelling properties of hydrogels

[0095] The freeze-dried hydrogel was weighed and immersed in PBS buffer (pH = 7.4) and placed in a constant temperature box at 37 ° C. The samples were taken out at different time points, and the excess water on the gel surface was wiped with filter paper and weighed again until the gel weight no longer changed, indicating that the gel swelling reached equilibrium. Three parallel samples were set for each group, and the gel weight changes were recorded respectively. The average value was taken and the swelling rate was calculated according to the formula:

[0096]

[0097] Among them, W d ——Mass of dry hydrogel, unit: g

[0098] 5. M-HPCS / HA-PEG and M-HPCS / HA-PEG+Mg 2+ Photothermal properties of hydrogels

[0099] M-HPCS / HA-PEG and M-HPCS / HA-PEG+Mg 2+ Hydrogel (1 mL) was heated at different power densities (0.5, 0.75, 1, 1.25, 1.5 W / cm 2 ) for 10 min, and the M-HPCS / HA-PEG hydrogel was heated by near-infrared laser irradiation (808 nm, 1 W / cm 2 The thermal stability of the film was evaluated by 5 cycles of heating (5 min) and cooling naturally (5 min). Meanwhile, an infrared thermal imager was used to monitor the temperature changes and capture thermal images.

[0100] 6. M-HPCS / HA-PEG and M-HPCS / HA-PEG+Mg 2+ Antioxidant properties of hydrogels

[0101] (1) DPPH scavenging ability determination

[0102] Different ratios of M-HPCS / HA-PEG and M-HPCS / HA-PEG+Mg 2+ After the hydrogel is gelled, it is freeze-dried and ground into powder. 20 mg of dry gel is weighed and added to 2 mL of DPPH working solution. After the reaction is carried out on a shaker at 37 ° C in the dark for 30 minutes, the absorbance of the hydrogels with various ratios is adjusted to zero with ethanol at 515 nm, and the absorbance of the hydrogels with various ratios is measured and recorded as A1. After the M-HPCS / HA-PEG hydrogels with different ratios are gelled, they are freeze-dried and ground into powder. 20 mg of dry gel is weighed and added to 2 mL of ethanol solution as the control group. After the reaction is carried out on a shaker at 37 ° C in the dark for 30 minutes, the absorbance of the hydrogels with various ratios is adjusted to zero with ethanol at 515 nm, and the absorbance of the hydrogels with various ratios is measured and recorded as A2. 1 mL of ethanol is measured and added to 2 mL of DPPH working solution as the control group. The absorbance of the hydrogels with various ratios is adjusted to zero with ethanol at 515 nm, and the absorbance is measured in turn and recorded as A0. The DPPH free radical scavenging rate is calculated according to the following formula:

[0103]

[0104] (2) ABTS scavenging ability determination

[0105] Weigh 1 mg of M-HPCS / HA-PEG and M-HPCS / HA-PEG+Mg in different ratios 2+ Dissolve the dried gel powder in 2 mL of ABTS+ working solution and mix thoroughly. Incubate the mixture at room temperature in the dark for 30 minutes. Adjust the absorbance to zero with PBS buffer at 734 nm. Measure the absorbance of the hydrogels at various ratios, and record it as A1. Three samples are run in parallel for each group. For the control group, add 1 mL of PBS buffer to 2 mL of ABTS+ working solution and mix thoroughly. Adjust the absorbance to zero with PBS buffer at 734 nm, and measure its absorbance, and record it as A0. Calculate the ABTS free radical scavenging rate using the following formula:

[0106]

[0107] 7. Cytocompatibility of hydrogels

[0108] Preparation of M-HPCS / HA-PEG hydrogel extract: Preparation of M-HPCS7 / HA-PEG3 hydrogel and M-HPCS3 / HA-PEG7+Mg 2+ The hydrogel was freeze-dried, weighed, and placed in a centrifuge tube for irradiation sterilization. Serum-free DMEM medium was added at a ratio of 0.1 g / mL and incubated in a cell culture incubator for 72 hours to obtain the M-HPCS / HA-PEG hydrogel extract. The extract was filtered twice through a 0.22 μm filter membrane, sealed, and stored at 4°C until ready for use.

[0109] HUVEC cells and L929 cells in the logarithmic growth phase were digested and resuspended in DMEM complete medium to a dilution of 2×10 4 200 μL of cell suspension was inoculated into a 96-well plate, i.e. 4000 cells per well, and cultured in a cell culture incubator at 37°C and 5% CO2 for 24 h to allow them to adhere to the wall. The cell culture medium in the well plate was discarded, and different concentrations of M-HPCS7 / HA-PEG3 and M-HPCS3 / HA-PEG7+Mg were added again. 2+ Hydrogel extracts (20%, 40%, 60%, 80%, 100%) were added to the blank and control groups. Fresh DMEM complete medium was replaced at the same time. The cells were cultured in a cell culture incubator for 24 and 48 hours, respectively, and the cell status was observed under a microscope. The MTT assay was used to evaluate the effect of the hydrogel on cell proliferation. After observation and photography, 20 μL of 0.5 mg / mL MTT solution was added to each well. After incubation in an incubator for 4 hours, the hydrogel extract and MTT solution in the well plate were aspirated and discarded. 150 μL of DMSO solution was added to each well. The absorbance was read using a microplate reader, and the relative cell proliferation rate was calculated according to the following formula:

[0110]

[0111] Among them, A 实验组 is the average absorbance value of the experimental group, A 空白组 is the average absorbance value of the blank control group, A 对照组 is the average absorbance value of the control group.

[0112] 8. Live / dead cell detection in hydrogels

[0113] Live / dead cell staining was used to further evaluate the effect of hydrogel on L929 cell morphology and proliferation. After digestion, the cells were resuspended in complete culture medium and diluted to 4 × 10 4 cells / mL, 0.5 mL of L929 cell suspension was inoculated into a 48-well plate, i.e. 2 × 10 4 cells / well. After culturing for 24 hours, the cells adhered to the wall and the culture medium was discarded. 0.5 mL of hydrogel extract was added to each well of the experimental group, and 0.5 mL of fresh complete culture medium was added to the control group. The cells were placed in a cell culture incubator at 37°C and 5% CO2 and incubated for 1, 3, 5, and 7 days. The original culture medium was discarded, and the cells were washed with sterile PBS buffer. 200 μL of freshly prepared cell live-dead staining working solution was added to each well. The cells were stained in the dark at 37°C for 20 minutes, and then the cell staining was observed under a fluorescence microscope with the excitation wavelength set to 560 nm and the emission wavelength set to 600 nm.

[0114] 9. Cell migration assay in hydrogels

[0115] The cell scratch method was used to study the migration-promoting ability of hydrogels. L929 cells and HUVECs cells in the logarithmic growth phase were digested and centrifuged, and then resuspended and diluted to 3×10 in DMEM culture medium containing 10% fetal bovine serum. 5 cells / mL, 1 mL was inoculated into a 24-well plate, i.e. 3×10 cells / mL per well. 5 Cells were cultured in a cell culture incubator at 37°C and 5% CO2 until the cells adhered to the wall and covered the entire bottom of the well. The old culture medium was discarded, and a 10 μL sterile pipette tip was used to scratch the well plate perpendicularly to create cell scratches. The cells were then washed with sterile PBS solution to clean the cell debris. Then, the medium containing M-HPCS7 / HA-PEG3, M-HPCS3 / HA-PEG7+Mg was added again. 2+ The extract was added to serum-free DMEM medium. The control group was added with serum-free DMEM medium and cultured in a 37°C, 5% CO2 cell culture incubator. Cells were taken out after 0, 12, 24, and 36 hours, observed under a microscope, and photographed. The cell images were analyzed using Image J. The cell migration rate was calculated using the following formula:

[0116]

[0117] Among them, S0 is the initial wound area before cell migration, and St is the wound area after a certain period of cell migration.

[0118] 10. Hydrogel cell angiogenesis experiment

[0119] First, use a pre-cooled pipette tip to draw 50 μL of Matrigel into a 96-well plate, and then place the plate in 37°C for 30 minutes to allow Matrigel to solidify. Then, after digesting the HUVEC cells in good condition, dilute them with FBS-free DMEM high-glucose medium and use 3×10 4 Cells were evenly seeded onto the pre-gelled Matrigel plates at a density of 100 μL per well. 100 μL of FBS-free M-HPCS / HA-PEG hydrogel extract was added to each well of the experimental group, while the blank control group received FBS-free DMEM high-glucose medium. After seeding, the plates were incubated in a 37°C, 5% CO2 incubator for 2, 4, 6, and 8 hours. Vascular formation was then observed under an optical microscope and photographed. Image J software was used to count and analyze the vascular nodes and vessels in each experimental group to determine the number of newly formed blood vessels.

[0120] 11. Cell-level antioxidant experiment of hydrogel

[0121] (1) Establishment of H2O2-induced L929 cell injury model

[0122] L929 cells in the logarithmic growth phase were digested and centrifuged, and resuspended in DMEM culture medium containing 10% FBS and diluted to 2×10 4 cells / mL, 200 μL was taken and plated in a 96-well plate, i.e., 4000 cells per well, and cultured in a cell culture incubator at 37°C, 5% CO2 for 24 h. After the cells attached, the old medium was discarded and 200 μL of DMEM medium containing different concentrations of H2O2 (H2O2 concentrations of 0, 10, 25, 50, 100, 200, 400, 600, and 800 μM) was added. The cells were cultured at 37°C for 2 hours to stimulate the production of reactive oxygen species. After the incubation period, 20 μL of MTT detection solution was added and incubated at 37°C, 5% CO2 for 4 hours. The absorbance value was measured using a microplate reader to detect cell viability.

[0123] (2) ROS scavenging ability assessment

[0124] In order to evaluate the protective effect of hydrogel in scavenging ROS on cells, L929 cells in the logarithmic growth phase were digested and centrifuged, and then resuspended and diluted to 2×10 in DMEM culture medium containing 10% FBS. 4 cells / mL, 200 μL was taken and inoculated into a 96-well plate, i.e., 4000 cells per well, and cultured in a cell culture incubator at 37°C and 5% CO2 for 24 hours. After the cells attached, the old culture medium was discarded and 200 μL of hydrogel extract with a H2O2 concentration of 500 μM (20%, 40%, 60%, 80%, 100%) was added. The control group was replaced with fresh serum-free DMEM medium without H2O2, and the negative control group was only added with serum-free DMEM medium with a H2O2 concentration of 500 μM. After incubation at 37°C for 2 hours, 20 μL of MTT detection solution was added and incubated at 37°C and 5% CO2 for 4 hours. The absorbance value was measured using a microplate reader to detect cell viability.

[0125] (3) DCFH-DA fluorescence staining

[0126] To further evaluate the ROS scavenging ability of the hydrogel, the ROS probe DCFH-DA was used to observe cell labeling. The hydrogel was cut into 3m×3m×3m blocks (n=3), disinfected with alcohol and sterilized by ultraviolet irradiation before use. The well-grown L929 cells were digested and centrifuged and then resuspended in DMEM complete medium and diluted to 4×10 4 cells / mL, 500 μL was inoculated into a 48-well plate, i.e. 2×10 cells / mL per well. 4Cells were cultured in a 37°C, 5% CO2 incubator for 24 hours to allow attachment. The old medium was aspirated and then replaced with 500 μL of serum-free DMEM medium containing 500 μM H2O2 in the 48-well plate. For the blank control group, fresh serum-free DMEM medium without H2O2 was used. For the experimental group, after adding the medium, the sterilized hydrogels were carefully placed into the well plate and incubated at 37°C for an additional 2 hours. The solid hydrogels were then gently removed, the old medium was aspirated and washed with sterile PBS. 200 μL of 2',7'-dichlorofluorescein diacetate (DCFH-DA) was added and incubated in a dark incubator for 20 minutes. The cells were then washed with serum-free medium to remove unloaded DCFH-DA. Finally, intracellular fluorescence was observed and photographed using a fluorescence microscope or laser confocal scanning microscopy.

[0127] 12. Establishment of diabetic rat model, burn wound model and skin wound healing study

[0128] Preparation of STZ solution: Accurately weigh a certain amount of STZ powder and add citric acid buffer to prepare a 10 mg / mL STZ solution. Store in the dark and use within 30 minutes. After purchasing male SD rats (8-10 weeks old, 200-250g), raise them for one week and monitor their blood glucose levels to confirm that they are normal rats. After the rats have adapted to the environment, fast without food or water for 12 hours. The diabetic mouse model is induced by intraperitoneal injection of STZ solution (dose: 65 mg / kg; concentration: 40 mg / mL) into male SD rats. After injection, fast without water for 2 hours. The blood glucose levels of the rats are measured every 3 days. If the blood glucose concentration is above 16.7mM, the rat is considered to have diabetes. If the model is successful, subsequent surgery can be performed.

[0129] The diabetic SD rats were randomly divided into six groups, with 25 rats in each group, namely blank control group, positive control group (hemostatic guaiac gauze), M-HPCS7 / HA-PEG3 hydrogel group, M-HPCS7 / HA-PEG3 hydrogel+NIR group, M-HPCS3 / HA-PEG7+Mg 2+ Hydrogel group, M-HPCS3 / HA-PEG7+Mg 2+Hydrogel + NIR group. After the diabetic rats were anesthetized with 3% sodium pentobarbital (1mL / kg), the hair on the back of the rats was shaved and the hair was removed with a depilatory cream. The back was disinfected with 75% alcohol. After the skin surface was dry, the rat's back skin was pressed for 15 seconds under the conditions of 80°C and 500g using a constant temperature burn instrument to establish a wound diameter of 1.6cm deep second-degree burn wound model. After carefully cleaning the necrotic skin on the back of the rat, each group of hydrogel dressings was applied to the wound and ensured that the wound was completely covered. The blank control group used normal saline, and then covered the wound surface with a dressing and a bandage. Finally, it was wrapped with medical tape to prevent the rat from gnawing on the wound and the hydrogel from falling off. The hydrogel dressing was changed every two days after the operation, and the wound healing condition was recorded and the wound size was measured and recorded by taking pictures. The wound area was measured by Image J software, and the wound healing rate was calculated as shown below:

[0130]

[0131] Where S0 and S t The initial wound area on day 0 and the wound area on the designated date, respectively.

[0132] 13. In vivo photothermal conversion research

[0133] 0.2 mL of M-HPCS / HA-PEG hydrogel was applied to the burn wound of rats at different power densities (1, 1.25, 1.5 W / cm 2 ) for 5 min, and the M-HPCS / HA-PEG hydrogel was heated by near-infrared laser irradiation (808 nm, 1 W / cm 2 The thermal stability of the film was evaluated by 5 cycles of heating (5 min) and cooling naturally (5 min). Meanwhile, an infrared thermal imager was used to monitor the temperature changes and capture thermal images.

[0134] 14. HE staining, Masson staining and immunofluorescence staining of tissue sections

[0135] On days 3, 7, 14, 21, and 28 after treatment, five rats were randomly selected from each treatment group. Skin tissue from the burn wound was excised and fixed in 4% paraformaldehyde for 24 hours before subsequent staining. After fixation, the tissue blocks were rinsed with water for 12 hours, then trimmed into regular shapes and dehydrated for 2 hours using 50%, 70%, and 95% ethanol, and anhydrous ethanol I and II, respectively. The dehydrated tissues were soaked in xylene for 15 minutes, followed by another 15-minute soak in fresh xylene to make them transparent. Finally, the tissue blocks were soaked in paraffin wax I, paraffin wax II, and paraffin wax III, sequentially for 1 hour, before being embedded. The embedded tissue blocks were fixed on a tissue slicer and cut into 5 μm sections. The sections were spread in 40°C deionized water, removed with a slide, and allowed to dry overnight. After drying at 62°C for 1–2 h, sections were dewaxed with xylene I and xylene II for 10 min, respectively, and immersed in a xylene / anhydrous ethanol mixture (1:1 by volume) for 5 min. Sections were then immersed in anhydrous ethanol I and II, 95%, 85%, and 70% ethanol, and distilled water for 2 min, respectively. Sections were then stained with hematoxylin for 10 min, rinsed with distilled water for 3 min, differentiated with 1% hydrochloric acid alcohol for 1.5 s, and slowly rinsed with tap water for 10 min. Sections were then treated with 70%, 85%, and 95% ethanol for 2 min, stained with eosin for 1 s, and immersed in 95% ethanol I, 95% ethanol II, 95% ethanol III, anhydrous ethanol I, II, and III, xylene I, and xylene II for 5 min, respectively. Sections were then mounted with neutral gum, air-dried in a fume hood, and observed and photographed under an optical microscope.

[0136] The above tissue sections were sliced ​​and Masson staining was performed according to the instructions of the kit. Finally, the sections were sealed with neutral gum and dried. Then, the collagen fibers in the wounds of each group were observed under a microscope.

[0137] After 7 days of treatment, the skin tissue sections were sliced ​​and the inflammation and angiogenesis at the wound were determined by immunofluorescence staining of CD206, iNOS, CD31, and α-SMA to evaluate the effect of M-HPCS7 / HA-PEG3 hydrogel and

[0138] M-HPCS3 / HA-PEG7+Mg 2+ The inflammation-modulating and angiogenesis-promoting abilities of hydrogels in vivo.

[0139] Experimental results analysis:

[0140] 1. M-HPCS / HA-PEG and M-HPCS / HA-PEG+Mg 2+The hydrogel formation conditions are as follows: When the M-HPCS solution is mixed with the HA-PEG solution, the phenolic hydroxyl groups in the melanin interact with the amino groups on the HA-PEG chain through non-covalent bonds, and the hyaluronic acid and chitosan also produce chain entanglement, hydrogen bonding and other interaction forces, so that the M-HPCS / HA-PEG solution forms a 3D network structure hydrogel. 2+ After that, it chelates with the phenolic group and phenolic hydroxyl group on the melanin, M-HPCS3 / HA-PEG7+Mg 2 + The solution can also form a hydrogel with a 3D network structure.

[0141] Under certain temperature conditions, the hydrophilic-hydrophobic balance changes, and thus the solubility of the entire polymer network changes, causing a sol-gel phase transition. Figure 2 As can be seen from the figure, before heating in a water bath, all M-HPCS / HA-PEG mixtures of various concentrations exhibited a certain degree of fluidity, meaning they remained in a solution state. However, after 30 minutes in a 60°C water bath, all M-HPCS / HA-PEG mixtures with different volume ratios, except for the 1:9 volume ratio, which still retained fluidity but had not yet formed a hydrogel, had already formed a gel. The results indicate that M-HPCS3 / HA-PEG7, M-HPCS1 / HA-PEG1, M-HPCS7 / HA-PEG3, and M-HPCS9 / HA-PEG1 solutions all formed hydrogels upon heating.

[0142] At room temperature, various concentrations of M-HPCS / HA-PEG+Mg 2+ The mixed solution was still in solution state, but formed hydrogel after heating in water bath. Table 2 shows that M-HPCS3 / HA-PEG7+Mg 2+ 、M-HPCS1 / HA-PEG1+Mg 2+ 、M-HPCS7 / HA-PEG3+Mg 2+ The gelation temperatures of the hydrogels were 45℃, 60℃, and 60℃, respectively. In comparison, M-HPCS3 / HA-PEG7+Mg 2+ The gelation temperature is significantly reduced, which is due to the 2+ The addition of Mg reacts with the hydroxyphenol group of melanin, so that the mixture can form a stable network structure at a lower temperature. M-HPCS1 / HA-PEG1 and M-HPCS7 / HA-PEG3 can form a stable network structure at a lower temperature without adding Mg. 2+ In the case of Mg 2+ When the ratio of phenolic hydroxyl group to amino group is unbalanced, part of phenolic hydroxyl group is mixed with Mg 2+Interaction occurs, however, M-HPCS3 / HA-PEG7+Mg 2+ The proportion of phenolic hydroxyl groups available in the hydrogel is relatively high, so M-HPCS3 / HA-PEG7+Mg 2+ The gelation temperature of the hydrogel decreases, while Mg 2+ The addition of has little effect on the gelation temperature of M-HPCS1 / HA-PEG1 and M-HPCS7 / HA-PEG3 hydrogels.

[0143] Table 2

[0144]

[0145] 2. Micromorphology of M-HPCS / HA-PEG hydrogel

[0146] The pore size of the hydrogel is very important for wound healing. Figure 3 As shown in Figure 1-A, the hydrogels all exhibited porous structures. The M-HPCS9 / HA-PEG1 hydrogel had a loose internal structure, forming an irregular network. However, the hydrogels with the three ratios of M-HPCS3 / HA-PEG7, M-HPCS1 / HA-PEG1, and M-HPCS7 / HA-PEG3 all exhibited porous network structures with uniform void distribution. Furthermore, as the proportion of M-HPCS increased, the hydrogel structure became denser and the pore size decreased. The average pore sizes for M-HPCS3 / HA-PEG7, M-HPCS1 / HA-PEG1, and M-HPCS7 / HA-PEG3 were 435.26 μm, 191.22 μm, and 128.04 μm, respectively. A higher degree of cross-linking in a hydrogel results in more cross-linking points, which in turn leads to a decrease in the porosity and pore size of the hydrogel. Compared with M-HPCS3 / HA-PEG7 hydrogel, M-HPCS1 / HA-PEG1 and M-HPCS7 / HA-PEG3 hydrogels have lower pore size and porosity, can better maintain their stable structure, and have better mechanical strength.

[0147] Studies have shown that when the pore size of the hydrogel is around 125 μm, it is conducive to the migration of contractile fibroblasts and promotes wound repair. 2+ SEM images of hydrogels Figure 3 -D, M-HPCS / HA-PEG+Mg 2+ The hydrogels all showed porous structures, and the pore size and porosity were obtained by measurement. 2+ , M-HPCS1 / HA-PEG1+Mg 2+ 、M-HPCS7 / HA-PEG3+Mg 2+The average pore sizes of the hydrogels were 103.44 μm, 128.04 μm, and 97.59 μm, respectively. 2+ The hydrogel cross-linking is relatively loose and the structure is not stable enough, while M-HPCS3 / HA-PEG7+Mg 2+ 、M-HPCS1 / HA-PEG1+Mg 2+ The hydrogel is tightly cross-linked and has uniform pore distribution. 2+ With the addition of M-HPCS3 / HA-PEG7+Mg 2+ 、M-HPCS1 / HA-PEG1+Mg 2+ The porosity and pore size of the hydrogel were significantly reduced, and compared with M-HPCS1 / HA-PEG1+Mg 2+ Hydrogel, M-HPCS3 / HA-PEG7+Mg 2+ The average pore size of the hydrogels was significantly different. The above results show that Mg 2+ The addition of makes the cross-linking of molecules in the hydrogel tighter.

[0148] 3. Adhesion of M-HPCS / HA-PEG hydrogel

[0149] like Figure 4 As shown in the figure, after the finger is bent at different angles, the hydrogel still adheres firmly to the finger and does not fall off. After the hydrogel adheres to the skin for several minutes, the hydrogel can still be completely torn off from the skin without tearing the skin or causing secondary damage to the tissue. The adhesion ability of the hydrogel on pig skin was measured by a universal testing machine. Figure 5 As shown in the figure, the adhesion strengths of M-HPCS3 / HA-PEG7, M-HPCS1 / HA-PEG1, and M-HPCS7 / HA-PEG3 hydrogels were 3511.9 Pa, 3942.81 Pa, and 4613.39 Pa, respectively. It can be seen that the addition of melanin imparts good adhesion to the hydrogels, and the adhesion of the hydrogels gradually increases with increasing melanin content. Furthermore, compared with M-HPCS3 / HA-PEG7 and M-HPCS1 / HA-PEG1 hydrogels, M-HPCS7 / HA-PEG3 hydrogels exhibits the best adhesion, and this adhesion strength is suitable for wound repair.

[0150] like Figure 5 As shown, when Mg is added 2+ Afterwards, M-HPCS3 / HA-PEG7+Mg 2+ 、M-HPCS1 / HA-PEG1+Mg 2+The adhesion strength of the hydrogels increased, and the adhesion forces were 6701.42 and 4457.02 Pa, respectively. 2+ The adhesion of hydrogels changed the most, which was due to the 2+ The addition of Mg 2+ The chelation reaction with the phenolic hydroxyl groups of melanin occupies a large number of hydroxyl groups, which reduces the cross-linking degree of the entire network structure, as shown by the results of microstructural observation. 2+ 、M-HPCS1 / HA-PEG1+Mg 2+ These two hydrogels can not only adhere stably to the skin, but also avoid secondary damage to the wound and surrounding skin during the peeling process.

[0151] 4. Rheological properties of M-HPCS / HA-PEG hydrogel

[0152] like Figure 6 As shown in (A), starting from 25℃, the storage modulus G' of the three hydrogels is higher than the loss modulus G", and the change trend is the same. The difference between the storage modulus G' and the loss modulus G" increases at 62℃, 61.91℃, and 60℃, respectively, indicating that these temperatures are the gelation temperatures of the three hydrogels.

[0153] M-HPCS3 / HA-PEG7+Mg 2+ 、M-HPCS1 / HA-PEG1+Mg 2+ 、M-HPCS7 / HA-PEG3+Mg 2+ The gelation temperature of the hydrogel was determined by Figure 6 As shown in (B), similar to the M-HPCS / HA-PEG hydrogel, the storage modulus G' of these three hydrogels is higher than the loss modulus G", and the change trend is the same between 25°C and 60°C. This is because the viscosity of the mixed M-HPCS solution and HA-PEG solution is similar to that of the hydrogel. The difference between the storage modulus G' and the loss modulus G" of these three hydrogels at 46.55°C, 59.85°C, and 61.9°C, respectively, becomes larger, indicating that these temperatures are the gelation temperatures of the three hydrogels.

[0154] from Figure 7As can be seen from (A), within a certain frequency range, the storage modulus G' of M-HPCS3 / HA-PEG7, M-HPCS1 / HA-PEG1, and M-HPCS7 / HA-PEG3 hydrogels are all higher than the loss modulus G", indicating that the M-HPCS / HA-PEG hydrogels are in a good gel state. Compared with the M-HPCS3 / HA-PEG7 hydrogel, the storage modulus G' of M-HPCS1 / HA-PEG1 and M-HPCS7 / HA-PEG3 hydrogels are much higher than the loss modulus G", which indicates that the M-HPCS3 / HA-PEG7 hydrogel has a lower degree of cross-linking and its network structure is more easily destroyed under certain external forces, thus turning into a solution state. In contrast, the M-HPCS1 / HA-PEG1 and M-HPCS7 / HA-PEG3 hydrogels have a high degree of cross-linking and a more stable hydrogel structure, which is not easily destroyed. In addition, the storage modulus G' of these three hydrogels can reach 339.48, 1120.6, and 7492.75 Pa, respectively. The G' value of M-HPCS7 / HA-PEG3 hydrogel is significantly higher than that of M-HPCS3 / HA-PEG7 and M-HPCS1 / HA-PEG1 hydrogels. This is because with the increase of melanin content, the cross-linking density increases and the viscoelasticity of the hydrogel is enhanced, indicating that the addition of melanin improves the density of the hydrogel network.

[0155] At the same time, the rheometer was used to study the change of the modulus of the hydrogel with strain, and further evaluate the stability of the network structure of the hydrogel. The oscillatory strain scan is mainly used to determine the linear viscoelastic region (LVR) of the material. The LVR can reflect the structural properties of the hydrogel system and characterize the stability of the hydrogel. When a certain range of oscillatory strain is applied to the hydrogel, the hydrogel will undergo reversible deformation. At this time, the structure of the hydrogel is not destroyed. Once the applied force or deformation exceeds the LVR, a certain degree of damage will occur inside the hydrogel, and even structural collapse will occur. Figure 7 It can be clearly seen in (B) that in the strain range of 1%-100%, the storage modulus G' of M-HPCS3 / HA-PEG7, M-HPCS1 / HA-PEG1, and M-HPCS7 / HA-PEG3 hydrogels are all higher than the loss modulus G", and the LVR region of M-HPCS7 / HA-PEG3 hydrogel is wider than that of M-HPCS3 / HA-PEG7 hydrogel and M-HPCS1 / HA-PEG1 hydrogel.

[0156] The viscoelasticity of a hydrogel is mainly determined by its storage modulus G' and loss modulus G". When the storage modulus is greater than the loss modulus, the viscoelasticity of the hydrogel is better, and the larger the storage modulus value, the better the viscoelasticity of the hydrogel.

[0157] M-HPCS / HA-PEG+Mg 2+The dynamic rheological results of the hydrogel showed that ( Figure 7 (C)), within a certain range, the storage modulus G' is greater than the loss modulus G", indicating that M-HPCS / HA-PEG+Mg 2+ The hydrogel is in a gel state within this range and has a certain viscoelasticity; M-HPCS3 / HA-PEG7+Mg 2+ 、M-HPCS1 / HA-PEG1+Mg 2+ 、M-HPCS7 / HA-PEG3+Mg 2+ The storage modulus G' of the hydrogel in the oscillation frequency range of 1-100 Hz is approximately 938.7 Pa, 933.23 Pa, and 297.92 Pa. In this range, M-HPCS3 / HA-PEG7+Mg 2+ The storage modulus of the hydrogel is large, indicating that M-HPCS3 / HA-PEG7+Mg 2+ The hydrogel has good mechanical properties.

[0158] from Figure 7 As can be seen in (D), when M-HPCS3 / HA-PEG7+Mg 2+ 、M-HPCS1 / HA-PEG1+Mg 2+ 、M-HPCS7 / HA-PEG3+Mg 2+ When the strain of the hydrogel exceeded 761%, 429%, and 180%, respectively, the storage modulus G' and loss modulus G" showed a reverse trend, indicating that when the strain strength was exceeded, the internal structure of the hydrogel was destroyed. Compared with M-HPCS1 / HA-PEG1+Mg 2+ and M-HPCS7 / HA-PEG3+Mg 2+ For hydrogel, M-HPCS3 / HA-PEG7+Mg 2+ When the internal structure of the hydrogel is destroyed, the external force required is greater, which indicates that M-HPCS3 / HA-PEG7+Mg 2+ The hydrogel's internal network is highly cross-linked, has better toughness, and can withstand greater deformation without being destroyed.

[0159] 5. Swelling properties of M-HPCS / HA-PEG hydrogel

[0160] like Figure 8As shown in (A), after immersion for 24 h, the swelling ratios of M-HPCS3 / HA-PEG7, M-HPCS1 / HA-PEG1, and M-HPCS7 / HA-PEG3 hydrogels were 54.15, 35.47, and 26.94, respectively. The swelling ratios of M-HPCS1 / HA-PEG1 and M-HPCS7 / HA-PEG3 hydrogels were significantly lower than those of M-HPCS3 / HA-PEG7 hydrogel and were within an appropriate range. This indicates that M-HPCS1 / HA-PEG1 and M-HPCS7 / HA-PEG3 hydrogels can quickly absorb wound exudate in skin wounds, avoid tissue fluid leakage, maintain a moist environment for wound tissue, and prevent hydrogel swelling and shedding. This is consistent with microstructural observations. The M-HPCS3 / HA-PEG7 hydrogel exhibits larger network pores, indicating looser crosslinking between its molecular chains, allowing water molecules to more easily enter the hydrogel network. Consequently, the M-HPCS3 / HA-PEG7 hydrogel exhibits higher swelling and is more prone to disintegration. In contrast, the M-HPCS1 / HA-PEG1 and M-HPCS7 / HA-PEG3 hydrogels exhibit lower swelling. This is partly due to the benzene ring structure of melanin, which enhances the hydrophobicity of the molecular chains. It also reflects the higher degree of crosslinking and smaller pores in these two hydrogel groups. The swelling performance results further demonstrate that as melanin content increases, the hydrogel exhibits tighter crosslinking and better performance.

[0161] The swelling ratio of the hydrogel evaluated the ability of the hydrogel to absorb wound exudate. Figure 8 (B)

[0162] M-HPCS / HA-PEG+Mg 2+ The water absorption of hydrogel within 24 hours, due to Mg 2+ The internal structure of the hydrogel is also more compact after immersion for 24 hours. 2+ 、M-HPCS1 / HA-PEG1+Mg 2+ 、M-HPCS7 / HA-PEG3+Mg 2+ The swelling ratios of the hydrogels were 31.10, 28.03, and 21.23, respectively. The results showed that M-HPCS / HA-PEG+Mg 2+ The hydrogels all have good water absorption properties.

[0163] 6. Photothermal properties of M-HPCS / HA-PEG hydrogel

[0164] Hot water baths or mild heat stimulation can promote angiogenesis, increase the vascular density of granulation tissue in skin wounds, and improve wound healing rate. Figure 8The temperature rise of M-HPCS / HA-PEG hydrogel under near-infrared light irradiation of different powers is shown in Figure 2. 2 After irradiation with near-infrared light for 10 minutes, the temperatures can be raised to 41.3, 43.4, 47.9, 51.5, and 56°C, respectively. It can be seen that the M-HPCS / HA-PEG hydrogel has a high photothermal conversion efficiency, and its temperature rise shows a certain power dependence. The greater the power, the faster the heating rate of the same hydrogel and the higher the temperature that can be reached. On this basis, 1W / cm 2 The power was used to determine the temperature rise of hydrogels with different proportions. Under near-infrared light irradiation, the temperatures of M-HPCS3 / HA-PEG7, M-HPCS1 / HA-PEG1, and M-HPCS7 / HA-PEG3 hydrogels rose rapidly in a short period of time and then tended to be stable. The maximum temperatures reached 47.9, 51.3, and 55.45°C after 10 minutes. This shows that melanin gives M-HPCS / HA-PEG hydrogel good photothermal properties, and with the increase of melanin content, the photothermal performance of M-HPCS / HA-PEG hydrogel improves. Compared with M-HPCS3 / HA-PEG7 and M-HPCS1 / HA-PEG1 hydrogels, M-HPCS7 / HA-PEG3 hydrogel has the highest photothermal conversion efficiency. Figure 8 (H) M-HPCS3 / HA-PEG7, M-HPCS1 / HA-PEG1, and M-HPCS7 / HA-PEG3 hydrogels at a power of 1 W / cm 2 From the temperature-increasing thermal imaging diagram below, it can be seen intuitively that with the increase of time, the high-temperature spot in the area irradiated by near-infrared light becomes larger, indicating that the temperature in this area increases. M-HPCS / HA-PEG hydrogel has good photothermal properties and can be used in photothermal therapy for wound repair.

[0165] The temperature changes of M-HPCS / HA-PEG hydrogel after five consecutive near-infrared light irradiation cycles were as follows: the first highest temperature was 51°C, the second temperature was 50.2°C, the third temperature was 51°C, the fourth temperature was 51.5°C, and the fifth temperature was 51.5°C. The temperature change of M-HPCS / HA-PEG hydrogel after five irradiations was not obvious, indicating that M-HPCS / HA-PEG hydrogel has good photothermal stability.

[0166] 1 mL of hydrogel was placed in a 1.5 mL centrifuge tube and the photothermal properties of the hydrogel were measured under 808 nm NIR laser irradiation. Figure 8 It can be seen that as time goes by, the temperature of the hydrogel rises rapidly and then tends to be stable. After 10 minutes of NIR irradiation, the temperature of M-HPCS3 / HA-PEG7+Mg2+ 、M-HPCS1 / HA-PEG1+Mg 2+ 、M-HPCS7 / HA-PEG3+Mg 2+ The highest temperatures of the hydrogels can reach 46.17, 48.6, and 50.1 °C, respectively. The results are similar to those of the photothermal results of M-HPCS / HA-PEG hydrogels. 2+ The photothermal properties of the hydrogel are somewhat dependent on the content of melanin, and Mg 2+ The addition of Mg had no effect on the photothermal properties of the hydrogel. 2+ 、M-HPCS1 / HA-PEG1+Mg 2 + 、M-HPCS7 / HA-PEG3+Mg 2+ Hydrogels have good photothermal conversion capabilities.

[0167] The photothermal stability of the hydrogel was further determined, e.g. Figure 8 As shown, after 5 irradiation cycles, the M-HPCS / HA-PEG+Mg 2+ The highest temperature of the hydrogel can reach about 47.4℃, indicating that

[0168] M-HPCS / HA-PEG+Mg 2+ The hydrogel has good photothermal stability.

[0169] 7. Antioxidant properties of M-HPCS / HA-PEG hydrogel

[0170] Depend on Figure 9 As shown in (A), the DPPH scavenging rates of M-HPCS3 / HA-PEG7, M-HPCS1 / HA-PEG1, and M-HPCS7 / HA-PEG3 hydrogels were 71.99%, 74.98%, and 77.16%, respectively, indicating that M-HPCS / HA-PEG hydrogels have good antioxidant properties, and their antioxidant properties will increase with the increase of melanin content; similarly, the ABTS scavenging experiment also showed that M-HPCS / HA-PEG hydrogels have good antioxidant properties. The ABTS scavenging rates of M-HPCS3 / HA-PEG7, M-HPCS1 / HA-PEG1, and M-HPCS7 / HA-PEG3 hydrogels were 72.18%, 73.23%, and 74.59%, respectively, and their antioxidant properties also increased with the increase of melanin content. The experiment showed that the addition of melanin promoted the strong antioxidant properties of M-HPCS / HA-PEG hydrogels, and their antioxidant properties also became stronger with the increase of melanin content.

[0171] like Figure 9 (A), M-HPCS3 / HA-PEG7+Mg 2+ , M-HPCS1 / HA-PEG1+Mg 2+ 、M-HPCS7 / HA-PEG3+Mg 2+ The DPPH scavenging rates of the hydrogels were 84.10%, 84.13%, and 85.26%, respectively, indicating that M-HPCS / HA-PEG+Mg 2+ The hydrogel still has good antioxidant properties, and its antioxidant properties are enhanced due to the addition of magnesium ions; similarly, Figure 9 As shown in (B), ABTS clearance experiments also showed that M-HPCS / HA-PEG+Mg 2+ The hydrogel has good antioxidant properties, M-HPCS3 / HA-PEG7+Mg 2+ , M-HPCS1 / HA-PEG1+Mg 2+ 、M-HPCS7 / HA-PEG3+Mg 2+ The ABTS clearance rates of the hydrogels were 73.91%, 75.19%, and 76.82%, respectively. The ABTS clearance abilities of the hydrogels were all enhanced compared with those of the M-HPCS / HA-PEG hydrogels. The results showed that the addition of magnesium ions enhanced the

[0172] M-HPCS / HA-PEG+Mg 2+ The antioxidant property of hydrogel is enhanced, M-HPCS / HA-PEG+Mg 2+ The hydrogels all have good free radical scavenging ability.

[0173] 8. Evaluation of hydrogel cell compatibility

[0174] from Figure 10 As can be seen in the results, the relative proliferation rates of cells after 24 and 48 hours of culture were both above 85%, and the relative proliferation rate after 48 hours was higher than that after 24 hours, both exceeding 90%. According to the cytotoxicity rating, the hydrogel extracts at all concentrations were non-toxic to cells.

[0175] At the same time, the growth status of cells was observed by optical microscopy after culturing cells with hydrogel extracts of different concentrations for 24 and 48 h, and the biocompatibility of the hydrogel was further evaluated. Figure 11 As shown in the figure, under co-culture with hydrogel extracts of various concentrations, the growth status of L929 cells and HUVECs cells was good. Most of the L929 cells were spindle-shaped, and most of the HUVECs cells were cobblestone-like. The cell outlines were clearly visible, and there was no obvious atrophy or death. In addition, the cell number increased significantly after 48 hours of culture, indicating that the hydrogel had no cytotoxicity and good biocompatibility.

[0176] The same method was used to evaluate the effect of M-HPCS3 / HA-PEG7+Mg 2+ The biocompatibility of hydrogels is determined by Figure 12 It can be seen that the relative proliferation rate of L929 cells and HUVECs cells exceeded 80% after 24h or 48h of co-culture, indicating that the addition of magnesium ions was non-toxic to the cells and M-HPCS3 / HA-PEG7+Mg 2+ The hydrogel has good biocompatibility. Figure 13 It can be seen that after 24h and 48h of co-culture, L929 cells and HUVECs cells can grow and proliferate normally, with good cell morphology, and the cell number is the same as that without M-HPCS3 / HA-PEG7+Mg 2+ There was no significant difference in the cells extracted from the hydrogel, and no cell necrosis or apoptosis was found.

[0177] 9. Live / dead cell analysis in hydrogels

[0178] Live / dead cell staining was used to observe the cell morphology and proliferation of L929 cells after co-culture with hydrogels. Calcein-AM and PI dyes were used to stain live cells and dead cells, respectively. Green fluorescent signals represent live cells, and red fluorescent signals represent dead cells. Figure 14 It can be seen that after the first day of co-culture, the number of cells in the hydrogel group was not significantly different from that in the control group. The cells all showed a plump spindle shape and no obvious red fluorescence was observed. It can also be clearly observed that compared with the first day, the number of cells increased significantly after the third, fifth, and seventh days of co-culture, and the cell morphology was normal. There was no obvious red fluorescence representing dead cells. In addition, the number of cells in the experimental group was slightly more than that in the control group, indicating that M-HPCS7 / HA-PEG3 hydrogel and M-HPCS3 / HA-PEG7+Mg 2+ Hydrogels have good ability to promote cell growth.

[0179] 10. Cell migration analysis of hydrogels

[0180] like Figure 15 and 16 As shown, for the HUVEC cell group, at 24 h, the confluence rate of the M-HPCS7 / HA-PEG3 hydrogel group reached 55.35%, and the M-HPCS3 / HA-PEG7+Mg 2+ The confluence rate of the hydrogel group reached 57.55%, which was significantly different from that of the control group. At 36 h, the confluence rate of the hydrogel group was close to 90%, almost completely confluent, while that of the control group was only 44.11%, indicating that the M-HPCS7 / HA-PEG3 hydrogel and M-HPCS3 / HA-PEG7+Mg 2+The hydrogels all had significant ability to promote HUVEC cell migration; and for L929 cells, after co-culture with the hydrogel extract for 12 hours and 24 hours, the confluence rate of the hydrogel group was significantly different from that of the control group, and after 36 hours, the confluence of the hydrogel group was close to 50%, while the confluence rate of the control group was only 18.02%, and there was a significant difference between the hydrogel group and the control group. The results showed that compared with the control group, the hydrogel group significantly accelerated the migration speed of HUVEC cells and also had a relatively good promoting effect on the migration of L929 cells, indicating that M-HPCS7 / HA-PEG3 hydrogel and M-HPCS3 / HA-PEG7+Mg 2+ Hydrogels can promote cell migration and accelerate wound healing during the wound healing process.

[0181] 11. Angiogenesis Analysis of Hydrogels

[0182] like Figure 17 As shown in the figure, compared with the blank control group, the hydrogel group showed angiogenesis after 2 hours, a small amount of angiogenesis in the hydrogel group after 4 hours, and obvious tubular structures were observed in the hydrogel group after 8 hours, indicating that new blood vessels had been formed, while the control group showed only a slight trend of angiogenesis. The new blood vessels were quantified by counting the number of vascular nodes and the number of blood vessels. Figure 5-8 As shown in (B) and (C), at 8 h, the average number of nodes and blood vessels in the M-HPCS7 / HA-PEG3 hydrogel group was 48 and 78, respectively. 2+ The average number of nodes and blood vessels in the hydrogel were 46 and 77, respectively, which were twice that of the control group, indicating that the hydrogel could promote the formation of new blood vessels.

[0183] 12. Analysis of the antioxidant capacity of hydrogel

[0184] The damage degree of H2O2 to L929 cells was studied by MTT method. Figure 18 (A) It can be seen that excessively high H2O2 concentrations can lead to severe cell apoptosis, while low H2O2 concentrations have no significant effect on cell activity. That is, after stimulating L929 cells with H2O2 at concentrations of 0, 10, 25, 50, 100, 200, and 400 μM for 2 hours, the cell viability was still above 80%. When the H2O2 concentrations were 600 and 800 μM, the cell viability was 13.42% and 9.60%, respectively. Since the cell viability was higher at a H2O2 concentration of 400 μM and lower at a H2O2 concentration of 600 μM, a H2O2 concentration of 500 μM was subsequently selected to study the protective effect of M-HPCS / HA-PEG hydrogel on L929 cells.

[0185] like Figure 18As shown in (B), when the H2O2 concentration was 500μM, the cell viability of L929 cells was 67.36% after 2 hours of stimulation. Under the antioxidant effect of different concentrations of M-HPCS7 / HA-PEG3 hydrogel extract, the cell viability of L929 cells was significantly increased. When the concentrations of M-HPCS7 / HA-PEG3 hydrogel extract were 20%, 40%, 60%, 80%, and 100%, the cell viability was 94.71%, 99.87%, 92.59%, 98.41%, and 98.41%, respectively, indicating that M-HPCS7 / HA-PEG3 hydrogel has a protective effect on cells damaged by H2O2. 2+ It can reduce the influx of calcium ions across the membrane, protect the membrane, and reduce the generation of free radicals. Therefore, M-HPCS3 / HA-PEG7+Mg 2+ The cell viabilities when the hydrogel extract concentrations were 20%, 40%, 60%, 80%, and 100% were 98.15%, 107.67%, 106.35%, 105.99%, and 103.57%, respectively, indicating that M-HPCS3 / HA-PEG7+Mg 2+ The hydrogel also has a protective effect on cells damaged by H2O2, and its antioxidant effect is stronger than that of M-HPCS7 / HA-PEG3 hydrogel.

[0186] from Figure 18 As can be seen in (C), the blank group without H2O2 and hydrogel had almost no obvious green fluorescence, indicating that no large amount of reactive oxygen species were produced in the cells without stimulation, and the cell morphology was good; in the control group with only H2O2 added, a strong green fluorescence signal was found, indicating that H2O2 entered the cells and stimulated the cells to produce a large amount of ROS, and under bright field, it can be observed that the cell morphology of a large number of L929 cells has changed, shrinking and becoming round, indicating that the cells are damaged and apoptotic. Under the intervention of hydrogel, M-HPCS7 / HA-PEG3, M-HPCS3 / HA-PEG7+Mg 2+ Compared to the control group, both hydrogel groups showed no strong green fluorescence, indicating that no significant amounts of reactive oxygen species were produced within the cells. Furthermore, most cells remained in good condition, exhibiting a spindle-shaped morphology. This result is consistent with the antioxidant activity, demonstrating that the hydrogel can interfere with H2O2 stimulation of cells, regulate intracellular reactive oxygen species levels, balance oxidation and antioxidant effects, prevent oxidative stress, and thus protect cells.

[0187] 13. Observation of wound healing

[0188] like Figure 19As shown, deep second-degree burns were inflicted on the backs of rats using a burn instrument. Immediately after the burn, the wound surface turned pale, slightly swollen, and showed no exudation of body fluid. Three days after treatment with the different treatment methods, reddening of the wound edges was observed in each group. The wound surface in the control group remained pale, without scab formation, and was more swollen than immediately after the burn, with slight suppuration, indicating severe inflammation on the wound surface. In the positive control group, a partial scab had formed, with no swelling and a slight reduction in wound size. In the experimental group, a complete scab had formed, with a smooth and regular surface slightly higher than normal skin, and no swelling, with a reduction in wound size. On the 7th day, the wounds in the blank group were scabby and still swollen. It can be seen that the scab surface was significantly higher than normal skin and extremely uneven. The color was slightly yellow-green, different from that of the other groups, indicating that the wound was still severely inflamed. In contrast, both the control and experimental groups had completely formed a flat and regular hard scab, with no swelling, no obvious inflammation, and normal color. The scabs at the edges of the wounds in both groups were raised. As can be seen from the figure, the wound area in the experimental group was smaller than that in the control and positive control groups, and the wound areas in the two NIR groups were significantly smaller than those in the two groups, indicating that NIR can promote wound healing. On the 14th day, the wounds in the blank group were slightly swollen, with no obvious inflammation, and the wounds were slightly smaller. However, the wound edges in the positive control and experimental groups were slightly raised, with no obvious changes in color or texture. The wound size was significantly smaller than that on the 7th day. On day 21, the wound surface of the control group was dark red, indicating congestion on the epidermis, and the wound area was much larger than that of the experimental group. The scabs in the positive control group had fallen off, and there was also slight congestion on the surface. The wound size was slightly smaller than that of the control group. The scabs in the experimental group had mostly fallen off, and the skin surface was smooth and pink after shedding, indicating the formation of new granulation tissue. New hair was also observed around the wound. On day 28, some scabs in the control group were still not completely shed, and the wound surface was not fully healed. The wound surface of the positive control group was also not completely healed, and the skin was red. In the experimental group, the wounds were completely healed, and some of the skin was indistinguishable from the surrounding normal tissue, with no scar formation.

[0189] 14. Analysis of wound healing rate

[0190] like Figure 20 As shown in Figure 2, on the 3rd day, the wound healing rates of each group were low, with the healing rate of the control group being only 2%, while the healing rates of the other groups were around 15%. On the 7th day, the healing rate of the control group was 14.87%, while the healing rates of the hydrogel groups showed significant differences, with M-HPCS7 / HA-PEG3 hydrogel and M-HPCS3 / HA-PEG7+Mg 2+ The healing rate of the hydrogel group was about 30%, and the healing rate of the hydrogel + NIR group was about 38%. On the 14th day, the healing rates of all groups increased significantly, with the healing rate of the control group being 40.32%, and the healing rates of the other groups ranging from 52.73% to 63.75%. 2+The wound healing rates of the hydrogel + NIR group were the highest, at 62.25% and 63.75% respectively. It can be seen that from the 7th day to the 14th day, the healing rate of the blank group increased faster, which may be due to the M-HPCS7 / HA-PEG3 and M-HPCS3 / HA-PEG7+Mg 2+ The hydrogel has anti-inflammatory and inhibitory effects on excessive proliferation of fibroblasts. The hydrogel can make the wound normally enter the proliferation stage from the inflammatory stage. Therefore, the healing rate of the hydrogel group was higher on the 7th day. Afterwards, in order to avoid the formation of scar tissue, the hydrogel will inhibit the excessive proliferation of fibroblasts, resulting in a slight decrease in the proliferation rate of fibroblasts in the wound in the hydrogel group. On the 21st day, except for the control group, the healing rate of the wound in each group was greater than 90%. On the 28th day, the healing rate of the wound in the M-HPCS7 / HA-PEG3 hydrogel + NIR and M-HPCS3 / HA-PEG7 + Mg 2+ The wound healing rate in the hydrogel + NIR group reached nearly 100%, achieving complete healing, while some wounds in the control and positive groups were still not completely healed. The results show that hydrogels can effectively promote wound healing, and photothermal therapy can effectively promote wound healing on top of the use of functional dressings.

[0191] 15. In vivo photothermal conversion analysis

[0192] In the in vivo wound healing experiment, a combination of hydrogel and photothermal therapy was used, namely, M-HPCS7 / HA-PEG3 hydrogel+NIR, M-HPCS3 / HA-PEG7+Mg 2+ The optimal power used in photothermal therapy was studied in the preliminary experiment. M-HPCS7 / HA-PEG3 hydrogel, M-HPCS3 / HA-PEG7+Mg 2+ The hydrogel was applied to the wound surface and different optical densities were used, 1.0W / cm 2 , 1.25W / cm 2 , 1.5W / cm 2 The hydrogel was irradiated with 808nm near-infrared light for 5 minutes, and the temperature changes at the wound site were recorded with an infrared camera. Figure 21 As shown, at an optical density of 1W / cm 2 The M-HPCS7 / HA-PEG3 hydrogel group and the M-HPCS3 / HA-PEG7+Mg 2+ The highest temperature of the hydrogel group can reach about 42℃; at an optical density of 1.25W / cm 2 Under 808nm near-infrared light irradiation, the M-HPCS7 / HA-PEG3 hydrogel group and the M-HPCS3 / HA-PEG7+Mg 2+The highest temperature of the hydrogel group can reach about 43℃ within 5 minutes; 2 Under 808nm near-infrared light irradiation, the M-HPCS7 / HA-PEG3 hydrogel group and the M-HPCS3 / HA-PEG7+Mg 2+ The temperature of the hydrogel group reached about 45°C after 3 minutes of irradiation, and the highest temperature within 5 minutes reached about 50°C. The results showed that the optical density was 1.5W / cm 2 M-HPCS7 / HA-PEG3 hydrogel group and M-HPCS3 / HA-PEG7+Mg 2+ The temperature that the hydrogel can reach is suitable for wound healing. Therefore, in the subsequent treatment process, M-HPCS7 / HA-PEG3 hydrogel+NIR, M-HPCS3 / HA-PEG7+Mg 2+ The hydrogel + NIR group used an optical density of 1.5W / cm 2 The skin was irradiated with 808nm near-infrared light for 5 minutes until the treatment was completed.

[0193] 16. Histopathological analysis of wound repair

[0194] (1) Analysis of HE staining results

[0195] like Figure 22As shown in the figure, on the third day after wound treatment, the skin epidermis in all groups was damaged and its structure was blurred. The collagen fibers in the dermis were severely degenerated and disordered, with a few remaining hair follicles and damaged appendages such as the sebaceous glands, consistent with the pathological tissue of a second-degree burn. There was obvious congestion. On the seventh day, the wound surface was infiltrated with inflammatory cells, vacuoles appeared in the dermis, and the tissue was completely necrotic. The inflammatory cells in the dermis of the control group and the positive control group were significantly fewer than those in the experimental group. The collagen fibers in the dermis of each group were thick and disordered. On the 14th day, no epidermal formation was observed in the control group, but a large number of inflammatory cells were present in the dermis, and the collagen fibers were still extremely disordered. Re-epithelialization had not yet begun in the positive control group, but the dermis was also infiltrated with a large number of inflammatory cells. However, a thinner epidermis had formed in the experimental group, and a large number of capillaries and fibroblasts were generated in the dermis. Inflammatory cell infiltration was still present, but significantly reduced. On day 21, the epidermis in the control group had not yet fully re-epithelialized; it continued to thicken, with fibrous proliferation visible in the dermis. No obvious hair follicles, sebaceous glands, or other appendages were observed. In the experimental group, the scabs on the wound surface had largely fallen off, and epithelial cells had regenerated. The epidermis thickened, with a distinct papillary layer and a structure almost resembling that of normal skin. The epidermal-dermal boundary was also more distinct. In both the experimental and positive control groups, the collagen fibers in the dermis were more uniformly arranged, forming a basket-like pattern. New granulation tissue was evident, with an increase in fibroblasts and new blood vessels. Hair follicles and sebaceous glands were visible in some areas. On day 28, congestion was still present in the control group, while the wounds in the experimental group were completely healed, with a thinner epidermis, relatively complete differentiation, and a well-organized structure. The dermis showed a uniform distribution of skin appendages, and the collagen fibers were neatly arranged, resembling the tissue structure of normal skin.

[0196] (2) Analysis of Masson staining results

[0197] like Figure 23As shown in the results of HE staining, on day 3, the epidermis of the wounds in all groups was damaged and structurally blurred. The collagen fibers in the dermis were severely degenerated and disorganized, with a few remaining hair follicles. The sebaceous glands and other appendages were destroyed, consistent with the pathological histology of second-degree burns. On day 7, the wound tissue was completely necrotic, with inflammatory cell infiltration and vacuoles in the dermis. The collagen fibers in the dermis of all groups were thick and disorganized. On day 14, the necrotic tissue in the control group was still not cleared and had not entered the proliferation stage. However, the dermis of the hydrogel group contained a large number of inflammatory cells and disorganized collagen fibers, but a large number of capillaries, fibroblasts, and new collagen fibers were observed in the dermis. On day 21, the epidermis in the control group had not yet fully re-epithelialized, and the epidermis continued to thicken. Fiber proliferation was observed in the dermis, but no obvious hair follicles, sebaceous glands, or other appendages were observed. In the hydrogel group, the scabs on the wound surface largely fell off, revealing a largely intact epidermal structure with a distinct papillary layer and a more distinct boundary between the epidermis and dermis. In both the experimental and positive control groups, the collagen fibers in the dermis were arranged more neatly than before, forming a basket-shaped pattern. The dermis had been completely repaired, with hair follicles and sebaceous glands visible in some areas. On day 28, congestion was still present in the control group, and some wounds had not yet healed, remaining in the proliferation stage. In the hydrogel group, the wounds were completely healed, with relatively complete epidermal differentiation and good structure. The dermis showed a uniform distribution of skin appendages and neatly arranged collagen fibers, mirroring the structure of normal skin tissue.

[0198] (3) Immunofluorescence staining analysis

[0199] like Figure 24 As shown, the wounds treated with only saline (control group) had a higher iNOS (red) fluorescence level, indicating that the wounds in the control group were still in a severe inflammatory stage, while the red fluorescence signals in the wounds of the other groups were reduced compared with the control group, indicating that the positive control group, M-HPCS7 / HA-PEG3, M-HPCS3 / HA-PEG7+Mg 2+ The inflammatory response at the wound site of the hydrogel and NIR groups was at normal levels, indicating that the two hydrogels can synergistically promote the polarization of M1 macrophages to M2 macrophages to regulate inflammation and promote wound repair.

[0200] like Figure 25 As shown, it can be found that M-HPCS7 / HA-PEG3 hydrogel, M-HPCS3 / HA-PEG7+Mg 2+ The green fluorescence signals of the two hydrogel groups were stronger than those of the control group. After combined photothermal treatment, the green fluorescence signals of M-HPCS7 / HA-PEG3 hydrogel+NIR and M-HPCS3 / HA-PEG7+Mg 2+There were more green fluorescence signals in the wound sites treated with hydrogel + NIR, which indicated that near-infrared light irradiation could promote the transformation of macrophages to M2 type and the wound had entered the proliferation stage normally. 2+ Hydrogel treatment enabled diabetic skin wounds to successfully transition from the inflammatory stage to the proliferative stage. In contrast, the CD206 fluorescence signal in the wound tissue after treatment in the control group was weak, indicating that the number of M2 macrophages in the wound was small and the wound was still in a prolonged inflammatory stage. The results once again confirmed that M-HPCS7 / HA-PEG3 hydrogel, M-HPCS3 / HA-PEG7+Mg 2+ The hydrogel can promote the transformation of macrophages to the M2 type, effectively regulate the inflammatory response in diabetic skin wounds, thereby promoting the formation of granulation tissue in the wounds and accelerating the healing of diabetic skin wounds. In addition, iNOS and CD206 immunofluorescence staining showed that the therapeutic effect of the hydrogel can be enhanced after combined photothermal therapy.

[0201] CD31 is a transmembrane protein expressed in the early stage of angiogenesis, and α-SMA is a cytoplasmic protein expressed in the late stage of angiogenesis. It marks the maturation of vascular smooth muscle cells and is associated with tissue fibrosis and wound contraction. Figure 26 and 27 It was observed that on day 7, M-HPCS7 / HA-PEG3 hydrogel and M-HPCS3 / HA-PEG7+Mg 2+ The fluorescence signals of CD31 and α-SMA in the hydrogel were stronger, and in the group treated with combined photothermal therapy, the fluorescence signal levels of CD31 and α-SMA were higher. The results showed that M-HPCS7 / HA-PEG3 hydrogel and M-HPCS3 / HA-PEG7+Mg 2+ Hydrogels can promote angiogenesis during skin wound healing, and photothermal therapy can further promote angiogenesis. Both hydrogels can promote the healing of diabetic wounds by regulating macrophage polarization and promoting angiogenesis. Photothermal therapy of the wound surface further enhances the therapeutic effect of the hydrogel.

[0202] Finally, although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A melanin-hydroxypropyl chitosan / hyaluronic acid-bisamino polyethylene glycol composite hydrogel, characterized in that: The composite hydrogel is formed by mixing hydroxypropyl chitosan grafted with melanin and hyaluronic acid-bisamino polyethylene glycol; the preparation method of the composite hydrogel comprises the following steps: (1) Hydrophilic modification of chitosan was performed by etherification reaction to obtain hydroxypropyl chitosan (HPCS), and then melanin was grafted onto HPCS by amide reaction to prepare M-HPCS; hyaluronic acid-bisamino polyethylene glycol (HA-PEG) was prepared by amide reaction; (2) preparing an M-HPCS solution and a HA-PEG solution, and then mixing the M-HPCS solution and the HA-PEG solution evenly and placing them in a water bath to prepare a composite hydrogel; in the step (2), the prepared M-HPCS and HA-PEG are dissolved in deionized water to prepare solutions with concentrations of 7% and 4% (w / v), respectively; the volume ratio of the M-HPCS solution to the HA-PEG solution is 3-7:7-3.

2. The melanin-hydroxypropyl chitosan / hyaluronic acid-bisamino polyethylene glycol composite hydrogel according to claim 1, characterized in that: In step (2), the volume ratio of the M-HPCS solution to the HA-PEG solution is 7:3; and the water bath temperature is 45°C-60°C.

3. An M-HPCS / HA-PEG / soluble metal ion composite hydrogel, characterized in that: The composite hydrogel is prepared by mixing the hydroxypropyl chitosan solution grafted with melanin and the hyaluronic acid-bisamino polyethylene glycol solution according to claim 1 with a soluble metal ion solution. The preparation method of the composite hydrogel is as follows: (1) Hydrophilic modification of chitosan was performed by etherification reaction to obtain hydroxypropyl chitosan (HPCS), and then melanin was grafted onto HPCS by amide reaction to prepare M-HPCS; hyaluronic acid-bisamino polyethylene glycol (HA-PEG) was prepared by amide reaction; (2) preparing an M-HPCS solution and a HA-PEG solution, then uniformly mixing the M-HPCS solution and the HA-PEG solution, and adding a soluble metal ion solution dropwise while mixing, that is, placing the solution in a water bath for a certain period of time to obtain the composite hydrogel; in the step (2), the prepared M-HPCS and HA-PEG are dissolved in deionized water to prepare solutions with concentrations of 7% and 4% (w / v), respectively; the volume ratio of the M-HPCS solution: HA-PEG solution is 3-7:7-3; the concentration of the added soluble metal ion solution is 4% (w / v), and the volume percentage of the added solution is 5%.

4. The M-HPCS / HA-PEG / soluble metal ion composite hydrogel according to claim 3, wherein: In the step (2), the ratio of the M-HPCS solution to the HA-PEG solution is 3:7 (v / v).

5. Use of the M-HPCS / HA-PEG composite hydrogel according to claim 1 in the preparation of products having antioxidant effects, promoting cell growth, promoting angiogenesis, photothermal properties, or promoting skin wound healing.

6. Use of the M-HPCS / HA-PEG / soluble metal ion composite hydrogel according to claim 3 in the preparation of products having antioxidant effects, promoting cell growth, promoting neoangiogenesis, photothermal properties, or promoting skin wound healing.

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