Multi-layer multifunctional hydrogel microsphere for diabetic wounds as well as preparation method and application of multi-layer multifunctional hydrogel microsphere

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to a multi-layer multifunctional hydrogel microsphere for diabetic wounds as well as a preparation method and application of the multi-layer multifunctional hydrogel microsphere. The preparation method comprises the following steps: firstly, synthesizing a chitosan derivative with proper amino and aldehyde group contents, oxidized macromolecules and modified chitin; then preparing photocuring hydrogel microspheres by utilizing a microfluidic technology, and sequentially immersing the photocuring hydrogel microspheres into an oxidized polymer solution, a modified chitin solution, a tannic acid solution and a metal salt solution, so as to construct the hydrogel microspheres with a multifunctional layer structure. The invention further provides specific application intervals of parameters such as solvents used by various solutions, concentration ranges and soaking time. The preparation method provided by the invention has expansibility and universality, various types of microspheres can be constructed for sequential release of bioactive substances, and the requirements of tissue repair are met.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to multi-layered multifunctional hydrogel microspheres for diabetic wounds, a preparation method thereof, and applications thereof. Background Art

[0002] The continued rise in diabetes prevalence worldwide has directly led to a rapid increase in the incidence of diabetic wounds. Compared to physiological wounds, the microenvironment of diabetic wounds is characterized by persistent inflammatory infiltration (abnormally elevated levels of pro-inflammatory cytokines such as IL-6 and TNF-α), imbalanced redox homeostasis (significantly increased levels of ROS), and an increased risk of infection with multidrug-resistant bacteria. These pathological mechanisms ultimately hinder wound repair by inhibiting cell migration, angiogenesis, extracellular matrix deposition, and granulation tissue formation.

[0003] Hydrogels facilitate cell adhesion, proliferation, and extracellular matrix deposition, and have been widely used in the development of diabetic wound dressings. Currently, hydrogel preparation methods include self-assembly, photocuring, electrolyte soaking, and various physical / chemical crosslinking methods. To obtain bioactive hydrogels, researchers have incorporated photothermal and piezoelectric materials, or bioactive molecules such as cells, exosomes, and proteins.

[0004] Based on this, researchers have applied Schiff base hydrogels based on amino and aldehyde crosslinking, as well as metal-polyphenol networks, to diabetic wound repair. However, the preparation technology for these gel dressings faces the following challenges: it is difficult to prepare multilayer hydrogel microspheres based on natural polymers that can promote tissue repair. Summary of the Invention

[0005] To address the existing problem of being unable to prepare multilayer hydrogel microspheres based on natural polymers that can promote tissue repair, the present invention provides multilayer multifunctional hydrogel microspheres for diabetic wounds, as well as a preparation method and application thereof. To achieve the above objectives, the present invention employs the following technical solutions.

[0006] One of the purposes of the present invention is to provide a method for preparing multilayer multifunctional hydrogel microspheres for treating diabetic wounds, comprising the following steps: A chitosan modification with an amino content greater than 10% was prepared, and then the chitosan modification was dissolved in PBS using the modified chitosan as a raw material to prepare a chitosan modification PBS solution with a mass concentration of 1% to 4%. An oxidized polymer with an aldehyde content greater than 15% was prepared, and then the oxidized polymer was dissolved in PBS using the modified chitosan as a raw material to prepare an oxidized polymer solution with a mass concentration of 1% to 4%. A chitin modification with an amino content greater than 10% was prepared, and then the modified chitosan modification was dissolved in PBS using the modified chitosan as a raw material to prepare a chitosan modification solution with a mass concentration of 0.5% to 1.5%.

[0007] Preparation of tannic acid solution and metal salt solution.

[0008] Preparation of multilayer multifunctional hydrogel microspheres: Using microfluidic technology, the polysaccharide modified PBS solution and photoinitiator are used as the aqueous phase, and mineral oil containing a surfactant is used as the oil phase. Microspheres are photocured under ultraviolet irradiation to obtain chitosan modified hydrogel microspheres.

[0009] The chitosan modified hydrogel microspheres are then immersed in the oxidized polymer solution, the chitin modified solution, the tannic acid solution and the metal salt solution in sequence, thereby constructing hydrogel microspheres with a multi-layer multifunctional structure, and obtaining the multi-layer multifunctional hydrogel microspheres for diabetic wounds.

[0010] The present invention provides a preparation method for multi-layer multifunctional hydrogel microspheres for diabetic wounds. The method uses methacrylic acid-modified chitosan microspheres containing sufficient amino groups as the core, and based on Schiff base reaction and electrostatic interaction, sequentially immerses the microspheres in an oxidized polymer solution, a chitin modification solution, a tannic acid solution and a metal salt solution to finally construct a hydrogel microsphere with a multifunctional layer structure.

[0011] Preferably, the chitosan modification includes at least one of methacrylate carboxymethyl chitosan, methacrylate sulfonated chitosan, and methacrylate quaternary ammonium salt chitosan.

[0012] Preferably, the oxidized polymer includes at least one of oxidized carboxymethyl starch, oxidized hyaluronic acid and oxidized dextran.

[0013] Preferably, the chitosan modification comprises at least one of quaternized chitosan and carboxymethyl chitosan.

[0014] Preferably, the metal salt solution includes at least one of a copper chloride solution, a nitrate solution and a zinc chloride solution.

[0015] Preferably, the ratio of the mass of the chitosan modified hydrogel microspheres to the volume of the oxidized polymer solution is 0.8g~1.2g:10mL; the ratio of the mass of the microspheres obtained after immersing in the oxidized polymer solution to the volume of the chitin modified solution is 0.8g~1.2g:10mL; the ratio of the mass of the microspheres obtained after immersing in the chitin modified solution to the volume of the tannic acid solution is 0.8g~1.2g:10mL; the ratio of the mass of the microspheres obtained after immersing in the tannic acid solution to the volume of the metal salt solution is 0.8g~1.2g:10mL.

[0016] Preferably, the aqueous phase is obtained by compounding the polysaccharide modified product PBS solution and the photoinitiator; the ratio of the mass of the photoinitiator to the volume of the chitosan modified product PBS solution is 1.8g-2.2g:100mL. More preferably, the ratio of the mass of the photoinitiator to the volume of the chitosan modified product PBS solution is 2.0g:100mL, that is, the mass concentration of the photoinitiator in the chitosan modified product PBS solution is 0.2%; the photoinitiator is LAP. That is, in a specific embodiment of the present invention, the aqueous phase is a PBS solution of 3% w / v methacrylate carboxymethyl chitosan and 0.2% w / v LAP as a photoinitiator.

[0017] Preferably, the oil phase is mineral oil containing 2% w / v of span 80.

[0018] The second purpose of the present invention is to provide multi-layer multifunctional hydrogel microspheres for diabetic wounds prepared by the preparation method.

[0019] The third object of the present invention is to provide the use of the multi-layered multifunctional hydrogel microspheres for diabetic wounds in the preparation of drugs for preventing and / or treating diabetic wounds.

[0020] Preferably, the antibacterial drug uses the multi-layer multifunctional hydrogel microspheres for diabetic wounds as an active ingredient.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides a method for preparing multilayer multifunctional hydrogel microspheres for diabetic wounds. The method for preparing the multilayer multifunctional hydrogel microspheres for diabetic wounds provided by the present invention includes preparing chitosan modifications, oxidized polymers, and chitin modifications; preparing tannic acid solutions and metal salt solutions; preparing polysaccharide modifications in PBS, oxidized polymer solutions, and chitin modifications; utilizing microfluidics technology to photocure microspheres under ultraviolet irradiation to obtain chitosan modified hydrogel microspheres; then, sequentially immersing the chitosan modified hydrogel microspheres in the oxidized polymer solution, chitin modified solution, tannic acid solution, and metal salt solution. Based on Schiff base reaction and electrostatic interaction, the hydrogel microspheres having a multifunctional layer structure are sequentially immersed in the oxidized polymer solution, chitin modified solution, tannic acid solution, and metal salt solution to obtain multilayer multifunctional hydrogel microspheres for diabetic wounds. Through the mutual coordination of the above steps, hydrogel microspheres with controllable thickness and multi-layer structure can be obtained, which can be anti-inflammatory, antioxidant, antibacterial and thus promote tissue repair, thereby solving the problem in the existing technology that it is impossible to prepare multi-layer hydrogel microspheres that can promote tissue repair based on natural polymers.

[0022] The multifunctionality of the multilayered multifunctional hydrogel microspheres prepared by the present invention refers to the fact that the hydrogel microspheres have the biological activities of antibacterial, anti-inflammatory, anti-oxidative stress, cell migration promotion, angiogenesis promotion and collagen maturation promotion.

[0023] 2. The multilayered, multifunctional hydrogel microspheres prepared by this invention for diabetic wounds exhibit biological activity, including antibacterial, anti-inflammatory, anti-oxidative stress, cell migration, angiogenesis, and collagen maturation, thereby promoting diabetic wound healing. These multilayered, multifunctional hydrogel microspheres are not only useful for diabetic wound management but also offer new insights into the design of hydrogel structures for subsequent wound dressings and even tissue repair.

[0024] 3. This invention utilizes modified chitosan, oxidized polymers, modified chitin, tannic acid, and metal salt solutions to synthesize multilayered, multifunctional hydrogel microspheres. Mechanistically, the methacrylated chitosan-modified hydrogel microspheres provide a foundational platform for subsequent reactions and sufficient amino groups. By controlling the degree of substitution, molecular weight, concentration, and immersion time of each subsequent molecule, a controllable multilayer structure can be formed on the hydrogel surface. This multilayer structure can be used to encapsulate bioactive substances (such as cells and drugs) in multiple layers, achieving sequential delivery and enhancing therapeutic efficacy. Furthermore, the tannic acid-metal chelate in these microspheres is primarily distributed in the hydrogel surface. Its anti-inflammatory, antioxidant, and angiogenic properties are well-suited to the challenges faced by wounds in the early stages of wound healing. As the wound heals, the outermost layer of the hydrogel microspheres degrades, and the natural polymer scaffold within provides adhesion sites for migrating cells, promoting collagen secretion and maturation, ultimately optimizing wound healing outcomes in diabetic patients.

[0025] 4. The multi-layer multifunctional hydrogel microspheres for diabetic wounds provided by the present invention are a medical material for diabetic wounds, which have the functions of antibacterial, anti-inflammatory, anti-oxidative stress, promotion of cell migration, promotion of angiogenesis and promotion of collagen maturation, etc., and are in line with the current clinical situation.

[0026] 5. The multi-layered multifunctional hydrogel microspheres for diabetic wounds provided by the present invention provide a method for preparing the multi-layered hydrogel microspheres.

[0027] 6. The polymers and metals in the multi-layered multifunctional hydrogel microspheres for diabetic wounds proposed in the present invention can be customized according to needs, are suitable for sequential delivery of active substances, may be applied to other tissue repair applications, and are more in line with personalized medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the synthesis, cross-linking relationship and function of multi-layered multifunctional hydrogel microspheres in an embodiment of the present invention; wherein, Figure 1Figure A shows the interaction between polymers and the schematic diagram of the layer-by-layer deposition of multilayer structures: Figure 1 Figure B shows the biological properties of multilayered multifunctional hydrogel microspheres.

[0029] Figure 2 The morphology of the multilayer multifunctional hydrogel microspheres in the embodiment of the present invention under ordinary microscope and scanning electron microscope; wherein, Figure 2 Figure A shows the microscopic images of carboxymethyl methacrylate chitosan hydrogel microspheres (CSMA), quaternary ammonium methacrylate chitosan / oxidized carboxymethyl starch microspheres (CO), quaternary ammonium methacrylate chitosan / oxidized carboxymethyl starch / quaternary ammonium chitin microspheres (COQ), quaternary ammonium methacrylate chitosan / oxidized carboxymethyl starch / quaternary ammonium chitin / tannic acid microspheres (COQT), and quaternary ammonium methacrylate chitosan / oxidized carboxymethyl starch / quaternary ammonium chitin / tannic acid / copper microspheres (COQTCu) (scale bar = 500 μm); Figure 2 Figure B shows the general view of CSMA, CO, COQ, COQT, and COQTCu; Figure 2 Figure C in the figure is the diameter of the microsphere; Figure 2 Figure D in the figure shows the porosity of the microspheres; Figure 2 Figure E in the figure is the SEM image of the freeze-dried microspheres; Figure 2 Figure F shows the SEM image of COQTCu and the corresponding EDS elemental analysis; Figure 2 Figure G shows the surface content ratio of COQTCu.

[0030] Figure 3 The bright field fluorescence images of the multi-layered multifunctional hydrogel microspheres in the embodiment of the present invention at different stages using fluorescent labeling molecules to participate in the synthesis of microspheres further prove the formation of the multi-layer structure; wherein, Figure 3 Figure A in Figure 3 Figure B in Figure 3 Panel C in the figure shows the bright field and fluorescence images of CO, COQ, and COQT obtained after immersion in the outermost fluorescent labeling solution for 1 minute, 5 minutes, and 30 minutes, respectively; Figure 3 Panel D shows bright field and fluorescence images of COQO (COQ subsequently immersed in OCMS-DAPI); Figure 3 Panel E shows the bright field and fluorescence images of COQ after storage in the dark at room temperature for 30 days; Figure 3 Panel F shows AO / PI staining of CO and COQ cultured in fibroblasts. In this figure, the microsphere names are color-coded to indicate the corresponding fluorescent-labeled molecule solution used. Scale bar = 200 µm.

[0031] Figure 4The general view and relevant statistical data of the multi-layered multifunctional hydrogel microspheres in the embodiments of the present invention in promoting diabetic wound healing; wherein, Figure 4 Figure A shows the gross appearance of wounds in the gauze, CSMA, COQ, and COQTCu groups within 12 days; Figure 4 Panel B shows H&E staining of skin tissue on day 12 at 25x (scale bar = 1 mm) and 400x magnification (scale bar = 50 μm); Figure 4 Panel C shows CD31 immunofluorescence staining images of wound tissue on day 12 (scale bar = 50 μm); Figure 4 Figure D is a quantitative analysis of the gross area of ​​wounds in different groups at different times; Figure 4 Panel E in the figure is the quantification of granulation tissue width; Figure 4 Panel F in the figure is the quantification of granulation tissue thickness; Figure 4 Figure G in the figure is the quantification of epidermal thickness; Figure 4 Panel H in the figure is the quantitative counting of fibroblasts; Figure 4 Figure I shows the quantification of blood vessel number. DETAILED DESCRIPTION

[0032] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0033] Example 1 A method for preparing multilayer multifunctional hydrogel microspheres for treating diabetic wounds comprises the following steps: 1. Preparation of polymers (1) Preparation of methacrylate carboxymethyl chitosan: First, carboxymethyl chitosan is prepared, wherein the degree of deacetylation of the carboxymethyl chitosan is 99%, the total degree of carboxymethyl substitution of the carboxymethyl chitosan is 95%, and the amino content of the carboxymethyl chitosan is 33%.

[0034] 1 g of carboxymethyl chitosan was dissolved in 50 mL of deionized water, and 205 μL of methacrylic anhydride was added at a rate of 0.5 mL / min. After reacting at 60°C for 4 hours, the reaction mixture was dialyzed in deionized water for 3 days and freeze-dried to obtain methacrylic acid carboxymethyl chitosan.

[0035] The prepared methacrylate carboxymethyl chitosan has a methacrylate substitution degree of 23% and an amino content of 10%.

[0036] (2) Preparation of oxidized carboxymethyl starch: Corn starch is used as raw material, carboxymethyl starch is firstly produced through alkalization and etherification reaction, and then oxidized carboxymethyl starch is produced through oxidation reaction.

[0037] Wherein, the method for synthesizing carboxymethyl starch is as follows: 80 mL of 95% by volume ethanol was added to 20 g of corn starch, followed by the addition of 11.6 g of sodium hydroxide and 23 g of chloroacetic acid to obtain a reaction mixture. The reaction mixture was heated at 40°C for 10 hours and then dried to obtain carboxymethyl starch.

[0038] The method for preparing oxidized carboxymethyl starch is as follows: Dissolve 10 g of carboxymethyl starch (70% carboxymethyl substitution) in 100 mL of deionized water, and add sodium periodate at a molar ratio of 2:1 between sodium periodate and anhydrous glucose units. Stir and react at room temperature for 2 hours in the dark. After the reaction, place the reaction solution in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyze against deionized water for 7 days. The resulting mixture is then freeze-dried to yield oxidized carboxymethyl starch.

[0039] The aldehyde content of oxidized carboxymethyl starch was determined to be 27% by alkali consumption method.

[0040] (3) Preparation of quaternary ammonium salt chitosan: β-Chitin with a deacetylation degree of 5% was isolated and purified from squid pens according to a previous study (Chen Z, et al. Macromol Biosci. 2022;22(3):e2100418). One gram of the purified β-Chitin powder was dispersed in a mixture of potassium hydroxide, urea, and water in a mass ratio of 20:4:75 to prepare a 1% suspension. The suspension was stirred at −30°C for approximately 30 minutes to obtain a clear and viscous chitin solution.

[0041] Subsequently, 2,3-epoxypropyltrimethylammonium chloride (in a molar ratio of 8:1 to glucosamine units) was added to the chitosan solution and stirred at 45°C for 24 hours. After the reaction, the mixture was neutralized with hydrochloric acid. The resulting quaternary ammonium chitosan solution was dialyzed against distilled water for 7 days through a dialysis membrane with a molecular weight cutoff of 3500 Da. The solution was then freeze-dried to obtain the quaternary ammonium chitosan for later use.

[0042] The deacetylation degree of the quaternary ammonium salt chitosan obtained above is 36%, and the quaternary ammonium salt substitution degree of the quaternary ammonium salt chitosan is 35%.

[0043] 2. Preparation of multilayer multifunctional hydrogel microspheres (1) Preparation of methacrylate carboxymethyl chitosan PBS solution: 3 g of methacrylate carboxymethyl chitosan was dissolved in 100 mL of PBS to obtain methacrylate carboxymethyl chitosan PBS solution.

[0044] (2) Preparation of oxidized carboxymethyl starch solution: Oxidized carboxymethyl starch was dissolved in PBS to obtain an oxidized carboxymethyl starch solution with a w / v concentration of 3%.

[0045] (3) Preparation of quaternary ammonium salt chitosan solution: The quaternary ammonium salt chitosan was dissolved in PBS to obtain a quaternary ammonium salt chitosan solution with a w / v concentration of 1%.

[0046] (4) Preparation of tannic acid solution: Tannic acid was dissolved in deionized water to obtain a tannic acid solution with a w / v concentration of 1%.

[0047] (5) Preparation of copper chloride solution: Copper chloride was dissolved in deionized water to obtain a copper chloride solution with a w / v concentration of 0.1%.

[0048] (6) Preparation of methacrylate carboxymethyl chitosan hydrogel microspheres: A monodisperse droplet generation system was constructed according to a previous study (Xiao Z, et al. Small 2022;18 (29): e2202596). The aqueous phase of the microfluidic system consisted of a PBS solution containing 3% w / v carboxymethyl methacrylate chitosan and 0.2% w / v LAP as a photoinitiator, and the oil phase consisted of a mineral oil containing 2% w / v Span 80, flowing through the outer coaxial dispensing needle. Syringe pumps controlled the flow rates of the aqueous and oil phases at 0.7 mL / h and 6 mL / h, respectively. Microspheres were photocured by irradiation with ultraviolet light (405 nm, 10 mW / cm²) for 10 seconds, resulting in microspheres immersed in oil.

[0049] The microspheres immersed in oil obtained in the previous step were then replaced with the oil phase by the demulsifier perfluorooctanol and washed repeatedly with PBS to obtain methacrylate carboxymethyl chitosan hydrogel microspheres (CSMA).

[0050] The preparation method of the LAP photoinitiator with a w / v ratio of 0.2% is as follows: 0.2g LAP is dissolved in 100mL PBS. LAP: Aladdin, L157759.

[0051] Mineral oil containing 2% w / v Span 80 is prepared by dissolving 2 g of Span 80 in 100 mL of mineral oil. Span 80: Aladdin, S110840. Mineral oil: Aladdin, M274343.

[0052] (7) Preparation of multilayer multifunctional hydrogel microspheres: The collected methacrylate carboxymethyl chitosan hydrogel microspheres were immersed in oxidized carboxymethyl starch solution for 30 minutes, and then washed to obtain methacrylate carboxymethyl chitosan / oxidized carboxymethyl starch hydrogel microspheres (CO).

[0053] The methacrylate carboxymethyl chitosan / oxidized carboxymethyl starch hydrogel microspheres obtained in the previous step were immersed in a quaternary ammonium salt chitin solution for 20 minutes, and then washed to obtain methacrylate carboxymethyl chitosan / oxidized carboxymethyl starch / quaternary ammonium salt chitin hydrogel microspheres (COQ).

[0054] The methacrylate carboxymethyl chitosan / oxidized carboxymethyl starch / quaternary ammonium salt chitin hydrogel microspheres obtained in the previous step were immersed in tannic acid solution for 5 minutes, and then washed to obtain methacrylate carboxymethyl chitosan / oxidized carboxymethyl starch / quaternary ammonium salt chitin / tannic acid hydrogel microspheres (COQT).

[0055] The methacrylate carboxymethyl chitosan / oxidized carboxymethyl starch / quaternary ammonium salt chitosan / tannic acid hydrogel microspheres obtained in the previous step were immersed in a copper chloride solution for 3 minutes, and then washed to obtain methacrylate carboxymethyl chitosan / oxidized carboxymethyl starch / quaternary ammonium salt chitosan / tannic acid / copper hydrogel microspheres (COQTCu), thereby obtaining multilayer multifunctional hydrogel microspheres for diabetic wounds. The schematic diagram of the preparation process and final biological function of the multilayer multifunctional microspheres obtained in this example is shown in FIG. Figure 1 shown.

[0056] It should be noted that during the preparation process, all microspheres obtained in the above steps need to be washed repeatedly with the solvent of the subsequent solution until it is confirmed under a microscope that no flocs or particles are generated when the eluate is mixed with the next solution.

[0057] Example 2 A method for preparing multilayer multifunctional hydrogel microspheres for treating diabetic wounds comprises the following steps: 1. Preparation of polymers (1) Preparation of methacrylic acid sulfonated chitosan: First, sulfonated chitosan was prepared according to previous literature (ménez-Arriagada, Daniela et al. Virology journalvol. 19,1 52. 24 Mar. 2022), with a deacetylation degree of 98%, a total sulfate group substitution degree of 110%, and an amino content of 28% for the sulfonated chitosan.

[0058] 1 g of sulfonated chitosan was dissolved in 50 mL of deionized water, and 125 μL of methacrylic anhydride was added at a rate of 0.5 mL / min. After reacting at 60°C for 4 hours, the reaction mixture was dialyzed in deionized water for 3 days and freeze-dried to obtain methacrylic acid sulfonated chitosan.

[0059] The methacrylic acid substitution degree of the methacrylic acid sulfonated chitosan obtained above is 10%, and the amino content is 18%.

[0060] (2) Preparation of oxidized dextran: 2 g of dextran was dissolved in 200 mL of deionized water and oxidized with 1.0 g of sodium periodate at 25°C for 2.5 hours. The reaction was terminated by adding 0.5 mL of ethylene glycol, and stirring was continued for another hour. After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 3500 Da and dialyzed against deionized water for 7 days. The resulting mixture was then freeze-dried to obtain oxidized carboxymethyl starch.

[0061] The aldehyde content of oxidized carboxymethyl starch was determined to be 22% by alkali consumption method.

[0062] (3) Preparation of carboxymethyl chitin: β-chitosan with a deacetylation degree of 5% was isolated and purified from squid pens using the same method as described in the previous “Preparation of quaternized chitosan”.

[0063] One gram of purified β-chitosan powder was dispersed in a mixed solution of potassium hydroxide, urea, and water in a mass ratio of 20:4:75 to prepare a 1% suspension. The suspension was stirred at −30°C for approximately 30 minutes to obtain a clear and viscous chitosan solution.

[0064] Subsequently, chloroacetic acid (at a molar ratio of 10:1 to glucosamine units) was added to the chitosan solution and stirred at 0°C for 72 hours. After the reaction, the mixture was neutralized with hydrochloric acid. The resulting carboxymethyl chitosan solution was dialyzed against distilled water for 7 days through a 3500 Da dialysis membrane and then freeze-dried to obtain carboxymethyl chitosan for later use.

[0065] The deacetylation degree of the obtained carboxymethyl chitin is 32%, and the carboxymethyl substitution degree is 28%.

[0066] 2. Preparation of multilayer multifunctional hydrogel microspheres (1) Preparation of methacrylic acid sulfonated chitosan PBS solution: 3 g of methacrylate sulfonated chitosan was dissolved in 100 mL of PBS to obtain a 2% w / v methacrylate sulfonated chitosan PBS solution.

[0067] (2) Preparation of oxidized dextran solution: Oxidized dextran was dissolved in PBS to obtain a 2% w / v oxidized dextran solution.

[0068] (3) Preparation of carboxymethyl chitosan solution: Carboxymethyl chitosan was dissolved in PBS to obtain a 1% w / v carboxymethyl chitosan solution.

[0069] (4) Preparation of tannic acid solution: Tannic acid was dissolved in deionized water to obtain a 1% w / v tannic acid solution.

[0070] (5) Preparation of nitrate solution: Silver nitrate was dissolved in deionized water to obtain a 0.1% w / v nitrate solution.

[0071] (6) Preparation of methacrylic acid sulfonated chitosan hydrogel microspheres: A monodisperse droplet generation system was constructed according to a previous study (Xiao Z, et al. Small 2022;18 (29): e2202596). The aqueous phase of the microfluidic system consisted of a 2% w / v methacrylated chitosan PBS solution and a 0.2% w / v LAP photoinitiator flowing through a coaxial dispensing needle. The oil phase consisted of a 2% w / v span 80 mineral oil flowing through a coaxial dispensing needle. Syringe pumps controlled the flow rates of the aqueous and oil phases at 0.7 mL / h and 5 mL / h, respectively. Microspheres were photocured by irradiation with ultraviolet light (405 nm, 10 mW / cm²) for 10 seconds, resulting in microspheres immersed in oil.

[0072] The microspheres immersed in oil obtained in the previous step were then replaced with the oil phase by the demulsifier perfluorooctanol and washed repeatedly with PBS to obtain methacrylate sulfonated chitosan hydrogel microspheres (SCMA).

[0073] (7) Preparation of multilayer multifunctional hydrogel microspheres: The collected methacrylate sulfonated chitosan hydrogel microspheres were immersed in oxidized dextran solution for 20 minutes, and then washed to obtain methacrylate sulfonated chitosan / oxidized dextran hydrogel microspheres (SO).

[0074] The methacrylate sulfonated chitosan / oxidized dextran hydrogel microspheres obtained in the previous step were immersed in a carboxymethyl chitin solution for 20 minutes, and then washed to obtain methacrylate sulfonated chitosan / oxidized dextran / carboxymethyl chitin hydrogel microspheres (SOC).

[0075] The methacrylate sulfonated chitosan / oxidized dextran / carboxymethyl chitin hydrogel microspheres obtained in the previous step were immersed in tannic acid solution for 5 minutes, and then washed to obtain methacrylate sulfonated chitosan / oxidized dextran / carboxymethyl chitin / tannic acid hydrogel microspheres (SOCT).

[0076] The methacrylate sulfonated chitosan / oxidized dextran / carboxymethyl chitin / tannic acid hydrogel microspheres obtained in the previous step were immersed in a silver nitrate solution for 2 minutes, and then washed to obtain methacrylate sulfonated chitosan / oxidized dextran / carboxymethyl chitin / tannic acid / silver hydrogel microspheres (SOCTAg), namely, multilayer multifunctional hydrogel microspheres for diabetic wounds.

[0077] It should be noted that during the preparation process, all microspheres obtained in the above steps need to be washed repeatedly with the solvent of the subsequent solution until it is confirmed under a microscope that no flocs or particles are generated in the eluate when it is mixed with the next solution.

[0078] In order to illustrate the effect of the preparation method of the multilayer multifunctional hydrogel microspheres for diabetic wounds provided by the present invention, the following experiments were conducted: 1. Morphology of multilayered multifunctional hydrogel microspheres All microspheres were photographed using an Olympus IX73 microscope, and their diameters were measured using NIH ImageJ software. The morphology of the freeze-dried microspheres was observed using a scanning electron microscope (SEM, TESCAN MIRA LMS). Subsequently, the elemental composition of the COQTCu samples was analyzed using SEM-EDS. The porosity of the hydrogel microspheres was determined using the liquid replacement method.

[0079] The morphology of hydrogel microspheres is shown in Figure 2 shown. Figure 2 Figure A shows microscopic images of hydrogel microspheres (CSMA, CO, COQ, COQT, and COQTCu) immersed in PBS, respectively, which show obvious morphological differences. Compared with CSMA, the edge of CO and the surrounding PBS is more blurred. In contrast, the boundary of COQ is clearer than that of CO. A large number of gully-like structures were observed in the outer layer of COQT. For COQTCu, the number of green gullies accumulated in the outer layer increased, and the edges appeared more uneven instead of forming a complete arc. Overall, the changes in morphology indicate that the composition of the microspheres has changed, especially in the surface layer. Overall observation of the hydrogel microspheres showed obvious color changes ( Figure 2 (Figure B in the figure). COQ appears pale white, consistent with the color of the QC solution. COQT appears pink, consistent with the color of previously reported QC / TA hydrogels (Xie F, et al. Food Hydrocolloids. 2025; 159: 110636). In contrast, COQTCu appears gray-green, consistent with the appearance of TA / Cu nanoparticles in earlier research (Tao L, et al. Nano Today. 2024; 54: 102071). These color changes confirm the successful preparation of the hydrogel microspheres and the corresponding chemical reactions.

[0080] The diameters of CSMA, CO, COQ, COQT and COQTCu hydrogel microspheres decrease in sequence ( Figure 2 Figure C in the figure), where the diameter of COQTCu is about two-thirds of the initial CSMA (526.1 μm vs. 784.0 μm). Similarly, the porosity of the hydrogel microspheres also showed a downward trend ( Figure 2 (D in Figure 1). The rapid decrease in diameter indicates that water expulsion is primarily driven by the Schiff base reaction between OCMS and CSMA. Furthermore, when CSMA, OCMS, and QC were dissolved in deionized water, the diameter of the hydrogel microspheres decreased even more significantly.

[0081] Subsequently, the structures of the hydrogel microspheres (CSMA, CO, COQ, COQT, and COQTCu) were observed using scanning electron microscopy (SEM). Figure 2 Figure E in the figure). Due to the large specific surface area of ​​the microspheres, they are prone to water loss and shrinkage during the separation and freeze-drying process, so the SEM images cannot accurately reflect their true size. Nevertheless, the surface morphology revealed by SEM provides valuable insights into their structural details. The CSMA surface exhibits a porous structure, while the angular substances observed on the surface may be due to the deposition of phosphate crystals during the manufacturing process. The CO surface appears smoother than CSMA, and the COQ surface structure is smoother and denser, both of which are attributed to the Schiff base reaction. For COQT, no obvious porous structure was observed on the surface of the microspheres, which may be due to the physical cross-linking of TA and QC through electrostatic interactions and hydrogen bonds. Finally, the COQTCu surface exhibits many wrinkles, similar to nanoflowers, which is mainly attributed to the chelation reaction between Cu²⁺ and TA, resulting in surface shrinkage and structural changes. SEM-EDS images show that copper is evenly distributed on the surface of COQTCu ( Figure 2 The F graph and Figure 2 (Figure G in Figure 1). TA reacts with QC, distributing it extensively on the COQT surface. During the COQTCu preparation process, Cu²⁺ chelates with TA, resulting in a relatively uniform distribution of copper on the microsphere surface.

[0082] 2. Structural evaluation of multilayered multifunctional hydrogel microspheres 2.1 Synthesis of DAPI-functionalized OCMS and FITC-functionalized QC To synthesize DAPI-functionalized OCMS (OCMS-DAPI), 200 mg of OCMS was dissolved in 10 mL of deionized water. 0.3 mmol of EDC and NHS were then added to the homogeneous solution. After incubation for 30 minutes, 2 mg of DAPI, previously dissolved in 1 mL of deionized water, was added. The mixture was stirred at 25°C in the dark for 12 hours, dialyzed against distilled water for 7 days, and then freeze-dried. To synthesize FITC-functionalized QC (QC-FITC), 50 mg of QC was dissolved in 5 mL of deionized water to form a homogeneous solution. Then, 0.1 mg of FITC, previously dissolved in 50 μL of methanol, was added. The mixture was stirred at 25°C in the dark for 12 hours, dialyzed against distilled water for 7 days, and then freeze-dried.

[0083] 2.2 Preparation of Fluorescently Labeled Hydrogel Microspheres To elucidate the structure of the hydrogel microspheres, OCMS, QC, or TA were replaced with OCMS-DAPI, QC-FITC, or TA-Cy5 solutions during the immersion phase. The remaining experimental conditions were consistent with the standard microsphere preparation method. All fluorescently labeled microspheres were imaged using an Olympus IX73 microscope or a confocal microscope (Leica, Germany, TCS SP8).

[0084] First, CO, COQ, and COQT microspheres were prepared, and their outermost layers were fluorescently labeled. In this step, the microspheres were immersed in OCMS-DAPI, QC-FITC, or TA-Cy5 solutions for 1, 5, and 30 minutes, respectively, and then observed. Second, CSMA microspheres were sequentially immersed in OCMS-DAPI (30 minutes), QC-FITC (20 minutes), and TA-Cy5 (5 minutes) to prepare COQT multicolor microspheres. Third, non-fluorescent COQ microspheres were immersed in OCMS-DAPI for 30 minutes to prepare COQO microspheres.

[0085] 2.3 Preparation of cell-laden hydrogel microspheres First, L929 cells were suspended in OCMS or QC solution. Subsequently, non-fluorescent CSMA or CO microspheres were immersed in OCMS or QC solution containing L929 cells for 30 or 20 minutes, respectively. After thorough washing, the cell-laden hydrogel microspheres were cultured in DMEM complete medium for 24 hours. Acridine orange / propidium iodide (AO / PI) stain (Epizyme Biotech, China) was used to assess cell viability and spatial distribution.

[0086] The results of the evaluation of the multi-layered multifunctional hydrogel microsphere structure are as follows Figure 3As shown in Figure 2, fluorescent labeling molecules (OCMS-DAPI, QC-FITC, and TA-Cy5) were used to evaluate the detailed structure of the hydrogel microspheres. The fluorescence distribution accurately reflects the spatial location of the new components, while the fluorescence intensity corresponds to their relative content. Figure 3 In the figure, the letters in the microsphere names are color-coded to indicate the corresponding fluorescent labeling molecule solution used. The single fluorescent-labeled microspheres CO, COQ, and COQT were prepared by immersing the non-fluorescent CSMA, CO, and COQ in OCMS-DAPI, QC-FITC, and TA-Cy5 solutions, respectively, for different time periods.

[0087] For single fluorescent labeled CO microspheres ( Figure 3 (Figure A in the figure) The blue color is relatively uniform throughout, with similar blue fluorescence intensities observed at 5 and 30 minutes, and significantly stronger than at 1 minute. The fluorescence distribution and intensity indicate that OCMS-DAPI rapidly penetrates and uniformly distributes within the microspheres, primarily within the first 5 minutes.

[0088] For a single fluorescently labeled CoQ ( Figure 3 (B) Green fluorescence is consistently confined to the outer layer of the microspheres, with intensity gradually increasing within 1, 5, and 30 minutes after immersion. This indicates that QC-FITC permeates the microspheres relatively slowly, forming a distinct layered structure within 30 minutes.

[0089] For a single fluorescently labeled COQT ( Figure 3 (C) Red fluorescence initially localizes to the thinner outer layer of the microsphere; however, over time (from 1 minute to 5 minutes to 30 minutes), the intensity gradually increases toward the inner layer. The present invention proposes that TA preferentially binds to the outer QC due to electrostatic interactions between the positively charged QC and the negatively charged TA.

[0090] The present invention also provides a novel method for preparing microspheres with multiple additional layers. Non-fluorescent COQ was immersed in OCMS-DAPI solution for 30 minutes to prepare COQO ( Figure 3 Figure D in Figure ). With CO ( Figure 3 Unlike the uniform blue fluorescence observed in Figure 1 (A), COQO exhibits a distinct blue ring structure on its outer circumference. This fluorescence pattern suggests that more layers can be prepared by sequentially immersing in solutions of macromolecules containing amino and aldehyde groups using Schiff base reactions and electrostatic interactions. The stability of the multilayered hydrogel microspheres was further verified using a single fluorescently labeled COQ (CO / QC-FITC) and stored in the dark at room temperature for 30 days. Figure 3 The preserved green fluorescence distribution indicates that the QC layer structure remained intact and largely unchanged during this period.

[0091] In the present invention, non-fluorescent CSMA or CO was immersed in OCMS or QC solution containing resuspended L929 cells, and then cultured for 24 hours. AO / PI staining showed very few red (dead) cells, and most cells were stained green (live). Most of the live cells were located in the outer layer of the hydrogel microspheres ( Figure 4 These results indicate that the microspheres have good biocompatibility and their multilayer structure provides a new strategy for the sequential delivery of bioactive substances.

[0092] 3. Multi-layered multifunctional hydrogel microspheres promote diabetic wound healing All BALB / c mice were induced into a diabetic model by a single intraperitoneal injection of streptozotocin (200 mg / kg). A successful model was defined as a blood glucose level exceeding 16.8 mmol / L on three consecutive measurements. The diabetic wound model in mice was established by creating two symmetrical circular wounds on the back of the mice using a hole punch (7 mm diameter) after anesthesia, hair removal, and skin preparation. A silicone ring frame (20 mm outer diameter, 10 mm inner diameter) was then secured around the wounds. The diabetic mice were randomly divided into four groups: Group 1 had its wounds covered with gauze as a control group; Group 2 had its wounds covered with CSMA; Group 3 had its wounds covered with COQ; and Group 4 had its wounds covered with COQTCu. All dressings were applied on days 0 and 3, and wound images were taken on days 0, 3, 6, 9, and 12. Mice were sacrificed by dislocation on day 12 after surgery, and wound tissue was collected.

[0093] Wound tissue specimens collected on postoperative day 12 were fixed with 4% paraformaldehyde, embedded in paraffin, and stained with H&E. Embedded wound tissue specimens were sectioned along the long axis of the mouse (parallel to the direction of tension). Sections were imaged using an Olympus IX73 microscope or a polarizing microscope (Nikon Eclipse Ci-POL, Nikon). Granulation tissue clearance, granulation tissue thickness, epidermal thickness, and fibroblast counts were analyzed using ImageJ software based on H&E staining. Dewaxed wound tissue sections were sequentially dewaxed, hydrated, incubated with hydrogen peroxide, and blocked with bovine serum albumin. Subsequently, the specimens were incubated overnight with an anti-CD31 (3528, CST) primary antibody, followed by a Cy3-conjugated secondary antibody. Cell nuclei were counterstained with DAPI.

[0094] The results of multi-layered multifunctional hydrogel microspheres promoting diabetic wound healing are as follows Figure 4.. To evaluate the wound healing effect of multilayered hydrogel microspheres, an excisional wound splint model was established on the back of diabetic mice, and the splint was firmly fixed using glue and interrupted 4-0 sutures. This fixation method can prevent the wound edge from shrinking and better simulate the human wound healing process, which mainly relies on granulation tissue formation and tissue remodeling. The mice were randomly divided into four groups: control group, CSMA group, COQ group and COQTCu group. The wound was defined as an area with obvious color compared with the surrounding normal skin. On the 12th day after surgery, the CSMA group, COQ group and COQTCu group all showed accelerated wound healing, indicating that the hydrogel microspheres promoted repair ( Figure 4 Figure A and Figure 4 Figure D in the figure).

[0095] Notably, wound area in the COQ group was smaller than in the CSMA group on days 9 and 12, suggesting that the addition of OCMS and QC to the multilayered microspheres further promoted healing. The COQTCu group had the smallest wound area on day 12, with only 36.7% remaining, compared to 71.5% in the control group.

[0096] In addition, the wounds in the COQTCu group achieved re-epithelialization on the 6th day, while granulation tissue was still visible in the wounds of the control group, but no epidermal formation was observed. On the 12th day after surgery, H&E staining was used for histological examination to further evaluate the wound healing ( Figure 4 Diabetic wounds often fail to close properly due to insufficient myofibroblast differentiation, restricted fibroblast migration, and reduced collagen secretion. In the present invention, the granulation tissue gaps in the COQTCu group were the narrowest, which is consistent with the gross observations ( Figure 4 Figure E in the figure).

[0097] In addition, the granulation tissue in the COQTCu group was the thickest, indicating that COQTCu hydrogel microspheres most effectively promoted the formation of granulation tissue ( Figure 4 Although impaired diabetic wound healing is often associated with insufficient epithelial regeneration, persistent inflammation and oxidative stress can also lead to pathological epidermal thickening; however, no significant differences in epidermal thickness were found between the groups ( Figure 4 Another characteristic of diabetic wounds is the defect in the number and function of fibroblasts and blood vessels. CD31 is a specific marker for vascular endothelial cells; therefore, a higher number of CD31-positive cells indicates a higher density of blood vessels in the tissue ( Figure 4 Notably, the COQTCu group had the highest number of fibroblasts and blood vessels ( Figure 4 The H graph and Figure 4 Figure 1), further demonstrating its effectiveness in enhancing wound repair.

[0098] The above experimental results show that the present invention can adopt multiple cross-linking strategies to prepare multilayer hydrogel microspheres (COQTCu), which can effectively promote diabetic wound healing in vivo and exhibit multiple biological properties including anti-inflammation, promotion of cell migration, and promotion of angiogenesis.

[0099] In summary, the methacrylate carboxymethyl chitosan / oxidized carboxymethyl starch / quaternized chitin / tannic acid / copper hydrogel microspheres prepared by the preparation method for diabetic wounds provided by the present invention have a multilayer structure of multilayer microspheres, which mainly rely on the Schiff base reaction between polymers and the imine bond cross-linking formation. Therefore, the requirements for the chitosan modification, oxidized polymer, and chitin modification are that they can provide sufficient amino or aldehyde groups; the tannic acid-metal coordination cross-linking network has anti-inflammatory, antioxidant, and angiogenesis-promoting capabilities.

[0100] In addition, the structure and function of the multilayer multifunctional hydrogel microspheres for diabetic wounds provided by the present invention are highly dependent on the concentration and immersion time of each molecular solution. The present invention provides a series of relatively stable parameters, but the selection of polymers, the concentration of each molecular solution and the immersion time can also be changed to prepare new microspheres, thereby broadening their application in tissue repair.

[0101] It should be noted that when the present invention involves a numerical range, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes a preferred embodiment.

[0102] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once the basic inventive concepts become known, and all such changes and modifications fall within the scope of the present invention.

Claims

1. A method for preparing multi-layer multifunctional hydrogel microspheres for diabetic wounds, characterized in that: The steps include: Prepare a chitosan modified product with an amino content greater than 10%, and then use it as a raw material to prepare a chitosan modified product PBS solution; prepare an oxidized polymer with an aldehyde content greater than 15%, and then use it as a raw material to prepare an oxidized polymer solution; prepare a chitin modified product with an amino content greater than 10%, and then use it as a raw material to prepare a chitin modified product solution; Preparation of tannic acid solution and metal salt solution; Utilizing microfluidic technology, the polysaccharide modification PBS solution and a photoinitiator are used as the aqueous phase, and a mineral oil containing a surfactant is used as the oil phase. Under ultraviolet irradiation, photocuring is performed to generate microspheres to obtain chitosan modification hydrogel microspheres. The chitosan modification hydrogel microspheres are then immersed in the oxidized polymer solution, the chitin modification solution, the tannic acid solution, and the metal salt solution in sequence to construct hydrogel microspheres with a multilayer multifunctional structure, thereby obtaining the multilayer multifunctional hydrogel microspheres for diabetic wounds.

2. The preparation method according to claim 1, characterized in that The chitosan modification comprises at least one of methacrylate carboxymethyl chitosan, methacrylate sulfonated chitosan and methacrylate quaternary ammonium salt chitosan.

3. The preparation method according to claim 1, characterized in that The oxidized polymer includes at least one of oxidized carboxymethyl starch, oxidized hyaluronic acid and oxidized dextran.

4. The preparation method according to claim 1, characterized in that The chitosan modification comprises at least one of quaternized chitosan and carboxymethyl chitosan.

5. The preparation method according to claim 1, characterized in that The metal salt solution includes at least one of a copper chloride solution, a nitrate solution and a zinc chloride solution.

6. The preparation method according to claim 1, characterized in that The ratio of the mass of the chitosan modified hydrogel microspheres to the volume of the oxidized polymer solution is 0.8g~1.2g:10mL; the ratio of the mass of the microspheres obtained after immersion in the oxidized polymer solution to the volume of the chitin modified solution is 0.8g~1.2g:10mL; the ratio of the mass of the microspheres obtained after immersion in the chitin modified solution to the volume of the tannic acid solution is 0.8g~1.2g:10mL; and the ratio of the mass of the microspheres obtained after immersion in the tannic acid solution to the volume of the metal salt solution is 0.8g~1.2g:10mL.

7. The preparation method according to claim 1, characterized in that The aqueous phase is obtained by compounding the polysaccharide modified material PBS solution and the photoinitiator; the ratio of the mass of the photoinitiator to the volume of the chitosan modified material PBS solution is 1.8g-2.2g:100mL.

8. Multi-layered multifunctional hydrogel microspheres for diabetic wounds prepared by the preparation method of claim 1.

9. Use of the multi-layered multifunctional hydrogel microspheres for treating diabetic wounds according to claim 8 in the preparation of a medicament for preventing and / or treating diabetic wounds.

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