A hydrogel for promoting vascularization of diabetic wounds and a method of preparing the same

Abstract

The application relates to the field of biological medical materials, and mainly relates to a diabetic wound vascularization regeneration hydrogel and a preparation method thereof. A preparation method of a diabetic wound vascularization regeneration hydrogel (MoS2 QDs / HM) comprises the following steps: mixing methyl methacryl group-substituted hydroxypropyl chitosan (HM) and MoS2 QDs solution to obtain a precursor solution; the concentration of the MoS2 QDs is 0.1-0.5 mg / mL; the concentration of the HM is 35-45 mg / mL; the precursor solution and a photoinitiator are stirred and mixed; and after light irradiation, the MoS2 QDs / HM is obtained. The MoS2 QDs / HM provided in the application has good anti-swelling capacity, mechanical performance and biocompatibility, realizes the dual biological activities of inhibiting wound inflammatory polarization and promoting vascular endothelial cell function repair, and significantly accelerates the vascularization regeneration and healing process of a diabetic chronic wound.

Description

Technical Field

[0001] This application relates to the field of biomedical materials, and mainly to a hydrogel that promotes vascularization and regeneration of diabetic wounds and its preparation method. Background Technology

[0002] Currently, hydrogel materials used for healing diabetic wounds suffer from uncontrollable swelling effects and poor mechanical properties, easily leading to wound compression and hydrogel damage. Furthermore, due to the hyperglycemic microenvironment of the wound, the repair of diabetic wounds faces interconnected problems such as insufficient angiogenesis, dysregulation of the immune-inflammatory response, and impaired wound contraction and closure. Existing hydrogel materials cannot comprehensively address these issues in one go, limiting their effectiveness in promoting diabetic wound healing. Therefore, developing hydrogels with anti-swelling and flexible properties, as well as inherent pro-angiogenic, immunomodulatory, and wound-constriction-promoting properties, is of significant research importance for the clinical treatment of diabetic wounds. Summary of the Invention

[0003] In view of the shortcomings of the prior art, the purpose of this application is to provide a hydrogel that promotes vascularization and regeneration of diabetic wounds and its preparation method, aiming to provide an injectable photocrosslinked hydrogel dressing with good anti-swelling and vascularization and regeneration capabilities for diabetic wounds.

[0004] The technical solution of this application is as follows: A method for preparing a hydrogel that promotes vascularization and regeneration of diabetic wounds includes the following steps: A precursor solution was obtained by mixing methacrylated hydroxypropyl chitosan and MoS2QDs solution. The concentration of MoS2QDs in the MoS2QDs solution is 0.1-0.5 mg / mL; the concentration of methacrylyl-substituted hydroxypropyl chitosan in the precursor solution is 35-45 mg / mL. The precursor solution and photoinitiator are stirred and mixed under light-protected conditions for 18-30 hours; after light irradiation, a hydrogel that promotes vascularization and regeneration of diabetic wounds is obtained.

[0005] Furthermore, the method for preparing the MoS2QDs solution includes the following steps: mixing MoS2QDs with water to obtain a MoS2QDs solution.

[0006] Furthermore, the preparation method of the MoS2QDs includes the following steps: Ammonium tetrathiomolybdate, hydrazine, and water are mixed to obtain a homogeneous solution; the homogeneous solution contains 0.1-0.2 M ammonium tetrathiomolybdate and 2-4 M hydrazine. The homogeneous solution was reacted at a temperature of 170-190℃ for 24-48 hours; Solid-liquid separation was performed, and the precipitate was washed and dried to obtain the MoS2QDs.

[0007] Furthermore, the preparation method of the methacryl-substituted hydroxypropyl chitosan includes the following steps: Hydroxypropyl chitosan and buffer solution are mixed to obtain the first mixed solution; Methacrylamide and the first mixed solution are stirred and mixed at 45-55℃ for 3-6 hours to obtain a second mixed solution; the ratio of the amount of hydroxypropyl chitosan to the amount of methacrylic anhydride is 1g: (0.05-0.2)mL; After dialysis and drying, the second mixed solution yields the methacryl-substituted hydroxypropyl chitosan.

[0008] Furthermore, the buffer solution includes PBS; The ratio of the amount of hydroxypropyl chitosan to the amount of buffer solution is 1 g : (35-45) mL.

[0009] Furthermore, the dialysis includes: dialysis for 4-6 days in a buffer solution at 40-50°C, followed by dialysis in water for 1-2 days.

[0010] Furthermore, the precipitate is washed with NaOH, ethanol, and water, and then dried under vacuum at 55-65°C for 12-24 hours to obtain the MoS2QDs.

[0011] Furthermore, the final concentration of the photoinitiator is 0.01-0.2% (w / v).

[0012] Furthermore, the illumination uses blue light with an intensity of 9-11 mW / cm². 2 .

[0013] This application also provides a hydrogel that promotes vascularization and regeneration of diabetic wounds.

[0014] The purpose of this application is to formulate a photocrosslinked injectable hydrogel with anti-swelling properties, which is formed by crosslinking molybdenum disulfide quantum dots (MoS2QDs) with methacrylic acid-substituted hydroxypropyl chitosan (HM). This hydrogel significantly improves angiogenesis, reduces inflammation, and promotes vascular regeneration and healing of diabetic wounds. This application utilizes MoS2QDs / HM to form a hydrogel with anti-swelling properties, while also having drug-free angiogenesis and anti-inflammatory capabilities, achieving precise control of the wound microenvironment, and possessing good injectability, in-situ molding capability, and low production cost.

[0015] Compared with the prior art, this application has the following beneficial effects: This application develops an injectable, anti-swelling hydrogel based on the self-assembly of molybdenum disulfide quantum dots and methacrylyl-substituted hydroxypropyl chitosan. Utilizing the electrostatic attraction between the two, a stable hierarchical sea urchin-like microstructure is constructed, forming a hydrogel with excellent anti-swelling ability and mechanical properties under photocrosslinking. It also exhibits good biocompatibility, achieving dual bioactivity of inhibiting wound inflammatory polarization and promoting vascular endothelial cell function repair, significantly accelerating the vascularization and healing process of diabetic chronic wounds. Attached Figure Description

[0016] Figure 1 For pure HM system ( Figure 1 Figure A in the paper) and the MoS2 / HM composite system of this application ( Figure 1 Molecular dynamics (MD) simulations and related data (Figure B in the figure); Figure 1 Plot C in the figure represents the mean square displacement (MSD) analysis, which shows the movement of molecules over time. The slope of the MSD reflects the speed of molecular motion; the larger the slope, the faster the motion. The decrease in diffusion is due to aggregation. Figure 1 The D diagram in the figure represents the intermolecular van der Waals interaction energy; Figure 1 The E diagram in the figure represents the intermolecular electrostatic interaction energy; System 1 is a pure HM system, and system 2 is a MoS2 / HM composite system; HM-HM represents the intermolecular force of HM, and HM-MoS2 represents the force between HM and MoS2.

[0017] Figure 2 A diagram illustrating the self-assembly process of the MoS2 / HM composite system. Figure 2 (In Figure A, located in the top row), energy spectrum analysis diagram ( Figure 2 (in Figure A, located in the bottom row) and Zeta potential analysis plot ( Figure 2 (Figure B in the text) Figure 2 In Figure A, the first row from left to right corresponds to the different states of the MoS2 / HM composite system at the initial stage of self-assembly, in progress, and after completion. Figure 2 In Figure B, MoS2 / HM1 represents the MoS2 / HM composite system with a MoS2 QDs concentration of 0.125 mg / mL, diluted 20 times with pure water; MoS2 / HM2 represents the MoS2 / HM composite system with a MoS2 QDs concentration of 0.25 mg / mL, diluted 20 times with pure water; and MoS2 / HM3 represents the MoS2 / HM composite system with a MoS2 QDs concentration of 0.5 mg / mL, diluted 20 times with pure water.

[0018] Figure 3 SEM images of pure HM gel (first from left) and MoS2QDs / HM; The second from the left corresponds to a MoS2QDs concentration of 0.125 mg / mL MoS2QDs / HM, the third from the left corresponds to a MoS2QDs concentration of 0.25 mg / mL MoS2QDs / HM, and the fourth from the left corresponds to a MoS2QDs concentration of 0.5 mg / mL MoS2QDs / HM.

[0019] Figure 4 TEM images, SEAD diagrams, energy spectra, and particle size distributions of MoS2 QDs quantum dots.

[0020] Figure 5 The images show physical models of the MoS2 / HM composite system and MoS2QDs / HM.

[0021] Figure 6 The graph shows the detection data of swelling behavior of pure HM gel and MoS2QDs / HM.

[0022] Figure 6 In the legend, HM corresponds to pure HM gel, MoS2QDs / HM1 and MoS2 / HM1 correspond to MoS2 QDs concentration of 0.125 mg / mL, MoS2QDs / HM2 and MoS2 / HM2 correspond to MoS2 QDs concentration of 0.25 mg / mL, and MoS2QDs / HM3 and MoS2 / HM3 correspond to MoS2 QDs concentration of 0.5 mg / mL. G′ is the storage modulus, and G″ is the loss modulus; Figure 6 Figure A in the figure shows the weight swelling ratio data of hydrogels in PBS for each group; Figure 6 Figure B in the figure shows actual images of the swelling test results of the hydrogels in each group; Figure 6 Figure C in the figure shows the compressive stress-strain curves of the hydrogels in each group; Figure 6 Figures D and E in the figure show the dynamic rheological test results of the hydrogels in each group.

[0023] Figure 7 Figure showing the in vitro angiogenesis-promoting effects of hydrogels in each group; Figure 7In the legend, MoS2 / HM1 corresponds to a MoS2 QDs concentration of 0.125 mg / mL MoS2 QDs / HM, MoS2 / HM2 and MoS2 / HM correspond to a MoS2 QDs concentration of 0.25 mg / mL MoS2 QDs / HM, and MoS2 / HM3 corresponds to a MoS2 QDs concentration of 0.5 mg / mL MoS2 QDs / HM. Figure 7 Figure A in the diagram shows the results of the hemolysis experiment; Figure 7 Figure B in the diagram shows the results of the cell compatibility experiment. Figure 7 Figure C in the figure shows the results of the HUVEC scratch test; Figure 7 Figure D in the figure represents the results of the transwell experiment. Figure 7 Figure E in the figure shows the results of the tube forming experiment; Figure 7 Figure F in the figure is a statistical chart of the healing area in the HUVEC scratch test; Figure 7 The G-plot in the image is a statistical graph of cell counts in the transwell experiment. Figure 7 Figure I in the image is an immunofluorescence image of CD31 (angiogenesis marker); Figure 7 The J-figure in the figure is a statistical graph of CD31 fluorescence intensity; Figure 7 The K-plot in the figure shows the qPCR results for ANG1, HIF-1α, and VEGF. Figure 7 The L-figure in the image is a flow cytometry result of the EDU cell proliferation experiment.

[0024] Figure 8 Images related to animal experiments; Figure 8 In the legend, C corresponds to the PBS group, 3M corresponds to the 3M commercial dressing group, HM corresponds to the pure HM gel group, and MoS2 / HM corresponds to the MoS2QDs / HM group with a concentration of 0.25 mg / mL. Figure 8 Figure A in the diagram is a flowchart of animal experiments; Figure 8 Figure B in the diagram shows the results of the wound healing experiment. Figure 8 Figure C in the diagram is a schematic diagram of the wound healing area; Figure 8 Figure D in the diagram shows the H&E staining results; Figure 8The E-plot in the figure represents experimental data on wound closure rate. Figure 8 The F-plot in the figure represents Masson's trichrome staining. Figure 8 The G-graph in the figure represents the relative collagen content data.

[0025] Figure 9 A double immunofluorescence staining image of CD31 and α-SMA in skin tissue ( Figure 9 Figure A in the middle), data chart ( Figure 9 (Figures B and C in the diagram). Figure 9 In the legend, C and Control correspond to the PBS group, 3M corresponds to the 3M commercial dressing group, HM corresponds to the pure HM gel group, and MoS2 / HM and MoS2@HM correspond to the MoS2QDs / HM group with a concentration of 0.25 mg / mL. Detailed Implementation

[0026] This application provides a hydrogel that promotes vascularization and regeneration of diabetic wounds, and a method for preparing the same. To make the objectives, technical solutions, and effects of this application clearer and more explicit, the following provides a more detailed description. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0027] This application provides a method for preparing a hydrogel that promotes vascularization and regeneration of diabetic wounds, comprising the following steps: Step 1: Prepare MoS2QDs solution, including the following steps: Step 1a: Synthesize MoS2QDs via a one-pot hydrothermal method, including the following steps: Ammonium tetrathiomolybdate [(NH4)2MoS4] and hydrazine (N2H4) were dissolved in deionized water and ultrasonically treated for 20-40 minutes to form a homogeneous solution.

[0028] Regarding the content, the homogeneous solution contains 0.1-0.2M (NH4)2MoS4 and 2-4M hydrazine. The prepared homogeneous solution is transferred to a reaction vessel, sealed, and reacted at 170-190℃ for 24-48 hours.

[0029] After the reaction was complete, the mixture was allowed to cool naturally to room temperature, and then filtered to obtain a black precipitate.

[0030] Step 1b: Wash the precipitate sequentially with 0.5-1.5 mol / L NaOH, anhydrous ethanol, and deionized water, and then dry it in a vacuum environment at 55-65℃ for 12-24 h to obtain negatively charged MoS2QDs.

[0031] Step 1c: Disperse MoS2QDs in water by ultrasonication to obtain a MoS2QDs solution.

[0032] Step 2: Preparation of methacryl-substituted hydroxypropyl chitosan (HM), including the following steps: Step 2a: Add hydroxypropyl chitosan (HCS) to a buffer solution (pH 7.2-7.4), stir continuously until the solution is homogeneous, and heat to 45-55℃ to obtain the first mixed solution.

[0033] The preferred buffer solution is PBS.

[0034] The ratio of hydroxypropyl chitosan to PBS is 1g:(35-45)mL.

[0035] Step 2b: Slowly add methacrylic anhydride to the first mixed solution and stir continuously at 45-55℃ for 3-6 hours, then cool to room temperature to obtain the second mixed solution.

[0036] The ratio of hydroxypropyl chitosan to methacrylic anhydride is 1g: (0.05-0.2)mL.

[0037] Step 2c: Dialyze the second mixed solution in a buffer solution using a dialysis bag to remove unreacted methacrylic anhydride at a temperature of 40-50°C for 4-6 days.

[0038] After dialyzing in deionized water for 1-2 days, freeze-dry to obtain a white flocculent HM solid with a positive charge (+20~40mV).

[0039] Step 3: Mix HM with MoS2QDs solution to prepare precursor solution.

[0040] In the MoS2QDs solution, the concentration of MoS2QDs is 0.1-0.5 mg / mL.

[0041] In the precursor solution, the HM concentration is 35-45 mg / mL.

[0042] Strongly negatively charged quantum dots and positively charged HM molecular chains are rapidly attracted by strong electrostatic attraction, supplemented by hydrogen bonds and coordination bonds, spontaneously assembling into highly ordered hierarchical urchin-like hybrid microstructures; the molecular dynamics (MD) simulation diagram of this process is shown in [reference]. Figure 1 Figure B in the diagram corresponds to a MoS2QDs concentration of 0.5 mg / mL.

[0043] The pure HM system is a mixture with an HM concentration of 40 mg / mL and water as the solvent. The pure HM system is used for comparison. Figure 1 Figure A in the figure is a molecular dynamics (MD) simulation of the pure HM system.

[0044] All molecular dynamics (MD) simulations were performed using the LAMMPS software package. VMD software was used for trajectory visualization and analysis. A single HM molecule consists of 1163 atoms, and a single MoS2 quantum dot contains 30 atoms. The simulation systems were divided into two groups: the first group (pure HM system) contained 20 HM molecular chains; the second group (MoS2 / HM composite system, i.e., precursor solution) added 20 MoS2 quantum dots to the first group. The simulation box size was set to 14.9 nm × 14.9 nm × 14.9 nm. The OPLS-AA force field parameters for HM were derived from published simulation studies. The universal force field was used for MoS2. Nonbonded van der Waals interactions were simulated using a 12-6 Lennard-Jones potential, while electrostatic interactions such as long-range Coulomb interactions were handled using a particle-particle-particle mesh technique. During the production phase, the time step was set to 1 fs, and data was collected every 10 ps. The system first employs the steepest descent method to minimize energy while maintaining isotropic box length. Both systems are run at 298 K for 100 ps under the NVT ensemble to reach equilibrium. Subsequently, they are run for another 50 ns under the NVT ensemble at 298 K to observe aggregation and obtain system parameters. Periodic boundary conditions are applied in all directions.

[0045] The assembly mechanism of the system was further analyzed using molecular dynamics (MD) simulations. In the pure HM system, the HM chains initially exist as random coils, and gradually form loose clusters under the influence of intermolecular hydrogen bonds. Figure 1 (See Figure A in the image). In contrast, in the MoS2 / HM composite system, HM chains rapidly aggregate towards MoS2QDs within a short time, forming a dense hybrid spherical structure. A magnified view shows that polar functional groups such as –OH and –NH2 on the chitosan backbone form a dense short-range interaction network with the MoS2 surface. Figure 1 Figure B in the diagram shows that the polymer chains in the pure HM system have high mobility, while in the MoS2 / HM composite system, the MSD of the HM chains is significantly reduced, indicating that MoS2 QDs have a significant anchoring effect on the polymer chains. Figure 1 (See Figure C in the diagram). Further energy analysis shows that in the presence of MoS2, both the electrostatic interaction energy and the van der Waals interaction energy in the system are significantly enhanced. The absolute value of the electrostatic interaction energy is significantly higher than that of the van der Waals interaction energy, indicating that electrostatic interaction plays a dominant role in the stability of the composite system. Figure 1 (Figures D and E in the middle).

[0046] Based on the combined experimental characterization and simulation results, the following assembly model can be proposed: MoS2QDs and HM with opposite charges first rapidly combine through electrostatic attraction, and then gradually solidify into a stable hierarchical spherical structure under the combined action of hydrogen bonding and van der Waals interactions. These self-assembled structures act as key physical crosslinking nodes in the subsequent photocrosslinking process, thereby significantly improving the mechanical properties and swelling resistance of the hydrogel on a macroscopic scale.

[0047] The addition of MoS2QDs promoted the formation of micropores (pore size <50μm) on the nanocomposite material, resulting in a more compact fibrous network structure and a higher degree of physical cross-linking in the subsequently formed hydrogel that promotes vascularization and regeneration of diabetic wounds. As an effective physical cross-linking agent, MoS2QDs promoted the assembly and cross-linking of HM, significantly improving the cross-linking density of the hydrogel and forming a fibrous network.

[0048] The self-assembly process, energy dispersive spectroscopy (EDS) spectrum, and zeta potential analysis of the MoS2 / HM composite system are shown in the reference diagram. Figure 2 .

[0049] The self-assembly behavior of MoS2QDs mixed with HM was observed at different stages using TEM. In the initial stage, the system exhibited high-contrast MoS2QDs discretely distributed against a low-contrast HM polymer chain background, forming amorphous clusters. As the assembly process progressed, these primary clusters gradually aggregated and evolved into more uniform spherical structures. Ultimately, the system formed a well-defined, structurally ordered urchin-like spherical structure. EDS elemental distribution results showed that Mo and S elements were uniformly distributed throughout the structure, indicating that MoS2QDs were not simply embedded, but rather served as indispensable building blocks in the composite structure.

[0050] To investigate the driving force of self-assembly, zeta potential analysis was performed on MoS2QDs, HM and their composite systems. Figure 2 (See Figure B in the diagram). The pristine MoS₂QDs exhibit a strong negative potential (approximately -60 mV), which may be related to their edge defects and sulfur end groups; HM exhibits a positive potential (approximately +20 mV) due to amine matrix protonation. When the two are mixed, the zeta potential of the composite system remains positive but is significantly lower than that of pure HM, gradually approaching neutrality with increasing MoS₂QDs concentration. This potential change indicates that electrostatic interactions play a crucial role in the self-assembly of MoS₂QDs and HM.

[0051] Step 4: After the precursor solution is completely dissolved, add the photoinitiator LAP at a final concentration of 0.01-0.2% (w / v), and stir at room temperature for 18-30 hours under light-protected conditions to obtain the third mixed solution.

[0052] After the third mixed solution was allowed to stand and degassed, it was then irradiated with blue light to obtain a hydrogel that promotes vascularization and regeneration of diabetic wounds (MoS2QDs / HM).

[0053] Preferably, the blue light is 405nm with a light intensity of 9-11mW / cm². 2 .

[0054] Upon irradiation with blue light (preferably 405nm), LAP absorbs photons and decomposes, generating highly reactive free radicals. These free radicals rapidly attack the methacrylamide (MA) double bonds at the ends of the HM molecular chains, initiating a chain polymerization reaction. The originally discrete "sea urchin-like" hybrid units are tightly bound together by covalent bonds between HM chains, forming a complex three-dimensional interconnected porous network (see reference). Figure 3 (Second to fourth from the left). Short-range interactions (hydrogen bonds, van der Waals forces) and polymer chain entanglement further solidify the structure into a stable hierarchical sea urchin spherical structure. This structure increases the active sites of MoS2QDs / HM and enhances the mechanical properties of MoS2QDs / HM by dispersing external forces through relative slip between molecules and chains; its dense network restricts the penetration and physical contact of water molecules, thereby inhibiting excessive swelling; at the same time, the enhanced hydrogen bonds, acting as sacrificial bonds, efficiently dissipate energy, giving the material excellent toughness and fatigue resistance.

[0055] The present application will be further described below through specific embodiments.

[0056] Example 1 This application provides a method for preparing a hydrogel that promotes vascularization and regeneration of diabetic wounds, comprising the following steps: MoS₂QDs were synthesized via a one-pot hydrothermal method. (NH₄)₂MoS₄ (Sigma-Aldrich) and N₂H₄ (Sigma-Aldrich) were dissolved in 80 mL of deionized water, and the solution was sonicated for 30 minutes to form a homogeneous solution. The homogeneous solution contained 0.15 M (NH₄)₂MoS₄ and 3 M N₂H₄.

[0057] The prepared homogeneous solution was transferred to a reaction vessel, sealed, and reacted at 180°C for 24 hours. After the reaction was complete, it was allowed to cool naturally to room temperature, then filtered to obtain a black precipitate. The black precipitate was washed with 1 mol / L NaOH, anhydrous ethanol, and deionized water, and then dried under vacuum at 60°C for 12 hours to obtain negatively charged MoS₂QDs.

[0058] TEM images, SEAD diagrams, energy spectrum, and particle size distribution of MoS2 QDs quantum dots (reference). Figure 4 .

[0059] Transmission electron microscopy (TEM) was used to characterize the morphology, dispersion, and size distribution of MoS2QDs. The results showed that the MoS2QDs were uniformly dispersed, and clear, parallel lattice fringes were observed under high magnification, indicating high crystallinity. Selected area electron diffraction (SAED) patterns revealed a series of clear concentric diffraction rings, indicating that the MoS2QDs are a typical polycrystalline structure. Their interplanar spacings were approximately 0.28 nm and 0.16 nm, corresponding to the (100) and (110) crystal planes of the 2H phase MoS2, respectively. Size statistics showed that the particle size of the MoS2QDs was mainly distributed between 2.5 and 5.0 nm, with an average diameter of approximately 3.7 nm.

[0060] MoS2QDs were ultrasonically dispersed in water to obtain a MoS2QDs solution.

[0061] 5 g of hydroxypropyl chitosan (HCS) was added to 200 mL of PBS (pH=7.4), and the mixture was stirred continuously until homogeneous. The solution was then heated to 50 °C to obtain the first mixed solution. 0.75 mL of methacrylic anhydride was slowly added to the first mixed solution, and the mixture was stirred continuously at 50 °C for 4 h. The mixture was then cooled to room temperature to obtain the second mixed solution. The second mixed solution was dialyzed in PBS using a 10,000 MW dialysis bag at 45 °C for 5 days to remove unreacted methacrylic anhydride. After dialyzing in deionized water for 1 day, the HM solution was freeze-dried to obtain a positively charged (approximately +20 mV) white flocculent HM solid.

[0062] HM was mixed with MoS2QDs solutions of different concentrations (0.125, 0.25, 0.5 mg / mL), i.e., 40 mg HM was added to 1 mL of MoS2QDs solution to prepare a precursor solution with an HM concentration of 40 mg / mL.

[0063] After the precursor solution is completely dissolved, a final concentration of 0.1% (w / v) of photoinitiator LAP is added, and the mixture is stirred at room temperature for 24 hours under light-protected conditions to obtain the third mixed solution.

[0064] After the third mixed solution was allowed to stand and degas, it was then subjected to 405nm illumination (light intensity 10mW / cm²). 2 Irradiation yielded a hydrogel (MoS2QDs / HM) that promotes vascularization and regeneration of diabetic wounds.

[0065] Reference Figure 5The uncured MoS2 QDs at a concentration of 0.25 mg / mL MoS2 / HM composite system (first from left) exhibited good flowability, allowing for easy injection molding, and rapidly cured into a stable hydrogel after 5 min of 405 nm light irradiation (second from left) (third and fourth from left). This injectability and in-situ photocrosslinking property enable it to adapt to complex and irregular wound morphologies, meeting the needs of clinical applications.

[0066] This application provides an injectable, anti-swelling hydrogel based on the self-assembly of molybdenum disulfide quantum dots and methacrylyl-substituted hydroxypropyl chitosan. The electrostatic attraction between the two components constructs a stable hierarchical, spherical microstructure resembling sea urchins. Under photocrosslinking, this forms a hydrogel with excellent anti-swelling ability and mechanical properties. It also exhibits good biocompatibility, achieving dual bioactivity by inhibiting wound inflammation polarization and promoting vascular endothelial cell function repair, significantly accelerating the vascularization and healing process of diabetic chronic wounds.

[0067] Preparation of pure HM gel: A final concentration of 0.1% (w / v) of photoinitiator LAP was added to the pure HM system, and the mixture was stirred at room temperature for 24 hours under light-protected conditions to obtain a homogeneous solution. After static degassing, the solution was irradiated with 405 nm light (10 mW / cm²). 2 Irradiation yielded pure HM gel. SEM images of the pure HM gel are shown below. Figure 3 (First from the left).

[0068] about Figure 6 Among the many key properties of hydrogel dressings, swelling behavior is one of the core factors affecting their clinical feasibility. Excessive swelling may lead to the destruction of the three-dimensional network structure, causing a decrease in mechanical properties or even material disintegration. It may also cause overhydration and mechanical compression of surrounding healthy tissue, thus hindering wound healing. Therefore, this study systematically evaluated the swelling kinetics and final equilibrium swelling ratio of different hydrogels in PBS. The results showed that the weight swelling ratio of all hydrogels in PBS first increased and then tended to stabilize over time. Compared with pure HM gel, composite hydrogels with added MoS2QDs exhibited superior anti-swelling properties, with MoS2 / HM2 (MoS2QDs at a concentration of 0.25 mg / mL) showing the most outstanding performance. Figure 6 (Figure A in the figure). The volume change results are consistent with the trend of weight swelling; MoS2 / HM2 only underwent slight volume expansion within 48 h and remained stable. Figure 6 (See Figure B in the diagram). The equilibrium swelling ratio of pure HM hydrogel is approximately 150%, while the introduction of MoS2QDs further effectively inhibits the swelling behavior of the hydrogel, reducing its swelling ratio to 1.1-1.2.

[0069] The mechanical properties of hydrogels are crucial in tissue engineering and regenerative medicine applications. To evaluate the enhancing effect of MoS2QDs HM hydrogels on mechanical properties, compression tests and rheological analyses were performed on different samples. The compressive stress-strain curves showed that all samples exhibited typical nonlinear elastic behavior, i.e., stress increased exponentially with increasing strain. Figure 6 (See Figure C in the diagram). Compared to pure HM gel, the MoS2QDs / HM composite hydrogel exhibits significantly higher compressive stress under the same strain conditions, indicating that the introduction of MoS2QDs effectively enhances the hydrogel network strength. At 60% compressive strain, the MoS2QDs / HM2 exhibits the highest compressive strength, approximately 90 kPa, significantly higher than the pure HM group (approximately 20 kPa) and other composite groups. It is noteworthy that the compressive strength decreases slightly with further increases in MoS2QDs concentration. This may be related to the light-blocking effect of high-concentration quantum dots, leading to insufficient cross-linking within the hydrogel.

[0070] Further analysis of the hydrogel network structure at the microscopic level was conducted, and dynamic rheological tests were performed on the samples. Within the angular frequency range of 0.1–100 rad / s, the storage modulus (G′) of all samples was consistently significantly higher than the loss modulus (G″), and did not fluctuate significantly with frequency changes. Figure 6 The D-plot in the figure shows that a stable elastic gel network was formed in each system. Among them, MoS2QDs / HM2 had the highest G′ value, which is highly consistent with the compression test results. The strain scan results show that when the strain is less than 100%, the G′ of each sample remains basically stable, indicating that the hydrogel has good structural integrity; when the strain exceeds 100%, G′ drops sharply and crosses with G″, marking the destruction of the network structure. Figure 6 (See Figure E in the diagram). It is noteworthy that while maintaining a high modulus, the critical yield strain of MoS2QDs / HM2 did not decrease significantly, indicating that the introduction of MoS2QDs enhanced the material's strength without causing significant embrittlement. This is of great significance for mechanical matching in tissue engineering. In summary, the mechanical and rheological test results consistently demonstrate that the MoS2QDs / HM2 formulation achieves optimal mechanical property balance through nanoscale interfacial interactions.

[0071] about Figure 7 : Cell culture and establishment of a high glucose model: Human umbilical vein endothelial cells (HUVECs, ATCC® PCS-100-010) TMHUVECs were cultured in endothelial cell culture medium (ECM, ScienCell) containing 5% fetal bovine serum (FBS, ScienCell), 1% endothelial cell growth supplement (ECGS, ScienCell), and 1% penicillin-streptomycin (Gibco). Cells were incubated at 37°C in a 5% CO2 incubator. Cells from passages 2-5 were used in all experiments. To establish a high glucose model, HUVECs were pretreated for 24 hours in complete medium supplemented with 50 mM D-glucose before functional experiments. A normal glucose group (Health) and a high glucose control group (C, 50 mM D-glucose) were also established.

[0072] Preparation of hydrogel extract: Hydrogels with different MoS2QDs concentrations (0.125, 0.25, 0.5 mg / mL) were immersed in serum-free culture medium (surface area to culture medium volume ratio 1 cm²). 2 The solution was incubated at 37°C for 24 hours ( / mL). After incubation, the supernatant was collected and filtered through a 0.22 μm filter membrane to obtain the hydrogel extract. When used, the extract was prepared as a complete culture medium for cell treatment.

[0073] Cell proliferation experiment: HUVECs were 1×10 4 Cells were seeded at a density of 1 cell / well on the surface of a hydrogel in a 96-well plate and cultured for 24, 48, and 72 hours, respectively. Cell viability was assessed using a penicillin-streptomycin kit (Dojindo): 10 mL of cck-8 reagent was added to each well, and after incubation for 2 hours, absorbance was measured at 450 nm (n=5).

[0074] Scratch healing experiment: A scratching insert (coolrun) was attached to the bottom of a 24-well plate. HUVECs were cultured in the 24-well plate until confluence, then the insert was removed to create standard scratches. After washing with PBS, culture medium containing 1% serum and prepared with hydrogel extract was added. At 0 and 24 hours, images were taken at five predetermined locations in each well using a microscope (Agilent Technologies, CYTATION 5), and the scratch width was measured using ImageJ software. Migration rate (%) = [(W0-W 24 ) / W 24 ]×100(n=5).

[0075] Transwell migration experiments: HUVECs pretreated with hydrogel extract for 24 hours were resuspended in serum-free ECM (2×10⁻⁶). 5200 μL of cell suspension was added to the upper chamber of the Transwell (8 μm pore size, Corning), and 600 μL of ECM containing 10% FBS was added to the lower chamber. After culturing for 24 hours, cells that migrated to the membrane surface of the lower chamber were fixed with 4% paraformaldehyde, stained with 0.5% crystal violet, observed under a stereomicroscope (Leica, S9i), and counted in 5 random fields of view (n=5).

[0076] Angiogenesis and Tube Formation Experiment: Matrigel (50 μL / well, Corning), with reduced growth factor dosage, was polymerized in a 96-well plate at 37°C for 30 min. The treated HUVECs (2 × 10⁻⁶) were then... 4 (Numbers / well) were inoculated onto the Matrigel surface. After 4 hours of incubation, the formed tubular network was photographed. The total lumen length and the number of branch points were quantified using the Angiogenesis Analyzer plugin of ImageJ (n=5).

[0077] A core pathological feature of diabetic chronic wounds is impaired angiogenesis. Therefore, this study used human umbilical vein endothelial cells (HUVECs) as a model cell to systematically evaluate the regulatory effect of MoS2 / HM hydrogel (MoS2QDs / HM) on angiogenesis in vitro. Prior to this, the biocompatibility of the materials was assessed. Hemolysis experiments showed that the hemolysis rates of both HM and MoS2 / HM were less than 1%, with no significant difference compared to the PBS control group (code C). Figure 7 Figure A in the diagram shows that the hydrogel system has good blood compatibility. Subsequently, the cell compatibility of HUVECs with HM and different MoS2 / HM hydrogel extracts was evaluated using a CCK-8 assay. The results showed that, compared with the control group, HUVECs cultured in all groups of hydrogel extracts maintained high cell viability, and no significant cytotoxicity was observed. Figure 7 (See Figure B in the table). Notably, the MoS2 / HM treatment group significantly promoted the proliferation of HUVECs at 24, 48, and 72 h, suggesting that MoS2 / HM not only has good cell compatibility but may also play a positive regulatory role in endothelial cell proliferation.

[0078] To further evaluate the effect of MoS2 / HM on endothelial cell migration behavior, scratch assays and Transwell migration assays were performed. The scratch assay results showed that after 24 h of culture, all composite hydrogels containing MoS2QDs significantly promoted HUVEC migration, with the MoS2 / HM2 group exhibiting the highest scratch healing rate (91.6 ± 8.5%). Consistent with this, the Transwell migration assay results indicated that compared to the control group, the number of migrating cells significantly increased after MoS2 / HM2 treatment, by more than 5 times. These results collectively demonstrate that MoS2 / HM, especially the MoS2 / HM2 formulation, can significantly enhance the migration ability of endothelial cells. Based on this, its angiogenesis potential was further evaluated through an in vitro tube formation assay. After HUVECs were seeded on Matrigel and cultured for 6 h, the formed tubular structures were observed and quantitatively analyzed. The results showed that the number of tubular structures, the number of branch nodes, and the total tube length in the MoS2 / HM treatment group were significantly higher than those in the control and HM groups, with the MoS2 / HM2 group showing the most significant promoting effect. In contrast, the control group and the HM group only formed incomplete or sparse tubular networks. These results indicate that MoS2 / HM can effectively promote endothelial cell migration and tubular structure formation in vitro, thereby enhancing angiogenesis. Based on the above results, MoS2 / HM2 was used as the representative formulation in subsequent in vitro and in vivo experiments, and will be referred to as MoS2 / HM below.

[0079] Given the CCK-8 assay results suggesting that MoS2 / HM has the potential to promote HUVEC proliferation, its proliferative effect was further verified using an EdU assay. Cells without EdU were used as a negative control (Blank), and normal cultured cells without high glucose treatment were used as a healthy control group (Health). The results showed that high glucose treatment significantly inhibited HUVEC proliferation, with only 11.8% of cells entering S phase, significantly lower than the 26.2% in the Health group. HM treatment alleviated the high glucose-induced proliferation inhibition to some extent (19.8%), but failed to completely restore cell proliferation capacity. In contrast, MoS2 / HM treatment significantly increased the proportion of cells entering S phase to 35.8%, effectively reversing the proliferation inhibition under high glucose conditions and even exceeding the proliferation level of the Health control group.

[0080] CD31 is a classic marker of vascular endothelial cells, and its expression level is usually closely related to angiogenesis activity. Therefore, the expression of CD31 in HUVECs under different treatment conditions was analyzed by immunofluorescence staining. The results showed that the MoS2 / HM treatment group exhibited a more concentrated CD31 fluorescence signal, indicating that the angiogenesis phenotype of endothelial cells was significantly activated. Quantitative analysis based on the mean fluorescence intensity per cell (IntDen / cell number) further confirmed that MoS2 / HM treatment significantly upregulated the expression level of CD31 compared with the control group and the HM group. Further qRT-PCR analysis of the transcriptional levels of angiogenesis-related genes such as CD31, VEGF, and ANG1 showed that MoS2 / HM significantly upregulated the expression of CD31, VEGF, and ANG1. These results indicate that MoS2 / HM can significantly promote the proliferation, migration, and activation of angiogenesis-related phenotypes of endothelial cells under high glucose conditions.

[0081] about Figure 8 : H&E staining results ( Figure 8 Figure D in the diagram shows the differences in epithelial regeneration capacity among the different treatment groups. On postoperative day 7, the wound area in the MoS2 / HM group had formed a continuous and well-structured epithelial layer with tight adhesion between the epidermis and dermis, and numerous neovascular structures were observed within the dermis. In contrast, the control group (code C) and the 3M hydrogel group were still dominated by granulation tissue infiltrated with inflammatory cells, with incomplete epithelial coverage and sparse vascular structures. The epithelial integrity in the HM group was improved compared to the control group, but significant angiogenesis was not yet observed. By postoperative day 14, complete epithelium had formed in all groups, but the wound width in the MoS2 / HM and HM groups was significantly reduced, and regeneration of skin appendages such as hair follicles and sebaceous glands was observed in some sections, suggesting that the skin structure was recovering to a mature state.

[0082] Masson tricolor staining ( Figure 8 Figure F in the figure further reveals the process of collagen deposition and matrix remodeling. On postoperative day 7, the MoS2 / HM group showed significantly enhanced collagen deposition, and quantitative analysis showed that its collagen content was more than twice that of the control group. Figure 8 (See Figure G in the original text). By day 14 post-surgery, the collagen fiber area in the MoS2 / HM group reached 56%, and in the HM group it reached 45%, with both groups exhibiting regular collagen fiber arrangement and a typical bundle-like structure. In contrast, the collagen fiber distribution in the control group was more disordered, with an overall accumulation level of only 32%. These results indicate that MoS2 / HM can significantly promote the transformation of diabetic wounds from early granulation tissue to a structurally ordered mature matrix.

[0083] Postoperative wound photos were taken on days 0, 3, 7, 10, and 14. Figure 8Figure B in the diagram), and calculate the wound closure rate based on the initial wound area ( Figure 8 (See Figure E in the table). The results showed that, compared to the PBS group (code C), both HM and MoS2 / HM treatments accelerated the wound closure process to some extent. Specifically, the MoS2QDs / HM group consistently exhibited a faster healing rate throughout the observation period. By day 10 post-surgery, the wound closure rate of the MoS2QDs / HM group exceeded 85%, significantly higher than both the PBS and HM groups; by day 14, the wounds in the MoS2QDs / HM group were almost completely closed, while significant unhealed areas were still observed in the PBS group. These results indicate that MoS2QDs / HM can significantly accelerate the wound closure process under diabetic conditions. Notably, the HM group also showed a certain healing-promoting trend in the mid-to-late stages compared to the PBS group, but the extent of this promotion was significantly lower than that in the MoS2QDs / HM group. This phenomenon suggests that HM may primarily exert a limited auxiliary effect on wound repair by improving the local microenvironment and providing physical support, while the introduction of MoS2 further endows the material with more active bioregulatory capabilities.

[0084] about Figure 9 Given that angiogenesis occurs throughout the entire wound healing process, further analysis was conducted on angiogenesis and maturation. Dual immunofluorescence staining of CD31 (green) and α-SMA (red) showed that on postoperative day 3, the control group showed almost no vascular-related signals in the wound area, while the MoS2 / HM group exhibited high-density CD31 positive signals, accompanied by simultaneous upregulation of α-SMA expression, suggesting that it can rapidly initiate the construction of microvascular networks in the early stages of healing. By postoperative day 7, the MoS2 / HM group had formed a denser vascular network, while the control group only showed scattered vascular structures. Notably, on postoperative day 14, CD31 expression levels in all groups tended to converge, but the MoS2 / HM group showed higher α-SMA signal, indicating that it not only promotes angiogenesis but also further drives the transformation of newly formed blood vessels into a mature and stable state.

[0085] It should be understood that the application of this application is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of this application.

Claims

1. A method for preparing a hydrogel that promotes vascularization and regeneration of diabetic wounds, characterized in that, Includes the following steps: A precursor solution is obtained by mixing methacrylated hydroxypropyl chitosan and MoS2 QDs solution. The concentration of MoS2 QDs in the MoS2 QDs solution is 0.1-0.5 mg / mL; the concentration of methacrylyl-substituted hydroxypropyl chitosan in the precursor solution is 35-45 mg / mL. The precursor solution and photoinitiator are stirred and mixed under light-protected conditions for 18-30 hours; after light irradiation, a hydrogel that promotes vascularization and regeneration of diabetic wounds is obtained.

2. The method for preparing the hydrogel for promoting vascularization and regeneration of diabetic wounds according to claim 1, characterized in that, The method for preparing the MoS2 QDs solution includes the following steps: mixing MoS2 QDs with water to obtain a MoS2 QDs solution.

3. The method for preparing the hydrogel for promoting vascularization and regeneration of diabetic wounds according to claim 2, characterized in that, The preparation method of the MoS2 QDs includes the following steps: Ammonium tetrathiomolybdate, hydrazine, and water are mixed to obtain a homogeneous solution; the homogeneous solution contains 0.1-0.2 M ammonium tetrathiomolybdate and 2-4 M hydrazine. The homogeneous solution was reacted at a temperature of 170-190℃ for 24-48 hours; Solid-liquid separation was performed, and the precipitate was washed and dried to obtain the MoS2 QDs.

4. The method for preparing the hydrogel for promoting vascularization and regeneration of diabetic wounds according to claim 1, characterized in that, The preparation method of the methpropylene-substituted hydroxypropyl chitosan includes the following steps: Hydroxypropyl chitosan and buffer solution are mixed to obtain the first mixed solution; Methacrylamide and the first mixed solution are stirred and mixed at 45-55℃ for 3-6 hours to obtain a second mixed solution; the ratio of the amount of hydroxypropyl chitosan to the amount of methacrylic anhydride is 1g: (0.05-0.2)mL; After dialysis and drying, the second mixed solution yields the methacryl-substituted hydroxypropyl chitosan.

5. The method for preparing the hydrogel for promoting vascularization and regeneration of diabetic wounds according to claim 4, characterized in that, The buffer solution includes PBS; The ratio of the amount of hydroxypropyl chitosan to the amount of buffer solution is 1 g : (35-45) mL.

6. The method for preparing the hydrogel for promoting vascularization and regeneration of diabetic wounds according to claim 4, characterized in that, The dialysis process includes: dialysis for 4-6 days in a buffer solution at 40-50°C, followed by dialysis in water for 1-2 days.

7. The method for preparing the hydrogel for promoting vascularization and regeneration of diabetic wounds according to claim 3, characterized in that, The precipitate was washed with NaOH, ethanol, and water, and then dried under vacuum at 55-65℃ for 12-24 hours to obtain the MoS2 QDs.

8. The method for preparing the hydrogel for promoting vascularization and regeneration of diabetic wounds according to claim 1, characterized in that, The final concentration of the photoinitiator is 0.01-0.2% (w / v).

9. The method for preparing the hydrogel for promoting vascularization and regeneration of diabetic wounds according to claim 1, characterized in that, The illumination uses blue light with an intensity of 9-11 mW / cm². 2 .

10. A hydrogel for promoting vascularization and regeneration of diabetic wounds prepared by the method of preparing the hydrogel for promoting vascularization and regeneration of diabetic wounds according to any one of claims 1-9.

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