Preparation method and application of metabolism programming hydrogel

Abstract

The invention belongs to the technical field of hydrogel preparation, and relates to a preparation method and application of metabolism programming hydrogel, and the preparation method comprises the following steps: 1, oxidizing dextran Dex through sodium periodate NaIO4 to generate oxidized dextran ODex rich in aldehyde; 2, under the protection of nitrogen, carrying out amino dynamic Schiff base reaction of APTC and carrying out covalent binding with an aldehyde group of oxidized dextran ODex to form ODex-APTC; 3, mixing ODex-APTCC and CMC GOx solutions at room temperature to form hydrogel based on hemiacetal crosslinking and a hydrogen bond mechanism; according to the invention, autonomous'antibacterial repair 'dual-phase switching is realized through metabolic microenvironment reprogramming; in the stage of wound surface high-glucose infection, the hydrogel utilizes a diabetes wound surface high-glucose environment as an endogenous fuel, and glucose reduction, active oxygen mediated sterilization and microenvironment acidification are realized at the same time through reaction of glucose oxidase and glucose; when glucose oxidase reacts, the acid environment triggers a controlled H2S release program, and wound healing is accelerated by relieving oxidative stress and activating a regeneration signal channel.

Description

Technical Field

[0001] This invention belongs to the field of hydrogel preparation technology, specifically relating to a method for preparing and applying metabolically programmed hydrogels. Background Technology

[0002] Chronic diabetic wounds, also known as diabetic foot ulcers or diabetic refractory wounds, are a serious complication of diabetes caused by prolonged hyperglycemia. Specifically, they refer to wounds that fail to heal properly for extended periods (usually more than 4-6 weeks) due to diabetic neuropathy and varying degrees of lower extremity vascular disease, resulting in foot infections, skin breakdown, and the formation of wounds that do not heal normally. Chronic diabetic wounds have received widespread attention in the healthcare and medical fields, with hyperglycemia being a key pathogenic factor. Sustained hyperglycemia not only induces microcirculatory dysfunction but also triggers chronic inflammatory responses and immunosuppression, ultimately leading to severe infections. Furthermore, hyperglycemia causes oxidative stress and the accumulation of advanced glycation end products (AGEs), leading to cellular senescence / apoptosis and exacerbating tissue damage.

[0003] To address the hyperglycemic microenvironment unique to diabetic wounds, glucose oxidase (GOx) therapy employs glucose starvation and generates hydrogen peroxide for antibacterial effects. However, this process induces persistent acidity and exacerbated oxidative stress in the mid-to-late stages of wound healing. Excessive ROS accumulation perpetuates the inflammatory response, disrupts physiological healing timelines, and ultimately hinders wound repair. Furthermore, a single strategy of increasing or decreasing ROS cannot ensure dynamic equilibrium because spatiotemporally programmed ROS homeostasis is a prerequisite for optimizing tissue repair kinetics.

[0004] Hydrogen sulfide (H2S), as a pleiotropic biomodulator, not only directly neutralizes reactive oxygen species (ROS) through sulfidation modification but also coordinates redox homeostasis by simultaneously stimulating the secretion of anti-inflammatory cytokines and the expression of angiogenic factors. This dual therapeutic paradigm has driven the development of hydrogen sulfide donors, from inorganic salts to thiourea derivatives. However, existing systems struggle to align hydrogen sulfide release kinetics with the dynamic wound healing process in both time and space. For example, traditional inorganic sulfide donors with burst release kinetics risk inducing toxic concentration peaks, while existing organic hydrogen sulfide donors lack pH-activation specificity in the diabetic microenvironment.

[0005] Therefore, a glucose-reactive organic donor is needed to initiate pH-catalyzed H2S generation using pathological hyperglycemia, thereby achieving sustained release synchronized with the GOx-mediated antibacterial cycle to solve the aforementioned technical problems. Summary of the Invention

[0006] This invention aims to develop a smart programmable hydrogel to achieve a phased treatment plan synchronized with the blood glucose level fluctuations of diabetic patients. This hydrogel achieves autonomous "antibacterial repair" biphasic switching through metabolic microenvironment reprogramming. During the hyperglycemic infection phase of the wound, the hydrogel utilizes the hyperglycemic environment of the diabetic wound as an endogenous fuel. Through the reaction of glucose oxidase (GOx) with glucose, it simultaneously reduces glucose, induces reactive oxygen species (ROS)-mediated sterilization, and acidifies the microenvironment. Simultaneously with the glucose oxidase reaction, the acidic environment triggers a controlled H2S release program, accelerating wound healing by alleviating oxidative stress and activating regeneration signaling pathways.

[0007] This invention provides the following technical solution: a method for preparing a metabolically programmed hydrogel, comprising the following steps:

[0008] Step S1: Oxidize dextran Dex with sodium periodate (NaIO4) to generate aldehyde-rich oxidized dextran ODex;

[0009] Step S2: Under nitrogen protection, APTC is covalently bonded to the aldehyde group of oxidized dextran ODex via the amino-dynamic Schiff base reaction to form ODex-APTC;

[0010] Step S3: At room temperature, ODex-APTCC and CMC GOx solutions are mixed to form a hydrogel based on hemiacetal crosslinking and hydrogen bonding mechanisms.

[0011] Preferably, step S1 specifically includes: dissolving dextran Dex in deionized water, then adding NaIO4, stirring at room temperature in the dark for 3-4 hours, then adding ethylene glycol and stirring for 0.5-1 hours to terminate the reaction, dialyzing the solution through a cellulose membrane for 65-75 hours, and then freeze-drying for 45-50 hours to obtain the white product ODex.

[0012] More preferably, in step S1, the mass ratio of dextran Dex to NaIO4 is 1:0.9-1.1; the mass-to-volume ratio of dextran Dex to deionized water is 1g:20-30mL; and the volume ratio of ethylene glycol to deionized water is 3:120-130.

[0013] More preferably, in step S1, the cellulose membrane is of type MWCO 8000-14000Da.

[0014] Preferably, step S2 specifically includes: dissolving ODex in deionized water, dispersing APTC in DMSO and adding it dropwise into the ODex solution, stirring for 3-5 hours at 45-55°C under N2 atmosphere, and then precipitating the resulting solution multiple times with cold ethanol to obtain ODex-APTC.

[0015] More preferably, in step S2, the mass-to-volume ratio of ODex to deionized water is 1g:8-12mL; and the mass-to-volume ratio of APTC to DMSO is 1g:70-90mL.

[0016] More preferably, in step S2, the cold ethanol precipitation is performed at least four times.

[0017] Preferably, step S3 specifically includes: dissolving CMC in deionized water, adding GOx to the solution and stirring for 0.5 to 1.5 hours until GOx is completely dispersed, then dissolving ODex-APTC in deionized water, mixing and stirring the two solutions evenly, and crosslinking at room temperature for 5 to 7 hours.

[0018] More preferably, in step S3, the mass-to-volume ratio of CMC to deionized water is 1g:12-16mL; the mass ratio of GOx to CMC is 3:45-55; the mass ratio of ODex-APTC to CMC is 1:1-1.5; and the mass-to-volume ratio of ODex-APTC to deionized water is 1g:7-8mL.

[0019] The present invention also discloses a metabolic programming hydrogel for the treatment of chronic diabetic wounds, which is prepared by the above-described method for preparing metabolic programming hydrogels.

[0020] The beneficial effects of this invention are:

[0021] This invention achieves autonomous "antibacterial repair" biphasic switching through metabolic microenvironment reprogramming. During the high glucose infection stage of the wound, the hydrogel utilizes the high glucose environment of the diabetic wound as an endogenous fuel, and simultaneously achieves glucose reduction, ROS-mediated sterilization, and microenvironment acidification through the reaction of glucose oxidase (GOx) with glucose. At the same time as the glucose oxidase reaction, the acidic environment triggers a controlled H2S release program, which accelerates wound healing by alleviating oxidative stress and activating regeneration signaling pathways. Attached Figure Description

[0022] Figure 1 This is a schematic diagram illustrating the preparation steps of a metabolic programming hydrogel according to the present invention and its application.

[0023] Figure 2The images show the preparation and characterization of the Odex-APTC@CMC-GOx hydrogel of this invention; (A) the preparation process of the OACG hydrogel; (B) the modification procedure of ODexAPTC; (C) the 1H NMR spectra of Dex, ODex, and ODex-APCC (from top to bottom); (D) the FTIR spectra of the CMC, ODex-APTC, GOx, and OACG hydrogels; (E) the XPS measurement spectra of the OC and OACG hydrogels; (F) the high-resolution C1s XPS spectra of CMC, ODex, and CMC-ODex; (G) the high-resolution O1s XPS spectra of CMC, ODex, and CMC-ODex; and (H) the SEM image of the OACG hydrogel (arrows indicate incorporated GOx), scale bar: 200 μm. For magnified images, scale bar: 50 μm; (I) Pore size distribution of OACG hydrogel; (J) EDS spectrum of OACG hydrogel; (K) SEM image and elemental distribution image of OACG hydrogel, scale bar: 60 μm;

[0024] Figure 3 The following images illustrate the rheological properties, self-healing properties, and adhesive properties of the Odex-APTC@CMC-GOx hydrogel of this invention: (A) Image of the gelation process of the OACG hydrogel; (B) Gel time of OA and OACG hydrogels at different ODex concentrations; (C) Injection performance of the hydrogel; (D) Modulus of the hydrogel at different ODex concentrations; (E) Rheological analysis of the storage modulus (G′) and loss modulus (G″) of the hydrogel at different ODex concentrations; (F) Strain scan of the hydrogel, with a strain range of 1% to 1000% (1Hz); (G) Rheological behavior of the hydrogel under alternating strains switching from 1% to 10% over three cycles. 00%; (H) Photograph of the macroscopic healing ability of hydrogel; (I) Schematic diagram of the tensile test process of self-healing hydrogel; (J) Tensile curves of hydrogel after different self-healing times; (K) Schematic diagram of the hydrogel healing mechanism; (L) Photograph of the adhesion behavior of different material surfaces; (M) Photograph of the adhesion behavior of different material surfaces; (N) Schematic diagram of hydrogel adhesion experiment on pigskin; (O) Adhesion strength of ODex-containing hydrogel on pigskin at different concentrations; (P) Maximum adhesion strength of hydrogel; (Q) Maximum adhesion strength of hydrogel on different substrates; (R) Tough adhesion caused by the increase of interfacial layer bonding density and the synergistic energy dissipation of bulk region through hydrogen bond interaction and reversible covalent bonds. Statistical data are expressed as mean ± standard deviation (SD);

[0025] Figure 4This is a diagram illustrating the ROS regulation of the Odex-APTC@CMC-GOx hydrogel of the present invention; wherein, (A) is a schematic diagram of programmable ROS regulation mediated by the hydrogel in diabetic chronic wounds; (B) studies the ability of the OCG hydrogel to generate hydrogen peroxide (H2O2) and acidify the microenvironment; (C) the H2S generation capacity of the hydrogel under different pH conditions; (D) the spatiotemporal regulation of ROS by the hydrogel under simulated hyperglycemic conditions; (EF) the H2O2 scavenging capacity of hydrogels with different compositions; (GH) the scavenging capacity of DPPH hydrogels with different compositions; (IL) ABTS· + The clearance capacity of hydrogels with different compositions is statistically expressed as mean ± standard deviation (SD);

[0026] Figure 5 The UV-Vis spectra of ODex, APTC, and ODex-APTC are shown. Characteristic absorption peaks of ODex and APTC were observed in the ODex-APTC spectrum, indicating successful conjugation between the two components.

[0027] Figure 6 The images show the FT-IR spectra of Dex, ODex, APTC, and ODex-APTC. Compared with Dex, the appearance of the aldehyde characteristic peak in ODex indicates that Dex oxidation was successful. In ODex-APTC, the decrease in aldehyde peak intensity and the appearance of APTC-specific functional groups confirm the covalent conjugation between ODex and APTC.

[0028] Figure 7 SEM images and pore size distribution analysis diagrams for different groups; OC and OAC groups exhibit smooth surfaces, while OCG and OACG exhibit densely distributed nanoscale protrusions, which is attributed to the incorporation of GOX. Notably, all four hydrogels show comparable pore size distributions, indicating that neither APTC nor GOX significantly alters the internal porous structure of the hydrogel matrix.

[0029] Figure 8 Tensile curves of CMC-ODex hydrogels with different ODex concentrations are shown; the tensile strength of the hydrogels gradually increases with increasing ODex content.

[0030] Figure 9 The tensile strength of CMC-ODex hydrogels with different ODex concentrations is shown; the tensile strength of the hydrogels gradually increases with increasing ODex content.

[0031] Figure 10 The tensile modulus graph shows the tensile modulus of CMC-ODex hydrogels with different ODex concentrations; the tensile modulus of the hydrogel gradually increases with increasing ODex content.

[0032] Figure 11The tensile fracture energy diagram shows the tensile fracture energy of CMC-ODex hydrogels with different ODex concentrations; as the ODex content increases, the tensile fracture energy of the hydrogel gradually increases.

[0033] Figure 12 The following is a representative tensile stress-strain curve of the hydrogel; stress-strain curves obtained from the same specimen during cyclic tensile tests (selected cycles: 1, 5, 10, 15, 20) show the material's response to repeated deformation.

[0034] Figure 13 The figure shows the effect of cyclic stretching on the tensile strength of the hydrogel; in repeated stretching cycles, the tensile strength remains moderate but gradually decreases (1-20 cycles), revealing the fatigue resistance of the material to cumulative structural damage.

[0035] Figure 14 This image demonstrates the adhesion of the hydrogel to mouse organs; the hydrogel maintained strong adhesion to the surfaces of various organs, including skeletal muscle, heart, liver, kidney, and spleen.

[0036] Figure 15 The graph shows the ability of GOx to consume glucose; the GOx-infused hydrogel exhibits significant glucose consumption, while the control group shows negligible glucose consumption activity under the same conditions.

[0037] Figure 16 The graph shows the H2O2 production of OCG hydrogels with different GOx concentrations under high glucose (20 mM) conditions. At a fixed glucose concentration (20 mM), the H2O2 generation capacity of the hydrogels increases with increasing glucose oxidase (GOx) concentration.

[0038] Figure 17 Figure 1 shows the H2O2 production of OCG hydrogel at different glucose concentrations; at a fixed Gox concentration (3 mg / mL), the H2O2 generation capacity of the hydrogel increases with increasing glucose concentration. Detailed Implementation

[0039] The related technologies of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0040] like Figures 1-4 As shown, this embodiment provides a method for preparing and applying a metabolically programmed hydrogel, including the following steps:

[0041] Preparation of ODex (oxidized dextran). First, 5g of dextran (Dex) was completely dissolved in 125mL of deionized water, then 5g of NaIO4 was added. The mixture was stirred at room temperature in the dark for 3.5h, then 3mL of ethylene glycol was added and stirred for 0.5h to terminate the reaction. The solution was dialyzed through a cellulose membrane (MWCO 8000-14000Da) for 72h, and then freeze-dried for 48h to obtain the white product ODex.

[0042] ODex-APTC is formed via a Schiff base reaction between an aldehyde and an amino group. First, 0.5 g of ODex was completely dissolved in 5 mL of deionized water. Then, 0.05 g of APTC was dispersed in 4 mL of DMSO and added dropwise to the ODex solution. After stirring for 4 h at 50 °C under N2 atmosphere, the resulting solution was precipitated four times with cold ethanol to obtain the pale yellow product ODex-APTC.

[0043] To synthesize the OACG hydrogel, firstly, 0.25 g of CMC was dissolved in 3.5 mL of deionized water. Then, 15 mg of GOx was added to the solution, and the mixture was stirred for 1 h until the GOx was completely dispersed. Next, 0.2 g of ODex-APTC was dissolved in 1.5 mL of deionized water. Subsequently, the two solutions were mixed and stirred thoroughly, and crosslinked at room temperature for 6 h.

[0044] Synthesis and Identification of Hydrogels

[0045] This specific embodiment constructs an acid-sensitive H2S-releasing hydrogel system (OACG) using a one-pot method. This system is formed through physicochemical crosslinking between oxidized dextran-grafted 2-aminopyridine-5-thiourea (ODex-APTC) and GOx-loaded carboxymethyl cellulose (CMC). Figure 2 A). The synthesis of ODex-APTC involves two steps ( Figure 2 B): First, dextran (Dex) is oxidized by sodium periodate (NaIO4) to generate aldehyde-rich oxidized dextran (ODex). Subsequently, under nitrogen protection, the amino group of APTC covalently bonds to the aldehyde group of ODex via a dynamic Schiff base reaction to form ODex-APTC. The chemical evolution of Dex, ODex, and ODex-APTC was systematically verified by 1H NMR. Figure 2 C) confirmed the oxidation of Dex and the grafting of APTC. UV-Vis spectroscopy further confirmed the formation of ODex and APTC; ODex exhibited strong absorbance at 197 nm, and APTC showed characteristic peaks at 278 and 296 nm. Figure 5 FT-IR analysis showed that ODex was at 1732 cm⁻¹. -1 There is strong C=O stretching vibration at this point, while ODexAPTC is at 1588 cm⁻¹. -1The presence of a conjugate C=N / pyridine C=C vibrational peak at this location supports the success of APTC grafting. Figure 6 ).

[0046] FT-IR and XPS analyses elucidated the crosslinking mechanism and functional synergy of the OACG hydrogel. FT-IR showed a C=O peak intensity of 1732 cm⁻¹. -1 The value decreased to 1111cm. -1 The presence of a new hemiacetal COC stretching vibration confirms the dynamic covalent crosslinking between -CHO in ODex and -OH in CMC. Figure 2 D). The blue shift of the -OH stretching vibration indicates a strong hydrogen bond between ODex-APTC and CMC, revealing a cooperative covalent hydrogen bond network. Notably, the retained 1588 cm⁻¹... -1 Peak (C=N / C=C) and 1018 cm -1 The CN vibration (GOx) at the site confirmed that APTC / GOx was integrated into the hydrogel. XPS detected distinct N1s (400 eV) and S2p (168 eV) signals from APTC / Gox. Figure 2 E), validating the functional loading. In a control experiment without APTC / GOx, the ODex@CMC (OC) hydrogel showed that, compared to ODex / CMC, the binding energies of C=O, CO, and -OH in OC increased (E). Figure 2 The reduced C=O / CO peak area in OC confirms the formation of a partial hemiacetal, consistent with FT-IR observations. In summary, the 3D network of OACG depends on dynamic hemiacetal crosslinking between ODex and CMC, enhanced by hydrogen bonding, while APTC / GOx is immobilized through covalent and physical interactions. Scanning electron microscopy (SEM) images of the lyophilized hydrogel reveal a highly porous network (average pore size: ~130 μm). Figure 2 H, 2I and Figure 7 OACG is an ideal candidate for fibroblast migration and nutrient diffusion. Notably, compared to the smooth surface of OC / OC, OACG exhibits dense nanoscale surface protrusions ODex-APTC@CMC (OAC), confirming the formation of GOx. EDS and SEM images showed a uniform C / O distribution (matrix integrity) and a uniform N / S signal (APTC / GOx distribution), confirming the success of functionalization. Figure 2 J and 2K).

[0047] Performance characterization of hydrogels

[0048] The hydrogel system was formed by mixing ODexAPTC and CMC GOx solutions at room temperature. Figure 3A). The synthesis of hydrogels is based on hemiacetal crosslinking and hydrogen bonding mechanisms. With increasing ODex concentration, the binding between the -CHO groups in the gelling agent and the -OH groups in the CMC is enhanced, and the gelation time is shortened. It was observed that the OC and OACG hydrogels had the shortest gelation times (5-10 minutes) when the ODex concentration reached 2 wt%. Figure 3 (B) This is a rapid gelation characteristic that is beneficial for future clinical surgery. Injectability and self-healing ability were systematically evaluated through rheological analysis and macroscopic testing. Shear-thinning experiments confirmed that the viscosity rapidly decreases with increasing shear rate. Figure 3 C), thus enabling precise deep tissue delivery via microneedles. To investigate the changes in mechanical properties caused by increased crosslinking density, rheological tests were conducted to evaluate the modulus changes of the hydrogel at different ODex concentrations. Figure 3 D). As the ODex concentration gradient increased, the storage modulus (G') of the hydrogel gradually increased from 0.27 to approximately 0.33, 0.37, and 1.17 kPa, confirming the dose-dependent modulation of mechanical strength by ODex. Frequency scan analysis showed that G' consistently exceeded the loss modulus (G') throughout the entire monitoring frequency range. Figure 3 E) indicates viscoelastic solid properties. A comprehensive mechanical evaluation conducted through uniaxial tensile testing revealed the network reinforcement effect (E). Figure 8 This indicates that higher ODex concentrations correlate with higher tensile strength, elastic modulus, and loss modulus. Notably, after 20 tensile recovery cycles, the hydrogel strength only decreased slightly. Figure 12 and Figure 13 The excellent fatigue resistance of the hydrogel is attributed to stress dissipation through dynamic hemiacetal and hydrogen bonds. These results highlight the hydrogel's superior robustness, elongation, and fatigue resistance, which together provide sustained mechanical protection for wounds.

[0049] OACG hydrogels exhibit excellent self-healing properties and shape adaptability, which can extend service life and improve safety. Amplitude scanning revealed structural collapse at 200% critical strain. Figure 3 F). Subsequent stepwise strain tests with alternating strain (1%–1000%, 60-second intervals) showed that at 1000% strain, G "dropped below G", but in all cycles, it rapidly recovered to its original value at 1% strain. Figure 3 G). Macroscopic self-healing behavior indicates that the cleaved hydrogel with a molten interface completely recovers its shape ( Figure 3 H), involving polymer chain diffusion, reversible hemiacetal bond breaking / recombining, and the synergistic effect of hydrogen bond networks. Quantitative tensile testing ( Figure 3(I and 3J) showed that the fracture strength recovered by more than 90% after 30 minutes of healing, approaching the performance of the original sample. This rapid self-healing ability not only maintains the integrity of the dressing and reduces the need for clinical dressing changes, but also reduces the risk of secondary infection through a sustained barrier effect, providing an innovative solution for chronic wound management.

[0050] Interfacial adhesion is a key functional parameter for wound sealing and infection prevention. Hydrogels exhibit general adhesion to wood, skin, metal, glass, and other substrates. Figure 3 K and 3L), which is attributed to the multifunctional interaction between surface hydroxyl / aldehyde groups and various materials. Adhesion test of porcine tissues / organs ( Figure 14 This has been confirmed to have effective binding with muscles, heart, kidneys, and liver. (For example...) Figure 3 As shown in Figure M, the hydrogel maintained tight skin adhesion under inversion, bending, and twisting conditions, with no edge lifting, demonstrating excellent adhesion properties and complete wound sealing. Overlap shear tests on pigskin showed that the adhesive strength increased with increasing ODex content. Figure 3 N), with a peak value of 9.68 kPa ( Figure 3 O and 3P), this may be due to the increased mechanical strength reducing hydrogel damage / deformation and increasing contact area. Substrate-dependent adhesion test ( Figure 3 Q) shows that the gradient decays as the surface functional group density and roughness decrease.

[0051] Detection of ROS modulation in ODex-APTC@CMC-GOx hydrogel

[0052] The complex microenvironment created by metabolic disturbances in hyperglycemic diabetic wounds significantly increases the risk of bacterial infection. Effective management requires simultaneous glucose depletion and elevated ROS during the hyperglycemic infection phase; however, persistent ROS accumulation hinders tissue repair during the healing phase, thus necessitating ROS reduction to alleviate oxidative stress. By integrating glucose oxidase (GOx) and an acid-labile H2S donor, an endogenous biochemical signaling (pH change)-driven autonomous feedback mechanism was developed to temporarily modulate ROS and hydrogen sulfide levels in hyperglycemic and normglycemic wounds. Figure 4 A).

[0053] Under hyperglycemic conditions (20 mM glucose), GOx continuously catalyzed the conversion of glucose to H2O2 and gluconic acid for approximately 10 hours. This process was accompanied by the gradual depletion of glucose, a decrease in the pH gradient from 5.9 to 4.8, and a linear accumulation of H2O2. Figure 4B and S11). Dynamic curves revealed a triple antibacterial mechanism of Gox during infection: glycemic regulation, microenvironment acidification, and the generation of antibacterial ROS. Notably, the catalytic activity of GOx exhibited a dual regulatory characteristic, depending on enzyme concentration and glucose level. By systematically adjusting the GOx loading (0-3.0 mg / mL) of ODex@CMC-GOx(OCG) hydrogel, this embodiment observed GOx concentration-dependent H2O2 generation, which was demonstrated by the increased proportion of the characteristic absorption peak at 405 nm corresponding to sustained H2O2 accumulation. Figure 16 This programmable ROS generation enables precise, on-demand antibacterial activation. Further studies at a fixed GOx concentration (3 mg / mL) demonstrated linear glucose concentration ROS generation kinetics. Figure 17 This study revealed a biphasic regulatory mechanism: 1) rapid H2O2 production catalyzed by excess glucose during hyperglycemia, followed by 2) automatic transition to a low-activity mode after glucose normalization. This self-limiting mechanism effectively prevents excessive ROS accumulation post-infection while maintaining therapeutic efficacy.

[0054] Excessive ROS accumulation can trigger inflammatory responses and severely hinder wound repair. Therefore, timely removal of locally accumulated ROS during periods of normal blood glucose levels is crucial for regulating tissue regeneration in diabetic wound healing. A pH-sensitive hydrogen sulfide donor can precisely regulate H2S release based on dynamic wound pH changes. By simulating the pH evolution (weakly alkaline to weakly acidic) in the microenvironment of chronic diabetic wounds, this embodiment observed a positive correlation between H2S release rate and environmental acidity. Figure 4 C). At pH 4, the hydrogel released approximately 90 μM H2S within 72 hours, exceeding the release under neutral conditions. This was attributed to the protonation effect induced by accelerated C=S bond breaking due to gluconic acid accumulation. Although the release kinetics of ROS and H2S have been elucidated, their spatiotemporal dynamic equilibrium remains unexplored. The spatiotemporal modulation of ROS / H2S by the OACG hydrogel was further evaluated in a simulated diabetic wound microenvironment. Figure 4 (D) Experimental results showed that the ROS concentration in the OACG group peaked at 10 hours and then decreased to physiological levels, while no significant ROS fluctuations were observed in the OC / OAC group, and the OCG group maintained a consistently high ROS level. This indicates that the sequential release of ROS and H2S is controlled by a series of endogenous biochemical signals: the hyperglycemic environment activates antibacterial activity through the GOx-H2O2 axis, while the self-induced acidic microenvironment triggers H2S release to counteract oxidative damage. This phase transition mechanism successfully encapsulates the natural timing of antioxidant protection during the disinfection phase of inflammation and the repair phase of physiological healing.

[0055] Besides the H2O2 produced by Gox catalysis, the endogenous ROS microenvironment in diabetic wounds also needs careful consideration. This invention systematically elucidates the efficacy and mechanism of OACG hydrogel in scavenging reactive oxygen species. H2O2 scavenging kinetic analysis shows that in the OAC group, there is a dose-dependent positive correlation between scavenging efficiency and ODex-APC C loading: when the ODex-APC concentration increased from 0 to 2 mg / mL, the 12-hour scavenging rate increased from 4.6% to 53.8%. Figure 4 E). Notably, Gox incorporation (OACG group) did not significantly alter the H2O2 scavenging ability, while the non-specific scavenging rate of the APTC-deficient OC / OCG group was less than 5%, which may be due to the solution dilution effect of material degradation. Figure 4 F). Furthermore, this embodiment confirms the nitrogen radical scavenging ability of the hydrogel. With increasing APTC content, the scavenging rate of 2,2-diphenyl-1-picrylhydrazyl (DPPH·) reached 70% within 12 hours, accompanied by a color change from deep purple to yellow, while the control group without APTC showed negligible activity. Figure 4 G and 4H). Interestingly, the scavenging test of 2,2'-azidobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS·+) showed distinct kinetic characteristics. Figure 4 I): The OAC group containing APTC achieved a clearance rate of >99% within 30 minutes, and the clearance efficiency was not significantly correlated with the ODex-APTC concentration. This difference may be related to the redox properties of ABTS·+, whose standard reduction potential exceeds that of DPPH· and H2O2, potentially enabling direct electron transfer of the C=S bond rather than the H2S-mediated pathway. Control experiments confirmed that within 30 minutes, the ABTS·+ clearance rate in the APTC-free OC group was <5%, while different ODex-APTC concentrations (0.5-2 mg / mL) showed a linear negative correlation between the complete clearance time (>99%) and the component concentration. Figure 4 These findings indicate that OACG hydrogels effectively scavenge multiple ROS types through pH-sensitive H2S release and APTC-mediated direct radical elimination mechanisms.

[0056] In summary, unlike traditional exogenous phase change therapy systems (such as light / heat triggering), this implementation establishes spatiotemporal ROS / H2S homeostasis at the wound site through the coordination of endogenous glucose and pH dual signals, avoiding the uncontrollability of external intervention and the delayed antioxidant effect of static scavengers, thus providing an innovative treatment paradigm for time-based treatment of diabetic wounds.

[0057] Example

[0058] Synthesis of hydrogels

[0059] To synthesize the OACG hydrogel, firstly, 0.25 g of CMC was dissolved in 3.5 mL of deionized water. Then, 15 mg of GOx was added to the solution, and the mixture was stirred for 1 h until the GOx was completely dispersed. Next, 0.2 g of ODex-APTC was dissolved in 1.5 mL of deionized water. Subsequently, the two solutions were mixed and stirred thoroughly, and crosslinked at room temperature for 6 h. For comparison, this example used the same assembly process to prepare OC hydrogels, OAC hydrogels, OCG hydrogels, OACG hydrogels, and OC hydrogels with different ODex contents. See Supplementary Table 1 for details.

[0060] CMC(g) ODex(g) ODex-APTC(g) Gox (mg) Water (mL) OC 0.25 0.2 - - 5 OAC 0.25 - 0.2 - 5 OCG 0.25 0.2 - 15 5 OACG 0.25 - 0.2 15 5 <![CDATA[OC 1％ ]]> 0.25 0.05 - 5 <![CDATA[OC 2％ ]]> 0.25 0.1 - - 5 <![CDATA[OC 3％ ]]> 0.25 0.15 - - 5 <![CDATA[OC 4％ ]]> 0.25 0.2 - - 5

[0061] Evaluation of the effects of hydrogels on H2O2 generation and scavenging

[0062] To simulate a high-glucose environment, a 20 mM glucose solution was used. To investigate the effect of GOx concentration on H2O2 formation, 0.5 mL of OCG hydrogel containing different GOx concentrations (0, 0.5, 1, 2, 3 mg / mL) was mixed with 3 mL of 20 mM glucose solution and incubated at 37°C for 6 hours using a constant-temperature shaker. Subsequently, 1 mL of the supernatant was collected and mixed with 2 mL of Ti(SO4)2 solution (prepared by dissolving 0.64 g Ti(SO4)2 and 16.66 mL H2SO4 in 100 mL of deionized water) and immersed for 30 minutes. The absorbance of the solution was measured using UV-Vis spectroscopy in the 300-600 nm range to quantify H2O2 production. To evaluate the effect of glucose concentration on H2O2 formation, 0.5 mL of OCG hydrogel was mixed with 3 mL of glucose solutions of different concentrations (0, 2, 4, 6, 8, 10 mM) and incubated under the same conditions. As described above, the supernatant was analyzed using the titanium sulfate colorimetric method.

[0063] The H2O2 scavenging capacity was evaluated by mixing 0.5 mL of hydrogel with 3 mL of 20 mM H2O2 solution at 37 °C. At specified time points, 50 μL of supernatant was collected, mixed with 100 μL of Ti(SO4)2 solution for 30 min, and the absorbance at 405 nm was measured to determine the consumption of H2O2.

[0064] Assessment of glucose consumption by hydrogels

[0065] The glucose-consuming capacity of the hydrogel was assessed by mixing 0.5 mL of hydrogel with 3 mL of 10 mM glucose solution at 37 °C. The supernatant was collected at predetermined time intervals, and the residual glucose concentration was determined using a glucose assay kit (catalog number S0201S, Beyotime Biotechnology).

[0066] Analysis of hydrogel scavenging of DPPH· and ABTS·+ free radicals

[0067] 3 mL of 0.1 mM DPPH· solution was mixed with 0.5 mL of hydrogel and incubated in the dark. The supernatant was collected at specified time intervals, and the absorbance at 517 nm was recorded by UV-Vis spectroscopy to assess DPPH· elimination. ABTS·+ solution was diluted to an absorbance of 0.78–0.8 at 734 nm. Then, 3 mL of the diluted ABTS·+ solution was mixed with 0.5 mL of hydrogel and incubated in the dark. The absorbance at 734 nm was measured at specific time points to quantify ABTS·+- clearance.

[0068] H2S release detection

[0069] The concentration of H2S released in acidic environments (pH 4 and 7) was analyzed using an H2S content assay kit (Solarbio, Beijing, China). Specifically, after mixing the solution with a zinc acetate / sodium acetate mixture, methylene blue (MB) was formed by adding N,N-dimethyl-p-phenylenediamine dihydrochloride and FeCl3. The H2S concentration was determined by measuring the absorbance of MB in the 550-750 nm range.

[0070] Mechanical performance testing

[0071] The tensile and self-healing properties of the hydrogel were evaluated using a universal testing machine (model) equipped with a 50 N load cell. Hydrogel specimens (10 mm wide × 50 mm long × 2 mm thick) were stretched at a rate of 20 mm·min⁻¹. Cyclic tensile tests were performed at 75% of the maximum strain. For self-healing assessment, cut hydrogel samples were allowed to stand for 15 or 30 minutes prior to the tensile test to measure the recovery efficiency.

[0072] Rheological and adhesive properties testing

[0073] Rheological properties were analyzed using a rheometer (HAKKE MARS 60). Hydrogel discs (20 mm diameter × 2 mm height) were placed between parallel plates. Steady-state shear tests measured the relationship between viscosity and shear rate (1–100 s⁻¹). Time-scan tests (1 Hz frequency, 1% strain amplitude) recorded the storage modulus (G′) to assess stiffness. Frequency-scan tests (1–100 rad / s, 1% strain) determined G′ and loss modulus (G″). Strain amplitude scans (1–1000%) determined the critical strain threshold. Self-healing behavior was evaluated using alternating strain steps of 1 rad / s (1% and 1000% strain, 60-second intervals). All tests were performed at 25 °C.

[0074] Macroscopic adhesion was evaluated on various substrates (wood blocks, pigskin, metal, glass, plastic, rubber) and rat tissues (muscle, heart, liver, kidney, spleen). Fresh pigskin was pretreated by soaking in PBS, removing excess fat, and blotting dry with filter paper. Adhesion was demonstrated by inversion, bending, and twisting. For the lap shear test, a hydrogel block (1 cm × 1 cm × 2 mm) was sandwiched between substrates (pigskin, wood, plastic, or glass), pressed for 1 minute, and tested using a universal testing machine at 10 mm / min. -1 The speed was tested.

[0075] In summary, this invention achieves autonomous "antibacterial repair" biphasic switching through metabolic microenvironment reprogramming. During the hyperglycemic infection phase, the hydrogel utilizes excess wound glucose as endogenous fuel, simultaneously reducing glucose, reactive oxygen species-mediated sterilization, and microenvironment acidification through glucose oxidase cascade metabolism. After glucose normalization, the acidic environment triggers a controlled H2S release program, accelerating wound healing by clearing oxidative stress and activating regeneration signaling pathways.

[0076] It should be emphasized that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A method of preparing a metabolically programmable hydrogel, comprising, The method comprises the following steps: Step S1, oxidizing dextran Dex by sodium periodate NaIO4 to generate aldehyde-rich oxidized dextran ODex; Step S2, covalently combining the aldehyde group of the oxidized dextran ODex with the amino dynamic Schiff base of APTC under nitrogen protection to form ODex-APTC; Step S3, mixing ODex-APTC and CMC GOx solution at room temperature to form a hydrogel based on a hemiacetal cross-linking and hydrogen bonding mechanism.

2. The method for preparing a metabolically programmed hydrogel according to claim 1, characterized in that, The step S1 specifically comprises: dissolving dextran Dex in deionized water, then adding NaIO4, stirring at room temperature for 3-4 hours in the dark, then adding ethylene glycol and stirring for 0.5-1 hour to terminate the reaction, dialyzing the solution with a cellulose membrane for 65-75 hours, and then freeze-drying for 45-50 hours to obtain a white product ODex.

3. The method for preparing a metabolically programmed hydrogel according to claim 2, characterized in that, In the step S1, the mass ratio of the dextran Dex to NaIO4 is 1:0.9-1.1; the mass-to-volume ratio of the dextran Dex to deionized water is 1g:20-30 mL; and the volume ratio of the ethylene glycol to deionized water is 3:120-130.

4. The method for preparing a metabolically programmed hydrogel according to claim 2, characterized in that, In the step S1, the cellulose membrane is of a type of MWCO 8000-14000 Da.

5. The method for preparing a metabolically programmed hydrogel according to claim 1, characterized in that, The step S2 specifically comprises: dissolving ODex in deionized water, dispersing APTC in DMSO and dropping it into the ODex solution, stirring at 45-55°C under N2 atmosphere for 3-5 hours, and then precipitating the obtained solution with cold ethanol for multiple times to obtain ODex-APTC.

6. The method of claim 5, wherein the metabolic programming hydrogel is prepared by, In the step S2, the mass-to-volume ratio of the ODex to deionized water is 1g:8-12 mL; and the mass-to-volume ratio of the APTC to DMSO is 1g:70-90 mL.

7. The method for preparing a metabolically programmed hydrogel according to claim 5, characterized in that, In the step S2, the cold ethanol precipitation is not less than four times.

8. The method of claim 1, wherein the metabolic programming hydrogel is prepared by, The step S3 specifically comprises: dissolving CMC in deionized water, adding GOx in the solution and stirring for 0.5-1.5 hours until the GOx is completely dispersed, then dissolving ODex-APTC in deionized water, and mixing and stirring the two solutions uniformly, and cross-linking at room temperature for 5-7 hours.

9. The method of claim 8, wherein the metabolic programming hydrogel is prepared by, In the step S3, the mass-to-volume ratio of the CMC to deionized water is 1g:12-16 mL; and the mass ratio of the GOx to CMC is 3:45-55. The mass ratio of the ODex-APTC to CMC is 1:1-1.5, and the mass-to-volume ratio of the ODex-APTC to deionized water is 1g:7-8 mL.

10. A metabolically programmed hydrogel for chronic diabetic wound treatment, characterized in that, The metabolic programming hydrogel is prepared by the preparation method of any one of the metabolic programming hydrogels in claims 1-9.

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