A double-responsive OPu hydrogel cross-linked by CA and loaded with carbon dots of radix isatidis and a preparation method and application thereof

Abstract

This application provides a CA-crosslinked, Isatis indigotica-derived carbon dots-loaded dual-responsive OPU hydrogel, its preparation method, and its applications. The hydrogel is prepared using oxidized pullulan as a backbone and chlorogenic acid as a crosslinking agent; carbon dots derived from Isatis indigotica are loaded into the hydrogel. This hydrogel exhibits excellent injectability and self-healing ability, effectively conforming to irregular wound surfaces. Under the stimulation of the acidic and oxidative microenvironment unique to infected wounds, the hydrogel gradually dissociates, achieving controlled release of Isatis indigotica carbon dots and chlorogenic acid. The Isatis indigotica carbon dots exhibit potent antibacterial activity, while chlorogenic acid synergistically works to efficiently scavenge excess reactive oxygen species and reactive nitrogen species, promoting tissue regeneration and reducing inflammatory responses. In vitro and in vivo experiments have confirmed that this hydrogel possesses excellent biocompatibility, exerting anti-infective, antioxidant, and anti-inflammatory effects in the early stages of healing, and promoting angiogenesis and accelerating wound healing in the later stages.

Description

Technical Field

[0001] This application belongs to the field of biomaterials technology, and in particular relates to a CA crosslinked and loaded with carbon dots from Isatis indigotica, a dual-responsive OPU hydrogel, its preparation method, and its application. Background Technology

[0002] Bacterial infection remains a key obstacle in clinical wound management. Once pathogens colonize the wound, they readily form biofilms that firmly anchor to the tissue surface and protect the encapsulated bacteria from host immune clearance. This biofilm-mediated protection significantly reduces treatment efficacy and contributes to persistent infection. The persistent bacterial load, in turn, drives an excessive inflammatory response, disrupting the normal wound healing process. Despite routine use of systemic antibiotics and topical antimicrobials, the emergence of multidrug-resistant strains and the complex, dynamic microenvironment of wounds are increasingly undermining their effectiveness.

[0003] Polysaccharide-based hydrogels have become an attractive dressing for treating infected wounds. Their inherent biodegradability, cytocompatibility, and low toxicity minimize the risk of adverse immune responses and long-term complications. Furthermore, these hydrogels possess a porous three-dimensional structure. This structure supports tissue regeneration while also serving as a physical barrier against external microbial invasion. Pullulan is a water-soluble, microbially derived polysaccharide obtained from the fermentation broth of *Brachystomata*. This polymer exhibits favorable biomedical properties, including enzymatic degradation, adhesion, and good biocompatibility. However, polysaccharide hydrogels typically lack inherent antibacterial activity, which significantly limits their application in treating infected wounds. To overcome this limitation, previous studies have incorporated traditional antibacterial agents (such as amoxicillin) or copper ions into hydrogels to impart antibacterial properties. While these strategies can enhance antibacterial properties, they also come with significant drawbacks. Excessive or prolonged exposure to amoxicillin may promote antibiotic resistance and potentially adversely affect host immune function. While copper ions are effective at killing bacteria, their accumulation in tissues can cause systemic or local toxicity. Furthermore, infection-driven inflammation induces oxidative stress at the wound site, leading to excessive production of reactive oxygen species (ROS), which in turn damage cells and extracellular matrix components, further delaying wound healing. Traditional hydrogel dressings have limited functionality and cannot meet the complex needs of repairing infected wounds. Summary of the Invention

[0004] This application is made in view of the above-mentioned issues, and its purpose is to provide an injectable, pH and ROS-responsive hydrogel dressing with safe and efficient antibacterial effect, as well as ROS scavenging ability to reduce oxidative damage and controlled drug release, so as to achieve rapid healing of infected wounds.

[0005] The first aspect of this application provides a CA-crosslinked and loaded Isatis indigotica carbon dots dual-responsive OPU hydrogel, comprising a hydrogel composed of OPU, 3-APBA and CA, and Isatis indigotica carbon dots encapsulated within the hydrogel; wherein the OPU concentration is 6-8% (w / v), and the molar ratio of 3-APBA and CA is 1:1-3.

[0006] In any embodiment, the dosage of 3-APBA is 3-10 mg / ml.

[0007] In any embodiment, the amount of CA added is 8-78 mg / ml.

[0008] In any embodiment, the amount of carbon dots from Isatis indigotica is 0.5-1.2 mg / mL.

[0009] A second aspect of this application also provides a method for preparing a CA-crosslinked and loaded Isatis indigotica carbon dots dual-responsive OPU hydrogel, comprising: mixing oxidized pullulan (OPu), 3-aminophenylboronic acid (3-APBA), chlorogenic acid (CA) and Isatis indigotica carbon dots (IR-CDs) in a formulation to obtain a hydrogel encapsulating Isatis indigotica carbon dots dual-responsive OPU hydrogel.

[0010] In any embodiment, the preparation method includes the following steps: 1) Add an appropriate amount of 3-aminophenylboronic acid (3-APBA) solution to an aqueous solution of oxidized pullulan polysaccharide (OPu), stir for 3-5 hours to obtain a mixed solution; 2) Add the carbon dots (IR-CDs) from Isatis root to the mixture in 1); 3) Add chlorogenic acid (CA) aqueous solution to 2) to obtain IR-CDs@OPAC.

[0011] In any embodiment, the concentration of OPU is 6-8% (w / v), the molar ratio of 3-APBA to CA is 1:1-3, the dosage of 3-APBA is 3-10 mg / ml, the dosage of CA is 8-78 mg / ml, and the dosage of Isatis indigotica carbon dots is 0.5-1.2 mg / mL.

[0012] In any embodiment, the IR-CDs are prepared as follows: Isatis root powder is dissolved in ultrapure water, the mixture is transferred to a reaction vessel, and reacted at 160-200℃ for 8-14 h, followed by natural cooling to room temperature; centrifugation is performed, and the supernatant is filtered through a 0.1-0.45 μm filter membrane to remove large particles; the filtrate is dialyzed in a 200-1000 Da cellulose dialysis bag for 24-72 h; finally, the dialyzed solution is freeze-dried, and the resulting solid product (IR-CDs) is stored at room temperature.

[0013] In any embodiment, the synthesis of the oxidized pullulan is as follows: sodium periodate and pullulan are dissolved in distilled water at a mass ratio of 0.6-1.2:1, and the mixture is stirred at room temperature for 6-10 h to obtain oxidized pullulan; then 5-8 mL of ethylene glycol is added to the mixture, and the reaction is terminated by stirring for 1.5-3 h; the obtained product is dialyzed in a 2500-4500 kDa dialysis bag for 48-96 h, and then freeze-dried to obtain OPU.

[0014] The third aspect of this application provides an application of a CA-crosslinked and loaded Isatis indigotica carbon dots dual-responsive OPU hydrogel, including the hydrogel of the first aspect of this application or the hydrogel obtained according to the preparation method of the second aspect of this application for use in wound dressings.

[0015] The beneficial effects of this application are: The IR-CDs@OPAC composite hydrogel prepared in this application provides a promising biomaterial strategy for repairing skin damage, particularly for treating infected wounds. First, IR-CDs derived from the traditional Chinese medicine Isatis indigotica were synthesized via a hydrothermal method. Subsequently, the IR-CDs were integrated into a hydrogel constructed through dual dynamic bonds, yielding IR-CDs@OPAC. This hydrogel network is constructed through dual reversible bonds, with CA acting as a bioactive cross-linking agent, thus preserving its inherent activity. The synergistic effect of CA and IR-CDs effectively realizes multiple functions of IR-CDs@OPAC, including injectability, self-healing ability, and dual pH / ROS responsiveness. These properties enable this hydrogel to serve as a smart wound dressing, inhibiting bacterial proliferation, reducing oxidative damage, stimulating angiogenesis and collagen deposition, and ultimately accelerating the healing of infected wounds. Furthermore, injectability and adhesion allow the hydrogel to closely contact irregular wound surfaces, meeting the practical needs of dynamic and complex tissue environments. Importantly, the cross-linked network of IR-CDs@OPAC enables microenvironment-responsive drug release, effectively targeting acidic and ROS-rich regions for controlled delivery of therapeutic components. In summary, the multifunctional IR-CDs@OPAC provides an effective biomaterial strategy for treating infected wounds and other complex skin lesions, indicating significant potential for clinical application.

[0016] This application presents a pH / ROS dual-responsive multifunctional hydrogel constructed based on dynamic Schiff bases and borate ester bonds. The hydrogel is prepared using oxidized pullulan as a backbone and chlorogenic acid, a bioactive polyphenol, as a cross-linking agent. Subsequently, carbon dots derived from Isatis indigotica were synthesized via a hydrothermal method and loaded into the hydrogel. This hydrogel exhibits excellent injectability and self-healing capabilities, effectively conforming to irregular wound surfaces. Under the stimulation of the acidic and oxidative microenvironment unique to infected wounds, the hydrogel gradually dissociates, achieving controlled release of Isatis indigotica carbon dots and chlorogenic acid. The Isatis indigotica carbon dots exhibit potent antibacterial activity, while chlorogenic acid synergistically works to efficiently scavenge excess reactive oxygen species and nitrogen species, thereby promoting tissue regeneration and reducing inflammatory responses. In vitro and in vivo experiments have confirmed that this hydrogel possesses excellent biocompatibility, exerting anti-infective, antioxidant, and anti-inflammatory effects in the early stages of healing, and promoting angiogenesis and accelerating wound healing in the later stages. This multifunctional hydrogel provides a promising and efficient biomaterial platform for the management of bacterial-infected wounds.

[0017] Injectability is a key property in wound dressings because it allows hydrogels to fill irregularly shaped defects. Furthermore, the excellent tissue adhesion and self-healing properties of hydrogels effectively prevent rupture caused by body movement, thereby reducing the risk of bacterial infection and frequent dressing changes. The reversible association and dissociation of the bidynamic covalent bonds in the hydrogel endow it with self-healing behavior and injectability. OPAC hydrogels exhibit excellent self-healing behavior, enabling rapid repair of fracture sites; this reconstruction capability stems from the synergistic effect of bidynamic bonds (including Schiff base bonds and borate ester bonds), which are rebuilt through interfacial diffusion and dynamic exchange between OPU, 3-APBA, and CA. Attached Figure Description

[0018] Figure 1 Characterization of the IR-CDs of this application; wherein, A is a transmission electron microscope image and a high-resolution transmission electron microscope image, B is a size distribution map, C is an X-ray diffraction spectrum, D is a Fourier transform infrared spectrum, E is a UV-Vis absorption and fluorescence spectrum, F is a full X-ray photoelectron spectrum, and G, H, and I are high-resolution XPS spectra of C1s, N1s, and O1s. Figure 2 Fourier transform infrared spectra of Pu and OPU in this application; Figure 3 This is a scanning electron microscope image of the hydrogel of this application; Figure 4 This is an EDS elemental mapping diagram of the hydrogel of this application; Figure 5 This is a diagram showing the water content of the hydrogel in this application; Figure 6Characterization of the hydrogel of this application; wherein, A is the formation of IR-CDs@OPAC within 5 minutes after the addition of CA, B is the macroscopic self-healing process of IR-CDs@OPAC, C is the adhesion performance of IR-CDs@OPAC on various substrates and the adhesion of the hydrogel to pigskin, D is the frequency scan test of IR-CDs@OPAC, E is the strain scan test of IR-CDs@OPAC, F is the continuous step strain test of IR-CDs@OPAC under repeated deformation at 1% and 1000% strain, G is the shear thinning behavior test of OPAC and its injectability, H is the swelling rate of the hydrogel, and I is the water retention performance of the hydrogel at room temperature. Figure 7 This is a Zeta potential diagram of the IR-CDs of this application; Figure 8 This diagram illustrates the response behavior, antioxidant properties, and antibacterial properties of the hydrogel in this application. A represents a macroscopic observation of the hydrogel's response to pH / reactive oxygen species; B and C represent the release kinetics of IR-CDs and IR-CDs@OPAC under different conditions; D represents the DPPH scavenging rate of the hydrogel; E represents the ABTS scavenging rate of the hydrogel; F represents the H2O2 scavenging rate of the hydrogel; G represents the ·OH scavenging rate of the hydrogel; H represents the antibacterial effect of the hydrogel against Escherichia coli and Staphylococcus aureus; and I and J represent the antibacterial rates of the hydrogel against Escherichia coli and Staphylococcus aureus. Figure 9 This is a diagram showing the coagulation status of the IR-CDs@OPAC in this application; Figure 10 The images show the biocompatibility and cell migration ability of the hydrogels in this application. Among them, A and B are representative images of hemolysis under different treatments and hemolysis rate graphs; C, D, and E are cell viability graphs of L929 cells, Raw264.7 cells, and HUVEC cells after co-incubation with different hydrogel extracts for 24 hours; F is the cell scratch healing rate graph within 36 hours; and G is the graph of HUVEC cell scratch experiment after incubation with different hydrogels for different times. Figure 11 This is a live in vivo infection wound healing experiment using hydrogels according to this application; wherein, A is a schematic diagram of the animal experiment process for infected wounds, B is a macroscopic photograph of wounds in different treatment groups on days 0, 3, 6, 9 and 13, C is a simulated stacked diagram of wound progression on days 0, 3, 6, 9 and 13, D is a graph showing the change in relative wound area for each group, E is a photograph of bacteria on LB agar plates after 3 days of different in vivo treatments, and F is a graph showing the antibacterial rate of different hydrogels against Staphylococcus aureus determined by the plating method. Figure 12Histological evaluation of wounds in different groups of the hydrogel of this application; wherein, A is the hematoxylin-eosin staining image of wounds in mice on day 6 and day 13 of treatment, B is the Masson staining image of wounds in mice on day 6 and day 13 of treatment, C is the epidermal thickness image of wounds in all groups on day 13, and D is the collagen deposition image of wounds in all groups on day 13. Figure 13 Immunohistochemical evaluation of wounds in different groups using the hydrogel of this application; wherein, A is an immunohistochemical staining image of CD206, B is an immunohistochemical staining image of CD31, and C and D are quantitative analysis images of CD206 and CD31. Detailed Implementation

[0019] The following detailed description, with appropriate reference to the accompanying drawings, discloses embodiments of the CA-crosslinked and Isatis indigotica-loaded dual-responsive OPU hydrogel, its preparation method, and its applications. However, unnecessary details may be omitted. For example, detailed descriptions of well-known facts and repetitive descriptions of essentially the same structures may be omitted. This is to avoid unnecessarily lengthy descriptions and to facilitate understanding by those skilled in the art. Furthermore, the accompanying drawings and the following description are provided for a full understanding of this application by those skilled in the art and are not intended to limit the subject matter of the claims.

[0020] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a specific parameter, it is expected that ranges of 60-110 and 80-120 are also included. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5. In this application, unless otherwise stated, the numerical range "ab" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0-5" indicates that all real numbers between "0-5" have been listed in this article; "0-5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0021] Unless otherwise specified, all embodiments and optional embodiments of this application can be combined to form new technical solutions.

[0022] Unless otherwise specified, all technical features and optional technical features of this application may be combined to form new technical solutions.

[0023] Unless otherwise specified, the terms "comprising" and "including" as used in this application can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other components not listed may also be included, or that only the listed components may be included.

[0024] Unless otherwise specified, the term "or" is inclusive in this application. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0025] Polysaccharide-based hydrogels have become an attractive dressing for treating infected wounds due to their inherent biodegradability, cytocompatibility, and low toxicity, minimizing the risk of adverse immune responses and long-term complications. This application selects pullulan, whose backbone contains multiple hydroxyl groups, making it suitable for various chemical modifications and enabling the synthesis of numerous derivatives with tunable mechanical strength, stability, and biological functions. However, pullulan hydrogels typically lack inherent antibacterial activity, significantly limiting their application in treating infected wounds. To overcome this limitation, previous studies have incorporated traditional antibacterial agents (such as amoxicillin) or copper ions into pullulan hydrogels to impart antibacterial properties. While these strategies can enhance antibacterial properties, they also come with significant drawbacks. Excessive or prolonged exposure to amoxicillin may promote antibiotic resistance and potentially adversely affect host immune function. Although copper ions are effective at killing bacteria, their accumulation in tissues can cause systemic or local toxicity. Furthermore, infection-driven inflammation induces oxidative stress at the wound site, leading to excessive production of reactive oxygen species (ROS), which in turn damages cells and extracellular matrix components, further delaying wound healing. Therefore, pullulan-based hydrogel dressings should not only provide safe and effective antibacterial effects but also possess ROS scavenging capabilities to mitigate oxidative damage.

[0026] Injectable hydrogels can be delivered minimally invasively in wound care, conforming to irregular wounds and adhering to tissue surfaces to achieve localized, sustained delivery of therapeutic drugs. Injectable self-healing hydrogels can be obtained by introducing reversible covalent bonds into a polymer network. However, these dynamic covalent bonds are unstable under specific stimuli (such as changes in pH, glucose concentration, temperature, reactive oxygen species (ROS) levels, or enzyme activity), allowing the network to dissociate on demand and control the release of encapsulated drugs. Chronic wounds are characterized by persistently high ROS levels due to ongoing inflammation, leading to cell damage and delayed repair. Furthermore, the wound microenvironment is typically acidic, with bacterial metabolism further lowering the surface pH to approximately 4.5–6.5. Therefore, hydrogels responsive to both ROS and acidity are ideal for treating infected wounds, as they can selectively degrade and release antibacterial and antioxidant agents where they are most needed.

[0027] Carbon dots (CDs), as a novel type of carbon-based nanomaterial, are valued for their excellent water solubility, low toxicity, and superior biocompatibility. Selecting carbon precursors with intrinsic bioactivity is a key strategy for preparing carbon dots that possess both biocompatibility and effective antibacterial properties. Traditional Chinese medicine (TCM) herbs are highly attractive candidate materials due to their abundant resources, low cost, and rich bioactive components. Isatis root (Banlangen) is a well-known TCM herb with inherent antibacterial properties. This application first synthesizes carbon dots (IR-CDs) derived from Isatis root via a hydrothermal method, retaining the green precursors of bioactive functional carbon dots.

[0028] For the repair of infected wounds, excessive ROS disrupts the balance between the oxidant and antioxidant systems and slows tissue regeneration. Therefore, it is essential to develop dressings with intrinsic antioxidant properties to scavenge ROS and restore redox homeostasis at the wound site. Chlorogenic acid (CA) is a naturally occurring phenolic compound that exhibits effective free radical scavenging activity while maintaining relatively low toxicity. In previous studies, chlorogenic acid has been introduced into hydrogels through physical loading or covalent bonding between its carboxyl groups and polymer amine groups via amide bonds. However, this chemical grafting route often suffers from low coupling efficiency and may require the use of crosslinking agents or catalysts, raising concerns about its residual toxicity. This application aims to develop a more efficient and safer method for preparing chlorogenic acid-containing hydrogels. Meanwhile, integrating multiple functions (such as injectability, self-healing properties, antibacterial activity, and antioxidant activity) into a single hydrogel system remains challenging, but is crucial for meeting the diverse needs of infected wound treatment.

[0029] In this application, a multifunctional hydrogel was designed by integrating dual dynamic covalent bonds into a network structure, achieving injectability, self-healing properties, potent antibacterial activity, and dual sensitivity to acidity and ROS. First, bioactive carbon dots (IR-CDs) were synthesized via a one-step hydrothermal method using Isatis indigotica root as a precursor, which helps retain the therapeutic functions derived from the herbal medicine. Simultaneously, a dynamically cross-linked oxidized pullulan (OPu) / 3-aminophenylboronic acid (APBA) / chlorogenic acid (CA) hydrogel (OPAC) was constructed, providing a mechanically stable and adaptable matrix. Embedding IR-CDs into OPAC resulted in the complex IR-CDs@OPAC, forming a stimulus-responsive delivery platform. Under the typical ROS-rich and acidic conditions of infected wounds, Schiff base and borate ester bonds gradually loosen, inducing network structure loosening and promoting the on-demand release of IR-CDs and CA. When injected into irregular wound sites, IR-CDs@OPAC can adhere to the defect area and locally release therapeutic agents to eradicate bacteria and alleviate oxidative stress. Meanwhile, we systematically characterized the rheological and mechanical properties, antibacterial efficacy, and cell compatibility of this hydrogel. Furthermore, we evaluated the in vivo wound healing performance of IR-CDs@OPAC in a C57BL / 6 mouse model of full-thickness skin defects caused by Staphylococcus aureus infection. The results showed that IR-CDs@OPAC effectively inhibited bacterial growth and significantly accelerated tissue regeneration. IR-CDs@OPAC hydrogel is a promising wound dressing for treating infected and complex skin wounds.

[0030] In one embodiment of this application, a dual-responsive OPU hydrogel with CA crosslinking and loaded with Isatis indigotica carbon dots is proposed, comprising a hydrogel composed of OPU, 3-APBA and CA, and Isatis indigotica carbon dots encapsulated within the hydrogel; wherein the OPU concentration is 6-8% (w / v), and the molar ratio of 3-APBA and CA is 1:1-3.

[0031] Excessive OPU concentration reduces self-healing ability, while excessively low concentration weakens mechanical properties.

[0032] Controlling the concentration of OPU is beneficial for constructing the three-dimensional framework of the hydrogel. As the main polymer matrix, if the concentration of OPU is too low, the number of aldehyde groups in the system is insufficient, resulting in too few cross-linking sites. This can lead to excessively long gelation time (or even failure to gel) or extremely low mechanical strength in the formed hydrogel, exhibiting a fluid-like state and unable to maintain a stable three-dimensional structure. If the concentration is too high, the solution viscosity increases sharply, leading to: poor mixing uniformity (when adding 3-APBA and CA, due to high diffusion resistance, localized cross-linking is prone to occur too quickly, resulting in uneven gelation); and increased brittleness (excessive polymer concentration will cause excessive cross-linking density, leading to decreased hydrogel toughness and easy brittle fracture, which is detrimental to its application in biological environments).

[0033] The molar ratio of 3-APBA to CA is crucial for maintaining the balance of dynamic covalent bonds (Schiff base bonds and borate ester bonds). A 1:1 ratio ensures a basic balance between the amino groups in 3-APBA and the aldehyde / hydroxyl groups in CA (or OPU). Increasing the proportion of CA provides a large number of hydroxyl (-OH) and amino (-NH2) groups. Excess CA ensures that the phenylboronic acid groups on 3-APBA have sufficient diol sites to bind, forming stable dynamic borate ester bonds. A greater number of CA sites also provides a greater buffering capacity for changes in the external chemical environment, enabling more precise and controlled release.

[0034] In some embodiments, the dosage of 3-APBA is 3-10 mg / ml.

[0035] 3-APBA acts as a "bridge" connecting OPU and CA, providing basic cross-chain connectivity and responsiveness. Low concentrations result in insufficient dynamic covalent bond density to support the entire network. Excessively high concentrations negatively impact biocompatibility.

[0036] In some embodiments, the CA dosage is 8-78 mg / ml.

[0037] CA modulates the viscoelasticity and gelation threshold of hydrogels. Too low a dosage results in a softer gel with high water content, suitable for drug delivery with high diffusion rate requirements; too high a dosage significantly increases the modulus of the gel. Since CA contains a large number of polar groups, increasing its content will enhance the hydrogen bonding between molecular chains, significantly improving the self-healing ability and mechanical stability of the gel.

[0038] In some embodiments, the amount of carbon dots from Isatis indigotica is 0.5-1.2 mg / mL.

[0039] Too low a concentration of carbon dots in Isatis indigotica can lead to insufficient biological activity (such as antibacterial effect); the surface of carbon dots usually has a large number of functional groups (such as -OH, -COOH, -NH2). Excessive carbon dots will competitively consume the active sites on OPU or 3-APBA, thereby interfering with the normal cross-linking between the main chains, resulting in unstable gel structure, and may also lead to cytotoxicity.

[0040] In one embodiment of this application, a method for preparing a CA-crosslinked and loaded Isatis indigotica carbon dots dual-responsive OPU hydrogel is proposed, comprising: mixing oxidized pullulan (OPu), 3-aminophenylboronic acid (3-APBA), chlorogenic acid (CA) and Isatis indigotica carbon dots (IR-CDs) in a formulation to obtain a hydrogel encapsulating Isatis indigotica carbon dots dual-responsive OPU hydrogel.

[0041] In some embodiments, the preparation method includes the following steps: 1) Add an appropriate amount of 3-aminophenylboronic acid (3-APBA) solution to an aqueous solution of oxidized pullulan polysaccharide (OPu), stir for 3-5 hours to obtain a mixed solution; 2) Add the carbon dots (IR-CDs) from Isatis root to the mixture in 1); 3) Add chlorogenic acid (CA) aqueous solution to 2) to obtain IR-CDs@OPAC.

[0042] First, OPU and 3-APBA are mixed. Oxidized pullulan (OPu) contains a large number of aldehyde groups (-CHO), while 3-APBA contains primary amino groups (-NH2). The purpose of mixing them first is to allow 3-APBA to be covalently attached to the OPU backbone via a Schiff base reaction; stirring allows sufficient time for the amino and aldehyde groups to collide and reach reaction equilibrium.

[0043] Chlorogenic acid (CA) contains ortho- and tho-dihydroxyl groups. As a cross-linking agent, CA's hydroxyl groups undergo a rapid esterification reaction with the phenylboronic acid groups grafted onto OPU in the first step. Since CA molecules typically contain multiple binding sites, they can act like "lockers" to pull together different OPU molecular chains, thereby transforming the solution from a fluid state into a hydrogel (OPAC) with a three-dimensional structure.

[0044] The carbon dots on the surface of Isatis root typically contain abundant carboxyl, hydroxyl, or amino groups. To ensure the carbon dots are well encapsulated within the hydrogel, they usually need to be added after the first step. Although these active groups may form bonds with the aldehyde group of OPU or the boric acid group of 3-APBA, thus competitively consuming crosslinking sites and leading to gelation failure or decreased strength, their low carbon dot content and the fact that their non-ortho-dihydroxy structures do not readily form borate ester bonds with phenylboronic acid significantly affect the overall hydrogel formation.

[0045] In some embodiments, the OPU concentration is 6-8% (w / v), the molar ratio of 3-APBA to CA is 1:1-3, the dosage of 3-APBA is 3-10 mg / ml, the dosage of CA is 8-78 mg / ml, and the dosage of Isatis indigotica carbon dots is 0.5-1.2 mg / mL.

[0046] Excessive deviation in the amount of the above-mentioned raw materials will affect the gelation effect and deteriorate the performance of the hydrogel, such as non-injectability, lower water retention, and poorer self-healing.

[0047] In some embodiments, the IR-CDs are prepared as follows: Isatis root powder is dissolved in ultrapure water, the mixture is transferred to a reaction vessel, and reacted at 160-200°C for 8-14 h, followed by natural cooling to room temperature; centrifugation is performed, and the supernatant is filtered through a 0.1-0.45 μm filter membrane to remove large particles; the filtrate is dialyzed in a 200-1000 Da cellulose dialysis bag for 24-72 h; finally, the dialyzed solution is freeze-dried, and the resulting solid product (IR-CDs) is stored at room temperature.

[0048] Excessively high or low temperatures, or excessively short times, will affect the yield of carbon points.

[0049] In some embodiments, the synthesis of the oxidized pullulan is as follows: sodium periodate and pullulan are dissolved in distilled water at a mass ratio of 0.2-1.2:1, and the mixture is stirred at room temperature for 6-10 h to obtain oxidized pullulan; then 2.5-6 mL of ethylene glycol is added to the mixture, and the reaction is terminated by stirring for 1.5-3 h; the obtained product is dialyzed in a 2800-4500 kDa dialysis bag for 48-96 h, and then freeze-dried to obtain OPU.

[0050] In one embodiment of this application, the application of a CA-crosslinked and loaded Isatis indigotica carbon dots dual-responsive OPU hydrogel is proposed, including the hydrogel of the first aspect of this application or the hydrogel obtained according to the preparation method of the second aspect of this application for use in wound dressings.

[0051] Example The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0052] Example 1 A method for preparing a CA-crosslinked and Isatis indigotica-loaded dual-responsive OPU hydrogel includes the following steps: 1) Preparation of IR-CDs Isatis root powder (1 g) was dissolved in 30 mL of ultrapure water. The mixture was transferred to a reaction vessel and reacted at 180 °C for 12 h, followed by natural cooling to room temperature. After centrifugation, the supernatant was filtered through a 0.22 μm filter membrane to remove large particles. The filtrate was dialyzed in a cellulose dialysis bag (500 Da) for 48 h. Finally, the dialyzed solution was freeze-dried, and the resulting solid products (IR-CDs) were stored at room temperature.

[0053] 2) Synthesis of oxidized pullulan (OPu) 1.5 g of sodium periodate and 2.5 g of pullulan were dissolved in 100 mL of distilled water and reacted with the solution at room temperature for 6 h to prepare oxidized pullulan. Then, 5 mL of ethylene glycol was added to the mixture, and the reaction was terminated by stirring for another 2 h. The resulting product was dialyzed in a dialysis bag (MWCO: 3500 kDa) for 72 h and then freeze-dried to obtain the final product.

[0054] 3) Preparation of IR-CDs@OPAC Prepare a 6% (w / v) aqueous solution of OPU, add 1 mg / mL IR-CDs, 5 mg / mL 3-APBA and 22 mg / mL CA aqueous solution, stir for 30 min to obtain IR-CDs@OPAC, and store in a sealed container for later use.

[0055] Example 2 A method for preparing a CA-crosslinked and Isatis indigotica-loaded dual-responsive OPU hydrogel includes the following steps: 1) Preparation of IR-CDs Isatis root powder (1 g) was dissolved in 30 mL of ultrapure water. The mixture was transferred to a reaction vessel and reacted at 160 °C for 12 h, followed by natural cooling to room temperature. After centrifugation, the supernatant was filtered through a 0.22 μm filter membrane to remove large particles. The filtrate was dialyzed in a cellulose dialysis bag (500 Da) for 48 h. Finally, the dialyzed solution was freeze-dried, and the resulting solid products (IR-CDs) were stored at room temperature.

[0056] 2) Synthesis of oxidized pullulan (OPu) 1.5 g of sodium periodate and 2.5 g of pullulan were dissolved in 100 mL of distilled water and reacted with the solution at room temperature for 6 h to prepare oxidized pullulan. Then, 5 mL of ethylene glycol was added to the mixture, and the reaction was terminated by stirring for another 2 h. The resulting product was dialyzed in a dialysis bag (MWCO: 3500 kDa) for 72 h and then freeze-dried to obtain the final product.

[0057] 3) Preparation of IR-CDs@OPAC Prepare a 6% (w / v) aqueous solution of OPU, add 1 mg / mL IR-CDs and 5 mg / mL 3-APBA solution, and stir for 4 h; then add 22 mg / mL CA aqueous solution and stir for 30 min to obtain IR-CDs@OPAC, which can be stored in a sealed container for later use.

[0058] Example 3 A method for preparing a CA-crosslinked and Isatis indigotica-loaded dual-responsive OPU hydrogel includes the following steps: 1) Preparation of IR-CDs Isatis root powder (1 g) was dissolved in 30 mL of ultrapure water. The mixture was transferred to a reaction vessel and reacted at 160 °C for 14 h, followed by natural cooling to room temperature. After centrifugation, the supernatant was filtered through a 0.35 μm filter membrane to remove large particles. The filtrate was dialyzed in a cellulose dialysis bag (800 Da) for 24 h. Finally, the dialyzed solution was freeze-dried, and the resulting solid products (IR-CDs) were stored at room temperature.

[0059] 2) Synthesis of oxidized pullulan (OPu) 1.5 g of sodium periodate and 2.5 g of pullulan were dissolved in 100 mL of distilled water and reacted with the solution at room temperature for 6 h to prepare oxidized pullulan. Then, 5 mL of ethylene glycol was added to the mixture, and the reaction was terminated by stirring for another 2 h. The resulting product was dialyzed in a dialysis bag (MWCO: 3500 kDa) for 72 h and then freeze-dried to obtain the final product.

[0060] 3) Preparation of IR-CDs@OPAC Prepare an 8% (w / v) aqueous solution of OPU, add 1.2 mg / mL IR-CDs, 10 mg / mL 3-APBA and 41 mg / mL CA aqueous solution to obtain IR-CDs@OPAC, and store in a sealed container for later use.

[0061] Example 4 A method for preparing a CA-crosslinked and Isatis indigotica-loaded dual-responsive OPU hydrogel includes the following steps: 1) Preparation of IR-CDs Isatis root powder (1 g) was dissolved in 30 mL of ultrapure water. The mixture was transferred to a reaction vessel and reacted at 200 °C for 14 h, followed by natural cooling to room temperature. After centrifugation, the supernatant was filtered through a 0.22 μm filter membrane to remove large particles. The filtrate was dialyzed in a cellulose dialysis bag (500 Da) for 48 h. Finally, the dialyzed solution was freeze-dried, and the resulting solid products (IR-CDs) were stored at room temperature.

[0062] 2) Synthesis of oxidized pullulan (OPu) 1.5 g of sodium periodate and 2.5 g of pullulan were dissolved in 100 mL of distilled water and reacted with the solution at room temperature for 8 h to prepare oxidized pullulan. Then, 5 mL of ethylene glycol was added to the mixture, and the reaction was terminated by stirring for another 2 h. The resulting product was dialyzed in a dialysis bag (MWCO: 3500 kDa) for 72 h and then freeze-dried to obtain the final product.

[0063] 3) Preparation of IR-CDs@OPAC Add 7 mg / mL of 3-aminophenylboronic acid (3-APBA) solution to 8% (w / v) oxidized pullulan polysaccharide (OPu) aqueous solution and stir for 3-5 h to obtain a mixture; then add 1 mg / mL of Isatis indigotica carbon dots (IR-CDs) and 32 mg / mL of chlorogenic acid (CA) aqueous solution to the mixture and mix for 5-30 min to obtain IR-CDs@OPAC.

[0064] Example 5 A method for preparing a CA-crosslinked OPU hydrogel includes the following steps: 1) Preparation of IR-CDs Isatis root powder (1 g) was dissolved in 30 mL of ultrapure water. The mixture was transferred to a reaction vessel and reacted at 160-200 °C for 8-14 h, followed by natural cooling to room temperature. After centrifugation, the supernatant was filtered through a 0.22 μm filter membrane to remove large particles. The filtrate was dialyzed in a cellulose dialysis bag (500 Da) for 48 h. Finally, the dialyzed solution was freeze-dried, and the resulting solid products (IR-CDs) were stored at room temperature.

[0065] 2) Synthesis of oxidized pullulan (OPu) 1.5 g of sodium periodate and 2.5 g of pullulan were dissolved in 100 mL of distilled water and reacted with the solution at room temperature for 6–10 h to prepare oxidized pullulan. Then, 5 mL of ethylene glycol was added to the mixture, and the reaction was terminated by stirring for another 2 h. The resulting product was dialyzed in a dialysis bag (MWCO: 3500 kDa) for 72 h and then freeze-dried to obtain the final product.

[0066] 3) Preparation of OPAC OPU was prepared into a 6% (w / v) aqueous solution, and 5 mg / mL 3-APBA solution was added. The mixture was stirred for 4 h. Separately, a 22 mg / mL CA aqueous solution was prepared and added to the above mixture to obtain OPAC hydrogel.

[0067] To demonstrate the above technical effects, this application provides the following technical characterization of the above embodiments: 1. Performance characterization of IR-CDs IR-CDs were synthesized via a hydrothermal method using Isatis indigotica as the carbon source. Transmission electron microscopy revealed that the IR-CDs were uniformly distributed, exhibiting a near-spherical morphology, with a particle size concentrated around an average of 2.9 ± 0.7 nm, and good dispersibility. Figure 1 (AB). High-resolution transmission electron microscopy revealed clearly discernible lattice fringes with an interplanar spacing of 0.21 nm, a typical characteristic of carbon domains in graphite. The structural features of IR-CDs were further confirmed by X-ray diffraction analysis. Figure 1As shown in Figure C, a broad diffraction peak was observed at 21.13°, corresponding to the (002) crystal plane of graphitic carbon, attributed to disordered carbon atoms. Fourier transform infrared spectra of IR-CDs ( Figure 1 D) shows that at 1654 cm -1 There is a C=O stretching vibration peak at 3278 cm⁻¹, and at 3278 cm⁻¹... -1 The prominent peaks nearby correspond to NH stretching vibrations, confirming the nitrogen doping effect in IR-CDs. Furthermore, at 2911 cm⁻¹... -1 and 1006 cm -1 The peaks at these locations are attributed to the bending vibrations of CH4 and CO, respectively. The optical properties of the IR-CDs were characterized using UV-Vis absorption spectroscopy and fluorescence spectroscopy. Figure 1 E). The UV-Vis absorption spectrum exhibits a distinct absorption peak at 283 nm, which can be attributed to π-π* transitions generated by conjugated carbon structures (such as aromatic or unsaturated domains). The fluorescence spectra of IR-CDs show that the optimal excitation wavelength is 395.6 nm, with maximum emission at 502 nm. A significant redshift between the excitation and emission wavelengths indicates that IR-CDs possess abundant surface states and functional groups, which not only stabilize electrons but also enhance water dispersibility. Furthermore, this electron-stabilizing surface structure may generate photothermal or photodynamic effects under light irradiation, thereby contributing to antibacterial activity. X-ray photoelectron spectroscopy analysis was employed, such as... Figure 1 As shown in F, IR-CDs are mainly composed of carbon, nitrogen, and oxygen. The high-resolution XPS spectrum of C 1s (…) Figure 1 G) shows three deconvolution peaks, corresponding to CO / C=N (286.54 eV), CN (284.8 eV), and CC (283.38 eV), respectively. N 1s energy spectrum ( Figure 1 The presence of two peaks at 400.40 eV and 398.51 eV indicates the presence of graphitic nitrogen and pyrrole nitrogen. Furthermore, the high-resolution XPS spectrum of O 1s (…) shows… Figure 1 I) Three deconvolution peaks were observed at 532.11 eV, 531.04 eV, and 529.97 eV, corresponding to CO, C=O, and OH, respectively. IR-CDs are rich in various surface functional groups, including a variety of polar groups and nitrogen-containing structures, which enhances their hydrophilicity and fluorescence properties.

[0068] 2. Characterization of relevant properties of hydrogels Oxidized pullulan is prepared by oxidizing pullulan with sodium periodate. For example... Figure 2 As shown, the structural modification of polysaccharides was investigated using Fourier transform infrared spectroscopy. The spectra were observed at 1732 cm⁻¹. -1A characteristic absorption peak appears at [location], corresponding to the stretching vibration of C=O, proving that the aldehyde group has been successfully introduced into the polymer backbone. The construction process of IR-CDs@OPAC is as follows: Figure 1 As shown in B. In short, IR-CDs (1 mg / mL) were first dispersed in an OPU solution under stirring. Then, 3-APBA was dissolved in ethanol and slowly added to the above solution. Finally, CA was added to initiate gel formation, yielding IR-CDs@OPAC. In this process, a dual-dynamic covalent network was constructed through two reversible bonds: a Schiff base bond between the residual aldehyde group on OPU and the amino group on 3-APBA, and a borate ester bond between the phenylboronic acid on 3-APBA and the vicinal diol on CA. Figure 6 Image A shows representative images before and after gelation. Scanning electron microscopy was used to further observe the internal microstructure of the hydrogel. Figure 3 As shown, the hydrogel exhibits a three-dimensional network structure with high-density pores, indicating its suitability as a drug delivery platform. Furthermore, as... Figure 4 As shown, energy dispersive spectroscopy (EDS) analysis reveals a uniform distribution of C, N, O, and B elements. These results further confirm the successful formation of a homogeneous hydrogel network.

[0069] For wound dressing applications, injectability is a key characteristic, as it allows hydrogels to fill irregularly shaped defects. In addition, the excellent tissue adhesion and self-healing properties of hydrogels effectively prevent rupture caused by body movement, thereby reducing the risk of bacterial infection and frequent dressing changes. The reversible association and dissociation of bidynamic covalent bonds in the hydrogel endow it with self-healing behavior and injectability. To further evaluate the self-healing behavior, such as... Figure 6 As shown in Figure B, after cutting a circular OPAC hydrogel in half and bringing the cut surfaces into close contact, stretching both ends of the sample after 1 minute confirmed that the fracture site was rapidly repaired. This reconstruction capability stems from the synergistic effect of dual dynamic bonds (including Schiff base bonds and borate ester bonds), which are reconstructed through interfacial diffusion and dynamic exchange between OPU, 3-APBA, and CA.

[0070] In addition, the adhesive properties of the hydrogel were evaluated, such as... Figure 6 As shown in C, this hydrogel exhibits strong adhesion to a variety of surfaces. Skin adhesion was tested using a pigskin overlap shear test, achieving an adhesion strength of 10.94 kPa, highlighting its potential application in wound dressings. This excellent adhesion is primarily attributed to the catechol groups in CA, which can interact with various surfaces through both covalent and non-covalent bonds. Furthermore, the catechol groups containing the o-quinone oxide structure facilitate covalent bonding with nucleophiles such as amine, thiol, and hydroxyl groups on tissue surfaces.

[0071] 3. Rheological property testing of OPAC hydrogel To ensure the structural integrity of the hydrogel during use, suitable mechanical properties are crucial. Therefore, rheological measurements were performed at different frequencies and strain amplitudes, where the storage modulus reflects the elastic properties of the hydrogel, while the loss modulus reflects its viscous properties. The rheological properties of the OPAC hydrogel were characterized at 37°C using a rotational rheometer (Antonpah AG, Austria, MCR-302). The rheological properties were determined by shear rate scans (0.1–100 s⁻¹). -1 The shear-thinning behavior of the hydrogel was investigated. The frequency scan range was 0.1–100 rad / s, with a fixed strain of 1%. To determine the linear viscoelastic region, amplitude scans were performed at a fixed angular frequency of 10 rad / s, with the oscillating strain gradually increased from 0.1% to 1000%. Furthermore, the self-healing properties of the hydrogel network were investigated by continuous step strain measurements: small strain (1%) and large strain (1000%) were alternately applied at a constant oscillation frequency of 1 Hz.

[0072] Frequency scan results as follows Figure 6 As shown in Figure D, within the angular frequency range of 0.1–100 rad / s, G' is consistently higher than G'', indicating that elastic behavior dominates and the hydrogel network has been successfully constructed. Notably, at low frequencies, the values ​​of G' and G'' are very close, suggesting that the hydrogel cross-linking is weak or in a fluid-like state. Figure 6 As shown in Figure E, the critical point for hydrogel collapse was evaluated using strain scanning. When the strain was approximately 425%, the G'' value exceeded the G' value, indicating the destruction of the three-dimensional network structure and its transformation into a flowing quasi-liquid state. Subsequently, continuous step strain tests were performed to demonstrate the self-healing properties of the hydrogel. Figure 6 As shown in Figure F, when the applied strain suddenly increases from 1% to 1000%, a sharp decrease in G' is observed, indicating severe damage to the network structure. However, when the strain recovers to 1%, both G' and G'' values ​​recover rapidly, indicating that the hydrogel achieves effective repair and confirming its excellent self-healing ability. Figure 6 As shown in G, the viscosity of the hydrogel decreases rapidly with increasing shear rate, indicating that the hydrogel exhibits shear-thinning properties. In summary, these rheological properties can be attributed to the dynamic Schiff base bonds and borate ester bonds within the polymer network, which continuously undergo bond dissociation and reconstruction under external mechanical stimulation.

[0073] 4. Moisture content and swelling performance test A sufficiently moist environment is widely considered a key factor in promoting efficient wound healing. To determine the initial water content of the hydrogel, the initial wet weight of the freshly prepared sample was obtained. The sample was then placed in a vacuum freeze dryer for 48 hours to completely remove residual moisture, and the dry weight was obtained after drying. The water content was calculated using the formula: Water content (%) = (W0 - W xThe swelling rate was calculated as Wt / W0 × 100%, where W0 and Wx represent the mass of the hydrogel before and after freeze-drying, respectively. The swelling behavior of the hydrogel was further evaluated by immersing it in PBS (pH 7.4) at 37°C. The initial weight of each hydrogel was recorded as W0. At predetermined time points, the hydrogels were removed, excess liquid was blotted off with filter paper, and the weight was recorded as Wt. The swelling rate was calculated using the formula: Swelling rate (%) = (Wt - W0) / W0 × 100%.

[0074] like Figure 5 As shown, all hydrogels have a water content exceeding 90%, a level comparable to the natural extracellular matrix, thus providing favorable moist conditions for damaged tissue. Such high hydration promotes cell activity and tissue regeneration at the wound site. Furthermore, swelling behavior is another important parameter for evaluating the suitability of hydrogels as wound dressings. Figure 6 As shown in Figure H, the hydrogel exhibits high water absorption and swells rapidly within the initial 6 hours. The swelling rate was observed to reach equilibrium within 24 hours. This swelling behavior indicates that the hydrogel can effectively absorb wound exudate while maintaining structural stability.

[0075] 5. Evaluation of water retention performance The water retention capacity of the hydrogel was further evaluated by monitoring its mass loss at room temperature and weighing it at specific time intervals. The initial weight of the hydrogel was recorded as W0. The hydrogel was placed in a room temperature environment, and its weight was recorded at specified time points, denoted as Wx. The formula for calculating water retention capacity is: Water retention rate (%) = Wx / W0 × 100%. Figure 6 As shown in Figure I, the hydrogel retained nearly 80% of its initial moisture content after 7 days. These results demonstrate that the hydrogel effectively manages wound exudate, mimics the extracellular matrix, and maintains a clean and moist healing environment. Therefore, this hydrogel shows great potential as a functional dressing material in wound repair applications.

[0076] 6. pH / ROS dual response behavior and drug release kinetics of IR-CDs@OPAC Based on dynamic Schiff base and boronic acid ester bonds, IR-CDs@OPAC exhibits responsive behavior to pH and reactive oxygen species. To investigate this dual-stimulus responsiveness, we studied the structural stability and degradation of IR-CDs@OPAC under different conditions. Figure 8As shown in Figure A, upon exposure to acidic PBS (pH 5.0), the gel integrity gradually decreased, and disintegration occurred progressively within 8 hours. Furthermore, similar response behavior was observed upon the addition of H₂O₂ (1 mM). The results indicate that under acidic and ROS-rich conditions, Schiff base and borate ester bonds undergo hydrolysis, leading to the disruption of the hydrogel network structure. Once the network structure collapses, the encapsulating components (IR-CDs and CA) are exposed to the external medium. Microenvironment-responsive drug release behavior is particularly advantageous for wound treatment as it enables localized, on-demand drug delivery. Accordingly, we analyzed the drug release kinetics of IR-CDs@OPAC in PBS (pH 7.5 and 5.0), with and without 1 mM H₂O₂. Figure 8 B and Figure 4 As shown in Figure C, neutral conditions (pH 7.4) significantly inhibited drug release throughout the initial burst and plateau phases, while acidic (pH 5.0) and ROS (1 mM H2O2) environments significantly accelerated the release of IR-CDs and CA. Notably, under conditions of both acidity and ROS (pH 5.0 + 1 mM H2O2), IR-CDs@OPAC achieved the fastest and most complete drug release, with cumulative release rates of 86.6% and 84.2% for IR-CDs and CA, respectively, within the test time. This hydrogel's dual response to pH and ROS stimulation stems from the dynamic borate ester bonds and Schiff base bonds that constitute its structure. In the inflammatory wound microenvironment (typically characterized by low pH and elevated oxidant levels), this network structure undergoes accelerated relaxation and partial collapse, thereby enabling timely release of the therapeutic drug. As tissue repair progresses and the local microenvironment becomes more temperate, the matrix is ​​expected to maintain greater stability, thus transitioning to a slower, more sustained release pattern and prolonging the duration of action of bioactive molecules in the later stages of healing.

[0077] 7. Determination of the antioxidant and antibacterial properties of IR-CDs@OPAC 1) Antioxidant capacity of hydrogels DPPH free radical scavenging ability: Dissolve the DPPH reagent in ethanol to prepare a 200 μM DPPH solution. Add 200 μL of OPAC, IR-CDs@OPAC, and IR-CDs (1 mg / mL) to the DPPH solution, respectively, and incubate at room temperature in the dark for 1 h. Record the absorbance of each mixture at 517 nm using a UV-Vis spectrophotometer. The DPPH scavenging rate is calculated as follows: DPPH scavenging rate (%) = (A0 - A x ) / A0×100%, where the absorbance of the control group is recorded as A0, and the absorbance of the material group is recorded as Ax .

[0078] ABTS free radical scavenging ability: ABTS working solution was prepared by reacting 7 mM ABTS solution with 2.45 mM potassium persulfate. Before use, the ABTS working solution was diluted to a final concentration of 200 μM. 200 μL of the diluted ABTS working solution was mixed with equal volumes of OPAC, IR-CDs@OPAC, and IR-CDs (1 mg / mL). After incubation at room temperature in the dark for 1 h, the absorbance of the samples was recorded at 734 nm. ABTS scavenging rate (%) = (A0 - A x ) / A0 × 100%, where A0 is the absorbance of the control group, A x The absorbance of the material group.

[0079] H2O2 removal ability: The H2O2 scavenging ability of the prepared hydrogel was evaluated using Ti(SO4)2. A Ti(SO4)2 working solution was prepared by mixing a Ti(SO4)2 solution (10% w / v, sulfuric acid as solvent) with H2O2 (5 mM). OPAC, IR-CDs@OPAC, and IR-CDs were added to the Ti(SO4)2 working solution and reacted for 1 h. The absorbance of the samples was measured at 415 nm using a UV-Vis spectrophotometer.

[0080] Hydroxyl radical (·OH) scavenging ability: The hydroxyl radical scavenging performance of the hydrogels was investigated using the Fenton reaction system. OPAC, IR-CDs@OPAC, and IR-CDs were added to a mixture containing salicylic acid (2 mM, ethanol as solvent), ferrous sulfate (1 mM, water as solvent), and H2O2 (1 mM), and incubated at room temperature for 1 h. The absorbance of each sample was recorded at 510 nm using a UV-Vis spectrophotometer.

[0081] Excessive accumulation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) is a key factor leading to persistent inflammation and hindered skin regeneration, primarily due to an imbalance in the immune response around infected wounds. Wound dressings with excellent antioxidant properties can effectively scavenge ROS and RNS, alleviate inflammation, and accelerate tissue repair. We first evaluated the RNS scavenging ability of hydrogels using DPPH and ABTS experiments. Figure 8 D and Figure 8As shown in Figure E, the IR-CDs@OPAC group exhibited the highest DPPH radical scavenging activity, reaching 86.4%, significantly higher than both the IR-CDs and OPAC groups. Furthermore, the ABTS radical scavenging capacity of IR-CDs@OPAC was 87.8%. Subsequently, we further evaluated the ability of this hydrogel to scavenge ROS (especially H2O2 and ·OH). Figure 8 F and Figure 8 As shown in Figure G, the IR-CDs@OPAC group exhibited a significant H2O2 scavenging rate of 87.9%. Furthermore, IR-CDs@OPAC also demonstrated significant ·OH scavenging ability, with a scavenging efficiency of 84.2%. These results, including the scavenging abilities of DPPH, ABTS, H2O2, and ·OH, highlight the strong antioxidant capacity of IR-CDs@OPAC. This significant free radical scavenging performance is mainly attributed to the synergistic antioxidant function of CA and IR-CDs. Notably, in all antioxidant assessments, IR-CDs alone demonstrated a scavenging efficiency exceeding 80%, which is closely related to its retention of the inherent antioxidant properties derived from the traditional Chinese medicine Isatis indigotica. Moreover, the catechol groups in CA not only participate in the dynamic cross-linking within the hydrogel network but also significantly contribute to free radical neutralization, thereby further amplifying the overall antioxidant capacity of the system.

[0082] 2) In vitro antibacterial test Antibacterial activity was evaluated using the agar plate method. Before the experiment, IR-CDs, OPAC, and IR-CDs@OPAC were sterilized by ultraviolet light for 4 h. Each sterilized sample was then mixed with 1 mL of bacterial suspension (10... 8 The bacterial suspension was mixed with CFU / mL and incubated at 37°C for 6 h. The suspension was then diluted with PBS. 100 μL of the diluted suspension was evenly spread onto an agar plate and incubated in a shaker incubator for 12 h. Finally, colony-forming units were recorded by photographing and bacterial counts were performed to assess the antibacterial effect.

[0083] In chronic wound environments, impaired immune function often leads to repeated microbial invasions, while excessive wound exudate creates favorable conditions for bacterial proliferation. Therefore, excellent antibacterial properties are a core requirement for advanced wound dressings. To evaluate the antibacterial ability of the hydrogel, *Escherichia coli* and *Staphylococcus aureus* were selected as representatives of Gram-negative and Gram-positive bacteria, respectively. The standard plate count method was used to assess the antibacterial effect of the hydrogel. Figure 8As shown in the HJ, compared with the control group, the number of surviving bacterial colonies was significantly reduced in all experimental groups after co-incubation. In particular, IR-CDs@OPAC exhibited the most significant antibacterial activity, achieving an inhibition efficiency of 85.8% against *Escherichia coli* and complete eradication (100%) against *Staphylococcus aureus*. In contrast, IR-CDs alone showed antibacterial rates of 64% and 100% against *Escherichia coli* and *Staphylococcus aureus*, respectively, while OPAC showed a more moderate inhibitory effect, with corresponding values ​​of 38.1% and 60.5%, respectively. These results indicate that the integrated hydrogel system outperforms its individual components. The enhanced antibacterial activity of IR-CDs@OPAC is mainly attributed to the synergistic effect of CA and IR-CDs. Previous reports have indicated that polyphenols like CA can disrupt bacterial liposome membranes, leading to increased membrane permeability, leakage of intracellular components, and ultimately bacterial death. Current results demonstrate that CA's antibacterial function is well preserved after integration into the hydrogel network through dynamic cross-linking with OPU and 3-APBA. Furthermore, Zeta potential measurements showed that IR-CDs carried a positive surface charge of +17.3 mV ( Figure 7 This may be related to nitrogen doping in its carbon framework. Figure 1 H). This positive charge enhances the electrostatic attraction between IR-CDs and the negatively charged bacterial cell membrane, promoting close contact and amplifying the antibacterial effect.

[0084] 8. Biocompatibility of IR-CDs@OPAC The hydrogel used for wound dressings must have excellent biocompatibility. First, the blood compatibility of the hydrogel was evaluated through a hemolysis test.

[0085] First, fresh rabbit red blood cells were collected and washed three times with PBS to dilute the cells to a concentration of 5% (v / v). 500 μL of IR-CDs, OPAC, and IR-CDs@OPAC dispersions were added to 500 μL of the diluted red blood cell suspension, respectively. Deionized water and physiological saline were used as positive and negative controls, respectively. The mixture was incubated at 37°C for 2 h, followed by centrifugation at 1500 rpm for 10 min. The supernatant was collected, and the absorbance was measured at 540 nm using a microplate reader. The hemolysis rate was calculated using the formula: Hemolysis rate (%) = (Ax - An) / (Ap - An) × 100%, where Ax - An is the hemolysis rate. x α represents the absorbance of the sample group, An represents the negative control group of physiological saline, and Ap represents the positive control group of deionized water.

[0086] like Figure 10As shown in Figure A, red blood cells in the water ruptured significantly, while those in the saline solution showed almost no rupture after incubation. Red blood cells in the IR-CDs and hydrogel groups exhibited similar behavior to those in the saline group, and the supernatant was observed to be clear and transparent after incubation. Furthermore, as... Figure 10 As shown in Figure B, the hemolysis rates of the saline group, IR-CDs group, OPAC group, and IR-CDs@OPAC group were all significantly lower than those of the water group, confirming that IR-CDs@OPAC and its components have excellent blood compatibility. Fibroblasts, macrophages, and endothelial cells are key cellular components of skin tissue and play a crucial role in the regulation of wound repair, especially in infected wounds.

[0087] In vitro biocompatibility evaluation Cell compatibility of the hydrogels was assessed using the CCK-8 assay. L929, Raw264.7, and HUVEC cells were selected as model cells and seeded at a density of 5000 cells per well in 96-well plates for 24 h. Subsequently, the culture medium was replaced with DMEM containing IR-CDs, OPAC extract, and IR-CDs@OPAC extract, respectively. Cells were incubated at 37°C in a 5% CO2 incubator for 24 h. After incubation, the culture medium was removed, and cells were washed with PBS to remove residual material. Finally, DMEM containing 10% CCK-8 was added to each well, and the cells were incubated at 37°C for 1 h. The absorbance at 450 nm was recorded using a microplate reader. Cell viability was calculated using the formula: Cell viability (%) = (OD2 / OD2) / (Cell viability + ... x - OD c ) / (ODm- OD c ) × 100%, where OD x ODm represents the absorbance of the sample group, and ODm represents the absorbance of the culture medium group. c The absorbance is for the CCK-8 group.

[0088] To further assess cell compatibility, the effect of the hydrogel on the viability of L929, Raw264.7, and HUVEC cells was detected using the CCK-8 assay. Figure 10 As shown in the CE, after treatment with 1 mg / mL IR-CDs, the prepared OPAC, and IR-CDs@OPAC for 24 hours, the CCK-8 assay results showed that the survival rate of all cell types was higher than 90%, and there was no significant difference between the experimental group and the control group, proving that the hydrogel is non-toxic. Cell migration plays a crucial role in wound closure, and endothelial cells promote angiogenesis throughout the healing process by releasing various vasoactive mediators.

[0089] Cell scratch assay HUVEC cells were spaced at 1 × 10⁶ cells per well. 6Cells were seeded at a density of [number] cells per well in 6-well plates and cultured until confluence exceeded 90%. After removing the culture medium, a straight scratch was made on the bottom of the culture dish using a 200 μL pipette tip. The plates were washed three times with PBS. IR-CDs, OPAC extract, and IR-CDs@OPAC extract were added, respectively, with DMEM as a control group. The scratches were observed using an inverted microscope at predetermined time points (0, 12, 24, and 36 h). The scratch healing rate was calculated using the formula: Scratch healing rate (%) = (A0 - A0) / [(A0 - A0)] ... x ) / A0× 100%, where A0 and A x These represent the scratch area before and after the intervention, respectively.

[0090] To investigate the effect of hydrogels on HUVEC cell migration, a scratch assay was performed. Figure 10 F and Figure 10 As shown in Figure G, treatment with IR-CDs, OPAC, and IR-CDs@OPAC extract significantly promoted cell migration compared to the control group. After 36 hours of incubation, the scratch healing rate in the IR-CDs@OPAC group was significantly increased to 96.4%, while the healing rate in the control group was only 40.7%. This significant improvement in migration behavior may be related to the sustained release of the bioactive components IR-CDs and CA, which together promote endothelial cell activity and support the wound healing process.

[0091] Polysaccharide-based materials have been extensively explored for hemostasis. In this study, the coagulation ability of IR-CDs@OPAC was tested using a simplified inversion method. CaCO3, a well-known hemostatic agent, was used as a reference material, as it accelerates local clot formation. Figure 9 As shown, whole blood mixed with PBS failed to clot spontaneously and remained fluid even after 3 minutes under inverted conditions. In contrast, samples treated with CaCO3 or IR-CDs@OPAC rapidly formed stable blood clots that retained their shape after inversion. These results indicate that IR-CDs@OPAC exhibits in vitro coagulation capabilities comparable to CaCO3, effectively acting as a hemostatic barrier. The hemostatic behavior of this hydrogel is primarily attributed to pullulan polysaccharide chains and catechol functional groups derived from CA. These components work together to create strong adhesive interactions with blood components and surrounding tissues, facilitating the physical closure of bleeding points and promoting rapid clot formation. This combination endows the hydrogel with significant hemostatic potential in wound care applications.

[0092] 9. Therapeutic effect of IR-CDs@OPAC in a Staphylococcus aureus infection wound model The excellent biocompatibility and antioxidant capacity exhibited by IR-CDs@OPAC in in vitro experiments indicate that this hydrogel is a promising candidate material for chronic wound management. Preliminary in vivo safety was confirmed through topical administration. After five consecutive days of dorsal administration, mice showed no obvious signs of erythema or edema. These observations validate the good biocompatibility of this hydrogel in vivo and support its suitability as a safe wound dressing.

[0093] Construction of a mouse model of full-thickness skin defect infection In addition, this application established a mouse model of infected full-thickness skin wound induced by Staphylococcus aureus to explore its therapeutic effect. All procedures and experiments involving animals were performed in accordance with the National Research Council's Guidelines for the Care and Use of Laboratory Animals. A full-thickness skin defect infection model was established using male C57BL / 6 mice to evaluate the in vivo therapeutic effect of the hydrogel. Mice were anesthetized by intraperitoneal injection, and the hair on their backs was removed using a power shaver. After anesthesia, a circular wound with a diameter of approximately 10 mm was made on the back of each mouse. 20 μL of Staphylococcus aureus suspension (10 μL / mL) was injected into each wound. 8 CFU / mL), incubated for 24 h to establish an infected wound model. Experimental groups were treated with OPAC and IR-CDs@OPAC, respectively, while the control group received no treatment. Wound photographs were taken on days 0, 3, 6, 9, and 13, and dressings were changed accordingly. The percentage of wound area was calculated using the following formula: Relative wound area (%) = A x / A0 × 100%, where A0 and A x These represent the wound area on days 0, 3, 6, 9, and 13, respectively.

[0094] The process of treating internal wounds, such as Figure 11 As shown in Figure A, mice were randomly divided into three groups. Untreated wounds served as the control group, while the other two groups were treated with OPAC and IR-CDs@OPAC, respectively. Wound site photographs were taken on days 0, 3, 6, 9, and 13 to record the wound healing process. In vivo antimicrobial efficacy was assessed by bacterial colony counting on day 3, and histological examination was performed on days 6 and 13 to evaluate tissue regeneration quality. Figure 11 As shown in B and C, compared with the control group, the wound closure speed was significantly faster in both hydrogel treatment groups. The group using dressings showed a higher wound area shrinkage rate than the control group, which may be related to the hydrogel's ability to maintain a moist microenvironment, thereby promoting the tissue repair process. Figure 11As shown in Figure D, the relative wound area in the IR-CDs@OPAC group decreased to 23.7% after 6 days, while the residual areas in the OPAC group and the control group were 32.6% and 49.7%, respectively. By day 13, the hydrogel-treated wounds had almost completely healed. Notably, throughout the healing period, the residual wound area in the IR-CDs@OPAC treatment group was consistently smaller than that in the OPAC-only treatment group, indicating that the synergistic effect of IR-CDs and CA surpasses the efficacy of single components in promoting the repair of infected wounds. The in vivo antibacterial properties of the hydrogel were evaluated by counting residual bacteria on the wound tissue using a smear method. Figure 11 As shown in E and F, a large number of bacterial colonies were detected in the control group, while only sparse colonies were observed in the samples from the OPAC group and the IR-CDs@OPAC group. The results confirm that this hydrogel can effectively eliminate bacteria in vivo. Furthermore, the excellent antibacterial properties of IR-CDs@OPAC are due to the combined effect of IR-CDs and CA, highlighting the advantages of their synergistic integration within the hydrogel matrix.

[0095] 10. Histological evaluation To gain a more detailed understanding of tissue regeneration, mice were sacrificed on days 6 and 13, and skin tissue was collected for subsequent histological and immunohistochemical analysis. Skin tissue surrounding the wound was excised and immediately fixed in 4% paraformaldehyde for 24 hours. After fixation, the specimens were dehydrated, embedded in paraffin, sectioned, and the wound site was stained with hematoxylin-eosin (HE) and Masson's stain. On day 6, the control group still showed significant inflammatory cell infiltration (…). Figure 12 A) indicates a prolonged inflammatory phase. In contrast, the OPAC and IR-CDs@OPAC groups showed a significant reduction in inflammatory cells, indicating accelerated inflammation resolution. At this stage, incomplete epidermal coverage was observed in all groups, reflecting ongoing re-epithelialization. By day 13, differences in tissue regeneration became apparent. Newly formed skin appendages were observed in the OPAC and IR-CDs@OPAC groups, while only limited appendage regeneration was observed in the control wound. Notably, the IR-CDs@OPAC group exhibited richer and better-structured appendages, indicating superior skin repair. Furthermore, the epidermal thickness of the wounds in the three groups was quantified ( Figure 12 C). The epidermal thickness in the control group was 86 μm, while the epidermal layers in the OPAC group (56 μm) and the IR-CDs@OPAC group (48 μm) were significantly thinner and more uniform. Thinner epidermis is generally associated with a more mature skin barrier with better functional recovery, while the increased epidermal thickness in the control group indicates a delayed and incomplete remodeling process. Masson staining was used to further examine collagen deposition and tissue arrangement. Figure 12B). By day 13, collagen accumulation had increased in all groups compared to the earlier stages. However, the degree and quality of collagen remodeling varied significantly. The IR-CDs@OPAC group showed the largest area of ​​collagen deposition ( Figure 12 D) The OPAC group exhibited a more uniform and well-organized fibrous structure, followed by the control group. In contrast, the control group showed sparse collagen fibers, reflecting poor extracellular matrix remodeling. In summary, these histological results indicate that both OPAC and IR-CDs@OPAC significantly promote wound healing, with IR-CDs@OPAC showing the most significant therapeutic effect. This enhanced performance is primarily attributed to the presence of IR-CDs, which effectively inhibits bacterial load at the wound site, thereby reducing inflammation. Furthermore, the addition of the bioactive small molecule CA endows the hydrogel with strong antioxidant capabilities, reducing oxidative stress in the wound microenvironment and further promoting tissue regeneration.

[0096] 11. Expression of CD31 and CD206 at the wound site Bacterial contamination at the wound site often leads to excessive oxidative stress and an exacerbated inflammatory cascade, both of which hinder the transition of the wound to the repair phase. Effective resolution of inflammation is closely related to macrophage polarization, particularly the shift from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype, a process that plays a crucial role in tissue regeneration. In this study, CD206 was used as a representative marker of M2 macrophages, and immunohistochemical analysis was employed to assess inflammatory regulation. Figure 13 A and Figure 13 As shown in Figure C, compared with the control group, CD206 expression was significantly increased in the OPAC group on day 6, indicating an improved inflammatory response. Notably, the IR-CDs@OPAC group exhibited even higher levels of CD206 expression, demonstrating the strongest anti-inflammatory activity and the most advanced early tissue repair among all groups. On day 13, CD206 expression in the OPAC group continued to increase relative to the control group, reflecting the sustained resolution of inflammation and the progress of tissue remodeling. The IR-CDs@OPAC group consistently showed the highest CD206 signal intensity, confirming its superior ability to inhibit inflammation and promote regeneration. This enhanced immunomodulatory effect can be attributed to multiple factors. First, IR-CDs possess potent antibacterial activity, reducing pathogen-induced inflammatory stimulation at the wound site. Second, the catechol groups in CA have significant antioxidant properties, enabling the hydrogel to effectively scavenge excess ROS, thereby further alleviating the inflammatory response.

[0097] Angiogenesis is another key factor for successful wound healing, as newly formed blood vessels deliver essential oxygen and nutrients for collagen synthesis and epidermal remodeling. To assess angiogenesis in wound tissue, immunohistochemical staining analysis was performed using CD31 as a specific endothelial cell marker. Figure 13 B and Figure 13 As shown in Figure D, CD31 expression levels in the OPAC group and the IR-CDs@OPAC group were significantly higher than those in the control group. Consistent with previous studies, the presence of CA in the hydrogel matrix contributes to angiogenesis. Notably, the IR-CDs@OPAC group exhibited the most significant CD31 expression, reflecting the highest degree of angiogenesis. This superior pro-angiogenic response is likely the result of a synergistic effect of multiple factors, including the bioactivity of CA, the potent antibacterial efficacy of IR-CDs, and the favorable physicochemical properties of the hydrogel, such as mechanical integrity, high hydration capacity, and effective absorption of wound exudate. These properties collectively create a supportive microenvironment conducive to endothelial cell migration, vascular maturation, and overall wound tissue regeneration.

[0098] It should be noted that this application is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments with the same structure and effect as the technical concept within the scope of this application are included in the technical scope of this application. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of this application, are also included in the scope of this application.

Claims

1. A dual-responsive OPu hydrogel cross-linked by CA and loaded with carbon dots of radix isatidis, characterized in that, The hydrogel is composed of OPu, 3-APBA and CA, and the IR-CDs encapsulated in the hydrogel; the concentration of OPu is 6-8% (w / v), and the molar ratio of 3-APBA to CA is 1:1-3.

2. The dual-responsive OPu hydrogel crosslinked by CA and loaded with carbon dots of radix isatidis according to claim 1, characterized in that, The feeding amount of 3-APBA is 3-10 mg / ml.

3. The dual-responsive OPu hydrogel of claim 1, wherein, The feeding amount of CA is 8-78 mg / ml.

4. The dual-responsive OPu hydrogel of claim 1, wherein, The feeding amount of IR-CDs is 0.5-1.2 mg / mL.

5. A method for preparing a CA cross-linked and Radix Isatidis carbon dots loaded dual-responsive OPu hydrogel, comprising: The formula of the OPu, 3-APBA, CA and IR-CDs is mixed to obtain the hydrogel encapsulating IR-CDs and the double-responsive OPu hydrogel.

6. The preparation method of the CA cross-linked and loaded with radix isatidis carbon dots dual-responsive OPu hydrogel according to claim 5, characterized in that, The steps of the preparation method include: 1) A proper amount of 3-APBA solution is added to the OPu aqueous solution, and stirred for 3-5 h to obtain a mixed solution; 2) IR-CDs are added to the mixed solution of 1); 3) A CA aqueous solution is added to 2), and mixed for 5-30 min to obtain IR-CDs@OPAC.

7. The preparation method of the dual-responsive OPu hydrogel cross-linked by CA and loaded with carbon dots of radix isatidis according to claim 5 or 6, characterized in that, The concentration of OPu is 6-8% (w / v), the molar ratio of 3-APBA to CA is 1:1-3, the feeding amount of 3-APBA is 3-10 mg / ml, the feeding amount of CA is 8-78 mg / ml, and the feeding amount of IR-CDs is 0.5-1.2 mg / mL.

8. The preparation method of the dual-responsive OPu hydrogel cross-linked by CA and loaded with carbon dots of radix isatidis according to claim 5 or 6, characterized in that, The preparation of IR-CDs: the root of isatis powder is dissolved in ultrapure water, and the mixed solution is transferred to a reaction kettle and reacted at 160-200℃ for 8-14 h, and then naturally cooled to room temperature; centrifugal treatment is performed, and the supernatant is filtered with a 0.1-0.45 μm filter membrane to remove large particles; the filtrate is dialyzed in a 200-1000 Da cellulose dialysis bag for 24-72 h; finally, the dialyzed solution is freeze-dried, and the obtained solid product (IR-CDs) is stored at room temperature.

9. The preparation method of the dual-responsive OPu hydrogel cross-linked by CA and loaded with carbon dots of radix isatidis according to claim 5 or 6, characterized in that, The synthesis of OPu: sodium periodate and pullulan are dissolved in distilled water at a mass ratio of 0.2-1.2:1, and stirred at room temperature for 6-10 h to prepare OPu; then 2.5-6 mL of ethylene glycol is added to the mixed solution, and the reaction is continued to be stirred for 1.5-3 h to terminate the reaction; the obtained product is dialyzed in a 2800-4500 KDa dialysis bag for 48-96 h, and freeze-dried to obtain OPu.

10. The application of the double-responsive OPu hydrogel cross-linked by CA and loaded with IR-CDs, including the hydrogel of any one of claims 1-4 or the hydrogel obtained by the preparation method of any one of claims 5-9 for wound dressing.

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