Injectable responsive hydrogel loaded with chitosan oligosaccharide-octyl gallate and its preparation method and application

Abstract

The application provides a kind of injectable responsive hydrogel loaded with chitosan oligosaccharide-gallic acid octyl ester and its preparation method and application, and the preparation method comprises: chitosan oligosaccharide-gallic acid octyl ester conjugate is added into carboxymethyl chitosan solution to obtain a mixed solution;The mixed solution and the oxidized tannic acid solution are mixed to form an injectable responsive hydrogel.The three-dimensional hydrogel network formed by carboxymethyl chitosan and oxidized tannic acid is constructed, chitosan oligosaccharide-gallic acid octyl ester is stably loaded in the gel system, can form a physical barrier in the local wound and provide sustained support, thereby significantly improving the retention capacity of antibacterial components in the wound site, prolonging its effective action time, reducing the need for repeated administration.Effectively solve the problem that antibacterial functional materials are difficult to stay in humid infected wounds for a long time and are easy to lose with exudate, can be applied and prepared into antibacterial drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and relates to a photoresponsive hydrogel material for antibacterial and infectious wound treatment and its construction method, particularly to an injectable responsive hydrogel loaded with chitosan oligosaccharide-octyl gallate and its preparation method and application. Background Technology

[0002] As the largest organ in the human body, the skin is a crucial physical barrier against external pathogens. When the skin is damaged due to trauma, burns, surgical incisions, or chronic diseases, pathogens can easily invade the wound and cause infection, severely hindering normal wound healing. Among various pathogens, methicillin-resistant Staphylococcus aureus (MRSA) has become one of the most common pathogens in infected wounds due to its strong drug resistance and ease of colonization. MRSA infection further exacerbates the inflammatory response of the wound, inhibits tissue repair, and significantly prolongs the healing period, severely impacting the patient's quality of life and recovery.

[0003] Traditional dry dressings rely solely on physical coverage for wound protection, failing to effectively prevent bacterial invasion. Furthermore, frequent dressing changes can cause secondary damage to newly formed wound tissue, making them unsuitable for the clinical treatment of infected wounds. Currently, clinical treatment for MRSA infections still heavily relies on antibiotics. However, with the widespread spread of drug-resistant strains, the efficacy of traditional antibiotics is significantly limited. Simultaneously, long-term or inappropriate use of antibiotics can further induce drug resistance, posing significant risks to effectiveness and safety, making it difficult to achieve long-term stable control of infected wounds. Derivatives based on chitosan oligosaccharide modification (such as chitosan oligosaccharide-octyl gallate conjugate, COS-OG) have shown promising application prospects in the field of anti-MRSA infection due to their excellent water solubility, amphiphilicity, and photodynamic antibacterial activity. However, when these functionalized nanomaterials are directly applied to open moist wounds, serious practical application problems still exist: powdered nanomedicines are difficult to remain on the wound surface for a long time and are easily lost with wound exudate, resulting in a short duration of effective local drug concentration; frequent administration not only increases the workload of medical staff, but may also interfere with the repair process of newly formed tissues and affect the wound healing effect.

[0004] Hydrogel dressings, with their unique three-dimensional network structure, can provide a suitable moist healing environment for wounds and solve the problems of drug retention and sustained release, thus attracting widespread attention in the treatment of infected wounds. However, existing commercially available and reported hydrogel dressings still have many significant shortcomings: First, poor tissue adaptability; prefabricated hydrogels are difficult to tightly adhere to irregular wounds with complex shapes or deep infections, easily forming gaps that can lead to bacterial growth; traditional in-situ gelation systems often rely on toxic chemical cross-linking agents such as glutaraldehyde, or have problems with excessively slow gelation rates, failing to meet the clinical need for rapid wound closure and immediate treatment. Second, lack of intelligent response capabilities; traditional hydrogels mostly physically encapsulate drugs, with drug release often being burst-release or only diffusion-controlled, unable to achieve on-demand release based on pH fluctuations in the wound infection microenvironment, external light stimulation, etc., lacking the ability to actively kill deep-seated drug-resistant bacteria, making it difficult to achieve precise treatment effects. Third, the therapeutic function is singular. Most current strategies for repairing infected wounds focus only on the single "bactericidal" effect, neglecting the adverse effects of the imbalance of the immune microenvironment of damaged tissue on the healing process. Simple antibacterial treatment cannot effectively reverse the persistent inflammatory state of chronic wounds. How to construct an integrated dressing that combines efficient COS-OG transport, photodynamic synergistic antibacterial action, immune microenvironment regulation, and tissue regeneration promotion is a technical problem that urgently needs to be solved in the field of biomedical materials.

[0005] In summary, existing treatments for MRSA infections suffer from antibiotic dependence, limited efficacy, and a high risk of drug resistance. Novel antibacterial materials exhibit poor wound retention and require frequent administration. Existing hydrogel dressings, on the other hand, suffer from poor tissue compatibility, insufficient biosafety, lack of intelligent responsiveness, and limited therapeutic function, failing to simultaneously achieve synergistic treatment of highly effective anti-drug-resistant bacterial infections, immune modulation, and tissue repair. Therefore, developing a novel hydrogel dressing with good biocompatibility, rapid in-situ gelation, stimulus-responsive intelligent drug release properties, and the ability to effectively inhibit MRSA infection, regulate the wound immune microenvironment, and promote tissue repair and regeneration is of great significance for improving the clinical treatment of infected wounds and meeting the clinical needs for safe, efficient, and precise treatment. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide an injectable responsive hydrogel supported on chitosan oligosaccharide-octyl gallate, its preparation method and application.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution adopted in this patent application: A method for preparing an injectable responsive hydrogel supported on chitosan oligosaccharide-octyl gallate, the method comprising the following steps: S1. Add the chitosan oligosaccharide-octyl gallate coupling compound to the carboxymethyl chitosan solution to obtain a mixed solution; S2. The mixed solution and the oxidized tannic acid solution are mixed to form an injectable responsive hydrogel.

[0008] The above preparation method, further, wherein the oxidized tannic acid solution in S2 is prepared by the following method: Tannic acid is dissolved in ultrapure water to obtain a tannic acid solution. The pH of the tannic acid solution is adjusted to 8.0-9.5 (preferably 8.5) with sodium hydroxide. The solution is stirred at 200-600 rpm (preferably 300 rpm) for 1-3 hours. The pH is then adjusted back to neutral to obtain oxidized tannic acid.

[0009] In the above preparation method, the mass fraction of tannic acid in the tannic acid solution is 1% to 10% (preferably 4%).

[0010] In the above preparation method, further, in step S1, the mass fraction of carboxymethyl chitosan in the carboxymethyl chitosan solution is 2% to 8% (preferably 5%).

[0011] In the above preparation method, further, in step S1, the concentration of the chitosan oligosaccharide-octyl gallate conjugate is 1 mg / mL to 4 mg / mL.

[0012] In the above preparation method, the final mass fraction of the oxidized tannic acid solution in the hydrogel in step S2 is 0.2% to 2.0% (preferably 0.8%).

[0013] Based on a general technical concept, the present invention also provides an injectable responsive hydrogel of chitosan oligosaccharide-octyl gallate prepared by the preparation method described above.

[0014] Based on a general technical concept, the present invention also provides the application of the aforementioned injectable responsive hydrogel loaded with chitosan oligosaccharide-octyl gallate in the preparation of antibacterial drugs.

[0015] In the above-described application, the antibacterial drug is further described as an anti-MRSA drug.

[0016] Compared with the prior art, the advantages of the present invention are as follows: (1) This invention provides an injectable responsive hydrogel loaded with chitosan oligosaccharide-octyl gallate. By constructing a three-dimensional hydrogel network formed by carboxymethyl chitosan and oxidized tannic acid, chitosan oligosaccharide-octyl gallate is stably loaded into the gel system, which can form a physical barrier and provide continuous support at the wound site, thereby significantly improving the retention capacity of antibacterial components at the wound site, prolonging their effective action time, and reducing the need for repeated administration. It effectively solves the problem that antibacterial functional materials are difficult to retain for a long time in moist infected wounds and are easily lost with exudate.

[0017] (2) This invention provides an injectable responsive hydrogel loaded with chitosan oligosaccharide-octyl gallate, constructing a ready-to-use in-situ gelling hydrogel system that requires no special storage conditions, overcoming the dependence of traditional hydrogels on strict storage conditions such as low temperature and light protection. Experiments show that injecting 5 wt% of carboxymethyl chitosan precursor solution and 4 wt% of oxidized tannic acid precursor solution together using a double-barrel syringe at a volume ratio of 4:1 can achieve rapid gelation within seconds. Furthermore, after loading chitosan oligosaccharide-octyl gallate, the system still maintains the above-mentioned ultrafast gelling kinetics. This on-demand mixing and in-situ gelling preparation method fundamentally solves the problem of poor long-term storage stability of traditional hydrogels, significantly improving the flexibility and operability of practical applications.

[0018] (3) This invention provides an injectable responsive hydrogel supported on chitosan oligosaccharide-octyl gallate, which significantly reduces the amount of tannic acid used while ensuring the gelling performance and stability of the hydrogel. In this invention, the amount of tannic acid used is only about 20% of the mass of carboxymethyl chitosan. Compared with the existing technical solutions that rely on high tannic acid content to construct hydrogel networks, this invention reduces the amount of tannic acid used to about 1.5 to 15.6 times that of the existing system by auto-oxidizing tannic acid under alkaline conditions. While maintaining the stability and functionality of the gel structure, it effectively reduces the risk of cell stimulation that may be caused by high doses of tannic acid, and significantly reduces the cost of raw materials, thereby improving the biosafety and engineering application feasibility of the system.

[0019] (4) This invention provides a method for preparing an injectable responsive hydrogel loaded with chitosan oligosaccharide-octyl gallate. A dynamic covalent network structure is constructed through the synergistic effect of Schiff base bonds and multiple hydrogen bonds formed between oxidized tannic acid and carboxymethyl chitosan, enabling effective antibacterial function and release regulation. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy results show that the quinone / aldehyde structure in oxidized tannic acid undergoes a Schiff base reaction with the amino groups on the carboxymethyl chitosan molecular chain, while multiple hydrogen bonds exist in the system, thus endowing the hydrogel with good structural stability and dynamic reversibility. In the micro-acidic environment unique to bacterial infected wounds, the Schiff base bonds can undergo reversible breakage, thereby triggering local collapse of the network structure and promoting the release of the loaded drug. This adaptive response to the infection microenvironment (pH changes) allows the hydrogel to accelerate the release of functional components during the bacterial colonization stage, improving local antibacterial efficiency.

[0020] (5) This invention provides a method for preparing an injectable responsive hydrogel supported on chitosan oligosaccharide-octyl gallate. The hydrogel system uses safe raw materials, has mild preparation conditions, does not rely on toxic chemical crosslinking agents, and does not rely on complex photoinitiation steps such as ultraviolet light, thus exhibiting good biocompatibility and application feasibility. This system is suitable for use in ready-to-use or in-situ administration, is easy to operate, has high repeatability and stability, and lowers the barriers to production and use, making it suitable for further scale-up preparation and clinical application.

[0021] (6) This invention provides the application of an injectable responsive hydrogel loaded with chitosan oligosaccharide-octyl gallate in the preparation of drugs for treating MRSA infection. The injectable responsive hydrogel loaded with chitosan oligosaccharide-octyl gallate not only has antibacterial activity but also can orderly improve the local inflammatory state of infected wounds, providing a favorable microenvironment for tissue repair. Under sustained-release conditions, the hydrogel loaded with chitosan oligosaccharide-octyl gallate can reduce the number of bacteria by approximately 3.33 log CFU / mL within 24 hours under dark conditions. Under blue light stimulation, the antibacterial effect is further enhanced, with the number of bacteria reduced by up to 5.96 log CFU / mL within 24 hours, achieving an antibacterial efficiency of over 99.99%, significantly superior to the control hydrogel system without the functional component. In vitro cell experiments show that even at a hydrogel concentration as high as 20 mg / mL, the survival rate of fibroblasts and macrophages remains above 80%, demonstrating good cell safety. Meanwhile, this hydrogel can promote the polarization of macrophages towards the repair phenotype (M2), reduce the expression of pro-inflammatory factors and enhance the secretion of anti-inflammatory factors, which is beneficial to improve the inflammatory microenvironment of infected wounds and promote the transformation of inflammation to the repair stage, thereby creating conditions for subsequent tissue regeneration and wound closure.

[0022] (7) This invention provides the application of an injectable responsive hydrogel loaded with chitosan oligosaccharide-octyl gallate in the preparation of drugs against MRSA infection. It exhibits excellent in vivo antibacterial, anti-inflammatory, and wound-healing properties in an animal model of MRSA infection wounds. In a mouse MRSA infection wound model, the hydrogel treatment group loaded with chitosan oligosaccharide-octyl gallate and combined with blue light irradiation achieved a bacterial clearance efficiency of 99.99% at the wound site on day 3. By day 13, the wound healing rate of this group reached 92.63%, significantly higher than the negative control group (55.49%) and the commercial 3M dressing group (67.18%). During the treatment, the mice continued to gain weight, and no obvious pathological damage was observed in major organs, indicating that this hydrogel system has good in vivo safety.

[0023] In summary, this invention, through the rational design of an injectable, responsive hydrogel carrier, achieves highly effective anti-MRSA infection while taking into account biosafety, cost control, and engineering feasibility, providing a novel hydrogel dressing solution with promising application prospects for the treatment of infected wounds. Attached Figure Description

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0025] Figure 1 The appearance of injectable responsive hydrogels loaded with chitosan oligosaccharide-octyl gallate at different concentrations and their effect on gel formation time.

[0026] Figure 2 Microstructures of injectable responsive hydrogels loaded with chitosan oligosaccharide-octyl gallate at different concentrations.

[0027] Figure 3 The swelling rate, water retention rate, cumulative TA release, and adhesion of the injectable responsive hydrogels loaded with chitosan oligosaccharide-octyl gallate at different concentrations to skin tissue were tested.

[0028] Figure 4 The results of the rheological properties investigation of injectable responsive hydrogels loaded with chitosan oligosaccharide-octyl gallate at different concentrations.

[0029] Figure 5 Spectra of injectable responsive hydrogels loaded with chitosan oligosaccharide-octyl gallate at different concentrations.

[0030] Figure 6 Image showing the anti-MRSA effect of an injectable responsive hydrogel loaded with chitosan oligosaccharide-octyl gallate (colony images and colony counts).

[0031] Figure 7 Effect of injectable responsive hydrogels loaded with chitosan oligosaccharide-octyl gallate on the survival of L929 and RAW264.7 cells.

[0032] Figure 8 The in vitro anti-inflammatory effect of an injectable responsive hydrogel loaded with chitosan oligosaccharide-octyl gallate is shown in the figure.

[0033] Figure 9 The in vivo antibacterial effect of an injectable responsive hydrogel loaded with chitosan oligosaccharide-octyl gallate is shown in the figure.

[0034] Figure 10 Image showing the effect of an injectable responsive hydrogel loaded with chitosan oligosaccharide-octyl gallate on promoting wound healing of MRSA-infected dogs in vivo.

[0035] Figure 11 To investigate the in vivo anti-inflammatory effects of an injectable responsive hydrogel loaded with chitosan oligosaccharide-octyl gallate.

[0036] Figure 12 The results of the in vivo biosafety assessment of the injectable responsive hydrogel loaded with chitosan oligosaccharide-octyl gallate. Detailed Implementation

[0037] The present invention will be further described below with reference to specific preferred embodiments, but this does not limit the scope of protection of the present invention.

[0038] The materials, reagents, and instruments used in the following examples are all commercially available. Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art.

[0039] Example 1 A method for preparing an injectable responsive hydrogel supported on chitosan oligosaccharide-octyl gallate, comprising the following steps: (1) Dissolve carboxymethyl chitosan (CMCS, 5% by mass) in ultrapure water to obtain a CMCS solution. Dissolve tannic acid (TA, 4% by mass) in ultrapure water to obtain a TA solution. Adjust the pH of the TA solution to 8.5 with sodium hydroxide, stir at 300 rpm for 2 hours (1 to 3 hours is also acceptable), and then adjust the pH back to neutral to obtain oxidized tannic acid (OTA).

[0040] (2) OTA (final mass fraction in hydrogel is 0.8%) is added to CMCS solution (mass fraction 4%) to form hydrogel, denoted as COT.

[0041] Example 2 A method for preparing an injectable responsive hydrogel supported on chitosan oligosaccharide-octyl gallate, comprising the following steps: (1) Dissolve carboxymethyl chitosan (CMCS, 5% by mass) in ultrapure water to obtain a CMCS solution. Add 1 mg / mL of COS-OG to the CMCS solution to form solution one. Dissolve tannic acid (TA, 4% by mass) in ultrapure water to obtain a TA solution. Adjust the pH of the TA solution to 8.5 with sodium hydroxide and stir at 300 rpm for 2 hours (1 to 3 hours is also acceptable). Then adjust the pH back to neutral to obtain oxidized tannic acid (OTA).

[0042] (2) Add OTA (final mass fraction in hydrogel is 0.8%) to mixed solution one (mass fraction 4%) to form hydrogel, denoted as COT1.

[0043] Example 3 A method for preparing an injectable responsive hydrogel supported on chitosan oligosaccharide-octyl gallate, comprising the following steps: (1) Dissolve carboxymethyl chitosan (CMCS, 5% by mass) in ultrapure water to obtain a CMCS solution. Add 2 mg / mL of COS-OG to the CMCS solution to form a mixture. Dissolve tannic acid (TA, 4% by mass) in ultrapure water to obtain a TA solution. Adjust the pH of the TA solution to 8.5 with sodium hydroxide and stir at 300 rpm for 2 hours (1 to 3 hours is also acceptable). Then adjust the pH back to neutral to obtain oxidized tannic acid (OTA).

[0044] (2) Add OTA (final mass fraction of 0.8% in hydrogel) to mixed solution 2 (mass fraction of 4%) to form hydrogel, denoted as COT2.

[0045] Example 4 A method for preparing an injectable responsive hydrogel supported on chitosan oligosaccharide-octyl gallate, comprising the following steps: (1) Dissolve carboxymethyl chitosan (CMCS, 5% by mass) in ultrapure water to obtain a CMCS solution. Add 4 mg / mL COS-OG to the CMCS solution to form a mixture. Dissolve tannic acid (TA, 4% by mass) in ultrapure water to obtain a TA solution. Adjust the pH of the TA solution to 8.5 with sodium hydroxide and stir at 300 rpm for 2 hours (1 to 3 hours is also acceptable). Then adjust the pH back to neutral to obtain oxidized tannic acid (OTA).

[0046] (2) Add OTA (final mass fraction of 0.8% in hydrogel) to mixed solution three (mass fraction of 4%) to form hydrogel, denoted as COT4.

[0047] Experiment 1: To investigate the appearance of injectable responsive hydrogels loaded with chitosan oligosaccharide-octyl gallate at different concentrations and their effect on gel formation time.

[0048] The gelation time of the hydrogel was determined using the inverted vial method. After freeze-drying, the cross-section of the hydrogel was fractured with liquid nitrogen and its pore structure was observed using a scanning electron microscope. The elemental composition of the hydrogel surface was characterized using an energy dispersive spectroscopy (EDS) system.

[0049] Figure 1 The appearance of CMCS-OTA hydrogels incorporating different concentrations of chitosan oligosaccharide-octyl gallate and their effect on gel formation time were shown in the figure. As can be seen from the figure: the gel time of CMCS is close to 0 seconds, indicating almost instantaneous gelation; the gel times of COT, COT1, and COT2 are all within 10 seconds, belonging to the rapid gelation system; the gel time of COT4 is significantly extended to approximately 120 seconds, much longer than the other samples, indicating a significantly slower gelation rate.

[0050] Figure 2 The microstructures of CMCS-OTA hydrogels incorporating different concentrations of chitosan oligosaccharide-octyl gallate are shown in the figures. As can be seen from the figures, COT exhibits a relatively dense pore structure with thicker walls, smaller pore sizes, a compact network skeleton, and low porosity. COT1 shows a looser pore structure with slightly larger pore sizes, thinner pore walls, and a finer network skeleton. COT2 exhibits a further looser pore structure with even larger pore sizes and thinner walls, presenting a more open porous network. COT4 has the most loose pore structure, with the largest pore sizes, thinnest pore walls, significantly increased porosity, and a highly fine network skeleton. Elemental distribution and composition analysis results show that green (C), purple (N), and yellow (O) are uniformly distributed in all four samples, with no obvious elemental aggregation or deficiency, indicating uniform chemical composition and no localized phase separation during the modification / crosslinking process.

[0051] Experiment 2: Swelling rate, water retention rate, cumulative TA release, and adhesion of the injectable responsive hydrogel loaded with chitosan oligosaccharide-octyl gallate to skin tissue.

[0052] Swelling rate test: 5 mL of hydrogel was immersed in 20 mL of PBS buffer (pH = 7.4) and incubated at 37°C to evaluate its swelling performance. At predetermined time intervals, the sample was carefully removed, the surface was gently blotted dry with moistened filter paper, and then weighed to record the mass change. The swelling rate of the hydrogel was calculated using the following formula: (1) Where Wt is the mass of the hydrogel at a specific time, and Wd is the initial mass.

[0053] Cumulative Tannic Acid Release Test: The hydrogel was immersed in 20 mL of PBS buffer at different pH values ​​(pH = 6 or pH = 7, 37℃) to investigate its pH-responsive release characteristics. At predetermined time intervals, 3 mL of supernatant was collected and immediately replenished with an equal volume of fresh PBS to maintain constant system volume and conditions. The absorbance of the collected supernatant was measured at 276 nm using a UV spectrophotometer. The cumulative release of tannic acid (TA) was calculated using the following formula: (2) Where Ve represents the replacement volume of PBS, V0 represents the total system volume, Ci represents the concentration of the solution released during the i-th replacement sampling, m0 represents the total mass of TA in the hydrogel, and n represents the cumulative count of PBS replacement events.

[0054] Figure 3 Swelling rate (A), water retention rate (B), cumulative TA release (C), and adhesion of the hydrogel to skin tissue were measured for injectable responsive hydrogels loaded with chitosan oligosaccharide-octyl gallate (D).

[0055] As shown in Figure A, the swelling rates of both COT and COT2 gradually increased over time, eventually stabilizing. The swelling rate of COT2 was significantly higher than that of COT, indicating that its gel network is more hydrophilic and has a looser pore structure (consistent with SEM results), enabling it to absorb more water and exhibit superior swelling capacity.

[0056] As shown in Figure B, the water retention rates of both COT and COT2 decreased slowly over time, indicating a continuous water loss process. COT2 consistently maintained a higher water retention rate than COT, suggesting that its network structure has a stronger ability to bind water molecules and better hydration stability. This may stem from more hydrophilic functional groups (such as carboxyl and hydroxyl groups) or a more uniform cross-linked network, reducing water loss.

[0057] As shown in Figure C, the cumulative TA release of all samples at pH 7 was significantly higher than that at pH 6, indicating that the materials exhibit a clear pH-responsive release characteristic. The increased swelling of the gel network or the weakened interaction between TA and the material (such as hydrogen bonding and electrostatic interactions) under weakly alkaline conditions accelerated drug diffusion. COT, COT1, and COT2 showed faster release rates and higher cumulative release amounts at pH 7, with COT2 exhibiting the steepest release curve.

[0058] As can be seen from D in the figure, COT2 can be bent at large angles such as 180°, 90°, -90°, and -180° without breaking or damaging, demonstrating excellent flexibility and deformability.

[0059] Experiment 3: Rheological property testing of injectable responsive hydrogels supported on chitosan oligosaccharide-octyl gallate.

[0060] Amplitude scanning (strain range 0.1%–1000%, frequency 1 Hz, temperature 25°C) was used to determine the linear viscoelastic region; frequency scanning (frequency range 0.1–100 Hz, strain 1%, temperature 25°C) was used to monitor the dynamic modulus (storage modulus G' and loss modulus G”); temperature scanning (temperature range 25–55°C, heating rate 5°C / min, strain 1%, frequency 1 Hz) was used to evaluate the thermal stability of the hydrogel; strain cycling test (strain switching between 1% and 600%, 60-second intervals, 5 cycles, temperature 25°C, frequency 1 Hz) was used to evaluate the self-healing properties of the hydrogel; shear rate scanning (strain 1%, frequency 1 Hz) was used to determine the shear thinning characteristics. The in-situ molding capability of the hydrogel precursor solution was demonstrated using a dual-barrel syringe.

[0061] Figure 4 The results show the rheological properties of the injectable responsive hydrogel supported on chitosan oligosaccharide-octyl gallate (material COT2 in the figure). Figure A is the strain scan, Figure B is the frequency scan, Figure C is the shear thinning test, Figure D is the temperature scan, Figure E is the cyclic strain scan, and Figure F is a schematic diagram of the injectable hydrogel. As shown in Figure A, the storage modulus (G') is consistently higher than the loss modulus (G”) within the strain range of 1% to 100%. When the strain reaches 428.4%, G” surpasses G', and the hydrogel undergoes a transition from a sol to a gel state. Figure B shows the continued dominance of G', confirming its robust three-dimensional network structure. Figure C shows that the system viscosity decreases significantly with increasing shear rate, exhibiting obvious shear-thinning behavior, which ensures that the hydrogel can be smoothly injected through a needle without clogging. Figure D shows that although the value of G' decreases slightly with increasing temperature, it still dominates, confirming the stability of the hydrogel network at physiological temperatures. Figure E shows that at high strain, G' is temporarily lower than the loss modulus G”, but when the strain recovers to 1%, G' rapidly recovers and becomes significantly higher than G”, demonstrating a reversible gel-sol transition. This strain-triggered phase transition remained effective after five consecutive cycles, indicating that the hydrogel possesses excellent self-healing capabilities. Figure F shows that instantaneous gelation can be achieved within seconds by simultaneously injecting the hydrogel precursor solution using a dual-barrel syringe. This hydrogel avoids the use of toxic chemical cross-linking agents (such as glutaraldehyde) and complex photoinitiation procedures (such as ultraviolet irradiation), and can be easily prepared using ordinary household syringes, thus significantly lowering the production threshold.

[0062] Experiment 4: Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy detection. Fourier transform infrared spectroscopy was performed using a FT-IR instrument with a range of 4000 cm⁻¹. 1 ~400 cm 1 The X-ray photoelectron spectra of the hydrogel were recorded using an X-ray electron spectrometer.

[0063] Figure 5 The images show the spectra of injectable responsive hydrogels loaded with chitosan oligosaccharide-octyl gallate. A in the figure is the Fourier transform infrared spectrum, B is the X-ray diffraction (XRD) spectrum of the CMCS, and C is the XRD spectrum of the hydrogel. From A, it can be seen that the TA (transformer in the chromatogram) is at 1713 cm⁻¹ among all hydrogel groups. 1 The characteristic absorption peaks (aromatic C=O stretching vibrations) at 1602 cm⁻¹ all disappeared, indicating that ionic interactions and hydrogen bonding occurred between the components. 1 The presence of characteristic peaks (C=N bonds) confirms the formation of Schiff base bonds in the gel network structure, similar to the peak at 1199 cm⁻¹. 1 The peak intensity at 2933 cm⁻¹ (phenolic hydroxyl C–O stretching vibration) is significantly reduced, indicating that the phenolic hydroxyl groups are partially consumed during the crosslinking process. Furthermore, compared to the COT hydrogel, the COT1 / COT2 hydrogel shows a significantly lower peak intensity at 2933 cm⁻¹. 1 The absorption peak intensity at 860 cm⁻¹ is significantly enhanced, and this peak represents the alkyl chain vibration of OG, which verifies the successful introduction of COS-OG into the hydrogel network. Furthermore, the absorption peak intensity at 860 cm⁻¹ is significantly enhanced. 1 The enhanced peak intensity further indicates that the Michael addition reaction between the quinone structure and the amino group continues. Figure B shows that the CMCS does not have a characteristic C=N absorption peak. Figure C shows that in the hydrogel sample, a characteristic C=N peak at 400.6 eV appears in the N 1s spectrum, providing direct evidence for the Schiff base reaction.

[0064] Example 5 The application of the injectable responsive hydrogel loaded with chitosan oligosaccharide-octyl gallate as described in Examples 1 to 4 in antibacterial activity includes the following method: applying 1 mL of the hydrogel to the wound surface and irradiating it with blue light for 20 min at a distance of 10 cm. Irradiation is repeated once daily for three consecutive days, with a light intensity of 45.2 mW / cm² per irradiation. 2 .

[0065] Experiment 5: In vitro antibacterial performance test. In sterile test tubes, 1 mL of hydrogel was mixed with 2 mL of bacterial suspension (concentration of 10⁸ CFU / mL) and irradiated with blue light before incubation at 37°C (irradiation time 20 min, irradiation distance 10 cm, irradiation at 0 h and 24 h after the start of the experiment). At specified time intervals, 100 μL of bacterial suspension was spread onto LB agar plates and incubated at 37°C for 24 h. Colony-forming units were counted using the standard serial dilution method to assess bacterial viability.

[0066] To observe bacterial morphology, after co-culturing for 24 h, the bacteria were centrifuged at 8000 rpm for 5 min, washed three times with PBS, fixed overnight with 2.5% glutaraldehyde, dehydrated with ethanol, critical point dried, sputter-coated with gold, and then imaged for observation. Following the previously described method, PI / SYTO 9 staining was performed, and fluorescence microscopy was used for observation and quantitative analysis to assess the integrity of the bacterial cell membrane.

[0067] Figure 6 The images show the anti-MRSA effect of the injectable responsive hydrogel loaded with chitosan oligosaccharide-octyl gallate (colony images and counts). The figures show abundant colony growth at 4 h, 16 h, and 24 h, indicating that bacteria can proliferate normally without material intervention. The COT group showed a high colony count at 4 h, a significant decrease at 16 h, and a small amount remaining at 24 h, demonstrating some antibacterial activity, but with limited effect. The COT2 group had fewer colonies at 4 h than the COT group, a significant decrease at 16 h, and almost no colonies at 24 h, showing a better antibacterial effect than COT. The COT2 BL group showed very few colonies at 4 h, almost no colonies at 16 h, and completely no colonies at 24 h, exhibiting the most thorough antibacterial effect. The antibacterial effect ranking is: COT2 BL > COT2 > COT > control group.

[0068] Experiment 6: Cytotoxicity test. L929 and RAW264.7 cells (5 × 10³ cells / well) were seeded in 96-well plates and incubated overnight. The culture medium was then replaced with different concentrations of hydrogel extract (the hydrogel was soaked in the medium for 24 hours and then filtered through a 0.22 µm sterile filter). After 24 hours of co-culture, cell viability was quantitatively assessed using the CCK-8 assay at 450 nm absorbance. Furthermore, cell viability was evaluated on days 1, 3, and 5 using a live / dead staining method.

[0069] Figure 7The effect of injectable responsive hydrogels loaded with chitosan oligosaccharide-octyl gallate on the survival of L929 (A) and RAW264.7 (B) cells was investigated. The figures show that at hydrogel concentrations of 0.1–20 mg / mL, the cell viability of both COT and COT2 remained above 80%, with viability approaching or exceeding 90%. This indicates that at low concentrations, both hydrogels exhibited minimal cytotoxicity and good cell compatibility. At a hydrogel concentration of 50 mg / mL, the cell viability of the COT group decreased to approximately 45%, and that of the COT2 group decreased to approximately 30%; the cell viability of the COT group decreased to approximately 35%, and that of the COT2 group decreased to approximately 25%. This indicates a significant decrease in cell viability at high concentrations, suggesting that excessively high material concentrations may affect cell metabolism or proliferation, exhibiting concentration-dependent cytotoxicity.

[0070] Experiment 7: In vitro anti-inflammatory capacity test. RAW264.7 macrophages (2.0 × 10⁵ cells / well) were seeded and then co-stimulated with lipopolysaccharide (LPS, 100 ng / mL) and interferon-γ (IFN-γ, 20 ng / mL) to induce M1 polarization. Cells were pretreated with the corresponding drugs for 1 h before polarization and then cultured for another 24 h. The cells were divided into the following groups: blank group (no treatment), LPS group (LPS + IFN-γ), DXM group (dexamethasone, positive control, final concentration 1 μM), COT group (10 mg / mL), and COT2 group (10 mg / mL). RT-qPCR, immunofluorescence staining, flow cytometry, and ELISA were used to analyze the treated cells.

[0071] For immunofluorescence staining, cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.5% Triton X-100 for 10 min, and then blocked with 10% bovine serum albumin (BSA) for 1 h. Subsequently, CD86 (1:200) and CD206 (1:200) were added and incubated overnight at 4°C. The next day, after washing, secondary antibody (Alexa 488 / 594, 1:200) was added and incubated for 1 h, followed by DAPI staining of the cell nuclei. Fluorescence intensity was quantitatively analyzed using ImageJ software.

[0072] In the enzyme-linked immunosorbent assay (ELISA) experiment, cell culture supernatant was collected, and the expression levels of IL-1β and IL-10 were detected according to the ELISA kit instructions.

[0073] Figure 8To illustrate the in vitro anti-inflammatory effect of injectable responsive hydrogels loaded with chitosan oligosaccharide-octyl gallate, Figure A shows immunofluorescence staining images, and Figure B shows quantitative analysis of different treatment groups (scale bar: 10 μm). Figure C shows ELISA analysis of IL-1β, and Figure D shows ELISA analysis of IL-10. Figures A and B show that the M2 / M1 ratio was low in the Blank group. The ratio further decreased in the LPS group, indicating that the M1 type was absolutely dominant and the inflammatory response was activated. The ratio in the COT group was higher than that in the LPS group, showing a certain anti-inflammatory polarization regulatory effect. The ratio in the COT2 group was significantly higher, approaching that of the DXM group, indicating that COT2 can efficiently promote the transformation of macrophages to the anti-inflammatory M2 type and reverse the LPS-induced pro-inflammatory phenotype. As shown in Figures C and D, the IL-1β concentration was significantly increased in the LPS group, validating the results of pro-inflammatory M1 polarization. The concentration in the COT group was lower than that in the LPS group, and the concentration in the COT2 group was further decreased, approaching that of the DXM group, indicating that COT2 can significantly inhibit the release of pro-inflammatory factors. The IL-10 concentration was slightly increased in the LPS group, but the increase was more significant in the COT group, and the concentration in the COT2 group was significantly increased, approaching that of the DXM group, indicating that COT2 can effectively promote the secretion of anti-inflammatory factors.

[0074] Experiment 8: In vivo application of injectable responsive hydrogels loaded with chitosan oligosaccharide-octyl gallate.

[0075] In vivo experiments were conducted using 6-8 week old male C57BL / 6 mice. After isoflurane anesthesia, the fur on the back of the mice was removed, and a circular wound with a diameter of 10 mm was made using a biopsy puncture instrument. The mice were then inoculated with methicillin-resistant Staphylococcus aureus (MRSA) (100 μL, 10...). 7 CFU / mL). Mice were randomly assigned to five groups (n=12 per group): control group (no drug treatment but otherwise the same as the treatment group), 3M group (using 3M Tegaderm™ film), COT group, COT2 group, and COT2 combined with BL group (denoted as COT2BL, BL was applied once daily for three consecutive days during treatment, then removed. Temperature was maintained within a certain range throughout the BL irradiation). Treatment (injection of hydrogel via a double-lumen syringe) began 24 hours post-infection (day 0). Mouse weight and wound area were monitored every other day. On days 1 and 3, wound exudate was collected with sterile swabs, soaked in sterile PBS, and used for inoculation and colony counting. On day 13, all remaining mice were euthanized under anesthesia by cervical dislocation. Wound skin tissue was collected for hematoxylin-eosin (H&E) staining and Masson's trichrome staining to assess wound healing. H&E staining analysis was performed on organ sections (heart, liver, spleen, lung, and kidney) to assess the in vivo biocompatibility of the hydrogel.

[0076] Figure 9 This study investigates the in vivo antibacterial effect of an injectable responsive hydrogel loaded with chitosan oligosaccharide-octyl gallate. Figure A shows colony images of tissue exudate on days 1 and 3, B shows the colony count on day 1, and C shows the colony count on day 3. The figures show that both the control and 3M groups exhibited abundant colony growth on days 1 and 3, indicating that bacteria can proliferate continuously without effective antibacterial intervention, and 3M (the control material) showed almost no antibacterial activity. In the COT group, the colony count on day 1 was lower than the control, but a significant number of colonies remained on day 3, demonstrating some short-term antibacterial activity, but with insufficient persistence. In the COT2 group, the colony count on day 1 was significantly lower than the COT group, and the colony count on day 3 was further reduced, indicating superior antibacterial effect and persistence compared to COT. In the COT2 BL group, the colony count on day 1 was extremely low, and almost no colonies were observed on day 3, demonstrating the most thorough and persistent antibacterial effect.

[0077] Figure 10 Images show the effect of injectable responsive hydrogels loaded with chitosan oligosaccharide-octyl gallate on promoting wound healing in dogs with MRSA infection in vivo. Figure A shows the change in body weight of mice during treatment; Figure B shows the wound closure rate of different treatment groups; Figure C shows the appearance of wound healing over time. Figure A shows that the body weight of animals in all groups (control, 3M, COT, COT2, COT2 BL) increased steadily over time, without significant decrease or abnormal fluctuations. This indicates that the treatment materials had no adverse effects on the overall health of the animals and had good biocompatibility. Figures B and C show that the COT2 BL group consistently had the highest wound closure rate, approaching 100% at day 13, and the fastest healing speed. The COT2 group had a significantly higher closure rate than COT, 3M, and the control group, with the second-highest healing effect. The COT group had a better closure rate than 3M and the control group, but weaker than COT2 and COT2 BL. The 3M group and the control group had the slowest healing speed and the lowest closure rate, indicating that the efficiency of traditional materials or natural repair is limited.

[0078] Figure 11 To demonstrate the in vivo anti-inflammatory effect of the injectable responsive hydrogel loaded with chitosan oligosaccharide-octyl gallate, Figure A shows hematoxylin-eosin (H&E) staining; Figure B shows Masson's trichrome staining; and Figure C shows quantitative analysis. The figures show that the collagen area ratio increases sequentially from the control group, 3M group, COT group, COT2 group to COT2 BL group, with the COT2 BL group exhibiting the highest collagen area ratio. This is completely consistent with the visual results of Masson staining, proving that it most effectively promotes collagen synthesis and deposition, accelerating tissue remodeling.

[0079] Figure 12The results of the in vivo biocompatibility assessment of the injectable responsive hydrogel loaded with chitosan oligosaccharide-octyl gallate are shown, specifically H&E stained sections of the heart, liver, spleen, lung, and kidney organs of mice after treatment. The images show that no obvious lesions or inflammatory cell infiltration were observed in the major organs of the mice, indicating that the hydrogel has excellent in vivo biocompatibility.

[0080] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention.

Claims

1. A method for preparing an injectable responsive hydrogel supported on chitosan oligosaccharide-octyl gallate, characterized in that, The preparation method includes the following steps: S1. Add the chitosan oligosaccharide-octyl gallate coupling compound to the carboxymethyl chitosan solution to obtain a mixed solution; S2. Mix the mixed solution with the oxidized tannic acid solution to obtain an injectable responsive hydrogel.

2. The preparation method according to claim 1, characterized in that, The oxidized tannic acid solution in S2 is prepared by the following method: Tannic acid is dissolved in ultrapure water to obtain a tannic acid solution. The pH of the tannic acid solution is adjusted to 8.0-9.5 with sodium hydroxide. The solution is stirred at a speed of 200-600 rpm and then the pH is adjusted back to neutral to obtain oxidized tannic acid.

3. The preparation method according to claim 2, characterized in that, The tannic acid solution contains 2% to 8% tannic acid by mass.

4. The preparation method according to claim 2, characterized in that, The stirring time is 1 to 3 hours.

5. The preparation method according to any one of claims 1 to 4, characterized in that, In S1, the mass fraction of carboxymethyl chitosan in the carboxymethyl chitosan solution is 2% to 8%.

6. The preparation method according to any one of claims 1 to 4, characterized in that, In S1, the concentration of the chitosan oligosaccharide-octyl gallate conjugate is 1 mg / mL to 4 mg / mL.

7. The preparation method according to any one of claims 1 to 4, characterized in that, The final mass fraction of the oxidized tannic acid solution in S2 in the injectable responsive hydrogel is 0.2% to 2.0%.

8. An injectable responsive hydrogel loaded with chitosan oligosaccharide-octyl gallate prepared by the preparation method according to any one of claims 1 to 7.

9. The use of the injectable responsive hydrogel of chitosan oligosaccharide-octyl gallate as described in claim 8 in the preparation of antibacterial drugs.

10. The application according to claim 9, characterized in that, The antibacterial drug is an anti-MRSA drug.

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