Modified chitosan compound capable of promoting healing of seawater soaking injury as well as preparation method and application of modified chitosan compound

Abstract

The invention discloses a modified chitosan compound with a function of promoting healing of seawater soaking wounds. The modified chitosan compound DGCS prepared by the invention has good biological activity, has a remarkable promotion effect on cell proliferation, and is enhanced along with the increase of concentration; the inflammation level in cells can be reduced; the healing of a seawater soaking wound can be promoted. An antibacterial experiment shows that the DGCS has good antibacterial activity on escherichia coli, bacillus subtilis, staphylococcus aureus, pseudomonas aeruginosa, enterococcus faecalis, vibrio vulnificus and vibrio parahaemolyticus.

Description

Technical Field

[0001] This invention belongs to the field of marine biomedicine technology, specifically, it relates to a modified chitosan complex that promotes the healing of seawater immersion wounds, its preparation method, and its application. Background Technology

[0002] The incidence of seawater immersion injuries has increased significantly in modern times. Seawater has a complex composition, rich in high concentrations of salt, various microorganisms, and harmful substances, creating a hypertonic and highly bacterial environment that easily leads to severe infection, tissue edema, and makes debridement far more difficult than for ordinary wounds [Carbohydr Polym. 2025;362:123656.]. Wounds immersing in seawater are prone to mixed infections from marine and terrestrial bacteria, thus requiring the use of antibacterial dressings. Simultaneously, to accelerate wound healing, the dressings must also possess anti-inflammatory and healing-promoting properties. Wound healing is a complex process involving the continuous repair of tissues, and many factors can influence its progress and final outcome. Wounds caused by seawater immersion during offshore operations tend to heal for longer periods and are more difficult to treat. Currently, there are few research reports on the treatment of seawater immersion wounds. Hydrogel dressings and gauze dressings offer poor protection. Biological treatments include adipose-derived stem cell therapy, but these are complex to operate and have poor environmental tolerance. Physical methods include vacuum-assisted therapy, but these require transport to the hospital and cannot be used for emergency treatment. In terms of chitosan, only chitosan dressings containing antibiotics are available, but their synthesis is difficult and costly. All these treatment methods have limitations, making the development of special dressings for seawater immersion wounds of great significance.

[0003] Chitosan is a polycationic polysaccharide obtained by deacetylation of the natural polysaccharide chitin. It has a variety of functions, including hemostasis, antibacterial properties, non-toxicity and biodegradability, promotion of wound healing and inhibition of scar formation, which meet the ideal characteristics of wound dressing. In addition, chitosan is also a multifunctional material with good biocompatibility, no immunogenicity and no irritation [Carbohydr Polym. 2025;363:123761.].

[0004] With in-depth research on the structure of chitosan and the increasing demand for biodegradable multifunctional hydrogel wound dressings, chitosan has shown great application potential in wound dressings. However, most current research on chitosan in wound dressings focuses on its interaction with other compounds, such as sodium alginate and gelatin, to form mixtures that work together at the wound site. There are relatively few applications that directly modify the structure of chitosan [Carbohydr Polym. 2025;360:123604.].

[0005] Currently, the chitosan dressings commonly used in the market are mainly low-viscosity chitosan and its derivatives, with limited research on the application of high-viscosity chitosan. High-viscosity chitosan refers to chitosan with a viscosity greater than 500 mPa•s in an acidic solution containing 1% chitosan. Its advantages are: firstly, good film-forming properties, enabling the formation of tough and stable films; and secondly, superior mechanical properties, with high strength and toughness. When used as a biomedical material, it can provide a stable supporting environment for cell growth and tissue repair. Utilizing the high film-forming properties and excellent mechanical properties of high-viscosity chitosan may become a new research direction [State-of-the-art in natural hydrogel-based wound dressings: Design,functionalization, and fabrication approaches. Adv Colloid Interface Sci. 2025, Publication Date 04.25.].

[0006] Modifying chitosan itself based on high-viscosity chitosan is a very important research direction. Chitosan has abundant amino and hydroxyl groups, and the main methods for modifying chitosan include chemical reactions such as alkylation, carboxymethylation, oxidation, and reduction at the 2- and 6-positions [J Nanobiotechnology. 2025;23(1):162.]. Through these reactions, different types of chitosan derivatives are generated, thereby improving the application performance of chitosan and expanding its application range.

[0007] Dopamine molecules possess a biomimetic mussel adhesion mechanism, which can form covalent bonds, hydrogen bonds, and metal coordination with various interfaces through catechol groups and amino groups, enabling materials to achieve strong adhesion in humid environments. At the same time, it improves cell adhesion to promote the spread and proliferation of related cells. The catechol structure of dopamine can inhibit the excessive activation of neutrophils to regulate the inflammatory microenvironment and shorten the inflammatory period of wounds. Meanwhile, its catechol groups and chitosan amino groups synergistically exert antibacterial effects and inhibit bacterial biofilm formation. In addition, the natural molecular structure of dopamine is compatible with human metabolic pathways and can reduce immunogenicity [CarbohydrPolym. 2025;355:123360.].

[0008] The incorporation of glutamic acid can significantly enhance the hydrophilicity and swelling properties of chitosan, increasing its solubility in neutral or weakly alkaline environments and enabling controllable swelling behavior, thereby broadening its application scenarios. The biocompatibility and cell affinity of the modified material are significantly improved. Its natural amino acid structure can promote the adhesion and proliferation of fibroblasts and other cells, indirectly inducing collagen synthesis to accelerate wound repair. At the same time, glutamic acid itself has the ability to regulate inflammatory responses, and research on the participation of polyglutamic acid in wound dressings has been reported [Biomaterials. 2025;318:123134.].

[0009] Currently, there are no literature reports on the preparation of high-viscosity modified chitosan hydrogels with good antibacterial and anti-inflammatory properties using glutamic acid as a molecular bridge connecting dopamine and chitosan. Summary of the Invention

[0010] The purpose of this invention is to provide a modified chitosan complex that promotes the healing of seawater immersion wounds.

[0011] Another object of the present invention is to provide a method for preparing the modified chitosan complex that promotes the healing of seawater immersion wounds.

[0012] Another object of the present invention is to provide the application of the modified chitosan complex having the effect of promoting the healing of seawater immersion wounds in the preparation of dressings for promoting the healing of seawater immersion wounds.

[0013] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0014] In a first aspect, the present invention provides a modified chitosan complex that promotes the healing of seawater-immersed wounds, the structure of which is shown below:

[0015] .

[0016] A second aspect of the present invention provides a method for preparing the modified chitosan complex that promotes healing of seawater immersion wounds, comprising the following steps:

[0017] Step 1, Preparation of Compound 2:

[0018] Under an argon atmosphere, compound 1, tert-butyldimethylchlorosilane and imidazole in a molar ratio of 1:1 to 4:1 to 6 are dissolved in dry dichloromethane and stirred vigorously at room temperature for 1 to 24 hours (preferably 16 hours). After post-treatment, compound 2 is obtained.

[0019] The second step is the preparation of compound 4:

[0020] Compound 3, compound 2, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine in a molar ratio of 1:1:1~3:0.01~0.3 were dissolved in dry dichloromethane and stirred at room temperature for 1~5 hours (preferably 2 hours). After post-treatment, compound 4 was obtained.

[0021] The third step is the preparation of compound 5:

[0022] Under an argon atmosphere, palladium on carbon is added to a methanol solution of compound 4, with a molar ratio of palladium on carbon to compound 4 of 0.001 to 0.1:1. Under conditions of 30 to 40°C (preferably 35°C) and 101.325 kPa, hydrogen is introduced and the mixture is stirred for 1 to 18 hours (preferably 12 hours). After post-treatment, compound 5 is obtained.

[0023] Step 4, preparation of compound 7:

[0024] Compound 5, compound 6, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine in a molar ratio of 1:1:1~3:0.01~0.3 were dissolved in dry N,N-dimethylformamide and stirred for 1~18 hours (preferably 12 hours). After post-treatment, compound 7 was obtained.

[0025] Step 5, preparation of compound 8:

[0026] Excess trifluoroacetic acid is added to a dichloromethane solution of compound 7, and the mixture is reacted at room temperature for 1 to 5 hours (preferably 3 hours). After post-treatment, the modified chitosan complex that promotes the healing of seawater immersion wounds is obtained.

[0027] The molar ratio of compound 1, tert-butyldimethylchlorosilane, and imidazole is 1:2.5:4.

[0028] The molar ratio of compound 3, compound 2, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 1:1:2:0.1.

[0029] The molar ratio of palladium on carbon to compound 4 is 0.03:1.

[0030] The molar ratio of compound 5, compound 6, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 1:1:2:0.1.

[0031] A third aspect of the present invention provides the application of the modified chitosan complex having the effect of promoting healing of seawater immersion wounds in the preparation of a dressing for promoting healing of seawater immersion wounds.

[0032] In a fourth aspect, the present invention provides a hydrogel prepared from the modified chitosan complex that promotes healing of seawater immersion wounds.

[0033] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0034] The modified chitosan complex DGCS prepared in this invention possesses excellent biological activity, significantly promoting cell proliferation with increasing concentration; it can reduce cellular inflammation levels; and it can promote the healing of wounds soaked in seawater. Antibacterial experiments showed that DGCS exhibited good antibacterial activity against *Escherichia coli*, *Bacillus subtilis*, *Staphylococcus aureus*, *Pseudomonas aeruginosa*, *Enterococcus faecalis*, *Vibrio vulnificus*, and *Vibrio parahaemolyticus*.

[0035] The modified chitosan complex DGCS prepared in this invention exhibits a significant wound-healing effect under a seawater immersion injury model and can be used to prepare dressings for promoting wound healing in seawater immersion. Attached Figure Description

[0036] Figure 1 This is a schematic diagram of the proton NMR spectrum of compound 4.

[0037] Figure 2 This is a schematic diagram of the mass spectrum of compound 4.

[0038] Figure 3 This is a schematic diagram of the proton NMR spectrum of compound 5.

[0039] Figure 4 This is a schematic diagram of the hydrogen nuclear magnetic resonance spectrum of compound 7.

[0040] Figure 5 This is a schematic diagram of the carbon NMR spectrum of compound 8.

[0041] Figure 6 This is a schematic diagram of the proton NMR spectrum of compound 8.

[0042] Figure 7 This is a schematic diagram of the infrared spectra of DGCS and HVCS.

[0043] Figure 8 This is a schematic diagram of the ultraviolet spectra of DGCS and HVCS.

[0044] Figure 9 This is a schematic diagram of the in vitro cytotoxicity results of DGCS tested using the CCK8 method.

[0045] Figure 10This is a schematic diagram of the in vitro cell anti-inflammatory results of DGCS.

[0046] Figure 11 This is a schematic diagram of the results of a rat in vivo DGCS-induced wound healing experiment involving seawater immersion.

[0047] Figure 12 This is a schematic diagram of the in vitro antibacterial activity results of the modified chitosan complex DGCS prepared in this invention. Detailed Implementation

[0048] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0049] Example 1

[0050] A method for preparing a modified chitosan complex that promotes healing of seawater-immersed wounds includes the following steps:

[0051] Step 1, Preparation of Compound 2:

[0052] TBS is an abbreviation for tert-butyldimethylsilyl.

[0053] Under an argon atmosphere, dopamine hydrochloride (compound 1) (5.0 g, 26.4 mmol), tert-butyldimethylchlorosilane (9.95 g, 66.0 mmol), and imidazole (7.18 g, 105.6 mmol) were dissolved in 60 mL of dry dichloromethane. The mixture was stirred vigorously at room temperature for 16 hours. The resulting suspension was filtered, and the filtrate was concentrated under reduced pressure. The crude product was dissolved in diethyl ether and washed successively with saturated sodium bicarbonate aqueous solution, water, and brine. The organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain a pale yellow oily substance, compound 2, which could be used without further purification.

[0054] The second step is the preparation of compound 4:

[0055] Under an argon atmosphere, compound 3 (5.39 g, 16.0 mmol), compound 2 (6.1 g, 16.0 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (6.13 g, 32.0 mmol), and 4-dimethylaminopyridine (195.5 mg, 1.6 mmol) were dissolved in 200 mL of dry dichloromethane and stirred at room temperature for 2 hours. The reaction solution was washed successively with water and brine, the organic layer was dried over sodium sulfate, filtered, concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 5:1) to give 9.86 g of compound 4, 88% yield, a colorless viscous liquid, ¹H NMR spectrum as shown below. Figure 1 As shown, Figure 1 This is a schematic diagram of the proton NMR spectrum of compound 4. 1 H NMR (400 MHz, Chloroform-d) δ 7.22-7.10 (m, 5H), 6.56 (d, J = 8.0 Hz, 1H), 6.49-6.37 (m, 2H), 5.63 (s, 1H), 5.12 (d, J = 7.5 Hz, 1H), 5.05-4.90 (m, 2H), 4.11 (s, 1H), 3.24 (q, J = 6.6 Hz, 2H), 2.48 (t, J =7.0 Hz, 2H), 1.98 (s, 3H), 1.83-1.68 (m, 1H), 1.24 (s, 9H), 0.79 (s, 18H). Figure 2 This is a schematic mass spectrum of compound 4. The calculated LCMS (ESI) value is 723.38, which is close to the measured value of 723.40, proving that the molecular composition of the compound is consistent with the expected (C... 37 H 60 The results are consistent with N2O7Si2Na, which proves that compound 4 was successfully prepared.

[0056] The third step is the preparation of compound 5:

[0057] Under an argon atmosphere, palladium on carbon (13 mg, 0.00611 mmol) was carefully added to 13 mL of a methanol solution of compound 4 (135 mg, 0.19 mmol). The mixture was stirred for 12 hours under hydrogen gas at 35 °C and 101.325 kPa. The resulting suspension was filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 5:1) to give 125 mg of compound 5, 90% yield, as a colorless viscous liquid. The 1H NMR spectrum of compound 5 is shown below. Figure 3 As shown, Figure 3 This is a schematic diagram of the proton NMR spectrum of compound 5. 1 H NMR (400 MHz, Chloroform-d) δ 6.66-6.71(m, 3H), 4.10-4.14 (t, J = 6 Hz, 3H), 3.38-3.65 (m, 4H), 2.64-2.68 (t, J =7.2Hz 1H), 1.91-2.28 (m, 4H), 1.23-1.43 (m, 9H), 0.79-0.96 (m, 18H).

[0058] Step 4, preparation of compound 7:

[0059] Compound 5 (61 mg, 0.1 mmol), chitosan (compound 6) (16.1 mg, 0.1 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (38.3 mg, 0.2 mmol), and 4-dimethylaminopyridine (1.2 mg, 0.01 mmol) were dissolved in 5 mL of dry N,N-dimethylformamide and stirred for 12 hours. The solution was concentrated, and the residue was purified by silica gel column chromatography (eluent: petroleum ether: ethyl acetate = 5:1) to give 12.8 mg of compound 7, yield 79.5%, as a colorless viscous oil. Figure 4 This is a schematic diagram of the hydrogen nuclear magnetic resonance spectrum of compound 7. 1 H NMR (500 MHz, Methanol-d4)δ 6.77(d, J = 8.1 Hz, 1H), 6.73-6.66 (m, 2H), 4.66-4.53 (m, 1H), 3.94 (t, J = 7.1Hz, 1H), 3.75 (dd, J = 14.5, 7.2 Hz, 1H), 3.34 (dd, J = 8.7, 7.0 Hz, 3H), 3.29 (d, J = 6.7 Hz, 1H), 2.67 (t, J = 7.4 Hz, 2H), 2.56 (t, J = 7.7 Hz, 2H), 2.39 (s, 6H), 2.29 (t, J = 7.2 Hz, 2H), 2.11-1.99 (m, 1H), 1.80 (p, J = 7.0Hz, 3H), 1.25 (t, J = 7.0 Hz, 3H), 0.99 (d, J = 4.6 Hz, 19H), 0.19 (d, J =7.3 Hz, 12H).

[0060] Step 5, preparation of compound 8:

[0061] Trifluoroacetic acid (10.0 mL, 130.6 mmol) was added to 50 mL of dichloromethane solution of compound 7 (1.18 g), and the reaction was carried out at room temperature for 3 hours. The reaction progress was monitored by TLC. After the reaction was completed, the mixture was concentrated under reduced pressure. The crude product was purified by column chromatography using a C18 column to obtain 509 mg of pale yellow solid. The modified chitosan complex, compound 8, abbreviated as DGCS, was a pale yellow solid. Figure 5 This is a schematic diagram of the carbon NMR spectrum of compound 8. Figure 6 This is a schematic diagram of the proton NMR spectrum of compound 8. 1 ¹H NMR (500 MHz, DMSO-d⁶) δ 8.67–8.74 (d, J = 3.5 Hz, 2H), 8.31 (s, 1H), 7.87–7.89 (m, 1H), 6.42–6.63 (m, 3H), 4.04–4.05 (t, J = 0.5 Hz, 1H), 3.01–3.18 (m, 4H), 2.51–2.74 (s, 6H), 2.13–2.19 (m, 6H), 1.75–2.19 (m, 6H). The ¹H NMR spectrum from DGCS shows the signal at 8.31–8.74 at aromatic hydrogens on the benzene ring, -NH₂ and -NH₃ at 6.42 and 7.89 respectively, and the signal at 4.04–4.05 at aliphatic hydrogens on the sugar ring bonded to two oxygen atoms. The carbon spectrum shows that the two signals at 171.32 and 174.70 are attributed to ester groups, and the six signals from 116.77 to 157.06 are attributed to benzene rings; the signals from 42.33 to 115.38 belong to sugar rings, with the carbon atom bonded to two oxygen atoms located at 115.38; the remaining peaks belong to aliphatic carbon chains.

[0062] The infrared and ultraviolet spectra of DGCS and HVCS (High Viscosity Chitosan, viscosity 1281 mPa·s) were measured. The infrared results for DGCS and HVCS are as follows: Figure 7 As shown, Figure 7 This is a schematic diagram of the infrared spectra of DGCS and HVCS. As can be seen from the figure, the peak of the -OH bond on HVCS is at 3442 cm⁻¹. -1 The -OH bond peak on DGCS is at 3400 cm⁻¹. -1 At this point, these peaks shift to lower frequencies due to hydrogen bonding. Compared to HVCS, DGCS shows a lower frequency shift at 1708 cm⁻¹. -1 and 1771cm -1The absorption peaks are attributed to the amide bonds formed by the reactions of HVCS with glutamate and dopamine with glutamate. Compared to HVCS, the corresponding peaks of DGCS shift to higher frequencies due to the oxidation of the hydroxyl groups on dopamine to form hydrazone bonds.

[0063] The ultraviolet spectra of DGCS and HVCS are as follows: Figure 8 As shown, Figure 8 This is a schematic diagram of the ultraviolet spectra of DGCS and HVCS. As can be seen from the figure, an absorption peak exists at 280 nm, which is a characteristic peak of the benzene ring in dopamine, proving that the dopamine-glutamate complex was successfully grafted onto chitosan.

[0064] I. In vitro cytotoxicity test of the modified chitosan complex DGCS prepared in this invention:

[0065] Prepare an aqueous solution of DG (dopamine glutamate condensation product, (S)-2-amino-5-(3,4-dihydroxyphenethoxy)-5-oxovalerate) with a concentration of 100 μg / ml.

[0066] Prepare a high-viscosity (viscosity 1281 mPa.s) chitosan 1% acetic acid aqueous solution with a concentration of 100 μg / ml.

[0067] Prepare DGCS aqueous solutions with concentrations of 1 μg / ml, 10 μg / ml, 100 μg / ml, and 1000 μg / ml.

[0068] In vitro cytotoxicity was assessed using the CCK8 assay with Hacat cells (human immortalized epidermal cells). Cells were seeded in 96-well plates, and each group's sample solution was used instead of cell culture medium. The cells were incubated for 24 hours, followed by the addition of CCK8 reagent and incubation at 37°C for 1 hour. OD values ​​were then measured using a microplate reader. 450 The value was used, with only cells in the culture medium as the control group.

[0069] The cells were divided into nine groups: blank group (culture medium was added but no cells were added), control group (no treatment was given), NC group (1% acetic acid reagent was added as a negative control), 100 μg / mL DG group (100 μg / mL DG solution), 100 μg / mL HVCS group (100 μg / mL HVCS solution), 1 μg / mL DGCS group (1 μg / mL DGCS solution), 10 μg / mL DGCS group (10 μg / mL DGCS solution), 100 μg / mL DGCS group (100 μg / mL DGCS solution), and 1000 μg / mL DGCS group (1000 μg / mL DGCS solution).

[0070] Cell survival rate is calculated relative to the survival rate of control group cells, and the calculation formula is as follows:

[0071] Cell survival rate % = [(OD test –OD blank ) / (OD control –OD blank )]×100%

[0072] OD test OD blank OD control The absorbance at 450 nm represents the absorbance of the test group, blank group, and control group, respectively.

[0073] Figure 9 This is a schematic diagram of the in vitro cytotoxicity results of DGCS tested using the CCK8 assay (**P<0.01, ***P<0.001, ****P<0.0001). As can be seen from the figure, cell viability was generally higher than the control group at DGCS concentrations ranging from 1 to 1000 μg / mL, and there was no effect on the blank group (cell-free) and the NC group (acetic acid control), demonstrating that the modified chitosan complex DGCS prepared in this invention has good biocompatibility and no cytotoxicity. Further comparison of cell viability revealed that the cell viability of the 100 μg / mL HVCS group and the 100 μg / mL DGCS group was not significantly different from that of the control group, indicating no significant effect on cell proliferation. However, the cell viability of the 1 μg / mL DGCS group, the 10 μg / mL DGCS group, the 100 μg / mL DGCS group, and the 1000 μg / mL DGCS group all increased significantly, indicating that the modified chitosan complex DGCS prepared in this invention has a significant promoting effect on cell proliferation. Among them, the DGCS concentration of 1000 μg / mL had the greatest effect on cell viability, increasing it by 1.5 times compared with the control group. The DGCS groups with concentrations of 1, 10, and 100 μg / mL also achieved a 1.2-fold increase. Therefore, the modified chitosan complex DGCS prepared in this invention has a significant promoting effect on cell proliferation, and this effect increases with increasing concentration.

[0074] II. In vitro anti-inflammatory activity test of the modified chitosan complex DGCS prepared in this invention:

[0075] Hacat cells were seeded in culture plates and, after adhesion, an inflammation model was established. Cells were cultured for 6 hours in medium containing 1 μg / mL TNF-α and INF-γ. After establishing the inflammation model, the medium was replaced with medium containing the samples from each group and cultured for 24 hours. After discarding the medium, 50 μl of buffer containing a NO fluorescent probe was added to each well, and the cells were incubated in the dark for 30 minutes. Unbound probes were removed by washing with PBS. The fluorescence intensity at excitation wavelength of 488 nm and emission wavelength of 515 nm was measured using a microplate reader. Changes in fluorescence intensity reflected intracellular NO levels. Background fluorescence was subtracted from the experimental results, and statistical analysis was performed.

[0076] The experiment was divided into 10 groups: control group (no treatment), NC group (negative control group, with 1% acetic acid solution added), model group (inflammatory model group), DXMS group (dexamethasone, positive control group, 10 μg / mL added), 100 μg / mL DG group (100 μg / mL DG solution), 100 μg / mL HVCS group (100 μg / mL HVCS solution), 1 μg / mL DGCS group (1 μg / mL DGCS solution), 10 μg / mL DGCS group (10 μg / mL DGCS solution), 100 μg / mL DGCS group (100 μg / mL DGCS solution), and 1000 μg / mL DGCS group (1000 μg / mL DGCS solution).

[0077] Figure 10 This is a schematic diagram of the in vitro cellular anti-inflammatory results of DGCS (*P<0.05, ****P<0.0001). The figure shows that NO release was significantly increased in the inflammation model group, proving the successful establishment of the inflammation model. NO release in the negative control group, the 100 μg / mL DGCS group, and the 100 μg / mL HVCS group was comparable to that in the model group, indicating that it had virtually no effect on inflammation. It was observed that the positive control group had lower NO release, showing a significant inhibitory effect on inflammation. NO release was significantly reduced when the DGCS concentration was between 1 and 1000 μg / mL, with the most significant reduction observed at a DGCS concentration of 1000 μg / mL. This demonstrates that the modified chitosan complex DGCS prepared in this invention can reduce cellular inflammation levels.

[0078] III. In vivo test to determine the effect of the modified chitosan complex DGCS prepared in this invention on promoting wound healing after seawater immersion:

[0079] The rats used in the experiment were SD rats, weighing about 200g. The seawater was taken from the East China Sea to simulate the seawater immersion environment.

[0080] Rats were anesthetized by intraperitoneal injection of 3% sodium pentobarbital (60 mg / kg), their back hair was shaved, and the skin was disinfected with povidone-iodine. A full-thickness skin defect (1.5 × 1.5 cm) was created in the midline of the back using a scalpel. 2 (Deep into the muscle layer) to ensure consistent wound area and depth; immerse rats in seawater at 20℃ for 2 hours to simulate a polluted seawater environment; after immersion, gently dry the wound surface with sterile gauze, apply dressings to the wounds, and transfer the rats to clean cages for individual housing and regular observation.

[0081] The experiment was divided into 5 groups: the model group (seawater immersion without treatment), the CMCS group (Commercial Chitosan, commercially available chitosan dressing), the Silvadene group (silver sulfadiazine, used as a positive control), the HVCS group, and the DGCS group.

[0082] The HVCS group uses high-viscosity chitosan hydrogel. The preparation method of high-viscosity chitosan hydrogel is as follows: Due to the high viscosity of high-viscosity chitosan, it can form a gel on its own without the need for a cross-linking agent. Dissolve 2g of high-viscosity chitosan in 100mL of 1% acetic acid aqueous solution and stir at room temperature for 4 hours to obtain high-viscosity chitosan hydrogel.

[0083] The DGCS group used modified chitosan complex DGCS hydrogel. The preparation method of modified chitosan complex DGCS hydrogel is as follows: 2g of modified chitosan complex DGCS was dissolved in 100mL of water and stirred at room temperature for 30 minutes to obtain modified chitosan complex DGCS hydrogel.

[0084] The positive control group used commercially available chitosan dressings and clinically used silver sulfadiazine cream, while the experimental group used HVCS and DGCS.

[0085] The results are as follows Figure 11 As shown, Figure 11This is a schematic diagram of the results of a rat in vivo DGCS-induced seawater immersion wound healing assay. As can be seen from the diagram, the wound area gradually decreased in each group during the postoperative period. On day 1, the wounds in all groups were bright red and moist with exudate. Since the inflammatory response began simultaneously at the initial stage of trauma, the differences between groups were not significant, but the wound contraction in the DGCS group accelerated from day 3. By day 3 of treatment, the wound contraction rate in the DGCS group was significantly faster, forming a thin, dry scab at the wound edge. In contrast, the other groups still had thick scabs covering the wounds, adhering to exudate, and their wound area reduction was significantly delayed. In the DGCS group, the wound area decreased by more than 60% compared to the initial measurement, and a light pink continuous epithelial layer was visible at the wound edge. The positive control group treated with silver sulfadiazine cream also showed promoted wound healing, but its epithelial formation rate was significantly slower than that of the DGCS group. By day 10, the wounds treated with DGCS had almost completed epithelial formation, the color of the new tissue was close to that of the surrounding skin, and there was very little scarring; in contrast, the other groups had residual unclosed wounds or formed obvious hypertrophic scars. The above demonstrates that the modified chitosan complex DGCS prepared in this invention can promote the healing of wounds immersed in seawater.

[0086] IV. In vitro antibacterial activity test of the modified chitosan complex DGCS prepared in this invention:

[0087] Five common pathogenic bacteria (Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis) and two specific pathogenic bacteria found in seawater (Vibrio vulnificus and Vibrio parahaemolyticus) were detected. The experiment employed the disk disc method, where seven indicator bacteria (Staphylococcus aureus, Bacillus subtilis, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Vibrio parahaemolyticus, and Vibrio vulnificus) were cultured in liquid medium until the optical density (OD) at 600 nm was reached. 600 The concentration was 0.6. DGCS, DG, HVCS, the positive control drug ampicillin, and the negative control solvent (1% acetic acid) were each prepared into 10 μg / mL solutions. Blank controls and solvents for dissolving the samples were also prepared. The test samples were sequentially pipetted onto sterile and dried circular filter paper discs (1 cm in diameter). After the discs absorbed the liquid and were air-dried to a semi-dry state, they were placed on agar plates coated with 50 μL of indicator bacteria. The plates were placed at room temperature for 20 minutes, then inverted and incubated in an oven at 28°C for 16–18 hours to observe the formation of the inhibition zone.

[0088] The results are shown in Table 1. It can be seen that DGCS has an inhibitory effect on common pathogens and special pathogens in seawater. The antibacterial activity of DGCS against the seven tested bacteria is significantly stronger than that of HVCS, and its performance is comparable to that of the positive control drug ampicillin.

[0089] Table 1

[0090]

[0091] Figure 12 This is a schematic diagram illustrating the in vitro antibacterial activity results of the modified chitosan complex DGCS prepared in this invention. As can be seen from the figure, DGCS exhibits good antibacterial activity against Vibrio vulnificus, Vibrio parahaemolyticus, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis, Escherichia coli, and Bacillus subtilis. Compared to HVCS, DGCS demonstrates antibacterial activity against two specific seawater-borne pathogenic Vibrio species (Vibrio vulnificus and Vibrio parahaemolyticus), and its antibacterial activity against other common pathogenic bacteria is also superior to HVCS.

[0092] 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 some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A modified chitosan complex that promotes healing of seawater immersion wounds, characterized in that, The structure is as follows: 。 2. A method for preparing the modified chitosan complex of claim 1 that promotes healing of seawater immersion wounds, characterized in that, Includes the following steps: Step 1, Preparation of Compound 2: ； Under an argon atmosphere, compound 1, tert-butyldimethylchlorosilane and imidazole in a molar ratio of 1:1~4:1~6 were dissolved in dry dichloromethane and stirred vigorously at room temperature for 1~24 hours. After post-treatment, compound 2 was obtained. The second step is the preparation of compound 4: ； Compound 3, compound 2, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine were dissolved in dry dichloromethane in a molar ratio of 1:1:1~3:0.01~0.3 under an argon atmosphere and stirred at room temperature for 1~5 hours. After post-treatment, compound 4 was obtained. The third step is the preparation of compound 5: ； Under an argon atmosphere, palladium on carbon was added to a methanol solution of compound 4, with a molar ratio of palladium on carbon to compound 4 of 0.001 to 0.1:

1. Under conditions of 30 to 40°C and 101.325 kPa, hydrogen was introduced and the mixture was stirred for 1 to 18 hours. After post-treatment, compound 5 was obtained. Step 4, preparation of compound 7: ； Compound 5, compound 6, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine in a molar ratio of 1:1:1~3:0.01~0.3 were dissolved in dry N,N-dimethylformamide and stirred for 1~18 hours. After post-treatment, compound 7 was obtained. Step 5, preparation of compound 8: ； Excess trifluoroacetic acid was added to a dichloromethane solution of compound 7, and the mixture was reacted at room temperature for 1 to 5 hours. After post-treatment, the modified chitosan complex that promotes the healing of seawater immersion wounds was obtained.

3. The method for preparing the modified chitosan complex for promoting healing of seawater immersion wounds according to claim 2, characterized in that, The molar ratio of compound 1, tert-butyldimethylchlorosilane, and imidazole is 1:2.5:

4.

4. The method for preparing the modified chitosan complex that promotes healing of seawater immersion wounds according to claim 2, characterized in that, The molar ratio of compound 3, compound 2, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 1:1:2:0.

1.

5. The method for preparing the modified chitosan complex for promoting healing of seawater immersion wounds according to claim 2, characterized in that, The molar ratio of palladium on carbon to compound 4 is 0.03:

1.

6. The method for preparing the modified chitosan complex for promoting healing of seawater immersion wounds according to claim 2, characterized in that, The molar ratio of compound 5, compound 6, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and 4-dimethylaminopyridine is 1:1:2:0.

1.

7. The use of the modified chitosan complex of claim 1, which promotes healing of seawater immersion wounds, in the preparation of a dressing that promotes healing of seawater immersion wounds.

8. A hydrogel, characterized in that, It is prepared from the modified chitosan complex of claim 1, which promotes the healing of seawater immersion wounds.

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