Near-infrared responsive hydrogel preparation as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of wound healing medicines, and particularly relates to a near-infrared responsive hydrogel preparation as well as a preparation method and application thereof. The near-infrared responsive hydrogel preparation provided by the invention comprises a hydrogel matrix and drug-loading particles loaded in the hydrogel matrix, the hydrogel matrix is carboxymethyl chitosan, and the drug-loading particles comprise polydopamine nanoparticles loaded with a PAD4 inhibitor; the PAD4 inhibitor has a structure as shown in a formula 1. The near-infrared responsive hydrogel preparation provided by the invention has good biocompatibility and high bioavailability, so that formation of NETs can be effectively inhibited, and chronic inflammation can be relieved; meanwhile, the antibacterial effect is enhanced through the photothermal effect, and cell adhesion and proliferation are promoted; the medicine has an excellent treatment effect on diabetic wounds.

Description

Technical Field

[0001] The present invention belongs to the technical field of wound healing drugs, and in particular relates to a near-infrared responsive hydrogel preparation, a preparation method and an application thereof. Background Art

[0002] In the medical field, chronic wound healing has always been a major problem that has troubled people, especially diabetic wound healing.

[0003] Diabetic ulcers are one of the most serious complications of diabetes, with an incidence rate as high as 15%. They are difficult to heal, prone to infection, and result in high disability rates, severely impacting patients' quality of life. Traditional treatments, such as debridement, antibiotic therapy, and the use of growth factors, face challenges in clinical practice, including low drug delivery efficiency, inadequate control of local inflammation, and prolonged healing periods.

[0004] Recent studies have revealed that excessive release of neutrophil extracellular traps (NETs), mediated by protein arginine deiminase 4 (PAD4), is a key pathological mechanism leading to chronic inflammation in diabetic wounds. Inhibiting PAD4 activity can effectively reduce NET formation and alleviate local inflammatory responses, offering a new approach for the treatment of diabetic ulcers. However, existing PAD4 inhibitors suffer from poor biocompatibility, low bioavailability, and insufficient antibacterial activity, limiting their clinical application. Summary of the Invention

[0005] The purpose of the present invention is to provide a near-infrared responsive hydrogel preparation, a preparation method and application thereof. The near-infrared responsive hydrogel preparation provided by the present invention has good biocompatibility and high bioavailability, thereby being able to effectively inhibit the formation of NETs and alleviate chronic inflammation; at the same time, it enhances the antibacterial effect through the photothermal effect, promotes cell adhesion and proliferation; and has excellent therapeutic effects on diabetic wounds.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a near-infrared responsive hydrogel preparation, comprising a hydrogel matrix and drug-loaded particles loaded in the hydrogel matrix, wherein the hydrogel matrix is ​​carboxymethyl chitosan, and the drug-loaded particles include polydopamine nanoparticles loaded with a PAD4 inhibitor; the PAD4 inhibitor has a structure shown in Formula 1:

[0008]

[0009] Preferably, the mass ratio of the PAD4 inhibitor to the polydopamine nanoparticles is ≤0.85:1.

[0010] Preferably, the particle size of the near-infrared responsive hydrogel preparation is 350 to 600 nm.

[0011] Preferably, the absolute value of the Zeta potential of the near-infrared responsive hydrogel preparation is >30 mV.

[0012] The present invention provides a method for preparing the near-infrared responsive hydrogel preparation described in the above technical solution, comprising the following steps:

[0013] The PAD4 inhibitor, polydopamine nanoparticles and water are mixed and incubated to obtain polydopamine nanoparticles loaded with the PAD4 inhibitor;

[0014] The polydopamine nanoparticles loaded with the PAD4 inhibitor, carboxymethyl chitosan and water are mixed to obtain the near-infrared responsive hydrogel preparation.

[0015] Preferably, the mass ratio of the PAD4 inhibitor to the polydopamine nanoparticles is ≤1:1.

[0016] Preferably, the mass ratio of the polydopamine nanoparticles loaded with the PAD4 inhibitor to the carboxymethyl chitosan is (15-20):70.

[0017] The present invention provides the use of the near-infrared responsive hydrogel preparation described in the above technical solution or the near-infrared responsive hydrogel preparation prepared by the preparation method described in the above technical solution in the preparation of a drug for promoting wound healing.

[0018] Preferably, the drug that promotes wound healing is a drug that promotes skin wound healing.

[0019] Preferably, the drug for promoting wound healing is a drug for promoting diabetic wound healing.

[0020] The present invention provides a near-infrared responsive hydrogel preparation (Gel-4B), comprising a hydrogel matrix and drug-loaded particles loaded in the hydrogel matrix, wherein the hydrogel matrix is ​​carboxymethyl chitosan (CMCS), and the drug-loaded particles include polydopamine nanoparticles loaded with a PAD4 inhibitor; the PAD4 inhibitor has a structure shown in Formula 1. The present invention successfully constructs a hydrogel preparation by combining PAD4 inhibitors with polydopamine nanoparticles (PDANPs) and CMCS, which has good biocompatibility, thereby improving the bioavailability of the PAD4 inhibitor, thereby alleviating chronic inflammation by inhibiting the formation of NETs. At the same time, the present invention utilizes the photothermal effect of PDANPs to enhance the antibacterial properties of the hydrogel preparation, and utilizes the natural biological activity of CMCS to promote cell adhesion and proliferation in wounds. Thus, the near-infrared responsive hydrogel preparation provided by the present invention achieves efficient loading and controllable release of the PAD4 inhibitor. Combining anti-inflammatory, antibacterial, photothermal, and biocompatibility benefits, the material has demonstrated excellent biocompatibility, antibacterial properties, and wound healing effects through in vitro and in vivo experiments, providing an innovative strategy for the treatment of chronic diabetic wounds. The multi-mechanism synergistic effects of Gel-4B, provided by this invention, highlight the potential application of intelligent biomaterials in precision medicine and possess significant scientific significance and clinical translational value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The synthetic route of the PAD4 inhibitor 4B of the present invention is shown in FIG.

[0022] Figure 2 is the mass spectrum of the PAD4 inhibitor 4B of the present invention;

[0023] Figure 3 is the PAD4 inhibitor 4B of the present invention 1 H NMR spectrum (300 MHz, DMSO-d6);

[0024] Figure 4 is a synthetic route for polydopamine nanoparticles in the present invention;

[0025] Figure 5 is a scanning electron microscope image of the polydopamine nanoparticles of the present invention;

[0026] Figure 6 is a scanning electron microscope image of the carboxymethyl chitosan of the present invention;

[0027] Figure 7 This is a photothermal effect diagram of the Gel prepared in Comparative Example 1 of the present invention;

[0028] Figure 8 is the in vitro release curve of Gel-4B in the present invention (4B release amount);

[0029] Figure 9 This is the MTT assay for Gel and Gel-4B in the present invention;

[0030] Figure 10 This is the antibacterial experiment of Gel and Gel-4B against Escherichia coli;

[0031] Figure 11 This is the antibacterial experiment of Gel and Gel-4B against Staphylococcus aureus;

[0032] Figure 12 The results of the in vivo wound healing experiment in the present invention;

[0033] Figure 13 This is the experimental result of TNF-α level in the present invention. DETAILED DESCRIPTION

[0034] The present invention provides a near-infrared responsive hydrogel preparation, comprising a hydrogel matrix and drug-loaded particles loaded in the hydrogel matrix, wherein the hydrogel matrix is ​​carboxymethyl chitosan, and the drug-loaded particles include polydopamine nanoparticles loaded with a PAD4 inhibitor; the PAD4 inhibitor has a structure shown in Formula 1:

[0035]

[0036] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well known to those skilled in the art.

[0037] The near-infrared responsive hydrogel preparation provided by the present invention includes a hydrogel matrix. In the present invention, the hydrogel matrix is ​​carboxymethyl chitosan. The hydrogel matrix formed by the carboxymethyl chitosan has a continuous and flexible three-dimensional network architecture. The skeleton of the hydrogel matrix provided by the present invention is composed of fibrous structures of varying thicknesses interwoven with each other. The surface of the fibrous structure is relatively smooth, and the uniformity of thickness fluctuates within a certain range. The present invention utilizes the natural biological activity of CMCS to enable the near-infrared responsive hydrogel preparation to promote cell adhesion and proliferation in wounds.

[0038] The near-infrared responsive hydrogel preparation provided by the present invention includes drug-loaded particles loaded in the hydrogel matrix. In the present invention, the drug-loaded particles include polydopamine nanoparticles loaded with a PAD4 inhibitor. The PAD4 inhibitor (abbreviated as 4B) has a structure shown in Formula 1. The mass ratio of the PAD4 inhibitor to the polydopamine nanoparticles is preferably ≤0.85:1, and in the embodiment it can be 0.843:1. In the present invention, PDANPs have a photothermal effect, so that when the near-infrared responsive hydrogel preparation is irradiated with near-infrared light, the temperature can be stably maintained at a maximum of about 40°C. This temperature level can effectively promote the antibacterial performance without producing a strong burning sensation, thereby having good safety and applicability in biological application scenarios. In the present invention, the PAD4 inhibitor is electrostatically adsorbed on the polydopamine nanoparticles.

[0039] In the present invention, the particle size of the near-infrared responsive hydrogel preparation is preferably 350 to 600 nm, and in the embodiment, it can be 482.4 ± 105.2 nm. The absolute value of the zeta potential of the near-infrared responsive hydrogel preparation is preferably > 30 mV, and in the embodiment, it can be 32.1 mV. In the present invention, the zeta potential of the near-infrared responsive hydrogel preparation is preferably -32.1 mV.

[0040] The present invention provides a method for preparing the near-infrared responsive hydrogel preparation described in the above technical solution, comprising the following steps:

[0041] The PAD4 inhibitor, polydopamine nanoparticles and water are mixed and incubated to obtain polydopamine nanoparticles loaded with the PAD4 inhibitor;

[0042] The polydopamine nanoparticles loaded with the PAD4 inhibitor, carboxymethyl chitosan and water are mixed to obtain the near-infrared responsive hydrogel preparation.

[0043] The present invention mixes a PAD4 inhibitor, polydopamine nanoparticles and water for incubation to obtain polydopamine nanoparticles loaded with a PAD4 inhibitor. The present invention has no special requirements for the source of the PAD4 inhibitor (abbreviated as 4B), and either a commercially available product or a homemade product can be used. The present invention has no special requirements for the preparation method of the PAD4 inhibitor. In the specific embodiment of the present invention, Figure 1 The present invention has no special requirements on the source of the polydopamine nanoparticles, and commercial products or homemade products can be used. In the present invention, the Zeta potential of the PAD4 inhibitor is preferably 15.72. The present invention has no special requirements on the preparation method of the polydopamine nanoparticles. In the specific embodiment of the present invention, the polydopamine nanoparticles are prepared according to Figure 4The preparation process shown can be used. In the present invention, the particle size of the polydopamine nanoparticles is preferably 765.3±143.6nm. The zeta potential of the polydopamine nanoparticles is preferably -37.36. In the present invention, the mass ratio of the PAD4 inhibitor to the polydopamine nanoparticles is preferably ≤1:1, and in the embodiment, it can be 1:1. The water is preferably deionized water, and the present invention has no special requirements for the amount of water used.

[0044] In the present invention, the mixing preferably includes dispersing the PAD4 inhibitor and polydopamine nanoparticles in water. The incubation temperature is preferably room temperature, and the incubation time is preferably 12 to 24 hours. The incubation is preferably carried out on a shaker. After the incubation is completed, an incubation reaction liquid is obtained. In the present invention, the incubation reaction liquid is preferably subjected to solid-liquid separation, and the resulting solid phase product is sequentially washed and dried to obtain the polydopamine nanoparticles loaded with the PAD4 inhibitor. In the present invention, the solid-liquid separation is preferably performed by centrifugation. The number of washes is preferably two. The drying is preferably freeze-drying.

[0045] After obtaining polydopamine nanoparticles loaded with a PAD4 inhibitor, the present invention mixes the polydopamine nanoparticles loaded with a PAD4 inhibitor, carboxymethyl chitosan, and water to obtain the near-infrared responsive hydrogel preparation. In the present invention, the mass ratio of the polydopamine nanoparticles loaded with a PAD4 inhibitor and the carboxymethyl chitosan is preferably (15-20):70, and in the embodiment, it can be 18.4:70. The water is preferably deionized water. The present invention has no special requirements for the amount of water used. The temperature of the mixing is preferably room temperature. The mixing preferably includes stirring and mixing and standing in sequence. The stirring and mixing time is preferably 5-10 minutes. The standing time is preferably 10-15 minutes.

[0046] The present invention provides the use of the near-infrared responsive hydrogel preparation described in the above technical solution or the near-infrared responsive hydrogel preparation prepared by the preparation method described in the above technical solution in the preparation of a drug for promoting wound healing.

[0047] In the present invention, the drug for promoting wound healing is preferably a drug for promoting skin wound healing.

[0048] In the present invention, the drug for promoting wound healing is preferably a drug for promoting the healing of chronic wounds of the skin.

[0049] In the present invention, the drug for promoting wound healing is preferably a drug for promoting diabetic wound healing. Diabetic wounds preferably include diabetic ulcers.

[0050] In the present invention, the near-infrared responsive hydrogel is preferably used under near-infrared irradiation. The wavelength of the near-infrared irradiation is 808 nm. The power of the near-infrared irradiation is preferably 0.5 to 1.5 W, more preferably 1 W.

[0051] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0052] The experimental materials and instruments used in the following examples include:

[0053] (1) Experimental materials and reagents

[0054] Dichloromethane (DCM), methanol (CH3OH), ethyl acetate (EA), petroleum ether (PE), and anhydrous ethanol were purchased from Beijing Reagent Company (analytical grade); N-tert-butyloxycarbonyl-N'-benzyloxycarbonyl-L-ornithine (Boc-Orn(cbz)-OH) was purchased from Sigma-Aldrich Trading Co., Ltd.; benzylamine was purchased from Aladdin Reagent Co., Ltd.; N,N-diisopropylethylamine (DIPEA) was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; 2-chloroacetimidate ethyl ester hydrochloride was purchased from Suzhou Amate Biotechnology Co., Ltd.; N-hydroxyphenyltriazole (HOBt), dicyclohexylcarbodiimide (DCC), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were purchased from Shanghai Jier Biochemical Co., Ltd.; Tetrahydrofuran (THF) was purchased from Beijing Chemical Plant; Sodium bicarbonate (NaHCO3), potassium bisulfate (KHSO4), sodium chloride (NaCl), and anhydrous sodium sulfate (NaSO4) were purchased from Beijing Chemical Plant; Palladium carbon (Pb / C) was purchased from Aladdin Reagent Co., Ltd.; Silica gel H (200-300 mesh) and thin layer chromatography silica gel GF254 were purchased from Qingdao Ocean Chemical Co., Ltd.; N-methylmorpholine (NMM) was purchased from Sinopharm Centralized Chemical Reagent Co., Ltd.; Dimethyl sulfoxide-d6 (DMSO-d6) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; Carboxymethyl chitosan (CMCS) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; Dialysis bag (1000D) was purchased from Beijing Kulaibo Technology Co., Ltd.

[0055] (2) Experimental instruments

[0056] Constant-temperature magnetic stirrer: RCTbasic, IKA, Germany; electronic balances: BS 423S, Max 220 g, d = 0.001 g, Sartorius, Germany; BS124S, Max 120 g, d = 0.0001 g, Sartorius, Germany; portable UV lamp: WFH-204B, Shanghai Jingke Industrial Co., Ltd., China; circulating multi-purpose vacuum pump: SHB-S III, Zhengzhou Great Wall Technology Industry and Trade Co., Ltd., China; rotary evaporator: Laborota 4000, Heidolph, Germany; ultrasonic cleaner: KQ-500E, Kunshan Ultrasonic Instrument Co., Ltd., China; water purifier: Milli-q Integral, Merck, France; mass spectrometer: Micromass Quattro micro™ API, Waters, USA; nuclear magnetic resonance spectrometer: Avance II 300 MHz NMR, Bruker, Germany.

[0057] (3) Solution preparation

[0058] Sodium bicarbonate (NaHCO3) was prepared into a saturated solution; potassium bisulfate (KHSO4) was prepared into a saturated solution; sodium chloride (NaCl) was prepared into a saturated solution; thin layer chromatography silica gel GF254 was added with a 7% sodium carboxymethyl cellulose (CMC-Na) aqueous solution (homemade) and mixed evenly, and the air was expelled to prepare a silica gel small plate TLC.

[0059] Example 1

[0060] This embodiment provides a method for preparing CMCS-PDANPs-4B gel, which specifically includes the following steps:

[0061] (1) According to Figure 1 The synthetic route shown is to prepare 4-carboxyphenylboronic acid-Orn(Cl)-NBzl (i.e., PAD4 inhibitor 4B): Figure 1 i) DCC, HOBt, NMM, THF; ii) 4M HCl / EA; iii) DCC, HOBt, NMM, THF; iv) H2, Pd / C; v) DIPEA, MeOH.

[0062] 1) Preparation of Boc-Orn(cbz)-Nbzl

[0063] Dissolve 3.664 g (10 mmol) of N-tert-butyloxycarbonyl-N'-benzyloxycarbonyl-L-ornithine (Boc-Orn(cbz)-OH) in 80 mL of anhydrous tetrahydrofuran (THF). Add 1.458 g (11 mmol) of N-hydroxybenzotriazole (HoBt) and 2.226 g (11 mmol) of dicyclohexylcarbodiimide (DCC) under ice-cooling. Stir to dissolve. Remove the ice-cooling bath and activate at room temperature for half an hour. Add 1.179 g (11 mmol) of benzylamine to the reaction mixture under ice-cooling. Adjust the pH to 9 with N-methylmorpholine (NMM). Remove the ice-cooling bath and allow the reaction to proceed overnight at room temperature. The reaction was monitored by TLC (volume ratio: dichloromethane:methanol = 20:1). After completion of the reaction, the mixture was filtered under reduced pressure to remove dicyclohexylurea (DCU). The filtrate was then dried under reduced pressure, reconstituted with ethyl acetate (EA), and added to a separatory funnel. The organic layer was washed three times with saturated aqueous NaHCO₃, saturated aqueous NaCl, saturated aqueous KHSO₄, saturated aqueous NaCl, saturated aqueous NaHCO₃, and saturated aqueous NaCl. The organic phase was separated and dried over anhydrous NaSO₄ for 2 h. The desiccant was removed by filtration under reduced pressure, and the filtrate was concentrated to dryness to obtain the crude product Boc-Orn(cbz)-Nbzl. The crude product was purified by column chromatography (gradient elution with dichloromethane and methanol) to obtain Boc-Orn(cbz)-Nbzl, weighing 3.9 g, with a yield of 86.28%.

[0064] 2) Preparation of HCl·H-Orn(Cbz)-NBzl

[0065] 1.6 g (3.5 mmol) of Boc-Orn(cbz)-Nbzl was dissolved in 15 mL of dry 4N HCl / EtOAc under ice-cooling. A drying tube was placed at the bottom of the bottle and the reaction was continued under ice-cooling. The reaction was monitored by TLC (dichloromethane:methanol = 20:1). After the reaction was complete (i.e., disappearance of the Boc-Orn(cbz)-Nbzl spot), the solvent was removed by water pump, dry ethyl acetate was added, and the reaction was again pumped dry. This was repeated three times to obtain HCl·H-Orn(Cbz)-NBzl, weighing 1.23 g, with a yield of 89.05%.

[0066] 3) Preparation of 4-Carboxyphenylboronic acid-Orn(Cbz)-NBzl

[0067] Dissolve 4.30 g (11 mmol) of 4-carboxyphenylboronic acid in 80 mL of anhydrous tetrahydrofuran (THF). Add 1.458 g (11 mmol) of N-hydroxybenzotriazole (HoBt) and 2.226 g (11 mmol) of dicyclohexylcarbodiimide (DCC) under ice-cooling. Stir to dissolve. Remove the ice-cooling bath and activate at room temperature for half an hour. Add 1.66 g (10 mmol) of HCl·H-Orn(Cbz)-NBzl to the reaction mixture under ice-cooling. Adjust the pH to 9 with N-methylmorpholine (NMM). Remove the ice-cooling bath and allow the reaction to proceed overnight at room temperature. The reaction was monitored by TLC (dichloromethane:methanol, volume ratio = 20:1). After completion (i.e., disappearance of the HCl·H-Orn(Cbz)-NBzl spot), the product was filtered under reduced pressure to remove dicyclohexylurea (DCU). The filtrate was then dried under reduced pressure, reconstituted with ethyl acetate (EA), and added to a separatory funnel. The organic layer was washed three times with saturated aqueous NaHCO3, saturated aqueous NaCl, saturated aqueous KHSO4, saturated aqueous NaCl, saturated aqueous NaHCO3, and saturated aqueous NaCl. The organic phase was separated and dried over anhydrous NaSO4 for 2 h. The desiccant was removed by filtration under reduced pressure, and the filtrate was concentrated to dryness to obtain the crude product 4-carboxyphenylboronic acid-Orn(Cbz)-NBzl. The crude product was purified by column chromatography (dichloromethane & methanol gradient elution, specifically using a dichloromethane-methanol system with a volume ratio of dichloromethane to methanol of 1:99, 5:95, 10:90, and 15:85 for elution, C18 column) to obtain 4-carboxyphenylboronic acid-Orn(Cbz)-NBzl, weighing 4.48 g, with a yield of 89.07%.

[0068] 4) Preparation of 4-carboxyphenylboronic acid-Orn-NBzl

[0069] Dissolve 1.51 g (3 mmol) of 4B-Orn(cbz)-Nbzl in an appropriate amount of methanol, add 0.151 g of Pd / C, remove air from the reaction flask using a pump, and then introduce hydrogen via a hydrogen line. Keep the hydrogen line connected and allow the reaction to proceed at room temperature. Monitor the reaction by TLC (methylene chloride:methanol = 20:1, by volume). After completion (i.e., disappearance of 4B-Orn(cbz)-Nbzl spots), remove the Pd / C by filtration under reduced pressure. The filtrate is then concentrated to dryness under reduced pressure to yield 0.83 g of 4-carboxyphenylboronic acid-Orn-NBzl, with a yield of 74.77%.

[0070] 5) Preparation of 4-Carboxyphenylboronic acid-Orn(Cl)-NBzl (4B)

[0071] 1.85 g (5 mmol) of 4B-Orn-Nbzl was dissolved in an appropriate amount of anhydrous methanol by stirring. 3.92 g (25 mmol) of ethyl 2-chloroacetimidate hydrochloride was added under ice-cooling. The pH was adjusted to 10 with N,N-diisopropylethylamine (DIPEA). The reaction was allowed to react at room temperature for 10 h and monitored by TLC (volume ratio: methanol: water: glacial acetic acid = 10 mL: 1 mL: 2 drops). After completion of the reaction, the solvent was evaporated to obtain the crude product 4-carboxyphenylboronic acid-Orn(Cl)-NBzl. Purification by column chromatography (methanol & water gradient elution, using a dichloromethane-methanol system with a volume ratio of 1:99, 5:95, 10:90, and 15:85, sequentially, on a C18 column) yielded 4-carboxyphenylboronic acid-Orn(Cl)-NBzl (4B), weighing 1.87 g with a yield of 84.20%. ESI-MS (m / z): 445.2 [M+H] + ; 1 H-NMR (300MHz, DMSO-d6): δ (ppm) = 9.59 (s, 1H), 8.71-8.56 (m, 2H), 8.23 ​​(s, 1H), 7.90 (q, J = 7.9Hz, 4H), 7.26 (dt, J = 10.7, 7.5Hz, 5H), 4.6 0-4.39(m,1H),4.47(s,2H),4.30(d,J=5.6Hz,2H),3.58-3.51(m,2H),1.89(dq,J=12.6,7.1Hz,2H),1.65(ddt,J=23.8,15.8,7.1Hz,2H).

[0072] Figure 2 This is the mass spectrum of 4B prepared in Example 1. Figure 3 4B prepared in Example 1 1 HNMR spectrum (300MHz, DMSO-d6). Figure 2 )and 1 H-NMR ( Figure 3 ) were used for structural identification, which showed that the structure of 4B prepared in this example was correct.

[0073] (2) According to Figure 4 The preparation process shown in the figure is used to prepare polydopamine nanoparticles (PDANPs), which specifically includes the following steps:

[0074] To a mixture of 2.4 mL of ethanol and 5.4 mL of deionized water, add 450 μL of 25% NH₃·H₂O and stir to mix thoroughly. Weigh 30 mg of dopamine hydrochloride and dissolve it in 600 μL of deionized water. Slowly add the solution dropwise to the mixture and allow to react at room temperature for 12 hours. Dialyze the mixture using a 10 kDa dialysis bag for 48 hours, then freeze-dry to remove the solvent to obtain polydopamine nanoparticles (PDANPs).

[0075] (3) Preparation of PDA NPs-CMCS smart hydrogel loaded with PAD4 inhibitor 4B (4B-PDA NPs-CMCS, Gel-4B), specifically comprising the following steps:

[0076] 10 mg of 4B (4-carboxyphenylboronic acid-Orn(Cl)-NBzl) and 10 mg of polydopamine nanoparticles (PDANPs) were added to a 1.5 mL centrifuge tube, 1 mL of deionized water was added, and the mixture was incubated overnight on a laboratory shaker. The supernatant was removed by centrifugation, reconstituted with deionized water, and centrifuged again. This process was repeated twice before freeze-drying to obtain polydopamine nanoparticles (PDANPs) loaded with 4B (4-carboxyphenylboronic acid-Orn(Cl)-NBzl), referred to as 4B-PDANPs. 18.4 mg of the freeze-dried 4B-PDANPs and 70 mg of carboxymethyl chitosan (CMCS) were added to a 1.5 mL centrifuge tube, 1 mL of deionized water was added, and the mixture was stirred for 5 minutes and allowed to stand for 10 minutes to obtain a 4B-PDANPs-CMCS hydrogel.

[0077] Comparative Example 1:

[0078] This comparative example provides the preparation of PDANPs-CMCS (Gel), which specifically includes the following steps:

[0079] 10 mg of polydopamine nanoparticles (PDANPs) prepared in Example 1 and 70 mg of carboxymethyl chitosan were added to a 1.5 mL centrifuge tube, 1 mL of deionized water was added, and the mixture was stirred for 5 min and then allowed to stand for 10 min to obtain a non-drug-loaded PDANPs-CMCS hydrogel system.

[0080] Test Example 1: Structural Identification and Characterization of Drugs

[0081] The experimental materials and instruments used in this test example include: experimental reagents and consumables include PBS buffer, centrifuge tubes, and silicon wafers; experimental instruments include a laser nanoparticle size analyzer: Nano-ZS90, Malvern, a scanning electron microscope: S-4800, Hitachi, and an 808nm laser: MHPL-808-5-MM-B.

[0082] The experimental method of this test case includes

[0083] (1) Zeta potential and particle size characterization

[0084] Appropriate amounts of 4B, PDANPs, Gel, and Gel-4B were weighed and diluted to a desired concentration with ultrapure water. Ultrasonic dispersion was then performed to uniformly disperse the sample. 1 mL of the sample was precisely pipetted into a dedicated test dish and placed in a Malvern laser nanoparticle size analyzer. The particle size and zeta potential of the sample solution were measured and plotted. The data were tabulated and summarized in Table 1.

[0085] Table 1 Zeta potential and particle size of different structures

[0086] Object Zeta(mV) Particle size (nm) 4B 15.72 1255±121.5 PDANPs -37.36 765.3±143.6 Gel -38.11 397.2±136.3 Gel-4B -32.1 482.4±105.2

[0087] The analytical test results in Table 1 show that 4B has a zeta potential of +15.72 mV (positive charge), which is due to its surface containing cationic groups such as imino groups (-NH-). The zeta potentials of PDANPs, Gel, and Gel-4B are all negative, due to the presence of anionic groups on their surfaces, such as carboxyl groups (-COOH), hydroxyl groups (-OH), or the phenolic hydroxyl groups of polydopamine (PDA). The zeta potential of Gel-4B (-32.1 mV) is closer to zero than that of Gel (-38.11 mV), presumably due to the partial neutralization of the negative charge of Gel by the positively charged groups of 4B. This suggests that the two may bind through electrostatic interactions, leading to 4B adsorption into the Gel. The larger particle size of 4B in Table 1 is due to its poor water solubility, resulting in aggregation in water and a larger measured particle size. The smaller particle size of Gel-4B indicates that 4B is more evenly dispersed in the gel system.

[0088] The absolute value of the potential of 4B is only 15.72 mV, indicating weak electrostatic repulsion and easy aggregation due to van der Waals forces, consistent with its maximum particle size (1255 nm). After drug loading to form Gel-4B, the absolute value of the zeta potential is high (>30 mV), indicating strong electrostatic repulsion between particles, good dispersion stability, and resistance to agglomeration.

[0089] (2) Scanning electron microscopy (SEM) characterization

[0090] Figure 5 is the scanning electron microscopy image of PDANPs; Figure 6 This is a scanning electron microscope image of carboxymethyl chitosan. Figure 5 It can be seen that the polydopamine nanoparticles present a relatively regular spherical morphology, with a relatively uniform size distribution, and the particle size is mostly concentrated around 80nm. Figure 6 As can be seen from the left picture, carboxymethyl chitosan has a continuous and flexible three-dimensional network structure after forming a gel. From the details, the skeleton of the hydrogel is composed of fibrous structures of varying thicknesses interwoven with each other. The surface of these fibrous structures is relatively smooth, and the thickness uniformity fluctuates within a certain range. Figure 6 The right image shows a large number of pores distributed throughout the network structure, each of which is approximately circular in shape. This complex porous structure gives the carboxymethyl chitosan hydrogel a large specific surface area, offering potential advantages for applications such as adsorption and material transport. Furthermore, this structural characteristic also imparts good flexibility and mechanical strength, enabling it to maintain a certain degree of morphological stability.

[0091] (3) Investigation of drug loading rate

[0092] 4B and PDANPs were mixed at different feed ratios to form drug-loaded nanoparticles (4B not loaded on the nanoparticles was removed by centrifugation and washed three times). The 4B content was measured by high-performance liquid chromatography, and the drug loading efficiency was calculated, as shown in Table 2.

[0093] Table 2 Screening of feed ratios of PDANPs loaded with 4B

[0094]

[0095]

[0096] One-way ANOVA and Tukey's post hoc test showed that when the 4B input amount was in the range of 250μg to 10000μg, there was no statistically significant difference in the drug loading efficiency (DLE) (P>0.05); however, when the 4B input amount was 10000μg, the drug loading content (DLC) per unit carrier reached its peak. Based on the above results, subsequent experiments selected a mass feed ratio of 4B to PDANPs of 1:1 for preparation. After preparation, the actual mass ratio of 4B to PDANPs obtained was 0.843:1 (the dosage was converted based on the actual content of 4B).

[0097] (4) Photothermal effect analysis

[0098] In the experiment to explore the photothermal effect of thermosensitive hydrogel, the gel prepared in Comparative Example 1 was irradiated with 808nm near-infrared light of different intensities for 15 minutes, and the temperature was recorded every 15 seconds with a thermal imager to draw a temperature change curve.

[0099] Figure 7 This is the photothermal effect of Gel. Figure 7Experimental data showed that when the near-infrared light intensity was 1W, the gel temperature could be stably maintained at a maximum of around 40°C. This temperature level effectively promoted the antibacterial properties without causing a strong burning sensation, ensuring good safety and applicability in biological applications. Based on this, subsequent experiments used a near-infrared light intensity of 1W as the standard condition, laying the foundation for in-depth research on the properties and applications of thermosensitive hydrogels.

[0100] Test Example 2: In vitro release study

[0101] In the in vitro drug release experiment, starting from 0 minutes, the near-infrared light source was turned on with an intensity of 1W, and the Gel-4B was continuously and stably irradiated for 10 minutes. After 10 minutes, the near-infrared light was turned off, and the Gel-4B entered a 20-minute phase without near-infrared light irradiation. This was repeated three times. The drug release amount at different time points was measured and recorded, and a time-drug release curve was plotted, as shown in Figure 2. Figure 8 shown. Figure 8 This is the in vitro release curve of Gel-4B (4B release amount). Figure 8 The results showed that the drug was released rapidly when exposed to light, while the drug release slowed down in the absence of light. These results confirm that the gel system has near-infrared light-responsive drug release properties, and its release behavior can be dynamically controlled by external light stimulation, showing clear application potential in on-demand drug delivery systems.

[0102] Test Example 3: In vitro activity evaluation

[0103] The present invention used the MTT assay to evaluate the cytotoxic activity of Gel and Gel-4B, using human skin fibroblasts (HSF) and human immortalized keratinocytes (HaCaT) for activity evaluation. The present invention then used a 96-well plate antimicrobial susceptibility test to evaluate the antibacterial effects of Gel and Gel-4B against Escherichia coli (E. coli) and Staphylococcus aureus (SA).

[0104] 1 In vitro cytotoxicity test

[0105] The MTT assay is a cell viability assay widely used in fields such as cell biology, pharmacology, and toxicology. Its core principle is to indirectly reflect cell survival and proliferation by detecting succinate dehydrogenase (SDH) activity in the mitochondria of living cells. MTT, chemically known as 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, is a yellow, water-soluble compound that is reduced by mitochondrial enzymes within living cells to water-insoluble purple formazan particles.

[0106] 1.1 Experimental Materials and Instruments

[0107] (1) Experimental materials and consumables

[0108] 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Tianjin Xiens Biochemical Technology Co., Ltd., China;

[0109] DMEM medium (including double antibodies) was purchased from Jiangsu KeyGen Biotechnology Co., Ltd., China;

[0110] PBS buffer was purchased from Jiangsu KeyGen Biotech Co., Ltd., China;

[0111] Fetal bovine serum (FBS, 35081006, Fetal Bovine Serum New Zealand Origin) was purchased from Corning, China;

[0112] Trypsin-EDTA digestion solution: KGY0012 was purchased from Jiangsu KeyGen Biotech Co., Ltd., China;

[0113] Dimethyl sulfoxide (DMSO, CAS No.: 67-68-5) was purchased from Sigma, USA;

[0114] Cryotubes, centrifuge tubes, 96-well cell culture plates, and culture flasks were purchased from Wuhan Sevier Biotechnology Co., Ltd., China;

[0115] The straws were purchased from Zhejiang Pamet Medical Plastics Co., Ltd.

[0116] (2) Experimental instruments

[0117] Automatic high-pressure steam sterilizer: SX-700, TOMY, USA;

[0118] Centrifuge: TDZ5-WS, Hunan Pingfan Technology Co., Ltd., China;

[0119] Ultrapure water machine: Milli-QA10, MERCK (MILLIPORE), USA;

[0120] Biological safety cabinet: 1300SERIESA2, Thermo, USA;

[0121] Inverted microscope: Zeiss, Germany;

[0122] Water bath: YLE-1000, Beijing Tianyou Technology Development Co., Ltd., USA;

[0123] Incubator: HERACELL 160i, Thermo, USA;

[0124] Microplate reader: Molecular Devices SpectraMax M3, Thermo, USA.

[0125] (3) Preparation of 5 mg / mL MTT solution

[0126] Pre-sterilize the sterile workstation with UV light for 30 minutes. Place all necessary pipettes, pipette tips, and electronic balance weighing paper on the workstation to ensure a sterile working environment. Use an electronic balance to accurately weigh 50 mg of MTT powder. Weigh as quickly as possible to avoid exposing the MTT powder to air for extended periods. Transfer the weighed MTT powder to a sterile EP tube. Use a graduated cylinder to measure 10 mL of PBS buffer and slowly add it to the EP tube containing the MTT powder. Wrap the EP tube in tin foil and sonicate to fully dissolve the MTT powder. This process may take 10-15 minutes. Observe carefully during this time to ensure that the MTT is completely dissolved and the solution is a uniform yellow color. Filter the dissolved MTT solution through a 0.22 μm filter membrane to sterilize. You can use a syringe connected to the filter membrane to slowly inject the solution into a sterile brown reagent bottle. The purpose of filtration is to remove bacteria and impurities from the solution and ensure the sterility of the solution. Store the filtered solution in a refrigerator at 4°C until needed.

[0127] (4) Test samples

[0128] Control group: PBS buffer;

[0129] Experimental Group: Gel-4B (based on the amount of 4B contained therein) was dissolved in PBS buffer to prepare a 4 mM solution, and then gradiently diluted with PBS buffer solution to prepare a maximum concentration of 100 μM. Six concentration gradients were set, and the half-dilution method was used between each concentration (according to the operation, after adding it to the well to be tested, it will be diluted 5 times by the cell fluid, so the actual concentration is 20 μM).

[0130] The concentrations of PDANPs and CMCS in the Gel group and the Gel-4B group were comparable.

[0131] (5) Cell lines

[0132] Human skin fibroblasts (HSF) and human immortalized keratinocytes (HaCaT) were cultured in DMEM medium containing 10% FBS.

[0133] 1.2 Experimental methods

[0134] The in vitro cell proliferation and toxicity of different test drugs on human skin fibroblasts (HSF) and human immortalized keratinocytes (HaCaT) were evaluated by the following methods:

[0135] To resuscitate cells: Add 4 mL of culture medium to a 15 mL centrifuge tube. Rapidly thaw the cell suspension in a 37°C warm water bath and add it to the centrifuge tube to mix thoroughly. Centrifuge for 5 minutes, discard the supernatant, add 1 mL of culture medium, and pipette evenly. Add 3-4 mL of culture medium to the culture flask, then add the cell suspension to the flask, pipette evenly and shake in a figure-8 pattern to mix thoroughly. Observe under a microscope and place in an incubator at 37°C, 5% CO2 for 24 hours. Observe cell growth under a microscope and select subculture based on cell number.

[0136] Cell passaging: Take the cell culture flask out of the incubator and observe the number and morphology under a microscope to see if it can be passaged (the cells must fill more than 80% of the culture flask). Pour out the culture medium in the culture flask, rinse twice with PBS buffer, then add 1-2 mL of trypsin and place in the incubator for digestion for about 2 minutes. Take out the culture flask and observe under a microscope whether the adherent cells have flowed. Add culture medium twice the volume of trypsin to stop digestion, mix well and add to a 15 mL centrifuge tube. Place in a centrifuge and centrifuge for 5 minutes, then discard the liquid, add 1 mL of new culture medium and pipette evenly. Add 3-4 mL of culture medium to the culture flask, take 1 mL of cell fluid and add it to the culture flask, pipette evenly and shake in an "8" shape to mix well, place in the incubator and culture at 37°C and 5% CO2 for 24 hours, then observe the cell growth under a microscope.

[0137] To seed cells: Pour off the culture medium from the flask, wash twice with PBS, and trypsinize the cells. After digestion, add twice the volume of culture medium and centrifuge. After centrifugation, discard the liquid and re-add 5 mL of culture medium, pipette well, and centrifuge again. After completion, add 1 mL of culture medium and dilute 100 μL of the cell suspension tenfold with 900 μL of culture medium. Count the cells. First, spray the cell counting plate with alcohol, wipe it clean with a cotton ball or paper towel, and place a coverslip on it. Take 10 μL of the diluted 1 mL of cell suspension and quickly inject it through the gap in the coverslip to ensure there are no bubbles. Count the cells under a microscope. Seed 3,000 to 5,000 cells per well of a 96-well plate and seal the outermost circle of the plate with PBS buffer. Incubate the seeded 96-well plate in a 37°C, 5% CO2 incubator for 4 hours.

[0138] Drug administration: After the cells adhere to the wall, add 25 μL of the prepared drug solution to each well. After adding, tap the side of the plate to confirm that the drug solution has not adhered to the wall, and then place it in the incubator for 24 hours.

[0139] Post-treatment: Add 25 μL of the prepared 5 mg / mL MTT solution to each well and incubate in a 37°C, 5% CO2 incubator for 4 hours. Remove and discard the supernatant, add 150 μL of DMSO to each well, shake on a shaker for 15 minutes, and measure the OD value of each well at 490 nm using a microplate reader.

[0140] Cell viability = (average OD value of the test drug group - average OD value of the Blank group) / (average OD value of the Blank group - average OD value of the Blank group) × 100%.

[0141] 1.3 Experimental Results and Discussion

[0142] The experiment evaluated the effects of hydrogel (Gel) and drug-loaded hydrogel (Gel-4B) on the proliferation of human epidermal immortalized cells (HaCaT) and fibroblasts (HSF). Figure 9 shown. Figure 9 This is the MTT assay for Gel and Gel-4B.

[0143] The experimental results showed that as the concentrations of Gel and Gel-4B gradually increased, they promoted the proliferation of HaCaT cells and HSF, which provided a strong experimental basis for their application in the field of wound healing.

[0144] 2 In vitro antibacterial experiments

[0145] The 96-well plate antimicrobial susceptibility test (AST) is a high-throughput assay based on broth microdilution that can determine the sensitivity (or tolerance) of pathogenic microorganisms to various antimicrobial drugs in vitro. Based on this, the present invention systematically explored its antimicrobial properties against Escherichia coli (E. coli) and Staphylococcus aureus (SA).

[0146] 2.1 Experimental Materials and Instruments

[0147] (1) Experimental materials and consumables

[0148] Tryptone and yeast extract were purchased from Oxoid Limited, UK;

[0149] Sodium chloride (NaCl) was purchased from Beijing Chemical Plant, China;

[0150] Purified agar powder was purchased from Sinopharm Chemical Reagent Co., Ltd., China;

[0151] Culture dishes were purchased from Wuhan Saiweier Biotechnology Co., Ltd., China.

[0152] (2) Experimental instruments

[0153] Automatic high-pressure steam sterilizer: SX-700, TOMY, USA;

[0154] Ultrapure water machine: Milli-QA10, MERCK (MILLIPORE), USA;

[0155] Biological safety cabinet: 1300SERIESA2, Thermo, USA;

[0156] Plate shaker: MS2 Minishaker, IKA, Germany;

[0157] Electronic balance: BS 423S, Max 220 g, d = 0.001 g, Sartorius, Germany; BS124S, Max 120 g, d = 0.0001 g, Sartorius, Germany;

[0158] Microplate reader: Molecular Devices SpectraMaxM3, Thermo, USA.

[0159] (3) Preparation of LB liquid culture medium

[0160] Use a balance to weigh 10g of tryptone, 5g of yeast extract, and 10g of sodium chloride (NaCl) into a 1L reagent bottle. Add 1L of ultrapure water and mix thoroughly by ultrasonic vibration for 15 minutes. Transfer to an automatic high-pressure steam sterilizer for sterilization. Once completed, transfer to a 4°C refrigerator for storage.

[0161] (4) Preparation of LB solid culture medium

[0162] Using a balance, weigh 10g of tryptone, 5g of yeast extract, 10g of sodium chloride (NaCl), and 20g of agar into a 1L reagent bottle. Add 1L of ultrapure water and mix thoroughly by ultrasonication for 15 minutes. Then transfer to an automatic high-pressure steam sterilizer for sterilization. After sterilization, turn on the clean bench fan, wipe your hands and countertop with 75% (v:v) alcohol, and light an alcohol burner. Remove the sterilized plate and use a sterile graduated cylinder or pipette to draw up the culture medium. Pour the medium slowly along the inner wall of the plate, avoiding bubbles. Immediately cover the plate and gently shake the plate to evenly distribute the culture medium. Place the plate horizontally on a table and let it sit until the agar has completely solidified. After solidification, label the culture medium name and date on the bottom of the plate with a marker. Invert the plate (bottom-side up) and store in a refrigerator at 4°C until ready for use.

[0163] (5) Test samples

[0164] Control group: LB liquid medium;

[0165] Experimental group: Gel-4B (based on the amount of 4B contained therein) was dissolved in LB liquid medium to prepare a 4 mM solution, and then gradiently diluted with LB liquid medium to prepare a maximum concentration of 100 μM. Six concentration gradients were set, and the half-fold dilution method was used between each concentration.

[0166] The concentrations of PDANPs and CMCS in the Gel group and the Gel-4B group were comparable.

[0167] (6) Bacterial strains

[0168] Escherichia coli (E. coli) and Staphylococcus aureus (SA) were cultured and passaged in LB liquid medium.

[0169] 2.2 Experimental methods

[0170] The in vitro antibacterial effects of different test drugs on Escherichia coli (E.coli) and Staphylococcus aureus (SA) were evaluated by the following methods:

[0171] Resuscitate bacteria: Quickly remove the glycerol tube from the -80°C freezer and thaw quickly. Using a sterile pipette, draw 50 μL of the glycerol tube into a culture tube containing 10 mL of LB liquid medium and gently mix. Incubate the tube in a 37°C incubator at 200 rpm until the logarithmic growth phase begins and the culture becomes turbid.

[0172] Bacterial subculture: Take 100 μL of the bacterial solution in the logarithmic growth phase (OD600≈0.6~1.0) and transfer it to a new 10 mL LB liquid medium.

[0173] Inoculation of bacteria: Dilute the bacterial solution in the logarithmic growth phase to an appropriate concentration, inoculate into a 96-well plate, add 100 μL of bacterial solution to each well, and seal the outermost circle of the plate with LB liquid culture medium.

[0174] Dosing: Add 100 μL of the prepared drug solution to each well. After addition, gently tap the side of the plate to ensure that the drug adheres to the wall. Measure the OD value of each well at a wavelength of 600 nm using a microplate reader as background. Then, incubate in an incubator for 24 hours. Post-treatment: Measure the OD value of each well at a wavelength of 600 nm using a microplate reader.

[0175] Antibacterial efficacy = (average OD value of the test drug group - average OD value of the test drug group background) / (average OD value of the blank group - average OD value of the blank group background) × 100%.

[0176] 2.3 Experimental Results and Discussion

[0177] In the antibacterial experiments on hydrogel (Gel) and drug-loaded hydrogel (Gel-4B), the present invention systematically explored their antibacterial properties against Escherichia coli (E. coli) and Staphylococcus aureus (SA). The experimental results are as follows: Figure 10 and Figure 11 shown. Figure 10 This is the antibacterial experiment of Gel and Gel-4B against Escherichia coli. Figure 11 This is an antibacterial experiment of Gel and Gel-4B against Staphylococcus aureus. The results show that the antibacterial efficiency of hydrogels and drug-loaded hydrogels increases significantly with increasing concentration. When the set highest concentration (4mM) is reached, the antibacterial efficiency of hydrogels and drug-loaded hydrogels is close to 100%. At this concentration, the survival of bacteria such as Escherichia coli and Staphylococcus aureus is almost unobservable. This result fully demonstrates the powerful antibacterial efficacy of hydrogels and drug-loaded hydrogels at high concentrations, providing broad prospects for their application in the antibacterial field, and is expected to be developed into a new type of highly effective antibacterial product.

[0178] Test Example 4: In vivo activity evaluation

[0179] 1 In vivo wound healing experiment

[0180] The db / db mouse is a classic model of type 2 diabetes. Due to a mutation in the leptin receptor (LepR) gene, it leads to obesity, insulin resistance, and hyperglycemia. Its wound healing ability is significantly weaker than that of wild-type mice, making it suitable for simulating chronic, non-healing wounds in diabetes. This study established a full-thickness skin defect model to evaluate the efficacy of Gel and Gel-4B on diabetic wound healing, including gross observation, statistical analysis of wound area, and histological analysis.

[0181] 1.1 Experimental Materials and Instruments

[0182] (1) Experimental reagents and consumables

[0183] The reagents and consumables required for cell culture are the same as those in Test Example 3;

[0184] 0.9wt% normal saline.

[0185] (2) Experimental instruments

[0186] The experimental instruments required for cell culture are the same as those in Test Example 3;

[0187] 1mL syringe, ophthalmic punch, ophthalmic scissors, and ophthalmic forceps.

[0188] (3) Experimental animals

[0189] SPF male db / db mice (6-8 weeks old) were purchased from Beijing Weitonglihua Animal Experiment Technology Co., Ltd. and housed in the animal barrier of Capital Medical University.

[0190] (4) Test drug

[0191] Positive control: 4B solution

[0192] Negative control: 0.9wt% NaCl solution

[0193] Test groups: Gel and Gel-4B

[0194] 1.2 Experimental methods

[0195] The experiment selected 8-week-old db / db male mice, and made wound models of the same size on their backs. The experiment used the normal saline group (NS) as the negative control, and each group was treated with different drugs. The NS group was only given an equal volume of normal saline, while the experimental group applied the same dose of 4B, hydrogel and drug-loaded hydrogel to the wound, and set up the two groups to undergo additional near-infrared light irradiation after administration. During the experimental observation period, the present invention regularly photographed and recorded the mouse wounds, and measured the wound area. After the experiment, the mouse blood was taken and the tumor necrosis factor α (TNF-α) of each group of mice was measured using an ELISA kit.

[0196] 1.3 Experimental Results and Analysis

[0197] Figure 12 In vivo wound healing experiment. Figure 12 The results showed that the wound healing rate in the Gel-4B group was significantly faster than that in the control group. Especially in the early stages of the experiment, the drug-loaded hydrogel effectively protected the wound from infection and helped promote scab formation. Over time, granulation tissue grew rapidly at the wound site, and epithelial cells rapidly proliferated to cover the wound. This experimental result demonstrates that the drug-loaded hydrogel can effectively promote wound healing in diabetic mice, providing a new potential solution for diabetic wound treatment.

[0198] Figure 13 This is the result of the TNF-α level test. The ELISA test results showed that compared with the normal saline group, the PAD4 inhibitor 4B showed good anti-inflammatory ability, and Gel-4B further enhanced its anti-inflammatory ability. The expression of TNF-α protein in the wound tissue of mice in the Gel-4B and near-infrared light irradiated Gel-4B groups was significantly reduced (P<0.001). As an important pro-inflammatory cytokine, the decrease in TNF-α levels indicates that the excessive local inflammatory response in the wounds of the experimental group has been effectively alleviated, which may be related to the intervention measures regulating the immune system and inhibiting abnormal inflammatory activation. The results preliminarily suggest that the intervention measures adopted may reduce the inflammatory damage of the wounds of diabetic mice by downregulating TNF-α expression, thereby creating a favorable microenvironment for promoting wound healing and providing a new experimental basis for the clinical treatment of diabetic refractory wounds.

[0199] From the above examples, it can be seen that the present invention successfully synthesized the PAD4 inhibitor 4B, and 1 Its structure and purity were verified by H NMR, MS, and HPLC. A near-infrared (NIR)-responsive hydrogel, Gel-4B, was constructed based on polydopamine nanoparticles (PDANPs) and carboxymethyl chitosan (CMCS). Characterization results showed that the average particle size of PDANPs was 80±5 nm, the zeta potential was +22.3 mV, and the drug loading capacity for 4B reached 84.3%. Gel-4B exhibited a stable photothermal effect under 1 W NIR light intensity, maintaining a temperature of approximately 40°C, combining the mild thermal stimulation required for antibacterial efficacy with biosafety. In vitro release experiments confirmed that the system could release drugs on demand in response to NIR light, achieving spatiotemporal controllable drug delivery. In vitro cytotoxicity experiments demonstrated that Gel-4B exhibited a viability exceeding 90% in both human skin fibroblasts (HSF) and human immortalized keratinocytes (HaCaT) at concentrations ranging from 50 to 100 μM, demonstrating excellent biocompatibility. The hydrogel significantly promoted the migration and proliferation of HaCaT cells and HSFs, providing a cytological basis for epidermal re-epithelialization and granulation tissue formation during wound healing. Antibacterial experiments revealed that Gel-4B exhibited concentration-dependent antibacterial activity against both Escherichia coli and Staphylococcus aureus, reaching nearly 100% inhibition at a high concentration (4 mM). Combined with photothermal effects, its antibacterial properties were further enhanced, providing a dual safeguard for controlling diabetic wound infection. In vivo experiments using a db / db diabetic mouse model demonstrated significantly faster wound healing in the Gel-4B-treated group compared with the control group. Gross observations, wound area statistics, and histological analyses revealed that Gel-4B accelerated epidermal re-epithelialization, promoted granulation tissue formation, and reduced levels of the inflammatory cytokine TNF-α. Its mechanism of action involves inhibiting PAD4-mediated neutrophil extracellular trap (NET) formation, modulating the local inflammatory microenvironment, and synergistically promoting extracellular matrix remodeling and angiogenesis through photothermal effects to aid antibacterial activity. These results confirm the effectiveness and multi-pathway synergistic advantages of Gel-4B in diabetic wound treatment.

[0200] In summary, the present invention combines the PAD4 inhibitor 4B with PDANPs for the first time, and uses CMCS to construct an NIR-responsive hydrogel, thereby achieving local precision delivery and light-controlled release of drugs. The present invention solves the problems of poor water solubility and insufficient targeting of traditional PAD4 inhibitors, and provides a new strategy for the delivery of small molecule drugs. The Gel-4B provided by the present invention integrates the advantages of anti-inflammatory, antibacterial, photothermal effects and biocompatibility: it reduces chronic inflammation by inhibiting the formation of NETs, ​​enhances the antibacterial effect by using the photothermal effect of PDANPs, and promotes cell adhesion and proliferation through the natural biological activity of CMCS. This multi-dimensional synergistic effect targets the core pathological mechanisms of diabetic wounds (inflammatory disorders, difficult-to-control infections, and delayed healing), and demonstrates therapeutic potential that is superior to that of single functional materials.

[0201] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A near-infrared responsive hydrogel preparation, characterized in that: The invention comprises a hydrogel matrix and drug-loaded particles loaded in the hydrogel matrix, wherein the hydrogel matrix is ​​carboxymethyl chitosan, and the drug-loaded particles comprise polydopamine nanoparticles loaded with a PAD4 inhibitor; the PAD4 inhibitor has a structure shown in Formula 1:

2. The near-infrared responsive hydrogel preparation according to claim 1, characterized in that The mass ratio of the PAD4 inhibitor to the polydopamine nanoparticles is ≤0.85:

1.

3. The near-infrared responsive hydrogel preparation according to claim 1 or 2, characterized in that The particle size of the near-infrared responsive hydrogel preparation is 350-600 nm.

4. The near-infrared responsive hydrogel preparation according to claim 1 or 2, characterized in that The absolute value of the zeta potential of the near-infrared responsive hydrogel preparation is greater than 30 mV.

5. The method for preparing the near-infrared responsive hydrogel preparation according to any one of claims 1 to 4, characterized in that: The following steps are involved: The PAD4 inhibitor, polydopamine nanoparticles and water are mixed and incubated to obtain polydopamine nanoparticles loaded with the PAD4 inhibitor; The polydopamine nanoparticles loaded with the PAD4 inhibitor, carboxymethyl chitosan and water are mixed to obtain the near-infrared responsive hydrogel preparation.

6. The preparation method according to claim 5, characterized in that The mass ratio of the PAD4 inhibitor to the polydopamine nanoparticles is ≤1:

1.

7. The preparation method according to claim 5, characterized in that The mass ratio of the polydopamine nanoparticles loaded with the PAD4 inhibitor to the carboxymethyl chitosan is (15-20):

70.

8. Use of the near-infrared responsive hydrogel preparation according to any one of claims 1 to 4 or the near-infrared responsive hydrogel preparation prepared by the preparation method according to any one of claims 5 to 7 in the preparation of a drug for promoting wound healing.

9. The use according to claim 8, characterized in that The medicine for promoting wound healing is a medicine for promoting skin wound healing.

10. The use according to claim 8 or 9, characterized in that: The medicine for promoting wound healing is a medicine for promoting diabetic wound healing.

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