A temperature-sensitive PAD4 inhibitor-loaded hydrogel and a preparation method and application thereof

Abstract

The application provides a temperature-sensitive PAD4 inhibitor-loaded hydrogel as well as a preparation method and application thereof, and belongs to the technical field of biological medicines.The temperature-sensitive PAD4 inhibitor-loaded hydrogel comprises a temperature-sensitive hydrogel base and a PAD4 inhibitor loaded in the temperature-sensitive hydrogel base; the components of the temperature-sensitive hydrogel base comprise chitosan and beta-glycerophosphate disodium; and the PAD4 inhibitor has a structure shown in formula 1.The PAD4 inhibitor is loaded in the hydrogel, the PAD4 inhibitor is slowly released through local application, the formation of NETs is inhibited for a long time, the chronic inflammation is reduced, and finally the healing process of a diabetic wound is accelerated.The temperature-sensitive PAD4 inhibitor-loaded hydrogel provided by the application shows a significant therapeutic effect in experiments, and overcomes the limitations of traditional drug treatment to a certain extent.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a thermosensitive hydrogel carrying a PAD4 inhibitor, its preparation method, and its application. Background Technology

[0002] Peptidylarginine deiminase 4 (PAD4) is a calcium-dependent enzyme responsible for deiminizing arginine residues in proteins to produce citrulline. This enzyme is expressed in various immune cells, including neutrophils, monocytes, and macrophages. One of PAD4's best-known functions is its involvement in the formation of extracellular traps (NETs) on neutrophils. NETs are a network of DNA and proteins that traps and kills pathogens. Under normal circumstances, NETs are an important component of the immune system, preventing infection by capturing and neutralizing pathogens. However, excessive NET formation can damage host tissues, leading to inflammation and tissue damage. In the diabetic setting, NETs are often over-formed, further exacerbating chronic inflammation and closely associated with poor wound healing. Studies have found that PAD4 protein expression in neutrophils of diabetic patients is upregulated fourfold, and NET levels in blood and wound tissue are significantly elevated. In the diabetic setting, PAD4 exacerbates the inflammatory response by promoting NET formation. Upon PAD4 activation, histones in neutrophils undergo deiminolation, chromatin loosens, and nuclear DNA is released, forming NETs. This process plays a positive role in anti-infection, but in the wounds of diabetic patients, excessive accumulation of NETs can actually delay healing. Existing PAD4 inhibitors, such as F-amidine, Cl-amidine, and TDFA, generally have limited specificity and also exhibit some inhibitory effects on other members of the PAD family, potentially leading to organ toxicity. Summary of the Invention

[0003] In view of this, the purpose of this invention is to provide a thermosensitive PAD4 inhibitor-loaded hydrogel, its preparation method, and its application. The thermosensitive PAD4 inhibitor-loaded hydrogel provided by this invention can significantly accelerate the healing of diabetic wounds.

[0004] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0005] This invention provides a thermosensitive hydrogel loaded with a PAD4 inhibitor, comprising a thermosensitive hydrogel matrix and a PAD4 inhibitor loaded in the thermosensitive hydrogel matrix;

[0006] The thermosensitive hydrogel matrix comprises chitosan and disodium β-glycerophosphate;

[0007] The PAD4 inhibitor has the structure shown in Formula 1:

[0008]

[0009] Preferably, the mass ratio of chitosan to disodium β-glycerophosphate is 1:2 to 4.

[0010] Preferably, the loading of PAD4 inhibitor in the temperature-sensitive PAD4 inhibitor-loaded hydrogel is 0.1–10 mg / mL.

[0011] This invention provides a method for preparing the above-mentioned temperature-sensitive PAD4 inhibitor-loaded hydrogel, comprising the following steps:

[0012] Chitosan was mixed with hydrochloric acid solution to obtain a chitosan solution;

[0013] The chitosan solution, β-glycerophosphate disodium and PAD4 inhibitor solution were mixed to obtain a thermosensitive PAD4 inhibitor-loaded hydrogel.

[0014] Preferably, the concentration of the hydrochloric acid solution is 1 mol / L;

[0015] The chitosan solution contains 2-3% chitosan by mass.

[0016] Preferably, the β-glycerophosphate disodium is added in solution form, and the mass concentration of the β-glycerophosphate disodium solution is 30-60%.

[0017] Preferably, the concentration of the PAD4 inhibitor solution is 10-100 mg / mL, and the solvent is PBS buffer.

[0018] Preferably, the mixing is vortex mixing, and the rotation speed of the vortex mixing is 1000-3000 rpm, and the time is 1-10 min.

[0019] This invention provides the application of the above-mentioned thermosensitive PAD4 inhibitor-loaded hydrogel in the preparation of diabetic wound dressings.

[0020] The present invention provides a diabetic wound dressing comprising the above-mentioned thermosensitive PAD4 inhibitor-loaded hydrogel and pharmaceutically acceptable excipients.

[0021] This invention provides a thermosensitive PAD4 inhibitor-loaded hydrogel, comprising a thermosensitive hydrogel matrix and a PAD4 inhibitor loaded within the thermosensitive hydrogel matrix; the thermosensitive hydrogel matrix comprises chitosan and disodium β-glycerophosphate; the PAD4 inhibitor has the structure shown in Formula 1 (denoted as PAD4 inhibitor YJ-2). The PAD4 inhibitor YJ-2 in this invention exhibits stronger specificity for PAD4. Loading it into the hydrogel allows for local application and slow release of the PAD4 inhibitor, thereby inhibiting the formation of NETs for a prolonged period, reducing chronic inflammation, and ultimately accelerating the healing process of diabetic wounds. The thermosensitive PAD4 inhibitor-loaded hydrogel provided by this invention has demonstrated significant therapeutic effects in experiments, overcoming to some extent the limitations of traditional drug treatments. Furthermore, the thermosensitive PAD4 inhibitor-loaded hydrogel provided by this invention is safe, convenient, and highly effective, with good biocompatibility. It can transform from a liquid to a solid gel at body temperature (37°C) and release the drug sustainably at the wound site, providing a more effective treatment option for diabetic patients. Attached Figure Description

[0022] Figure 1 The proton NMR spectrum of the PAD4 inhibitor YJ-2;

[0023] Figure 2 The UV spectrum of the PAD4 inhibitor YJ-2;

[0024] Figure 3 These are photographs of the hydrogels obtained in Example 1 and Comparative Example 1 at different temperatures.

[0025] Figure 4 These are scanning electron microscope images of the hydrogels obtained in Example 1 and Comparative Example 1;

[0026] Figure 5 Fourier transform infrared spectra of the hydrogels obtained from YJ-2, Example 1, and Comparative Example 1;

[0027] Figure 6 The drug release curve of the drug-loaded thermosensitive CS / β-GP hydrogel;

[0028] Figure 7 The results of MTT assays for YJ-2 with different hydrogels;

[0029] Figure 8 Images of HaCaT cells migrating at 12 hours;

[0030] Figure 9 The healing rate of YJ-2-induced migration scratch test;

[0031] Figure 10 Confocal images for anti-NETs experiments;

[0032] Figure 11 Here is a fluorescence intensity diagram of H3cit;

[0033] Figure 12 A statistical graph showing the wound area of ​​mice treated with hydrogel. Detailed Implementation

[0034] The present invention provides a thermosensitive hydrogel loaded with a PAD4 inhibitor, comprising a thermosensitive hydrogel matrix and a PAD4 inhibitor loaded in the thermosensitive hydrogel matrix;

[0035] The thermosensitive hydrogel matrix comprises chitosan and disodium β-glycerophosphate;

[0036] The PAD4 inhibitor has the structure shown in Formula 1, and is denoted as PAD4 inhibitor YJ-2:

[0037]

[0038] The present invention does not have any special requirements on the source of the PAD4 inhibitor YJ-2; commercially available PAD4 inhibitor YJ-2 or self-prepared inhibitors can be used.

[0039] In this invention, the viscosity of the chitosan is preferably 100-400 mPa·s, more preferably 200-300 mPa·s; the mass ratio of the chitosan to β-glycerophosphate is preferably 1:2-4, more preferably 1:3-4. At this mass ratio, the hydrogel has good fluidity and a suitable gelation time.

[0040] In this invention, the loading of PAD4 inhibitor in the thermosensitive PAD4 inhibitor-loaded hydrogel is preferably 0.1-10 mg / mL, more preferably 1-8 mg / mL, further preferably 2-6 mg / mL, and even more preferably 3-5 mg / mL.

[0041] This invention provides a method for preparing the above-mentioned temperature-sensitive PAD4 inhibitor-loaded hydrogel, comprising the following steps:

[0042] Chitosan was mixed with hydrochloric acid solution to obtain a chitosan solution;

[0043] The chitosan solution, β-glycerophosphate disodium and PAD4 inhibitor solution were mixed to obtain a thermosensitive PAD4 inhibitor-loaded hydrogel.

[0044] This invention involves mixing chitosan with a hydrochloric acid solution to obtain a chitosan solution. Preferably, the concentration of the hydrochloric acid solution is 1 mol / L, and the mass concentration of chitosan in the chitosan solution is 2-3%. This invention does not impose any special requirements on the mixing method; any mixing method well-known in the art can be used, such as stirring.

[0045] After obtaining the chitosan solution, the present invention mixes the chitosan solution, β-glycerophosphate disodium, and PAD4 inhibitor solution to obtain a temperature-sensitive PAD4 inhibitor-loaded hydrogel. In this invention, the β-glycerophosphate disodium is preferably added in solution form, and the solvent is preferably deionized water; the mass concentration of the β-glycerophosphate disodium solution is preferably 30-60%, more preferably 50%. In this invention, the concentration of the PAD4 inhibitor solution is preferably 10-100 mg / mL, more preferably 20-80 mg / mL, more preferably 40-60 mg / mL, and the solvent of the PAD4 inhibitor solution is preferably PBS buffer.

[0046] Before mixing, the present invention preferably places the chitosan solution, β-glycerophosphate disodium and PAD4 inhibitor solution in an ice bath for cooling, and the cooling time in the ice bath is preferably 5 minutes. This cooling method ensures that the hydrogel remains liquid during the preparation process.

[0047] In this invention, the volume ratio of the chitosan solution, β-glycerophosphate disodium solution, and PAD4 inhibitor solution is preferably 5 to 10:1:1.

[0048] In this invention, the mixing is preferably vortex mixing, the rotation speed of the vortex mixing is preferably 1000-3000 rpm, the time is preferably 1-10 min, more preferably 2-8 min, and even more preferably 4-6 min.

[0049] This invention provides the application of the above-mentioned thermosensitive PAD4 inhibitor-loaded hydrogel in the preparation of diabetic wound dressings.

[0050] This invention provides a diabetic wound dressing comprising the aforementioned thermosensitive PAD4 inhibitor-loaded hydrogel and pharmaceutically acceptable excipients. This invention does not impose any special requirements on the type of excipients; excipients conventionally used in the art can be employed.

[0051] The following detailed description, in conjunction with embodiments, illustrates the thermosensitive PAD4 inhibitor-loaded hydrogel, its preparation method, and its applications provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0052] Example 1

[0053] (1) Synthesis of PAD4 inhibitor YJ-2

[0054] The synthetic route for the PAD4 inhibitor YJ-2 is shown in Formula A:

[0055]

[0056] In formula A, i) benzylamine, DCC, HoB; ii) hydrochloric acid gas, ice bath; iii) p-hydroxybenzoic acid, DCC, HoBt; iv) H2, Pd / C; v) 2-chloroacetyliminoethyl ester, anhydrous methanol, DIPEA.

[0057] 1. Preparation of Boc-Orn(Cbz)-NBzl:

[0058] 10 mmol of Boc-Orn(Cbz)-OH was dissolved in 20 mL of anhydrous tetrahydrofuran (THF). 12 mmol of N-hydroxybenzotriazole (HOBt) was added under ice bath conditions and allowed to dissolve completely. 12 mmol of dicyclohexylcarbonyldiimide (DCC) was slowly added and stirred for 30 min to obtain reaction solution A. 12 mmol of benzylamine was dissolved in 20 mL of anhydrous THF and added to reaction solution A under ice bath conditions. 1 mL of N-methylmorpholine (NMM) was added, and the pH was adjusted to 8–9. The mixture was stirred under ice bath conditions for 1 h, then at room temperature for 48 h. TLC (by volume ratio, dichloromethane:methanol = 20:1) was performed. 1) The Boc-Orn(Cbz)-OH layer was observed to disappear. Dicyclohexylurea (DCU) was filtered off, and THF was removed by vacuum distillation. The residue was dissolved in 50 mL of ethyl acetate (EA). The resulting solution was washed three times each with saturated NaHCO3 aqueous solution, saturated NaCl aqueous solution, saturated KHSO4 aqueous solution, saturated NaCl aqueous solution, saturated NaHCO3 aqueous solution, and saturated NaCl aqueous solution. The ethyl acetate layer was dried with anhydrous Na2SO4, and the drying agent was removed by vacuum filtration. The filtrate was concentrated to dryness under vacuum to obtain the compound Boc-Orn(Cbz)-NBzl.

[0059] 2. Preparation of HCl·H-Orn(Cbz)-NBzl:

[0060] Boc-Orn(Cbz)-NBzl (10 mmol) was dissolved in a small amount of anhydrous ethyl acetate. A 4 mol / L HCl / EtOAc solution was added with stirring in an ice bath. TLC (EA:H2O:HAc = 4:1:0.1 by volume) showed the disappearance of the starting material spot. The solution was dried by a water pump, and anhydrous ethyl acetate was added. The reaction mixture was then dried by a water pump again. This process was repeated three times to obtain HCl·H-Orn(Cbz)-NBzl.

[0061] 3. Preparation of p-hydroxybenzoic acid-Orn(Cbz)-NBzl:

[0062] 10 mmol of p-hydroxybenzoic acid was dissolved in 20 mL of anhydrous tetrahydrofuran (THF). Under ice bath conditions, 12 mmol of N-hydroxybenzotriazole (HOBt) was added and allowed to dissolve completely. Then, 12 mmol of dicyclohexylcarbonyldiimide (DCC) was slowly added and stirred for 30 min to obtain reaction solution A. Under ice bath conditions, 12 mmol of... HCl·H-Orn(Cbz)-NBzl was dissolved in 20 mL of anhydrous THF and added to reaction solution A. 1 mL of N-methylmorpholine (NMM) was added, and the pH was adjusted to 8-9. The mixture was stirred in an ice bath for 1 h, and then stirred at room temperature for 48 h. TLC (dichloromethane:methanol = 20:1 by volume) showed that p-hydroxybenzoic acid disappeared. Dicyclohexylurea (DCU) was filtered off, and THF was evaporated under reduced pressure. The residue was dissolved in 50 mL of ethyl acetate (EA). The resulting solution was washed three times each with saturated NaHCO3 aqueous solution, saturated NaCl aqueous solution, saturated KHSO4 aqueous solution, saturated NaCl aqueous solution, saturated NaHCO3 aqueous solution, and saturated NaCl aqueous solution. The ethyl acetate layer was dried with anhydrous Na2SO4, and the drying agent was removed by vacuum filtration. The filtrate was concentrated to dryness under reduced pressure to obtain p-hydroxybenzoic acid-Orn(Cbz)-NBzl.

[0063] 4. Preparation of p-hydroxybenzoic acid-Orn-NBzl:

[0064] 10 mmol of p-hydroxybenzoic acid-Orn(Cbz)-NBzl was dissolved in methanol with stirring. An appropriate amount of Pd / C was added. The reaction system was kept sealed, air was evacuated, and a hydrogen cylinder was connected to maintain a hydrogen environment. The reaction was stirred at room temperature until the starting material spot disappeared. The reaction progress was monitored by TLC. After the reaction was completed, Pd / C was removed by filtration under reduced pressure. The filtrate was concentrated to dryness under reduced pressure to obtain p-hydroxybenzoic acid-Orn-NBzl.

[0065] 5. Preparation of PAD4 inhibitor YJ-2 (p-hydroxybenzoic acid-Orn(Cl)-NBzl):

[0066] 1 mmol of p-hydroxybenzoic acid-Orn-NBzl was dissolved in anhydrous methanol with stirring. Under ice-water bath conditions, 5 mmol of 2-chloroacetylimine ethyl ester hydrochloride was added, and the pH was adjusted to 10 with N,N-diisopropylethylamine (DIPEA). The mixture was stirred at room temperature for 12 h. TLC (by volume ratio, EA:H2O:HAc = 4:1:0.1) showed that 4-carboxyphenylboronic acid-Orn-NBzl disappeared. The solution was concentrated to dryness under reduced pressure and purified by C18 column chromatography to obtain the PAD4 inhibitor YJ-2 (p-hydroxybenzoic acid-Orn(Cl)-NBzl).

[0067] The proton NMR spectrum of the obtained PAD4 inhibitor YJ-2 is as follows: Figure 1 As shown, the ultraviolet spectrum is as follows Figure 2 As shown.

[0068] (2) The preparation of thermosensitive PAD4 inhibitor-loaded hydrogels is carried out using the following steps:

[0069] ① Dissolve chitosan in 0.1M HCl solution to obtain a 2% (w / v) CS solution;

[0070] ② Dissolve disodium β-glycerophosphate in deionized water to obtain a 50% (w / v) β-GP solution;

[0071] ③ Dissolve the PAD4 inhibitor YJ-2 in PBS buffer to obtain a drug solution with a concentration of 10 mg / mL;

[0072] ④ Place the CS solution, β-GP solution, and drug solution in an ice bath for 5 minutes;

[0073] ⑤ Under vortex conditions, 100 μL of β-GP solution was added dropwise to 1 mL of CS solution, followed by 100 μL of drug solution. The vortex was vortexed for 6 min at a rotation speed of 3000 rpm to obtain a thermosensitive PAD4 inhibitor-loaded hydrogel, which is denoted as drug-loaded thermosensitive CS / β-GP hydrogel.

[0074] Comparative Example 1

[0075] Following the method of Example 1, the difference is that the drug solution is replaced with an equal amount of PBS buffer to obtain an empty-loaded temperature-sensitive CS / β-GP hydrogel.

[0076] Structural characterization

[0077] (1) Physical images of the hydrogels obtained in Example 1 and Comparative Example 1 at different temperatures are shown below. Figure 3 As shown. Figure 3 In the image, A is a physical image of an unloaded thermosensitive CS / β-GP hydrogel at 0℃; B is a physical image of a drug-loaded thermosensitive CS / β-GP hydrogel at 0℃; C is a physical image of an unloaded thermosensitive CS / β-GP hydrogel at 37℃; and D is a physical image of a drug-loaded thermosensitive CS / β-GP hydrogel at 37℃.

[0078] Depend on Figure 3 It can be seen that both hydrogels have good temperature sensitivity.

[0079] (2) Scanning electron microscope images of the hydrogels obtained in Example 1 and Comparative Example 1 are shown below. Figure 4 As shown, Figure 4 In the images, A represents an unloaded thermosensitive CS / β-GP hydrogel, and B represents a drug-loaded thermosensitive CS / β-GP hydrogel. Scanning electron microscopy (SEM) images show that the hydrogels have a regular porous structure. The morphology of the hydrogels did not change significantly after loading YJ-2, and YJ-2 particles were visible within them.

[0080] (3) The Fourier transform infrared spectra of the hydrogels obtained in YJ-2, Example 1, and Comparative Example 1 are as follows: Figure 5 As shown. Fourier transform infrared spectroscopy revealed that after the addition of YJ-2, the CS / β-GP thermosensitive hydrogel reached a wavelength of 966.16 cm⁻¹. -1 The absorption peak at [location] red-shifted to 951.70 cm⁻¹. -1 This may be due to hydrogen bonding between YJ-2 and the hydrogel; at 1633.41 cm⁻¹ -1 Up to 1257.36cm -1 The peak intensity increased at 1603.52 cm⁻¹. -1 A new absorption peak was added, possibly related to YJ-2 at 1606.41 cm⁻¹. -1 The addition of YJ-2 is related to the vibration of the aromatic skeleton. The infrared spectrum of the CS / β-GP thermosensitive hydrogel did not change significantly after the addition of YJ-2, confirming that YJ-2 has a relatively small effect on the hydrogel. This is consistent with the results of scanning electron microscopy, verifying the feasibility of using the CS / β-GP thermosensitive hydrogel as a YJ-2 carrier.

[0081] Performance testing

[0082] (1) Drug release experiment

[0083] YJ-2 exhibits a specific absorption peak at 254 nm. To ensure a suitable concentration of YJ-2 in the supernatant during drug release testing, a larger drug loading was employed. 2 mL of the empty thermosensitive CS / β-GP hydrogel (Comparative Example 1) was added to a 15 mL EP tube, followed by the addition of 20 mg of YJ-2. The mixture was stirred thoroughly and then placed in a 37°C water bath to allow gelation. 10 mL of PBS was slowly added around the hydrogel at room temperature. 1 mL of the supernatant was collected daily, and an equal volume of PBS was added. The absorbance at 254 nm of the YJ-2 standard solution and the daily supernatant was measured using a UV spectrophotometer to obtain a standard curve, which was then used to calculate the YJ-2 release curve. The drug release curve of the thermosensitive CS / β-GP hydrogel is shown below. Figure 6 As shown.

[0084] This invention utilizes ultraviolet spectroscopy to detect the ultraviolet absorption at 254 nm in liquid collected at different times, and then uses the measured YJ-2 standard curve to calculate the drug release curve of the CS / β-GP / YJ-2 thermosensitive hydrogel over 14 days. The results show that the hydrogel begins to release the drug on day 1, with a relatively rapid release rate in the first 3 days, a slowing release rate starting on day 4, and reaching the maximum release amount—approximately 65% ​​of the drug loading—around day 8.

[0085] (2) MTT test

[0086] Preparation of hydrogel extract: Add 2 mL of blank hydrogel of Comparative Example 1 to a 15 mL EP tube, place it in a water bath at 37 °C to gel, use a spatula to remove it from the bottom of the tube, add 10 mL of PBS, and after 24 h, take the supernatant and filter it with a 0.22 μm sterile filter in a biosafety cabinet to obtain the hydrogel extract.

[0087] Preparation of the mixed solution (used to simulate the drug-loaded hydrogel extract of Example 1): Prepare a 400 μmol / mL YJ-2 solution in PBS in a 1.5 mL EP tube. Take seven 1.5 mL EP tubes and dilute them 2 / 3 times to obtain eight different concentrations of solutions ranging from 400 μmol / mL to 3.125 μmol / mL. Then, use a pipette to add the same volume of hydrogel extract as the solution in each EP tube and mix thoroughly by pipetting.

[0088] The effect of hydrogel on in vitro cell proliferation was assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol bromide). HaCaT cells were trypsinized, centrifuged, and resuspended in complete culture medium to a concentration of 40,000 cells / mL. 100 μL of each cell was seeded into 96-well plates and incubated in a CO2 incubator for 12 h. Different concentrations of YJ-2, hydrogel extract (Gel), and a mixture of both (YJ-2 / Gel) were added to each 96-well plate at 25 μL, and incubated for 24 h. Then, 25 μL of MTT solution (5 mg / mL) was added to each 96-well plate and incubated for 4 h. After removing the liquid from the wells, 150 μL of LDMSO was added to each well, and the plates were shaken for 15 min. The absorbance at 490 nm and 570 nm was measured using a microplate reader.

[0089] The MTT assay results of YJ-2 with different hydrogels are as follows: Figure 7 As shown in the figure. MTT assay results showed that YJ-2 had a slight promoting effect on HaCaT cells compared to the hydrogel extract.

[0090] (3) Cell migration ability assay

[0091] HaCaT cells play an important role in wound healing. Scratch assays were performed on monolayers of HaCaT cells to evaluate the effects of CS / β-GP thermosensitive hydrogel and YJ-2 on HaCaT cell migration and their impact on wound healing. HaCaT cells were trypsinized, centrifuged, and resuspended in complete culture medium to a concentration of 5 × 10⁻⁶. 5Cells were seeded at a density of 2 mL / mL into 6-well plates and incubated in a CO2 incubator for 24 h. Cells were confirmed to have reached confluence in the wells under an inverted microscope. A 200 μL pipette tip was used to sweep the cell layer perpendicularly across the plate. The culture medium was aspirated, and floating cells were washed away with PBS. A photograph was taken as a 0 h control. DMEM medium containing 2% FBS was added to each well, followed by 500 μL of YJ-2, hydrogel extract, or a mixture of both (YJ-2 + hydrogel). The cells were incubated in a CO2 incubator, and a photograph was taken at 12 h. The scratch area for each group was measured using imagineJ at different time points.

[0092] Migration images of HaCaT cells at 12h are shown below. Figure 8 As shown, the healing rate of YJ-2 in the scratch experiment was as follows: Figure 9 As shown in the figure. The scratch assay results showed that both YJ-2 alone and YJ-2+ hydrogel extract could significantly promote HaCaT cell migration, indicating that YJ-2 has the potential to promote wound healing.

[0093] (4) In vitro anti-NETs experiment

[0094] Culture dishes were treated with poly-L-lysine overnight incubation. ICR mice were euthanized by cervical dislocation after anesthesia. The hind leg bones were isolated, and the bone marrow was exposed by cutting off both ends of the leg bones. The bone marrow was flushed out using a syringe filled with PBS, passed through a cell sieve, and centrifuged. The supernatant was discarded, and neutrophils were isolated using a kit. Cells were cultured at 5 × 10⁻⁶ cells / day. 5 Cells were seeded into 5 culture dishes and randomly divided into control, PMA, YJ-2, hydrogel extract, and a mixture of the two groups. YJ-2, hydrogel extract, and a mixture of the two groups were added to three dishes, while the remaining dishes were added with an equal volume of PBS. After incubation for two hours, except for the control group, an equal amount of NETs inducer PMA was added and incubated for another 2 hours. Centrifuge at 500×g for 5 min, discard the supernatant, wash with PBS, discard the supernatant again; fix with 4% paraformaldehyde for 15 min, centrifuge at 500×g for 5 min, discard the supernatant; wash once with PBST and once with PBS, centrifuge at 500×g for 10 min, discard the supernatant; block with 5% BSA at room temperature for 30 min; discard the supernatant, add primary antibody and incubate overnight at 4℃; recover the primary antibody, wash once with PBST and once with PBS, centrifuge at 500×g for 5 min, discard the supernatant; add secondary antibody and incubate at room temperature in the dark for 2 h; recover the secondary antibody, wash once with PBST and once with PBS, centrifuge at 500×g for 5 min, discard the supernatant; stain with DAPI in the dark for 5 min, wash once with PBS, add mounting medium. Use a laser confocal microscope to capture fluorescence images. Confocal images of the anti-NETs experiment are shown below. Figure 10 As shown. H3cit fluorescence intensity as Figure 11 As shown.

[0095] Figure 10 In the confocal images, red represents H3cit and blue represents chromatin. Chromatin depolymerization and neutrophil rupture were clearly observed in the PMA-induced and hydrogel-incubated groups, while no obvious chromatin depolymerization was observed in the two groups incubated with YJ-2.

[0096] Depend on Figure 11 It can be seen that the average fluorescence intensity of H3cit in the PMA-induced and hydrogel-incubated groups was significantly higher than that in the control group, while the average fluorescence intensity in the two groups incubated with YJ-2 was significantly lower. The experimental results show that YJ-2 reduces the expression of H3cit in mouse neutrophils, confirming that YJ-2 has an inhibitory effect on NETs.

[0097] (5) Mouse wound healing experiment

[0098] After anesthetizing mice, four 8mm diameter holes were punched in the skin on their backs using a puncher. After photographing, 0.1 mL of drug was administered to each mouse per hole in each group. The administration was divided into three groups: a control group, an empty thermosensitive CS / β-GP hydrogel group (denoted as Gel), a YJ-2 group (concentration 5 mg / mL), a drug-loaded thermosensitive CS / β-GP hydrogel group (denoted as YJ-2 / Gel, drug loading 1 mg / mL), and a drug-loaded thermosensitive CS / β-GP hydrogel group (denoted as YJ-2 / Gel, drug loading 5 mg / mL). Starting from day 3, mice were anesthetized every other day, and the drugs were administered after photographing. Mice were euthanized on day 11, and skin tissue and organs were collected. The wound area of ​​the mice treated with hydrogels is statistically analyzed as follows. Figure 12 As shown in the figure, from day 3 onwards, the wounds of diabetic mice treated with drug-loaded thermosensitive CS / β-GP hydrogel showed obvious closure, and by day 11, the wounds were almost completely closed. The figure shows a curve of wound area, with the wound area of ​​diabetic mice treated with drug-loaded thermosensitive CS / β-GP hydrogel being significantly smaller than that of the control group.

[0099] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A temperature-sensitive hydrogel loaded with a PAD4 inhibitor, characterized in that, It includes a thermosensitive hydrogel matrix and a PAD4 inhibitor loaded in the thermosensitive hydrogel matrix; The thermosensitive hydrogel matrix comprises chitosan and disodium β-glycerophosphate; The PAD4 inhibitor has the structure shown in Formula 1:

2. The thermosensitive PAD4 inhibitor-loaded hydrogel according to claim 1, characterized in that, The mass ratio of chitosan to β-glycerophosphate disodium is 1:2 to 4.

3. The thermosensitive PAD4 inhibitor-loaded hydrogel according to claim 1 or 2, characterized in that, The PAD4 inhibitor loading capacity in the thermosensitive PAD4 inhibitor-loaded hydrogel is 0.1–10 mg / mL.

4. The method for preparing the thermosensitive PAD4 inhibitor-loaded hydrogel according to any one of claims 1 to 3, characterized in that, Includes the following steps: Chitosan was mixed with hydrochloric acid solution to obtain a chitosan solution; The chitosan solution, β-glycerophosphate disodium and PAD4 inhibitor solution were mixed to obtain a thermosensitive PAD4 inhibitor-loaded hydrogel.

5. The preparation method according to claim 4, characterized in that, The concentration of the hydrochloric acid solution is 1 mol / L; The chitosan solution contains 2-3% chitosan by mass.

6. The preparation method according to claim 4 or 5, characterized in that, The β-glycerophosphate disodium is added in solution form, and the mass concentration of the β-glycerophosphate disodium solution is 30-60%.

7. The preparation method according to claim 4, characterized in that, The concentration of the PAD4 inhibitor solution is 10–100 mg / mL, and the solvent is PBS buffer.

8. The preparation method according to claim 4, characterized in that, The mixing is vortex mixing, with a rotation speed of 1000-3000 rpm and a time of 1-10 min.

9. The application of the thermosensitive PAD4 inhibitor-loaded hydrogel according to any one of claims 1 to 3 or the thermosensitive PAD4 inhibitor-loaded hydrogel prepared by the preparation method according to any one of claims 4 to 8 in the preparation of diabetic wound dressings.

10. A diabetic wound dressing, characterized in that, The product includes the thermosensitive PAD4 inhibitor-loaded hydrogel according to any one of claims 1 to 3 or the thermosensitive PAD4 inhibitor-loaded hydrogel prepared by the preparation method according to any one of claims 4 to 8, and pharmaceutically acceptable excipients.

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