Temperature-sensitive PAD4 inhibitor loaded hydrogel as well as preparation method and application thereof

Abstract

The invention provides 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 type PAD4 inhibitor loaded hydrogel provided by the invention comprises a temperature-sensitive type hydrogel matrix and a PAD4 inhibitor loaded in the temperature-sensitive type hydrogel matrix, the temperature-sensitive type hydrogel matrix is prepared from the following components: chitosan and beta-glycerophosphate disodium; the PAD4 inhibitor has a structure as shown in a formula 1. The PAD4 inhibitor is loaded in the hydrogel, and the PAD4 inhibitor is slowly released through local application, so that formation of NETs is inhibited for a long time, chronic inflammation is relieved, and finally the healing process of diabetic wounds is accelerated. The temperature-sensitive PAD4 inhibitor loaded hydrogel provided by the invention shows a remarkable treatment effect in experiments, and overcomes the limitation of traditional drug treatment to a certain extent.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a temperature-sensitive PAD4 inhibitor-loaded hydrogel and a preparation method and application thereof. Background Art

[0002] Peptidylarginine deiminase 4 (PAD4) is a calcium-dependent enzyme responsible for deiminating arginine residues in proteins to form citrulline. This enzyme is expressed in a variety of immune cells, including neutrophils, monocytes, and macrophages. One of PAD4's best-known functions is its involvement in the formation of neutrophil extracellular traps (NETs). NETs are meshwork-like structures composed of DNA and protein that capture and kill 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 overproduced, further exacerbating chronic inflammatory responses and being closely associated with impaired wound healing. Studies have found that PAD4 protein expression is upregulated fourfold in neutrophils of diabetic patients, and NET levels are significantly elevated in blood and wound tissue. In the diabetic setting, PAD4 promotes NET formation, thereby exacerbating the inflammatory response. Upon PAD4 activation, histone deiminylation and chromatin loosening in neutrophils lead to the release of nuclear DNA and the formation of NETs. This process plays a positive role in combating infection, but in diabetic wounds, excessive accumulation of NETs can actually delay healing. Existing PAD4 inhibitors, such as F-amidine, Cl-amidine, and TDFA, have limited specificity and 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 the present invention is to provide a thermosensitive PAD4 inhibitor-loaded hydrogel and its preparation method and application. The thermosensitive PAD4 inhibitor-loaded hydrogel provided by the present invention can significantly accelerate the healing of diabetic wounds.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a thermosensitive PAD4 inhibitor-loaded hydrogel, comprising a thermosensitive hydrogel matrix and a PAD4 inhibitor loaded in the thermosensitive hydrogel matrix;

[0006] The components of the thermosensitive hydrogel matrix include chitosan and disodium β-glycerophosphate;

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

[0008]

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

[0010] Preferably, the drug loading amount of the PAD4 inhibitor in the thermosensitive PAD4 inhibitor-loaded hydrogel is 0.1 to 10 mg / mL.

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

[0012] mixing chitosan with a hydrochloric acid solution to obtain a chitosan solution;

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

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

[0015] The mass concentration of chitosan in the chitosan solution is 2-3%.

[0016] Preferably, the β-glycerophosphate disodium is added in the form of a solution, 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, the rotation speed of the vortex mixing is 1000-3000 rpm, and the time is 1-10 minutes.

[0019] The present invention provides use of the temperature-sensitive PAD4 inhibitor-loaded hydrogel in preparing a diabetic wound dressing.

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

[0021] The present 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 of the present invention has a stronger specificity for PAD4. Loading it into the hydrogel, it is then applied topically to slowly release the PAD4 inhibitor, thereby inhibiting the formation of NETs for a long time, alleviating chronic inflammation, and ultimately accelerating the healing process of diabetic wounds. The thermosensitive PAD4 inhibitor-loaded hydrogel provided by the present invention has demonstrated significant therapeutic effects in experiments, overcoming the limitations of traditional drug treatments to a certain extent. Furthermore, the thermosensitive PAD4 inhibitor-loaded hydrogel provided by the present invention is safe, convenient, and highly effective to use, has good biocompatibility, and can transform from a liquid to a solid gel at body temperature (37°C) and slowly release the drug at the wound site, providing a more effective treatment option for diabetic patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the hydrogen spectrum of the PAD4 inhibitor YJ-2;

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

[0024] Figure 3 The actual pictures 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 FTIR spectra of the hydrogels obtained from YJ-2, Example 1, and Comparative Example 1;

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

[0028] Figure 7 The results of MTT experiments of YJ-2 and different hydrogels;

[0029] Figure 8 Migration pictures of HaCaT cells at 12 h;

[0030] Figure 9 The healing rate of the YJ-2 migration-promoting scratch assay;

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

[0032] Figure 11 is the H3cit fluorescence intensity graph;

[0033] Figure 12 Statistical graph of the wound area in mice treated with hydrogel. DETAILED DESCRIPTION

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

[0035] The components of the thermosensitive hydrogel matrix include chitosan and disodium β-glycerophosphate;

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

[0037]

[0038] The present invention has no special requirements on the source of the PAD4 inhibitor YJ-2. The PAD4 inhibitor YJ-2 commercially available in the art or prepared by the user can be used.

[0039] In the present 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 disodium 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 the present invention, the drug loading amount of the 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] The present invention provides a method for preparing the above-mentioned temperature-sensitive PAD4 inhibitor-loaded hydrogel, comprising the following steps:

[0042] mixing chitosan with a hydrochloric acid solution to obtain a chitosan solution;

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

[0044] The present invention mixes chitosan with a hydrochloric acid solution to obtain a chitosan solution. In the present invention, the concentration of the hydrochloric acid solution is preferably 1 mol / L, and the mass concentration of chitosan in the chitosan solution is preferably 2-3%. The present invention has no particular requirements for the mixing method, and any mixing method 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 a PAD4 inhibitor solution to produce a temperature-sensitive PAD4 inhibitor-loaded hydrogel. In the present invention, the β-glycerophosphate disodium is preferably added as a solution, and the solvent is preferably deionized water. The mass concentration of the β-glycerophosphate disodium solution is preferably 30-60%, more preferably 50%. In the present invention, the concentration of the PAD4 inhibitor solution is preferably 10-100 mg / mL, more preferably 20-80 mg / mL, and even more preferably 40-60 mg / mL. The solvent for the PAD4 inhibitor solution is preferably PBS buffer.

[0046] Prior to mixing, the chitosan solution, β-glycerophosphate disodium, and PAD4 inhibitor solution are preferably cooled in an ice bath, preferably for 5 minutes. This cooling ensures that the hydrogel remains liquid during the preparation process.

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

[0048] In the present invention, the mixing is preferably vortex mixing, the rotation speed of the vortex mixing is preferably 1000 to 3000 rpm, the time is preferably 1 to 10 minutes, more preferably 2 to 8 minutes, and further preferably 4 to 6 minutes.

[0049] The present invention provides use of the temperature-sensitive PAD4 inhibitor-loaded hydrogel in preparing a diabetic wound dressing.

[0050] The present invention provides a diabetic wound dressing comprising the aforementioned thermosensitive PAD4 inhibitor-loaded hydrogel and pharmaceutically acceptable excipients. The present invention has no particular requirements for the type of excipients, and excipients commonly used in the art may be used.

[0051] The temperature-sensitive PAD4 inhibitor-loaded hydrogel provided by the present invention, its preparation method and application are described in detail below with reference to the examples, but they 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 of 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-chloroacetimidylethyl 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 completely dissolved. 12 mmol of dicyclohexylcarbodiimide (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 under ice bath conditions 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 at room temperature for 48 h. TLC (by volume ratio, dichloromethane:methanol = 20: 1) Boc-Orn(Cbz)-OH disappeared, dicyclohexylurea (DCU) was filtered out, THF was evaporated under reduced pressure, and the residue was dissolved with 50 mL of ethyl acetate (EA). The resulting solution was washed three times with saturated aqueous NaHCO3, saturated aqueous NaCl, saturated aqueous KHSO4, saturated aqueous NaCl, saturated aqueous NaHCO3, and saturated aqueous NaCl, respectively. The ethyl acetate layer was dried over anhydrous Na2SO4, and the desiccant was removed by filtration under reduced pressure. The filtrate was concentrated to dryness under reduced pressure 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, and 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 that the starting material spot disappeared. The solution was pumped dry with a water pump, and anhydrous ethyl acetate was added. The reaction solution was pumped dry again with a water pump. This 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), 12 mmol of N-hydroxybenzotriazole (HOBt) was added under ice bath conditions and completely dissolved, 12 mmol of dicyclohexylcarbodiimide (DCC) was slowly added, and stirred for 30 min to obtain reaction solution A; 12 mmol of N-hydroxybenzotriazole (HOBt) was added under ice bath conditions and completely dissolved, and 12 mmol of dicyclohexylcarbodiimide (DCC) was slowly added and stirred for 30 min to obtain reaction solution A; 12 mmol of N-hydroxybenzotriazole (HOBt) was added under ice bath conditions and completely dissolved, and 12 mmol of dicyclohexylcarbodiimide (DCC) was added and stirred for 30 min to obtain reaction solution A. 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 the disappearance of p-hydroxybenzoic acid. Dicyclohexylurea (DCU) was filtered out 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 aqueous NaHCO3, saturated aqueous NaCl, saturated aqueous KHSO4, saturated aqueous NaCl, saturated aqueous NaHCO3, and saturated aqueous NaCl. The ethyl acetate layer was dried over anhydrous Na2SO4 and filtered to remove the desiccant under reduced pressure. 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 by stirring, and an appropriate amount of Pd / C was added. The reaction system was kept sealed, the 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 spots disappeared. The reaction progress was monitored by TLC. After the reaction, the Pd / C was removed by filtration under reduced pressure, and 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. 5 mmol of ethyl 2-chloroacetimidate hydrochloride was added under ice-water bath, and the pH was adjusted to 10 with N,N-diisopropylethylamine (DIPEA). The mixture was stirred at room temperature for 12 h. TLC (EA:H2O:HAc=4:1:0.1 by volume) showed the disappearance of 4-carboxyphenylboronic acid-Orn-NBzl. The mixture 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 hydrogen spectrum of the obtained PAD4 inhibitor YJ-2 is as follows Figure 1 As shown, the UV spectrum is Figure 2 shown.

[0068] (2) Preparation of thermosensitive PAD4 inhibitor-loaded hydrogels using the following steps:

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

[0070] ② Dissolve β-glycerophosphate disodium 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, and then 100 μL of drug solution was added and vortexed for 6 min at a vortex speed of 3000 rpm to obtain a thermosensitive PAD4 inhibitor-loaded hydrogel, which was recorded as drug-loaded thermosensitive CS / β-GP hydrogel.

[0074] Comparative Example 1

[0075] The method of Example 1 was followed, except that the drug solution was replaced with an equal amount of PBS buffer to obtain an empty temperature-sensitive CS / β-GP hydrogel.

[0076] Structural characterization

[0077] (1) The actual pictures of the hydrogels obtained in Example 1 and Comparative Example 1 at different temperatures are as follows Figure 3 shown. Figure 3 In the figure, A is a physical picture of the empty thermosensitive CS / β-GP hydrogel at 0℃; B is a physical picture of the drug-loaded thermosensitive CS / β-GP hydrogel at 0℃; C is a physical picture of the empty thermosensitive CS / β-GP hydrogel at 37℃; D is a physical picture of the 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) The scanning electron microscope images of the hydrogels obtained in Example 1 and Comparative Example 1 are as follows: Figure 4 As shown, Figure 4 In the figure, A is an empty thermosensitive CS / β-GP hydrogel, and B is a drug-loaded thermosensitive CS / β-GP hydrogel. Scanning electron microscopy images show that the hydrogel has a regular pore structure. The loading of YJ-2 does not significantly change the hydrogel morphology, and YJ-2 particles are visible.

[0080] (3) The Fourier transform infrared spectra of the hydrogels obtained from YJ-2, Example 1, and Comparative Example 1 are as follows: Figure 5 Fourier transform infrared spectroscopy showed that after adding YJ-2, the CS / β-GP thermosensitive hydrogel had a high molecular weight at 966.16 cm -1 The absorption peak at 951.70 cm-1 was red-shifted to 951.70 cm-1. -1 , which may be caused by the hydrogen bonding between YJ-2 and hydrogel; at 1633.41cm -1 to 1257.36cm -1 The peak intensity at 1603.52 cm -1 A new absorption peak was found at 1606.41 cm -1 After adding YJ-2, the infrared spectrum of the CS / β-GP thermosensitive hydrogel did not change significantly, confirming that YJ-2 had little effect on the hydrogel. This is consistent with the results of scanning electron microscopy, verifying the feasibility of using CS / β-GP thermosensitive hydrogel as a YJ-2 carrier.

[0081] Performance Testing

[0082] (1) Drug release experiment

[0083] YJ-2 has a special absorption peak at 254nm. During the drug release test, in order to keep the YJ-2 concentration in the supernatant within an appropriate range, a larger drug loading method was adopted. 2mL of the empty thermosensitive CS / β-GP hydrogel of Comparative Example 1 was added to a 15mL EP tube, and then 20mg of YJ-2 was added to the hydrogel and stirred evenly, and placed in a water bath at 37°C to gel it. At room temperature, 10mL of PBS was slowly added around the hydrogel, 1mL of supernatant was taken every day and supplemented with an equal volume of PBS, and the absorbance of the YJ-2 standard solution and the supernatant every day at 254nm was measured using an ultraviolet spectrophotometer to obtain a standard curve and calculate the YJ-2 release curve. The drug release curve of the drug-loaded thermosensitive CS / β-GP hydrogel is shown in Figure 2. Figure 6 shown.

[0084] The present invention used an ultraviolet spectrometer to measure the UV absorbance at 254 nm in liquids collected at different times. The drug release profile of the CS / β-GP / YJ-2 thermosensitive hydrogel over 14 days was calculated using the measured YJ-2 standard curve. The results showed that the hydrogel began releasing the drug on the first day, with a rapid release rate during the first three days. The release rate slowed from the fourth day onward, reaching a maximum release rate of approximately 65% ​​of the drug loading around the eighth day.

[0085] (2) MTT assay

[0086] Preparation of the hydrogel extract group: 2 mL of the blank hydrogel from Comparative Example 1 was added to a 15 mL EP tube and placed in a 37°C water bath to gel. The hydrogel was separated from the bottom of the tube with a spatula and 10 mL of PBS was added. After 24 hours, the supernatant was collected and filtered through a 0.22 μm sterilizing filter in a biosafety cabinet to obtain a hydrogel extract.

[0087] Preparation of a mixed solution of the two (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 the solution 1 / 2 times seven times to obtain eight solutions with different concentrations ranging from 400 μmol / mL to 3.125 μmol / mL. Then, use a pipette to add an equal volume of hydrogel extract to each EP tube and mix thoroughly by pipetting.

[0088] The effect of the hydrogel on cell proliferation in vitro was assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). HaCaT cells were trypsinized and centrifuged, then resuspended in complete culture medium to a concentration of 40,000 cells / mL. The cells were seeded into 96-well plates at a concentration of 100 μL per well and incubated in a CO2 incubator for 12 hours. YJ-2 at varying concentrations, the hydrogel extract (Gel), and a mixture of the two (YJ-2 / Gel) were then added to the 96-well plates at a concentration of 25 μL per well and incubated for 24 hours. MTT solution (5 mg / mL) was then added to the 96-well plates at a concentration of 25 μL per well and incubated for 4 hours. The liquid was aspirated, and 150 μL of DMSO was added to each well. After shaking for 15 minutes, the absorbance at 490 nm and 570 nm was measured using a microplate reader.

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

[0090] (3) Cell migration ability assay

[0091] HaCaT cells play an important role in wound healing. A scratch assay was performed on a monolayer of HaCaT cells to evaluate the effects of CS / β-GP thermosensitive hydrogels 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 5 / mL, inoculated into a 6-well plate, 2mL per well, incubated in a CO2 incubator for 24h, and confirmed under an inverted microscope that the cells have grown all over the wells. Use a 200μL pipette tip to scratch the cell layer perpendicular to the well plate. Aspirate the culture medium, wash away the floating cells with PBS, and take pictures as a 0h control. Add DMEM culture medium containing 2% FBS to each well, and then add 500μL YJ-2, hydrogel extract (hydrogel), and a mixture of the two (YJ-2+hydrogel) respectively. Incubate in a CO2 incubator and take pictures at 12h. ImagineJ measured the scratch area of ​​each group at different times.

[0092] 12h HaCaT cell migration pictures Figure 8 As shown in Figure 2, the healing rate of the YJ-2 migration-promoting scratch test is as follows: Figure 9 The scratch test 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] Incubate the culture dish overnight with poly-lysine. After anesthesia, the ICR mice were killed by cervical dislocation. The hind leg bones were isolated and the bone marrow was exposed by cutting the ends of the leg bones. The bone marrow was flushed out with a syringe filled with PBS, passed through a cell sieve, and centrifuged. The supernatant was discarded and neutrophils were isolated using a kit. 5×10 5 The resulting cells were seeded into five culture dishes at a density of 10 cells / well 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 were added to three dishes, and an equal volume of PBS was added to the remaining dishes. After incubation for two hours, an equal volume of the NETs inducer PMA was added to all dishes except the control group and incubated for 2 hours. After centrifugation at 500×g for 5 minutes, discard the supernatant, wash with PBS, and discard the supernatant; add 4% paraformaldehyde to fix for 15 minutes, and centrifuge at 500×g for 5 minutes, and discard the supernatant; wash once with PBST and PBS, and centrifuge at 500×g for 10 minutes, and discard the supernatant; block with 5% BSA at room temperature for 30 minutes; discard the supernatant, add the primary antibody and incubate overnight at 4°C; after recovering the primary antibody, wash once with PBST and PBS, and centrifuge at 500×g for 5 minutes, and discard the supernatant; add the secondary antibody and incubate at room temperature for 2 hours in the dark; after recovering the secondary antibody, wash once with PBST and PBS, and centrifuge at 500×g for 5 minutes, and discard the supernatant; add DAPI to stain for 5 minutes in the dark, wash once with PBS, and add the mounting medium. Fluorescence images were taken using a laser confocal microscope. Confocal images of the anti-NETs experiment are shown in the following figure. Figure 10 As shown. H3cit fluorescence intensity is Figure 11 shown.

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

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

[0097] (5) Mouse wound healing experiment

[0098] After anesthetizing the mice, four circular holes with a diameter of 8 mm were punched on the back skin with a hole puncher. After taking pictures, each group and each mouse were given 0.1 mL of drug per hole. The drugs were divided into blank group, empty thermosensitive CS / β-GP hydrogel group (denoted as Gel), YJ-2 group (concentration of 5 mg / mL), drug-loaded thermosensitive CS / β-GP hydrogel group (denoted as YJ-2 / Gel, drug loading of 1 mg / mL), and drug-loaded thermosensitive CS / β-GP hydrogel group (denoted as YJ-2 / Gel, drug loading of 5 mg / mL). Starting from the third day, the mice were anesthetized every other day, and the drugs were given after taking pictures. The mice were euthanized on the 11th day, and the skin tissues and organs were collected. The wound area of ​​hydrogel-treated mice is shown in the following table. Figure 12 As shown in the figure, the wounds of diabetic mice treated with the drug-loaded thermosensitive CS / β-GP hydrogel showed significant closure starting on day 3, and by day 11, the wounds were almost completely closed. The figure shows a graph of wound area, showing that the wound area of ​​diabetic mice treated with the drug-loaded thermosensitive CS / β-GP hydrogel was significantly smaller than that of the control group.

[0099] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

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

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

3. The temperature-sensitive PAD4 inhibitor-loaded hydrogel according to claim 1 or 2, characterized in that: The drug loading amount of the PAD4 inhibitor 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: The following steps are involved: mixing chitosan with a hydrochloric acid solution to obtain a chitosan solution; The chitosan solution, β-glycerophosphate disodium and PAD4 inhibitor solution are mixed to obtain a temperature-sensitive 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 mass concentration of chitosan in the chitosan solution is 2-3%.

6. The preparation method according to claim 4 or 5, characterized in that The β-disodium glycerophosphate is added in the form of a solution, and the mass concentration of the β-disodium glycerophosphate 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, the rotation speed of the vortex mixing is 1000-3000 rpm, and the time is 1-10 minutes.

9. Use 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 dressing.

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

Images