An inflammatory response hydrogel for promoting diabetic wound healing and preparation method thereof

Abstract

The invention discloses an inflammatory response hydrogel for promoting the healing of diabetic wounds and a preparation method thereof. The inflammatory response hydrogel for promoting the healing of diabetic wounds comprises a hydrogel and Prussian blue nanoparticles and vascular endothelial growth factor loaded on the hydrogel. The hydrogel uses HA-PBA-ALD as a skeleton, and forms borate bonds and acylhydrazone bonds after cross-linking with PVA / ADH. The gelation process can be completed rapidly within a few seconds without the need for additional catalysts, pH adjustment or the production of toxic byproducts. The dynamic covalent bonds give the hydrogel excellent injectability, self-healing and responsiveness to inflammatory stimuli, and have good prospects in clinical applications for the treatment of irregular wounds. With the enhancement of angiogenesis as a therapeutic target, the PBNPs loaded in the hydrogel improve the immune microenvironment by reducing local inflammation and oxidative stress on the wound, thereby protecting the exogenous VEGF activity, and ultimately achieving the effect of promoting the healing of diabetic wounds.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an inflammatory response hydrogel for promoting healing of diabetic wounds and a preparation method thereof. Background Art

[0002] Chronic refractory wounds are one of the most common complications of diabetes. Diabetic wounds are difficult to heal, prone to infection, and have a high amputation rate. Therefore, it is of great clinical significance to find an effective treatment plan to promote the healing of diabetic wounds.

[0003] The healing of normal wounds is a highly programmed process, including four overlapping stages: hemostasis, inflammation, proliferation, and remodeling, which coordinate with each other to promote wound repair. However, in diabetic wounds, the wound healing process faces many challenges. First, due to the inhibition of angiogenesis in diabetic patients, blood flow at the wound is reduced, resulting in insufficient supply of oxygen and nutrients. Secondly, due to the dysfunction of M2 polarization of macrophages, excessive proinflammatory factors and proteases are produced, causing diabetic wounds to remain in the inflammatory stage for a long time. The inflammatory cells recruited locally in the wound produce a large amount of reactive oxygen species (ROS), which aggravates the oxidative stress response. Oxidative stress not only damages cell membranes, proteins, and DNA, but also inhibits cell proliferation and differentiation, thereby delaying the wound healing process. In diabetic wounds, oxidative stress and inflammatory responses interact with each other to form a vicious cycle, aggravating wound damage.

[0004] Accelerating angiogenesis is important in diabetic wound healing because it can provide essential oxygen and nutrients to damaged tissues while helping to alleviate uncontrolled inflammatory responses.

[0005] In recent years, exogenous growth factors have become a research hotspot in the field of promoting angiogenesis and skin wound repair, showing great application potential and development prospects. Among them, vascular endothelial growth factor (VEGF) is believed to have a significant promoting effect on wound healing and angiogenesis. However, in actual clinical applications, VEGF has not shown obvious therapeutic effects. One of the key reasons is that diabetic wounds usually present a harsh microenvironment that is not conducive to regeneration, such as persistent chronic inflammation, high proteolysis, and severe oxidative stress. In this case, not only is the self-repair process of the wound tissue impaired, but the bioavailability of the exogenous angiogenesis factor VEGF is also greatly limited.

[0006] Nanozymes are small, enzyme-like nanomaterials that exhibit many clinical advantages due to their excellent enzyme stability, low cost, and simple preparation. As one of the representative nanozymes, Prussian blue nanoparticles (PBNPs) can mimic the activity of antioxidant enzymes such as peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD), remove excess ROS, and reduce inflammatory responses. In addition, PBNPs have good biocompatibility and have been used as detoxifiers for radioactive cesium and thallium. Based on the above advantages, PBNPs have been widely used in diseases related to inflammation and oxidative stress, such as pancreatitis, enteritis, etc., but they are still rare in the treatment of diabetic wounds.

[0007] As a type of highly hydrophilic biomaterial with a three-dimensional network structure, hydrogel has good moisture retention and air permeability. It can serve as a physical barrier for wounds and a drug carrier to achieve sustained release of drugs and improve the bioavailability of drugs, thereby improving the effect of wound treatment. It is considered to be one of the most ideal biomaterials for wounds. In particular, injectable self-healing hydrogels have many advantages. In addition to local drug delivery, they can fill irregularly shaped wounds and maintain integrity and functionality for a long time. Summary of the invention

[0008] The purpose of the present invention is to provide an inflammatory response hydrogel that promotes diabetic wound healing and a preparation method thereof. The hydrogel has good injectability and self-healing properties, and at the same time has inflammatory responsiveness. By loading two drugs, Prussian blue nanoparticles (PBNPs) and vascular endothelial growth factor (VEGF), under the stimulation of the inflammatory environment of the diabetic wound, the PBNPs can be quickly released to reduce the oxidative stress level and promote macrophage polarization to reprogram the immune microenvironment, thereby maximally retaining the activity of the released VEGF protein, and ultimately achieving the effect of promoting wound healing.

[0009] The invention firstly provides an inflammation response hydrogel for promoting diabetic wound healing, comprising a hydrogel and Prussian blue nanoparticles and vascular endothelial growth factor loaded on the hydrogel.

[0010] Preferably, the loading concentration of the Prussian blue nanoparticles is 150-250 μg / mL; the loading concentration of the vascular endothelial growth factor is 200-300 ng / mL. More preferably, the loading concentration of the Prussian blue nanoparticles is 200 μg / mL; the loading concentration of the vascular endothelial growth factor is 250 ng / mL.

[0011] Preferably, the vascular endothelial growth factor is of human, mouse, pig or rat origin.

[0012] The present invention further provides a method for preparing the inflammatory response hydrogel for promoting diabetic wound healing, comprising the following steps:

[0013] (1) coupling 3-aminomethylphenylboronic acid to hyaluronic acid to obtain hyaluronic acid-phenylboronic acid polymer (HA-PBA);

[0014] (2) oxidizing the vicinal diol on the hyaluronic acid phenylboronic acid polymer chain obtained in step (1) to obtain an oxidized hyaluronic acid phenylboronic acid polymer (HA-PBA-ALD);

[0015] (3) Loading Prussian blue nanoparticles and vascular endothelial growth factor onto the oxidized hyaluronic acid phenylboronic acid polymer obtained in step (2) to obtain the inflammatory response hydrogel that promotes diabetic wound healing.

[0016] Preferably, the method for preparing the Prussian blue nanoparticles comprises the following steps:

[0017] K 4 [Fe(CN) 6 ] and citric acid were dissolved in water to obtain solution A;

[0018] Fe(NO3) 3 Dissolve with citric acid in water to obtain solution B;

[0019] Solution B is added dropwise to solution A to obtain the Prussian blue nanoparticles through reaction.

[0020] Preferably, in step (1), the molar ratio of 3-aminomethylphenylboronic acid to hyaluronic acid is 1:1;

[0021] The coupling agents used in the coupling are 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride and EDC.

[0022] Preferably, in step (2), the oxidant used for oxidation is sodium periodate.

[0023] Preferably, in step (3), during loading, the oxidized hyaluronic acid phenylboronic acid polymer obtained in step (2) is first mixed into a buffer solution, and then the Prussian blue nanoparticles and vascular endothelial growth factor are mixed into the buffer solution to obtain a mixed solution A;

[0024] Mix polyvinyl alcohol (PVA) into a buffer solution, heat to dissolve, and then add adipic acid dihydrazide (ADH) to dissolve, to obtain a mixed solution B;

[0025] The mixed solution A and the mixed solution B are mixed and reacted to obtain the inflammation-responsive hydrogel for promoting diabetic wound healing.

[0026] The buffer can be a commonly used buffer, preferably a PBS buffer. The pH of the buffer is generally neutral to slightly alkaline, generally 7 to 7.8, specifically, for example, the pH is 7.4. Selecting a buffer with a suitable pH does not require adjusting the pH after the mixed solution A and the mixed solution B are mixed and reacted. If the pH of the buffer is not within a suitable range, it is necessary to adjust the pH after the mixed solution A and the mixed solution B are prepared, and then the two mixed solutions are mixed and reacted.

[0027] The present invention also provides the use of the inflammatory response hydrogel for promoting diabetic wound healing in the preparation of a wound healing medicament. Preferably, the wound is a diabetic wound.

[0028] The present invention prepares a hydrogel with intelligent controlled drug release in response to inflammation. The hydrogel uses HA-PBA-ALD as a skeleton, and forms borate bonds and acylhydrazone bonds after cross-linking with PVA / ADH. The gelation process can be completed rapidly within a few seconds without the need for additional catalysts, pH adjustment or the generation of toxic byproducts. The dynamic covalent bonds give the hydrogel excellent injectability, self-healing and responsiveness to inflammatory stimuli, and have good prospects in clinical applications for treating irregular wounds.

[0029] With the enhancement of angiogenesis as the therapeutic target, the PBNPs loaded in the hydrogel improve the immune microenvironment by reducing local inflammation and oxidative stress on the wound surface, thereby protecting the activity of exogenous VEGF and ultimately achieving the effect of promoting the healing of diabetic wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a schematic diagram of the preparation process of the present invention.

[0031] Figure 2 The results are H NMR and FTIR spectra, which confirm that the molecular structure of the synthesized product is the same as expected, where a is the synthesis route; b is the H NMR spectrum ( 1 H NMR); c is: Fourier transform infrared spectroscopy (FTIR).

[0032] Figure 3 Schematic diagram of the changes in the hydrogel gelation process.

[0033] Figure 4 Graph showing the injectable properties of the hydrogel.

[0034] Figure 5 This is a diagram of the self-healing performance of the hydrogel.

[0035] Figure 6 This is a diagram showing the inflammatory response performance of the hydrogel.

[0036] Figure 7 The drug release curve of the hydrogel.

[0037] Figure 8 This is a graph showing the antioxidant capacity of PBNPs at different concentrations in hydrogel.

[0038] Fig. 9 This is a diagram showing the effect of different concentrations of VEGF in hydrogel on promoting cell proliferation.

[0039] Fig.10 This is a diagram showing how hydrogel promotes vascular endothelial cell migration. Scale bar: 200 μm.

[0040] Fig.11 Diagram showing hydrogel promoting endothelial cell angiogenesis.

[0041] Fig.12 This is a diagram of hydrogel promoting wound healing in diabetic mice, where A is a wound photo with a scale of 5 mm; B is a schematic diagram of the wound range corresponding to A, and the last row is a schematic diagram of the wound range superimposed on the four rows above; C is a diagram of the wound healing ratio. DETAILED DESCRIPTION

[0042] Example 1

[0043] Figure 1 It is a schematic diagram of the preparation process of the present invention. Figure 2 a is the synthetic route map.

[0044] A method for preparing an inflammation-responsive hydrogel for promoting diabetic wound healing comprises the following steps:

[0045] 1. Preparation of Prussian blue nanoparticles (PBNPs).

[0046] At 60 °C, 0.01 mmol K 4 [Fe(CN) 6 ]·3H 2 O and 5mmol monohydrate citric acid were dissolved in 20mL pure water and stirred to form solution A. 0.05mmol Fe(NO3) 3 9H 2 O and 5mmol monohydrate citric acid were dissolved in 20mL pure water to form solution B. Solution B was slowly dripped into solution A in a titration manner for 10 minutes under stirring. The mixture immediately turned bright blue. The reaction was continued for 5 minutes, then cooled to room temperature under stirring and stirred for another 30 minutes. The above mixture was centrifuged at 9500rpm for 10 minutes and then washed with ethanol 3 times. Finally, it was dried under vacuum at 30°C overnight to obtain the product, namely PBNPs.

[0047] 2. Synthesis of hyaluronic acid phenylboronic acid polymer (HA-PBA).

[0048] HA-PBA was synthesized by linking 3-aminomethylphenylboronic acid (PBA) to hyaluronic acid (HA) with a molecular weight of 300,000 Daltons using 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride (DMTMM) as a coupling agent.

[0049] The specific reaction is as follows: 0.4g hyaluronic acid (converted to 1mmol according to the molecular weight of hyaluronic acid monomer) is dissolved in 80mL deionized water under constant stirring. After complete dissolution, 480mg DMTMM (1.5mmol) is added to the hyaluronic acid solution for reaction for 1h, and then 188mg PBA (1mmol) is added. After PBA is dissolved, the pH value of the reaction system is adjusted to 6.5 using 1M NaOH solution. All reaction mixtures are stirred at room temperature for 48 hours. Then, the mixture is transferred to a dialysis bag with a molecular weight cutoff of 8000Da, dialyzed at room temperature for 3 days, and the water is changed three times a day. The dialyzed solution is freeze-dried using a desktop freeze dryer to obtain HA-PBA.

[0050] 3. Synthesis of oxidized hyaluronic acid phenylboronic acid polymer (HA-PBA-ALD).

[0051] HA-PBA-ALD was synthesized by oxidizing the vicinal diol on the HA-PBA chain with sodium periodate.

[0052] The specific reaction is as follows: dissolve 1g of HA-PBA in 90mL of deionized water under constant stirring, take 0.6g of sodium periodate and dissolve it in 10mL of water, mix the above solutions, and react for 4h in the dark. Then add 5mL of ethylene glycol to the above reaction solution and stir for 2 hours to terminate the reaction. Then, transfer the mixture to a dialysis bag with a molecular weight cutoff of 3500Da and dialyze at room temperature for 3 days, changing the water three times a day. The dialyzed solution is freeze-dried using a desktop freeze dryer to obtain HA-PBA-ALD.

[0053] 4. Use HA-PBA-ALD and PVA-ADH to crosslink to form hydrogel.

[0054] The specific preparation steps of the inflammatory response hydrogel loaded with PBNPs and VEGF (Hydrogel@PB&VEGF) are as follows:

[0055] (1) A certain amount of HA-PBA-ALD was mixed into a PBS solution at pH 7.4 until the final concentration was 3 wt % to obtain a HA-PBA-ALD solution.

[0056] (2) A certain amount of PBNPs and VEGF (Recombinant Human VEGF165, purchased from PeproTech, catalog number 100-20) was added to the HA-PBA-ALD solution obtained in step (1) to obtain PB&VEGF@HA-PBA-ALD.

[0057] (3) Then, a certain amount of PVA (polyvinyl alcohol) was mixed into a PBS solution of pH 7.4, and heated at 90°C to completely dissolve it so that its final concentration was 6 wt%. Then, a certain amount of ADH (adipic acid dihydrazide) was added to the PVA solution so that the final concentration of ADH was 2 wt%, thereby obtaining PVA-ADH.

[0058] (4) The PB&VEGF@HA-PBA-ALD obtained in step (2) and the PVA-ADH obtained in step (3) were mixed in a volume ratio of 3:1. After slight shaking and stirring, the hydrogel was quickly formed. The final concentration of PBNPs in the hydrogel was 200 μg / mL, and the concentration of VEGF was 250 ng / mL.

[0059] In this gel system, hyaluronic acid HA is a natural polysaccharide with good biocompatibility and biodegradability, and is often used in drug delivery, medical implants, wound dressings and tissue engineering; polyvinyl alcohol PVA is a highly hydrophilic synthetic polymer with excellent mechanical properties; adipic acid dihydrazide ADH is a bifunctional small molecule cross-linking agent that can be cross-linked with hyaluronic acid as a protein drug carrier.

[0060] Example 2

[0061] The preparation process was the same as in Example 1, but the final concentration of PBNPs in the hydrogel was 150 μg / mL, and the concentration of VEGF was 300 ng / mL.

[0062] Example 3

[0063] The preparation process was the same as in Example 1, but the final concentration of PBNPs in the hydrogel was 250 μg / mL, and the concentration of VEGF was 200 ng / mL.

[0064] Example 4

[0065] The hydrogen nuclear magnetic resonance spectrum ( 1 H NMR) analysis. Take appropriate amounts of HA, HA-PBA and HA-PBA-ALD and dissolve them in deuterated water (D 2 O) was loaded for NMR acquisition, and the chemical shift was referenced to D 2 The solvent peak of O was 4.79 ppm for analysis.

[0066] Fourier transform infrared spectroscopy (FTIR) analysis. Take appropriate amounts of HA, HA-PBA and HA-PBA-ALD and a certain amount of potassium bromide (KBr) and put them into an agate mortar, grind and mix them evenly, press the mixture into a tablet and place it in an infrared spectrometer at a resolution of 4 cm -1 , scan times 32 times, 4000cm -1 ~400cm -1 The characteristic chemical structures of HA, HA-PBA and HA-PBA-ALD were determined by analyzing the peak wavenumber positions in the spectra.

[0067] 1 H NMR spectrum ( Figure 2 b), HA-PBA showed a characteristic peak attributable to the benzene ring of the phenylboronic acid group in the corresponding chemical environment, located between 7.4 and 7.7 ppm, which indicates that PBA was successfully modified on the HA main chain. In addition, the FTIR spectrum ( Figure 2 c) shows that HA-PBA has a peak at 1550 cm -1 and 700cm -1 There are C=C and CH bending vibrations of the benzene ring at 1340cm -1 The BO stretching vibration of boric acid appears at the position of the HA core, which also indicates that PBA has been successfully modified onto the HA backbone. The above indicates that HA-PBA has been successfully synthesized.

[0068] 1 H NMR spectrum ( Figure 2 b), compared with HA-PBA, due to the appearance of aldehyde groups, HA-PBA-ALD has new peaks at 4.9ppm, 5.0ppm and 5.1ppm, proving that the hydroxyl groups are oxidized to aldehyde groups. FTIR spectrum ( Figure 2 c) shows that HA-PBA-ALD has a peak at 1730 cm -1 The stretching vibration of the aldehyde C=O bond at HA-PBA indicates that sodium periodate has oxidized some of the hydroxyl groups in HA-PBA and HA-PBA-ALD has been successfully synthesized.

[0069] Example 5

[0070] Some intermediate products, i.e., the final products, during the preparation process of Example 1 were photographed. Figure 3 As shown, Figure 3 Shown are images of the hydrogel loaded with PBNPs and VEGF (Hydrogel@PB&VEGF) before and after formation. The prepared hydrogel is blue and transparent.

[0071] Example 6

[0072] The injectability test of the hydrogel was performed by loading the PBNPs and VEGF-loaded hydrogel (prepared in Example 1) into a 1 mL syringe with a 21-gauge needle, and manually pushing the syringe plunger to evaluate whether the hydrogel was squeezed through the needle into the PBS buffer or the surface of the culture dish.

[0073] Injectable hydrogels can be squeezed out through a syringe according to the shape of the wound to achieve precise coverage of the wound, which is particularly important for irregular or deep wounds because it ensures that the dressing fits the wound tightly, improves the sealing of the dressing on the wound, and reduces the risk of infection. We tested the injectability of the hydrogel, such as Figure 4 As shown, the hydrogel can be easily extruded into PBS buffer through a syringe or draw the letters "SSK" on the surface of a culture dish, and can maintain a stable gel state after the shear force is removed.

[0074] Example 7

[0075] To test the self-healing property of the hydrogel, two hydrogels of the same size (prepared in Example 1) were used, one of which was dyed blue with blue pigment. The hydrogel was cut in the middle and divided into four pieces. The four hydrogels were reassembled and placed in a culture dish. After 5 minutes, the self-healed hydrogel was lifted up with tweezers for observation to evaluate the ability of the four parts of the hydrogel to reconnect and self-heal.

[0076] Self-healing is another important property of hydrogels as wound dressings. Hydrogels with self-healing properties can quickly repair themselves after being damaged, which means that even if the dressing is damaged during use, it can maintain its integrity and functionality, which helps to extend the service life of the dressing and reduce the frequency of dressing changes. The double dynamic covalent bonds composed of borate bonds and acylhydrazone bonds can continuously break and recombine, thereby realizing the self-healing properties of the hydrogel. We tested the self-healing process of the hydrogel, such as Figure 5 As shown, the hydrogel was cut into four pieces and then reassembled together. After 5 minutes, it could be lifted up by tweezers, indicating that the four pieces of hydrogel can self-repair and withstand their own gravity without breaking.

[0077] Example 8

[0078] The hydrogel was prepared according to the method of Example 1, and then the pH value was lowered by adding 200 μL of PBS buffer with pH=5.5 to the surface of the hydrogel to evaluate the response characteristics of the hydrogel to pH.

[0079] By adding 200 μL of 1 mM HO to the hydrogel surface 2 O 2 To evaluate the response characteristics of hydrogels to ROS.

[0080] By adding 200 μL of 1 mM HO to the surface of the hydrogel2 O 2 , PBS buffer with pH = 5.5 was used to evaluate the dual response characteristics of the hydrogel to pH and ROS, namely, its inflammatory responsiveness.

[0081] Add the above-mentioned different buffers and shake evenly. After 8 hours, take a picture of the general shape of the hydrogel.

[0082] Based on the dual sensitivity of the phenylboronic acid ester bond and the acylhydrazone bond in the hydrogel to ROS and pH, we evaluated the inflammatory responsiveness of the hydrogel. Figure 6 As shown in Figure 2, after adding an acidic solution of pH 5.5 to the surface of the hydrogel, the hydrogel gradually dissolved and transformed from a gel state to a sol state. Similarly, after adding 1 mM H 2 O 2 Similar response behavior was also observed in the presence of both acid and H 2 O 2 The results showed that the hydrogel network structure was destroyed due to the hydrolysis of boronate bonds and acylhydrazone bonds in an inflammatory environment (low pH and high ROS). Once the three-dimensional network structure of the hydrogel collapsed, the PBNPs and VEGF loaded inside the hydrogel were quickly released into the external environment.

[0083] Example 9

[0084] Prepare PBS solution with pH=7.4 and PBS solution with pH=5.5, containing 1mM, pH=7.4H 2 O 2 PBS solution containing 1 mM HO 2 O 2 PBS solution with pH = 5.5 was used as the medium for dispersing and dialyzing the hydrogel. The hydrogel was placed in a dialysis bag and oscillated at a constant temperature of 37°C with an oscillation speed of 100 rpm. 100 μL of the solution was taken out at 3, 6, 12, 24, and 48 h, respectively. The cumulative drug release of PBNPs was determined by UV spectrophotometry, and the cumulative drug release of VEGF was determined by ELISA, and the drug release curve was prepared.

[0085] like Figure 7 As shown, under acidic conditions of pH 5.5 or 1 mM H 2 O 2Under oxidative conditions, PBNPs were rapidly released from the hydrogel, reaching 64.1% and 69.7% release at 12 h, respectively. In particular, under inflammatory conditions of acidic and oxidative environments, PBNPs showed the most sensitive and effective drug release performance, with a cumulative release rate of more than 60% in 6 h and 84.7% in 12 h. These results indicate that compared with the single responsiveness to ROS or pH, the hydrogel has an enhanced responsiveness to the inflammatory environment and more effective drug release. On the other hand, the release rate of loaded VEGF was slower than that of PBNPs, and the release rate of VEGF did not reach 77.2% until 24 h. This may be because the amino group of VEGF reacted with the aldehyde group of the polymer HA-PBA-ALD under Schiff base reaction, resulting in VEGF covalently bound to the hydrogel network structure.

[0086] Example 10

[0087] The in vitro antioxidant capacity of PBNPs was evaluated by scavenging 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) free radicals.

[0088] 100 μM DPPH and different concentrations of PBNPs solutions (0, 25, 50, 100, 200 μg / mL) were dispersed in 2 mL of ethanol and incubated for 12 h in the dark. Then, the absorbance value at 517 nm was recorded using a UV-visible spectrophotometer. The residual rate of DPPH was calculated by the following formula: DPPH residual rate = A sample / A blank × 100%.

[0089] PBNPs can remove excess ROS by simulating the activity of POD, SOD, and CAT antioxidant enzymes. Based on this characteristic, in order to evaluate the overall antioxidant effect of PBNPs, we conducted a DPPH free radical scavenging experiment. DPPH is a stable free radical that can be used to detect the antioxidant activity of nanomaterials. The results are shown in Figure 2. Figure 8 As shown, the ability of PBNPs to scavenge DPPH free radicals is concentration-dependent. When the concentration of PBNPs is 200 μg / mL, only about 50% of DPPH free radicals remain within 12 h, indicating that the prepared PBNPs have excellent antioxidant capacity.

[0090] Embodiment 11

[0091] Human umbilical vein endothelial cells (HUVECs) were seeded into 96-well plates at a density of 10,000 cells / well. After incubation for 24 hours in a medium containing VEGF at different concentrations (0, 5, 10, 50, 100, 250 ng / mL), the waste medium was discarded and the cells were washed once with PBS. A mixture of CCK-8 solution and serum-free medium at a volume ratio of 1:9 was added to each well. After incubation at 37°C for 2 hours, the absorbance at a wavelength of 450 nm was measured using an ELISA reader. The percentage of cell proliferation was calculated by comparing the absorbance of the sample group with that of the control group.

[0092] In order to determine the effective concentration of VEGF in the hydrogel, we performed a CCK-8 assay and calculated the cell proliferation rate after co-culture of HUVECs with different concentrations of VEGF for 24 h. Fig. 9 The results show that as the concentration of VEGF increases, its ability to promote cell proliferation gradually increases. When the concentration is 250ng / mL, the cell proliferation rate increases by 23.9%, which is significantly different from the control group (p<0.05).

[0093] Example 12

[0094] (1) Sample preparation: HUVECs cells were seeded in 6-well plates and divided into 5 groups: H 2 O 2 The cells were co-cultured with the culture medium at a volume ratio of 1:10 for 24 hours, and the pretreated cells were used for subsequent experiments.

[0095] (2) Draw a line on the bottom of the 6-well plate to mark the area in the same field of view when taking photos, which facilitates subsequent analysis and comparison of the wound healing status.

[0096] (3) Count the pretreated HUVECs and place them into a 6-well plate until they adhere to the plate and cover the entire bottom of the plate.

[0097] (4) Use a 1 mL pipette tip to create scratches on the cells perpendicular to the well plate, and try to ensure that the width of each scratch is consistent.

[0098] (5) Discard the cell culture medium and gently rinse the well plate three times with sterile PBS to wash away the cell debris generated when making cell scratches.

[0099] (6) Add serum-free culture medium and take photos. Continue culturing for 24 hours and take photos again.

[0100] Endothelial cell migration is a key step in angiogenesis and a hallmark of wound healing. The effect of Hydrogel@PB&VEGF on HUVECs cell migration was evaluated in vitro by a scratch healing experiment. Fig.10 As shown, compared with simple H 2 O 2 Compared with the Blank hydrogel group, the drug-loaded hydrogels can promote endothelial cell migration under oxidative stress, but the co-administration of PBNPs and VEGF can accelerate cell migration. The healing area reached 68% after 24 hours, which was significantly different from the other groups. The combined use of these two drugs showed a synergistic beneficial effect.

[0101] Embodiment 13

[0102] (1) Sample preparation: HUVECs cells were seeded in 6-well plates and divided into 5 groups: H 2 O 2 The cells were divided into three groups: blank hydrogel group, VEGF-loaded hydrogel group, PBNPs-loaded hydrogel group, and PBNPs- and VEGF-loaded hydrogel group. Each group was repeated for 3 wells. The hydrogel was first co-cultured with the culture medium at a volume ratio of 1:10 for 24 hours, and the pre-treated cells were used for subsequent experiments.

[0103] (2) One day before the experiment, place Matrigel in an ice box in the refrigerator and slowly melt it at 4°C. Before the experiment begins, keep Matrigel in an ice box and operate on ice. Use a pre-cooled pipette tip to mix the Matrigel.

[0104] (3) Dilute Matrigel with culture medium at a volume ratio of 1:1 and add 250 μl of Matrigel to each well of a 24-well plate, avoiding bubbles. Incubate the plate in a 37°C incubator for 30 minutes.

[0105] (4) The pretreated HUVECs cells were digested and incubated with 400 μM H 2 O 2 Resuspend the cells in culture medium, count and evenly plate them in each well, and incubate them in a 37°C incubator for 6 hours.

[0106] (5) After 6 hours, take 1 μl of 1 μM calcein AM and add it to 50 μl of culture medium to prepare a solution with a final concentration of 20 nM. Add an appropriate amount to the well plate and incubate with the cells at room temperature in the dark for 30 minutes.

[0107] (6) Slowly add PBS to avoid shocking the cells and wash three times.

[0108] (7) Immunofluorescence imaging analysis was performed using 485 nm / 529 nm wavelengths.

[0109] We also used matrigel to simulate the extracellular matrix and evaluated the angiogenesis-promoting ability of Hydrogel@PB&VEGF through angiogenesis experiments. Fig.11 As shown, HUVECs cells are labeled with green fluorescence. Consistent with the results of the scratch experiment, the Hydrogel@PB&VEGF group can significantly improve the ability of HUVECs cells to form vascular structures compared with the other four groups, while the Hydrogel@VEGF group has a very limited effect on blood vessel formation without improving the overall regenerative environment of the wound.

[0110] Embodiment 14

[0111] The db / db transgenic mouse is a hereditary type II diabetic, and its pathophysiological process is similar to that of clinical diabetic patients, including insulin resistance, insufficient insulin secretion, hyperglycemia and hyperinsulinemia. Compared with the wound model of STZ-induced mice, the db / db mouse wound model is more consistent with the actual situation of wound healing in clinical diabetic patients. Therefore, we selected db / db mice to establish a skin wound model for in vivo research.

[0112] The db / db mice used in the experiment were anesthetized with 1% sodium pentobarbital injected intraperitoneally at a dose of 40 mg / kg. After the mice entered a normal anesthesia state, the hair in the surgical area on the back was shaved with electric clippers, and the mice were fixed prone on the experimental table with their limbs spread out. The skin was then disinfected with alcohol and povidone iodine, and a disposable surgical disinfection drape was laid. On the middle side of the mouse's back, a circular full-thickness skin defect with a diameter of 1 cm was made with surgical scissors, and different hydrogel samples were placed on the wound, which was then covered with oiled gauze, sterile gauze and 3M transparent film in turn. The mice were free to eat and drink after waking up. Photos were taken on the 0th, 3rd, 7th and 14th days after surgery to record the changes in the wound.

[0113] In order to evaluate the effect of Hydrogel@PB&VEGF in promoting diabetic wound healing in vivo, we established a full-thickness skin defect model on the back of db / db mice. Gross images of the wound were recorded at different time points. The results are shown in Figure 2. Fig.12As shown in the figure, compared with the other four groups, Hydrogel@PB&VEGF can accelerate wound healing faster, and the wound is almost completely healed on the 14th day after injury, while only 56% of the wounds in the Control group and 64% of the wounds in the Blank hydrogel group have healed. In addition, we also observed that the healing speed of the Hydrogel@VEGF group and the Hydrogel@PB group was faster than that of the Control group but slower than that of the Hydrogel@PB&VEGF group, indicating that the combination of PBNPs and VEGF in the hydrogel can synergistically promote the healing of diabetic wounds, which is better than using them alone.

Claims

1. An inflammatory response hydrogel for promoting diabetic wound healing, characterized in that: The invention comprises a hydrogel and Prussian blue nanoparticles and vascular endothelial growth factor loaded on the hydrogel, wherein the loading concentration of the Prussian blue nanoparticles is 150-250 μg / mL; the loading concentration of the vascular endothelial growth factor is 200-300 ng / mL; the hydrogel uses HA-PBA-ALD as a skeleton, and forms a borate bond and an acylhydrazone bond after cross-linking with PVA / ADH; The amino groups of VEGF in the hydrogel react with the aldehyde groups of the polymer HA-PBA-ALD to form a Schiff base reaction, and VEGF is covalently bound to the hydrogel network structure, so the release rate of the loaded VEGF is slower than that of PBNPs. Prepared by the following steps: (1) coupling 3-aminomethylphenylboronic acid to hyaluronic acid to obtain hyaluronic acid-phenylboronic acid polymer; (2) oxidizing the vicinal diol on the hyaluronic acid phenylboronic acid polymer chain obtained in step (1) to obtain an oxidized hyaluronic acid phenylboronic acid polymer; (3) loading Prussian blue nanoparticles and vascular endothelial growth factor onto the oxidized hyaluronic acid phenylboronic acid polymer obtained in step (2) to obtain the inflammatory response hydrogel for promoting diabetic wound healing; In step (1), the molar ratio of 3-aminomethylphenylboronic acid to hyaluronic acid is 1:1; The coupling agents used in the coupling were 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride and EDC; In step (3), during loading, the oxidized hyaluronic acid phenylboronic acid polymer obtained in step (2) is first mixed into a buffer solution, and then the Prussian blue nanoparticles and vascular endothelial growth factor are mixed into the buffer solution to obtain a mixed solution A; Mix polyvinyl alcohol into a buffer solution, heat to dissolve, and then add adipic acid dihydrazide to dissolve to obtain a mixed solution B; The mixed solution A and the mixed solution B are mixed and reacted to obtain the inflammation-responsive hydrogel for promoting diabetic wound healing.

2. The inflammatory response hydrogel for promoting diabetic wound healing according to claim 1, characterized in that: The vascular endothelial growth factor is derived from human, mouse, pig or rat.

3. The inflammatory response hydrogel for promoting diabetic wound healing according to claim 1, characterized in that: The preparation method of the Prussian blue nanoparticles comprises the following steps: Dissolve K4[Fe(CN)6] and citric acid in water to obtain solution A; Dissolve Fe(NO3)3 and citric acid in water to obtain solution B; Solution B is added dropwise to solution A to obtain the Prussian blue nanoparticles.

4. The inflammatory response hydrogel for promoting diabetic wound healing according to claim 1, characterized in that: In step (2), the oxidant used for oxidation is sodium periodate.

5. Use of the inflammatory response hydrogel for promoting diabetic wound healing according to any one of claims 1 to 4 in the preparation of a wound healing medicament.

6. The use according to claim 5, characterized in that: The wound surface is a diabetic wound surface.

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