Preparation of nano composite hydrogel and application of nano composite hydrogel in acceleration of healing of diabetic wounds

Abstract

The invention belongs to the technical field of biomedical materials, and discloses a preparation method of nano composite hydrogel and application of the nano composite hydrogel in acceleration of healing of diabetic wounds. The invention constructs the hydrogel based on the epsilon-polylysine grafted by the oxidized hyaluronic acid and the phenylboronic acid and the ZIF-8. Due to the existence of the dynamic Schiff base, the boric acid ester bond and the coordination bond, the hydrogel has ideal mechanical property, strong viscosity, injectability, self-healing property and pH / ROS responsiveness. Due to the existence of chlorogenic acid and zinc ions, the hydrogel regulates macrophage phenotypes, relieves oxidative stress and accelerates healing of diabetic wounds through an NF-kappa B / JAK-STAT signal channel.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and particularly relates to the preparation of a nano-composite hydrogel and its use in accelerating the healing of diabetic wounds. Background Art

[0002] Hydrogels are a class of materials with a three-dimensional network structure and high water content. Similar to the extracellular matrix structure, they can simulate the microenvironment to promote the growth of cell tissues. In recent years, injectable hydrogels derived from natural polymers have been more attractive in tissue engineering due to their good biocompatibility and biodegradability.

[0003] However, traditional hydrogels have poor properties, such as lack of tissue adhesiveness, unsatisfactory mechanical and degradation properties, and no self-healing ability after injury, which limit their biomedical applications. Frequent dressing changes may also cause wound irritation, damage to newly grown tissues, and increase the risk of infection. In recent years, increasing attention has been paid to stimulus-responsive hydrogels because they can be regulated under the influence of external stimuli (including temperature, pH, and light). Considering the low pH and high ROS microenvironment at diabetic wound sites, developing a pH and ROS-responsive hydrogel is considered an effective strategy. This intelligent hydrogel can provide spatiotemporal control over drug release and effectively protect unstable drugs from degradation.

[0004] Nano-dynamic crosslinked hydrogels combine the injectability and self-healing properties of dynamic hydrogels with the versatility of nanomaterials, showing unique advantages. Incorporating nanomaterials as crosslinkers can improve the mechanical properties (strength, injectability, and shear-thinning properties) of hydrogels by strengthening the backbone and endowing it with multifunctionality. ZIF-8 is a widely used zinc-based MOF and has been extensively studied due to its favorable properties such as excellent biocompatibility, large pore size, high specific surface area, and pH responsiveness, making it an excellent candidate for drug delivery systems. Chlorogenic acid is a common antioxidant drug, and it has recently been reported to also have the ability to promote cell migration. In addition, ZIF-8 has acid-labile organic ligands that ionize in an acidic environment, causing ZIF-8 to decompose and release internal drugs and zinc ions, which is beneficial for cell proliferation, migration, and angiogenesis, accelerating the healing of diabetic wounds.

[0005] The present invention has developed a hydrogel containing ZIF-8, which has injectability, adhesiveness, self-healing ability, pH / ROS responsiveness, and excellent mechanical properties. The zinc ions and chlorogenic acid released by the hydrogel can relieve oxidative stress, promote cell proliferation, migration, and angiogenesis, and accelerate the healing of diabetic wounds. Summary of the Invention

[0006] The object of the present invention is to provide a nano-composite hydrogel, clarify its preparation process, and use it to accelerate the healing of diabetic wounds. The injection hydrogel has a novel formulation and excellent performance, and has broad application prospects in the biomedical industry.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The first aspect of the technical solution of the present invention provides a ZIF-8 hydrogel loaded with chlorogenic acid, which is characterized in that it is obtained by dynamic cross-linking of oxidized hyaluronic acid, ε-polylysine grafted with phenylboronic acid, and a metal-organic framework.

[0009] The metal-organic framework in the nano-composite hydrogel includes zeolitic imidazolate framework (ZIF-8).

[0010] The drug loaded in ZIF-8 in the nano-composite hydrogel includes chlorogenic acid.

[0011] In this nano-composite hydrogel system, the final concentration of ZIF-8 loaded with chlorogenic acid is 0.1 - 5000 μmol / L, and the preferred concentration is 500 - 3000 μmol / L; the final concentration of oxidized hyaluronic acid is 1 - 200 mg / mL, and the preferred concentration is 25 - 75 mg / mL; the final concentration of ε-polylysine grafted with phenylboronic acid is 10 - 500 mg / mL, and the preferred concentration is 75 - 150 mg / mL.

[0012] In the nano-composite hydrogel, the mass ratio of oxidized hyaluronic acid to ε-polylysine grafted with phenylboronic acid is 0.1:2 - 2:0.1, and the preferred mass ratio is 0.5:1.

[0013] In the nano-composite hydrogel, the oxidation degree of oxidized hyaluronic acid is 5% - 50%, and the preferred oxidation degree is 20% - 40%; the grafting rate of ε-polylysine grafted with phenylboronic acid is 1% - 30%, and the preferred grafting rate is 10% - 20%.

[0014] The second aspect of the technical solution of the present invention is to provide the application of the nano-composite hydrogel described in the first aspect in the preparation and acceleration of diabetic wound healing. This hydrogel regulates macrophage phenotype, alleviates oxidative stress, and accelerates the healing of diabetic wounds through the NF-κB / JAK-STAT signaling pathway. The steps are as follows:

[0015] (1) Dissolve 2 mg of chlorogenic acid and 80 mg of Zn(NO3)2·6H2O in 3.48 mL of dimethyl sulfoxide and 1.32 mL of H2O. After stirring for 30 min, add dropwise 4 mL of H2O containing 800 mg of 2-methylimidazole, and continue stirring for 30 min. Finally, obtain ZIF-8 loaded with chlorogenic acid by centrifugation (14,000 rpm, 10 min), and wash it three times with methanol and H2O;

[0016] (2) 1.25 mL of 10.72% (w / v) NaIO4 solution was added dropwise to 50 mL of 5% (w / v) hyaluronic acid solution, and the mixture was reacted at 25 °C and 600 rpm in the dark for 3.5 h. Then, 1.25 mL of ethylene glycol was added to the solution, and stirring was continued for 15 min. The solution was dialyzed using a dialysis bag (cut-off molecular weight of 3500), and the oxidized hyaluronic acid was obtained after lyophilization of the solution.

[0017] (3) 750 μL of 20 mg / mL ε-polylysine DMSO solution and 200 μL of 20 mg / mL 4-bromomethylphenylboronic acid DMSO solution were mixed, and then 10 μL of triethylamine was added. After reacting at 70 °C for 24 h, the phenylboronic acid-grafted ε-polylysine was obtained after dialysis and lyophilization.

[0018] (4) ZIF-8 loaded with chlorogenic acid was uniformly dispersed in the PBS solution of phenylboronic acid-grafted ε-polylysine. At the same time, the PBS solution of oxidized hyaluronic acid was obtained. The two solutions were completely mixed in proportion to obtain the nano-composite hydrogel.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. A hydrogel based on oxidized hyaluronic acid, phenylboronic acid-grafted ε-polylysine, and ZIF-8 was constructed. This hydrogel has injectability, adhesiveness, self-healing property, pH / ROS responsiveness, and excellent mechanical properties. Through coordination bond interaction, ZIF-8 loaded with chlorogenic acid acts as a cross-linking agent and nano-filler to enhance the mechanical properties of the hydrogel.

[0021] 2. Schiff base and borate ester bonds can respond to the low pH and high ROS at diabetic wound sites to regulate the inflammatory microenvironment; zinc and chlorogenic acid also have significant antioxidant effects; in addition, ZIF-8, chlorogenic acid, Schiff base, and borate ester bonds regulate macrophage phenotypes through the NF-κB / JAK-STAT signaling pathway, relieve oxidative stress, and accelerate the healing of diabetic wounds. Description of the Drawings

[0022] Figure 1 : Synthesis results of ZIF-8 loaded with chlorogenic acid, A is the particle size, B is the potential, and C is the transmission electron microscope;

[0023] Figure 2 : FT-IR results, A is oxidized hyaluronic acid, and B is phenylboronic acid-modified ε-polylysine;

[0024] Figure 3 : 1 1H NMR results, A is oxidized hyaluronic acid, and B is phenylboronic acid-modified ε-polylysine;

[0025] Figure 4 : Rheological results of OHA-PP@Z-CA hydrogel, A is frequency sweep, B is continuous strain sweep;

[0026] Figure 5 : Adhesion, injectability and self-healing results of OHA-PP@Z-CA hydrogel;

[0027] Figure 6 : In vitro swelling and degradation results of OHA-PP@Z-CA hydrogel, A is in vitro swelling result of OHA-PP hydrogel, B is in vitro swelling result of OHA-PP@Z-CA hydrogel; C is in vitro degradation result of OHA-PP hydrogel, D is in vitro degradation result of OHA-PP@Z-CA hydrogel;

[0028] Figure 7 : In vitro release results of OHA-PP@Z-CA hydrogel, A is Zn release result of OHA-PP@Z-CA hydrogel at pH 7.4, B is Zn release result of OHA-PP@Z-CA hydrogel at pH 6.5 + 1 mM H2O2, C is chlorogenic acid release result of OHA-PP@Z-CA hydrogel at pH 7.4, D is chlorogenic acid release result of OHA-PP@Z-CA hydrogel at pH 6.5 + 1 mM H2O2;

[0029] Figure 8 : Anti-inflammatory results of OHA-PP@Z-CA hydrogel, A is ROS scavenging result of OHA-PP@Z-CA hydrogel, B is macrophage phenotype regulation result of OHA-PP@Z-CA hydrogel, C is anti-inflammatory signaling pathway result of OHA-PP@Z-CA hydrogel;

[0030] Figure 9 : Results of OHA-PP@Z-CA hydrogel accelerating diabetic wound healing, A is blood glucose change result of rats during the experiment, B is wound change result of diabetic rats during the treatment, C is wound healing rate result of diabetic rats during the treatment. Specific implementation manners

[0031] The present invention will be further clarified below with reference to the accompanying drawings and specific embodiments. These embodiments are only used to illustrate the present invention and not to limit the scope of the present invention.

[0032] Example 1: Preparation of ZIF-8 loaded with chlorogenic acid

[0033] Dissolve 2 mg of chlorogenic acid (CA) and 80 mg of Zn(NO3)2·6H2O in 3.48 mL of dimethyl sulfoxide and 1.32 mL of H2O. After stirring for 30 min, add dropwise 4 mL of H2O containing 800 mg of 2-methylimidazole, and continue stirring for 30 min. Finally, obtain ZIF-8 loaded with chlorogenic acid by centrifugation (14,000 rpm, 10 min), and wash it three times with methanol and H2O to obtain ZIF-8 loaded with chlorogenic acid (Z-CA), the particle size of which is 160.3 nm( Figure 1 A), and the potential is 16.5 mV( Figure 1 B). Observe by transmission electron microscopy( Figure 1 C) that Z-CA shows a clear rhombic dodecahedron morphology with sharp edges and uniform particle size. The drug content (drug loading) of chlorogenic acid in Z-CA is 12.42 ± 1.31%.

[0034] Example 2: Synthesis of carrier material

[0035] This example includes the synthesis of oxidized hyaluronic acid and ε-polylysine grafted with phenylboronic acid

[0036] Add dropwise 1.25 mL of 10.72% (w / v) NaIO4 solution to 50 mL of 5% (w / v) hyaluronic acid solution, react at 25 °C and 600 rpm in the dark for 3.5 h, add 1.25 mL of ethylene glycol to the solution, continue stirring for 15 min, and dialyze using a dialysis bag (cut-off molecular weight is 3500). After the solution is freeze-dried, oxidized hyaluronic acid (OHA) is obtained.

[0037] Mix 750 μL of 20 mg / mL DMSO solution of ε-polylysine and 200 μL of 20 mg / mL DMSO solution of 4-bromomethylphenylboronic acid, add 10 μL of triethylamine, react at 70 °C for 24 h, and after dialysis and freeze-drying, obtain phenylboronic acid-modified ε-polylysine (PP).

[0038] Through FT-IR( Figure 2 ) and 1 H NMR( Figure 3 ) The results show that OHA and PP are successfully prepared.

[0039] Example 3: Preparation of nano-composite hydrogel

[0040] Prepare a 50 mg / mL OHA solution in PBS at pH 7.4. Disperse Z-CA evenly in PBS at pH 7.4, and then add PP to obtain a 75 mg / mL PP solution containing 30% (w / v) Z-CA. Mix the above two solutions in equal volumes to obtain a nano-composite hydrogel (OHA-PP@Z-CA). The hydrogel without Z-CA is denoted as OHA-PP hydrogel.

[0041] Example 4: Performance evaluation of OHA-PP@SeNPs hydrogel

[0042] This example includes evaluating the rheology, adhesiveness, injectability, self-healing property, in vitro swelling and degradation of the hydrogel.

[0043] (1) Rheology

[0044] Detect the storage modulus (G′) and loss modulus (G″) of the hydrogel at 37 °C using a rheometer. Perform a frequency sweep from 0.01 - 1 Hz at a constant strain of 0.5% in the linear viscoelastic region. G′ reveals the elastic properties of the hydrogel, and G″ reveals the viscous properties of the hydrogel. In the range of 0.01 - 1 Hz, G′ of each group is higher than G″, without a cross-over point ( Figure 4 A), indicating that the hydrogel forms stably and has good elasticity. The oscillation of G″ may be related to the breakage and recombination of dynamic Schiff base bonds, borate ester bonds and coordination bonds in the gel network. The G′ of the OHA-PP@Z-CA hydrogel group is higher than that of the OHA-PP hydrogel group, reflecting that the addition of Z-CA increases the mechanical properties of the hydrogel. Under high strain conditions, G″ is greater than G′, indicating that the network structure of the hydrogel has loosened or even been damaged, losing its integrity and mechanical stability. When the strain amplitude returns to low strain, G″ and G′ immediately return to their original values ( Figure 4 B).

[0045] (2) Adhesiveness, injectability and self-healing property

[0046] Use items of different materials to evaluate the adhesion of the OHA-PP@SeNPs hydrogel; to evaluate the injectability of the OHA-PP@Z-CA hydrogel, use a double-barrel single-needle syringe to spell different letters. As Figure 5 shown by the results, the good adhesiveness, injectability and self-healing property of the OHA-PP@Z-CA hydrogel are demonstrated.

[0047] (3) In vitro swelling and degradation

[0048] Perform a swelling experiment on the hydrogel in PBS at pH 7.4 at 37 °C. Take out the swollen hydrogel at different time points. After blotting the surface moisture with filter paper, the weight of the hydrogel at this time is denoted as W t。The initial weight of the freeze-dried OHA-PP@Z-CA hydrogel is denoted as M0. The freeze-dried OHA-PP hydrogel serves as the control group. Swelling ratio (%) = (M t -M0) / M0×100%. The results are shown in Figure 6 A and B, demonstrating the ideal in vitro swelling performance of the OHA-PP@Z-CA hydrogel.

[0049] The degradation of the hydrogel was tested in pH 7.4 PBS, pH 6.5 PBS, 1 mM H2O2 pH 7.4 PBS, and 1 mM H2O2 pH 6.5 PBS respectively. All samples were stored at room temperature for 14 days. At different time intervals, the degradation rate of the hydrogel was recorded. Figure 6 C and D are the in vitro degradation line graphs of the OHA-PP@Z-CA hydrogel, demonstrating the significant degradation performance and good pH and ROS sensitivity of the OHA-PP@Z-CA hydrogel.

[0050] (4) In vitro release

[0051] The in vitro release of the hydrogel was tested in pH 7.4 PBS and 1 mM H2O2 pH 6.5 PBS respectively. First, the release behavior of Zn 2+ was detected by ICP-MS. Compared with pH 7.4, under the condition of pH 6.5 + 1 mM H2O2, OHA-PP@Z-CA had a higher Zn 2+ release degree, and the release degree on the tenth day was 18.64% ± 1.42% ( Figure 7 A and B). Meanwhile, with the degradation of Z-CA, the internal CA was rapidly released ( Figure 7 C and D).

[0052] Example 5: Anti-inflammatory mechanism of OHA-PP@SeNPs hydrogel

[0053] (1) ROS scavenging

[0054] The ROS scavenging ability of the OHA-PP@SeNPs hydrogel was detected using DCFH-DA. RAW 264.7 cells were seeded in 12-well plates at a density of 1×10 5 cells / well. The cells were induced with 1 μg / mL LPS for 24 h. The cells were co-cultured with the hydrogel extract for 24 h. RAW 264.7 cells were incubated with 0.5 mL Hoechst 33342 for 10 min, and then incubated with 10 μmol / L DCFH-DA for 30 min. The results obtained by laser scanning confocal microscopy are shown in Figure 8As shown in Figure A, the OHA-PP@SeNPs hydrogel significantly reduced the ROS level in cells, indicating that the OHA-PP@SeNPs hydrogel has the property of scavenging excessive ROS.

[0055] (2) Macrophage phenotype

[0056] The phenotype of macrophages was detected by immunofluorescence staining. RAW 264.7 cells were seeded in 6-well plates at a density of 2×10 5 cells / well and cultured for 12 h. The cells were induced with 1 μg / mL LPS for 24 h. The cells were co-cultured with the hydrogel extract for 24 h. Next, the cells were fixed, permeabilized, and blocked. The cells were incubated with iNO (1:500) and CD163 (5 μg / ml) overnight at 4 °C, and then incubated with fluorescent secondary antibody for 1 h at 25 °C. The cells were co-incubated with DAPI dye for 10 min to stain the nuclei. The results obtained by laser scanning confocal microscopy are shown in Figure 8 Figure B. The OHA-PP@Z-CA hydrogel induced the transformation of macrophages from pro-inflammatory M1 type to anti-inflammatory M2 type, exerting an anti-inflammatory effect.

[0057] (3) NF-κB / JAK-STAT signaling pathway

[0058] RAW264.7 cells were seeded in 6-well plates at a density of 2×10 5 cells / well. The cells were induced with 1 μg / mL LPS for 24 h. The cells were co-cultured with the hydrogel extract for 24 h. The cells were collected by cell scraper and lysed in RIPA containing 1 mM PMSF in an ice bath. The total protein concentration was determined by BCA protein assay kit. The samples separated by SDS-PAGE were transferred onto 0.22 μm PVDF membranes. After blocking for 1 h, the PVDF membranes were incubated with primary antibodies overnight at 4 °C, and then incubated with fluorescent secondary antibody for 1 h at 25 °C. Finally, the PVDF membranes were imaged using ECL luminescent reagent in the dark environment, and the results are shown in Figure 8 Figure C. LPS induced the activation of NF-κB and JAK-STAT signaling pathways in macrophages. The phosphorylation of JAK and STAT was significantly reduced after treatment with the OHA-PP@Z-CA hydrogel ( Figure 8 Figure C). Meanwhile, the hydrogel could also reduce the expression of NF-κB pathway-related proteins (IKK, IκB-α, and P65), inhibiting NF-κB signal transduction. In addition, we found that the hydrogel reduced the phosphorylation level of P38. The Western blot results showed that the OHA-PP@Z-CA hydrogel could inhibit the activation of NF-κB and JAK-STAT signaling pathways induced by LPS, regulate macrophage phenotype, reduce ROS level, and exert an anti-inflammatory effect.

[0059] Example 6: OHA-PP@SeNPs hydrogel accelerates diabetic wound healing

[0060] Male Sprague-Dawley (SD) rats were selected as experimental subjects for skin wound healing experiments. A type I diabetic SD rat model was established by intraperitoneal injection of 60 mg / kg streptozotocin (citrate buffer, pH 4.5). When the fasting blood glucose of the rats continued to be higher than 16.7 mmol / L for one week, the type I diabetic rat model was considered successfully established. The rats were anesthetized by inhalation and the hair on the back was shaved off. Rats with intact skin were selected to establish a wound model. The back was shaved and a full-thickness wound model with a diameter of 8 mm was established on the back. The 60 successfully modeled SD rats were grouped and randomly divided into 5 groups, with 12 rats in each group, and they were housed separately in cages. They were named the model group, the Z-CA group (0.5 mL), the OHA-PP hydrogel group (0.5 mL), the OHA-PP@Z-CA hydrogel group (0.5 mL), and the positive group. The model group was not treated, and the positive group was covered with a commercially available Tegadron TM dressing (3M, USA). To evaluate the healing ability of the hydrogel, the optical images of the wounds at different times were recorded with a camera to calculate the wound healing rate, and the body weight was recorded. The wound contraction rate (%) was measured by Image J software. Another 6 healthy SD rats were set as the control group.

[0061] The blood glucose changes were as shown in Figure 9 A. During the whole administration process, the blood glucose level of the rats was higher than 16.7 mmol / L, proving that the diabetic model was successfully established.

[0062] The healing process of the diabetic wounds was as shown in Figure 9 B. In all groups, the wounds of the rats shrank over time. The wound healing rate of the model group was the lowest and scabbing occurred. Compared with the model group, the wound contraction of the OHA-PP@Z-CA hydrogel treatment group was significantly accelerated and the healing rate was the highest ( Figure 9 C). The results showed that the OHA-PP@Z-CA hydrogel had a better wound-healing effect than other groups (model, Z-CA, OHA-PP, and positive group).

[0063] The above-described embodiments are only preferred solutions of the present invention and do not impose any formal restrictions on the present invention. There are other improvements and variations without exceeding the prescribed amounts recorded in the claims.

Claims

1. A nano-composite hydrogel, characterized in that, It is obtained by dynamic crosslinking of oxidized hyaluronic acid, ε-polylysine grafted with phenylboronic acid, and metal-organic framework.

2. The nano-composite hydrogel according to claim 1, characterized in that, The metal-organic framework includes zeolitic imidazolate framework (ZIF-8).

3. The nano-composite hydrogel according to any one of claims 1-2, characterized in that, The drug loaded in ZIF-8 includes chlorogenic acid.

4. The nano-composite hydrogel according to claim 1, characterized in that, In the hydrogel system, the final concentration of ZIF-8 loaded with chlorogenic acid is 0.1 - 5000 μmol / L, and the preferred concentration is 500 - 3000 μmol / L; the final concentration of oxidized hyaluronic acid is 1 - 200 mg / mL, and the preferred concentration is 25 - 75 mg / mL; the final concentration of ε-polylysine grafted with phenylboronic acid is 10 - 500 mg / mL, and the preferred concentration is 75 - 150 mg / mL.

5. The nano-composite hydrogel according to claim 1, characterized in that, The mass ratio of oxidized hyaluronic acid to ε-polylysine grafted with phenylboronic acid is 0.1:2 - 2:0.1, and the preferred mass ratio is 0.5:

1.

6. The nano-composite hydrogel according to claim 1, characterized in that, The oxidation degree of oxidized hyaluronic acid is 5% - 50%, and the preferred oxidation degree is 20% - 40%; the grafting rate of ε-polylysine grafted with phenylboronic acid is 1% - 30%, and the preferred grafting rate is 10% - 20%.

7. Use of the nano-composite hydrogel according to any one of claims 1 - 6 in the preparation and acceleration of diabetic wound healing.

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