A cytokine hydrogel, its preparation method and application in skin injury repair

Abstract

The application provides a cytokine hydrogel and a preparation method and application thereof in skin damage repair, and belongs to the technical field of biological medicine. The method comprises the following steps: (1) adding beta-GP into a chitosan solution to obtain a CS-beta-GP pre-gel solution; (2) dissolving Pluronic F127 and hyaluronic acid in a PBS solution to obtain a F127-HA solution after complete transparency; (3) sequentially dissolving trehalose, cordycepin, a cytokine composition and collagen peptide in a PBS solution, incubating, obtaining a cytokine mixture, and mixing the cytokine mixture with the CS-beta-GP pre-gel solution and the F127-HA solution. A double temperature-sensitive system is established, the system can be injected at low temperature, forms a gel quickly under the condition of body temperature, the effective period of active ingredients is prolonged by using trehalose stabilization technology, the anti-inflammatory effect of cordycepin and the cell repair effect of collagen peptide are combined, and efficient repair of skin damage can be realized.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a cytokine hydrogel, a preparation method thereof, and an application thereof in repairing skin damage. Background Art

[0002] Skin injury repair is an important research direction in clinical medicine and regenerative medicine, involving multiple biological processes such as hemostasis, anti-infection, cell proliferation and tissue remodeling. Although traditional dressings (such as gauze and hydrocolloid dressings) can provide physical protection, they cannot dynamically respond to the wound microenvironment and lack the ability to accurately deliver active ingredients that promote repair. Hydrogel is a soft material composed of a hydrophilic polymer network that can absorb and retain a large amount of water (usually tens to hundreds of times its own weight) while maintaining three-dimensional structural stability and can load drugs. Due to its unique physicochemical properties, good biocompatibility and controllable mechanical properties, hydrogel has a wide range of applications in biomedicine, tissue engineering, drug delivery, wound dressing and other fields, and is an ideal skin repair material.

[0003] Thermosensitive hydrogels can remain liquid at low temperatures (below 25°C), making them easy to inject or apply, and quickly gel at body temperature (37°C) to fit the wound. Chitosan (CS) has been widely studied for its natural antibacterial, procoagulant and biodegradable properties, but its mechanical properties are poor, its gelling properties are unstable, and active ingredients such as cytokines are difficult to stably exist, resulting in a low wound repair rate. Therefore, this application hopes to provide a cytokine hydrogel with good gelling properties, which can ensure the activity of cytokines and improve the wound repair rate. Summary of the Invention

[0004] The purpose of the present invention is to provide a cytokine hydrogel that can quickly form a gel, ensure the activity of cytokines, and facilitate the repair of skin damage.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing a cytokine hydrogel, comprising the following steps:

[0007] (1) adding sodium β-glycerophosphate to a chitosan solution to obtain a CS-β-GP pregel solution;

[0008] (2) dissolving Pluronic F127 and hyaluronic acid in PBS solution until completely transparent to obtain F127-HA solution;

[0009] (3) dissolving trehalose and cordycepin in PBS solution, adding the cytokine composition and collagen peptide, and incubating to obtain a cytokine mixture;

[0010] (4) Filter the CS-β-GP pregel solution and F127-HA solution separately and mix them with the cytokine mixture.

[0011] Preferably, the chitosan solution in step (1) is prepared by dissolving chitosan in 0.1-0.2 M acetic acid at a pH of 4.5-5.0, and stirring with a magnetic stirrer at 4°C until completely dissolved, to obtain a chitosan solution with a final concentration of 10-20 g / L;

[0012] The addition amount of the β-sodium glycerophosphate is 80-120 g / L.

[0013] Preferably, in step (2), the final concentration of the Pluronic F127 in the F127-HA solution is 150-200 g / L; and the final concentration of the hyaluronic acid is 5-20 g / L.

[0014] Preferably, in step (3), the amount of trehalose added is 20-25 mg / mL; the amount of cordycepin added is 0.1-1 mg / mL; the amount of the cytokine composition added is 50-200 ng / mL; and the amount of the collagen peptide added is 10-15 mg / mL.

[0015] Preferably, the cytokine composition comprises: EGF, bFGF and VEGF, with a mass ratio of 1 to 3:1 to 2:1.

[0016] Preferably, the incubation conditions in step (3) are 0-5°C for 20-40 min.

[0017] Preferably, the filter membrane used in step (4) has a pore size of 0.2 to 0.3 μm.

[0018] Preferably, the volume ratio of the CS-β-GP pregel solution, F127-HA solution and cytokine mixture in step (4) is 1 to 3:1 to 3:1.

[0019] The present invention provides a cytokine hydrogel.

[0020] The present invention also provides application of the cytokine hydrogel in preparing a medicine for repairing skin damage.

[0021] Beneficial effects

[0022] The hyaluronic acid (HA) in the present invention can enhance the moisture retention and cell affinity of the gel and improve the ability of cell migration; trehalose as a protective agent can not only maintain a moist environment to accelerate epithelialization, but also effectively protect the loaded cytokines (such as epidermal growth factor EGF, basic fibroblast growth factor bFGF and endothelial growth factor VEGF), stabilize the activity of polypeptides, and prevent denaturation and inactivation during storage and delivery. Collagen peptides can promote cell proliferation and repair. Cordycepin has anti-inflammatory, antioxidant and immunomodulatory effects, can reduce inflammatory damage, and cooperate with cytokines to accelerate the repair of damaged skin.

[0023] The present invention establishes a dual temperature-sensitive system that can be injected at low temperatures and quickly gel at body temperature, maintaining a balance between injectability and gel strength. The cytokine hydrogel provided by the present invention uses trehalose stabilization technology to extend the effective period of the active ingredients. Its repair effect can cover the entire cycle of the skin repair process. At the same time, combined with the anti-inflammatory effect of cordycepin and the cell repair effect of collagen peptides, it can achieve efficient repair of skin damage. DETAILED DESCRIPTION

[0024] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0025] Example 1

[0026] (1) Chitosan (90% deacetylation) was dissolved in 0.1 M acetic acid at a pH of 5.0 and stirred at 4°C until completely dissolved to obtain a chitosan solution with a concentration of 20 g / L. Sodium β-glycerophosphate was slowly added at a concentration of 100 g / L under ice bath conditions and the pH was adjusted to 7 to obtain a CS-β-GP pregel solution.

[0027] (2) Pluronic F127 and hyaluronic acid were dissolved in PBS solution until completely transparent to obtain F127-HA solution. The amount of Pluronic F127 added was 150 g / L; the amount of hyaluronic acid added was 15 g / L.

[0028] (3) Trehalose, cordycepin, cytokine composition (EGF, bFGF and VEGF, mass ratio of 1.5:1.5:1) and collagen peptide were added to PBS solution in the following order: 25 mg / mL trehalose, 1 mg / mL cordycepin, 200 ng / mL cytokine composition and 15 mg / mL collagen peptide, and incubated at 4°C for 30 min to obtain a cytokine mixture;

[0029] (4) The CS-β-GP pregel solution and F127-HA solution were sterilized by filtration through a 0.22 μm filter membrane, and then mixed with the cytokine mixture at a volume ratio of 1:1:1.

[0030] Example 2

[0031] (1) Chitosan (90% deacetylation) was dissolved in 0.1 M acetic acid at a pH of 5.0 and stirred at 4°C until completely dissolved to obtain a chitosan solution with a concentration of 15 g / L. Sodium β-glycerophosphate was slowly added at a concentration of 100 g / L under ice bath conditions and the pH was adjusted to 7 to obtain a CS-β-GP pregel solution.

[0032] (2) Pluronic F127 and hyaluronic acid were dissolved in PBS solution until completely transparent to obtain F127-HA solution. The amount of Pluronic F127 added was 180 g / L; the amount of hyaluronic acid added was 15 g / L.

[0033] (3) Trehalose, cordycepin, cytokine composition (EGF, bFGF and VEGF, mass ratio of 1:1:1) and collagen peptide were added to PBS solution in the following order: 25 mg / mL trehalose, 0.8 mg / mL cordycepin, 150 ng / mL cytokine composition and 12 mg / mL collagen peptide, and the mixture was incubated at 4°C for 30 min to obtain a cytokine mixture.

[0034] (4) The CS-β-GP pregel solution and F127-HA solution were sterilized by filtration through a 0.22 μm filter membrane, and then mixed with the cytokine mixture at a volume ratio of 1:2:1.

[0035] Example 3

[0036] (1) Chitosan (90% deacetylation) was dissolved in 0.1 M acetic acid at a pH of 5.0 and stirred at 4°C until completely dissolved to obtain a chitosan solution with a concentration of 20 g / L. β-Sodium glycerophosphate was slowly added at an amount of 80 g / L under ice bath conditions and the pH was adjusted to 7 to obtain a CS-β-GP pregel solution.

[0037] (2) Pluronic F127 and hyaluronic acid were dissolved in PBS solution until completely transparent to obtain F127-HA solution. The amount of Pluronic F127 added was 150 g / L; the amount of hyaluronic acid added was 5 g / L;

[0038] (3) According to the addition amount of 20 mg / mL trehalose, 0.5 mg / mL cordycepin, 100 ng / mL cytokine composition, 10 mg / mL collagen peptide, trehalose, cordycepin, cytokine composition (EGF, bFGF and VEGF, mass ratio 2:1:1) and collagen peptide were sequentially added in PBS solution, and incubated at 4°C for 30 min to obtain a cytokine mixture;

[0039] (4) The CS-β-GP pre-gel solution and the F127-HA solution were filtered through a 0.22 μm filter membrane to remove bacteria, and then mixed with the cytokine mixture at a volume ratio of 1:1:1.

[0040] Comparative Example 1

[0041] Different from Example 1, no cytokine composition was added in this comparative example.

[0042] Comparative Example 2

[0043] Different from Example 1, no trehalose was added in this comparative example.

[0044] Comparative Example 3

[0045] Different from Example 1, no cordycepin was added in this comparative example.

[0046] Comparative Example 4

[0047] Different from Example 1, no collagen peptide was added in this comparative example.

[0048] Comparative Example 5

[0049] Different from Example 1, the combination of cytokines in this comparative example was epidermal growth factor (EGF), hepatocyte growth factor (HGF) and insulin growth factor-1 (IGF-1).

[0050] Comparative Example 6

[0051] Different from Example 1, the step of preparing F127-HA solution was not performed in this comparative example, and the volume ratio of CS-β-GP pre-gel solution to cytokine mixture in step (4) was 2:1.

[0052] Test Example 1 Wound Healing Rate

[0053] Two-month-old, SPF-grade healthy SD male rats weighing 200-250g were used as experimental subjects to establish a rat model of non-healing burn wounds. After anesthesia with intraperitoneal injection of 0.5mL of 3% sodium pentobarbital, the rats were depilated on their backs. A wound was created on the midline of the rats' backs. The wound was exposed to 95°C for 30 seconds using a 2cm diameter burn mold (aluminum block). 60μL of doxorubicin hydrochloride (2mg / mL) was injected subcutaneously 0.2cm from the wound edge. On the 28th day after the operation, the scabs on the rats' backs were removed to establish a rat model of non-healing burn wounds. Ten mice were housed normally as a blank control group (CK1).

[0054] The model rats were randomly divided into a model control group (CK2), Example 1 (T1), and comparative examples 1 to 6 (D1 to D6), with 10 rats in each group. Cytokine hydrogel (hydrogels prepared from Example 1 and comparative examples 1 to 6, respectively) was applied to the wound surface at a rate of 3 mg / cm 2 The control group received no medication and applied an equal amount of pure water.

[0055] On days 0, 4, 10, and 18 of treatment, digital cameras were used to photograph the wounds of the rats. Wound area was analyzed using digital image analysis software, and healing rates were calculated. Wound healing rate = (original wound area - unhealed wound area) / original wound area. The wound healing rates of each group of rats on days 4, 10, and 18 after treatment are shown in Table 1:

[0056] Table 1 Wound healing rate (%)

[0057] T1 D1 D2 D3 D4 D5 D6 CK2 Day 4 30.15 23.14 26.48 25.18 25.52 27.61 27.45 19.54 Day 10 70.16 45.84 56.25 51.48 53.01 64.25 60.81 31.85 Day 18 95.24 62.14 78.25 72.51 74.26 85.57 78.31 54.51

[0058] As can be seen from Table 1, at the same time, the wound healing rate of the cytokine hydrogel provided in the examples of the present application is higher than that of comparative examples 1 to 6, among which the cytokine composition has the greatest impact on the wound repair effect of the hydrogel. After the cytokine is used together with cordycepin and collagen peptide, the wound healing rate is significantly improved, indicating that the cordycepin and collagen peptide in the present application can work synergistically with the cytokine composition.

[0059] On day 8 of dosing, five mice were randomly selected from each group and fixed in an inverted position. The skin and artery of the neck were cut open, and blood was collected from the neck artery in a 10 mL centrifuge tube. The tube was allowed to stand for 2 hours and then centrifuged at 3000 rpm for 10 minutes. The upper serum layer was collected and frozen for future use. Serum levels of IL-6 and TNF-α were measured according to the test kit instructions.

[0060] Table 2

[0061] IL-6 (pg / mL) TNF-α (pg / mL) CK1 120.531 243.058 T1 135.251 261.245 D1 168.254 315.051 D2 146.624 291.153 D3 165.245 297.153 D4 158.352 287.248 D5 140.246 273.146 D6 145.254 281.241 CK2 180.254 330.242

[0062] As shown in Table 2, the present invention uses cordycepin and collagen peptides and other raw materials with anti-inflammatory and antioxidant effects in combination with cytokines, which can effectively alleviate the inflammatory response during skin damage and further enhance the repair effect of skin damage.

[0063] Test Example 2 Gel formation time

[0064] The experiment was divided into seven groups. Hydrogels were prepared according to the methods of Example 1 (T1) and Comparative Examples 1 to 6 (D1 to D6), with three replicates per group. The hydrogel solutions prepared in each group were placed at 37°C, and the time it took to form a solid gel was observed and recorded, with the average value taken. The time it took for the hydrogel solutions in each group to form a solid gel is shown in Table 3.

[0065] Table 3 Gel formation time (s)

[0066] T1 D1 D2 D3 D4 D5 D6 Gel formation time (s) 15.25 15.16 18.25 16.75 18.15 15.35 25.34

[0067] As shown in Table 3, the present invention has established a CS / β-GP+F127 dual temperature-sensitive system, which can quickly form a gel at body temperature.

[0068] 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 method for preparing a cytokine hydrogel, characterized in that: The following steps are involved: (1) adding sodium β-glycerophosphate to a chitosan solution to obtain a CS-β-GP pregel solution; (2) dissolving Pluronic F127 and hyaluronic acid in PBS solution until completely transparent to obtain F127-HA solution; (3) dissolving trehalose and cordycepin in PBS solution, adding the cytokine composition and collagen peptide, and incubating to obtain a cytokine mixture; (4) Filter the CS-β-GP pregel solution and F127-HA solution separately and mix them with the cytokine mixture.

2. The preparation method according to claim 1, wherein The chitosan solution in step (1) is prepared by dissolving chitosan in 0.1-0.2 M acetic acid at a pH of 4.5-5.0, and stirring with a magnetic stirrer at 4° C. until completely dissolved, to obtain a chitosan solution with a final concentration of 10-20 g / L. The addition amount of the β-sodium glycerophosphate is 80-120 g / L.

3. The preparation method according to claim 2, wherein In step (2), the amount of Pluronic F127 added to the F127-HA solution is 150-200 g / L; the amount of hyaluronic acid added is 5-20 g / L.

4. The preparation method according to claim 3, wherein In step (3), the amount of trehalose added is 20-25 mg / mL; the amount of cordycepin added is 0.1-1 mg / mL; the amount of the cytokine composition added is 50-200 ng / mL; and the amount of the collagen peptide added is 10-15 mg / mL.

5. The preparation method according to claim 4, wherein: The cytokine composition comprises EGF, bFGF and VEGF in a mass ratio of 1 to 3:1 to 2:

1.

6. The preparation method according to claim 5, characterized in that: The incubation conditions in step (3) are 0-5°C for 20-40 minutes.

7. The preparation method according to claim 6, characterized in that: The filter membrane used in step (4) has a pore size of 0.2 to 0.3 μm.

8. The preparation method according to claim 7, wherein: The volume ratio of the CS-β-GP pregel solution, F127-HA solution and cytokine mixture in step (4) is 1 to 3:1 to 3:

1.

9. A cytokine hydrogel prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the cytokine hydrogel according to claim 9 in preparing a drug for repairing skin damage.