Cytokine hydrogel as well as preparation method and application thereof in skin injury repair

Abstract

The invention provides cytokine hydrogel as well as a preparation method and application thereof in skin injury repair, and belongs to the technical field of biological medicines. Comprising the following steps: (1) adding beta-GP into a chitosan solution to obtain a CS-beta-GP pregel solution; (2) dissolving Pluronic F127 and hyaluronic acid in a PBS (Phosphate Buffer Solution) until the Pluronic F127 and the hyaluronic acid are completely transparent to obtain an F127-HA solution; and (3) sequentially dissolving trehalose, cordycepin, a cell factor composition and collagen peptide in a PBS solution, incubating to obtain a cell factor mixed solution, and mixing the cell factor mixed solution with the CS-beta-GP pregel solution and the F127-HA solution. According to the invention, a dual temperature-sensitive system is established, the gel can be quickly formed under the conditions of injection at low temperature and body temperature, the effective period of active ingredients is prolonged by utilizing a trehalose stabilization technology, and meanwhile, the anti-inflammatory effect of cordycepin and the cell repair effect of collagen peptide are combined, so that the efficient repair of skin injury can be realized.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a cytokine hydrogel, a preparation method thereof, and an application in skin injury repair. 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. Traditional dressings (such as gauze and hydrocolloid dressings) can provide physical protection, but they cannot dynamically respond to the wound microenvironment and lack the precise delivery ability of pro-repair active ingredients. Hydrogel is a soft material composed of a hydrophilic polymer network, which can absorb and retain a large amount of water (usually dozens to hundreds of times its own weight), while maintaining the stability of the three-dimensional structure and can load drugs. Due to its unique physical and chemical properties, good biocompatibility, and adjustable mechanical properties, hydrogel has a wide range of applications in the fields of biomedicine, tissue engineering, drug delivery, wound dressings, etc., and is an ideal skin repair material.

[0003] Thermosensitive hydrogels can remain liquid at low temperatures (below 25°C), which is convenient for injection or coating, and quickly gel at body temperature (37°C) to fit the wound surface. Chitosan (CS) has been widely studied due to its natural antibacterial, promoting blood coagulation, and biodegradability, but its mechanical properties are poor, the gelation property is unstable, and active ingredients such as cytokines are difficult to exist stably, and the wound repair rate is low. Therefore, this application hopes to provide a cytokine hydrogel with good gel properties, which can ensure the vitality 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 vitality of cytokines, and is beneficial to the repair of skin injuries.

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

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

[0007] (1) Add β-glycerophosphate to a chitosan solution to obtain a CS-β-GP pre-gel solution;

[0008] (2) Dissolve Pluronic F127 and hyaluronic acid in a PBS solution, and obtain an F127-HA solution after complete dissolution and transparency;

[0009] (3) Dissolve trehalose and cordycepin in a PBS solution, then add a cytokine composition and collagen peptide, and incubate to obtain a cytokine mixture;

[0010] (4) After filtering the CS-β-GP pre-gel solution and the F127-HA solution respectively, mix them with the cytokine mixture.

[0011] Preferably, the preparation method of the chitosan solution in step (1) is: dissolve chitosan in acetic acid with a concentration of 0.1 - 0.2 M, with a pH of 4.5 - 5.0, and stir magnetically 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-glycerophosphate is 80 - 120 g / L.

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

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

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

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

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

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

[0019] The present invention provides a cytokine hydrogel.

[0020] The present invention also provides the application of the cytokine hydrogel in the preparation of a drug for skin injury repair.

[0021] Beneficial effects

[0022] In the present invention, hyaluronic acid (HA) 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 vascular endothelial growth factor VEGF), stabilize the polypeptide activity, and prevent denaturation and inactivation during storage and delivery. Collagen peptide can promote cell proliferation and repair, and cordycepin has anti-inflammatory, antioxidant, and immunomodulatory effects, which can reduce inflammatory damage and synergistically accelerate the repair of damaged skin with cytokines.

[0023] The present invention establishes a dual thermosensitive system, which can be injected at low temperature and rapidly form a gel under body temperature conditions, maintaining the balance between injectability and gel strength. The cytokine hydrogel provided by the present invention uses trehalose stabilization technology to extend the validity period of 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 peptide, it can achieve efficient repair of skin damage. Detailed implementation mode

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

[0025] Example 1

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

[0027] (2) Pluronic F127 and hyaluronic acid were dissolved in PBS solution, and after complete dissolution and transparency, an F127-HA solution was obtained. The addition amount of Pluronic F127 was 150 g / L; the addition amount of hyaluronic acid was 15 g / L;

[0028] (3) Trehalose, cordycepin, cytokine composition (EGF, bFGF, and VEGF, mass ratio 1.5:1.5:1), and collagen peptide were sequentially added to PBS solution at addition amounts of 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) After filtering and sterilizing the CS-β-GP pre-gel solution and the F127-HA solution through a 0.22 μm filter membrane respectively, they are mixed with the cytokine mixture in a volume ratio of 1:1:1.

[0030] Example 2

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

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

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

[0034] (4) After filtering and sterilizing the CS-β-GP pre-gel solution and the F127-HA solution through a 0.22 μm filter membrane respectively, they are mixed with the cytokine mixture in a volume ratio of 1:2:1.

[0035] Example 3

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

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

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

[0039] (4) After filtering and sterilizing the CS-β-GP pre-gel solution and the F127-HA solution through a 0.22-μm filter membrane respectively, mix them with the cytokine mixture at a volume ratio of 1:1:1.

[0040] Comparative Example 1

[0041] Different from Example 1, this comparative example did not add the cytokine composition.

[0042] Comparative Example 2

[0043] Different from Example 1, this comparative example did not add trehalose.

[0044] Comparative Example 3

[0045] Different from Example 1, this comparative example did not add cordycepin.

[0046] Comparative Example 4

[0047] Different from Example 1, this comparative example did not add collagen peptide.

[0048] Comparative Example 5

[0049] Different from Example 1, the cytokine combination in this comparative example is: epidermal growth factor (EGF), hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1).

[0050] Comparative Example 6

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

[0052] Test Example 1 Wound healing rate

[0053] SPF - level SD healthy male rats at 2 - month - old with a body weight of 200 g - 250 g were used as experimental subjects to establish a refractory burn wound model in rats. After anesthetizing the rats by intraperitoneal injection of 0.5 mL of 3% sodium pentobarbital, the back hair of the rats was removed. A wound was created at the mid - line of the rat's back. A wound surface was made with a scalding mold (aluminum block) with a diameter of 2 cm in contact for 30 s at 95 °C, and 60 μL of doxorubicin hydrochloride (2 mg / mL) was subcutaneously injected within 0.2 cm of the subcutaneous layer at the edges of the wound surface. On the 28th day after the operation, after removing the eschar from the back wound of the rats, a refractory burn wound model in rats was obtained. Another 10 mice were normally raised as the blank control group (CK1).

[0054] The model rats were randomly divided into groups, namely the model control group (CK2), Example 1 (T1), Comparative Examples 1 - 6 groups (D1 - D6), with 10 rats in each group. Cytokine hydrogels (hydrogels prepared corresponding to Example 1 and Comparative Examples 1 - 6 respectively) were applied to the wound surface, and the application amount was 3 mg / cm 2 , and the control group was not given medicine and an equal amount of pure water was applied.

[0055] At 0, 4, 10, and 18 days after the drug administration treatment, a digital camera was used to take pictures to record the wound conditions of the rats. Digital image analysis software was used to analyze the wound area and calculate the healing rate. The wound healing rate = (original wound area - unhealed wound area) / original wound area. At 4, 10, and 18 days after the drug administration treatment of each group of rats, the wound healing rates of each group of rats 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 embodiments of the present application is higher than that of Comparative Examples 1 - 6. Among them, the cytokine composition has the greatest influence on the wound repair effect of the hydrogel. After the cytokine is used in combination with cordycepin and collagen peptide, the wound healing rate has been significantly improved, indicating that cordycepin and collagen peptide in the present application can work synergistically with the cytokine composition.

[0059] On the 8th day of drug administration, 5 mice were randomly selected from each group. The mice were fixed upside down, the neck skin and artery were cut open, and the carotid artery blood of the neck was collected with a 10 - mL centrifuge tube, placed for 2 h, centrifuged at 3000 rpm for 10 min. The upper - layer serum was taken and stored frozen for later use. The contents of IL - 6 and TNF - α in the serum were detected according to the detection instructions of the kit respectively.

[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 can be seen from Table 2, in the present invention, raw materials with anti-inflammatory and antioxidant effects such as cordycepin and collagen peptide are used in combination with cytokines, which can effectively relieve the inflammatory reaction during skin injury and further improve the repair effect of skin injury.

[0063] Test Example 2 Gel formation time

[0064] The experiment was divided into 7 groups, and hydrogels were prepared according to the methods of Example 1 (T1) and Comparative Examples 1-6 (D1-D6) respectively, with three replicates in each group. The hydrogel solutions prepared in each group were placed at 37 °C, and the time for them to form a solid gel was observed and recorded, and the average value was taken. The time 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 can be seen from Table 3, the present invention has established a CS / β-GP+F127 dual thermosensitive system, which can rapidly form a gel under body temperature conditions.

[0068] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing a cytokine hydrogel, characterized in that, It includes the following steps: (1) Add sodium β-glycerophosphate to the chitosan solution to obtain a CS-β-GP pre-gel solution; (2) Dissolve Pluronic F127 and hyaluronic acid in PBS solution, and after dissolving until completely transparent, obtain an F127-HA solution; (3) Dissolve trehalose and cordycepin in PBS solution, then add a cytokine composition and collagen peptide, and incubate to obtain a cytokine mixture; (4) Filter the CS-β-GP pre-gel solution and the F127-HA solution respectively, and then mix them with the cytokine mixture.

2. The preparation method according to claim 1, wherein The preparation method of the chitosan solution described in step (1) is: dissolve chitosan in acetic acid with a concentration of 0.1 - 0.2 M, with a pH of 4.5 - 5.0, and stir magnetically 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 sodium β-glycerophosphate is 80 - 120 g / L.

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

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

5. The preparation method according to claim 4, characterized in that, The composition of the cytokine composition is: EGF, bFGF, and VEGF, with a mass ratio of 1 - 3:1 - 2:

1.

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

7. The preparation method according to claim 6, wherein The filter membrane used for filtration in step (4) has a pore size of 0.2 - 0.3 μm.

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

1.

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

10. Use of the cytokine hydrogel according to claim 9 in the preparation of a drug for skin injury repair.