A platelet-like gel repair preparation, its preparation method, and its use in promoting wound healing

Abstract

This invention provides a platelet-inspired gel repair formulation, its preparation method, and its use in promoting wound healing. The formulation comprises the following growth factors: PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1. The platelet-inspired gel repair formulation of this invention exhibits excellent wound healing effects, providing a new option for clinical wound treatment.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to a platelet-inspired gel repair preparation, its preparation method, and its use in promoting wound healing. Background Technology

[0002] Platelets play a crucial role in physiological wound healing, angiogenesis, and coagulation. In the response to trauma, platelets are activated and migrate to the wound site. Platelet surface glycoproteins GP IIb / IIIa bind to fibrinogen in the plasma, leading to further platelet aggregation. Fibrinogen, under the action of thrombin, forms fibrin, thus stopping the bleeding. Subsequently, lysosomes exert antibacterial effects. Finally, various active substances within alpha granules are released, including platelet-derived growth factor (PDGF), epidermal growth factor (EGF), fibroblast growth factor (FGF), stromal cell-derived factor-1α (SDF-1α), vascular endothelial growth factor (VEGF), and insulin-like growth factor (IGF-1), enabling platelets to promote wound healing and the regeneration of the epidermis, blood vessels, and nerves. They are shared bioactive mediators among other cells involved in the regenerative cascade (endothelial cells, macrophages, neutrophils, and fibroblasts). Thrombin and fibrin work by causing platelets and white blood cells to form a gel at the wound site, which not only stops bleeding but also has an antibacterial effect.

[0003] Platelet gel (PG) plays an important role in treating chronic, slow-healing wounds, accelerating the healing of diabetic foot and venous ulcers, and promoting cartilage formation in osteoarthritis. However, the preparation of platelet gel requires extracting platelets from the patient's own blood and then processing them in vitro, which is not only time-consuming and laborious but also traumatic for the patient. Summary of the Invention

[0004] The purpose of this invention is to provide a platelet-like gel repair formulation that promotes wound healing.

[0005] The present invention relates to a platelet-derived gel repair formulation comprising growth factors PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1; wherein the weight ratio of PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 is (10-1000):(10-200):(1-100):(0.5-100):(0.01-50):(0.1-50).

[0006] Preferably, the formulation is a solution, wherein the final concentrations of PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 are (10-1000) ug / ml, (10-200) ug / ml, (1-100) ug / ml, (0.5-100) ug / ml, (0.01-50) ug / ml, and (0.1-50) ug / ml, respectively.

[0007] More preferably, the concentrations of PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 are 10 ug / ml, 200 ug / ml, 1 ug / ml, 100 ug / ml, 0.01 ug / ml, and 0.1 ug / ml, respectively; or, the concentrations of PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 are 1000 ug / ml, 10 ug / ml, 100 ug / ml, 0.5 ug / ml, 50 ug / ml, and 50 ug / ml, respectively.

[0008] More preferably, the formulation further includes fibrinogen and thrombin, with final concentrations of (8-12) mg / ml and (3-7) mg / ml, respectively. Particularly preferably, the final concentrations of fibrinogen and thrombin in the formulation are 10 mg / ml and 5 mg / ml, respectively.

[0009] Preferably, the formulation is a hydrogel, which is made by adding a growth factor solution to a hydrogel made of carbomer and triethanolamine as a matrix; the final contents of growth factors PDGF-BB, EGF, FGF-2, SDF-1α, VEGF and IGF-1 are (10-1000)ug / g, (10-200)ug / g, (1-100)ug / g, (0.5-100)ug / g, (0.01-50)ug / g and (0.1-50)ug / g, respectively.

[0010] More preferably, the weight ratio of carbomer to triethanolamine is 2:2.7; or, the final contents of the growth factors PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 are 10ug / g, 200ug / g, 5ug / g, 1ug / g, 0.1ug / g, and 0.1ug / g, respectively.

[0011] The aforementioned solution or growth factor solution uses physiological saline as a solvent.

[0012] The present invention also provides a method for preparing the aforementioned platelet-like gel repair agent that promotes wound healing, comprising the following steps: dissolving growth factor in water, adding fibrinogen and thrombin, mixing well to form a solution; or mixing the growth factor solution with a hydrogel matrix to form a hydrogel.

[0013] The present invention also provides the use of the aforementioned platelet-like gel repair formulation in a drug for promoting wound healing.

[0014] The growth factor of this invention is defined as follows:

[0015] PDGF-BB: Human platelet-derived growth factor-BB;

[0016] EGF: Epidermal Growth Factor;

[0017] FGF-2: Basic fibroblast growth factor;

[0018] SDF-1α: Stromal cell-derived factor-1α;

[0019] VEGF: Vascular endothelial growth factor;

[0020] IGF-1: Insulin-like growth factor 1.

[0021] Fibrinogen: A normal component of human blood, it is a glycoprotein (22v2) synthesized and secreted by liver cells and is an important protein involved in the coagulation and hemostasis process.

[0022] Thrombin: a white to grayish-white non-crystalline substance, which is a lyophilized powder.

[0023] The preparation method of the platelet-like gel repair agent of the present invention is simple, does not require extraction from platelets, has excellent wound healing effect, and has good prospects for clinical application.

[0024] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0025] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0026] Figure 1 Image showing the effect of wound repair.

[0027] Figure 2 Image showing the effect of wound repair. Detailed Implementation

[0028] All experimental materials and reagents used below are commercially available products. Human fibrinogen was purchased from Shanghai Yuanye Biotechnology Co., Ltd., and thrombin lyophilized powder was purchased from Zhejiang Hangkang Pharmaceutical Co., Ltd.

[0029] Example 1: The platelet-like gel repair formulation of the present invention

[0030] Prepare a solution of PDGF-BB, EGF, FGF-2, VEGF, SDF-1α, IGF-1, and fibrinogen dry powder reagents using physiological saline. Add thrombin to the prepared solution to obtain the final concentrations of cell growth factors PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1: 10 μg / ml, 200 μg / ml, 1 μg / ml, 100 μg / ml, 0.01 μg / ml, and 0.1 μg / ml, respectively. The final concentrations of fibrinogen and thrombin are 10 mg / ml and 5 mg / ml, respectively.

[0031] The repair preparation of this invention is a temporary preparation for use. Before use, it can be prepared into two raw material packages: raw material package 1 contains a solution of growth factors and fibrinogen, and raw material package 2 contains thrombin. The two are mixed together before use.

[0032] Example 2: The platelet-like gel repair formulation of the present invention

[0033] Add 2g of carbomer to 80ml of distilled water and stir until fully swollen. Add 2.7g of triethanolamine while stirring to prepare the hydrogel matrix. Separately, dissolve PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 in 2ml of physiological saline and add this solution to the hydrogel matrix while stirring. Add distilled water to a final volume of 100g and stir well. The final product contains PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 at concentrations of 10ug / g, 200ug / g, 5ug / g, 100ug / g, 0.01ug / g, and 0.1ug / g, respectively.

[0034] Example 3: The platelet-like gel repair formulation of the present invention

[0035] Prepare a solution of PDGF-BB, EGF, FGF-2, VEGF, SDF-1α, IGF-1, and fibrinogen dry powder reagents using physiological saline. Add thrombin to the prepared solution to obtain the final concentrations of cell growth factors PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1: 1000 ug / ml, 10 ug / ml, 100 ug / ml, 0.5 ug / ml, 50 ug / ml, and 50 ug / ml, respectively. The final concentrations of fibrinogen and thrombin are 10 mg / ml and 5 mg / ml, respectively.

[0036] The following examples illustrate the beneficial effects of the present invention:

[0037] Experimental Example 1: Repair Effect on Animal Skin Wounds

[0038] I. The Influence of Different Types of Growth Factors on Repair Efficacy

[0039] (I) Experimental Methods

[0040] 1. Product preparation

[0041] Product 1: Prepare a solution by mixing PDGF-BB, EGF, FGF-2, VEGF, SDF-1α, IGF-1, and fibrinogen dry powder reagents with physiological saline. Add thrombin to the prepared solution. The final concentrations of cell growth factors PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 are 10ug / ml, 200ug / ml, 1ug / ml, 100ug / ml, 0.01ug / ml, and 0.1ug / ml, respectively. The final concentrations of fibrinogen and thrombin are 10mg / ml and 5mg / ml, respectively. This product should be applied to the affected area immediately after preparation.

[0042] Product 2:

[0043] Add 2g of carbomer to 80ml of distilled water and stir until fully swollen. Add 2.7g of triethanolamine while stirring to prepare the hydrogel matrix. Separately, dissolve PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 in 2ml of physiological saline and add this solution to the hydrogel matrix while stirring. Add distilled water to a final volume of 100g and stir well. The final concentrations of cell growth factors PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 are 10ug / g, 200ug / g, 5ug / g, 1ug / g, 0.1ug / g, and 0.1ug / g, respectively.

[0044] Product 3:

[0045] The preparation method of this product is the same as that of product 2. The difference is that the types of raw materials are reduced, and only three cell growth factors are used. The final concentrations are: EGF, SDF-1α, and VEGF concentrations of 200ug / g, 1ug / g, and 0.1ug / g, respectively.

[0046] 2. Experimental Grouping

[0047] SPF-grade 2.5-3kg New Zealand rabbits were used as experimental animals and divided into product group 1 (using product 1), product group 2 (using product 2), product group 3 (using product 3) and control group (not using any drugs).

[0048] 3. Modeling and drug administration methods

[0049] Rabbits were anesthetized by injecting 0.5% sodium pentobarbital into the ear vein at a dose of 6 ml / kg. A 4*4 cm section of skin was removed from the rabbit's back. Products 1, 2, and 3 were applied to the wound once a day. The growth factor component in product group 1 was mixed with fibrinogen and thrombin before application to the wound. The healing of the rabbit's skin was observed every 3-5 days.

[0050] (II) Experimental Results

[0051] Fifty-five days after skin removal, the wounds in all four groups of animals had healed as expected. Figure 1 As shown in the diagram, the control group animals exhibited large fibrous connective tissue in their wounds (indicated by the red arrows). The animals using Product 3 had intact skin structure but lacked significant hair coverage. The animals using Products 1 and 2 had intact skin structure and hair coverage in their wounds. Product 1 showed better results than Product 2; the wounds of the animals using Product 1 healed completely, with hair almost completely covering the wound surface. The drug of this invention has excellent skin repair effects, and Product 1 is superior to Product 2 in wound repair.

[0052] The results showed that the platelet-like gel repair preparations (products 1 and 2) made by using the growth factor solution of the present invention (including PDGF-BB, EGF, FGF-2, VEGF, SDF-1α, IGF-1) with fibrinogen and thrombin or with hydrogel matrix could repair wounds well, while other growth solutions had poor repair effects.

[0053] II. The Influence of Different Concentrations of Growth Factors on Repair Efficacy

[0054] (I) Experimental Methods

[0055] 1. Product preparation

[0056] Product 1: Prepare a solution by mixing PDGF-BB, EGF, FGF-2, VEGF, SDF-1α, IGF-1, and fibrinogen dry powder reagents with physiological saline. Add thrombin to the prepared solution. The final concentrations of cell growth factors PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 are 10ug / ml, 200ug / ml, 1ug / ml, 100ug / ml, 0.01ug / ml, and 0.1ug / ml, respectively. The final concentrations of fibrinogen and thrombin are 10mg / ml and 5mg / ml, respectively. This product should be applied to the affected area immediately after preparation.

[0057] Product 4: The preparation and usage methods of this product are the same as those of Product 1. The difference lies in adjusting the amount and ratio of the raw materials to achieve the following final concentrations: PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 are 1000ug / ml, 10ug / ml, 100ug / ml, 0.5ug / ml, 50ug / ml, and 50ug / ml, respectively; and the final concentrations of fibrinogen and thrombin are 10mg / ml and 5mg / ml, respectively. This product should be applied to the affected area immediately after preparation.

[0058] 2. Experimental Grouping

[0059] SPF-grade 2.5-3kg New Zealand rabbits were used as experimental animals and were divided into product group 1 (using product 1) and product group 4 (using product 4).

[0060] 3. Modeling and drug administration methods

[0061] Rabbits were anesthetized by injecting 0.5% sodium pentobarbital into the ear vein at a dose of 6 ml / kg. A 4*4 cm section of skin was removed from the rabbit's back. The growth factor components from products 1 and 4 were mixed with fibrinogen and thrombin before application to the wound.

[0062] (II) Experimental Results

[0063] Observe the healing of the rabbit's skin every 3-5 days, and the results should be as follows: Figure 2 As shown:

[0064] After using the drug in product group 1, it was found that the wound was eventually covered by skin, but in the later stages, the skin still contracted and scars were visible.

[0065] After using the drug in product group 4, it was found that the skin matured within 10 days, basically healed within 20 days, and hair could grow on the wound within 30 days, with no obvious scarring.

[0066] The results showed that the platelet-like gel repair preparations (products 1 and 4) made from the two growth factor solutions of the present invention (including PDGF-BB, EGF, FGF-2, VEGF, SDF-1α, and IGF-1) with fibrinogen and thrombin could effectively repair wounds. Among them, product 4 had a particularly good repair effect, which was significantly better than that of product 1.

[0067] In summary, experiments have demonstrated that the platelet-like gel repair formulation prepared from the specific growth factor solution of this invention has an excellent effect on promoting wound repair, providing a new option for clinical wound repair.

Claims

1. A platelet-inspired gel repair preparation, characterized in that: The formulation is a solution, which consists of growth factors, fibrinogen, thrombin, and a solvent. The growth factors are PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1. The final concentrations of PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, IGF-1, fibrinogen, and thrombin are (10-1000) ug / ml, (10-200) ug / ml, (1-100) ug / ml, (0.5-100) ug / ml, (0.01-50) ug / ml, (0.1-50) ug / ml, (8-12) mg / ml, and (3-7) mg / ml, respectively.

2. The formulation according to claim 1, characterized in that: The concentrations of PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 are 10 ug / ml, 200 ug / ml, 1 ug / ml, 100 ug / ml, 0.01 ug / ml, and 0.1 ug / ml, respectively; or, the concentrations of PDGF-BB, EGF, FGF-2, SDF-1α, VEGF, and IGF-1 are 1000 ug / ml, 10 ug / ml, 100 ug / ml, 0.5 ug / ml, 50 ug / ml, and 50 ug / ml, respectively.

3. The formulation according to claim 1, characterized in that: In the formulation, the final concentrations of fibrinogen and thrombin are 10 mg / ml and 5 mg / ml, respectively.

4. The formulation according to any one of claims 1 to 3, characterized in that: The solution uses physiological saline as a solvent.

5. A method for preparing the formulation according to any one of claims 1 to 4, characterized in that: The steps are as follows: Dissolve growth factors and fibrinogen in water, then add thrombin, mix well, and form a solution.

6. Use of the formulation according to any one of claims 1 to 4 in the preparation of a medicament for promoting wound healing.

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