MYDGF-loaded hydrogel, preparation method and application of MYDGF-loaded hydrogel in wound repair drugs

Abstract

The invention provides MYDGF-loaded hydrogel, a preparation method and application of the MYDGF-loaded hydrogel in wound repair drugs, and belongs to the technical field of biomedical engineering. The preparation method of the MYDGF-loaded hydrogel comprises the following steps: dissolving PF-127 powder in a PBS (Phosphate Buffer Solution) at 0-8 DEG C to obtain PF-127 / PBS hydrogel; the preparation method comprises the following steps: dissolving MYDGF in water to obtain an MYDGF solution; at the temperature of 0-8 DEG C, the PF-127 / PBS hydrogel and the MYDGF liquid are mixed and stirred to be uniform, and the hydrogel loaded with the MYDGF is obtained. The MYDGF-loaded hydrogel disclosed by the invention has a good temperature-sensitive characteristic at 36-37 DEG C, and can be deformed and attached to a wound surface after being applied to the human skin wound surface, so that the biological adaptability of the hydrogel is improved. The MYDGF-loaded hydrogel disclosed by the invention can still keep the stability of the hydrogel structure, so that the MYDGF can still be continuously and stably released in a complex microenvironment, and the defect of unstable release of a traditional gel medicine is overcome. The MYDGF-loaded hydrogel can also improve the microcirculation of the wound surface, promote angiogenesis and accelerate the healing process of the wound surface.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical engineering, and more specifically, relates to a hydrogel loaded with MYDGF, a preparation method and an application thereof in wound repair medicine. Background Art

[0002] In the current field of wound repair, widely used hydrogel materials have performed well in wound protection and drug delivery due to their excellent biocompatibility and drug release regulation capabilities. However, traditional hydrogel materials still have significant deficiencies in promoting angiogenesis and rapid wound healing, especially in complex microenvironments such as difficult-to-heal ulcer wounds. There are problems such as unstable drug release, slow wound repair, and ineffective promotion of angiogenesis. The efficacy is limited and it is difficult to meet the clinical needs of difficult-to-heal wound repair. Summary of the invention

[0003] The purpose of the present invention is to provide a hydrogel loaded with MYDGF, a preparation method and its application in wound repair drugs, so as to solve the technical problems of unstable drug release, slow wound repair and ineffective promotion of angiogenesis in existing hydrogel materials in wound repair. The present invention designs a thermosensitive hydrogel system based on thermosensitive degradable Pluronic hydrogel (PF-127) loaded with myeloid growth factor (MYDGF), which can continuously and stably release MYDGF, effectively promote rapid wound repair, and promote angiogenesis.

[0004] To achieve the above object, the first aspect of the present invention provides a method for preparing a MYDGF-loaded hydrogel, comprising the following steps:

[0005] The PF-127 powder is dissolved in a PBS solution at 0 to 8° C. to obtain a PF-127 / PBS hydrogel; the PF-127 / PBS hydrogel is in a flowing liquid state at 4° C. and changes into a semisolid gel state within 1 to 5 minutes at 36 to 37° C.;

[0006] Dissolving MYDGF in water to obtain a MYDGF solution;

[0007] At 0-8°C, the PF-127 / PBS hydrogel and the MYDGF solution are mixed and stirred evenly to obtain a hydrogel loaded with MYDGF; the hydrogel loaded with MYDGF is in a flowing liquid state at 4°C, and changes into a semi-solid gel state within 1-5 minutes at 36-37°C.

[0008] Furthermore, in the PF-127 / PBS hydrogel, the mass percentage content of PF-127 is 20-30%.

[0009] Furthermore, in the PF-127 / PBS hydrogel, the mass percentage content of PF-127 is 25-30%.

[0010] Furthermore, in the MYDGF solution, the concentration of MYDGF is 0.1-0.2 ug / ul.

[0011] Furthermore, in the MYDGF-loaded hydrogel, the concentration of MYDGF is 50-200 ng / ml.

[0012] The second aspect of the present invention provides a MYDGF-loaded hydrogel prepared by any of the preparation methods described above.

[0013] The third aspect of the present invention provides an application of a MYDGF-loaded hydrogel in a wound repair drug, wherein the wound repair drug comprises a MYDGF-loaded hydrogel prepared by any of the preparation methods described above.

[0014] Compared with the prior art, the present invention has the following technical effects:

[0015] A preparation method of a MYDGF-loaded hydrogel of the present invention loads MYDGF into PF-127 / PBS hydrogel, and the prepared MYDGF-loaded hydrogel has good temperature-sensitive properties at 36-37°C, and can be deformed and fit the wound after being applied to the human skin wound, thereby improving the biocompatibility of the hydrogel. In addition, loading MYDGF into the PF-127 / PBS hydrogel can also optimize the release accuracy of MYDGF, so that the drug can be effectively released at the required time and position, thereby significantly enhancing the wound repair effect. Especially in the complex microenvironment of the wound such as diabetic foot ulcers or trauma, the MYDGF-loaded hydrogel prepared by the present invention can still maintain the stability of the hydrogel structure, so that it can still continuously and stably release MYDGF under the complex microenvironment, thereby overcoming the defect of unstable release of traditional gel drugs. On the other hand, the MYDGF-loaded hydrogel prepared by the present invention can also improve the microcirculation of the wound, promote angiogenesis, and accelerate the wound healing process. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0017] Figure 1 A comparison chart of wound healing conditions of four groups of mice on days 3, 7, and 14, respectively, provided in an embodiment of the present invention;

[0018] Figure 2 The MYDGF release curve of the MYDGF-loaded hydrogel prepared in Example 1 and Example 2 of the present invention at 37°C;

[0019] Figure 3 Fluorescence images of endothelial cells in skin specimens of each group of mice 5 days after surgery provided in the embodiments of the present invention;

[0020] Figure 4 A comparison chart of capillary density in skin specimens of each group of mice 1, 3, and 5 days after surgery provided in an embodiment of the present invention;

[0021] Figure 5 This is a comparison chart of the capillary density of endothelial cells of mice in group 4 cultured in MYDGF solutions of different concentrations for 1, 3, and 5 days under different MYDGF solution concentrations provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0022] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0023] The terms used in the embodiments of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. The singular forms "a", "said" and "the" used in the embodiments of the present invention and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0024] The weight of the relevant components mentioned in the description of the embodiments of the present invention may not only refer to the specific content of each component, but also indicate the weight ratio between the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the description of the embodiments of the present invention, it is within the scope disclosed in the description of the embodiments of the present invention. Specifically, the mass described in the description of the embodiments of the present invention may be μg, mg, g, kg, or other mass units known in the biochemical field.

[0025] The embodiment of the present invention provides a MYDGF-loaded hydrogel and a preparation method thereof, comprising the following steps:

[0026] (1) dissolving PF-127 powder in a PBS solution at 0 to 8° C. to obtain a PF-127 / PBS hydrogel;

[0027] (2) dissolving MYDGF in water to obtain a MYDGF solution;

[0028] (3) At 0-8°C, PF-127 / PBS hydrogel and MYDGF solution were mixed and stirred evenly to obtain a hydrogel loaded with MYDGF; the hydrogel loaded with MYDGF was in a flowing liquid state at 4°C and transformed into a semi-solid gel state within 1-5 minutes at 36-37°C.

[0029] Wherein, in the above step (1), PF-127 powder is F-127 powder, PBS solution is a phosphate buffer solution, and its pH is 7.2 to 7.4. The embodiment of the present invention studies the gelling properties of PF-127 / PBS hydrogels of different mass concentrations, and finds that when the mass percentage content of PF-127 in the PF-127 / PBS hydrogel is 20 to 30%, the initial gelling temperature of the hydrogel is 11 to 18°C, and the gelling time at 36 to 37°C is 1 to 5 minutes. Preferably, in the PF-127 / PBS hydrogel, the mass percentage content of PF-127 is 25 to 30%, so that at 36 to 37°C, the hydrogel can quickly gel within 3 minutes to better meet the application requirements.

[0030] In the process of preparing the PF-127 / PBS hydrogel in the embodiment of the present invention, by controlling the mass percentage content of PF-127 in the PF-127 / PBS hydrogel and selecting PBS as the solvent of PF-127, the prepared PF-127 / PBS hydrogel can be in a flowing liquid state at 4° C. and converted into a semi-solid gel state within 1 to 5 minutes at 36 to 37° C., so as to meet the application requirements in human wound healing.

[0031] In the above step (2), the embodiment of the present invention prepares a MYDGF solution by dissolving the MYDGF protein in a trace amount of water so that the MYDGF protein is in a liquefied state. In the MYDGF solution prepared in the embodiment of the present invention, the concentration of MYDGF is 0.1-0.2 ug / ul.

[0032] In the above step (3), in the hydrogel loaded with MYDGF, the mass of MYDGF added to each 100mL PF-127 / PBS hydrogel is controlled to be 5 to 20ug in the embodiment of the present invention. So that the hydrogel loaded with MYDGF can continuously and stably release MYDGF after being applied to the wound surface, and can play a role in rapidly promoting wound healing and promoting angiogenesis. In addition, it can also ensure that the addition of MYDGF will not affect the gelling properties of the PF-127 / PBS hydrogel itself, that is, the hydrogel loaded with MYDGF prepared in the embodiment of the present invention still remains in a flowing liquid state at 4°C, and is converted into a semi-solid gel state within 1 to 5 minutes at 36 to 37°C to meet its application requirements in human wound healing.

[0033] During the mixing process of step (3), MYDGF is incorporated into the grid of the PF-127 / PBS hydrogel by physical adsorption or chemical cross-linking, and is evenly distributed and stably incorporated into the PF-127 / PBS hydrogel.

[0034] The MYDGF component in the MYDGF-loaded hydrogel prepared in the embodiment of the present invention exhibits unique biological functions in promoting angiogenesis and wound healing. MYDG can not only promote the migration and proliferation of vascular endothelial cells, but also significantly reduce cell apoptosis and improve wound microcirculation, which is crucial for the repair of difficult-to-heal wounds. By combining MYDGF with the thermosensitive PF-127 / PBS hydrogel, the embodiment of the present invention successfully achieved the stable release and lasting effect of MYDGF at the wound, significantly accelerating the wound healing process, especially in difficult-to-heal wounds, showing excellent therapeutic effects.

[0035] The MYDGF-loaded hydrogel prepared in the embodiment of the present invention has a unique physical and chemical property design structure, which enables it to maintain the stable release and biological activity of MYDGF in a complex microenvironment. This property is crucial for the treatment of difficult-to-heal wounds, and can effectively promote angiogenesis, accelerate wound healing, and improve the overall repair quality of wounds.

[0036] A method for preparing a MYDGF-loaded hydrogel according to an embodiment of the present invention loads MYDGF into a PF-127 / PBS hydrogel, and the prepared MYDGF-loaded hydrogel has good temperature-sensitive properties at 36-37°C, and can be deformed and fit the wound after being applied to the human skin wound, thereby improving the biocompatibility of the hydrogel. In addition, loading MYDGF into the PF-127 / PBS hydrogel can also optimize the release accuracy of MYDGF, so that the drug can be effectively released at the required time and position, thereby significantly enhancing the wound repair effect. Especially in the complex microenvironment of the wound surface such as diabetic foot ulcers or trauma, the MYDGF-loaded hydrogel prepared by the embodiment of the present invention can still maintain the stability of the hydrogel structure, so that it can still continuously and stably release MYDGF under the complex microenvironment, thereby overcoming the defect of unstable release of traditional gel drugs. On the other hand, the MYDGF-loaded hydrogel prepared by the embodiment of the present invention can also improve the microcirculation of the wound surface, promote angiogenesis, and accelerate the wound healing process.

[0037] The following specific application examples are used to illustrate the MYDGF-loaded hydrogel, preparation method and application in wound repair medicine according to the embodiments of the present invention.

[0038] Example 1

[0039] Example 1 of the present invention provides a MYDGF-loaded hydrogel and a preparation method thereof, comprising the following steps:

[0040] (1) Dissolve 20 g of PF-127 powder in 100 ml of precooled 4°C PBS solution and stir magnetically at 4°C. Then filter and sterilize with a 0.22 μm filter membrane and store in a 4°C refrigerator for later use. The prepared 20% PF-127 / PBS hydrogel is a flowing liquid at 4°C and a semi-solid gel at 36-37°C.

[0041] (2) Take 100 ml of PF-127 / PBS hydrogel prepared in step (1), add 100 ul of MYDGF solution containing 10 ug of MYDGF protein, and mix by magnetic stirring at 4°C to obtain a MYDGF-loaded hydrogel, wherein the concentration of MYDGF is 100 ng / ml.

[0042] Example 2

[0043] Example 2 of the present invention provides a MYDGF-loaded hydrogel and a preparation method thereof, comprising the following steps:

[0044] (1) Dissolve 20 g of PF-127 powder in 100 ml of precooled 4°C PBS solution and stir magnetically at 4°C. Then filter and sterilize with a 0.22 μm filter membrane and store in a 4°C refrigerator for later use. The prepared 20% PF-127 / PBS hydrogel is a flowing liquid at 4°C and a semi-solid gel at 36-37°C.

[0045] (2) Take 100 ml of PF-127 / PBS hydrogel prepared in step (1), add 50 ul of MYDGF solution containing 5 ug of MYDGF protein, and mix by magnetic stirring at 4°C to obtain a MYDGF-loaded hydrogel, wherein the concentration of MYDGF is 50 ng / ml.

[0046] Example 3

[0047] Example 3 of the present invention provides an application of a MYDGF-loaded hydrogel in wound healing, comprising:

[0048] (1) Construction of animal model:

[0049] Experimental animals: Four-week-old male mice were selected and divided into mice with bone marrow cell-specific MYDGF knockout (labeled as MYDGF - / - ) and wild-type littermates (labeled with MYDGF f / f ).

[0050] Modeling method: After shaving the back hair, a full-thickness skin hole (8 mm in diameter) was punched to establish a full-thickness skin defect model, and a silicone ring was used to fix the wound edge to prevent skin sliding.

[0051] (2) Experimental grouping: The animal model was divided into four groups, with 32 mice in each group:

[0052] Group 1: MYDGF f / f +PF127 mice, using the PF-127 / PBS hydrogel prepared in step (1) of Example 1, without loading MYDGF;

[0053] Group 2: MYDGF f / f +MYDGF+PF127 mice, using the MYDGF-loaded hydrogel prepared in Example 1;

[0054] Group 3: MYDGF - / - +PF127 mice, using the PF-127 / PBS hydrogel prepared in step (1) of Example 1, without loading MYDGF;

[0055] Group 4: MYDGF - / - +MYDGF+PF127 mice, using the MYDGF-loaded hydrogel prepared in Example 1.

[0056] (3) Treatment implementation method:

[0057] After the animal modeling was successful, the wound and the area around the wound were rinsed with physiological saline, and each group was injected with 100ul of the PF-127 / PBS hydrogel prepared in step (1) of Example 1 or the MYDGF-loaded hydrogel prepared in Example 1 on the surface of the mouse wound. During injection, the hydrogels were all in a flowable liquid state. After injection, the wound was covered with a 3M medical film patch, and the edges were fixed with tape. After observation until the anesthesia was awakened, the mice were raised in separate cages. The wounds of each group of mice were treated only once, and the wound healing was observed daily for the next 15 days.

[0058] (4) Treatment results:

[0059] Wound healing observation: The wound area was photographed every day, and the wound recovery percentage was analyzed using ImageJ software (wound healing status = wound area on the first day - wound area on the nth day / wound area on the first day × 100%).

[0060] Figure 1 The wound healing status of the four groups of mice at 3, 7, and 14 days was shown. Figure 1 As seen in group 4 (MYDGF - / - +MYDGF+PF127) mice reached a wound healing rate of 94.9% on day 14, while group 3 (MYDGF - / -The wound healing rate of mice with MYDGF+PF127 was only 61.9% on the 14th day, and the wound healing rate of group 4 was increased by 53.31% compared with group 3. The comparison of healing conditions showed that the wound healing rate of the MYDGF-loaded hydrogel group was significantly faster than that of the other groups, that is, the use of MYDGF-loaded hydrogel was more conducive to promoting rapid wound healing.

[0061] Drug sustained release detection: The ELISA kit was used to detect the release rate of MYDGF at 37°C of the MYDGF-loaded hydrogels prepared in Example 1 (the mass concentration of MYDGF in the MYDGF-loaded hydrogel was 10%) and Example 2 (the mass concentration of MYDGF in the MYDGF-loaded hydrogel was 5%) of the present invention, and the obtained release curves were as follows: Figure 2 As shown. Figure 2 It can be seen that the MYDGF-loaded hydrogels prepared in Examples 1 and 2 of the present invention can both release MYDGF continuously and stably at 37°C.

[0062] Microvascular growth assessment: Laser Doppler technology was used to assess the microcirculatory blood flow of the wound surface daily. Skin specimens were collected 1, 3, and 5 days after surgery to count endothelial cells labeled with FITC-MYDGF and assess capillary density and angiogenesis. The test results were as follows: Figure 3 , Figure 4 , Figure 5 shown.

[0063] exist Figure 3 In the figure, the 10x micrographs of endothelial cells in the skin specimens of each group of mice 5 days after surgery are shown at 24 hours, 20x micrographs at 24 hours, 10x micrographs at 48 hours, and 20x micrographs at 48 hours.

[0064] Figure 4 , Figure 5 The OD value was measured by light transmittance to reflect the capillary density in the skin specimens of each group of mice 1, 3, and 5 days after surgery:

[0065] Figure 4 The capillary density in the skin specimens of each group of mice was measured at 1, 3, and 5 days after surgery when the concentration of MYDGF solution was 100 ng / mL.

[0066] Figure 5 The MYDGF solution concentrations were measured in four groups of mice (i.e. - / - +MYDGF+PF127) endothelial cells cultured in different concentrations of MYDGF solution for 1, 3, and 5 days.

[0067] Figure 3-Figure 5The test results can all indicate that mice treated with the MYDGF-loaded gel showed a higher capillary density, indicating that the MYDGF-loaded gel prepared in the embodiment of the present invention is effective in promoting angiogenesis.

[0068] The above embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the attached claims.

Claims

1. A method for preparing a hydrogel loaded with MYDGF, characterized in that: The following steps are involved: Dissolving PF-127 powder in a PBS solution at 0 to 8°C to obtain a PF-127 / PBS hydrogel; Dissolving MYDGF in water to obtain a MYDGF solution; At 0-8° C., the PF-127 / PBS hydrogel and the MYDGF solution were mixed and stirred evenly to obtain a hydrogel loaded with MYDGF.

2. The method for preparing the MYDGF-loaded hydrogel according to claim 1, wherein In the PF-127 / PBS hydrogel, the mass percentage content of PF-127 is 20-30%.

3. The method for preparing the MYDGF-loaded hydrogel according to claim 2, wherein: In the PF-127 / PBS hydrogel, the mass percentage content of PF-127 is 25-30%.

4. The method for preparing the MYDGF-loaded hydrogel according to claim 1, wherein In the MYDGF solution, the concentration of MYDGF is 0.1-0.2 ug / ul.

5. The method for preparing the MYDGF-loaded hydrogel according to claim 1, characterized in that: In the MYDGF-loaded hydrogel, the concentration of MYDGF is 50-200 ng / ml.

6. A MYDGF-loaded hydrogel, characterized in that: The method is prepared by any one of claims 1 to 5.

7. Application of MYDGF-loaded hydrogel in wound repair medicine, characterized in that: The wound repair drug comprises a MYDGF-loaded hydrogel prepared by the preparation method according to any one of claims 1 to 5.

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