Dual controllable slow-release functional nanofiber wound dressing and preparation method thereof

Abstract

The invention discloses a dual controllable slow-release functional nanofiber wound dressing and a preparation method thereof, the preparation method comprises the following steps: adding metformin and bioactive glass into absolute ethyl alcohol, fully stirring, centrifuging, discarding supernate, and freeze-drying to obtain metformin-loaded bioactive glass; the preparation method comprises the following steps: adding the bioactive glass loaded with metformin, liraglutide and a medical matrix material into hexafluoroisopropanol, and preparing the functional nanofiber through electrostatic spinning. According to the invention, the metformin which is easy to release suddenly is combined with the bioactive glass through an electrostatic adsorption principle, so that the controllable release of the metformin is realized; meanwhile, liraglutide is uniformly distributed on the surface of the nanofiber and forms a wide hydrogen bond network with a naturally-sourced matrix component, and the two active components show stable and consistent drug release characteristics and play a synergistic treatment effect of protecting endothelium and effectively controlling the glucose level of tissues, so that a new strategy is provided for local treatment of diabetic wounds.

Description

Technical Field

[0001] The invention relates to the technical field of medical dressings, and in particular to a dual controllable sustained-release functionalized nanofiber wound dressing and a preparation method thereof. Background Art

[0002] Diabetes affects the health of more than 400 million people worldwide. The global prevalence of diabetes has led to a high incidence of a series of diabetes-related complications, such as neuropathy, diabetic nephropathy and diabetic wounds, among which the latter is the most common. Wound dressings can cover the wound and provide a temporary barrier against external infection, and act as a scaffold to guide host cell migration and infiltration. They are an important means of local treatment of diabetic wounds in clinical practice.

[0003] Traditional wound dressings such as gauze and bandages have a single function. Their role in wound healing is limited to protecting the wound from external stimuli, and there is a risk of secondary damage caused by adhesion to the wound. Existing new wound dressings (such as hydrogels, sponges and nanofibers) can usually form a closed microenvironment locally on the wound surface to protect the wound bed, maintain a moist environment and isolate bacterial infection. However, their effectiveness in promoting diabetic wound repair is still very limited.

[0004] In-depth basic research has revealed the key influence of various adverse factors of diabetic wounds, including high tissue glucose levels, persistent inflammation, and blocked vascular regeneration, on delayed wound healing. Among them, high tissue glucose levels are the most prominent feature of diabetic wounds. A continuous high-sugar environment can damage local blood vessels, cause peripheral neuropathy and inhibit pain perception. At this time, various traumas can lead to wound formation, but patients often fail to discover it in time due to neuropathy and hypoalgesia, resulting in repeated occurrence and expansion of diabetic wounds. So far, existing research has focused on accelerating the repair of early diabetic wounds by improving specific adverse microenvironmental factors, and usually lacks control of tissue glucose levels. Considering the complex local microenvironmental disorder of diabetic wounds and the key initiating role of high tissue glucose levels, it is necessary to seek a functional dressing that can effectively regulate the regenerative microenvironment and take into account the control of glucose levels to close the wound and improve tissue healing. Summary of the invention

[0005] The purpose of the present invention is to provide a dual controllable sustained-release functionalized nanofiber wound dressing and a preparation method thereof in view of the deficiencies in the prior art.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] The first aspect is to provide a method for preparing a dual controllable sustained-release functionalized nanofiber wound dressing, comprising the following steps:

[0008] Step 1, adding metformin and bioactive glass into anhydrous ethanol, stirring sufficiently, centrifuging and discarding the supernatant, and obtaining the bioactive glass loaded with metformin by freeze drying;

[0009] Step 2: Add the bioactive glass loaded with metformin prepared in step 1, liraglutide and the medical matrix material into hexafluoroisopropanol, and stir them thoroughly to obtain a uniform electrospinning solution; prepare the electrospinning solution into dual-controllable sustained-release functionalized nanofibers through an electrospinning device, vacuum dry, cut and sterilize to obtain the dual-controllable sustained-release functionalized nanofiber wound dressing.

[0010] Furthermore, in step 1, the particle size of the bioactive glass in the bioactive glass loaded with metformin is 100-800 nm, wherein the mass ratio of metformin to the bioactive glass is 1:(1-4).

[0011] Furthermore, in step 2, the medical matrix material is one or more of PCL, PLGA, PU, ​​PGCL, PLCL, gelatin, chitosan, collagen, silk fibroin, and hyaluronic acid.

[0012] Furthermore, in the step 2, the mass ratio of metformin, liraglutide and the medical matrix material is 1:(0.4-1):(50-200).

[0013] Furthermore, in the step 2, the dosage ratio of the medical matrix material to hexafluoroisopropanol is (0.9-1.5) g:10 mL.

[0014] Furthermore, in the step 2, the electrospinning conditions are: the voltage is 8-12 kV, the distance from the needle tip to the aluminum receiving device is 12-18 cm, and the solution injection rate is 0.6-0.8 mL / h.

[0015] Furthermore, the thickness of the dual controllable sustained-release functionalized nanofibers is 60 to 120 μm.

[0016] The second aspect is to provide a dual controllable sustained-release functionalized nanofiber wound dressing prepared by the above-mentioned preparation method.

[0017] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0018] The present invention combines metformin, which is easy to release suddenly, with bioactive glass through the principle of electrostatic adsorption to enhance the function of nanofibers while achieving controlled release of metformin; in addition, liraglutide is evenly distributed on the surface of nanofibers and forms a wide range of hydrogen bond networks with matrix components of natural origin. The two active ingredients show stable and consistent drug release characteristics, thereby achieving a synergistic therapeutic effect of endothelial protection and effective control of tissue glucose levels; the preparation process of this dual-controllable sustained-release functionalized nanofiber is simple and efficient, the morphology is uniform, and the biological activity and compatibility are good, which provides a new strategy for the local treatment of diabetic wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The microscopic morphology characterization results of the functionalized nanofibers prepared in the present invention: (A) SEM images of PLGA / SF nanofibers and (B) PLGA / SF / Met@MBG / Lir nanofibers; (C) fiber diameter distribution (n=100) of PLGA / SF nanofibers and (D) PLGA / SF / Met@MBG / Lir nanofibers.

[0020] Figure 2 Drug release characteristics of the functionalized nanofibers prepared in the present invention: (A) release curve of metformin in PLGA / SF / Met nanofibers; (B) release curve of metformin in PLGA / SF / Met@MBG nanofibers; (C) release curve of liraglutide in PLGA / SF / Lir nanofibers (n=3).

[0021] Figure 3 The functionalized nanofibers prepared by the present invention were used for local treatment of the full-thickness skin wound model of diabetic rats, and the wound closure rate at different time points was quantitatively analyzed (n=3).

[0022] Figure 4 Representative CD31 immunohistochemical staining images of the functionalized nanofibers prepared in the present invention 14 days after topical treatment of the full-thickness skin wound model in diabetic rats. DETAILED DESCRIPTION

[0023] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.

[0024] Experimental reagents and equipment:

[0025] Metformin (Met, purity ≥98%), Shanghai Kemin Biotechnology Co., Ltd.;

[0026] Liraglutide (Lir, purity ≥98%), Shanghai Kemin Biotechnology Co., Ltd.;

[0027] Mesoporous bioactive glass (MBG) was synthesized by our laboratory with a particle size of 300-400 nm;

[0028] Poly(lactic-co-glycolic acid) (PLGA), Maiqi Biomaterials Co., Ltd.;

[0029] Hexafluoroisopropanol (HFIP, purity ≥99%), Adamas (Shanghai) Reagent Co., Ltd.;

[0030] Electrospinning device (SS-2533H), Beijing Yongkang Leye Technology Development Co., Ltd.

[0031] Example 1

[0032] This embodiment provides a method for preparing a dual controllable sustained-release functionalized nanofiber wound dressing, which specifically comprises the following steps:

[0033] Step 1: Metformin and mesoporous bioactive glass were added to anhydrous ethanol at a mass ratio of 1:1, and after being fully stirred for 24 hours, the mixture was centrifuged (10000 rpm / min, 10 min) and the supernatant was discarded, and the metformin-loaded mesoporous bioactive glass (Met@MBG) was obtained by freeze drying;

[0034] Step 2: PLGA (0.9 g) and silk fibroin (SF, 0.3 g) were weighed and added to a hexafluoroisopropanol (10 mL) solution and stirred thoroughly to obtain a uniform and clear solution; liraglutide (10 mg) and Met@MBG (20 mg) were then added to the above solution and stirred continuously at room temperature for 72 hours until completely dissolved to obtain an electrospinning solution;

[0035] The electrospinning solution was filled into a 10 mL needle tube, and the solution injection rate was set to 0.8 mL / h, the voltage was set to 10 kV, the spinning temperature was set to 25 ° C, and the ambient humidity was set to 20% on the electrospinning device. The ejected fibers were received by an aluminum receiving device, and the distance from the needle tip to the receiving device was 15 cm, thereby preparing dual controllable sustained-release functionalized nanofibers;

[0036] The prepared nanofibers were dried in a vacuum environment for 72 hours to remove the organic solvent, cut and sterilized to obtain a dual controllable sustained-release functionalized nanofiber wound dressing (PLGA / SF / Met@MBG / Lir).

[0037] Verification Example 1

[0038] The surface morphology of the nanofibers was observed by scanning electron microscopy (SEM). The nanofibers were cut into rectangles (5 mm × 5 mm) and attached to a sample stage with conductive glue. After gold spraying for 30 seconds, the fiber morphology was observed under an accelerating voltage of 10 kV. In addition, the average diameter of the nanofibers was quantified by image-J software.

[0039] Taking Example 1 as an example, the experimental results are as follows Figure 1 As shown in the figure, it can be seen that both PLGA / SF and PLGA / SF / Met@MBG / Lir nanofibers are composed of randomly oriented, bead-free and smooth fibers with uniform diameter distribution, indicating that each group of spinning solutions has good and stable spinnability, which provides a guarantee for its preparation process as a wound dressing.

[0040] Verification Example 2

[0041] The nanofibers were immersed in 10 mL of PBS solution and incubated at 37°C. A certain amount of supernatant was taken out at a set time point and an equal amount of PBS solution was added. The concentration of metformin or liraglutide in the supernatant was determined by UV-visible absorption spectroscopy. The cumulative release rate was calculated by the following formula:

[0042]

[0043] Wherein, C represents the concentration of metformin or liraglutide; V represents the volume of the solution; ∑w represents the mass of metformin or liraglutide consumed cumulatively in the sampling; and M represents the total mass of metformin or liraglutide contained in the nanofibers.

[0044] Taking Example 1 as an example, the experimental results are as follows Figure 2 In vitro drug release experiments showed that metformin loaded in PLGA / SF / Met nanofibers had an obvious burst release phenomenon, and the introduction of bioactive glass could effectively slow down its release curve, thus showing a stable and consistent release characteristic with liraglutide.

[0045] Verification Example 3

[0046] All animal experiments were approved by the Ethics Committee of the Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. Streptozotocin was injected into the abdomen of male SD rats to construct a diabetic model. After the rats were anesthetized with sodium phenobarbital, their back hair was shaved and disinfected with iodine. Afterwards, a full-thickness excision skin wound with a diameter of 15 mm was made on the back of each rat, and the wound was treated with the gauze to be tested or the nanofibers of each group. At the set time point, the wound of each group was recorded and the wound area was counted by Image-J software. The wound closure rate was calculated by the following formula:

[0047] Wound closure rate (%) = (S0–S) / S0×100%

[0048] Among them, S0 represents the initial wound area, and S represents the wound area at a set time point.

[0049] Taking Example 1 as an example, the experimental results are as follows Figure 3 As shown. According to the quantitative analysis of wound area, the wound area of ​​all groups gradually decreased over time. Compared with the blank control group and the non-drug-loaded nanofiber group, the PLGA / SF / Met@MBG / Lir nanofiber treatment group showed a significantly higher wound healing rate at each observation time point, confirming the improvement effect of the sustained co-release of metformin and liraglutide from nanofibers on diabetic wound healing.

[0050] Verification Example 4

[0051] On the 14th day after surgery, the rats were killed by cervical dislocation, and the skin tissue around the wound was obtained for further analysis. After the samples were fixed, the expression of CD31 was analyzed by immunohistochemical staining to evaluate the angiogenesis of the tissue.

[0052] Taking Example 1 as an example, the experimental results are as follows Figure 4 The results of immunohistochemical staining showed that on the 14th day after surgery, the expression of CD31 in the PLGA / SF / Met@MBG / Lir nanofiber treatment group was significantly stronger than that in the blank control group and the non-drug-loaded nanofiber group, which was attributed to the sustained and stable release of metformin and liraglutide from the nanofibers, which endowed the nanofibers with the dual functions of endothelial protection and effective control of tissue glucose levels.

[0053] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a dual controlled sustained-release functionalized nanofiber wound dressing, characterized in that: The steps include: Step 1, adding metformin and bioactive glass into anhydrous ethanol, stirring sufficiently, centrifuging and discarding the supernatant, and obtaining the bioactive glass loaded with metformin by freeze drying; Step 2, adding the metformin-loaded bioactive glass prepared in step 1, liraglutide and the medical matrix material into hexafluoroisopropanol, and stirring them thoroughly to obtain a uniform electrospinning solution; The electrospinning solution is prepared into dual-controllable sustained-release functionalized nanofibers by an electrospinning device, and vacuum dried, cut and sterilized to obtain the dual-controllable sustained-release functionalized nanofiber wound dressing.

2. The method for preparing the dual controlled sustained-release functionalized nanofiber wound dressing according to claim 1, characterized in that: In step 1, the particle size of the bioactive glass in the metformin-loaded bioactive glass is 100-800 nm, wherein the mass ratio of metformin to the bioactive glass is 1:(1-4).

3. The method for preparing the dual controlled sustained-release functionalized nanofiber wound dressing according to claim 1, characterized in that: In step 2, the medical matrix material is one or more of PCL, PLGA, PU, ​​PGCL, PLCL, gelatin, chitosan, collagen, silk fibroin, and hyaluronic acid.

4. The method for preparing the dual controllable sustained-release functionalized nanofiber wound dressing according to claim 1, characterized in that: In the step 2, the mass ratio of metformin, liraglutide and the medical matrix material is 1:(0.4-1):(50-200).

5. The method for preparing the dual controlled sustained-release functionalized nanofiber wound dressing according to claim 1, characterized in that: In the step 2, the dosage ratio of the medical matrix material to hexafluoroisopropanol is (0.9-1.5) g:10 mL.

6. The method for preparing the dual controlled sustained-release functionalized nanofiber wound dressing according to claim 1, characterized in that: In the step 2, the electrospinning conditions are: the voltage is 8-12 kV, the distance from the needle tip to the aluminum receiving device is 12-18 cm, and the solution injection rate is 0.6-0.8 mL / h.

7. The method for preparing the dual controlled sustained-release functionalized nanofiber wound dressing according to claim 1, characterized in that: The thickness of the dual controllable sustained-release functionalized nanofiber is 60-120 μm.

8. A dual controllable sustained-release functionalized nanofiber wound dressing prepared by the preparation method according to any one of claims 1 to 7.

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