Preparation method and application of composite nanofiber membrane prepared based on enzymolysis pectin

Abstract

The invention discloses a preparation method and application of a composite nanofiber membrane prepared on the basis of enzymolysis pectin, and belongs to the field of medical dressings. The technical problems that an existing gel dressing is not rigid, cannot protect a wound surface, and is not good for wound healing due to poor air permeability and antibacterial activity are solved. According to the method, the low-fat pectin is processed into the electrospun fibers, and the electrospun fibers can more effectively adsorb exogenous medicinal molecules (nano-silver, selenium and the like). The dressing based on the pectin material is prepared by simulating human skin, the outer layer can provide a rigid protection effect, the inner layer can adsorb medicinal molecules, and the dressing can effectively promote healing of diabetic wounds. According to the invention, waste reutilization is taken as a purpose, commercialization of sunflower discs is realized, higher value is given to agricultural wastes, a good dressing is provided for treatment of diabetic wounds, and the purpose of agricultural waste reutilization for helping agriculture and developing agriculture is also realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical dressings, and in particular relates to a preparation method and application of a composite nanofiber membrane prepared based on enzymatic pectin. Background Art

[0002] Diabetic wounds present with ischemia, persistent inflammation and epithelial dysfunction, which further lead to difficulty in forming granulation tissue and decreased wound tensile strength, resulting in prolonged or recurrent wound healing. The mechanism of delayed or difficult wound healing may be related to multiple factors, such as angiogenesis disorders, elevated levels of free radicals and oxidative stress, chronic inflammatory response, growth factor imbalance, immune metabolic disorders and abnormal expression of matrix metalloproteinases. These factors work together to make the wound healing process of diabetic patients complex and lengthy, and are prone to serious complications such as the spread of infection and amputation, causing persistent pain and health risks to patients, while also increasing medical costs and the difficulty of care.

[0003] In the field of natural polymer materials, pectin, a polysaccharide widely found in plant cell walls, is commonly used as a thickener, stabilizer, or gelling agent in the food industry due to its excellent biocompatibility, degradability, and safety. Using sunflower pectin to make biodegradable dressings can reduce the impact of medical waste on the environment, meeting the requirements of environmental protection and sustainable development. Furthermore, in Northeast China, the use of sunflower seed waste to prepare pectin materials not only achieves efficient recycling of agricultural waste and reduces environmental pollution, but also creates new economic growth points for local agriculture. It also promotes technological innovation and sustainable development, promotes the development of local specialty industries, increases the added value of agricultural production, and enhances the competitiveness of the local economy, with significant economic, social, and environmental benefits. However, natural pectin generally has a high degree of esterification (usually above 50%), resulting in weak cross-linking ability between its molecular chains, making it difficult to form fiber materials with high mechanical strength through conventional processing techniques (such as wet spinning). Therefore, the application of pectin in the existing technology is mainly limited to the gel state or the use in combination with other polymer materials. For example, gel products such as jelly and jam used as food dressings have low added value and fail to fully tap the application potential of pectin in high-end fields.

[0004] In recent years, with the growing demand for biomedical materials, pectin, due to its natural origin and bioactive properties, has been tried to expand into medical fields such as wound dressings. However, although traditional pectin-based hydrogels have certain liquid absorption and softness, their three-dimensional network structure lacks rigid support and cannot form an effective physical barrier to resist external mechanical stimulation in complex wounds (such as burns and chronic ulcers). In addition, the high water content of the gel leads to insufficient air permeability, which easily creates a localized moist environment on the wound surface, which is not conducive to cell migration and tissue regeneration.

[0005] Although previous studies have attempted to improve the mechanical and functional properties of pectin through chemical modification (such as low-esterification treatment and graft copolymerization) or compounding with synthetic polymers (such as polyvinyl alcohol and polyurethane), these methods often involve complex processes or the introduction of non-natural ingredients, resulting in increased material costs, reduced biodegradability, and even loss of pectin's natural advantages. Therefore, developing a new material based on pure natural pectin that combines high mechanical strength, excellent air permeability, and intrinsic antibacterial properties, and breaking through the technical bottleneck of its fiberization processing, is of great significance for increasing the added value of pectin and expanding its application in high-end medical fields.

[0006] Existing technologies have drawbacks: Most natural pectins have a high degree of esterification, resulting in poor filament formation, and are mostly used in gel form. Gel dressings lack rigidity, providing no protection for wounds, and lack breathability and antibacterial properties, hindering wound healing. Pectin has a low added value and is primarily used in food dressings. Summary of the Invention

[0007] The present invention aims to solve the technical problems that existing gel dressings have no rigidity, cannot protect the wound surface, and have poor air permeability and antibacterial properties, which are not conducive to wound healing. It provides a preparation method and application of a composite nanofiber membrane prepared based on enzymatic pectin.

[0008] The present invention provides a method for preparing low-fat pectin suitable for electrospinning. The method processes low-fat pectin into electrospun fibers that more effectively adsorb exogenous medicinal molecules (such as selenium and nanosilver). The invention also simulates human skin to prepare a pectin-based dressing, which has an outer layer that provides rigid protection and an inner layer that absorbs medicinal substances. This dressing can effectively promote the healing of diabetic wounds.

[0009] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0010] The object of the present invention is to provide a method for preparing a composite nanofiber membrane based on enzymatic hydrolysis of pectin, characterized in that it comprises the following steps:

[0011] Step 1: drying the sunflower disk into small pieces, crushing them, and sieving them to obtain sunflower disk powder;

[0012] Step 2: adding sodium citrate to distilled water, adjusting the pH to acidic with citric acid solution, and preparing an extract;

[0013] Step 3: Add the sunflower disk powder to distilled water, shake well, wash with water, centrifuge, collect the precipitate, add the extract, mix well, heat in a water bath, centrifuge, take the supernatant, concentrate, add anhydrous ethanol, and form a gel at 4°C overnight. Centrifuge the alcohol precipitate, add distilled water to re-dissolve the alcohol precipitate, evaporate the ethanol, concentrate, and freeze-dry to obtain crude pectin;

[0014] Step 4: mixing the crude pectin with water and adjusting the pH to acidic, adding pectinesterase, adjusting the pH to neutral after enzymatic hydrolysis, dialyzing the pectin solution, and freeze-drying to obtain low-esterification pectin with an esterification degree of less than 3%;

[0015] Step 5, dissolving a copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV) and polylactic acid (PLA) in a mixed solution of chloroform and DMF, stirring until completely dissolved to obtain an outer layer spinning solution, electrospinning, and standing to dry to obtain an outer layer nanofiber membrane;

[0016] Step 6: adding the pectin obtained in step 4 to distilled water and stirring until completely dissolved to obtain a pectin aqueous solution; adjusting the pH to neutral with a NaOH aqueous solution; adding a polyethylene oxide (PEO) solution; and then adding Triton X-10 (Triton X-100) and dimethyl sulfoxide; stirring evenly to obtain an inner layer spinning solution; electrospinning on the surface of the outer layer nanofibers; and drying; thereby obtaining the composite nanofiber membrane;

[0017] The PEO solution is a PEO aqueous solution, or a PEO solution prepared by adding PEO to a nanosilver solution.

[0018] It is further defined that the PEO solution is a PEO solution with a concentration of 5 wt.% prepared using an 8 mmol / L sodium selenite solution or a 10 ppm nanosilver solution.

[0019] Another embodiment of the present invention is: steps 1-4 are performed according to the above embodiment;

[0020] The pectin obtained in step 4 was added to distilled water and stirred until completely dissolved to obtain a pectin aqueous solution, the pH value was adjusted to neutral with a NaOH aqueous solution, the PEO solution was added, and then Triton X-10 (Triton X-100) and dimethyl sulfoxide were added, and the mixture was stirred evenly to obtain an inner layer spinning solution, electrospun, and allowed to stand to dry;

[0021] PHBV and PLA are dissolved in a mixed solution of chloroform and DMF, stirred until the outer layer spinning solution is completely dissolved, electrospinned on the surface of the inner layer nanofibers, and allowed to stand and dry, thereby obtaining the composite nanofiber membrane.

[0022] It is further defined that in step 1, the product is sieved through a 100-mesh sieve.

[0023] It is further defined that in step 2, sodium citrate is added to distilled water at a mass fraction of 0.74%.

[0024] It is further defined that in step 2, the concentration of the citric acid solution is 0.1 mol / L; and the pH value is adjusted to pH=3.3 using the citric acid solution.

[0025] It is further defined that in step 5, the mass ratio of PHBV to PLA is 2:1, the mass ratio of chloroform to DMF is 19:1, and the total mass concentration of PHBV and PLA is 5%.

[0026] It is further defined that in step 6, the concentration of the pectin aqueous solution is 5.5wt.%, the concentration of the PEO aqueous solution is 5wt.%, the PEO aqueous solution is added in a pectin to PEO mass ratio of 1:1, dimethyl sulfoxide accounts for 5wt.% of the inner layer spinning solution, and TritonX-100 accounts for 1wt.% of the inner layer spinning solution; the concentration of pectin and PEO in the inner layer spinning solution is maintained at 5.0wt.%.

[0027] The outer layer electrospinning parameters are further defined as follows: injection speed of 1.2 mL / h-2.0 mL / h, receiving distance of 15 cm, roller speed of 400 rpm-1000 rpm, voltage of 10 kV-17 kV, and humidity controlled at 20%-30%.

[0028] The inner layer electrospinning parameters are further defined as follows: injection speed of 1.0 mL / h-1.6 mL / h, receiving distance of 15 cm, roller speed of 400 rpm-1000 rpm, voltage of 7 kV-14 kV, and humidity controlled at 20%-30%.

[0029] It is further defined that in step 6, sodium selenite is added to the inner layer spinning solution, stirred evenly and then subjected to electrostatic spinning, and the concentration of sodium selenite in the spinning solution is 8 mmol / mL.

[0030] It is further defined that in step 6, the concentration of nanosilver in the inner layer spinning solution is 5 ppm.

[0031] Another object of the present invention is to provide a composite nanofiber membrane prepared by any of the above methods for use as a dressing for promoting diabetic wound healing.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] Heilongjiang Province is one of the main production areas of sunflower, an agricultural product. The present invention prepares agricultural waste sunflower disks into pectin, and then prepares the pectin into a new type of diabetic wound double-layer composite drug-loaded nanofiber dressing that simulates the skin structure based on the concept of tissue engineering. With the purpose of waste recycling, the commercialization of sunflower disks is realized, and agricultural waste is given higher value. It not only provides a good dressing for the treatment of diabetic wounds, but also achieves the purpose of agricultural waste recycling to help farmers and promote agriculture.

[0034] In order to further understand the features and technical contents of the present invention, please refer to the following detailed description of the present invention and the accompanying drawings. However, the accompanying drawings are provided for reference and illustration only and are not intended to limit the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 is a schematic diagram of the dressing structure;

[0036] Figure 2 It is the thermogravimetric analysis diagram;

[0037] Figure 3 is the Fourier infrared spectrum;

[0038] Figure 4 is an XRD crystallinity analysis diagram, a - blank group, b - sodium selenite, c - nanosilver;

[0039] Figure 5 It is the in vitro release profile of the loaded drug;

[0040] Figure 6 This is a diagram of wound healing. DETAILED DESCRIPTION

[0041] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art further understand the present invention and are not intended to limit the present invention in any way. It should be noted that those skilled in the art may make various modifications and improvements without departing from the scope of the present invention. These modifications and improvements are all within the scope of protection of the present invention.

[0042] Example 1: The preparation method of the dressing in this embodiment is achieved by the following steps:

[0043] Step 1. Preparation of low-fat pectin:

[0044] Step 1.1 Sunflower disk pretreatment:

[0045] Process the dried sunflower discs into small pieces, crush them with a vertical grinder, and pass them through a 100-mesh sieve for later use.

[0046] Step 1.2 Preparation of pectin extract:

[0047] An appropriate amount of sodium citrate was weighed and added to distilled water to prepare a sodium citrate solution with a mass fraction of 0.74%. The pH was adjusted to 3.3 with a 0.1 mol / L citric acid solution to obtain a pectin extract.

[0048] Step 1.3 Acid extraction of pectin:

[0049] Weigh 50 g of sunflower disc powder, add 2 L of distilled water at a liquid-to-solid ratio of 1:40 (g / mL), shake thoroughly, wash with water for 20 min, centrifuge at 4000 rpm for 15 min, remove the supernatant, and collect the precipitate for later use;

[0050] Add 1 L of the prepared extract to the precipitate at a liquid-to-solid ratio of 1:20 (g / mL), mix well, and heat in a water bath at 64.1°C for 80 min. Centrifuge at 4000 rpm for 20 min. Take the supernatant and concentrate it on a rotary evaporator. After concentration, add 3 times the volume of the sample solution of anhydrous ethanol and place in a 4°C refrigerator overnight to form a gel. Centrifuge the alcohol precipitate.

[0051] The alcohol precipitate was re-dissolved in an appropriate amount of distilled water, and the ethanol was evaporated on a rotary evaporator. The sample was concentrated and freeze-dried to obtain crude pectin (sunflower pectin).

[0052] Step 1.4 Enzymatic modification

[0053] The sunflower pectin is mixed with water, the pH is adjusted to acidic, and pectinesterase is added. After enzymatic hydrolysis, the pH is adjusted to neutral. The pectin solution is dialyzed and freeze-dried to obtain low-esterification pectin (esterification degree less than 3%).

[0054] Step 2 Dressing Preparation

[0055] Step 2.1 Preparation of outer nanofibers

[0056] PHBV and PLA were dissolved in a chloroform:DMF=19:1 solution at a ratio of 2:1 to prepare a 5% solution, and magnetic stirring was applied for 12 hours until completely dissolved to obtain the outer spinning solution. Electrospinning was performed using an electrospinning machine, and the outer spinning solution was extracted using a 10 mL syringe. A No. 7 needle was installed and placed on a micro pump. The injection speed was set to 1.8 mL / h, the distance between the needle and the roller was adjusted to 15 cm, the roller speed was 600 rpm, and a high voltage of 15 kV was applied to the needle. The humidity in the control cabinet was between 20-30%, electrospinning was performed at room temperature, and the solution was allowed to dry for 12 hours.

[0057] Step 2.2 Preparation of inner nanofibers

[0058] 5.5 wt.% sunflower pectin aqueous solution and 5 wt.% PEO aqueous solution were prepared respectively, and magnetic stirring was applied until they were completely dissolved. The pH of the pectin aqueous solution was adjusted to 7.0 using 1 wt.% sodium hydroxide aqueous solution.

[0059] An aqueous solution of sunflower pectin and an aqueous solution of PEO were mixed in a solute mass ratio of 1:1. 1 wt.% of the surfactant Triton X-100 and 5 wt.% of the co-solvent dimethyl sulfoxide were added to the solution in proportion. The solution was stirred for at least 12 hours to uniformly mix the components, thereby obtaining an inner spinning solution. The concentration of the polymer (pectin + PEO) in the solution was maintained between 4.5 wt.% and 5.0 wt.%. Sodium selenite powder was added to the inner spinning solution at a ratio of 8 mmol / mL and thoroughly stirred. A 10 mL syringe was then used to draw the solution, which was then fitted with a No. 7 needle and placed on a micropump. The injection rate was set to 1.2 mL / h, the distance between the needle and the roller was adjusted to 15 cm, the roller speed was adjusted to 600 rpm, and a high voltage of 12 kV was applied to the needle. The humidity in the control cabinet was maintained at 25%, and electrospinning was performed at room temperature. The solution was then allowed to dry for 12 hours.

[0060] Basic spinning preparation:

[0061] Prepare a 5.5 wt.% aqueous solution of sunflower pectin with varying degrees of esterification and a 5 wt.% aqueous solution of PEO. Stir magnetically until completely dissolved. Adjust the pH of the pectin solution to 7.0 using a 1 wt.% aqueous sodium hydroxide solution. Mix the two solutions in a 1:1 ratio of solute mass. Add 1 wt.% of the surfactant Triton X-100 and 5 wt.% of the co-solvent dimethyl sulfoxide (DMSO) to the solutions in proportion. Stir for at least 12 hours to ensure uniform mixing. Maintain a high polymer (pectin + PEO) concentration between 4.5 and 5.0 wt.%.

[0062] Electrospinning was performed using an electrospinning machine. A 10mL syringe was used to draw the spinning solution. A No. 7 needle was attached to the micropump. The injection rate was set to 0.2-0.7mL / h. The distance between the needle and the receiving screen was adjusted to 15cm. A high voltage of 7-13kV was applied to the needle. The humidity in the control cabinet was maintained between 20-30%. Electrospinning was performed at room temperature. Fiber formation, spinning stability, and fiber uniformity were observed under a microscope to determine the optimal esterification degree of the pectin for spinning. The above steps were repeated with pectins of varying molecular weights and the same esterification degree to identify the optimal molecular weight for spinning.

[0063] Example 2: This example differs from Example 1 in that 10 ppm nanosilver replaces sodium selenite powder, and the prepared PEO solution has a concentration of 5 wt.%; other steps and parameters are the same as those in Example 1.

[0064] 1. Trait Assessment

[0065] 1 Scanning electron microscope detection

[0066] The fibers were gold-sputtered for 120 seconds using an ion sputtering apparatus, and the morphology of the fibers was observed using a scanning electron microscope at an accelerating voltage of 20 kV.

[0067] 2 Fourier transform infrared spectroscopy

[0068] The Fourier transform infrared spectra of the materials were obtained using a Fourier transform infrared spectrometer.

[0069] 3 Crystallinity

[0070] X-ray diffractometer (XRD) was used to detect the crystallinity of the prepared composite nanofibers.

[0071] 4 Water contact angle

[0072] The contact angles of different films and solutions were measured using a contact angle meter. All samples were filmed for 600 seconds, with one frame every 60 seconds.

[0073] 5. Water vapor transmission rate

[0074] 30 ml of distilled water was added to the test tubes, and the composite nanofiber film was used to seal the tubes. These tubes were incubated in a 37°C incubator for 24 hours, and the amount of water evaporated was measured. The water vapor transmission rate was then determined using the formula:

[0075]

[0076] Where A and m represent the diameter of the test tube and the amount of water evaporation, respectively.

[0077] 6 Thermogravimetric analysis

[0078] The thermal properties of the electrospun biomimetic nanofiber membrane were evaluated using a thermal analyzer. The membrane sample (5.0 mg) was heated from 25°C to 800°C at a rate of 20°C / min.

[0079] 7 Drug release in vitro

[0080] The nanofiber membrane was immersed in phosphate-buffered saline (PBS) solution (pH 7.4) and incubated at 37°C with stirring. Every 10 minutes, 200 μL of released PBS was collected and replaced with 200 μL of fresh buffer. The drug concentration in the collected release medium was quantified using a UV spectrophotometer at an appropriate wavelength.

[0081] 2. Animal model preparation and experiments

[0082] 1STZ diabetes model

[0083] Mice were intraperitoneally injected with STZ at a concentration of 45 mg / kg in sodium citrate buffer (pH 4.3) to induce diabetes. Blood glucose levels were measured every three days after model establishment. Blood glucose levels were measured using an automated glucometer. A blood glucose level exceeding 16.7 mmol / L indicated successful model establishment and was used as a diabetic wound model.

[0084] 2. Creation of skin wound

[0085] Mice were anesthetized with 4% chloral hydrate (w / v) intraperitoneal injection, and a 2 cm × 2 cm dorsal area was shaved and disinfected with 75% ethanol solution. A full-thickness cortical injury model was prepared in mice using surgical scissors.

[0086] 3. Wound treatment

[0087] The mice were divided into three groups. The first group served as a control group, receiving no treatment. The second group received an unloaded nanofiber membrane, and the third group received a sodium selenite pentahydrate-loaded nanofiber membrane. The wounds were bandaged with gauze and medical tape. On days 0, 3, 6, 9, and 12, photographs were taken to record wound appearance and healing progress, and to estimate the percentage of wound healed area.

[0088] Dressing structure Figure 1 As shown in the figure, scanning electron microscopy results reveal that the inner layer exhibits a large number of beaded structures due to drug loading, while the outer layer of PLA / PHBV nanofibers is relatively smooth and has a coarser diameter than the inner layer, providing more stable mechanical properties. Comparison of water contact angles reveals that the outer layer is hydrophobic, while the inner layer is completely wetted, demonstrating that this dressing achieves the goal of a hydrophobic outer layer to prevent contamination and a hydrophilic inner layer to maintain wound moisture.

[0089] From Table 1 and Figure 2 It can be seen that after the two layers of materials are composited, the WVTR value of the nanofiber membrane is measured to be 0.2g / cm 2 Day, has good water vapor permeability. And through thermogravimetric analysis, the material only loses weight above 200°C, and has very good thermal stability in the human body environment.

[0090] Table 1

[0091]

[0092] Fourier infrared spectrum Figure 3 As shown by Figure 3It can be seen that in the Fourier infrared spectrum, compared with the infrared spectrum of the blank group (top), the infrared spectrum of the sodium selenite-loaded group (middle) and the infrared spectrum of the nanosilver-loaded group (bottom) only added the absorption peaks of sodium selenite and nanosilver, and the other waveforms remained basically the same, indicating that sodium selenite and nanosilver did not produce new chemical bonds with pectin and PEO, but were only loaded in the form of physical adsorption.

[0093] The XRD crystallinity analysis diagram is shown in Figure 4. According to the XDR crystallinity analysis, the diffraction image of the drug-loaded group ( Figure 4b 、 Figure 4c ) compared with the blank group ( Figure 4a ) did not appear with sodium selenite ( Figure 4b ) and nanosilver ( Figure 4c ) indicates that sodium selenite and nanosilver are in an amorphous phase within the material. Amorphous compounds typically have higher Gibbs free energy and solubility, making them more likely to diffuse into the wound and take effect.

[0094] In vitro drug release Figure 5 As shown in the figure, the nanofibers containing sodium selenite gradually released the substance within the first 30 minutes in PBS solution at 37°C. After this initial phase, the release rate slowed down significantly, and a stable release was achieved after about 120 minutes. Finally, a nearly complete cumulative release rate was obtained, indicating that sodium selenite has significant stability and rapid release potential on the electrospun biomimetic nanofiber membrane.

[0095] Wound healing Figure 6 As shown in the figure, based on the epigenetic analysis of the healing photos of mice on day 12, it can be found that the dressing treatment group and the dressing group loaded with sodium selenite and nanosilver are closer to the normal group than the model group, among which the therapeutic effect of the sodium selenite-loaded group is more obvious.

[0096] The above describes the specific embodiments of the present invention. It should be noted that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various variations or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A method for preparing a composite nanofiber membrane based on enzymatic pectin preparation, characterized in that: The following steps are involved: Step 1: drying the sunflower disk into small pieces, crushing them, and sieving them to obtain sunflower disk powder; Step 2: adding sodium citrate to distilled water, adjusting the pH to acidic with citric acid solution, and preparing an extract; Step 3: Add the sunflower disk powder to distilled water, shake well, wash with water, centrifuge, collect the precipitate, add the extract, mix well, heat in a water bath, centrifuge, take the supernatant, concentrate, add anhydrous ethanol, and form a gel at 4°C overnight. Centrifuge the alcohol precipitate, add distilled water to re-dissolve the alcohol precipitate, evaporate the ethanol, concentrate, and freeze-dry to obtain crude pectin; Step 4: mixing the crude pectin with water and adjusting the pH to acidic, adding pectinesterase, adjusting the pH to neutral after enzymatic hydrolysis, dialyzing the pectin solution, and freeze-drying to obtain low-esterification pectin with an esterification degree of less than 3%; Step 5, dissolving PHBV and PLA in a mixed solution of chloroform and DMF, stirring until completely dissolved to obtain an outer layer spinning solution, electrospinning, and standing to dry to obtain an outer layer nanofiber membrane; Step 6: adding the pectin obtained in step 4 to distilled water and stirring until completely dissolved to obtain a pectin aqueous solution; adjusting the pH to neutral with a NaOH aqueous solution; adding a PEO solution; and then adding Triton X-10 and dimethyl sulfoxide; stirring evenly to obtain an inner layer spinning solution; electrospinning on the surface of the outer layer nanofibers and drying; thereby obtaining the composite nanofiber membrane; The PEO solution is a PEO aqueous solution, or a PEO solution prepared by adding PEO to a nanosilver solution.

2. The method according to claim 1, characterized in that The pectin obtained in step 4 is added to distilled water and stirred until completely dissolved to obtain a pectin aqueous solution. The pH value is adjusted to neutral with a NaOH aqueous solution, and a PEO solution is added. Then, Triton X-10 and dimethyl sulfoxide are added and stirred evenly to obtain an inner layer spinning solution, which is electrospun and allowed to stand to dry. PHBV and PLA are then dissolved in a mixed solution of chloroform and DMF, stirred until completely dissolved to obtain an outer layer spinning solution, which is electrospun on the surface of the inner layer nanofibers and allowed to stand to dry to obtain the composite nanofiber membrane.

3. The method according to claim 1, characterized in that Sodium citrate was added into distilled water at a mass fraction of 0.74%.

4. The method according to claim 1, characterized in that The concentration of the citric acid solution is 0.1 mol / L.

5. The method according to claim 1, characterized in that: In step 5, the mass ratio of PHBV to PLA is 2:1, the mass ratio of chloroform to DMF is 19:1, and the total mass concentration of PHBV and PLA is 5%.

6. The method according to claim 1, characterized in that In step 6, the concentration of the pectin aqueous solution is 5.5wt.%, the concentration of the PEO aqueous solution is 5wt.%, and the PEO aqueous solution is added in a pectin to PEO mass ratio of 1:

1. Dimethyl sulfoxide accounts for 5wt.% of the inner layer spinning solution, and Triton X-100 accounts for 1wt.% of the inner layer spinning solution; the concentration of pectin and PEO in the inner layer spinning solution is maintained at 5.0wt.%.

7. The method according to claim 1, characterized in that: The electrospinning parameters of the outer layer are: injection speed of 1.2 mL / h-2.0 mL / h, receiving distance of 15 cm, roller speed of 400 rpm-1000 rpm, voltage of 10 kV-17 kV, and humidity controlled in steps of 20%-30%.

8. The method according to claim 1, characterized in that: The electrospinning parameters of the inner layer are: injection speed of 1.0 mL / h-1.6 mL / h, receiving distance of 15 cm, roller speed of 400 rpm-1000 rpm, voltage of 7 kV-14 kV, and humidity controlled at 20%-30%.

9. The method according to any one of claims 1 to 8, characterized in that: Step 5: adding sodium selenite to the inner spinning solution, stirring evenly and then performing electrostatic spinning. The concentration of sodium selenite in the inner spinning solution is 8 mmol / mL.

10. A composite nanofiber membrane prepared by the method according to any one of claims 1 to 9, used as a dressing for promoting diabetic wound healing.

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