A hydrophilic and hydrophobic Janus membrane, its preparation method and application

Abstract

A hydrophilic-phobic Janus membrane comprises a hydrophobic fiber layer and a hydrophilic fiber layer loaded with metal-ellagic acid nanoparticles. The metal-ellagic acid nanoparticles consist of ellagic acid and metal ions linked by coordination bonds. This invention synthesizes metal-ellagic acid nanoparticles via a self-assembly reaction and loads the particles onto the hydrophilic fiber layer using high-voltage electrospinning technology, obtaining a hydrophilic-phobic Janus membrane capable of continuously releasing ellagic acid and possessing antioxidant, antibacterial, and directional drainage functions. It can inhibit bacterial growth, reduce free radical levels, and simultaneously drain wound exudate. Furthermore, the Janus membrane loaded with metal-ellagic acid nanoparticles can effectively promote granulation tissue repair, wound collagen deposition, and accelerate epithelial migration, and can inhibit MMP-9 expression in diabetic wounds.

Description

Technical Field

[0001] This invention patent relates to the field of Janus membrane technology, and in particular to a hydrophilic / hydrophobic Janus membrane, its preparation method, and its application. Background Technology

[0002] Diabetic ulcers are a common complication of diabetes. Due to chronic hyperglycemia, diabetic patients experience a series of pathophysiological changes, leading to slow wound healing, harming their physical and mental health, and impacting their daily lives. Diabetic infected wounds are typically accompanied by significant exudation. Excessive pro-inflammatory factors in this exudate can lead to increased expression of matrix metalloproteinase 9 (MMP-9), resulting in the degradation of growth factors and affecting epithelialization, wound contraction, and matrix remodeling. Simultaneously, excessive pro-inflammatory factors can also trigger oxidative stress, increasing intracellular free radical levels and ultimately hindering wound healing.

[0003] The clinical management of diabetic infected wounds with excessive exudate is extremely challenging. Traditional hydrophilic dressings cannot drain wound exudate, increasing the risk of bacterial infection and hindering wound healing. Therefore, there is an urgent need to develop a Janus membrane with antioxidant, antibacterial, and directional drainage properties. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing hydrophilic dressings and provide a hydrophilic-hydrophobic Janus membrane with good cell compatibility, excellent antibacterial and antioxidant properties, and directional drainage function.

[0005] The present invention provides a hydrophilic-hydrophobic Janus membrane, comprising a hydrophobic fiber layer and a hydrophilic fiber layer carrying metal-ellagic acid nanoparticles. The metal-ellagic acid nanoparticles include ellagic acid and metal ions connected by coordination bonds. The metal-ellagic acid nanoparticles are loaded onto the hydrophilic fiber layer by electrospinning technology, so that the hydrophilic fibers form a beaded structure.

[0006] Preferably, the metal ion is selected from Bi. 3+ Cu 2+ Zn 2+ One of them.

[0007] Preferably, the metal-ellagic acid nanoparticles contain metal ions and ellagic acid in a molar ratio of 0.5-2:1, which undergo a self-assembly reaction.

[0008] The present invention provides a method for preparing a hydrophilic / hydrophobic Janus membrane, the method comprising the following steps:

[0009] Step (1): Electrospin the first electrospinning solution to obtain a hydrophobic fiber layer;

[0010] Step (2): The second electrospinning solution is electrospinned onto the surface of the hydrophobic fiber layer to form a hydrophilic fiber layer, thereby obtaining the hydrophilic-hydrophobic Janus membrane; the second electrospinning solution includes metal-ellagic acid nanoparticles and a hydrophilic electrospinning solution.

[0011] Preferably, the concentration of metal-ellagic acid nanoparticles in the second electrospinning solution is 1-5 mg / ml.

[0012] Furthermore, the preparation method of the metal-ellagic acid nanoparticles includes the following steps: weighing 0.26-0.52 mmol of metal salt and 0.26-0.52 mmol of ellagic acid respectively, adding 25 mL of N-methylpyrrolidone (NMP), vortexing until dissolved, and allowing the reaction to proceed at room temperature for 24-168 h. After the reaction is complete, centrifuging at 8000 rpm for 5 min, discarding the supernatant; washing twice with NMP and anhydrous ethanol, discarding the supernatant; drying the precipitate in an oven, and grinding to obtain metal-ellagic acid nanoparticles.

[0013] Preferably, the first electrospinning solution is a polyvinylidene fluoride (PVDF), polylactic acid (PLA), polycaprolactone (PCL), or polyurethane (PU) electrospinning solution.

[0014] Furthermore, in step (1), the electrospinning voltage is 10-20 kV, the needle specification is 18-22 G, the pushing speed is 0.0010-0.0030 mm / s, the electrospinning receiver is a roller receiver, the distance between the needle and the roller receiver is 20-25 cm, the rotation speed of the roller receiver is 200 rpm, and the spinning time is controlled at 20-30 min.

[0015] Furthermore, the mass concentration of the PVDF electrospinning solution is 10-12%.

[0016] Preferably, the hydrophilic electrospinning solution is a polyvinyl alcohol / polyethylene glycol (PVA / PEG), polyvinylpyrrolidone (PVP), or polyvinyl alcohol / chitosan (PVA / CS) electrospinning solution.

[0017] Furthermore, in step (2), the electrospinning voltage is 10-20 kV, the needle specification is 18-22 G, the pushing speed is 0.0010-0.0030 mm / s, the electrospinning receiver is a roller receiver, the distance between the needle and the roller receiver is 20-25 cm, the rotation speed of the roller receiver is 200 rpm, and the spinning time is controlled to be 10-30 min.

[0018] Furthermore, the mass concentration of PVA / PEG in the second electrospinning solution is 8-12%.

[0019] The application of the hydrophilic and hydrophobic Janus membrane provided by this invention in the preparation of products that promote wound healing.

[0020] Preferably, the wound includes a diabetic ulcer wound.

[0021] The beneficial effects of this invention are:

[0022] This invention synthesizes metal-ellagic acid nanoparticles through a self-assembly reaction and loads the particles onto a hydrophilic fiber layer using high-voltage electrospinning technology. This yields a hydrophilic-hydrophobic Janus membrane that can continuously release ellagic acid and has antioxidant, antibacterial, and directional drainage functions. It exhibits a liquid directional flow function, allowing liquid to be rapidly transported from the hydrophobic side to the hydrophilic side, but not from the hydrophilic side to the hydrophobic side. This demonstrates a good liquid "diode" function, enabling liquid to flow directionally from the hydrophobic side to the hydrophilic side.

[0023] The hydrophilic-hydrophobic Janus membrane provided by this invention can inhibit bacterial growth, reduce free radical levels, and draw out wound exudate. Furthermore, the Janus membrane loaded with metal-ellagic acid nanoparticles can effectively promote granulation tissue repair, wound collagen deposition, and accelerate epithelial migration. It can also inhibit MMP-9 expression in diabetic wounds and reduce the expression levels of inflammatory factors. Attached Figure Description

[0024] Figure 1 This is a scanning electron microscope (SEM) image of the M-EA particles obtained in Example 1.

[0025] Figure 2 The image shows the FTIR spectrum of the M-EA particles obtained in Example 1.

[0026] Figure 3 The images shown are scanning electron microscope (SEM) images of the hydrophilic fiber layer (HL) and hydrophobic fiber layer (HB) of the Janus membrane in Example 2. The scale bar of the hydrophilic fiber layer is 10 μm, and the scale bar of the hydrophobic fiber layer is 10 μm.

[0027] Figure 4 This is a series of real-time photos from the directional transmission performance experiment in Example 3. Figure 4 Group A has the hydrophobic fiber layer facing upwards, and the droplets are transported from the hydrophobic fiber layer to the hydrophilic fiber layer. Figure 4 Group B has the hydrophilic fiber layer facing upwards, and the droplets diffuse and are stored within the hydrophilic fiber layer.

[0028] Figure 5 This is a statistical graph showing the time taken for droplets with the hydrophobic fiber layer facing upwards to travel across the Janus membrane in the directional transport performance experiment of Example 3.

[0029] Figure 6The results are statistical results of Janus membrane EA release experiments in each group in Example 3.

[0030] Figure 7 This is a graph showing the experimental results of the intracellular antioxidant level of the Janus membrane in Example 3. Among them, Figure 7 A shows fluorescence images of each group of cells under an inverted fluorescence microscope, with a scale bar of 200 μm. Figure 7 B represents the statistical results of fluorescence intensity for each group.

[0031] Figure 8 This is a series of photographs of agar plates after 12 hours of incubation in each group of Example 3. Figure 8 A represents Staphylococcus aureus ( S.aureus (Image of a drop plate) Figure 8 B is a group of drop plate photographs of methoxyaureus (MRSA).

[0032] Figure 9 In Example 3 S.aureus Colony count statistics for each group. * P < 0.05, ** P < 0.01, *** P < 0.001.

[0033] Figure 10 This is a statistical chart of colony counts for each MRSA group in Example 3. * P < 0.05, ** P < 0.01, *** P < 0.001.

[0034] Figure 11 These are photographs of the instantaneous contact angle measurements of the Janus, BE-J, CE-J, and ZE-J films in Example 3.

[0035] Figure 12 These are representative images of the wounds and simulated wound healing diagrams on days 0, 3, 7, 10, and 14 after treatment in each group of Example 4.

[0036] Figure 13 These are photographs taken from wound exudate on days 0, 3, 7, 10, and 14 of Example 4, and then plated on agar plates and incubated for 12 hours.

[0037] Figure 14 The above are the statistical results of the changes in wound area ratio over time in each group in Example 4.

[0038] Figure 15 The images shown are H&E stained skin tissue sections from the wounds on days 7 and 14 of treatment in Example 4. The scale bar before magnification is 100 μm, and the scale bar after magnification is 10 μm. Black arrows indicate areas of inflammatory infiltration, red arrows indicate hair follicle appendages, and double-headed arrows indicate the width of granulation tissue in the wound.

[0039] Figure 16This is a statistical result graph of H&E staining images. Among them, Figure 16 A represents the statistical results of granulation tissue width in each group of wounds on days 7 and 14. Figure 16 B represents the statistical results of epidermal thickness in each group on day 14.

[0040] Figure 17 Masson-stained images of wound skin tissue sections from each group on days 7 and 14 of treatment in Example 4. The scale bar before magnification is 100 μm, and the scale bar after magnification is 10 μm.

[0041] Figure 18 This is a statistical result graph of the Masson staining images. Among them, Figure 18 A represents the statistical results of collagen volume fraction on day 7 of each group's wound. Figure 18 B represents the statistical results of collagen volume fraction on day 14 for each group of wounds. * P < 0.05, ** P < 0.01, *** P < 0.001.

[0042] Figure 19 These are immunohistochemical staining images of keratin in skin tissue sections from the wounds on days 7 and 14 of treatment in Example 4. The scale bar before magnification is 100 μm, and the scale bar after magnification is 10 μm.

[0043] Figure 20 This section presents statistical results of keratin immunohistochemical staining images. Among them, Figure 20 A is a representative image of MMP-9 immunohistochemical staining of wound skin tissue sections on day 7 of treatment in each group in Example 4, with a scale bar of 10 μm. Figure 20 B shows the normalized quantitative results of MMP-9 immunohistochemical staining. * P < 0.05, ** P < 0.01, *** P < 0.001. Detailed Implementation

[0044] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0045] All raw materials used in the embodiments of this invention were purchased commercially.

[0046] Example 1

[0047] 1.1 Preparation of Metal-Ellagic Acid Nanoparticles (M-EA)

[0048] Preparation of Bi-EA: Weigh 0.52 mmol of bismuth nitrate pentahydrate and 0.26 mmol of ellagic acid, add 25 mL of N-methylpyrrolidone (NMP), vortex until dissolved, and allow to stand at room temperature for 168 h. After the reaction is complete, centrifuge at 8000 rpm for 5 min and discard the supernatant; wash twice with NMP and anhydrous ethanol and discard the supernatant; dry the precipitate in a 65 ℃ oven, grind it in a mortar and pestle to obtain Bi-EA particles, and store at room temperature for later use.

[0049] Preparation of Cu-EA: Weigh 0.26 mmol of bismuth nitrate pentahydrate and 0.52 mmol of ellagic acid, add 25 mL of N-methylpyrrolidone (NMP), vortex until dissolved, and allow to stand at room temperature for 24 h. After the reaction is complete, centrifuge at 8000 rpm for 5 min and discard the supernatant; wash twice with NMP and anhydrous ethanol and discard the supernatant; dry the precipitate in a 65 ℃ oven, grind it in a mortar and pestle to obtain Cu-EA particles, and store at room temperature for later use.

[0050] Preparation of Zn-EA: Weigh 0.52 mmol of anhydrous zinc acetate, add 0.26 mmol of ellagic acid, and add 25 mL of N-methylpyrrolidone (NMP). Vortex until dissolved and allow to stand at room temperature for 24 h. After the reaction is complete, centrifuge at 8000 rpm for 5 min and discard the supernatant. Wash twice with NMP and anhydrous ethanol and discard the supernatant. Dry the precipitate in a 65 ℃ oven, grind it in a mortar and pestle to obtain Zn-EA particles, and store at room temperature for later use.

[0051] Three types of M-EA particles were dispersed in anhydrous ethanol to obtain corresponding metal-ellagic acid particle suspensions. The solutions were then dropped onto a silicon wafer using a dropper and allowed to air dry naturally before being sputter-coated with gold for 1 min. The microstructure of the particles was observed and recorded using a scanning electron microscope (SEM), such as... Figure 1 As shown, Bi-EA particles exhibit a nanoflower structure, Cu-EA particles exhibit a rhomboid structure, Zn-EA particles exhibit a "walnut-shaped" structure, and all three types of M-EA particles self-assemble into crystals with unique micromorphological morphologies.

[0052] like Figure 2 As shown, the changes in functional groups of M-EA particles before and after synthesis were determined by FTIR spectroscopy. The disappearance of the phenolic hydroxyl absorption peak and the appearance of the absorption peaks of bismuth-oxygen bond, copper-oxygen bond and zinc-oxygen bond proved that bismuth ions, copper ions and zinc ions complexed with phenolic hydroxyl groups, and three types of M-EA particles, Bi-EA, Cu-EA and Zn-EA, were synthesized.

[0053] The metal content in the three types of particles was measured using an atomic absorption spectrophotometer. The metal content of Bi-EA was 53%, Cu-EA was 29%, and Zn-EA was 11%.

[0054] 1.2 Preparation of Janus membranes

[0055] Step (1): Place 0.6 g of polyvinylidene fluoride (PVDF) powder in a 20 mL glass bottle, add 3 mL of N,N-dimethylformamide (DMF) and 2 mL of acetone, and dissolve by stirring at 500 rpm in a 60 ℃ water bath. After dissolution, continue stirring at 500 rpm at room temperature to obtain the first electrospinning solution. The electrospinning receiver is a roller receiver, and a ring of tin foil is placed on the receiver to receive the fiber membrane. Draw the first electrospinning solution with a 10 mL syringe, place it on the microfluidic pump of the electrospinning machine, set the needle to 18 G, voltage to 20 KV, push speed to 0.0030 mm / s, distance to 15 cm, rotation speed to 200 rpm, and control the spinning time to 10 min to obtain a hydrophobic fiber layer;

[0056] Step (2): Place 0.5 g of polyvinyl alcohol (PVA) and 0.5 g of polyethylene glycol (PEG) in a 20 mL glass bottle, add 10 mg / 30 mg of M-EA particles respectively, add 10 mL of pure water, and stir at 500 rpm in a 90 ℃ water bath to dissolve. After dissolution, continue stirring at 500 rpm at room temperature to obtain the second electrospinning solution. After the hydrophobic fiber layer is electrospinned, continue electrospinning the hydrophilic fiber layer. Use a 10 mL syringe to draw the second electrospinning solution and place it on the microfluidic pump of the electrospinning machine. Set the needle to 20G, voltage to 20 KV, push speed to 0.0030 mm / s, distance to 15 cm, rotation speed to 200 rpm, and control the spinning time to 30 min to obtain Janus membranes, which are denoted as 1%BE-J, 3%BE-J, 1%CE-J, 3%CE-J, 1%ZE-J, and 3%ZE-J respectively.

[0057] Janus membranes without added M-EA particles are denoted as Janus. Also included are 5% ZE-J and 5% CE-J, the difference being that the amount of M-EA particles added in step (2) is 50 mg.

[0058] Example 2 Characterization of Janus membrane

[0059] Cut a 0.5 cm × 0.5 cm Janus membrane, fix it on the sample stage, and sputter gold for 1 min. Use SEM to observe and record the fiber structure of the Janus membrane, and use ImageJ to count the fiber diameter. Use Origin to perform normal distribution analysis on the data results.

[0060] like Figure 3 As shown, the fiber structure on both hydrophilic and hydrophobic sides of the Janus membrane was observed by SEM. The hydrophilic fiber layer (HL) of the Janus membrane showed obvious beaded fibers with attached particles, indicating that M-EA particles were successfully loaded onto the hydrophilic fiber layer of the Janus membrane. The hydrophobic fiber layer (HB) was composed of multiple interwoven fibers with a normal distribution in diameter of about 0.5 μm, which is similar to the nanofiber structure of ECM, thus facilitating cell adhesion, proliferation, migration and differentiation.

[0061] Example 3: Performance of Janus Membrane

[0062] 3.1 Directional transport performance of Janus membranes

[0063] The Janus membrane was fixed at both ends on the same horizontal plane, making it taut with the hydrophilic and hydrophobic fiber layers facing upwards. Rhodamine B was used to adjust the saline solution to pink. A certain amount of liquid was drawn using a 5 mL syringe and fixed to a microfluidic pump. The microfluidic pump flow rate was set to 2.5 mL / min, with the microfluidic pump needle positioned above the Janus membrane, ensuring that the droplets falling from the needle accurately landed on the membrane. The droplet transport through the Janus membrane was recorded video. It was observed that the 5% ZE-J group with the hydrophobic fiber layer facing upwards still exhibited directional transport performance, while the 5% CE-J group did not.

[0064] Figure 4 A is a real-time photograph of droplets being transported through the Janus membrane. The hydrophobic fiber layer (HB) of the Janus membrane is placed upwards, and droplets are continuously transported from the microfluidic pump. The droplets first come into contact with the hydrophobic fiber layer (HB) and then drip from the hydrophilic fiber layer (HL) through directional transport, thus realizing the rapid flow-guiding performance of the Janus membrane. Figure 4 In B, the hydrophilic fiber layer (HL) of the Janus membrane is placed upwards. After the droplet comes into contact with the hydrophilic fiber layer (HL), it spreads rapidly and is eventually trapped inside the hydrophilic fiber layer (HL). Figure 5 The time required for droplets to travel through different Janus membranes with the hydrophobic fiber layer facing upwards was statistically analyzed. It was observed that the directional transport time of the Janus membrane loaded with M-EA particles increased, possibly because the particles were wrapped in the hydrophilic fiber layer (HL), which generated some resistance. There was no significant difference in transport time between the Janus membrane with 1% particle content and the Janus membrane without particles. The difference in transport time between the Janus membrane with 3% particle content and the Janus membrane without particles was acceptable, and unidirectional drainage was achieved in vitro.

[0065] 3.2 Performance of Janus membrane in releasing ellagic acid

[0066] Weigh 20 mg of 1%BE-J, 3%BE-J, 1%CE-J, 3%CE-J, 1%ZE-J and 3%ZE-J into 3 mL of PBS buffer solution and place them in a 37 ℃ constant temperature 200 rpm shaking incubator. Take samples at 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h and 72 h. After taking 1 mL of supernatant each time, add 1 mL of fresh PBS solution. Store the supernatant at -20 ℃.

[0067] The ellagic acid concentrations of all solutions were measured using an ultra-micro spectrophotometer. Figure 6 A and C are line graphs showing the release of EA from BE-J, CE-J, and ZE-J, respectively. It can be observed that all membranes can achieve sustained and slow release. The release amounts of 3% BE-J and 3% CE-J are significantly higher than those of 1% BE-J and 1% CE-J, indicating that BE-J, CE-J, and ZE-J can continuously release EA. Among them, 3% ZE-J has the highest total release, reaching 67 μg after 72 h.

[0068] 3.3 Intracellular antioxidant levels of Janus membrane

[0069] The antioxidant level of Janus membranes was verified using the DCFH-DA method. NIH-3T3 cells were seeded at a density of 100,000 cells per well in 12-well plates and cultured at 37 °C in a 5% CO2 incubator for 24 h, with continuous observation of cell growth. Samples (Janus, 1% BE-J, 3% BE-J, 1% CE-J, 3% CE-J, 1% ZE-J, and 3% ZE-J) were prepared into a 1 mg / mL extraction buffer using complete culture medium and incubated at 37 °C for 24 h. After discarding the plate solution, the plates were washed 1-2 times with PBS buffer, and 900 μL of sample solution was added to each well. The plates were then cultured at 37 °C in a 5% CO2 incubator for another 24 h. Cells were stimulated with 100 μL of the same material extract incubated with H2O2 for 6 h, with an H2O2 concentration of 75 μM in the well liquid. After discarding the plate solution, the cells were washed 1-2 times with PBS buffer, then incubated with 2.5 μM DCFH-DA for 5 min, followed by washing with PBS buffer and adding basal culture medium. Fluorescence intensity (λ) was detected using an inverted fluorescence microscope. ex = 488 nm, λ em = 525 nm), to assess intracellular ROS levels.

[0070] Figure 7A is a fluorescence photograph of the cells. The intensity of fluorescence is directly proportional to the level of reactive oxygen species in the cells. The positive control group and Janus group showed obvious green fluorescence, while the other groups did not show obvious fluorescence. This indicates that after co-incubation with the extracts of BE-J, CE-J, and ZE-J materials, the intracellular oxidative stress was significantly reduced and the amount of ROS generated was reduced. Figure 7 B statistically analyzed the fluorescence intensity of each group. The results showed that after adding the material extract, the fluorescence intensity of the cells was significantly reduced, showing a significant difference from the positive control group. However, the fluorescence intensity of the Janus membrane group alone did not change and showed no significant difference from the positive control group. The experimental results indicate that BE-J, CE-J, and ZE-J can reduce oxidative stress and ROS production at the cellular level.

[0071] 3.4 Antibacterial properties of Janus membrane

[0072] Staphylococcus aureus was collected using a sterile inoculation loop. S.aureus Methoxystaphylococcus aureus (MRSA) was incubated in LB broth at 37 °C with a shaking incubator at 180 rpm overnight. The LB broth was then centrifuged at 8000 rpm for 3 min, the supernatant was discarded, and the solution was diluted with sterile physiological saline. The absorbance of the bacterial suspension at 600 nm was measured using a UV spectrophotometer and found to be approximately 0.5, yielding 10... 8 CFU / mL S.aureus And MRSA bacterial suspension. After diluting 100 times, 10 6 Prepare a bacterial suspension at CFU / mL. Co-culture 1 mL of the bacterial suspension with 2.5 mg / 5 mg Janus, 1% BE-J, 3% BE-J, 1% CE-J, 3% CE-J, 1% ZE-J, and 3% ZE-J, shaking at 180 rpm for 4 h at 37 ℃. After incubation, dilute 10- ... 0 10 1 10 2 10 3 10 4 Take 5 μL of the sample and drop it onto an agar plate. Incubate the plate at 37 ℃ for 12 h and then observe and photograph it. When determining the anti-MRSA performance of the Janus membrane, ampicillin at a concentration of 50 μg / mL was selected as a control.

[0073] Janus membrane resistance S.aureus The effect is as follows Figure 8 As shown in Figure A, the diluted BE-J and ZE-J membranes exhibited significantly fewer bacterial colonies than the control group, while the CE-J group showed a strong antibacterial effect even when undiluted; the Janus membrane's anti-MRSA effect was as follows. Figure 8As shown in Figure B, MRSA is a common and highly virulent bacterium in clinical practice, and it is resistant to antibiotics such as ampicillin. Therefore, ampicillin was selected as a control. The colony photographs show that the CE-J group exhibits strong MRSA eradication capabilities, while the ampicillin group still shows significant colony survival.

[0074] The colony counts for each group after a 10,000-fold dilution were counted. For example... Figure 9 As shown S.aureus After co-incubation for 4 hours and 12 hours, the bacterial count results on the droplet plate showed a significant difference between the Janus membrane loaded with M-EA particles and the control group. Figure 10 The bacterial droplet count results after co-incubation with MRSA for 4 hours and culture for 12 hours are shown. The CE-J group has a strong ability to remove MRSA, while the ampicillin group still has obvious colony survival. The Janus membrane loaded with M-EA particles is significantly different from the control group and the ampicillin group.

[0075] Antibacterial test results confirmed the effectiveness of Janus membranes loaded with M-EA particles in scavenging. S.aureus And the significant effect of MRSA. Among them, the 3% CE-J membrane has excellent cleaning effect.

[0076] 3.5 Contact angle of Janus membrane

[0077] PVDF (F), PVA / PEG (A / G), Janus, 1%BE-J, 3%BE-J, 1%CE-J, 3%CE-J, 1%ZE-J, and 3%ZE-J were adhered to a glass slide with the hydrophilic and hydrophobic surfaces facing upwards, respectively. A 1 mL syringe filled with physiological saline was fixed to the contact angle instrument. After starting, droplets were added, and the changes in contact angle were recorded by photograph. Figure 11 The instantaneous contact angles of the Janus membrane were displayed using physiological saline as the liquid source. It was observed that the contact angle of the PVDF side (F) was >100°, exhibiting hydrophobicity, while the contact angle of the PVA-PEG side (A / G) was <50°, exhibiting hydrophilicity, demonstrating the successful preparation of the hydrophilic-hydrophobic Janus membrane.

[0078] Example 4: Application of Janus membrane in the repair of refractory wounds

[0079] 4.1 Establishment of a mouse model of diabetic wound infection

[0080] Based on the comprehensive performance test results, Janus membranes with a particle concentration of 3% were selected for further in vivo experiments.

[0081] Seventy-two male mice provided by the Beijing Vital River Laboratory Animal Center were selected to establish a diabetic wound infection model. At day -5, mice that had fasted for 12 hours were intraperitoneally injected with STZ and continued to be fed. Blood glucose levels were measured on day -2, and mice with blood glucose levels greater than 16.7 mmol / L were selected for the experiment. Two full-thickness skin excision wounds with a diameter of 6 mm were created on the back, and 20 μL of 1×10⁻⁶ STZ was administered. 7 CFU / mL S.aureus Bacterial solution was used to form infected wounds. At day 0, all mice were randomly divided into 6 groups: control group, J-HL group, J-HB group, BE-J group, CE-J group and ZE-J group, to study the therapeutic effect of Janus membrane on diabetic infected wounds.

[0082] In the control group, the medication was physiological saline. The J-HL group received a pure Janus membrane with its hydrophilic side in contact with the wound; the J-HB group received a pure Janus membrane with its hydrophobic side in contact with the wound; the BE-J group received a 3% Bi-EA Janus membrane with its hydrophobic side in contact with the wound; the CE-J group received a 3% Cu-EA Janus membrane with its hydrophobic side in contact with the wound; and the ZE-J group received a 3% Zn-EA Janus membrane with its hydrophobic side in contact with the wound. The volume of physiological saline was 20 μL, and the membrane size was 1.5 cm × 1.5 cm. The BE-J, CE-J, and ZE-J groups are collectively referred to as the material groups.

[0083] Administer the medication on days 0, 3, 7, and 10.

[0084] Samples were taken with moist sterile cotton swabs on days 0, 3, 7, 10, and 14. The swabs were stored in centrifuge tubes containing 10 mL of physiological saline. 70 μL of the liquid was spread evenly on an agar plate and incubated in a 37 ℃ incubator for 12 h. The samples were then photographed.

[0085] Wound photographs were taken on days 0, 3, 7, 10, and 14. ImageJ software was used to statistically analyze the wound size and create a wound trajectory simulation map. Wound area percentage was calculated.

[0086] Wound area percentage (%) = (wound area size at the time of photographing / wound area size at the time of initial modeling) × 100%.

[0087] On days 3, 7, and 14, one-third of the mice were randomly selected for skin and heart, liver, spleen, lung, and kidney sampling. Using sterile scissors, the skin and organs from the back wound were cut off, with each skin piece approximately 1 cm × 1 cm in size. The skin was washed with physiological saline and laid flat on filter paper. The skin was then divided in half. One half of the skin and organs was placed in an embedding cassette and immediately immersed in 4% paraformaldehyde for fixation, dehydration, embedding, and sectioning. The other half of the skin was placed in a centrifuge tube and immediately placed on dry ice, subsequently stored at -80 °C.

[0088] 4.2 Janus membrane promotes healing of diabetic infected wounds

[0089] from Figure 12 Photographs of the wound appearance and Figure 13 Based on the smear images, there was no significant difference in bacterial growth in the wounds of the different groups on day 0 before drug administration. After 3 days of drug administration, the amount of bacterial film and bacterial colonies in the wounds of the material group decreased, but the control group and the J-HL and J-HB groups still had a lot of bacterial film and colonies. On day 7, almost no bacterial film was observed in the wounds of the material group, and the colonies in the CE-J group almost disappeared. After 10 days of drug administration, the number of bacterial colonies in the wounds and on the smears of the material group was significantly less than that in the control group. On day 14, the wounds of the CE-J and ZE-J material groups were almost healed, and no obvious colonies appeared.

[0090] Figure 14 The changes in wound area percentage over time were statistically analyzed for each group. On day 3, the wound area of ​​the ZE-J group decreased by 50%. On day 7, the wound area of ​​the BE-J, CE-J, and ZE-J groups decreased to below 25%, while the wound area of ​​the control group, J-HL, and J-HB groups remained above 40%. On day 14, the wound area of ​​the BE-J, CE-J, and ZE-J groups was less than 5%, while the wound area of ​​the control group and J-HL group remained above 10%, and the wound area of ​​the J-HB group was 6.6%.

[0091] 4.3 Janus membrane promotes the growth of granulation tissue in wounds.

[0092] Skin tissue sections were stained with hematoxylin and eosin (H&E) on days 7 and 14, and images were acquired using an optical microscope. The results are as follows: Figure 15 As shown. On day 7, the control group and J-HL group showed significant inflammatory infiltration, while the material group showed less inflammation. Furthermore, the control group had the largest wound bed diameter, and the epithelium was not yet closed; while the material group showed a smaller wound bed diameter, with the CE-J group having the smallest wound diameter, and the ZE-J group already covered by the epidermis. On day 14, it was observed that the ZE-J group had completely healed wounds and appendages had fully grown, while the wound beds in the control group and J-HL group remained relatively prominent. Figure 16A statistical analysis of granulation tissue width showed that the CE-J group had the best healing performance on day 7, while the ZE-J group had the best healing performance on day 14. Figure 16 B statistically analyzed the epidermal thickness of the tissues. In the J-HB, BE-J and CE-J groups, cells continued to proliferate, and the thickness of the newly formed epidermis increased significantly, which was much higher than that of the control group and the J-HL group. In the ZE-J group, the wound healing entered the remodeling period, and the extracellular matrix was also continuously remodeled and adjusted. The epidermal thickness gradually tended to stabilize and approach the normal epidermal thickness.

[0093] 4.4 Janus membrane promotes collagen deposition in wounds

[0094] Skin tissue sections taken on days 7 and 14 were stained with Masson's trichrome, and images were acquired using an optical microscope. The results were then statistically analyzed. The formula for calculating the collagen volume fraction (CVF) is as follows:

[0095] Collagen volume fraction = (S1 / S) × 100%.

[0096] The results are as follows Figure 17 As shown. On day 7, no obvious blue collagen deposition was observed in the wound center of the control group and the J-HL group, while obvious blue collagen fibers were observed in the wound center of the material group. On day 14, the blue fibers in the wound center of the control group and the J-HL group were lighter in color, while the blue collagen fibers in the material group were more pronounced. Figure 18 It was also observed that the collagen volume fraction of the material group was the highest, which was significantly different from that of the control group, indicating that BE-J, CE-J, and ZE-J help the wound transition to the remodeling stage.

[0097] 4.5 Janus membrane promotes wound re-epithelialization

[0098] The results of skin tissue sections stained with immunohistochemical staining (IHC) on days 7 and 14 are as follows: Figure 19 The results showed that on day 7, the epithelium in the control group and the J-HL group was not completely closed and still had a large gap, while the epithelium in the other material groups was almost closed; on day 14, the epithelium in the control group and the J-HL group was still not completely closed, while the epithelium in the J-HB, BE-J, CE-J and ZE-J groups was completely closed on day 14; the results indicate that Janus dressing with directional drainage ability can promote the creep of the wound epithelial layer.

[0099] 4.5 Janus membrane inhibits MMP-9 expression in wound tissue

[0100] Further analysis of representative immunohistochemical (IHC) staining images from day 7, such as... Figure 20As shown, the expression of MMP-9 was relatively high in the control group, J-HL group, and J-HB group. However, due to its drainage effect, the J-HB group effectively drained the exudate from the diabetic wound, which slowed down the expression of MMP-9 to a certain extent, showing a significant difference from the control group. The J-HL group and the control group did not inhibit MMP-9. The expression of MMP-9 in the drainage Janus dressing group loaded with the three particles was relatively low. This was due to the drainage effect of the Janus dressing and the fact that the material could continuously release EA, which effectively inhibited the expression of MMP-9. The inhibitory effects were strongest in the CE-J group and the ZE-J group.

[0101] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A hydrophilic-hydrophobic Janus membrane, characterized in that, The membrane comprises a hydrophobic fiber layer and a hydrophilic fiber layer carrying metal-ellagic acid nanoparticles. The metal-ellagic acid nanoparticles consist of ellagic acid and metal ions linked by coordination bonds. The metal-ellagic acid nanoparticles are loaded onto the hydrophilic fiber layer using electrospinning technology, resulting in a beaded structure on the hydrophilic fibers. The hydrophilic-hydrophobic Janus membrane has a liquid-directing function, allowing liquid to be rapidly transported from the hydrophobic side to the hydrophilic side, but preventing it from being transported from the hydrophilic side to the hydrophobic side.

2. The hydrophilic-hydrophobic Janus membrane according to claim 1, characterized in that, The metal ions are selected from Bi. 3+ Cu 2+ Zn 2 + One of them.

3. The hydrophilic-hydrophobic Janus membrane according to claim 2, characterized in that, The metal-ellagic acid nanoparticles are fed with metal ions and ellagic acid in a molar ratio of 0.5-2:

1.

4. The method for preparing the hydrophilic-hydrophobic Janus membrane according to any one of claims 1-3, characterized in that, The preparation method includes the following steps: Step (1): Electrospin the first electrospinning solution to obtain a hydrophobic fiber layer; Step (2): The second electrospinning solution is electrospinned onto the surface of the hydrophobic fiber layer to form a hydrophilic fiber layer, thereby obtaining the hydrophilic-hydrophobic Janus membrane; the second electrospinning solution includes metal-ellagic acid nanoparticles and a hydrophilic electrospinning solution.

5. The method for preparing the hydrophilic-hydrophobic Janus membrane according to claim 4, characterized in that, The concentration of metal-ellagic acid nanoparticles in the second electrospinning solution is 1-5 mg / ml.

6. The method for preparing the hydrophilic-hydrophobic Janus membrane according to claim 4, characterized in that, The first electrospinning solution is one of PVDF, PLA, PCL or PU electrospinning solution.

7. The method for preparing the hydrophilic-hydrophobic Janus membrane according to claim 4, characterized in that, The hydrophilic electrospinning solution is one of PVA / PEG, PVP, or PVA / CS electrospinning solutions.

8. The method for preparing the hydrophilic-hydrophobic Janus membrane according to claim 7, characterized in that, In step (2), the voltage for electrospinning is 10-20 kV, the pushing speed is 0.0010-0.0030 mm / s, the electrospinning receiver is a roller receiver, the distance between the needle and the roller receiver is 20-25 cm, the rotation speed of the roller receiver is 200 rpm, and the spinning time is controlled to be 10-30 min.

9. The use of the hydrophilic and hydrophobic Janus membrane as described in any one of claims 1-3 in the preparation of wound healing products.

10. The application according to claim 9, characterized in that, The wounds include diabetic ulcers.

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