A biomimetic multilayer dressing for diabetic chronic wounds and method of preparation

Abstract

The present application relates to the field of biomedical materials, and discloses a kind of bionic multilayer dressing for chronic wound of diabetes and preparation method.The dressing constructs three-layer composite fiber structure using sequential coaxial electrospinning technology: long-term response layer (PLGA (85:15) / EGF / anti-scar drug), medium-term response layer (PLGA (50:50) / bFGF / VEGF) and fast response layer (PVA / levofloxacin / GM6001), after cross-linking solidification by glutaraldehyde vapor, form stable structure composite fiber membrane.The present application realizes precise time sequence controlled release of antibiotic rapid release anti-infection, growth factor medium-term release promotes angiogenesis, epidermal growth factor long-term release promotes epithelialization by the combination of three polymer materials with different degradation characteristics, effectively simulates and guides the natural healing process of wound, solves the technical problems of single function of traditional dressing and mismatch between drug release and healing stage, significantly improves the healing quality and efficiency of chronic wound of diabetes.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials, specifically to a biomimetic multilayer dressing for chronic wounds in diabetic patients and its preparation method. Background Technology

[0002] Diabetic wounds, especially diabetic foot ulcers, are notoriously difficult to heal due to the persistent hyperglycemia in patients. The recurrent infections, uncontrolled inflammation, cellular dysfunction, and growth factor deficiencies caused by hyperglycemia constitute the core factors contributing to their stubborn healing, and are the leading cause of amputation and non-traumatic amputation in diabetic patients, placing a heavy burden on global public health systems. Therefore, an ideal wound management strategy must go beyond static intervention and dynamically match the complex natural healing process. This means strong anti-inflammatory and anti-infection measures in the early stages, promoting cell proliferation and angiogenesis in the middle stages, and focusing on tissue remodeling and epithelialization in the later stages. However, existing clinical dressings are mostly passive coverings with limited functions, unable to actively respond to and intervene in this dynamically changing wound microenvironment. Therefore, this study aims to develop a smart multilayer fiber dressing based on controlled-release technology. By programmatically releasing different therapeutic drugs on demand, it can achieve proactive and precise management of the entire healing cycle of diabetic wounds, thereby breaking through the bottleneck in chronic wound treatment.

[0003] Currently, while the field of diabetic wound dressings has evolved from traditional gauze and foam dressings to functional dressings (such as hydrogels and silver ion dressings), significant limitations still exist. First, the problem of limited functionality is prominent; most dressings focus only on a single function, such as antibacterial or moisturizing, making it difficult to address the complex biological processes of wound healing, which involve multiple stages and diverse needs. Second, drug release behavior is uncontrollable; existing drug-loaded dressings mostly exhibit simple rapid or slow release, failing to achieve precise and sequential drug release within specific time windows. This may lead to missed opportunities for anti-infection or premature release of proliferative drugs, which are then destroyed by the inflammatory environment. Finally, there is a lack of dynamic adaptability; the function of traditional dressings remains static throughout their entire service life, unable to dynamically match the inflammatory, proliferative, and remodeling phases of wound healing. This severely restricts their therapeutic effect on chronic, refractory wounds. Therefore, the development of an intelligent dressing capable of automatically switching treatment modes according to the healing sequence is urgently needed. Summary of the Invention

[0004] The purpose of this invention is to address the aforementioned technical problems by providing a biomimetic multilayer dressing for chronic wounds in diabetic patients.

[0005] The dressing of this invention is composed of fast-response fibers, medium-response fibers, and long-response fibers layered from top to bottom:

[0006] The shell of the fast-response fiber contains polyvinyl alcohol, and the core layer contains antibiotics and protease inhibitors;

[0007] The shell of the intermediate-response fiber contains a first polylactic acid-glycolic acid copolymer (PLGA), and the core layer contains fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF).

[0008] The shell of the long-term responsive fiber is a second PLGA, and the core layer contains epidermal growth factor (EGF) and anti-scarring drugs;

[0009] The ratio of lactic acid to glycolic acid in the first polylactic acid-glycolic acid copolymer is 50:50;

[0010] The ratio of lactic acid to glycolic acid in the second polylactic acid-glycolic acid copolymer is 85:15;

[0011] The degradation rate of the first PLGA is faster than that of the second PLGA.

[0012] This invention also provides a method for preparing a biomimetic multilayer dressing for chronic diabetic wounds, specifically including the following steps:

[0013] (1) Preparation of functional spinning solutions: 8% polyvinyl alcohol aqueous solution was used as spinning solution A; Triton X-100 (0.5% by volume), 50 nM levofloxacin and 100 μg / mL protease inhibitor GM6001 were dissolved in 10 mM pH 7.4 PBS buffer as spinning solution B; 10% ethyl acetate solution of the first PLGA was used as spinning solution C; PBS solution containing 50 ng / mL FGF and 50 ng / mL VEGF was used as spinning solution D; 10% dichloromethane solution of the second PLGA was used as spinning solution E; PBS solution containing 30 ng / mL EGF and 10 μM mitomycin C was used as spinning solution F.

[0014] (2) Preparation of biomimetic multilayer dressing: A biodegradable polylactic acid film with a thickness of 0.2 mm was fixed on the receiving device of an electrospinning equipment. Coaxial electrospinning technology was used for sequential spinning. Spinning solution A was used as the shell layer and spinning solution B was used as the core layer for the first stage of spinning "fast response fiber". Spinning solution C was used as the shell layer and spinning solution D was used as the core layer for the second stage of spinning "medium response fiber". Spinning solution E was used as the shell layer and spinning solution F was used as the core layer for the third stage of spinning "long-term response fiber". Each stage was spun for 30 minutes according to the set spinning parameters. After spinning, a composite fiber membrane was obtained. Then, the composite fiber membrane was cross-linked with 25% glutaraldehyde vapor and then air-dried to obtain the biomimetic multilayer dressing.

[0015] Furthermore, the spinning parameters in step (2) are all set as follows: voltage 15 kV, receiving distance 11 cm, shell spinning speed 0.003 mm / s, and core spinning speed 0.001 mm / s.

[0016] Furthermore, the glutaraldehyde vapor crosslinking time in step (2) is 6 h.

[0017] The present invention also provides the application of a biomimetic multilayer dressing for diabetic chronic wounds in the preparation of drugs or medical devices for treating diabetic chronic wounds.

[0018] Furthermore, the dressing should adhere tightly to the wound during use due to its rapid-response fibers.

[0019] The advantages of this invention are:

[0020] 1. This invention, through ingenious material and structural design, combines rapidly water-soluble polyvinyl alcohol with PLGA of different degradation rates to construct a biomimetic multilayer dressing with "rapid-medium-long-term" triple drug release kinetics. This enables precise time-sequential controlled release of key therapeutic factors throughout the entire wound healing cycle, solving the technical problems of traditional dressings having single functions and mismatch between drug release behavior and healing stage.

[0021] 2. This invention employs a unique sequential coaxial electrospinning technology to encapsulate different functional therapeutic agents within the corresponding fiber core layers, successfully integrating multiple therapeutic functions such as anti-infection, proliferation promotion, and scar prevention into one, creating a "one-stop" intelligent dressing that can actively guide the healing process, breaking through the limitations of existing products that are mostly passively covered or have only a single function.

[0022] 3. The dressing structure of the present invention can dynamically respond to changes in the microenvironment of diabetic wounds. Its programmed release logic can adapt to the inflammatory phase, proliferation phase and remodeling phase of the healing process. It not only significantly improves the utilization efficiency and treatment targeting of drugs, but also effectively avoids healing stagnation or scar hyperplasia caused by improper drug release. It shows significant inventive advantages in achieving efficient and high-quality healing. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of a biomimetic multilayer dressing for chronic wounds of diabetic patients according to the present invention.

[0024] Figure 2 This is a morphological characterization of the dressing of the present invention.

[0025] Figure 3 Characterization of the drug release behavior of the dressing of the present invention: Figure 3 (a) shows the drug release behavior of the dressing prepared in Example 1; Figure 3 (b) shows the drug release behavior of the dressing prepared in Example 1;Figure 3 (c) shows the drug release behavior of the dressing prepared in proportion 2.

[0026] Figure 4 This is a characterization of the antibacterial properties of the dressing of the present invention.

[0027] Figure 5 This is a characterization of cell proliferation in the dressing of the present invention.

[0028] Figure 6 This is a wound healing characteristic of the dressing of the present invention. Detailed Implementation

[0029] The technical solutions described in this invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of this invention. Obviously, the embodiments described in this specification are only some feasible technical solutions of this invention. Other implementation methods obtained by those skilled in the art based on the embodiments of this invention without any creative effort should be considered to fall within the scope of protection of this invention.

[0030] Example 1: Preparation of biomimetic multilayer dressing

[0031] (1) Preparation of functional spinning solutions: 8% polyvinyl alcohol aqueous solution was used as spinning solution A; Triton X-100 (0.5% by volume), 50 nM levofloxacin and 100 μg / mL protease inhibitor GM6001 were dissolved in 10 mM pH 7.4 PBS buffer as spinning solution B; 10% ethyl acetate solution of the first PLGA was used as spinning solution C, in which the ratio of lactic acid to glycolic acid was 50:50, and PBS solution containing 50 ng / mL FGF and 50 ng / mL VEGF was used as spinning solution D; 10% dichloromethane solution of the second PLGA was used as spinning solution E, in which the ratio of lactic acid to glycolic acid was 85:15, and PBS solution containing 30 ng / mL EGF and 10 μM mitomycin C was used as spinning solution F.

[0032] (2) Preparation of biomimetic multilayer dressing: A biodegradable polylactic acid film with a thickness of 0.2 mm was fixed on the receiving device of an electrospinning device. Coaxial electrospinning technology was used for sequential spinning. Spinning solution A was used as the shell layer and spinning solution B was used as the core layer for the first stage of spinning "fast response fiber"; spinning solution C was used as the shell layer and spinning solution D was used as the core layer for the second stage of spinning "medium response fiber"; spinning solution E was used as the shell layer and spinning solution F was used as the core layer for the third stage of spinning "long-term response fiber"; each stage was spun for 30 minutes. The spinning parameters were set as follows: voltage 15 kV, receiving distance 11 cm, shell layer spinning speed 0.003 mm / s, core layer spinning speed 0.001 mm / s. After spinning, a composite fiber membrane was obtained. The composite fiber membrane was then crosslinked with glutaraldehyde vapor for 6 h and then air-dried to obtain the biomimetic multilayer dressing.

[0033] This embodiment prepares a biomimetic multilayer dressing for chronic diabetic wounds, the structure of which is as follows: Figure 1 As shown, fast-response fibers, medium-response fibers, and long-response fibers are stacked sequentially from top to bottom, with the fast-response fibers closely attached to the wound.

[0034] Comparative Example 1: Preparation of Mixed Drug-Loaded Dressing

[0035] The difference between this comparative example and Example 1 is that the three drugs (levofloxacin, FGF / VEGF, and EGF) were uniformly mixed and then encapsulated in a single PLGA (lactic acid and glycolic acid in a 50:50 ratio) fiber layer to form a dressing. The concentration parameters and preparation method are similar to those in Example 1.

[0036] Comparative Example 2: Preparation of Single Carrier Material Type Dressing

[0037] The difference between this comparative example and Example 1 is that the carrier material in all three layers of fiber is PLGA (lactic acid and glycolic acid in a ratio of 50:50).

[0038] Experimental Example 1: Morphological characterization of the dressing prepared in Example 1

[0039] The morphology of the dressing prepared in Example 1 was characterized using field emission scanning electron microscopy (SEM). After the samples were fixed with conductive adhesive, cross-sections were observed. The results are as follows: Figure 2 As shown, the fibers in each layer of the dressing are significantly different.

[0040] Experimental Example 2: Characterization of drug release behavior of the dressing prepared in Example 1

[0041] To evaluate the drug release behavior of the dressings prepared in Example 1, Comparative Example 1, and Comparative Example 2, each dressing was placed in 50 mL of pH 6.5 PBS buffer medium for drug release. At 1, 2, 4, 8, and 12 days, 3 mL of test solution was taken and replaced with an equal volume of fresh replenishment solution. The absorbance of the test solution at 515 nm was measured using a UV-spectrophotometer. The concentration of levofloxacin was detected by HPLC, and the concentrations of each growth factor were detected by ELISA. The cumulative release rate of each drug was calculated.

[0042] The results are as follows Figure 3 As shown in (a)-(c), the antibiotic levofloxacin in the dressing prepared in Example 1 was rapidly released within the initial 24 hours, with a cumulative release rate as high as 85.2%. Subsequently, FGF and VEGF entered a sustained release plateau, with cumulative releases reaching 71.3% and 53.8% respectively by day 7. EGF release was the slowest, with a cumulative release of only 48.7% by day 14, demonstrating its potential to support long-term tissue remodeling. This is because the different proportions of hydrophilic components in the shell layers of the three-layer fiber structure of the dressing prepared in Example 1 resulted in differences in the degradation capacity of each fiber layer. In contrast, Comparative Example 1 experienced a severe drug burst release within 24 hours, with the release of all three growth factors exceeding 70%, leading to a severe lack of kinetics in the later stages. Comparative Example 2, due to the convergence of shell degradation behavior, showed highly overlapping drug release curves and poor temporal sequence. In summary, the dressing prepared in Example 1, through the combination of differentiated materials, achieved control over the release kinetics of multiple therapeutic agents.

[0043] Experimental Example 3: Characterization of the antibacterial properties of the dressing prepared in Example 1

[0044] The antibacterial activity of the dressings prepared in Example 1, Comparative Example 1, and Comparative Example 2 was tested using the plate assay method. Suspensions of Staphylococcus aureus and Escherichia coli (10 μL) were collected. 6 The bacteria (CFU / mL) and dressing were co-cultured separately in pH 6.5 PBS buffer and shaken at 37°C for 24 hours. Gradual dilution was used to spread the bacteria onto plates, and colony growth was photographed and counted. Untreated bacteria served as a control group.

[0045] The results are as follows Figure 4As shown, the antibacterial rates of Example 1 against Staphylococcus aureus and Escherichia coli were as high as 99.5% and 99.1%, respectively, with no statistically significant difference from Comparative Example 2 (99.2% and 98.6%). However, there is a fundamental difference in their mechanisms of action: the potent antibacterial effect of Comparative Example 2 stems from its uncontrollable initial drug burst release, which is essentially a huge waste of drug and may weaken its subsequent activity due to premature release of growth factors; while the rapid antibacterial effect of Example 1 stems from the on-demand release of the antibiotic levofloxacin. Although Comparative Example 1 was loaded with the same amount of antibiotic, its cumulative antibiotic release rate within 24 hours was extremely low due to the difficulty in rapid degradation of the PLGA shell in the initial stage, resulting in a poorer antibacterial rate (25.8% and 32.6%). In summary, the dressing prepared in Example 1 has a good antibacterial effect through time-sequential drug release.

[0046] Experimental Example 4: Characterization of cell proliferation of the dressing prepared in Example 1

[0047] To evaluate the temporal effects of dressing release media on cell proliferation behavior, an in vitro cell proliferation experiment was conducted using the CCK-8 assay. First, conditioned medium was collected from the dressings of Example 1 and each comparative example on day 1 (early stage), day 3 (mid stage), and day 7 (late stage) of in vitro release. Subsequently, human dermal fibroblasts were cultured at a concentration of 5 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells per well in 96-well plates and cultured under standard conditions for 24 hours to allow complete cell adhesion. Afterward, the original culture medium was discarded, and conditioned medium was added to each well at the different time points mentioned above as experimental media. Wells containing fresh complete medium served as blank controls. Cells were cultured for another 24 hours, and then 10 μL of CCK-8 solution was added to each well. The cells were incubated at 37 °C for 2 hours. Finally, the absorbance of each well was measured at 450 nm using a microplate reader. All experiments were performed in triplicate (6 replicates each). The relative cell proliferation rate was calculated by comparing the absorbance values ​​of the experimental and control groups.

[0048] The results are as follows Figure 5As shown, on day 1 of culture, the cell proliferation rate of Example 1 group was similar to that of Comparative Example 2, reaching 112%, and significantly higher than that of Comparative Example 2 (103%). This is because the antibiotics released early by the rapid-response fibers effectively cleared the adverse factors in the simulated inflammatory environment, creating a good foundation for cell proliferation. On days 3 and 7 of culture, the proliferation-promoting effect of Example 1 group was significantly better than all comparative examples, with cell proliferation rates reaching 135% and 152%, respectively, demonstrating the strongest sustained promoting effect. This is mainly because the mid- and long-term response layers of the experimental group continuously and stably released growth factors such as FGF, VEGF, and EGF during this period, providing precise timing signal support for cell proliferation. In contrast, Comparative Example 1 showed a sharp weakening of its proliferation-promoting effect because growth factors were released in large quantities simultaneously with antibiotics in the early stages and were almost completely depleted in the mid- to late-stages; while Comparative Example 2, due to its chaotic drug release curve, could not provide effective concentrations at key time points. In summary, the dressings of Example 1, through a time-controlled release strategy, not only create a healthy cellular microenvironment in the early stages, but also efficiently and persistently drive cell proliferation during the subsequent critical healing stages, demonstrating that the dressings of the present invention have good biocompatibility.

[0049] Example 5: Wound Healing Characterization of the Dressing Prepared in Example 1 To evaluate the antibacterial and healing-promoting effects of the dressing prepared in Example 1 on infected diabetic wounds, a diabetic infected wound model was first established. Type 1 diabetes was induced in male C57BL / 6 mice by intraperitoneal injection of streptozotocin (STZ). After blood glucose stabilized above 16.7 mM, a full-thickness defect with a diameter of 8 mm was prepared on the back, and Staphylococcus aureus (S. aureus) bacterial suspension (10... 7 An infection model was established using CFU / mL. Mice were randomly divided into a control group and Example 1 (n=12 per group). The dressing was fixed to the wound with 3M adhesive and changed on days 0, 1, 3, 6, 9, and 15, and the wound healing was recorded by taking pictures.

[0050] The results are as follows Figure 6As shown, compared with the control group, the wounds in the Example 1 group exhibited superior healing throughout the observation period: In the early stage (days 3-6), the wounds in the Example 1 group were clean and slightly swollen, while the control group commonly showed exudate and significant inflammatory reactions, indicating that the rapid-release layer of the dressing effectively controlled the initial infection; in the middle stage (days 6-9), the wounds in the Example 1 group contracted rapidly and showed epithelial tissue regeneration, demonstrating strong tissue regeneration capacity, while the control group showed delayed healing and relatively open wounds; at the end of the experiment (day 15), the Example 1 group achieved almost complete epithelialization with minimal scarring, while the control group still had unhealed wounds. These significant differences in macroscopic morphology strongly demonstrate that this time-controlled release dressing, through an orderly procedure of "first anti-infection, then promoting regeneration," creates an ideal healing environment for the wound, thereby achieving high-quality tissue repair. In summary, the dressing prepared in Example 1 has excellent wound healing capabilities.

[0051] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the principle of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A biomimetic multilayer dressing for chronic diabetic wounds, characterized in that, The dressing is composed of fast-response fibers, medium-response fibers, and long-response fibers layered from top to bottom: The shell of the fast-response fiber contains polyvinyl alcohol, and the core layer contains antibiotics and protease inhibitors; The shell of the intermediate-response fiber contains a first polylactic acid-glycolic acid copolymer, and the core layer contains fibroblast growth factor and vascular endothelial growth factor. The shell of the long-term responsive fiber is a second polylactic acid-glycolic acid copolymer, and the core layer contains epidermal growth factor and anti-scar drugs; The degradation rate of the first polylactic acid-glycolic acid copolymer is faster than that of the second polylactic acid-glycolic acid copolymer.

2. The biomimetic multilayer dressing according to claim 1, characterized in that, The ratio of lactic acid to glycolic acid in the first polylactic acid-glycolic acid copolymer is 50:

50.

3. The biomimetic multilayer dressing according to claim 1, characterized in that, The ratio of lactic acid to glycolic acid in the second polylactic acid-glycolic acid copolymer is 85:

15.

4. The biomimetic multilayer dressing according to claim 1, characterized in that, The antibiotic in question is levofloxacin.

5. The method for preparing the biomimetic multilayer dressing according to any one of claims 1-4, characterized in that, The preparation method includes the following steps: (1) Preparation of functional spinning solution: 8% polyvinyl alcohol aqueous solution as spinning solution A, 0.5% Triton X-100, 50 nM levofloxacin and 100 μg / mL protease inhibitor GM6001 dissolved in 10 mM pH 7.4 PBS buffer as spinning solution B; 10% ethyl acetate solution of polylactic acid-glycolic acid copolymer as spinning solution C, and PBS solution containing 50 ng / mL FGF and 50 ng / mL VEGF as spinning solution D; A dichloromethane solution containing 10% by weight of the second polylactic acid-glycolic acid copolymer was used as spinning solution E, and a PBS solution containing 30 ng / mL EGF and 10 μM mitomycin C was used as spinning solution F. (2) Preparation of biomimetic multilayer dressing: A biodegradable polylactic acid film with a thickness of 0.2 mm was fixed on the receiving device of an electrospinning equipment. Coaxial electrospinning technology was used for sequential spinning. The voltage was 15 kV, the receiving distance was 11 cm, the shell spinning speed was 0.003 mm / s, and the core spinning speed was 0.001 mm / s. Spinning solution A was used as the shell and spinning solution B was used as the core to spin the first stage of fast-response fiber. Spinning solution C was used as the shell and spinning solution D was used as the core to spin the second stage of medium-response fiber. Spinning solution E was used as the shell and spinning solution F was used as the core to spin the third stage of long-response fiber. Each stage was spun for 30 minutes according to the set spinning parameters. After spinning, a composite fiber membrane was obtained. Then, the composite fiber membrane was cross-linked with 25% glutaraldehyde vapor for 6 h and then air-dried to obtain the biomimetic multilayer dressing.

6. Use of the dressing according to any one of claims 1-4 in the preparation of a medicament or medical device for treating chronic wounds of diabetes.

7. The use according to claim 6, characterized in that, When used, the dressing's fast-response fibers should adhere tightly to the wound.

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