Fiber membrane loaded with infinite coordination polymer nanoparticles as well as preparation method and application of fiber membrane

Abstract

The invention discloses a fibrous membrane loaded with infinite coordination polymer nanoparticles and a preparation method and application thereof, the fibrous membrane loaded with infinite coordination polymer nanoparticles comprises a nanofiber scaffold fused with polycaprolactone (PCL) and sericin Se, and curcumin-copper ICP loaded on the nanofiber scaffold. Accurate pH response release is shown, in an early infected / inflammatory wound (acidic microenvironment), curcumin Cur and Cu < 2 + > are released through rapid dissociation of curcumin-copper ICP, curcumin Cur clears ROS (reactive oxygen species) and inhibits inflammation, and Cu < 2 + > has a synergistic antimicrobial effect. Along with the transition of the wound to a neutral healing stage, the structure of the curcumin-copper ICP is re-stabilized, and the release rate is obviously slowed down. In the stage, low-dose curcumin Cur maintains the anti-inflammatory / anti-oxidation effect, and slow Cu < 2 + > release promotes angiogenesis. The intelligent controlled release provides a reference for designing an environmentally responsive drug delivery system.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a fiber membrane loaded with infinitely coordinated polymer nanoparticles, its preparation method, and its application. Background Technology

[0002] Diabetes is a prevalent chronic underlying disease. With changes in modern lifestyles, the number of people with diabetes is increasing, and the age range is broad. As the disease progresses, the body's physiological functions gradually decline, and the tissue repair process is hindered to varying degrees. This can lead to slow-healing wounds caused by trauma, surgery, or infection, severely impacting the patient's quality of life and even endangering their life. The long-term high blood sugar environment in diabetic patients affects the vitality of various cells and also has a certain impact on microvascular and immune responses. If a wound occurs, it is prone to chronic inflammation due to the large presence of inflammatory immune cells, inflammatory cytokines, reactive oxygen species (ROS), and proteases, as well as long-term and repeated bacterial infections, making it difficult to heal.

[0003] Inflammation is essential for tissue regeneration. When trauma occurs, danger signals at the wound site activate a series of intracellular signaling pathways, releasing chemokines and cytokines. This promotes the infiltration of inflammatory cells from the blood and their aggregation at the wound site, a crucial step in the initial inflammatory phase of wound healing. Under the influence of growth factors and cytokine-regulated signaling systems released by inflammatory cells, macrophages and other immune cells are stimulated to migrate towards the wound. If the cellular response is well-controlled, structural morphology equivalence to normal tissue can be re-established, achieving tissue regeneration. Conversely, if the cellular response is dysregulated, the healing process often deteriorates due to uncontrolled inflammation, leading to excessive fibrosis and an inability to restore normal function. In this competition between fibrosis and regeneration, macrophages exhibit high plasticity, differentiating into the classic pro-inflammatory M1 phenotype or the alternative anti-inflammatory M2 phenotype. Specifically, M1 macrophages accelerate inflammation by strongly releasing interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) within the host defense system. M2 macrophages antagonize M1 macrophage responses and regulate their anti-inflammatory activity by producing interleukin-4 (IL-4), interleukin-13 (IL-13), transforming growth factor-β1 (TGF-β1), and interleukin-10 (IL-10), thereby playing a role in wound healing and tissue repair. For some chronic wounds that remain in an inflammatory phase for an extended period, the shift from inflammation to proliferation is crucial for wound healing. Due to the hyperglycemic environment and compromised immune function in diabetic patients, dysfunctional immune cells disrupt the healing process, which is a major cause of delayed wound healing in diabetic patients. Therefore, one of the key factors affecting wound healing in diabetic patients is the shift in macrophage polarity within the wound.

[0004] During tissue repair, inflammatory immune cells also produce reactive oxygen species (ROS). In normal healing, appropriate concentrations of ROS can resist bacteria and other microorganisms. Conversely, if local hypoxia and inflammatory environment disturbances occur due to microvascular damage and immune dysfunction, excessive ROS will be produced, damaging ECM proteins and harming normal cells. This series of reactions further increases proteases and inflammatory factors, hindering tissue from entering the proliferative phase and prolonging the inflammatory cycle.

[0005] In addition, another major challenge in treating diabetic wounds is the long-term, recurrent bacterial infection. This is not only because the high blood sugar environment is conducive to bacterial growth, but also because impaired immune function prevents the body from relying on its own immunity to fight bacteria. Furthermore, the accumulation of immune cells can cause excessive inflammatory responses, damaging the body's normal tissue cells.

[0006] Recent studies have shown that incorporating metal ions into biomaterials to stimulate cellular responses and promote tissue regeneration is a highly promising strategy. Among bioactive ions, copper (Cu) is a prominent example. 2+ Cu ions can promote angiogenesis by stabilizing the expression of hypoxia-inducible factor (HIF-1α) and promoting the secretion of vascular endothelial growth factor (VEGF). The main mechanism is the artificial mimicking of hypoxia, which plays a crucial role in cell recruitment, differentiation, and angiogenesis. Meanwhile, Cu... 2+ It is also one of the antibacterial agents targeting Escherichia coli, methicillin-resistant Staphylococcus aureus, and Clostridium difficile. Therefore, Cu... 2+ Incorporating curcumin into wound-active dressings is of great significance for the healing of diabetic infected wounds. Curcumin, as a polyphenol, not only has synergistic antibacterial properties but also scavenges reactive oxygen species (ROS) and improves the inflammatory microenvironment through macrophage polarization. A series of studies have shown that curcumin also has good effects in treating wound healing, endothelial injury, and mucosal injury. It promotes collagen deposition and granulation tissue growth by enhancing fibroblast vitality and migration ability, and accelerates reepithelialization of the wound in the final stage of wound healing. Therefore, curcumin is considered a very promising wound-healing drug. However, due to its low hydrophilicity, poor physicochemical stability, and low bioavailability under physiological conditions, improving the loading rate and bioavailability of curcumin is an important prerequisite for its use in treating diabetic wounds. Summary of the Invention

[0007] The purpose of this invention is to provide a fiber membrane loaded with infinitely coordinated polymer nanoparticles, its preparation method, and its applications. A fiber membrane loaded with infinitely coordinated polymer nanoparticles exhibits precise pH-responsive release: in early-stage infection / inflammatory wounds (acidic microenvironment), the rapid dissociation of curcumin-copper ICP releases curcumin and Cu. 2+ Curcumin (Cur) scavenges ROS / inhibits inflammation, while Cu... 2+ Synergistic antimicrobial activity. As the wound transitions to the neutral healing phase, the structure of the curcumin-copper ICP re-stabilizes, significantly slowing the release rate. During this phase, low doses of curcumin (Cur) maintain anti-inflammatory / antioxidant effects, while the slow release of Cu... 2+ Releases substances that promote angiogenesis.

[0008] The objective of this invention is achieved as follows:

[0009] A fibrous membrane loaded with infinitely coordinated polymer nanoparticles includes a nanofiber scaffold fused with polycaprolactone (PCL) and sericin (Se) and curcumin-copper ICP loaded on the nanofiber scaffold.

[0010] A method for preparing a fiber membrane loaded with infinitely coordinated polymer nanoparticles includes the following steps:

[0011] S1. Preparation of freeze-dried sericin for later use;

[0012] S2, Preparation of curcumin-copper ICP: Curcumin-copper ICP was prepared by pH-controlled coordination-induced self-assembly for future use.

[0013] S3. Preparation of curcumin-copper / Se / PCL fiber by directional spinning: The freeze-dried sericin obtained in S1, the curcumin-copper ICP obtained in S2 and polycaprolactone are mixed to obtain a spinning solution. The fiber membrane curcumin-copper / Se / PCL is prepared by directional spinning. The fiber membrane curcumin-copper / Se / PCL is a fiber membrane loaded with infinitely coordinated polymer nanoparticles.

[0014] The specific operation of S1 is as follows: Extraction of sericin Se: The sericin is extracted by high temperature and high pressure method. The cleaned silkworm cocoon fragments are soaked in a certain amount of distilled water at a ratio of 10g / 250ml. Then, they are boiled in a pressure cooker (121℃, 0.1MPa) for 30 minutes to remove insoluble fibroin. After vacuum freeze drying, purified sericin can be obtained.

[0015] The specific operation of S2 is as follows:

[0016] S2.1. Weigh out CuCl2·2H2O and dissolve it in anhydrous ethanol to prepare a CuCl2 solution with a concentration of 17 mg / mL;

[0017] S2.2. Weigh out curcumin (Cur) and dissolve it in anhydrous ethanol to prepare a curcumin (Cur) solution with a concentration of 7 mg / mL;

[0018] S2.3. Take 10 mL of CuCl2 solution obtained in S2.1 and 10 mL of curcumin Cur solution obtained in S2.2 into a reaction flask to form an acidic precursor solution;

[0019] S2.4 A 10 mM Tris aqueous solution with pH 10.0 was rapidly added to the precursor solution, and the pH was adjusted to 7.4 at room temperature to obtain a mixture. The mixture was observed to turn dark brown. The mixture was placed on a magnetic stirrer in the dark and stirred for 6 hours to obtain the reaction solution.

[0020] S2.5 The reaction solution was transferred to an ultrafiltration tube to purify curcumin-copper ICP, and purified curcumin-copper ICP was obtained.

[0021] S2.6 The purified curcumin-copper ICP was collected by freeze-drying and stored at room temperature away from light.

[0022] In step S2.2, to ensure complete dissolution of curcumin, the curcumin solution is intermittently sonicated in a water bath three times, with each sonication lasting 10 seconds.

[0023] The specific operation of S3 is as follows:

[0024] S3.1. Dissolve the lyophilized sericin obtained in S1 in hexafluoroisopropanol (HFIP) as solvent and stir continuously for 24 hours to obtain a sericin Se solution with a concentration of 5% w / v, named solution a.

[0025] S3.2. Polycaprolactone was dissolved in hexafluoroisopropanol (HFIP) as solvent to obtain a polycaprolactone (PCL) solution with a concentration of 10% w / v, which was named solution b.

[0026] S3.3. Weigh 5 mg of curcumin-copper ICP obtained from S2 and add it to 10 mL of the mixture of the above solutions a and b to form a spinning solution, which is named solution c.

[0027] S3.4. Load the prepared solution c into a syringe, and place it on a micro-injection pump using a spinning needle with an inner diameter of 0.6 mm.

[0028] S3.5. Adjust the distance between the syringe needle and the high-speed roller receiving device for directional spinning to 10cm; the electrospinning conditions are: flow rate 1.2ml / h, voltage 16kV, roller speed 3000 rpm; to obtain a nanofiber film.

[0029] S3.6. Place the nanofiber film obtained in S3.5 in a fume hood and dry overnight to remove residual organic reagents, and obtain a curcumin-copper ICP oriented spun fiber membrane, which is a fiber membrane loaded with infinitely coordinated polymer nanoparticles.

[0030] In step S3.3, the volume ratio of solution a to solution b is 1:9.

[0031] Application of a fiber membrane loaded with infinitely coordinated polymer nanoparticles in a drug for treating diabetic wounds.

[0032] The beneficial effects of this invention are: the fiber membrane prepared by this invention has good biocompatibility and hydrophilicity. Due to the high specific surface area of ​​the fiber membrane, it can release drugs stably and for a long time, exerting its excellent antioxidant and angiogenic functions. In vitro antibacterial experiments have demonstrated that the CCSP fiber membrane possesses acid-triggered self-defense capabilities; acid stimulation accelerates the reversible decomposition of ICP released by the CCSP membrane, releasing a large amount of Cu. 2+ It exerts antibacterial function through the interaction with curcumin (Cur). Animal experiments have demonstrated that the CCSP membrane regulates macrophage polarization (upregulation of the M2 / M1 ratio) to improve the inflammatory environment, further promoting early angiogenesis (upregulation of CD31 and α-SMA) and collagen deposition, thus promoting the healing of diabetic infected wounds. The fibrous membrane of this invention can exert antibacterial, anti-inflammatory, and angiogenic functions, providing a promising strategy for wound repair treatment in diabetic patients. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the structure of the present invention, namely a schematic diagram of the process of synthesizing curcumin-copper ICP and a schematic diagram of the process of synthesizing a fiber membrane loaded with infinitely coordinated polymer nanoparticles.

[0034] Figure 2 The following are the detection results of this invention: A is the TEM result of the initial reaction solution of curcumin-copper, and no formed substances were observed; B and C are the TEM images of ICP nanoparticles formed after the curcumin-copper system was adjusted to neutral pH; D is the particle size analysis diagram; and E is the ESD mapping analysis diagram.

[0035] Figure 3 The image shows the detection results of this invention; A is the ultraviolet-visible spectrophotometer (UV-vis); B is the Fourier transform infrared spectrophotometer (FTIR).

[0036] Figure 4The above are XPS spectra of the present invention; A is the full XPS spectrum of Cur-Cu(II) ICPs; B is the high-resolution O1s XPS spectrum of Cur-Cu(II) ICPs; C is the high-resolution Cu2p XPS spectrum of CuCl2; D is the high-resolution Cu2p XPS spectrum of Cur-Cu(II) ICPs.

[0037] Figure 5 The image shows a microscopic view of the fiber membrane of the present invention; AC are high-magnification SEM images of the fiber membrane PCL, fiber membrane SP, and fiber membrane CCSP; D is a TEM image of a single fiber of the CCSP fiber scaffold under high electron beam intensity; the red circle indicates the oval ICP particles; and E is an ESD mapping analysis diagram of CCSP.

[0038] Figure 6 This is a schematic diagram showing the hydrophilicity of three groups of fiber membranes over time; A is a water contact angle image; B is a swelling performance diagram.

[0039] Figure 7 Curcumin Cur (standard curve);

[0040] Figure 8 Figure 1 shows the release efficiency of curcumin-copper ICP; Figure 2 shows the UV-vis results of curcumin Cur (Cur) released from curcumin-copper ICP as pH decreases; Figure 3 shows the in vitro release curves of curcumin Cur (Cur) from ICP at pH 7.4, pH 6.4, and pH 5.0.

[0041] Figure 9 This is the in vitro release curve of curcumin (Cur) from the CCSP membrane of this invention;

[0042] Figure 10 A is an image showing the antibacterial effect of the fiber membrane of the present invention; B is a colony photograph of Staphylococcus aureus and Escherichia coli after plating; C is a quantitative bacterial count of the Control group at pH=7.4 and pH=5.0; D is a quantitative count of Staphylococcus aureus;

[0043] Figure 11 The images show the live and dead fluorescence staining of Staphylococcus aureus and Escherichia coli.

[0044] Figure 12 The diagram shows the ability of the fibrous membrane of this invention to facilitate endothelial cell migration; A is a light micrograph of HUVECs after scratching and fibrous membrane treatment, and cultured for 24h and 36h; B is a microscopic image and related numerical analysis diagram of HUVECs seeded on Matrigel after each group of treatments and cultured for 24h; C is a quantitative diagram of related cell migration rate; D is a quantitative diagram of tubule branch count; E is a quantitative diagram of tubule length.

[0045] Figure 13Figure A shows the antioxidant properties of the fiber membrane of this invention. A is a fluorescence image of Raw264.7 cells after H2O2 treatment and after 1 hour of treatment with different fiber membranes. B is a DCFH-DA fluorescence staining image of Raw264.7 cells after H2O2 treatment and co-culture with different fiber membrane extracts for 1 hour. Cells treated with H2O2 alone are used as positive controls, and untreated cells are used as negative controls.

[0046] Figure 14 Figure A shows the effect of fibrous membrane on wound healing in diabetic rats; Figure B shows the establishment of a full-thickness skin resection infected wound model in diabetic rats; Figure C shows the wound photographs of rats in the Control, 3M, SP, and CCSP groups at different time points; Figure D shows the wound remaining area curves of the four groups of rats; Figure D shows the wound healing rate of the four groups of rats.

[0047] Figure 15 H&E staining images of wound tissue from each group;

[0048] Figure 16 Macrophage markers on day 9: iNOS (green, M1 type); CD206 (green, M2 type); CD68 (red, pan-macrophage marker); A is a representative image of CD68 and CD206 staining; B is a representative image of CD68 and iNOS staining; C is the ratio of CD68-positive macrophages in different groups; D is the ratio of iNOS (M1 type) positive macrophages to CD68 macrophages; E is the ratio of CD206 (M2 type) positive macrophages to CD68 macrophages; F is the ratio of CD206 (M2 type) to iNOS (M1 type) positive macrophages in different groups.

[0049] Figure 17 Immunofluorescence analysis of vascular cytokines CD31 and α-SMA in wound tissues of the Control, 3M, SP, and CCSP groups on postoperative days 6, 13, and 18. Detailed Implementation

[0050] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0051] A method for preparing a fiber membrane loaded with infinitely coordinated polymer nanoparticles includes the following steps:

[0052] S1. Preparation of freeze-dried sericin for later use;

[0053] The specific operation of S1 is as follows: Extraction of sericin Se: The sericin is extracted by high temperature and high pressure method. The cleaned silkworm cocoon fragments are soaked in a certain amount of distilled water at a ratio of 10g / 250ml. Then, they are boiled in a pressure cooker (121℃, 0.1MPa) for 30 minutes to remove insoluble fibroin. After vacuum freeze drying, purified sericin can be obtained.

[0054] S2, Preparation of curcumin-copper ICP: Curcumin-copper ICP was prepared by pH-controlled coordination-induced self-assembly for future use.

[0055] The specific operation of S2 is as follows:

[0056] S2.1. Weigh out CuCl2·2H2O and dissolve it in anhydrous ethanol to prepare a CuCl2 solution with a concentration of 17 mg / mL;

[0057] S2.2. Weigh out curcumin (Cur) and dissolve it in anhydrous ethanol to prepare a curcumin (Cur) solution with a concentration of 7 mg / mL. To ensure complete dissolution of curcumin (Cur), the curcumin (Cur) solution is intermittently sonicated in a water bath 3 times, each time for 10 seconds.

[0058] S2.3. Take 10 mL of CuCl2 solution obtained in S2.1 and 10 mL of curcumin Cur obtained in S2.2 into a reaction flask to form an acidic precursor solution;

[0059] S2.4 A 10 mM Tris aqueous solution with pH 10.0 was rapidly added to the precursor solution, and the pH was adjusted to 7.4 at room temperature to obtain a mixture. The mixture was observed to turn dark brown. The mixture was placed on a magnetic stirrer in the dark and stirred for 6 hours to obtain the reaction solution.

[0060] S2.5 The reaction solution was transferred to an ultrafiltration tube to purify curcumin-copper ICP, and purified curcumin-copper ICP was obtained.

[0061] S2.6 The purified curcumin-copper ICP was collected by freeze-drying and stored at room temperature away from light.

[0062] In S2.2,

[0063] S3. Preparation of curcumin-copper / Se / PCL fiber membrane by directional spinning of curcumin-copper ICP: The freeze-dried sericin obtained in S1, the curcumin-copper ICP obtained in S2 and polycaprolactone were mixed to obtain a spinning solution. The fiber membrane curcumin-copper / Se / PCL was prepared by directional spinning. The fiber membrane curcumin-copper / Se / PCL is a fiber membrane loaded with infinitely coordinated polymer nanoparticles.

[0064] The specific operation of S3 is as follows:

[0065] S3.1. Dissolve the lyophilized sericin obtained in S1 in hexafluoroisopropanol (HFIP) as solvent and stir continuously for 24 hours to obtain a sericin Se solution with a concentration of 5% w / v, named solution a.

[0066] S3.2. Polycaprolactone was dissolved in hexafluoroisopropanol (HFIP) as solvent to obtain a polycaprolactone (PCL) solution with a concentration of 10% w / v, which was named solution b.

[0067] S3.3. Weigh 5 mg of curcumin-copper ICP obtained from S2 and add it to 10 mL of the mixture of the above solutions a and b to form a spinning solution, named solution c; the volume ratio of solution a to solution b is 1:9.

[0068] S3.4. Load the prepared solution c into a syringe, and place it on a micro-injection pump using a spinning needle with an inner diameter of 0.6 mm.

[0069] S3.5. Adjust the distance between the syringe needle and the high-speed roller receiving device for directional spinning to 10cm; the electrospinning conditions are: flow rate 1.2ml / h, voltage 16kV, roller speed 3000 rpm; obtain nanofiber film.

[0070] S3.6. Place the nanofiber film obtained in S3.5 in a fume hood and dry overnight to remove residual organic reagents, and obtain a curcumin-copper ICP oriented spun fiber membrane, which is a fiber membrane loaded with infinitely coordinated polymer nanoparticles.

[0071] Curcumin (Cur) was purchased from Shanghai Aladdin Biochemical; copper chloride dihydrate (CuCl2·2H2O) was purchased from Shanghai Maclean Biochemical; polycaprolactone (PCL) was purchased from Shanghai Maclean Biochemical; Tris10.0 was purchased from Shanghai Maclean Biochemical; anhydrous ethanol was purchased from Shanghai Maclean Biochemical; and hexafluoroisopropanol (HFIP) was purchased from Shanghai Maclean Biochemical.

[0072] In vitro drug release

[0073] (1) The standard curve is plotted as follows:

[0074] Accurately weigh a certain amount of curcumin (Cur) and dissolve it in anhydrous ethanol to prepare a series of curcumin solutions with varying concentrations (5 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL). Measure the absorbance of the solutions at 530 nm using a fluorescence spectrophotometer. Plot a standard curve of curcumin in PBS using Origin software and derive the corresponding linear regression equation.

[0075] (2) The release characteristics of curcumin Cur in curcumin-copper ICP are as follows:

[0076] To analyze the reversible dissociation of curcumin-copper ICP, its spectral characteristics in PBS at different pH values ​​were measured using a UV-Vis spectrophotometer and a fluorescence spectrophotometer. To plot the in vitro release curve of curcumin (Cur) from curcumin-copper ICP particles, experiments were conducted in PBS environments at pH 7.4, pH 6.4, and pH 5.0. The prepared dried curcumin-copper ICP particles were resuspended in PBS. 5 mg of powder was added to 5 mL of PBS. The mixture was placed in a dialysis bag with a molecular weight cutoff of 10000 Da, clamped at both ends, and then placed in a dialysis fluid containing 3 L of deionized water. The bag was sealed and placed in a 37°C incubator. 1 mL samples were taken at 2 h, 4 h, 8 h, 16 h, 24 h, 32 h, 48 h, 72 h, and 96 h, with 1 mL of PBS added simultaneously. The samples were centrifuged at 16000 rpm, and the supernatant was collected. The absorbance at 530 nm was detected by fluorescence, and the drug release was calculated using the standard curve equation. The experiment was repeated three times, and the average value was used to plot the drug release curve.

[0077] (3) The characteristics of drug release in the fiber membrane are as follows:

[0078] The release characteristics of curcumin (Cur) in CCSP fiber membranes were analyzed using the methods described above.

[0079] Six-week-old male SD rats (230g-290g) were used in this experiment. All rats were purchased from the Chengbei Animal Center of Southwest Medical University. The animal research was approved by the Ethics Committee of the Southwest Medical University Research Institute. Rats were acclimatized for one week with free access to food and water.

[0080] (1) The establishment of a skin wound healing model in diabetic rats and wound treatment are as follows:

[0081] In animal experiments, a type 2 diabetic rat model was induced using streptozotocin (STZ). The method was as follows: Rats were fed a diabetic diet for one week; after fasting for 12 hours (with unlimited water), they were injected intraperitoneally with a single dose of freshly prepared streptozotocin (STZ, dissolved in a 0.1 mmol / L, pH 4.5 citrate-sodium citrate buffer) 60 mg / kg. Following the injection, they were given a high-sugar diet and water, and this injection was repeated for three consecutive days. 72 hours after administration, tail vein blood was measured using Roche blood glucose test strips. A blood glucose level ≥16.7 mmol / L was considered the standard for a successfully induced diabetic rat model. Two weeks after successful establishment of the diabetic rat model, 24 rats were selected for a skin wound model. Anesthesia was administered via intraperitoneal injection of 1% sodium pentobarbital (1 mL / kg). After anesthesia took effect, the rats were fixed in a prone position. After disinfection and skin preparation of the back, a 10 mm diameter full-thickness skin wound was created in the middle of the back, reaching the fascia. Wound hemostasis was achieved by pressure. Staphylococcus aureus (50 μL, 1.0 × 10⁻⁶) 6 Infected wounds were treated with CFU / mL dressings for 2 hours. Subsequently, dressings from each group (3M, SP, CCSP) were placed on the infected wound site, with saline serving as the control group. Gauze was used as a secondary dressing to prevent it from falling off, and the dressing was changed every 2 days. Wound photographs were taken on days 0, 3, 6, 9, 13, and 18 post-injury, and the wound area was measured using ImageJ software.

[0082] (2) The pathological study of newly formed wound tissue is as follows:

[0083] Newly formed skin tissue from the wound and surrounding tissue was harvested on postoperative days 6, 9, 13, and 18. The tissue was fixed in 4% paraformaldehyde for 24 hours, then embedded in paraffin and prepared into 5 μm sections using a microtome. The wound tissue sections were subjected to H&E staining, Masson staining, immunofluorescence staining (CD31, α-SMA, CD68, CD206, iNOS, IL-10, TNF-α), and immunohistochemistry (IL-6, TGF-1β) to characterize the physiological and pathological status of the newly formed tissue and the quality of healing. The stained sections were then observed and photographed using a scanner and an upright fluorescence microscope, and statistical analysis was performed using ImageJ.

[0084] Data Analysis: All experimental data were statistically processed using GraphPad Prism 8 software. All quantified data are expressed as mean ± standard deviation (Mean ± SD). Statistical significance was determined using a two-tailed t-test, and comparisons among multiple groups were corrected using analysis of variance. A p-value < 0.05 was considered statistically significant and marked with *; p < 0.01 was marked with **; p < 0.001 was marked with ***; and p < 0.0001 was marked with ****.

[0085] The conclusions and analysis are as follows:

[0086] The morphology and synthetic characterization of curcumin-copper ICP are as follows:

[0087] Currently, the general principle for the formation of infinite coordination polymerized nanoparticles (ICPs) is that metal ions and ligands first form oligomers, which continue to grow, and then anneal to form nanoparticles. Current preparation methods include room temperature self-polymerization, solvent-induced precipitation, solvothermal methods, and microemulsion methods. The final formation of ICPs depends not only on the types of ligands and metal ions, but also on the degree of polymerization, intermolecular forces, pH, and temperature.

[0088] This invention refers to the pH-controlled coordination self-polymerization method reported by Shen et al., inducing coordination between Cu(II) and curcumin (Cur). In this special nano-sizing process, due to the hydrolysis of CuCl2, the entire initial reaction system is acidic, and no formed material was observed under transmission electron microscopy (TEM). Figure 2 A) With the continuous addition of alkaline Tris, the pH of the reaction system quickly changed from acidic to neutral, triggering the nucleation and three-dimensional growth of curcumin-copper ICPs. TEM detection further revealed that curcumin-copper ICPs have a uniformly dispersed spherical morphology (Fig. 2B-C), and their particle size was measured to be approximately 5-10 nm (Fig. 2D). Elemental mapping analysis of curcumin-copper ICPs confirmed the uniform distribution of C, N, O, and Cu elements, providing direct evidence for the successful formation of curcumin-copper ICPs (Fig. 2E).

[0089] The formation of nanoparticles was further verified using ultraviolet-visible spectrophotometry (UV-vis). For example... Figure 3 As shown in Figure A, the characteristic peak of curcumin (Cur) is at 430 nm. In contrast, the peaks in the initial curcumin-copper reaction solution shift to higher wavelengths, indicating extended conjugation and coordination between the ligand and the metal center. The shoulder peak splitting at 450 nm can be attributed to ligand-metal charge transfer (LMCT). During pH-controlled reaction, the characteristic absorption peak of curcumin-copper ICP nanoparticles at approximately 388 nm appears with increasing pH, while the characteristic absorption peak of curcumin (Cur) at around 430 nm gradually disappears. For transition metal coordination polymers, the appearance and disappearance of characteristic absorption peaks in the UV-Vis spectrum signify the formation and destruction of coordination bonds in the coordination polymer. This proves the formation of coordination polymer nanoparticles. The coordination bonds of curcumin-copper ICP are further clarified by Fourier transform infrared spectroscopy (FTIR). Figure 3 As shown in B, the original Cur was at 1604 cm. -1A conjugated carbonyl absorption band is observed at this point, which shifts slightly to 1597 cm⁻¹ in curcumin-copper ICP. -1 This blue shift indicates that Cur and Cu 2+ Coordination exists between the carbonyl oxygen atoms of the ion because the electron donation of the carbonyl group to the metal center weakens the C=O bond. Furthermore, the original Cur at 3378 cm⁻¹... -1 The hydroxyl absorption peak at 0.05 cm⁻¹ shifts to 3246 cm⁻¹ in the complex. -1 This indicates that the phenolic hydroxyl groups also participate in the interaction with Cu. 2+ Coordination. Furthermore, in curcumin-copper ICP, at 1271 cm⁻¹ -1 and 1157 cm -1 Two new characteristic absorption bands appeared at [location], which were attributed to the C=O-Cu stretching vibration, providing direct evidence for metal-ligand bonding. Notably, the original Cur [band] at 1026 cm⁻¹... -1 and 962 cm -1 The significant trans C=C absorption peak at 420 cm⁻¹ is observed in curcumin-copper ICP. -1 The stronger absorption bands at this point mask the Cu-O vibrational mode. Overall, these FTIR results indicate that multiple functional groups of Cu (carbonyl, hydroxyl, and olefinic) are involved in the interaction with Cu. 2+ Coordination with the curcumin promotes the formation of a stable curcumin-copper ICP.

[0090] The elemental composition and valence changes of curcumin-copper ICP were verified by X-ray photoelectron spectroscopy (XPS). Figure 4 A shows the full spectrum of curcumin-copper ICP, with four distinct peaks at 288.78 eV, 399.53 eV, 531.98 eV, and 932.38 eV, corresponding to the C1s, N1s, O1s, and Cu2p orbitals, respectively. The O1s high-resolution XPS spectrum of curcumin-copper ICP (…) Figure 4 B) indicates the possible presence of C−O bonds (531.35 eV) and Cu−O bonds (533.00 eV). Furthermore, the high-resolution XPS spectrum of Cu2p... Figure 4 In D), a prominent peak for Cu2p3 / 2 was observed at 932.83 eV, and a corresponding peak for Cu2p1 / 2 was observed at 952.60 eV. Furthermore, these main peaks were accompanied by two satellite peaks detected at 934.49 eV and 954.36 eV. The presence of these characteristic peaks strongly suggests that the dominant valence state of Cu on the curcumin-copper surface is +2.

[0091] The morphology and characterization of the fibrous membrane are as follows:

[0092] Nanofiber scaffolds combining the advantages of polycaprolactone (PCL) and sericin (Se) were prepared using electrospinning technology. These scaffolds, serving as carriers for intracellular polymeric substances (ICPs), possess both the biological properties of natural materials and the physicochemical properties of synthetic polymers, thus improving the utilization rate of the dressing scaffold. Following the same method, pure PCL fiber membranes and sericin-modified fiber membranes were prepared, named PCL and Se / PCL (SP), respectively, as shown in Figures (5A-C). SEM results revealed a uniform and highly oriented topological structure of the three fiber membranes (PCL, SP, and CCSP). Furthermore, TEM images of CCSP fibers under high magnification (…) were also obtained. Figure 5 D) indicates that ICP is embedded in the nanofibers. EDS mapping ( Figure 5 E) The results showed that the curcumin-copper / Se / PCL fiber contained C, N and O elements, which came from the fiber's base material PCL, curcumin Cur and sericin Se; in addition, the uniformly distributed Cu element also indicated that the curcumin-copper ICP was successfully loaded into the fiber scaffold.

[0093] The hydrophilicity of the fibers was analyzed by comparing the water contact angles of the three groups. The smaller the contact angle, the better the hydrophilicity of the fibers. Experimental results ( Figure 6 A) indicates that when a water droplet contacts the blank PCL scaffold in the first second, the water contact angle is 106.0º±6.5º, indicating hydrophobicity. After the addition of sericin, the water contact angles of SP and CCSP decreased to 62.0º±3.2º and 72.0º±2.6º, respectively, indicating increased hydrophilicity. When the water droplet was completely absorbed, SP and CCSP both took 3 seconds, while PCL still showed no absorption after 10 seconds. The three membranes were placed in 37℃ SBF solution, and their hygroscopic properties were measured over 72 hours. The experimental results ( Figure 6The results showed that the moisture absorption rate of each group of membranes was relatively fast within the first 0.5-3 hours, and then gradually slowed down. S and CCSP reached near saturation in moisture absorption after about 20 hours, with water absorption rates of 640.0±50.0% and 501.3%±47.1%, respectively, demonstrating good moisture absorption capabilities. PCL also possessed some moisture absorption capacity, with a water absorption rate of 149.7±19.5% after 48 hours, but its moisture absorption capacity was far lower than that of SP and CCSP. These data indicate that doping with Se can improve the surface properties of the original PCL fibers because Se has abundant hydrophilic hydroxyl groups and high polarity, improving the hydrophilicity of the composite fiber scaffold. In addition, the three-dimensional network structure of the fibers provides a large specific surface area, allowing for significant space for water molecule adsorption. The uniform distribution of sericin during spinning enhances the hydrophilicity of the composite membrane while significantly improving its swelling and water retention rate. This characteristic adapts to the microenvironment requirements of moist wound healing, allowing it to absorb large amounts of wound exudate while maintaining a certain level of wound moisture. The addition of sericin effectively enhances the application value of dressing scaffolds. Therefore, sericin-modified scaffold groups (SP, CCSP) were selected for subsequent in vitro and in vivo validation of biological functions.

[0094] The characteristics of in vitro drug release are as follows:

[0095] (1) The results of the standard curve determination of curcumin Cur are as follows: Figure 7 Within the concentration range of 0.5-3.0 μg / mL, the absorbance y of curcumin Cur showed a good linear relationship with the mass concentration x, and the linear regression equation was obtained as y=0.2884*x-0.0462, R2=0.9992.

[0096] (2) The release characteristics of curcumin in curcumin-copper ICP nanoparticles are as follows: Figure 8 AUV-vis results showed that as pH decreased, the characteristic peak of curcumin-copper ICP disappeared around 388 nm, while a curcumin characteristic peak appeared around 427 nm, indicating that the coordination particles exhibited reversible pH-responsive dissociation. We further determined the sustained-release curves of curcumin (Cur) from the ICP particles at different pH values, as shown below. Figure 8 As shown in Figure B, approximately 43.1% of curcumin-copper ICP was released within the first 6 hours at pH 7.4; while at pH 6.5 and 5.0, 26.98% and 16.03% of curcumin-copper ICP were released, respectively. After 96 hours, the cumulative release of curcumin (Cur) in PBS at pH 7.4, 6.5, and 5.0 reached 34.55%, 60.16%, and 81.02%, respectively. At lower pH values, the release of curcumin (Cur) increased significantly (p < 0.05).

[0097] (3) Further evaluation of the drug delivery characteristics of the fiber dressing by in vitro release of curcumin from curcumin-copper / Se / PCL fiber membranes, by comparing the in vitro sustained-release curves of curcumin Cur in different fiber membranes ( Figure 9 It was found that drug release from the fiber membrane was accelerated at pH 5.0, and the release behavior of curcumin (Cur) from the fiber membrane was also pH-dependent. Notably, due to the release of ICP from electrospinning, approximately 32.3% of curcumin (Cur) was released from the fiber membrane in the first 6 hours, which was less than the release from individual ICP particles. However, because the electrospun fiber membrane has a high specific surface area and a porous three-dimensional structure that is conducive to long-acting drug release, it still showed a release trend after 96 hours.

[0098] The cell compatibility of the fibrous membrane is as follows:

[0099] Good cell compatibility is a fundamental requirement for medical wound dressings. The cell compatibility of the fibrous membrane was verified using the CCK-8 assay and cell viability / death staining. The cells used in this experiment were human umbilical vein endothelial cells (HUVECs) and the mouse fibroblast cell line (L929). With increasing culture time, the absorbance of cells in all three groups significantly increased, indicating that the cells in each group proliferated well, and there was no statistically significant difference in absorbance among the three groups (P > 0.05). With increasing culture time, the number of viable cells in all three groups significantly increased, with only a small number of dead cells observed, indicating good cell viability in all groups.

[0100] The in vitro antibacterial effects of the fiber membrane are as follows:

[0101] To verify the antibacterial properties of CCSP dressings, this invention employed Staphylococcus aureus and Escherichia coli for bacterial smear and live / dead bacterial staining experiments. For example... Figure 10 As shown in Figure A, the viable bacterial count in each group was determined using the Luria-Bertani agar plate method. For the Control group (untreated group), the viable bacterial count was lower under acidic conditions (pH=5.0) than under neutral conditions (pH=7.4) (p<0.05). Figure 10 B), the viable bacterial count of *S. aureus* was 75.45% ± 2.89%, and that of *E. coli* was 77.7% ± 7.51%, indicating that an acidic environment inhibited bacterial growth. At pH 7.4, the number of surviving bacteria in the three culture media showed a decrease in viable bacterial count after treatment with CCSP samples (p < 0.05). Figure 10(C, D) The remaining viable bacteria count of *S. aureus* was 84.27% ± 7.50%, and that of *E. coli* was 83.06% ± 2.09%. However, when the bacteria were cultured with the CCSP dressing in a solution at pH 5.0, the decrease in viable bacterial count was even more pronounced (P < 0.001, remaining viable bacteria count of *S. aureus* was 24.22% ± 3.40%, and that of *E. coli* was 25.48% ± 4.373%). These results indicate that the antibacterial ability of the CCSP fiber membrane is significantly enhanced under acidic conditions.

[0102] Further fluorescent staining of live and dead bacteria was performed to verify the bactericidal effect of the fibrous membrane. Bacteria with intact membrane structures fluoresced green, while bacteria with damaged membrane structures fluoresced red. For example... Figure 11 As shown, under neutral conditions, the bacteria in the Control group showed almost no red fluorescence, indicating good bacterial viability; under the same conditions, the CCSP group showed limited bactericidal effect. In acidic solution, the bacteria in the CON group showed a small amount of red fluorescence, indicating that the activity of both bacteria was somewhat damaged in the acidic environment; however, almost all bacteria showed obvious red staining under acidic conditions and after CCSP treatment, indicating that a large number of bacteria had been destroyed.

[0103] All the above results show that the CCSP group exhibited stronger antibacterial activity under acidic conditions than under neutral conditions. Both bacterial metabolism and the early stages of acute diabetic wounds acidify the local microenvironment. While the physiologically acidic environment of the wound itself provides some resistance to bacteria, its antibacterial ability is low, and it still cannot achieve satisfactory antibacterial effects when facing larger numbers and more virulent bacteria. Therefore, designing responsive intelligent dressings targeting the acidic environment of the wound is a highly promising strategy. In this study, the acid in the environment caused the CCSP dressing to release a large amount of copper ions (Cu) with broad-spectrum bactericidal activity. 2+ This results in the death of a large number of bacteria in contact with the material. This intelligent responsive release ensures that the dressing effectively releases antibacterial copper ions in the early stages of the wound to exert its antibacterial effect.

[0104] The evaluation of the fibrous membrane's ability to facilitate endothelial cell migration and its in vitro tube-forming capacity is as follows:

[0105] Angiogenesis involves numerous cells and cytokines, with endothelial cells playing a crucial role. The proliferation, migration, and differentiation capacity of endothelial cells directly affect the angiogenesis rate. This study investigated the various behaviors of HUVECs on different fibrous membranes during in vitro culture to reveal the pro-angiogenic mechanism of CCSP fibrous membranes. The scratch assay is a commonly used method for studying wound healing and assessing cell migration ability in vitro. Figure 12As shown in Figure A, after 24 hours of culture, cells in all groups migrated towards the central scratch site. CCSP treatment after scratching accelerated cell migration (p < 0.01), with a migration rate of 55.09% ± 3.636%. After 36 hours, exposure to CCSP treatment significantly promoted cell migration, with the highest migration rate of 84.71 ± 4.50% (p < 0.01).

[0106] The tubular formation capacity of endothelial cells is a crucial factor influencing angiogenesis at the wound site. Normally, after skin injury, cells migrate to the wound area, undergo morphological changes, and connect to form tubular structures, thus creating the prototype of new capillaries. In vitro tubular formation experiments of endothelial cells were conducted to illustrate this. Figure 12 As shown in Figure B, after co-culturing HUVECs with the dressing for 7 days, they were seeded onto the surface of Matrigel substrate and cultured for 8 hours. Angiogenesis was observed in all three groups, with the CCSP group showing obvious interconnected tubular structures. The number of tubular branching points and the length of tubules in the CCSP group were significantly higher than those in the blank group and the SP group (P<0.05). These results are mainly attributed to the sustainable Cu 2+ Released from the CCSP membrane, it gives endothelial cells a stronger angiogenic capacity.

[0107] The antioxidant properties of the fiber membrane are as follows:

[0108] Excessive ROS at the wound site can cause oxidative stress, leading to a prolonged inflammatory response that prevents the wound from transitioning to the proliferative phase, resulting in slow healing. H2O2 is an endogenous ROS and one of the most important oxidative factors in acute and chronic trauma, capable of causing oxidative stress and damaging cells. It is also a typical oxidative stress inducer. This study used H2O2 to induce oxidative stress in Raw264.7 macrophages to verify the in vitro antioxidant capacity of CCSP membranes.

[0109] First, the protective effect of CCSP membrane on Raw264.7 cells under oxidative stress was evaluated. The results of live / dead fluorescence staining of Raw264.7 cells were used to assess this effect. Figure 13A) It can be seen that, compared with the blank Control group, the cells in the H2O2-induced group showed severe survival inhibition, the SP group had no antioxidant function, and almost all cells were stained with red fluorescence, while the CCSP group maintained a large number of green-stained viable cells. These results indicate the effectiveness of the CCSP membrane in protecting cells from oxidative stress damage. Further investigation was conducted using a (2',7'-dichlorodihydrofluorescein diacetate, DCFH-DA) fluorescent probe to investigate the intracellular ROS levels in Raw264.7 cells co-cultured with the fiber membranes of each group. DCFH-DA enters the cell via passive transport, and ROS can undergo a redox reaction with the non-fluorescent DCFH-DA to generate dichlorofluorescein (DCF), which has strong green fluorescence. As shown in Figure (13B), bright green fluorescence was produced in the H2O2-induced group and the SP group, while the CCSP group showed a lower fluorescence intensity than the Control group. These results indicate that the fiber membrane has a good ability to scavenge intracellular ROS.

[0110] Effects of fibrous membranes on wound healing in diabetic rats

[0111] The gross observation of the wound and the analysis of the healing rate are as follows:

[0112] To evaluate the in vivo antibacterial capacity and wound-healing effect of the fibrous membrane, a diabetic rat model of S. aureus infection was established. Figure 14 A represents the animal model creation and treatment process. In the diabetic control group, rat wounds were treated only with saline solution, while conventional 3M dressings served as a control to evaluate the healing-promoting function of SP and CCSP fibrous membranes in this experiment. Wound images from different time points for each group are shown below. Figure 14 B) and wound area statistics ( Figure 14 C) It can be seen that during the entire wound healing monitoring period, the CCSP group had the best healing status, while the untreated group had the worst healing status.

[0113] During the first 6 days of the healing phase, the wound gradually scabs over and shrinks. The CCSP fibrous membrane showed a significant advantage, with the most pronounced wound shrinkage. Images from day 9 show further wound shrinkage, with visible granulation tissue covering the wound. Thickened keratinized layers on some epithelial surfaces easily peel off, exposing red, newly formed epithelial tissue. The Control group showed a small amount of bloody exudate, the 3M group showed scab formation, and the SP group showed yellowish-white exudate. The CCSP group showed virtually no exudate, with the most significant wound contraction. The newly formed epithelium on the wound surface became more mature and lighter in color. By day 13, most of the scabs had fallen off, and the skin was pale red. In the diabetic Control group, the wound contraction trend began to decrease on day 13, and the healing rate slowed. The healing trend in the 3M and SP groups slowed, and the inflammatory phase was difficult to transition to the proliferative phase. The slow growth of the proliferative phase led to delayed healing. In contrast, the CCSP group showed the most significant wound area shrinkage, with most of the wound having completed re-epithelialization. By day 18, the CCSP wounds had largely completed re-epithelialization, and the wounds in the CCSP-treated group promptly entered the proliferative phase, resulting in the most significant wound area reduction (P<0.05). Conversely, in the other three groups, especially the untreated group, wound contraction almost stopped, and the skin color darkened. Figure (14D) shows the wound closure rate at 18 days. The healing rate of untreated Staphylococcus aureus-infected wounds was reduced to 66.34%±1.53%, while the healing rates of 3M and SP wounds were 82.13%±3.20% and 80.53%±2.336%, respectively. In contrast, the wound healing rate in the CCSP-treated group increased to 95.31%±0.92%. This indicates that CCSP dressings have a positive effect on the healing of diabetic skin wounds and can shorten the healing process to some extent.

[0114] H&E and Masson staining are used to assess wound healing quality as follows:

[0115] Newly formed tissue was harvested from the wound site on postoperative days 9 and 18 and stained with Hematoxylin and Escherichia coli (H&E) and Masson's stain for detailed histomorphological studies to reflect reepithelialization and granulation tissue formation in the infected diabetic wound. The H&E staining results (…) Figure 15As can be seen, the untreated Control group of diabetic mice showed poor healing. On day 9, epidermal migration was incomplete, the basal layer was unclear, fibroblasts were significantly fewer, inflammatory cell infiltration was more abundant, and collagen fibers were sparse. The 3M and SP groups showed partial epithelial migration, but inflammatory infiltration remained significant. In contrast, the CCSP group showed greater contraction, less dermal inflammatory cell infiltration, more fibroblasts, a clearer tissue structure, and initial epithelial formation was observed, while the epidermis was relatively loose. By day 18, subcutaneous inflammatory infiltration had decreased in all groups. The CCSP group further formed new skin, which tended to thin and gradually become denser, with clearly visible hair follicles, sebaceous glands, capillaries, and other skin appendages. However, in the untreated Control group, the wound remained wide. Although most of the inflammation had cleared, the tissue structure was sparse, and there was no further healing or contraction. The 3M and SP groups showed a small number of skin appendages, and significant inflammation remained. In terms of progression at each stage, the other three groups were slightly slower than the CCSP group. Masson staining was used to analyze collagen deposition (blue staining area) in skin grafts. On day 9 post-surgery, a small amount of fibrous deposition spreading from the normal tissue interface was observed in all groups, with the CCSP group showing deeper and wider blue staining of collagen. On day 18 post-surgery, a large number of blue collagen fibers were visible in the dermis of the wound skin of the CCSP group rats. The collagen was abundant and strongly stained, densely and regularly arranged, with orderly direction and uniform distribution. No obvious nodular or whorled collagen arrangement was observed. The color and arrangement of collagen deposition in the newly formed tissue were more similar to normal tissue. A small amount of fibrous deposition was observed in the 3M and SP groups, with a loose arrangement. The untreated control group showed the lightest color and the least amount of newly formed collagen deposition.

[0116] The proliferative phase begins with the migration of fibroblasts and myofibroblasts. Granulation tissue, composed of a complex of numerous fibroblasts, granulocytes, macrophages, and blood vessels, along with collagen bundles, partially restores the structure and function of the damaged skin. Fibroblasts play a central role in granulation tissue formation. These results indicate that wounds in the untreated diabetes control group have difficulty entering the proliferative phase, thus delaying healing. 3M and SP offer simple barrier protection but result in poor healing; the CCSP group exhibits the best healing effect. This healing advantage stems from early and timely vascularization of the wound, entering the proliferative phase, promoting the regeneration of new tissue, and thus achieving wound healing contraction.

[0117] Macrophage polarization and regulation of the inflammatory microenvironment in wounds: Long-term chronic inflammation is one of the key factors contributing to the difficulty in healing diabetic wounds. To detect the anti-inflammatory effect of CCSP fiber membranes and its relationship with macrophage (M1, M2) polarization in vivo, iNOS (M1 marker), CD206 (M2 marker), and CD68 (pan-macrophage marker) indicators were detected by immunofluorescence staining.Figure 16 Nine days post-surgery, there was no significant difference in CD68-positive cell counts among the groups. The expression level of iNOS in the CCSP group was lower than in other groups, while the expression level of CD206 was higher. Furthermore, the M2 / M1 ratio was highest in the CCSP group (p<0.05). We further assessed the levels of representative inflammatory cytokines in the wound. On day 9, compared with other groups, the CCSP group had lower TNF-α levels and significantly increased IL-10 levels. These results indicate that the fibrous membrane can reduce macrophage M1 polarization and increase macrophage M2 polarization. This ability derives from the release of Cur from the CCSP membrane, exhibiting macrophage regulatory effects and improving the inflammatory microenvironment in diabetic rats.

[0118] In vivo angiogenesis potential assessment is crucial for evaluating wound angiogenesis capacity, a key factor influencing wound healing rate. Newly formed capillaries provide nutrients and oxygen to wound-associated tissue cells for proliferation and growth. Newly formed tissue samples were harvested from the wound at 6, 13, and 18 days post-surgery. Dual staining for endothelial markers (CD31) and α-smooth muscle actin (α-SMA) was performed to analyze the angiogenesis potential within the tissues. Results are as follows: Figure 17 As shown, α-SMA exhibits green fluorescence, while CD31 exhibits red fluorescence. By day 6, CCSP already showed a large number of new blood vessels, compared to other groups. By day 13, the total number of new blood vessels in each group increased, with the CCSP group still showing the highest, indicating excellent angiogenesis potential. By day 18, the number of blood vessels in each group showed a decreasing trend, indicating tissue remodeling and vascular degeneration.

[0119] The proliferative phase includes sub-phases such as granulation tissue formation, angiogenesis, and re-epithelialization. The proliferative phase begins with the migration of fibroblasts and myofibroblasts. Granulation tissue is composed of a complex of numerous fibroblasts, granulocytes, macrophages, and blood vessels, accompanied by collagen bundles, partially restoring the structure and function of the damaged skin. Fibroblasts play a central role in the formation of granulation tissue. In the entire wound healing process of diabetic rats in each group, the untreated diabetic control group showed the worst wound healing, and the wound healing process in the 3M and SP groups was also significantly slower. The simple barrier protection of 3M and SP could not improve the inflammatory environment, making it difficult for the wound to enter the proliferative phase, leading to delayed healing. Conversely, the CCSP group showed the best healing effect, demonstrating a healing advantage from the early stages. The mechanism may be that the dressing's active immune regulation led to controlled inflammation in the wound, which is conducive to early angiogenesis, fibroblast proliferation and migration, timely transition from the inflammatory state to the proliferative phase, and promotion of new tissue regeneration, thereby achieving wound healing contraction.

[0120] Diabetic wounds, with their dysregulated "infection-inflammation-angiogenesis" microenvironment, face challenges such as prolonged healing, recurrent infections, and poor wound quality. Existing monofunctional therapies (single-node targeting, no targeted delivery) fail to match the dynamics of wound healing. Therefore, developing intelligent controlled release systems (pH-responsive, multi-target) tailored to the wound microenvironment is crucial.

[0121] This invention designs a CCSP nanofiber membrane loaded with pH-adaptive curcumin-copper ICP. It exhibits precise pH-responsive release: in early-stage infection / inflammatory wounds (acidic microenvironment), the curcumin-copper ICP rapidly dissociates, releasing curcumin (Cur) and Cu. 2+ Curcumin (Cur) scavenges ROS / inhibits inflammation, while Cu... 2+ Synergistic antimicrobial activity. As the wound transitions to the neutral healing phase, the structure of the curcumin-copper ICP re-stabilizes, significantly slowing the release rate. During this phase, low doses of curcumin maintain anti-inflammatory / antioxidant effects, while the slow release of Cu... 2+ Release promotes angiogenesis. This "intelligent controlled release" provides a reference for designing environmentally responsive drug delivery systems.

[0122] This invention demonstrates the efficacy and translational potential of CCSP through in vitro / in vivo experiments. In vitro, CCSP exhibits potent antibacterial activity against Staphylococcus aureus (acidic conditions), antioxidant capacity, and modulated macrophage polarization (upregulating M2 and downregulating M1). It also supports pro-angiogenic potential through endothelial migration / luminal formation. In vivo, in a diabetic rat model with Staphylococcus aureus-infected wounds, CCSP accelerates healing and improves healing quality. Histological / immunofluorescence analysis shows that CCSP accelerates re-epithelialization and increases M2 macrophages, α-SMA+ cells, and CD31+ endothelial cells, consistent with its in vitro "anti-inflammatory-pro-angiogenic" mechanism.

[0123] This invention proposes a novel "pH-adaptive (curcumin-copper ICP) + electrospun PCL / serine scaffold" strategy, CCSP. Leveraging "intelligent controlled release" and "multi-target synergy," CCSP provides a safe, efficient, and multifunctional platform for diabetes trauma management. Its design also offers a multifunctional framework for controlled-release therapy in multifactorial diseases.

Claims

1. A fiber membrane loaded with infinitely coordinated polymer nanoparticles, characterized in that, This includes nanofiber scaffolds fused with polycaprolactone (PCL) and sericin (Se), and curcumin-copper ICP loaded on the nanofiber scaffolds.

2. A method for preparing a fiber membrane loaded with infinitely coordinated polymer nanoparticles, characterized in that, Includes the following steps: S1. Preparation of freeze-dried sericin for later use; S2, Preparation of curcumin-copper ICP: Curcumin-copper ICP was prepared by pH-controlled coordination-induced self-assembly for future use. S3. Preparation of curcumin-copper / Se / PCL fiber membrane by directional spinning of curcumin-copper ICP: The freeze-dried sericin obtained in S1, the curcumin-copper ICP obtained in S2 and polycaprolactone were mixed to obtain a spinning solution. The fiber membrane curcumin-copper / Se / PCL was prepared by directional spinning. The fiber membrane curcumin-copper / Se / PCL is a fiber membrane loaded with infinitely coordinated polymer nanoparticles.

3. The method for preparing a fiber membrane loaded with infinitely coordinated polymer nanoparticles according to claim 1, characterized in that, The specific operation of S1 is as follows: Extraction of sericin: The high temperature and high pressure method is used to extract sericin. The cleaned silkworm cocoon fragments are soaked in a certain amount of distilled water at a ratio of 10g / 250ml. Then, they are boiled in a pressure cooker for 30 minutes to remove insoluble fibroin. After vacuum freeze drying, purified sericin can be obtained.

4. The method for preparing a fiber membrane loaded with infinitely coordinated polymer nanoparticles according to claim 2, characterized in that, The specific operation of S2 is as follows: S2.

1. Weigh out CuCl2·2H2O and dissolve it in anhydrous ethanol to prepare a CuCl2 solution with a concentration of 17 mg / mL; S2.

2. Weigh out curcumin (Cur) and dissolve it in anhydrous ethanol to prepare a curcumin (Cur) solution with a concentration of 7 mg / mL; S2.

3. Take 10 mL of CuCl2 solution obtained in S2.1 and 10 mL of curcumin Cur obtained in S2.2 into a reaction flask to form an acidic precursor solution; S2.4 A 10 mM Tris aqueous solution with pH 10.0 was added to the precursor solution, and the pH was adjusted to 7.4 at room temperature to obtain a mixed solution. The mixed solution was observed to turn dark brown. The solution was protected from light and placed on a magnetic stirrer for 6 hours to obtain the reaction solution. S2.5 The reaction solution was transferred to an ultrafiltration tube to purify curcumin-copper ICP, and purified curcumin-copper ICP was obtained. S2.6 The purified curcumin-copper ICP was collected by freeze-drying and stored at room temperature away from light.

5. The method for preparing a fiber membrane loaded with infinitely coordinated polymer nanoparticles according to claim 1, characterized in that, In step S2.2, to ensure complete dissolution of curcumin, the curcumin solution is intermittently sonicated in a water bath three times, with each sonication lasting 10 seconds.

6. The method for preparing a fiber membrane loaded with infinitely coordinated polymer nanoparticles according to claim 2, characterized in that, The specific operation of S3 is as follows: S3.

1. Dissolve the lyophilized sericin obtained in S1 in hexafluoroisopropanol (HFIP) as solvent and stir continuously for 24 hours to obtain a sericin solution with a concentration of 5% w / v, named solution a. S3.

2. Polycaprolactone was dissolved in hexafluoroisopropanol (HFIP) as solvent to obtain a polycaprolactone (PCL) solution with a concentration of 10% w / v, which was named solution b. S3.

3. Weigh 5 mg of curcumin-copper ICP obtained from S2 and add it to 10 mL of the mixture of the above solutions a and b to form a spinning solution, which is named solution c. S3.

4. Load the prepared solution c into a syringe, and place it on a micro-injection pump using a spinning needle with an inner diameter of 0.6 mm. S3.

5. Adjust the distance between the syringe needle and the high-speed roller receiving device for directional spinning to 10cm; the electrospinning conditions are: flow rate 1.2ml / h, voltage 16kV, roller speed 3000 rpm; to obtain a nanofiber film; S3.

6. Place the nanofiber film obtained in S3.5 in a fume hood and dry overnight to remove residual organic reagents, and obtain a curcumin-copper ICP oriented spun fiber membrane, which is a fiber membrane loaded with infinitely coordinated polymer nanoparticles.

7. The method for preparing a fiber membrane loaded with infinitely coordinated polymer nanoparticles according to claim 2, characterized in that, In step S3.3, the volume ratio of solution a to solution b is 1:

9.

8. An application of a fiber membrane loaded with infinitely coordinated polymer nanoparticles, characterized in that, Application in medications for treating diabetic wounds.

Images