Preparation method of PLGA-PEI-VK1 nanoparticles and application of PLGA-PEI-VK1 nanoparticles in diabetes wound treatment

Abstract

The invention discloses a preparation method of PLGA-PEI-VK1 nano-particles and application of the PLGA-PEI-VK1 nano-particles in diabetic wound treatment, and belongs to the field of nano-drug delivery and diabetic complication treatment. The method comprises the following steps: dissolving PLGA and PEI in an organic solvent to prepare a mixed polymer solution, modifying VK1 to prepare an inclusion compound solution, mixing the inclusion compound solution with the polymer solution, ultrasonically preparing primary emulsion, preparing multiple emulsion, centrifuging, washing, and freeze-drying to obtain the nanoparticles. The obtained nanoparticles play antioxidant and anti-inflammatory roles through a targeted FSP1-mediated ferroptosis pathway, VK1 can be efficiently delivered, the water solubility and bioavailability of VK1 are remarkably improved, diabetic foot ulcer healing is accelerated, and the preparation process is simple and convenient, mild in condition, suitable for large-scale production and good in clinical transformation prospect.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine delivery and treatment of diabetic complications, specifically relating to a method for preparing PLGA-PEI-VK1 nanoparticles and their application in the treatment of diabetic wounds. Background Technology

[0002] Diabetes mellitus (DM) is a major global public health challenge. The International Diabetes Federation (IDF) predicts that approximately 783 million adults worldwide will be affected by it by 2045. Diabetic foot ulcer (DFU), as one of the most serious complications of diabetes, will affect nearly a quarter of diabetic patients at some point in their lives. Its pathological characteristics include persistent inflammation, impaired angiogenesis, and delayed epithelial regeneration, often developing into chronic, intractable ulcers. In severe cases, amputation may be necessary, placing a huge physical and psychological burden on patients and creating a heavy medical and economic burden.

[0003] The core mechanisms underlying impaired wound healing in diabetes involve the accumulation of advanced glycation end products (AGEs), persistent oxidative stress, and dysregulation of cell death pathways. Recent studies have confirmed that ferroptosis, as an iron-dependent regulated cell death pathway, is closely related to diabetic complications. It is driven by iron homeostasis imbalance and impaired antioxidant defense, leading to excessive accumulation of lipid peroxides, and is characterized by glutathione (GSH) depletion and glutathione peroxidase 4 (GPX4) inactivation. In the diabetic wound microenvironment, AGEs can induce ferroptosis in endothelial cells, keratinocytes, and fibroblasts, and downregulate the expression of ferroptosis inhibitor 1 (FSP1), further exacerbating ferroptosis and impairing angiogenesis. Therefore, targeting the FSP1-mediated ferroptosis pathway has become an innovative direction for the treatment of diabetic wounds.

[0004] In addition to its classic functions of regulating blood clotting and bone metabolism, vitamin K1 (VK1, phylloquinone) also exhibits significant antioxidant, anti-inflammatory, and cytoprotective activities. It can reduce cell damage through the ferroptosis pathway and has potential value in the treatment of diabetic wounds. However, the poor water solubility, limited bioavailability, and short plasma half-life of VK1 severely hinder its clinical translation.

[0005] Therefore, overcoming the core technological bottlenecks of VK1's poor water solubility and low bioavailability, constructing a drug delivery carrier that combines high loading efficiency, excellent stability, and targeting, and then exerting a therapeutic effect by regulating the FSP1-mediated ferroptosis pathway, has become the key to promoting the application of VK1 in diabetic wound treatment and improving clinical translation efficiency. Summary of the Invention

[0006] To address the problems of poor water solubility and low bioavailability in existing VK1 formulations, as well as the lack of effective targeted treatments for diabetic wounds, this invention provides a stable and easy-to-operate method for preparing PLGA-PEI-VK1 nanoparticles. It also discloses the application of these nanoparticles in the treatment of diabetic wounds, achieving efficient delivery of VK1 and synergistically exerting antioxidant and anti-inflammatory effects by targeting the FSP1-mediated ferroptosis pathway, thereby accelerating the healing of diabetic wounds.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A method for preparing PLGA-PEI-VK1 nanoparticles includes the following steps:

[0009] S1 Preparation of mixed polymer solution: Weigh polylactic acid-glycolic acid copolymer (PLGA) and polyethyleneimine (PEI) at a weight ratio of 10:1, dissolve them together in an organic solvent, and stir until completely dissolved to obtain a mixed polymer solution;

[0010] Preparation of S2 VK1 primary emulsion: Weigh VK1 and hydroxypropyl-β-cyclodextrin (HP-β-CD) at a mass ratio of 1:3. Dissolve HP-β-CD in deionized water and stir until completely dissolved to obtain an HP-β-CD aqueous solution. Add VK1 to the HP-β-CD aqueous solution, stir, and filter to obtain a VK1-HP-β-CD inclusion complex solution. Slowly add the inclusion complex solution to the mixed polymer solution obtained in step S1, and sonicate to form a uniformly dispersed oil phase primary emulsion.

[0011] S3 Preparation of double emulsion: The primary emulsion obtained in step S2 is slowly added dropwise to the polyvinyl alcohol solution and stirred to form a stable water-oil-water double emulsion;

[0012] S4 Particle Collection and Drying: The double emulsion was centrifuged and the precipitate was collected; the precipitate was washed with deionized water and then freeze-dried to obtain white powdery PLGA-PEI-VK1 nanoparticles.

[0013] This is a further optimization of a method for preparing PLGA-PEI-VK1 nanoparticles.

[0014] Preferably, in step S1, the organic solvent is dichloromethane, the stirring method is magnetic stirring, and the stirring time is 25~35 min.

[0015] Preferably, in step S1, the molecular weight of PLGA is 30,000-60,000, and the ratio of lactic acid to glycolic acid is 50:50; the molecular weight of PEI is 25,000.

[0016] Preferably, in step S2, after adding HP-β-CD aqueous solution to VK1, the stirring conditions are magnetic stirring at 25~30℃ for 2.5~3.5h.

[0017] Preferably, in step S2, the ultrasonic time is 5-10 minutes.

[0018] Preferably, in step S3, the concentration of the polyvinyl alcohol solution is 2% (w / v), the stirring method is magnetic stirring, the stirring speed is 500~600 rpm, and the stirring time is 60~80 min.

[0019] Preferably, in step S4, the centrifugation parameters are: centrifugation at 8000-10000 rpm for 15-20 min; washing is performed 2-3 times, with the centrifugation conditions after each washing being the same as the initial centrifugation conditions; freeze-drying time is 20-24 h; and the obtained nanoparticles are sealed and stored in a refrigerator at 4°C.

[0020] The application of PLGA-PEI-VK1 nanoparticles prepared by the aforementioned method in the preparation of drugs for treating diabetic wounds.

[0021] This invention also protects the application of the PLGA-PEI-VK1 nanoparticles prepared by the above method in the preparation of a drug for treating diabetic wounds, preferably diabetic foot ulcers. As the active pharmaceutical ingredient, these nanoparticles inhibit ferroptosis in the microenvironment of diabetic wounds by targeting the FSP1-mediated ferroptosis pathway, while simultaneously exerting the antioxidant and anti-inflammatory activities of VK1, improving impaired angiogenesis, and accelerating epithelial regeneration, thereby promoting wound healing. The drug is administered subcutaneously at a daily dose of 50 μL for 12 consecutive days.

[0022] Beneficial effects

[0023] (1) Breakthrough in the water solubility bottleneck of VK1: Through the key emulsification and stabilization effect of polyvinyl alcohol, the water-oil interfacial tension is effectively reduced, preventing the aggregation of oil phase dispersed droplets, enabling hydrophobic VK1 to achieve stable dispersion in the aqueous phase through nanocarriers, significantly improving the water solubility of the formulation, and solving the technical problem that traditional VK1 is difficult to make into water-based formulations.

[0024] (2) Excellent formulation stability: Polyvinyl alcohol and PLGA-PEI form a synergistic stabilizing system, resulting in nanoparticles with uniform particle size and good dispersibility. They are not prone to agglomeration during storage and administration, ensuring the stability of drug delivery efficiency.

[0025] (3) Significant therapeutic effect: The nanocarrier achieves efficient targeted delivery of VK1, inhibits cell ferroptosis by upregulating FSP1 expression, and synergistically exerts antioxidant and anti-inflammatory effects, effectively improving the microenvironment of diabetic wounds, accelerating angiogenesis and epithelial regeneration, and significantly improving the healing rate of chronic ulcers;

[0026] (4) The process is safe and reliable: PLGA, PEI and polyvinyl alcohol used are all biocompatible materials. There are no toxic or harmful substances left in the preparation process. The process steps are simple and the conditions are mild. It is easy to scale up production and has good prospects for clinical translation. Attached Figure Description

[0027] Figure 1 A schematic diagram illustrating the application of PLGA-PEI-VK1 nanoparticles in the treatment of diabetic wounds;

[0028] Figure 2 Characterization of PLGA-PEI-VK1 nanoparticles: (A) Transmission electron microscopy (TEM) image of PLGA-PEI-VK1 nanoparticles; (B) Average diameter of PLGA-PEI-VK1; (C) Release rate of PLGA-PEI-VK1; (D) Stability curve of PLGA-PEI-VK1 nanoparticles over time; (E) Zeta potential of PLGA-PEI and PLGA-PEI-VK1; Representative images and quantitative analysis of hemolysis rate (FG), 2,2'-adiazonium bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt cationic radical (ABTS·+) scavenging rate (HI), and 1,1-diphenyl-2-trinitrophenylhydrazine radical (DPPH) scavenging rate (JK).

[0029] Figure 3 (A) Experimental protocol and animal grouping for PLGA-PEI-VK1 to improve wound healing in diabetic patients; (B) Representative images of skin wound healing in mice in the diabetes (DM) group, diabetes plus PLGA-PEI group, and diabetes plus PLGA-PEI-VK1 group; (C) Quantitative analysis of wound closure rate over time; (D) Representative images and quantitative analysis of hematoxylin-eosin (HE) staining; (E) Immunohistochemical staining of α-smooth muscle actin (α-SMA) and collagen-1 at the wound site;

[0030] Figure 4To demonstrate that PLGA-PEI-VK1 promotes angiogenesis in diabetic mice and endothelial cells treated with AGEs, (A) enrichment analysis of vascular-related biological processes (BP) of VK1 in diabetic wounds; (B) representative laser speckle images of blood flow in the wound area; (C, D) representative staining images of platelet endothelial cell adhesion molecule-1 (CD31) (C) and vascular endothelial growth factor A (VEGFA) (D) in wound tissue.

[0031] Figure 5 PLGA-PEI-VK1 was used to improve AGEs-induced abnormal proliferation and migration of fibroblasts; (F, G) 5-ethynyl-2'-deoxyuridine (EdU) assay showed cell proliferation and quantitative results; (H, I) Transwell migration assay; (J) Western blotting was used to detect the expression of N-cadherin and vimentin.

[0032] Figure 6 Key targets for PLGA-PEI-VK1 in wound repair: (A) Gene Ontology (GO) cellular component enrichment analysis of overlapping targets; (B) Molecular docking model demonstrating the binding interaction between VK1 and FSP1; (C) Cell thermal transfer analysis (CETSA)-Western blot analysis showing that VK1 enhances the thermostability of FSP1; (DE) Immunofluorescence analysis of FSP1 expression at mouse wound sites on days 3 and 7; (FG) Western blot analysis and quantification of FSP1 protein expression in the AGEs-induced mouse fibroblast cell line (L929). Detailed Implementation

[0033] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to examples and comparative examples. The following content is merely illustrative and explanatory of the concept of the present invention. Those skilled in the art can make various modifications or additions to the described specific embodiments or use similar methods to replace them, as long as they do not depart from the inventive concept, all of which should fall within the protection scope of the present invention. The following examples and experimental results further explain the invention.

[0034] Example 1

[0035] The preparation of PLGA-PEI-VK1 nanoparticles includes the following steps:

[0036] (1) Preparation of mixed polymer solution: Weigh 100mg PLGA and 10mg PEI, dissolve them together in 5mL dichloromethane, and stir magnetically for 30min until completely dissolved to obtain a mixed polymer solution;

[0037] (2) Preparation of VK1 primary emulsion: Weigh VK1 and HP-β-CD at a mass ratio of 1:3. Accurately weigh 12.2 mg VK1 and 36.6 mg HP-β-CD. Dissolve HP-β-CD in 5 mL of deionized water and stir magnetically until completely dissolved to obtain an HP-β-CD aqueous solution. Add VK1 to the above aqueous solution and stir magnetically at 30 °C for 3 h. Filter through a 0.22 μm filter membrane to obtain a VK1-HP-β-CD inclusion complex solution. Slowly add the inclusion complex solution to the mixed polymer solution obtained in step (2) and treat with ultrasound for 10 min to form a uniformly dispersed oil phase primary emulsion.

[0038] (3) Preparation of double emulsion: The above primary emulsion was slowly added dropwise to 10 mL of 2% (w / v) polyvinyl alcohol solution and stirred at 500 rpm for 60 min on a magnetic stirrer to form a stable water-oil-water double emulsion;

[0039] (4) Particle collection and drying: Place the double emulsion in a centrifuge tube and centrifuge at 10,000 rpm for 20 min to collect the precipitate; wash the precipitate three times with deionized water, with the same centrifugation conditions each time; place the washed precipitate in a freeze dryer and freeze dry for 24 h to obtain white powdery PLGA-PEI-VK1 nanoparticles, which are then sealed and stored at 4°C.

[0040] Example 2

[0041] Animal experiments on the treatment of diabetic wounds with PLGA-PEI-VK1 nanoparticles:

[0042] (1) Grouping of experimental animals: Eight-week-old male C57BL / 6 mice weighing 18-20 grams (purchased from Guizhou Medical University) were selected and randomly divided into four groups after one week of acclimatization: type 1 diabetic mouse group (DM group), DM+PLGA-PEI group, DM+PLGA-PEI-VK1 group and DM+comparative example 1 product group, with 6 mice in each group. All experimental procedures were strictly performed in accordance with the ARRIVE guidelines and relevant animal experimental ethics.

[0043] (2) Diabetes model construction: After fasting for 12 hours, mice were injected intraperitoneally with 100 mg / kg streptozotocin. After fasting for another 4 hours, blood was collected from the tail vein to detect blood glucose levels. Mice with blood glucose levels exceeding 11.6 mmol / L were considered to have successfully constructed a diabetes model.

[0044] (3) Construction of diabetic wound model: One week after successful modeling, all diabetic mice underwent hair removal on their backs, and the skin on their backs was disinfected with 75% alcohol. A full-thickness skin defect was created in the center of the back using a 1cm diameter biopsy puncture device, and disinfected with povidone-iodine after hemostasis.

[0045] (4) Drug administration: The DM+PLGA-PEI group was injected subcutaneously with 50 μL of blank PLGA-PEI nanoparticle solution daily, the DM+PLGA-PEI-VK1 group was injected subcutaneously with 50 μL of PLGA-PEI-VK1 nanoparticle solution prepared in Example 1 daily, the DM+Comparative Example 1 product group was subjected to subcutaneous injection after ultrasonic dispersion of the Comparative Example 1 product in 50 μL of physiological saline, and the DM group was injected with an equal volume of physiological saline. The administration was carried out for 12 consecutive days.

[0046] The experimental results are explained below with reference to the accompanying diagrams in the instruction manual:

[0047] Figure 1 A schematic diagram illustrating the application of PLGA-PEI-VK1 nanoparticles in diabetic wound treatment; this diagram visually demonstrates the targeted therapy mechanism. It clearly depicts the pathway by which the nanoparticles, after locally releasing VK1 in diabetic wounds, inhibit ferroptosis in endothelial cells and fibroblasts by upregulating the expression of ferroptosis inhibitor protein 1 (FSP1), while simultaneously exerting antioxidant and anti-inflammatory effects, promoting angiogenesis and epithelial regeneration, and ultimately accelerating wound healing.

[0048] Figure 2 Characterization spectra of PLGA-PEI-VK1 nanoparticles; (A) TEM image: visually presents the morphological characteristics of the nanoparticles, showing that the particles are regularly spherical with a uniform particle size distribution, proving that the preparation process of this invention can obtain nanocarriers with regular morphology. (B) Average diameter: quantitatively shows the particle size range of the nanoparticles, which meets the particle size requirements of drug delivery carriers, ensuring that the particles can be retained locally in the wound and taken up by cells.

[0049] (C) Release rate curve: Reflects the in vitro release pattern of VK1 from nanoparticles, exhibiting a characteristic of "slow release in the initial stage and stable release in the later stage," proving that this carrier can achieve sustained and controlled release of VK1, avoiding excessively high local concentrations caused by sudden drug release and prolonging the duration of drug efficacy. (D) Stability curve: Shows the particle size change trend of nanoparticles in storage or physiological environment. The curve fluctuates little, proving that the synergistic stabilizing system formed by polyvinyl alcohol and PLGA-PEI can effectively prevent particle aggregation and ensure the stability of the formulation. (E) Zeta potential: Comparing the potential values ​​of blank PLGA-PEI particles and drug-loaded particles, cationic PEI imparts a positive potential to the particles, which is conducive to the binding of particles to negatively charged cell membranes and improves cellular uptake efficiency. (FG) Hemolysis rate: Verifies the biosafety of nanoparticles. The hemolysis rate is lower than the safety threshold, proving that the carrier materials used (PLGA, PEI, HP-β-CD) have good biocompatibility and no obvious cytotoxicity. (HI) ABTS·+ scavenging rate and (JK) DPPH free radical scavenging rate: In vitro antioxidant experimental data demonstrate that the drug-loaded nanoparticles have a significantly better free radical scavenging ability than blank particles and free VK1, directly verifying their antioxidant activity and providing experimental evidence for in vivo anti-inflammatory and anti-ferroptosis effects.

[0050] Figure 3 Animal experimental results of PLGA-PEI-VK1 improving wound healing in diabetic patients; (A) Experimental protocol and grouping: The design logic of the animal experiment was clearly defined. A control group was formed by setting up a diabetic model group (DM group), a blank particle group (DM+PLGA-PEI group), and a drug-loaded particle group (DM+PLGA-PEI-VK1 group) to ensure the scientific validity of the experimental results. (B) Representative images of wound healing: The wound healing status of mice in different groups at different time points after drug administration was intuitively displayed. The wound area of ​​the drug-loaded particle group shrank significantly faster than that of the other two groups, which intuitively proved its therapeutic advantage. (C) Quantitative analysis of wound closure rate: The wound healing process was quantitatively analyzed. The wound closure rate of the drug-loaded particle group was significantly higher than that of the control group at all time points. The therapeutic effectiveness of the nanoparticles of this invention was verified by statistical difference. (D) HE staining: The morphology of wound tissue was observed in pathological sections. The infiltration of inflammatory cells at the wound site of the drug-loaded particle group was reduced, the new formation of granulation tissue was vigorous, and the repair speed of the epithelial layer was fast, which proved that the nanoparticles can improve the wound microenvironment. (E) Immunohistochemical staining of α-SMA and collagen-1: α-SMA is a marker of myofibroblasts and collagen-1 is a major component of the extracellular matrix. The staining results of the two groups showed that the positive expression of the drug-loaded particles was significantly enhanced, proving that nanoparticles can promote granulation tissue formation and tissue remodeling.

[0051] Figure 4Experimental evidence of PLGA-PEI-VK1 promoting angiogenesis: (A) Enrichment analysis of vascular-related blood flow (BP): Bioinformatics analysis was used to screen for angiogenesis-related targets of VK1 in the treatment of diabetic wounds, providing molecular-level evidence of the mechanism of action of nanoparticles in promoting angiogenesis. (B) Laser speckle blood flow images: These images visually show the blood perfusion in the wound area. The blood flow signal was stronger in the drug-loaded particle group, indicating more vigorous local angiogenesis. (C) CD31 staining, (D) VEGFA staining: CD31 is a marker of vascular endothelial cells, and VEGFA is vascular endothelial growth factor. The expansion of the positive staining area proves that nanoparticles can significantly promote endothelial cell proliferation and angiogenesis, addressing the core issue of insufficient blood supply in diabetic wounds.

[0052] Figure 5 Experimental data on the improvement of fibroblast proliferation and migration by PLGA-PEI-VK1; (FG) EdU assay: EdU can label proliferating cells, and the results showed that the proliferation rate of fibroblasts in the drug-loaded particle group was significantly higher than that in the control group, proving that nanoparticles can reverse the inhibitory effect of AGEs on fibroblast proliferation. (HI) Transwell migration assay: The results showed the difference in the number of fibroblasts passing through the chambers. The fibroblasts in the drug-loaded particle group had stronger migration ability, proving that they can promote the recruitment of fibroblasts to the wound site and accelerate granulation tissue formation. (J) Western blotting detection of N-cadherin and vimentin: Both are cell migration-related markers. Upregulation of their expression levels proves that nanoparticles can enhance the migration ability of fibroblasts and provide a cellular basis for wound healing.

[0053] Figure 6 Validation of key targets of PLGA-PEI-VK1 action; (A) GO cell component enrichment analysis: Through enrichment analysis of differentially expressed genes, the core pathways of nanoparticle action were identified as being related to ferroptosis, cell proliferation, and angiogenesis, and their molecular action network was clarified.

[0054] (B) Molecular docking model: Demonstrating the binding site and binding energy of VK1 and FSP1 proteins, demonstrating that VK1 can directly target and bind to FSP1 and enhance its stability, elucidating the mechanism of targeting the ferroptosis pathway at the molecular level. (C) CETSA-Western blot analysis: Cell heat transfer experiments showed that VK1 treatment reduced the degradation of FSP1 protein at high temperatures, demonstrating that VK1 can enhance the thermal stability of FSP1 and improve its protein activity. (DE) FSP1 immunofluorescence analysis: Quantitative results of FSP1 fluorescence intensity in mouse wound tissue showed that FSP1 expression was significantly upregulated in the drug-loaded particle group, directly verifying its role in targeting the ferroptosis pathway in vivo. (FG) AGEs-induced FSP1 protein expression in L929 cells: In vitro cell experiments showed that drug-loaded particles could reverse the downregulation of FSP1 expression by AGEs, demonstrating the effectiveness of its anti-ferroptosis effect.

Claims

1. A method for preparing PLGA-PEI-VK1 nanoparticles, characterized in that, Includes the following steps: S1 Preparation of mixed polymer solution: Weigh PLGA and PEI at a weight ratio of 10:1, dissolve them together in an organic solvent, and stir until completely dissolved to obtain a mixed polymer solution; Preparation of S2 VK1 primary emulsion: Weigh VK1 and HP-β-CD at a mass ratio of 1:

3. Dissolve HP-β-CD in deionized water and stir until completely dissolved to obtain an HP-β-CD aqueous solution. Add VK1 to the HP-β-CD aqueous solution, stir, and filter to obtain a VK1-HP-β-CD inclusion complex solution. Slowly add the inclusion complex solution to the mixed polymer solution obtained in step S1, and sonicate to form a uniformly dispersed oil phase primary emulsion. S3 Preparation of double emulsion: The primary emulsion obtained in step S2 is slowly added dropwise to the polyvinyl alcohol solution and stirred to form a stable water-oil-water double emulsion; S4 Particle Collection and Drying: The double emulsion was centrifuged and the precipitate was collected; the precipitate was washed with deionized water and then freeze-dried to obtain white powdery PLGA-PEI-VK1 nanoparticles.

2. The preparation method according to claim 1, characterized in that, In step S1, the organic solvent is dichloromethane, the stirring method is magnetic stirring, and the stirring time is 25~35 min.

3. The preparation method according to claim 1, characterized in that, In step S1, the molecular weight of PLGA is 30,000-60,000, and the ratio of lactic acid to glycolic acid is 50:50; the molecular weight of PEI is 25,000.

4. The preparation method according to claim 1, characterized in that, In step S2, after adding HP-β-CD aqueous solution to VK1, the stirring conditions are magnetic stirring at 25~30℃ for 2.5~3.5h.

5. The preparation method according to claim 1, characterized in that, In step S2, the ultrasound duration is 5-10 minutes.

6. The preparation method according to claim 1, characterized in that, In step S3, the concentration of the polyvinyl alcohol solution is 2% (w / v), the stirring method is magnetic stirring, the stirring speed is 500~600 rpm, and the stirring time is 60~80 min.

7. The preparation method according to claim 1, characterized in that, In step S4, the centrifugation parameters are: centrifugation at 8000-10000 rpm for 15-20 min; washing is performed 2-3 times, with the centrifugation conditions after each washing being the same as the first centrifugation conditions; freeze-drying time is 20-24 h; and the obtained nanoparticles are sealed and stored in a refrigerator at 4°C.

8. The application of the PLGA-PEI-VK1 nanoparticles prepared by the method according to any one of claims 1-7 in the preparation of a drug for treating diabetic wounds, characterized in that, The diabetic wound is a diabetic foot ulcer; the drug is administered via subcutaneous injection at a daily dose of 50 μL for 12 consecutive days; the nanoparticles target the FSP1-mediated ferroptosis pathway, inhibiting ferroptosis in the microenvironment of the diabetic wound, exerting antioxidant and anti-inflammatory activities, improving impaired angiogenesis, accelerating epithelial regeneration, and promoting the healing of the diabetic wound.

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