Self-assembly of PEG-PPS polymers and LL-37 peptide nanomicelles improves the oxidative microenvironment and promotes angiogenesis to facilitate chronic wound healing

Abstract

Refractory diabetic wounds are associated with high incidence, mortality, and recurrence rates and are a devastating and rapidly growing clinical problem. However, treating these wounds is difficult owing to uncontrolled inflammatory microenvironments and defective angiogenesis in the affected areas, with no established effective treatment to the best of our knowledge. Herein, we optimized a dual functional therapeutic agent based on the assembly of LL-37 peptides and diblock copolymer poly(ethylene glycol)-poly(propylene sulfide) (PEG-PPS). The incorporation of PEG-PPS enabled responsive or controlled LL-37 peptide release in the presence of reactive oxygen species (ROS). LL-37@PEG-PPS nanomicelles not only scavenged excessive ROS to improve the microenvironment for angiogenesis but also released LL-37 peptides and protected them from degradation, thereby robustly increasing angiogenesis. Diabetic wounds treated with LL-37@PEG-PPS exhibited accelerated and high-quality wound healing in vivo. This study shows that LL-37@PEG-PPS can restore beneficial angiogenesis in the wound microenvironment by continuously providing angiogenesis-promoting signals. Thus, it may be a promising drug for improving chronic refractory wound healing.

Keywords: LL‐37@PEG–PPS nanomicelles; angiogenesis; diabetic wound healing; inflammatory microenvironment.

FIGURE 1

Synthesis diagram and characterization of nanomicelles. (a) The preparation of ROS‐responsive LL‐37@PEG–PPS in vitro and therapeutic mechanism of LL‐37@PEG–PPS in chronic wounds. (b) Representative TEM images of PEG–PPS and LL‐37@PEG–PPS nanomicelles. Scale bars: 200 nm. (c) The dry diameter of PEG–PPS and LL‐37@PEG–PPS nanomicelles was measured using Image J software. Data are expressed as mean ± SD (n = 50). *p < 0.05, **p < 0.01 (Student's t‐test). (d) The LC of LL‐37 in LL‐37@PEG–PPS nanomicelles was measured via HPLC (n = 3). (e) In vitro release rate curve of LL‐37 from LL‐37@PEG–PPS with 2.5 mM H₂O₂ and 250 μM H₂O₂ at predetermined timepoints (n = 3). HPLC, high‐performance liquid chromatography; LC, loading capacity; PEG–PPS, poly(ethylene glycol)–poly(propylene sulfide); ROS, reactive oxygen species; TEM, transmission electron microscope.

FIGURE 2

ROS responsiveness of LL‐37@PEG–PPS nanomicelles. (a) The hydrodynamic diameter distribution of LL‐37@PEG–PPS nanomicelles in PBS solution and 2.5 mM H₂O₂ was measured using DLS. (b) TEM images of LL‐37@PEG–PPS nanomicelles following treatment with 2.5 mM H₂O₂ for 0, 1, 2, and 10 h. Scale bar: 100 nm. (c) The number of LL‐37@PEG–PPS nanomicelles with a dry diameter of ≥28 nm were counted at different timepoints following treatment with 2.5 mM H₂O₂ (n = 3). (d) After coculture with FITC@LL‐37@PEG–PPS for 0.5, 1, and 2 h, confocal microscopy was performed to assess the amount of released FITC@LL‐37 in HUVECs. Scale bar: 50 μm (n = 3). DLS, dynamic light scattering; FITC, fluorescein isothiocyanate; HUVECs, human umbilical vein endothelial cells; PBS, phosphate‐buffered saline; PEG–PPS, poly(ethylene glycol)–poly(propylene sulfide); ROS, reactive oxygen species; TEM, transmission electron microscope.

FIGURE 3

Alleviating effect of LL‐37@PEG‐PPS on reactive oxygen species and apoptosis in vitro. (a) Reactive oxygen species (ROS) level in untreated and LL‐37@PEG–PPS‐treated HUVECs was quantified following incubation with 250 μM H₂O₂. Scale bar = 50 μm. (b) ROS levels in HUVECs under different treatments were analyzed via flow cytometry (n = 3). (c) Cell death (both apoptosis and necrosis) under different treatment conditions was assessed via flow cytometry. Data in each group are expressed as mean ± SD (n = 3). *p < 0.05, **p < 0.01, n.s., no significance (one‐way ANOVA). HUVECs, human umbilical vein endothelial cells; PEG–PPS, poly(ethylene glycol)–poly(propylene sulfide).

FIGURE 4

Promoting effect of LL‐37@PEG–PPS on cell migration and angiogenesis. (a) Representative images of scratch tests for HUVECs under PBS, LL‐37, PEG–PPS, and LL‐37@PEG–PPS treatments and (b) quantitative analysis of cell migration (n = 3). (c) Experimental schematic diagram of the migration effect of LL‐37@PEG–PPS on HUVECs. (d) Representative crystal violet staining images of HUVEC migration under different treatments via Transwell assays and (e) quantitative analysis (n = 5). (f) The angiogenic effects of PEG–PPS, LL‐37, and LL‐37@PEG–PPS on HUVECs. (g) Quantitative analysis of the number of nodes. Data are represented as mean ± SD (n = 3). *p < 0.05, **p < 0.01, n.s., no significance, (one‐way ANOVA). HUVECs, human umbilical vein endothelial cells; PEG–PPS, poly(ethylene glycol)–poly(propylene sulfide).

FIGURE 5

Therapeutic effects of LL‐37@PEG–PPS for diabetic wound healing. (a) Schematic diagram of the establishment and treatment of diabetic wounds. (b) Representative images of wound healing in diabetic mice on days 0, 3, 6, 9, and 12 after injury with different treatments. (c) Statistical analysis results of wound closure rate on days 0, 3, 6, 9, and 12 after injury with different treatments (n = 4). (d) Trace of wound closure following different treatments on days 0, 3, 6, 9, and 12 after the surgery. (e) On day 12 after injury, the length of the new epidermis in the different treatment groups is indicated by the green dashed line. (f) Data analysis of the neoepidermis length in the different treatment groups at day 12 (n = 4). (g) The ratio of the neoepidermis length in the different treatment groups to the neoepidermis length in the control group at day 12 (n = 4). The data in (c), (f), and (g) are expressed as mean ± SD. *p < 0.05, **p < 0.01, n.s., no significance (one‐way ANOVA). PEG–PPS, poly(ethylene glycol)–poly(propylene sulfide).

FIGURE 6

Therapeutic effects of LL‐37@PEG–PPS for diabetic wound healing. (a) Representative images of Masson staining for diabetic wounds under different treatments. (b) Statistical analysis of the total collagen volume fraction on day 12 following injury in the different treatment groups (n = 4). (c) CD31 and DAPI staining results of diabetic wound tissue in the different groups (scale bar: 50 μm). (d) Statistical analysis of the number of blood vessels in the wound tissue on day 12 (n = 4). (e) Confocal representative images of DHE (red) staining in the different treatment groups on day 9 (scale bar: 100 μm). (f) Fluorescence intensities of DHE (red) in the different treatment groups were analyzed using Image J software on day 9 (n = 4). The data in (b), (d), and (f) are expressed as mean ± SD. *p < 0.05, **p < 0.01 (one‐way ANOVA). DHE, dihydroethidium; PEG–PPS, poly(ethylene glycol)–poly(propylene sulfide).

FIGURE 7

Immunohistochemical staining of the inflammatory factors TNF‐α and IL‐1β in diabetic wounds. (a) Immunohistochemical staining of TNF‐α in diabetic wounds under different treatments. (b) Immunohistochemical staining of IL‐1β of diabetic wounds under different treatments. (c) Statistical analysis of the percentage of TNF‐α‐ and IL‐1β‐positive area in wound tissue (n = 4). The data are expressed as mean ± SD. *p < 0.05, **p < 0.01, n.s., no significance (one‐way ANOVA). IL, interleukin; TNF, tumor necrosis factor.

FIGURE 8

Biocompatibility of LL‐37@PEG–PPS nanomicelles in vitro and in vivo. (a) Typical photographs and hemolysis rate of red blood cells incubated with LL‐37@PEG–PPS in vitro at different concentrations (n = 3). (b) On day 12 after surgery, the toxicity evaluation of LL‐37@PEG–PPS in the major organs (heart, liver, spleen, lungs, and kidneys) was performed. Scale bar: 50 μm. (c–g) Routine blood analysis of mice treated with PBS and LL‐37@PEG–PPS. (h) Analysis of liver function in mice treated with PBS and LL‐37@PEG–PPS. (i and j) Renal function analysis of mice treated with PBS and LL‐37@PEG–PPS (n = 5). PEG–PPS, poly(ethylene glycol)–poly(propylene sulfide).