Antimicrobial Effects of Thonningianin a (TA)-Loaded Chitosan Nanoparticles Encapsulated by a PF-127 hydrogel in Diabetic Wound Healing

Abstract

Background and purpose: Diabetic wounds are serious chronic complications of diabetes and can lead to amputation and death. Although considerable progress has been made in drugs and materials for treating it, it's still an urgent clinical problem as the materials and drugs have potential therapeutic drawbacks, such as low delivery efficiency and poor tissue permeability. To promote diabetic wound healing, a composite of thonningianin A (TA)-loaded chitosan nanoparticles (CNPS) encapsulated by a Pluronic F-127 (PF-127) hydrogel (TA-CNPS-PF) was developed in this study.

Methods: TA-CNPS was prepared by ionic gelation method and TA-CNPS was thoroughly dispersed into PF-127 hydrogel to prepare TA-CNPS-PF. The particle size, hydrogel structure, encapsulation ratio, release ratio, antimicrobial properties of TA-CNPS-PF were determined and the effect of TA-CNPS-PF on diabetic wounds was assessed. The effect of TA on macrophage polarization was also examined in vitro.

Results: The particle size was approximately 100 nm of TA-CNPS-PF and the hydrogel had a homogeneous three-dimensional reticulation structure. The encapsulation efficiency of TA in the CNPS were 99.3% and the release ratio of TA-CNPS-PF was approximately 86% and has antimicrobial properties. TA-CNPS-PF promoted diabetic wound healing significantly. Histopathology confirmed that TA-CNPS-PF promoted complete re-epithelialization and adequate collagen deposition. TA promoted the polarization of M1 macrophages into M2 macrophages via light microscopy, immunocytometry and flow cytometry. TA-CNPS-PF also promoted an increase in the number of M2 macrophages in diabetic wounds.

Conclusion: TA promotes diabetic wound healing by promoting the polarization of M1 macrophages into M2 macrophages and TA-CNPS-PF has good antimicrobial activity and a good drug release ratio in this study, which provides a new direction for the treatment of diabetic wounds and is expected to be highly advantageous in clinical diabetes wound therapy.

Keywords: M1 macrophages; M2 macrophages; TA nanoparticle hydrogel; TA-CNPS-PF; diabetic wound; thonningianin A. TA.

Graphical abstract

Figure 1

Characterization and antimicrobial properties of TA-CNPS-PF. (A) Appearance of the TA working solution and appearance of saline, PF-127, lyophilized CNPS, CNPS-PF, lyophilized TA-CNPS and TA-CNPS-PS; (B) Scanning electron microscopy image of the morphology of the CNPS, TA-CNPS and measured particle sizes (30,000× magnification, the red lines show the nanoparticles and measured size); (C) Scanning electron microscopy images of the morphology of the CNPS-PF and TA-CNPS-PF hydrogels (200× and 400×); (D) TA release behavior from TA-CNPS-PF at 37 °C (n = 3); (E) Control, saline, hydrogel, CNPS-PF and TA-CNPS-PF on E. coli and S. aureus inhibition zone images; TA-CNPS-PF had better in vitro antimicrobial properties (n = 3). Hydrogels also exhibit certain antibacterial abilities. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 2

Wound healing efficacy of TA-CNPS-PF in vivo. (A) Flowchart of the animal experiment; (B) Random blood glucose and body weight data for each group of rats. A high-sugar and high-fat diet was fed for 4 weeks, and STZ (30 mg/kg) was intraperitoneally injected into the rats to establish a diabetes mellitus model. The random blood glucose levels of the rats in each group were measured at 0, 2, 3, 4, 5, 6, 7, and 14 days and the body weights of the rats in each group were measured at 0 and 14 days. According to the results, the random blood glucose level was greater than 16.7 mmol/L, and the body weight decreased significantly, whereas the blood glucose level in the control group did not change significantly, but the body weight increased at 14 d (n=5). (C) Macroscopic images of the whole body of rats in the control and diabetic groups and panoramic images of the healing of skin wounds in different treatment groups. One deep circular wound was produced in the skin of the back of each rat, d=2 cm, and was replaced with a new dressing at 3 d. The skin of the back of the rats had healed at 3, 7 and 14 d (n=5). (D) Quantitative analysis of the WHR of each group based on the wound area measured from the pictures taken at 0, 3, 7 and 14 d (n=5). *P<0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 3

Histological analyses of regenerated skin tissues. (A) HE staining and Masson staining of microscopy images at 0 d. The dermis and fat layer of the skin in the control group were thicker than those in the diabetic group. The collagen fibers in the dermis of the control group were tighter and more regularly arranged than those in the dermis of the diabetic group. The black arrows indicate the fat layer in the samples. Magnified images of the corresponding area indicated by the black rectangular box (4×, 10×) (n=5); (B) Microscopy images of HE staining at 7 and 14 days. Blue arrows: neovascularization; yellow arrows: inflammatory cell infiltration; magnified images of the corresponding regional areas are indicated by black rectangular boxes (4×, 10×) (n=5). (C) Microscopy images of Masson staining at 7 and 14 days. Blue indicates collagen fibers, red indicates muscle fibers, and magnified images of the corresponding areas are indicated by the black rectangular box (4×,10×) (n=5). *P<0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 4

TAs promote the polarization of M1 macrophages into M2 macrophages. (A) Flowchart of the effects of TA on RAW264.7 cells; (B) Appearance of 20 mm TA and measurement of the effects of different concentrations of TA at 0, 1, 2.5, 5, 10, and 20 μM on RAW264.7 cells via the CCK8 assay. The cellular activity decreased with increasing TA concentration. The IC₅₀ of TA on RAW264.7 cells was calculated, and the concentration of TA administered was determined to be 2 μM (n=3). (C) Light microscopy images of the control, high-glucose, high-glucose + 2 μM TA and 2 μM TA groups. The morphology of the macrophages in the high glucose+ 2 μM TA group changed from round to pike, and a small number of pike cells were observed in the 2 μM TA group. In the control and high-glucose groups, the cells were round (40×) (n=3). Immunocytometry of CD86 (green fluorescence, an M1 macrophage marker), CD206 (red fluorescence, an M2 macrophage marker), and nuclei (blue fluorescence, a nuclear marker) in the high-glucose group induced M1 macrophages, and TA induced the polarization of M1 macrophages into M2 macrophages (400 ×) (n=3). (D) Flow cytometry was used to detect the effect of TA on the expression of CD206. RAW264.7 cells in the M0 state were treated with 40 mmol/L glucose for 24 h in the presence of TA and labeled with an APC-coupled antibody against CD206 for flow cytometric analysis (n=3). (E) Immunofluorescence was used to detect the expression of CD206 in rat skin tissues on Day 7. CD206 (red fluorescence, M2 macrophage marker) and nuclei (blue fluorescence, nuclear marker) were detected (n=5). *P<0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.