Tetrahedral framework nucleic acids promote diabetic wound healing via the Wnt signalling pathway

Abstract

Objectives: To determine the therapeutic effect of tetrahedral framework nucleic acids (tFNAs) on diabetic wound healing and the underlying mechanism.

Materials and methods: The tFNAs were characterized by polyacrylamide gel electrophoresis (PAGE), atomic force microscopy (AFM), transmission electron microscopy (TEM), dynamic light scattering (DLS) and zeta potential assays. Cell Counting Kit-8 (CCK-8) and migration assays were performed to evaluate the effects of tFNAs on cellular proliferation and migration. Quantitative polymerase chain reaction (Q-PCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the effect of tFNAs on growth factors. The function and role of tFNAs in diabetic wound healing were investigated using diabetic wound models, histological analyses and western blotting.

Results: Cellular proliferation and migration were enhanced after treatment with tFNAs in a high-glucose environment. The expression of growth factors was also facilitated by tFNAs in vitro. During in vivo experiments, tFNAs accelerated the healing process in diabetic wounds and promoted the regeneration of the epidermis, capillaries and collagen. Moreover, tFNAs increased the secretion of growth factors and activated the Wnt pathway in diabetic wounds.

Conclusions: This study indicates that tFNAs can accelerate diabetic wound healing and have potential for the treatment of diabetic wounds.

FIGURE 1

Preparation, characterization and cellular uptake of tFNAs. (A) Schematic illustration of the synthesis of tFNAs. (B) PAGE examination of the molecular weights. (C) AFM evaluation of the tFNAs. The scale bar is 500 nm. (D) TEM evaluation of the tFNAs. The scale bar is 100 nm. (E) Size distribution of the tFNAs. (F) Zeta potential distribution of the tFNAs. (G) Cellular uptake of tFNAs and ssDNAs (Cy5‐tFNAs and Cy5‐ssDNAs: red; nucleus: blue; cytoskeleton: green). Scale bars are 25 μm

FIGURE 2

The effects of tFNAs on cellular proliferation, migration and growth factors. (A,B) In vitro wound‐healing assay of HaCaTs and semi‐quantification of the migration areas. The scale bar is 200 μm. (c) CCK‐8 results from HaCaTs. (D,E) In vitro wound‐healing assay of HDFs and semi‐quantification of the migration areas. The scale bar is 200 μm. (F) CCK‐8 results from HDFs. (G,H) In vitro wound‐healing assay of HUVECs and semi‐quantification of the migration areas. The scale bar is 100 μm. (I) CCK‐8 results from HUVECs. (J) ELISA results of bFGF, TGF‐β1 and VEGF‐A secretion at 24 h. (K) Transcript‐level expression of bFGF, TGF‐β1 and VEGF‐A by Q‐PCR at 24 h. Data are presented as the mean ± standard deviation (SD) (n = 5). Significance: *p < 0.05, **p < 0.01, ***p < 0.001

FIGURE 3

tFNAs facilitated diabetic wound healing, and increased epithelialization and collagen deposition in diabetic wounds. (A) Photographs of cutaneous wounds in diabetic mice treated with saline or 250 nM tFNAs at different time points. The scale bar is 5 mm. (B) Calculation and comparison of the wound healing area percentages between the control and tFNAs groups at different time points. (C) H&E staining of the control and tFNAs groups on day 21 after surgery. Scale bars are 250 or 50 μm. (D) Comparison of the average regenerative epidermal thickness between the control and tFNAs groups. (E) Comparison of the deposited collagen percentages between the control and tFNAs groups. (F) Masson staining of the control and tFNAs groups on day 21 after surgery. Scale bars are 250 or 50 μm. (G) Picrosirius red staining of the control and tFNAs groups on day 21 after surgery. The scale bar is 25 μm. Data are presented as the mean ± SD (n = 4). Significance: *p < 0.05, **p < 0.01, ***p < 0.001

FIGURE 4

tFNAs increased vascularization in diabetic wounds. (A) Immunofluorescence staining of CD31 and α‐SMA expression of the control and tFNAs groups (white arrow: vessel; nucleus: blue; CD31: red; α‐SMA: green). Scale bars are 50 μm. (B) Immunohistochemical staining of CD31 expression of the control and tFNAs groups (black arrow: vessel). The scale bar is 50 μm. (C) Quantification analysis of the number of CD31‐ and α‐SMA‐positive blood vessels between the control and tFNAs groups. (D) Quantification analysis of the number of CD31‐positive blood vessels between the control and tFNAs groups. Data are presented as the mean ± SD (n = 3). Significance: *p < 0.05, **p < 0.01, ***p < 0.001

FIGURE 5

tFNAs regulated the secretion of growth factors and the Wnt signalling pathway in diabetic wounds. (A) ELISA results of bFGF, TGF‐β1 and VEGF‐A secretion in the control and tFNAs groups. (B) Immunofluorescence to check for the expression of LEF1 and TCF1 in the control and tFNAs groups (nucleus: blue; LEF1: red; TCF1: pink). Scale bars are 50 μm. (C) Western blot detection of β‐catenin, c‐Myc and cyclin D1 expression in the control and tFNAs groups. (D) Semi‐quantification of Wnt signalling pathway protein levels in (C). Data are presented as the mean ± SD (n = 3). Significance: *p < 0.05, **p < 0.01, ***p < 0.001

FIGURE 6

Graphical abstract of the therapeutic effects of tFNAs in diabetic wound healing (created with BioRender.com, Agreement number: AO23WPVP88)