Tetrahedral framework nucleic acids-based delivery of MicroRNA-22 inhibits pathological neovascularization and vaso-obliteration by regulating the Wnt pathway

Abstract

The objective of this study was to investigate the effects and molecular mechanisms of tetrahedral framework nucleic acids-microRNA22 (tFNAs-miR22) on inhibiting pathological retinal neovascularization (RNV) and restoring physiological retinal vessels. A novel DNA nanocomplex (tFNAs-miR22) was synthesised by modifying microRNA-22 (miR22) through attachment onto tetrahedral frame nucleic acids (tFNAs), which possess diverse biological functions. Cell proliferation, wound healing, and tube formation were employed for in vitro assays to investigate the angiogenic function of cells. Oxygen-induced retinopathy (OIR) model was utilised to examine the effects of reducing pathological neovascularization (RNV) and inhibiting vascular occlusion in vivo. In vitro, tFNAs-miR22 demonstrated the ability to penetrate endothelial cells and effectively suppress cell proliferation, tube formation, and migration in a hypoxic environment. In vivo, tFNAs-miR22 exhibited promising results in reducing RNV and promoting the restoration of normal retinal blood vessels in OIR model through modulation of the Wnt pathway. This study provided a theoretical basis for the further understanding of RNV, and highlighted the innovative and potential of tFNAs-miR22 as a therapeutic option for ischemic retinal diseases.

FIGURE 1

Synthesis and characterisation of tFNAs‐miR22. (A) The synthesis of tFNAs‐miR22 is illustrated in a schematic diagram. (B) Successful generation of tFNAs and tFNAs‐miR22 was confirmed using high‐performance capillary electrophoresis (HPCE). (C) Polyacrylamide gel electrophoresis (PAGE) was used to detect the molecular weights of the synthesised tFNAs and tFNAs‐miR22. (D) Transmission electron microscope (TEM) image shows the molecular structure of the synthesised tFNAs and tFNAs‐miR22, with scale bars measuring 200 nm. (E) High‐performance liquid chromatography (HPLC) was performed on purified tFNAs to remove mismatched bases or single strands, as shown in the results. (F) Dynamic light scattering (DLS) analysis characterises the properties of both tFNAs and tFNAs‐miR22. (G) Zeta potential analysis measures the stability of both tFNAs and tFNAs‐miR22. H. After 8 h, HUVECs show uptake of Cy5‐loaded‐tFNAS‐miR22, indicated by red fluorescence, while blue fluorescence represents nuclei staining and green fluorescence represents cytoskeleton staining. Scale bars measure 25 μm. (I) Flow cytometry is used to observe uptake of miR‐22 without transfection reagent as well as uptake of tFNAs‐miR22 by HUVECs.

FIGURE 2

tFNAs‐miR22 inhibit angiogenesis in vitro. (A) The cell proliferation assay results of HUVECs after various treatments were evaluated. The scale bars used in the images are 500 μm. (B) Tube formations of HUVECs were examined at 12 h following different treatments. The scale bars used in the images are 500 and 200 μm. (C) Binary Image analysis was performed on scratch‐wound assays using HUVECs at 0, 24, and 48 h post‐treatment to assess wound healing progress. The scale bars used in the images are 375 μm. (D) The percentage of nuclei that were positive for Hoechst (blue) and also colocalized with EdU (red) was determined through quantification analysis. The data are presented as the mean ± standard deviation (n = 6). (E) Statistical analysis was performed on the measurements of capillary lengths, and the data are presented as the mean ± SD (n = 6). (F) Branch points were quantified and analysed statistically to evaluate angiogenesis potential. Data are presented as mean ± SD (n = 6). (G) and (H) Wound healing area rates at both 24 and 48 h post‐treatment were calculated. Data are presented as mean ± SD (n = 6). Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 result from ANOVA test.

FIGURE 3

tFNAs‐miR22 reduce avascular area and inhibit pathological angiogenesis in OIR model on retinal flat‐mount. (A) Explanation of the in vivo experiment using the OIR model to study retinal anatomy. OIR refers to oxygen‐induced retinopathy. (B) Illustrative images of the entire retinal tissue stained with isolectin B4 (IB4). The vaso‐obliterated area (VO) is indicated by a yellow dotted line. The scale bars represent 1 mm. (C) Representative visuals displaying the neovascular area. Scale bars: 1 mm. The second row highlights the neovascular region, depicted by a white line. (D) Assessment of both vaso‐obliterated area (VO) and neovascular area. The data are presented as mean ± SD (n = 3). Statistical analysis: the ANOVA test was applied, ***p ≤0.001, **p ≤0.01, and *p <0.05.

FIGURE 4

tFNAs‐miR22 reduce the number and mean length of filopodia in OIR model on retinal flat‐mount. (A) Representative images of filopodia (dashed boxes in the second row) from Normoxia, OIR, OIR + AFL, OIR + tFNAs, OIR + miR22, and OIR + tFNAs‐miR22 retinas. The magnified images of filopodia in the second and bottom row. (Red line and blue line). Scale bars are: 50 μm (Top row); 10 μm (second row); 10 μm (Bottom row). (B) Analysis of count, total lengths and average length of filopodia. Data are presented as mean ± SD (n = 6). All error bars represent SEM. Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 result from ANOVA test.

FIGURE 5

tFNAs‐miR22 prevent HUVECs proliferation and reduce avascular area via the Wnt pathway. (A) The expression levels of FZD4, GSK3β, β‐catenin, and c‐Myc were analysed using Western blotting (with β‐Actin as the internal control). (B) A summary diagram illustrating the synthesis process of tFNAs‐miR22 and its role in reducing retinal vascular abnormalities through the Wnt pathway. (C) The protein expression intensity of FZD4, GSK3β, β‐catenin, c‐Myc, and β‐actin was quantified relative to their respective controls. Mean ± SD values are presented (n = 3). (D) The mRNA expression intensity of FZD4, GSK3β, β‐catenin, c‐Myc, and β‐actin was measured relative to their respective controls. Mean ± SD values are presented (n = 3).Statistical analysis: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 result from ANOVA test.