A Novel Bioswitchable miRNA Mimic Delivery System: Therapeutic Strategies Upgraded from Tetrahedral Framework Nucleic Acid System for Fibrotic Disease Treatment and Pyroptosis Pathway Inhibition

Abstract

There has been considerable interest in gene vectors and their role in regulating cellular activities and treating diseases since the advent of nucleic acid drugs. MicroRNA (miR) therapeutic strategies are research hotspots as they regulate gene expression post-transcriptionally and treat a range of diseases. An original tetrahedral framework nucleic acid (tFNA) analog, a bioswitchable miR inhibitor delivery system (BiRDS) carrying miR inhibitors, is previously established; however, it remains unknown whether BiRDS can be equipped with miR mimics. Taking advantage of the transport capacity of tetrahedral framework nucleic acid (tFNA) and upgrading it further, the treatment outcomes of a traditional tFNA and BiRDS at different concentrations on TGF-β- and bleomycin-induced fibrosis simultaneously in vitro and in vivo are compared. An upgraded traditional tFNA is designed by successfully synthesizing a novel BiRDS, carrying a miR mimic, miR-27a, for treating skin fibrosis and inhibiting the pyroptosis pathway, which exhibits stability and biocompatibility. BiRDS has three times higher efficiency in delivering miRNAs than the conventional tFNA with sticky ends. Moreover, BiRDS is more potent against fibrosis and pyroptosis-related diseases than tFNAs. These findings indicate that the BiRDS can be applied as a drug delivery system for disease treatment.

Keywords: bioswitchable miR inhibitor delivery system; gene delivery; miRNAs; pyroptosis; skin fibrosis; tetrahedral framework nucleic acids.

Figure 1

Successful synthesis of bioswitchable miRNA mimic delivery system (BiRDS) and inhibition of skin fibrosis. Three miRNA molecules formed a tetrahedral structure with nucleic acid core based on Watson‐Crick base pairing rules. Under the action of RNase H, BiRDS is converted to darts and released miRNA in cells and skin tissues. It protected miRNA mimics, reduced skin thickness, maintained epithelial polarity, and inhibited smad and pyroptosis pathway.

Figure 2

Synthesis, verification, characteristics, and stability of BiRDS. A) BiRDS were synthesized with one‐pot annealing, composing of “sun core” (inner blue kernel) and “immortal birds” (outer red wings). B) Agarose gel electrophoresis experiments illustrated the successful package of tFNA and BiRDS. The proportional growth of fluorescence intensity proved the stepwise polymer of the materials. The nude miR‐27a without the protection of sun core only exhibited weak fluorescence. C) The detection by transmission electron microscope and atomic force microscope showed the tetrahedral shape of tFNA and BiRDS, with the same diameter order of magnitude as data in Figure 2D. Scale bars are 100 nm. D) Size and Zeta potential distribution of BiRDS and tFNA by number, which demonstrated the nanoscale diameters and negative electric charge of materials. E,F) BiRDS exhibited excellent stability when reacting with 0%–10% FBS for 2 h under 37 °C. It also maintained prominent stability in 1% FBS for at least 24 h. G) BiRDS has reliable storage stability at room temperature (25 °C) and hardly breaks down in 7 days. H) Enzyme can specifically recognize DNA–RNA hybrid identification and cut side arms of BiRDS. Data are presented as mean ± standard deviation (SD) (n = 3).

Figure 3

Cellular uptake of BiRDS and tFNA, cell proliferation, and effects on epithelial–mesenchymal transition (EMT) inhibition. A) The hybridization regions of BiRDS can hydrolyze in the presence of Rnase H. Each BiRDS molecule converted to a dart and released three miRs in cytosol. B) BiRDS and tFNA were largely uptaken into HaCaT cells after incubated for 12 h. Cy5 fluorescence signals were located at cytoplasm near the nucleus. C) Relative intensity of Cy5 labeling single strands, tFNA and BiRDS entering cells. D) Cell viability results via CCK8 experiments showed tFNA and BiRDS have positive effects on proliferation of HaCaT cells. E,I) Immunofluorescence (IF) results and 3D surface plots of αSMA expression after cells treated with TGF‐β, and its data analysis. F,J) IF staining and its statistical analysis of E‐cadherin. G,K) IF staining and its statistical analysis of Collagen‐I. H) Westernblot (WB) results were for testing protein expressions of αSMA and Collagen I, taking GAPDH as the internal standard. L,M) Data analysis of WB results in (H). Scale bars are 50 or 7 µm as labeled at the corner of images. Data are presented as mean ± standard deviation (SD) (n = 3). Statistical analysis: single tailed <0.05, double tailed <0.01, triplex tailed <0.001. * means “compared with the target in TGF‐β group”, and # means “compared with the target in BiRDS group”.

Figure 4

BiRDS inhibited Smad2/3 and pyroptosis pathways. A) WB results of fibronectin and Smad2/3 for analyzing protein expression levels. B,C) IF images and 3D surface plots of fibronectin and Smad2/3. D,E) Statistical analysis of WB and IF experiments in (A–C). F) Schematic illustration of pyroptosis pathway and BiRDS effects on different molecules and phases, from cell rupture to complete integration. G) BiRDS and tFNA inhibited pyroptosis pathway‐related proteins in WB results. H,I) IF images and 3D surface plots of Cleaved caspase‐1 and IL‐1β. J–N) Semi‐quantitative analysis of pyroptosis‐related protein expression in WB and IF experiments. All scale bars are 50 µm or 15 µm, as labeled at the corner of images. Statistical analysis: single‐tailed <0.05, double‐tailed <0.01, triple‐tailed <0.001. * represents the “compared with target in TGF‐β group,” and # represents the “compared with target in BiRDS group”.

Figure 5

BiRDS inhibited skin fibrosis, maintained normal structures, and protected miR‐27a in vivo. A) Experimental scheme of in vivo experiments, including fibrosis model establishing, injection, molecule detection, and data analysis. B) Masson staining of skin tissues in each experiment group on day 21. C) Data analysis of skin thickness on days 0, 7, 14, and 21 showed 250 nm BiRDS had the most potent inhibition effect on skin fibrosis. D) Hydroxyproline content in each group was tested on day 21. E) HE‐staining images demonstrated change in skin tissues. Solid and dashed boxes magnified represented epithelial and subcutaneous tissue structures, respectively. (Yellow arrows: epithelial spikelike structure; blue arrows: glandular sebacea; green arrows: adipose tissue; yellow arrows: blood capillary.) F) Fluorescence signal intensity was detected within 120 min injection of the miR‐27a mimics, BiRDS and tFNA, and the semi‐quantitive analysis of fluorescence intensity was conducted. G) The tissues’ signals were detected at the end of 2 h. Scale bars are 400 or 100 µm, as labeled at the corner of images. Statistical analysis: single‐tailed <0.05, double‐tailed <0.01, triple‐tailed <0.001. *represents the “compared with target in bleomycin (B) group,” and # represents the “compared with target in 250 nm BiRDS group”.

Figure 6

BiRDS inhibited skin fibrosis, maintained normal structures, and protected miR‐27a in vivo. A) Experimental scheme of in vivo experiments, including fibrosis model establishment, injection, molecule detection, and data analysis. B) Masson staining of skin tissues in each experiment group on day 21. C) Data analysis of skin thickness on days 0, 7, 14, and 21 showed 250 nm BiRDS had the most potent inhibition effect on skin fibrosis. D) Hydroxyproline content in each group was tested on day 21. E) Hematoxylin and eosin H,E) staining images demonstrated change in skin tissues. The solid and dashed boxes magnified represented epithelial structures and subcutaneous tissue structures, respectively. (Yellow arrows: epithelial spikelike structure; blue arrows: glandular sebacea; green arrows: adipose tissue; yellow arrows: blood capillary), F,G) Fluorescence signal intensity was detected within 120 min of injection of miR‐27a mimics, BiRDS, and tFNA, and tissue signals were detected after 2 h. Scale bars are 400 or 100 µm, as labeled at the corner of images. Statistical analysis: single‐tailed <0.05, double‐tailed <0.01, triple‐tailed <0.001. * represent the “compared with target in bleomycin (B) group,” and # represents the “compared with target in 250 nm BiRDS group”.

Figure 7

BiRDS inhibited bleomycin‐induced EMT and Smad pathway. A) Schematics illustrating TGF‐β activation, EMT, and Smad2/3 pathway. HaCaT cells converted to myofibroblasts under propulsion of EMT, which could be reversed by BiRDS. B,C) Immunohistochemical staining experiments of skin tissues were conducted to analyze protein levels of TGF‐β and αSMA, which were highly expressed in bleomycin groups and inhibited in group receiving 250 nm BiRDS. D) Double fluorescence staining images for evaluating expression levels of Smad and fibronectin (red: Smad; green: fibronectin). E,F) Semi‐quantitative analysis and statistical tests of proteins demonstrated in Figure 6B–D. Scale bars are 300 or 50 µm as labeled at the corner of images. Statistical analysis: single‐tailed <0.05, double‐tailed <0.01, triple‐tailed <0.001. * represents the “compared with target in bleomycin (B) group,” and # represents the “compared with target in 250 nm BiRDS group”.