Tetrahedral framework nucleic acids inhibit pathological neovascularization and vaso‐obliteration in ischaemic retinopathy via PI3K/AKT/mTOR signalling pathway

Abstract This study aimed to explore the effect and the molecular mechanism of tetrahedral framework nucleic acids (tFNAs), a novel self‐assembled nanomaterial with excellent biocompatibility and superior endocytosis ability, in inhibition of pathological retinal neovascularization (RNV) and more importantly, in amelioration of vaso‐obliteration (VO) in ischaemic retinopathy. tFNAs were synthesized from four single‐stranded DNAs (ssDNAs). Cell proliferation, wound healing and tube formation assays were performed to explore cellular angiogenic functions in vitro. The effects of tFNAs on reducing angiogenesis and inhibiting VO were explored by oxygen‐induced retinopathy (OIR) model in vivo. In vitro, tFNAs were capable to enter endothelial cells (ECs), inhibit cell proliferation, tube formation and migration under hypoxic conditions. In vivo, tFNAs successfully reduce RNV and inhibit VO in OIR model via the PI3K/AKT/mTOR/S6K pathway, while vascular endothelial growth factor fusion protein, Aflibercept, could reduce RNV but not inhibit VO. This study provides a theoretical basis for the further understanding of RNV and suggests that tFNAs might be a novel promising candidate for the treatment of blind‐causing RNV.

known as one of the most metabolically active tissues, consuming O 2 more rapidly than even the brain. 8 Maintenance of an adequate oxygen supply is critical for retinal function. The high oxygen demands of the retina, and the relatively sparse nature of the retinal vasculature, are thought to contribute to the particular vulnerability of the retina to vascular disease. A large proportion of retinal blindness is associated with diseases having a vascular component, and disrupted oxygen supply to the retina is likely to be a critical factor. 9 Currently, RNV patients with advanced ROP, PDR or RVO have been treated with retinal photocoagulation to decrease oxygen demand and relieve hypoxia, 10,11 or anti-vascular endothelial growth factor (VEGF) therapy to inhibit neovascularization, [12][13][14] while due to laser-induced loss of retinal tissue, retinal photocoagulation can lead to complications, such as decreased visual acuity, diminished night vision and persistent visual field constriction. 15 Anti-VEGF therapy is the first-line treatment for retinal neovascular diseases. It has been used clinically to treat hypoxia-induced RNV in patients with ROP, DR, as well as RVO. 11,[16][17][18][19] However, anti-VEGF drug has potential drawbacks. First, adverse effects associated with the blockade of VEGF signalling, including impairments of normal retinal vascular growth and retinal function, were suggested. Second, recurrence of RNV after intravitreal anti-VEGF administration, due to the persistence of the ischaemic/non-perfusion conditions, 11,20 is frequent in premature babies or diabetic patients. 11,21 Thus, there are highly desirable and, as-yet, unmet medical needs, for alternative therapeutic strategies for treating RNV.
Recently, DNA nanotechnology has been developed and employed in a variety of fields owing to the extensive biological functions of DNA nanostructures. [22][23][24][25][26] Tetrahedral framework nucleic acids (tFNAs) are synthesized by four isometric single-stranded DNAs (ssDNAs) through a simple, rapid and reliable process [27][28][29] which have been identified as three-dimensional (3D) DNA nanostructures with a stable structure and superior mechanical properties. [30][31][32][33][34] The tFNAs have been used extensively in various biological fields with considerable merits, including biocompatibility, structural stability and programmability. [35][36][37][38][39][40][41][42] For example, tFNAs could prevent apoptosis of neurons caused by oxygen-glucose deprivation/reoxygenation through interfering with ischemia in vitro and effectively ameliorate the microenvironment of the ischaemic hemisphere by upregulating expression of erythropoietin and inhibiting inflammation. 43 We also found that tFNAs could promote angiogenesis in vitro and in vivo, via activation of Notch signalling, JAK/STAT signalling, as well as Akt/Nrf2/ HO-1 Pathway. [44][45][46][47] Furthermore, tFNAs were found to be effective in ocular diseases prevention and treatment, for instance, for repairing corneal injury and suppress the oxidative stress in retinal ganglion cells. [48][49][50] In addition, we previously showed that tFNAs, as vectors, inhibit choroidal neovascularization by polarizing macrophages. 51 Hence, in this work, we chose tFNAs to explore the therapeutic effect and underlying mechanism in the treatment of RNV. Therefore, we ventured to speculate that tFNAs can enter human umbilical vein endothelial cells (HUVECs) and regulate against neovascularization, for therapeutic purposes. Meanwhile, we also investigated the therapeutic effects of tFNAs on oxygen-induced retinopathy (OIR) mouse models as well as the underlying mechanisms. Our results showed that tFNAs could effectively enter HUVECs and inhibit angiogenesis under hypoxic environment. More interestingly, we observed that tFNAs treatment on the ischaemic retina in OIR mouse models is beneficial not only for inhibiting pathological neovascularization, but also for ameliorating retinal avascular area and inhibiting retinal vaso-obliteration (VO) area by modulating the PI3K/AKT/mTOR signalling pathway, suggesting that it may be a promising agent for the treatment of ischaemic RNV.

| Synthesis of tFNAs
Four ssDNA were synthesized and characterized by Genescript (Nanjing, China). First, TM buffer with a pH of 8.0 containing 10 mM Tris-HCl and 50 mM MgCl 2 was prepared. Then, equal concentrations of the four ssDNAs were poured into TM buffer. After thorough mixing and centrifugation, the mixed solution was heated to 95 C for 10 min and then cooled to 4 C for 20 min to obtain tFNAs. The sequences of the four ssDNAs are shown in Table 1. Five hundred microlitres of tFNAs sample was concentrated to 100 μL with a 10 kD ultrafiltration tube and added to a high-performance liquid chromatography (HPLC). Then, tFNAs were purified with a DNA Pac™PA100 (Thermo Scientific, USA) chromatographic column at a flow rate of 1 mL min À1 with different mobile phases (mobile phase A:25 mM Tris-HCL, mobile phase B: 25 mM Tris-HCL + 375 mM NaClO 4 ). Next, the main peak of the sample was collected, and polyacrylamide gel electrophoresis (PAGE) was used for purity detection.

| Characterization of tFNAs
We conducted experiments using the methods described in previous studies to verify the successful synthesis of tFNAs. Briefly, we used PAGE and high-performance capillary electrophoresis (HPCE) to detect the difference in the molecular weights of tFNAs and then used a nanoparticle size analyser to detect the difference between the zeta potential and particle sizes of the two reagents to determine whether the synthesis was successful.

| Cell culture and treatment
HUVECs were purchased from ATCC. We incubated the cells with

| Uptake of Cy5-loaded-tFNAs
In this experiment, we tested whether tFNAs successfully entered HUVEC cells. We modified one of the ssDNAs (S2) labelled with Cy5 fluorescence in the tFNAs. HUVEC cells were cultured on a 6-well plate at the density of 4 Â 10 5 cells per well. After the cells were cultured for 24 h, the Cy5-tFNAs were added and cultured for 8-24 h, respectively. We then measured the fluorescence intensity at 8 h and compared it with the blank control group. And samples meeting the requirements of flow cytometry were collected. Flow cytometry (Attune NxT, ThermoFisher Scientific, USA) was used to spot the intracellular fluorescence intensity at 24 h.

| Cell immunofluorescence assay
Cell immunofluorescence staining was conducted to evaluate the expression of proteins related to angiogenesis and cell apoptosis.
HUVECs were plated in 6-well plates (Corning, USA) at a density of 9.6 Â 10 5 cells and treated as mentioned above. Then, the cells were   Table 2 lists the corresponding primers for the genes (VEGF, HIF-1α, PI3K, AKT and mTOR), all of which are BLAST search design with β-actin amplification as the control.

| Protein extraction and western blot analysis
HUVECs were cultured, divided into groups, and treated accordingly.
Then, tobramycin eye ointment was applied to prevent infection after the operation.

| Whole-mounted retinal immunofluorescence
Mice were euthanized at P17, and eyes were enucleated and fixed area and total retinal area were quantified using ImageJ. The area in each clock hour exhibiting RNV was measured and added together as the total RNV area per retina and expressed as a percent of the total area of the retina.

| Retinal frozen sectional immunofluorescence
To make retinal frozen sections, eyes of mice were enucleated and fixed in 4% PFA for 1 h at RT. Next, eyes were dehydrated in 10%, The frozen retinal sections were then analysed by confocal microscopy (Carl Zeiss, Oberkochen, Germany).

| Synthesis and characterization of tFNAs
Four ssDNAs in equimolar amounts shown in Table 1 were successfully synthesized into tFNAs. Each ssDNA was further divided into three small fragments self-assembled to form one triangle facet and hybridized with the other three strands according to highly specific complementary base-pairing. A brief description of the synthesis of tFNAs is provided ( Figure 1A). Among the ssDNAs, the fluorescent molecule Cy5 designed on the S2 chain was used to show the localization of tFNAs in subsequent experiments. The results of 8% polyacrylamide gel electrophoresis (PAGE) verified that we had succeeded in synthetizing tFNAs. According to the theoretical value in previous studies that tFNAs are comprised of four ssDNA molecules ( Figure 1B). 56 In addition, high-performance capillary electrophoresis (HPLC) was used to detect the successful synthesis of tFNAs ( Figure 1C), and the results were consistent with those obtained by PAGE.
Next, we examined the characterization of tFNAs by transmission electron microscopy (TEM) ( Figure 1D). The result indicated that, similar to the results in previous studies, 35,49 the successfully synthesized tFNAs were triangle like. The particle size of tFNAs was approximately 19.30 nm. As nucleic acids have a negative charge, their zeta potential was approach to À6.41 mV ( Figure 1E), confirming the stability of the synthesized tFNAs. The results showed that the purity of tFNAs was effectively improved ( Figure 1F).

| Cellular uptake of tFNAs
We validated the ability of tFNAs to enter HUVECs. HUVECs were treated with Cy5-loaded-tFNAs for 8 h and determined the localization of tFNAs via immunofluorescence assay. It could be observed that tFNAs were already present in the cytoplasm ( Figure 1G). Cells were then collected for flow cytometry analysis after 24 h Cy5-loaded-tFNAs treatment, and the results showed that the entry rate of tFNAs into cells was 99.867% while the entry rate in control group was only 0.176% (p < 0.001, Figure 1H).

| tFNAs reduce angiogenesis and inhibit the VO in OIR model
The (OPL). 57 In this study, all the three plexuses were unremarkable in three normoxia groups, even after AFL or tFNAs treatment ( Figure 4B). Moreover, RNV sprouted breakthrough the inner limiting membrane (ILM) in the vehicle-treated OIR group. All three layers of vascular structures disappeared in central area ( Figure 4B). In AFLtreated OIR mice, no vessels were noted in central area. Significant decrease of RNV was found in MP area with disappearance of IP and DP. In tFNAs-treated OIR mice, regenerated SP could be seen, with some IP and DP. In the MP area, fewer RNV was noted if compared with AFL in the MP area. In addition, there were no significant difference in the FP area among all groups ( Figure 4B).

| tFNAs ameliorate cell proliferation, migration and reduce RNV via the PI3K/AKT/mTOR pathway
To confirm whether tFNAs ameliorate cell proliferation and migration via the PI3K/AKT/mTOR/S6K pathway in this study, the expression levels of VEGFR2, AKT, PI3K subunits, mTOR and S6K in normoxic or hypoxic HUVECs were detected by western blot analysis ( Figure 5A, B). In our results, HIF-1α, VEGFR2, p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR and p-S6K/S6K increased conspicuously under hypoxia ( Figure 5A-D), and dramatically decreased after AFL and tFNAs treatment (all p < 0.05). Meanwhile, increased S6K immunofluorescence (red, Figure 5E) was detected in vehicle-treated hypoxic HUVECs, but significant decreased in AFL or tFNAs-treated cells ( Figure 5E). However, p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR and p-S6K/S6K deceased significantly more in tFNAs group, when compared with those in AFL group (p < 0.05 in p-AKT/AKT and p-S6K/S6K, p < 0.01 in p-PI3K/PI3K and p-mTOR/mTOR) ( Figure 5A-D), while VEGFR2 was inhibited more in AFL group, when compared with that in tFNAs group (p< 0.05, Figure 5C,D). Further confocal micrograph showed the extensions of filopodia at the leading edge of growing blood vessels at the edge of VO and we found that the number of filopodia (10.33 ± 3.01) and tip cells (5.17 ± 1.47) were significantly increased in tFNAs-treated cells, but not in AFL-treated cells ( Figure 5F, G). All the three plexuses were unremarkable in three normoxia groups in C, MP and FP areas. In nomoxia mice, SP were located in NFL, IP in IPL and DP in OPL (Columns 1-3). In vehicletreated OIR mice, no plexus was found in C. RNV sprouted breakthrough ILM. IP and DP were absent in FP (Column 4). In AFL-treated OIR mice, absence of three plexuses were noted in C. However, in tFNAs, vessels were found regenerated with relative normal structures (Columns 5 and 6).

| DISCUSSION
RNV is one of the leading irreversible causes of serious visual impairment and blindness in millions of patients worldwide. It is the cause of ROP, PDR and RVO. [2][3][4][5][6][7] Current mainstream therapies such as anti-VEGF drugs and photo coagulation has their potential drawbacks due to complications caused by blockade of VEGF signalling, persistence of the ischaemic/non-perfusion condition and loss of retinal tissue. [11][12][13][14][15]20 In our study, we explored the treatment effect and the molecular mechanism of tFNAs in ischaemic retinopathy. In consistent with previous studies, DNA nanomaterials also exhibit excellent stability and biological applications among similar materials. 35,49,56 To better demonstrate the effect of tFNAs and exclude confounding factors, we purified the synthesized tFNAs before experiments and tested their purity using HPLC before and after purification. After purification, supernumerary and mismatched bases and single strands were removed, which may be responsible for the discrepancy in the best concentration between in vivo experiments in this study and those (250 nmol L À1 ) in previous studies. 51 We confirmed in different ways that tFNAs were successfully synthesized and taken up by cells, which was the foundation for our subsequent experiments. As previously reported, DNA nanostructures have many excellent characteristics, which make them widely used in biological and biomedical applications. 58,59 The most outstanding characteristic of tFNAs compared to that of ssDNA and other spatial nanostructures, is that they enhanced endocytosis remarkably. 42 Importantly, tFNAs were noted to penetrate the cellular membrane without the help of transfection agents. Moreover, Fan et al. first used the single-particle tracking technique to report that Cy3-labelled tFNAs adjust their orientation so that their corners attach to the cell membrane, minimizing charge repulsion and leading to charge redistribution; this attachment is followed by the endocytosis of tFNAs via the caveolin-mediated pathway and their entry into lysosomes in a microtubule-dependent manner. 24,42,60 In our study, dose-effect experiment was conducted, and VEGF was most inhibited at the con- The OIR mouse model of hypoxia-induced NV is a wellestablished model for investigating novel putative antiangiogenic compounds. 52,67 In the in vivo animal experiment, a mouse model was conducted as previously described by our study. In this study, we herein demonstrated that consistent with the previous studies, [68][69][70] AFL showed the ability to reduce RNV, but not to inhibit VO. However, tFNAs dramatically inhibited VO in OIR retinas, which was superior to AFL. Interestingly, we found that VO area was significantly decreased after tFNAs treatment and regenerated superficial vascular plexus could be seen.
In an effort to better understand the molecular and cellular mechanisms underlying the effects of tFNAs in the treatment of ischaemic retinopathy, we attempted to study the PI3K/AKT/mTOR signalling pathway. Western blot and cell immunofluorescence staining analysis of the expression of proteins related to the PI3K/AKT/mTOR signalling pathways in vitro confirmed that tFNAs might inhibit angiogenesis through this signalling pathway. Our previous studies showed that tFNAs could activate the PI3K/AKT/mTOR pathway to enhance the autophagy, proliferation and migration of cells and promote angiogenesis, wound healing and neuroprotective effects. 38,56,[71][72][73][74] In our study, we found that the number of filopodia and tip cells were significantly increased in tFNAs-treated cells, but not in AFL- proliferation in vitro. Even more interestingly, as aforementioned, tFNAs-treated OIR mice were beneficial even more for ameliorating retinal VO and promoting the normalization of disrupted vasculature.
Based on these, we speculated that tFNAs might act as a novel and potential mTOR inhibitor. Our results showed that tFNAs reduced the extent of pathological retinal neovascular tufts significantly, which were consistent with those in previous studies on direct/indirect mTOR inhibitor (rapamycin or valproic acid, respectively). 78