Tetrahedral framework nucleic acid carrying miRNA in embedded mode and preparation method and application of tetrahedral framework nucleic acid

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to embedded miRNA-carried tetrahedral framework nucleic acid as well as a preparation method and application thereof. The miRNA is embedded into a tetrahedral framework nucleic acid (tFNA), ribonucleotide extends from the tail end of each miRNA chain to serve as a biological switch, the enzyme-responsive biological switch can be triggered by RNase H in a cell to realize opening of a three-dimensional structure, meanwhile, a foothold-mediated strand displacement reaction domain is arranged on the miRNA, combination of the miRNA and a target in the cell is facilitated, and the targeting effect of the miRNA is improved. The specific recognition on the cells is realized. Therefore, the invention provides a miRNA delivery platform with high carrying efficiency, high stability and good biological compatibility, and the miRNA delivery platform can effectively treat acute liver injury and acute kidney injury and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a tetrahedral framework nucleic acid embedded with miRNA and a preparation method and use thereof. Background Art

[0002] A variety of factors can lead to liver and kidney failure, causing severe damage, massive cell necrosis, and synthesis, secretion, transformation and detoxification of the liver. In severe cases, it can lead to kidney dysfunction or body incompensation. miRNA plays an important role in a series of physiological and pathological processes, and stands out among many nucleic acid drugs. In addition, the functions of miRNA are very extensive. A miRNA can show different biological functions in different tissues through direct or indirect regulation. For example, miRNA-125 can directly regulate kelch-like ECH-associated protein 1 in the liver, thereby upregulating the expression of nuclear factor-E2-related factor 2, a known regulator of acute liver failure (ALF), thereby improving ALF; while in the kidney, miRNA-125 can directly inhibit the protein expression of p53, which not only leads to the upregulation of CDK1 and Cyclin B1 to rescue G2 / M phase arrest, but also regulates Bcl-2 and Bax to inhibit apoptosis and treat acute kidney injury (AKI).

[0003] Due to the limitation of miRNA's own structure, it is easy to be degraded by nucleic acid nucleases in the blood when used in vivo, which also makes miRNA therapy unable to be widely used. Although miRNA delivery can be achieved through viral packaging, liposomes or dendrimers, the potential biosafety and uncertain delivery efficiency also make most miRNA therapies stop at the preclinical stage.

[0004] Tetrahedral framework nucleic acids (tFNAs) are a new type of nucleic acid nanomaterial. They are self-assembled into a tetrahedral structure by four prefabricated single-stranded DNAs. They have excellent in vivo properties, including low immunogenicity, good biocompatibility, and excellent stability. In addition, tFNAs are easy to prepare and have high yields, which support their use as the key to solving the problem of miRNA delivery in vivo. tFNAs can passively target the liver and kidneys in vivo and are highly enriched in these two organs, which means that they may be able to reduce off-target effects and improve the efficacy of miRNA therapy by delivering miRNAs related to liver and kidney damage. In addition, tFNAs also have anti-inflammatory and antioxidant effects and can activate pro-survival pathways. However, in the past, when tFNAs were used to transport miRNAs, siRNAs, or aptamers, the cargo was often loaded onto the apex of the tFNA or suspended on the side arms of the tFNA. However, this model not only increases the size of the nanostructure, but also exposes the transported nucleic acid drugs to a large number of nucleases in the blood, bringing more uncertainty to the cargo carried. In addition, this static delivery mode makes the release of miRNA uncontrollable.

[0005] Therefore, in order to improve the delivery efficiency of miRNA and ensure its stable and intact delivery to the target organs, it is urgent to develop a miRNA delivery platform with excellent transport efficiency, high stability and good biological compatibility, so as to open up new avenues for the innovative treatment of liver and kidney related diseases. Summary of the invention

[0006] In view of the problems of the prior art, the present invention provides a tetrahedral framework nucleic acid embedded with miRNA and a preparation method and use thereof.

[0007] A functionalized tetrahedral framework nucleic acid is a tetrahedral framework nucleic acid embedded with miRNA; the tetrahedral framework nucleic acid is assembled from three DNA single strands and three miRNA strands through base complementary pairing; the miRNA strand includes miRNA and 2-6 ribonucleotides at the end; 4-8 bp of ribonucleotides on the miRNA do not participate in the formation of the tetrahedral framework nucleic acid; the 2-6 deoxyribonucleotides at the end of the DNA single strand are complementary to the 2-6 ribonucleotides at the end of the miRNA strand, and the portion of the DNA single strand complementary to the miRNA is a ribonucleotide.

[0008] Preferably, in the miRNA chain, the length of the miRNA is 22-24 bp, and the number of terminal ribonucleotides is 2-6;

[0009] Preferably, in the miRNA chain, the length of the miRNA is 22 bp and the number of terminal ribonucleotides is 4.

[0010] Preferably, in the miRNA chain, the miRNA is selected from miRNA125; and / or the terminal ribonucleic acid is cuua.

[0011] Preferably, the miRNA chain is shown as SEQ ID NO.4.

[0012] Preferably, in the miRNA chain, the 4-8 bp ribonucleotides starting from the 8th to 11th ribonucleotides of the miRNA do not participate in forming the tetrahedral framework nucleic acid, and the remaining ribonucleotides complement the ribonucleotides on the DNA single strand to form the tetrahedral framework nucleic acid;

[0013] Preferably, in the miRNA chain, the 6 bp ribonucleic acid starting from the 9th ribonucleotide of miRNA does not participate in the formation of tetrahedral framework nucleic acid, and the 1st to 8th ribonucleotides and the 15th to 22nd ribonucleotides of miRNA complement the ribonucleotides on the DNA single strand to form a tetrahedral framework nucleic acid.

[0014] Preferably, the structure of the single-stranded DNA is RNA 1-deoxyribonucleic acid 2-RNA 3-deoxyribonucleic acid 4, wherein RNA 1 and RNA 3 are complementary to miRNA, and deoxyribonucleic acid 4 is complementary to the ribonucleotide at the end of miRNA; the lengths of RNA 1 and RNA 3 are (7-10) bp respectively, the length of deoxyribonucleic acid 4 is (2-6) bp, and the total length of the single-stranded DNA is 39-111 bp;

[0015] Preferably, the lengths of RNA 1 and RNA 3 are 8 bp respectively, the length of deoxyribonucleic acid 4 is 4 bp, and the total length of the DNA single strand is 63 bp.

[0016] Preferably, the nucleotide sequences of the three DNA single strands are shown as SEQ ID NO.1-SEQ ID NO.3.

[0017] The present invention also provides a method for preparing the above-mentioned functionalized tetrahedral framework nucleic acid, comprising the following steps: mixing three single-stranded DNAs and three miRNA chains, treating and incubating them in a thermal cycler, and obtaining the functionalized tetrahedral framework nucleic acid; the molar ratio of the three single-stranded DNAs is 1:1:1, and the molar ratio of any one single-stranded DNA to any one miRNA chain is 1:1.

[0018] The present invention also provides use of the functionalized tetrahedral framework nucleic acid in preparing a drug for treating acute liver injury and / or acute kidney injury.

[0019] The present invention also provides a medicine for treating acute liver injury and / or acute kidney injury, which is prepared by taking the above-mentioned functionalized tetrahedral framework nucleic acid as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

[0020] The structure of the tetrahedral framework nucleic acid of the present invention is as follows Figure 1 The tetrahedral framework nucleic acid is assembled by three DNA single strands and three miRNA strands through complementary base pairing, and the three miRNA strands form one face of the tetrahedral structure.

[0021] RNase H is an endoribonuclease that specifically decomposes the RNA chain in the RNA-DNA hybrid. The tetrahedral framework nucleic acid of the present invention is provided with a biological switch that depends on RNase H. Specifically, four ribonucleotides are connected to the end of the miRNA chain, and four complementary deoxyribonucleotides are provided at the end of the corresponding complementary DNA single strand, and the two complementarily pair to form an RNA-DNA hybrid. After the tetrahedral framework nucleic acid of the present invention enters the cell, RNase H can recognize the RNA-DNA hybrid to open the three-dimensional structure.

[0022] In the present invention, the miRNA chain has 6 ribonucleotides that do not participate in the formation of the tetrahedral framework nucleic acid, which are used as toehold-mediated chain displacement reaction domains. The toehold-mediated chain displacement reaction domain can be complementary paired with the nucleic acid sequence inside the cell, thereby releasing the miRNA chain and achieving specific recognition of the cell.

[0023] The present invention takes miRNA125 as an example, and embeds miRNA into the interior of tetrahedral framework nucleic acid (tFNA), thereby giving miRNA sufficient protection. The present invention extends ribonucleotides at the end of miRNA as a biological switch. This enzyme-responsive biological switch will be triggered by RNase-H after the nanostructure is delivered to the destination, realizing the opening of the three-dimensional structure. At the same time, there is a foothold-mediated chain displacement reaction domain on the miRNA, which is conducive to the binding of miRNA to intracellular targets and realizing specific recognition of cells. The delivery platform (NP) of the present invention has high delivery efficiency, high stability and good biological compatibility, can reduce inflammation, maintain ROS homeostasis, block hepatocyte apoptosis, and can effectively treat liver damage; at the same time, NP can significantly reverse cell apoptosis, effectively treat kidney damage, and open up new ways for innovative treatment of liver and kidney related diseases.

[0024] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0025] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of NP production.

[0027] Figure 2 AGE shows the stepwise synthesis of NP and tFNA. MiRNA-125 was successfully loaded onto NP, as indicated by enhanced Cy5 fluorescence (lane 1: tFNA-S1; lane 2: tFNA-S1+tFNA-S2; lane 3: tFNA-S1+tFNA-S2+tFNA-S3; lane 4: tFNA-S1+tFNA-S2+tFNA-S3+tFNA-S4; lane 5: S1; lane 6: S1+S2; lane 7: S1+S2+S3; lane 8: S1+S2+S3+Cy5-miRNA-125; lane 9: S1+S2+S3+2×Cy5-miRNA-125; lane 10: S1+S2+S3+3×Cy5-miRNA-125); CGE shows the stepwise preparation of NP and tFNA.

[0028] Figure 3 Schematic diagram of NPs when matched and mismatched. In AGE, matched NPs exhibit minimal Cy5 fluorescence, indicating successful integration of the structure.

[0029] Figure 4 The AFM images show the small size of the NPs and the TEM images show that the small-sized NPs are evenly distributed.

[0030] Figure 5 Hydrodynamic size analysis showed that NP and tFNA were similar in size.

[0031] Figure 6 is the negatively charged NP potential.

[0032] Figure 7 (a) Schematic diagram of NP opening in response to stimulation. (b) AGE showing conformational changes of NP in response to stimulation.

[0033] Figure 8 The CGE analysis confirmed the structural changes observed in AGEs. Data were analyzed using one-sided Student's t-test between two groups or one-way ANOVA and post hoc analysis (Sidak, test) for multiple comparisons. Data are expressed as mean ± standard deviation (SD) (n≥3), ***P<0.001.

[0034] Fig. 9Design of Cy5 (acceptor) and Cy3 (donor) in NP.

[0035] Fig.10 (a) Relative fluorescence intensity showing FRET results. (b) AGE also confirmed the FRET results. ***p<0.001.

[0036] Fig.11 The change in relative fluorescence intensity (FRET / Cy5) shows the metabolic state of NPs in vivo.

[0037] Fig.12 The changes of relative fluorescence intensity (FRET / Cy5) in liver and kidney. *p<0.05; **p<0.01; ***p<0.001.

[0038] Fig.13 MiRNA-qPCR results show passive targeting of NP. ***p<0.001.

[0039] Fig.14 To evaluate the metabolism of miRNA, Entranster and NP by using the changes in in vivo fluorescence signals.

[0040] Fig.15 Relative fluorescence intensity of the visceral organs, liver and kidney of mice at 4h, 8h and 24h.

[0041] Fig.16 Statistical analysis of relative fluorescence intensity. ***P<0.001 compared with miRNA; p<0.05, p<0.01 compared with Entranster.

[0042] Fig.17 Fluorescence imaging of tissue sections 24 hours after injection of miRNA, Entranster and NP.

[0043] Fig.18 Schematic diagram of miRNAscope.

[0044] Fig.19 In both liver and kidney tissue sections, a large number of red dots were observed in the NP group, and some red dots were also accumulated in the Entranster group, but almost no red dots were observed in the miRNA group.

[0045] Fig. 20 Statistical analysis of red dots in liver and kidney cells. ***P<0.001 compared with miRNA; p<0.001 compared with Entranster.

[0046] Fig.21(a) Storage stability test of NP and tFNA. (b, c) Serum stability test of NP and tFNA. (d) DNase I tolerance test of NP and tFNA; Data were analyzed using one-sided Student's t-test between two groups or one-way ANOVA and post hoc analysis (Sidak, test) for multiple comparisons, and data are expressed as mean ± SD (n ≥ 3).

[0047] Fig. 22 The therapeutic effect of NP on acute liver failure in mice. A) Schematic diagram of drug treatment methods and time nodes in vivo for ALF. B) Gross morphology of liver in each group. C) Kaplan-Meier survival analysis of mice after different treatments. D) Liver HE staining images (scale bars: 1 mm, 300 μm, 100 μm). Green asterisk: normal hepatocytes; red arrows: vacuolar degeneration of hepatocytes; blue arrows: lipid droplets, indicating fatty degeneration of hepatocytes; orange arrows: necrotic hepatocytes; N: necrotic area. E, F) Analysis of serum ALT and AST levels. G, I) Representative images of Ly6G and CD68 expression in the liver based on immunohistochemical staining (scale bars: 50 μm, 100 μm and 250 μm). H) Histopathological scoring of liver sections based on HE staining. J) Quantitative analysis of the percentage of Ly6g-positive cells. K) Immunofluorescence images of liver DHE staining (ROS: red; nucleus: blue; 3D thermal imaging: reconstructed fluorescence intensity of ROS (scale bars: 1.5 mm and 250 μm). L) Quantitative analysis of CD68 expression levels. M) Quantitative analysis of relative fluorescence intensity of ROS. All data were analyzed by one-way ANOVA and post hoc analysis (Sidak, test) and are expressed as mean ± standard deviation (n ≥ 3). Error bars represent standard deviation. Statistical analysis: (*) compared to the control group; *P<0.05, **P<0.01, ***P<0.001.( # ) compared to the AKI group; # P<0.05, ## P<0.01, ### P<0.001.( & ) compared to the tFNA group; & P<0.05, && P<0.01, &&& P<0.001.

[0048] Fig.23 Schematic diagram of the protective mechanism of NP on APAP-poisoned hepatocytes.

[0049] Fig.24(a) RT-qPCR expression analysis of Keap1 and its downstream related genes. (b) Western blotting analysis of Keap1, Nrf2, and HO-1 protein expression. (c) Quantitative analysis of related protein expression based on western blotting results.

[0050] Fig.25 Representative images of Keap1, Nrf2 and HO-1 expression in the liver based on immunohistochemical staining (scale bars: 50 μm, 100 μm and 200 μm) and quantitative analysis of Keap1, Nrf2 and HO-1 expression levels based on immunohistochemical results.

[0051] Fig.26 Immunofluorescence images of TUNEL-stained liver (TUNEL: green; nucleus: blue; 3D thermal imaging: reconstruction of TUNEL fluorescence intensity (scale bar: 200 μm).

[0052] Fig. 27 To quantitatively analyze the relative fluorescence intensity of TUNEL based on TUNEL staining and the percentage of TUNEL-positive cells in total cells based on TUNEL staining.

[0053] Fig.28 Western blotting analysis was performed for the protein expression of apoptosis-related proteins Bax, Bcl-2, and Caspase-3, and the expression of apoptosis-related proteins was quantitatively analyzed based on the western blotting results.

[0054] Fig.29 To quantitatively analyze the relative fluorescence intensity of Bax, Bcl-2, and Caspase-3 and the immunofluorescence images of Bax, Bcl-2, and Caspase-3 expression (Bax, Bcl-2, and Caspase-3: green; nucleus: blue; 3D thermal imaging: reconstructed fluorescence intensity of Bax, Bcl-2, and Caspase-3 (scale bar: 200 μm). All data were analyzed by one-way ANOVA and post hoc analysis (Sidak, test) and are expressed as mean ± standard deviation (n ≥ 3). Error bars represent standard deviation. Statistical analysis: (*) compared to the control group; *P<0.05, **P<0.01, ***P<0.001.( # ) compared to the AKI group; # P<0.05, ## P<0.01, ### P<0.001.( & ) compared to the tFNA group;& P<0.05, && P<0.01, &&& P<0.001.

[0055] Fig.30 Figure NP treats renal injury in the I / R-induced AKI mouse model in terms of pathology, serology, microstructure, and protein expression. A) Schematic diagram of drug treatment methods and time nodes in AKI in vivo. B, D) HE and PAS stained kidney images (scale bars: 100 μm, 300 μm, and 500 μm). Green arrows: healthy tubules; red arrows: coagulative necrosis of tubules; red asterisks: transparent casts; red triangles: mucus exudation; blue arrows: tubular dilatation; orange arrows: loss of borders. C, E) Analysis of serum CREA and urea levels. F) Histopathological scoring of renal sections based on HE staining. G) Representative transmission electron microscopy images of kidney mitochondria in different groups (scale bars: 5 μm and 1.5 μm). Green arrows: healthy mitochondria; red arrows: mitochondrial fragmentation with extravasation of matrix material; red asterisks: disordered or missing mitochondrial cristae; orange arrows: swollen mitochondria. H) Histopathological scoring of renal sections based on PAS staining. I) Immunohistochemical images of CD68 expression (scale bars: 250 μm and 100 μm). J) Immunohistochemical images of KIM-1 expression (scale bars: 250 μm and 100 μm). K) Quantitative analysis of CD68 expression based on immunohistochemical results. L) Western blotting analysis of KIM-1 protein expression. M) Quantitative analysis of KIM-1 expression based on western blotting results. N) Quantitative analysis of KIM-1 expression based on immunohistochemical results. All data were analyzed by one-way ANOVA and post hoc analysis (Sidak, test) and expressed as mean ± standard deviation (n≥3). Error bars represent standard deviation. Statistical analysis: (*) compared to the control group; *P<0.05, **P<0.01, ***P<0.001.( # ) compared to the AKI group; # P<0.05, ## P<0.01, ### P<0.001.( & ) compared to the tFNA group; & P<0.05, && P<0.01, &&& P<0.001.

[0056] Fig.31 Schematic diagram of the protective mechanism of NP against I / R-induced AKI.

[0057] Fig.32 Western blotting analysis of p53 protein and quantitative analysis of p53 expression based on western blotting results.

[0058] Fig.33 Quantitative analysis of p53 expression and immunohistochemical images of p53 expression for immunohistochemical results (scale bars: 250 μm and 100 μm).

[0059] Fig.34 The results of flow cytometry were quantitatively analyzed and the percentage of apoptosis of RTECs in each group was detected by flow cytometry.

[0060] Fig.35 Immunofluorescence images of kidneys stained with TUNEL (TUNEL: green; nuclei: blue; three-dimensional thermal imaging: reconstruction of the fluorescence intensity of TUNEL (scale bar: 200 μm), quantitative analysis of the relative fluorescence intensity of TUNEL based on TUNEL staining, and quantitative analysis of the percentage of TUNEL-positive cells in total cells based on TUNEL staining.

[0061] Fig.36 Western blotting analysis was performed for the protein expression of apoptosis-related proteins Bax, Bcl-2, and Caspase-3, and the expression of apoptosis-related proteins was quantitatively analyzed based on the western blotting results.

[0062] Fig.37 Immunohistochemical images of Bax expression (scale bars: 250 μm and 100 μm) and quantitative analysis.

[0063] Fig.38 The protein expression of cell cycle related proteins: Cyclin B1 and PCNA and the quantitative analysis of the expression of cell cycle related proteins based on western blotting results.

[0064] Fig.39 Immunohistochemical images of PCNA expression (scale bar: 250 μm and 100 μm) and quantitative analysis. All data were analyzed by one-way ANOVA and post hoc analysis (Sidak, test) and expressed as mean ± standard deviation (n ≥ 3). Error bars represent standard deviation. Statistical analysis: (*) compared to the control group; *P<0.05, **P<0.01, ***P<0.001.( # ) compared to the AKI group; # P<0.05, ## P<0.01, ###P<0.001.( & ) compared to the tFNA group; & P<0.05, && P<0.01, &&& P<0.001. DETAILED DESCRIPTION

[0065] In the following examples and experimental examples, reagents and raw materials not specifically described are all commercially available products.

[0066] Example 1: Tetrahedral framework nucleic acid (NP) embedded with miRNA 125 and preparation method thereof

[0067] This embodiment provides a tetrahedral framework nucleic acid (NP) embedded with miRNA, which is assembled by three single-stranded DNAs (S1-S3) and three miRNA chains through base complementary pairing ( Figure 1 ).

[0068] Taking miRNA125 as an example, the nucleotide sequence of single-stranded DNA S1 is shown in SEQ ID NO.1, the nucleotide sequence of single-stranded DNA S2 is shown in SEQ ID NO.2, the nucleotide sequence of single-stranded DNA S3 is shown in SEQ ID NO.3, and the nucleotide sequence of miRNA125 chain is shown in SEQ ID NO.4, as shown in Table 1.

[0069] Table 1

[0070]

[0071]

[0072] Four ribonucleotides extend from the end of each miRNA chain, serving as biological switches. In the base-pairing region between any single-stranded DNA and miRNA, the deoxyribonucleotides of the single-stranded DNA are replaced with ribonucleotides, which are called RNA binding domains. The part that is complementary to the biological switch is still designed as deoxyribonucleotides, thus ensuring that the biological switch can be triggered by RNase H, which is widely present in mammalian cells, and ultimately realizing the opening of this delivery platform. At the same time, at the vertex corners of each miRNA125, there are 6 ribonucleotide bases that do not participate in base-pairing ( Figure 1 ), thereby enabling miRNA125 to undergo a toehold-mediated strand displacement reaction (TMSD) with its target.

[0073] Preparation method: Mix three single-stranded DNAs (molar ratio of 1:1:1) and three miRNA chains to obtain a mixed solution, wherein the molar ratio of any one single-stranded DNA to any one miRNA chain is 1:1. The mixed solution is fully mixed with TM buffer (50mM MgCl2-6H2O and 10mM Tris-HCl), heated to 95°C, and then quickly cooled to 4°C for 30min to obtain the product.

[0074] Comparative Example 1

[0075] This comparative example provides the control samples used in the experiment:

[0076] 1. tFNA

[0077] The sample is assembled from four ssDNAs based on complementary base pairing. The preparation method is the same as that of Example 1. Four single-stranded DNAs (tFNA-S1-~tFNA-S4), wherein the nucleotide sequence of tFNA-S1 is shown in SEQ ID NO.7, the nucleotide sequence of tFNA-S2 is shown in SEQ ID NO.8, the nucleotide sequence of tFNA-S3 is shown in SEQ ID NO.9, and the nucleotide sequence of tFNA-S4 is shown in SEQ ID NO.10, see Table 1.

[0078] 2. miRNA

[0079] The sample is miRNA125.

[0080] The nucleotide sequence is shown in SEQ ID NO.4, see Table 1.

[0081] The technical solution of the present invention is further illustrated by experiments below.

[0082] Experimental Example 1 Characterization of tetrahedral framework nucleic acid (NP) embedded with miRNA125

[0083] In this experimental example, the NPs prepared in Example 1 were characterized.

[0084] 1. Experimental Methods

[0085] Capillary gel electrophoresis, atomic force microscopy, and transmission electron microscopy were used to observe the morphology of the NPs; dynamic light scattering was used to accurately quantify the hydrodynamic size of the NPs.

[0086] 2. Experimental Results

[0087] like Figure 2NP also showed similar relative fluorescence intensity to tFNA in capillary gel electrophoresis, proving that the two have similar structures. The 3' ends of miR125 and mismatched miR125 were modified using Black Hole Quencher-2 (BHQ-2), where the nucleotide sequence of Matched-miRNA125 is shown in SEQ ID NO.5, and the nucleotide sequence of Mismatched-miRNA125 is shown in SEQ ID NO.6. Matched-NP and Mismatched-NP ( Figure 3 Due to the successful synthesis of the NP structure and the complete closure of the framework, the relative fluorescence intensity of the Cy5 channel of the matched-NP decreased to 3% of that of the mismatched-NP ( Figure 3 Atomic force microscopy and transmission electron microscopy were used to observe the morphology of NPs. Figure 4 It can be seen that the NP size is very small (9.24±0.86nm) and evenly distributed. The dynamic light scattering method was then used to accurately quantify the hydrodynamic size of the NP. The results showed that after encapsulating miRNA125 inside the nanoparticles, it had a similar particle size to the 21bp side length tFNA ( Figure 5 ). Since RNA replaces the original DNA backbone in the structure, NP is more negatively charged than tFNA ( Figure 6 In order to facilitate the binding of miRNA125 loaded in NP to the intracellular target, the bio-switchable structure in NP can be triggered by RNase H which is commonly found in mammalian cells, degrading the RNA part of the DNA-RNA hybrid chain in the bio-switchable structure, thereby opening the NP ( Figure 7 a). Then, the opening of the nanostructure was simulated in the extracellular environment. As the enzyme activity units of RNase H in the incubation environment increased, the migration rate of the nanostructure gradually slowed down, which also confirmed that the three-dimensional conformation of NP changed ( Figure 7 b). This result was also confirmed by capillary gel electrophoresis, indicating that after the nanostructure enters the cell with a relatively small size, it is triggered by RNase H to open the three-dimensional structure, which is conducive to binding with the intracellular target ( Figure 8 ).

[0088] Experimental Example 2 Metabolism and distribution of NP in vivo

[0089] 1. Experimental Methods

[0090] 1. Agarose gel electrophoresis

[0091] 2. miRNA-QPCR

[0092] 2. Experimental Results

[0093] like Fig. 9 As shown in Figure 2, the 5' and 3' ends of miRNA125 are modified with Cy5 (acceptor) and cy3 (donor), respectively. When the structure of NP is intact, the acceptor (Cy5) and donor (cy3) undergo Resonance energy transfer (FRET), the fluorescence of the acceptor is enhanced, while the autofluorescence intensity of the donor is attenuated. When the NP is disintegrated, the fluorescence of the acceptor will not be enhanced. The change in the fluorescence intensity of the acceptor helps to clearly observe the metabolism and distribution of the NP in the body. Cy3-NP, Cy5-NP, and FRET-NP were prepared separately, and Cy3-NP and Cy5-NP were mixed in equal proportions to prepare Cy3+5-NP. The relative fluorescence intensity of the four samples was measured. The relative fluorescence intensity of FRET-NP was weaker than that of the other groups at 570nm, while the relative fluorescence intensity at 670nm was stronger, indicating that FRET occurred between the acceptor and donor of FRET-NP ( Fig.10 a). Agarose gel electrophoresis also confirmed this result ( Fig.10 b). Subsequently, FRET-NP and Cy3+5-NP were injected into mice, and the changes in relative fluorescence intensity (Channel1-FRET / Channel2-Cy5) were used to observe the metabolism of NP in vivo and the percentage of NP intactness ( Fig.11 ). Within 8 hours after injection, the proportion of intact NPs in the liver gradually decreased; while in the kidney, the proportion of intact NPs reached a peak at 0.5-1 hour and then gradually decreased ( Fig.12 ). At the 24-hour time point, there were no intact NPs in the liver and kidneys. In order to preliminarily evaluate the delivery efficiency of NPs, naked miRNA and NPs were injected intraperitoneally, and the liver and kidneys were collected at 24 hours. Finally, miRNA-QPCR was used to evaluate the expression level of miRNA125. The results of miRNA-qPCR found that the expression level of miRNA-125 in the NP group was higher than that of the blank group in the liver, and higher than that of the blank group in the kidneys, but direct injection of naked miRNA was almost ineffective ( Fig.13 ), which confirmed that NPs can greatly improve the delivery efficiency of miRNA.

[0094] To further demonstrate the great potential of NP in delivering miRNA, we combined it with another in vivo RNA transfection reagent, Entranster TMThe miRNA, Entranster and NP groups were prepared using cy5-modified miRNA-125, and the in vivo imaging system ( Fig.14 ). Direct injection of miRNA-125 showed almost no fluorescence within 45 minutes. The relative fluorescence intensity of the Entranster group and the NP group was similar within 15 minutes, but the relative fluorescence intensity of the NP group at 30 minutes was significantly higher than that of the Entranster group. The relative fluorescence intensity of the visceral organs, liver and kidney of mice was observed at 4h, 8h and 24h ( Fig.15 The results showed that there were statistically significant differences between the NP group and the Entranster group in the liver at 8h and 24h and the kidney at 24h ( Fig.16 ). Observing the fluorescence of liver and kidney slices at 24h, the Cy5 fluorescence of NP group was significantly stronger than that of Entranster group, while the fluorescence signal of miRNA group was almost undetectable ( Fig.17 miRNAscope is a stable RNA in situ hybridization assay that detects the spatial distribution of miRNA-125 in tissues through highly specific binding of probes to miRNA-125 ( Fig.18 In the liver and kidney tissue sections, a large number of red dots appeared in the NP group, and some red dots also appeared in the Entranster group, but almost no red dots were observed in the miRNA group ( Fig.19 ). Analyzing the number of red dots in each cell, it was found that there were statistical differences between the NP group and the Entranster group in both liver and kidney samples. These results also confirmed that NP has a higher miRNA delivery efficiency than Entranster, a commercially available RNA drug delivery carrier ( Fig. 20 ).

[0095] The miRNA in the miRNA delivery vehicle must be protected; therefore, the stability of NP was evaluated. In the storage stability test, NP and tFNA showed similar stability at room temperature, but NP showed outstanding stability at 37°C, which may be related to the presence of RNA double-stranded structure in its internal composition ( Fig.21 a). In the serum stability test, NP showed similar stability results to tFNA at both 2% and 10% serum concentrations ( Fig.21 b). In addition, after 12h and 24h incubation, the stability results of NPs were similar to those of tFNA with increasing serum concentrations ( Fig.21 c). In the DNase I tolerance test, when the DNase I concentration was greater than 5U / mL, NP was more stable than tFNA. The advantage of NP in efficient delivery of miRNA undoubtedly promotes the development prospects of miRNA-related drugs that are limited by unstable miRNA ( Fig.21 d).

[0096] Experimental Example 3 The therapeutic effect of NP on acute liver failure (ALF) in mice

[0097] 1. Experimental Methods

[0098] 1. ALF mouse model: Fig. 22 As shown in A, Balb / C mice were intraperitoneally injected with 350 mg / kg acetaminophen (APAP) and given saline, tFNA, or NP (100 μL) every 6 h to establish the ALF model. Serum and tissue samples were collected 24 h later.

[0099] 2. Experimental groups:

[0100] Ctrl Group: no APAP injection;

[0101] ALF Group: APAP injection without follow-up treatment;

[0102] tFNAGroup: APAP injection combined with tFNA treatment;

[0103] NP Group: APAP injection combined with NP treatment;

[0104] In the experimental results, “1” represents the Ctrl Group, “2” represents the ALF Group, “3” represents the tFNA Group, and “4” represents the NP Group.

[0105] 3. Serological indicators: Analyze the ALT and AST levels in serum.

[0106] 4. HE staining: Perform histopathological analysis on liver sections.

[0107] 5. Ly6G staining: Neutrophils can be identified and quantitatively analyzed by staining with antibodies against Ly6G.

[0108] 6. IHC staining.

[0109] 7. Detection of reactive oxygen species by DHE staining of liver.

[0110] 8. Western blotting was used to analyze the expression of Keap1, Nrf2, HO-1, Bax, Bcl-2, and Caspase-3 proteins.

[0111] 9. PCR analysis of gene expression of Keap1, Nrf2, and HO-1.

[0112] 10. Immunohistochemical staining was used to analyze the expression of Keap1, Nrf2 and HO-1 in the liver.

[0113] 11. TUNEL staining was used to evaluate the anti-apoptotic ability of NP.

[0114] 12. Immunofluorescence staining was used to analyze the fluorescence intensity of Bax, Bcl-2, and Caspase-3.

[0115] 2. Experimental Results

[0116] The mortality rates of mice in different groups were Fig. 22 As shown in C, Kaplan-Meier survival curve analysis showed that the 24-hour survival rate of the ALF group was 0%, which increased significantly to 50% after NP administration, indicating that mice treated with NP had a significant survival advantage. The gross morphological results of the liver in each group showed ( Fig. 22 B), the liver surface of the control group was smooth and shiny, with uniform texture and bright and ruddy color. However, the liver of the APAP-treated mice showed an uneven, rough, and atrophic appearance, with a hard texture and dark color, indicating that the ALF model was successfully established. Both tFNA and NP treatments can alleviate the liver pathological state caused by APAP, and the liver of the NP group is significantly healthier. Similarly, the key serological indicators of ALF showed that the liver function of the ALF group was severely impaired, such as Fig. 22 As shown in E and F, the ALF group had the highest levels of AST and ALT. Only the NP-treated group showed a significant decrease in ALT and AST levels, indicating that NP effectively prevented APAP-induced liver injury, thereby improving the survival rate of mice.

[0117] The liver tissue was then Fig. 22 HE staining of D visually shows APAP-induced hepatotoxicity and the therapeutic effect of NP on ALF. Histologically, severe liver damage was observed in the livers of the ALF group, with liver structural destruction, large areas of necrosis accompanied by a large number of vacuolar degeneration and fatty degeneration of hepatocytes. Liver necrosis was reduced after tFNA intervention, but it was still severe, while the NP group had less damage, healthy tissue pathology, and was accompanied by a small number of lipid droplets. Histopathological scoring of liver sections according to the severity of liver damage further showed that NP treatment did help alleviate APAP-induced liver failure ( Fig. 22 H). Necroinflammation is considered a common indicator of ALF. To evaluate the infiltration of inflammatory cells into the liver injury site, the infiltration of neutrophils was monitored by Ly6G staining, and the expression of the macrophage surface-specific marker CD68 was monitored by IHC. Fig. 22As shown in G and I, Ly6G-positive cells and CD68-positive cells (dark brown staining) represent neutrophils and macrophages infiltrating the liver tissue, respectively. Compared with the control group, the liver of the ALF group showed severe enhancement of neutrophil infiltration and upregulated CD68 expression, indicating aggravated necrotic inflammation. Both tFNA and NP treatments can improve liver inflammation, and the effect of NP treatment is more obvious, with a significant decrease in the percentage of Ly6G-positive cells and the expression level of CD68. Statistical analysis of histological sections further verified these conclusions ( Fig. 22 J and L).

[0118] Excessive production of ROS can lead to DNA and mitochondrial damage, rapid development of inflammation, massive release of ALT, and ultimately hepatocyte necrosis. To determine whether NPs can help clear APAP-induced ROS, DHE staining of liver tissues was performed. Red fluorescence showed that ROS levels increased sharply after APAP injection and decreased significantly after NP treatment, which confirmed the significant protective ability of NPs against oxidative damage in vivo ( Fig. 22 K and M). In summary, the results of serological and histological analyses indicated that NP effectively inhibited liver necrosis, alleviated inflammation, and maintained ROS homeostasis.

[0119] like Fig.23 As shown in the figure, the Keap1 / Nrf2 signaling pathway is associated with a variety of oxidative stress diseases and can be used as an important therapeutic target for ALF. Studies have shown that miRNA-125 is an effective regulator of the Keap1 / Nrf2 signaling pathway, which can target Keap1 protein, promote the dissociation of Keap1 and Nrf2, and then activate its downstream anti-ROS pathway, ultimately achieving the effect of treating APAP-induced ALF. Therefore, regulating the Keap1 / Nrf2 pathway by increasing the expression of intracellular miRNA-125 is a potential method for treating ALF.

[0120] To investigate the mechanism of action of NP on APAP-induced ALF mice, western blotting and IHC staining were used to evaluate the role of Keap1 / Nrf2 axis. Fig.24 a) showed that NP upregulated anti-inflammatory and antioxidant genes downstream of the Keap1 / Nrf2 pathway, such as Ugt1a6, Gclc, Nqo1, and Gsta2, indicating that this pathway was activated. Western blotting ( Fig.24 b, c) show that ALF mice have elevated Keap1 and decreased Nrf2 and HO-1. NP treatment reversed these effects by inhibiting Keap1 and restoring Nrf2 and HO-1 levels, helping to eliminate ROS. Fig.25Immunohistochemical staining further confirmed that Keap1 expression increased in the ALF group and decreased after NP treatment. Nrf2 and HO-1 levels were highest in the NP group, consistent with the results of western blotting. This suggests that the therapeutic effect of NP on ALF is due to the activation of the Keap1 / Nrf2 signaling pathway.

[0121] Oxidative stress and the extensive apoptosis of hepatocytes caused by it are the main pathological mechanisms of ALF progression. The two complement each other and jointly mediate hepatocyte necrosis. Therefore, TUNEL staining was used to evaluate the anti-apoptotic ability of NP ( Fig.26 A large number of DNA chain breaks (corresponding to apoptotic cells) were observed in the liver cells of the ALF group, which emitted green fluorescence under the catalysis of terminal deoxynucleotidyl transferase. tFNA reduced the production of green fluorescence to a certain extent, but the effect was far less than that of NP. Fig. 27 Quantitative analysis of TUNEL fluorescence results confirmed that NP treatment could significantly reduce the apoptosis of damaged liver cells, and the green fluorescence intensity and the proportion of apoptotic cells were significantly decreased.

[0122] The effective anti-apoptotic activity of NP may be related to its good ROS scavenging properties and the beneficial anti-apoptotic effect of miRNA-125. Western blotting was used to detect its role in the mitochondrial apoptosis pathway and explore the expression levels of apoptosis-related proteins in each group of cells ( Fig.28 ), NP treatment effectively reversed the upregulation of Bax and Caspase-3 in APAP-intoxicated hepatocytes, inhibited the downregulation of Bcl-2, and was beneficial to alleviate cell apoptosis. Immunofluorescence staining of these proteins also confirmed these results ( Fig.29 ). Bax and Caspase-3 showed the brightest green fluorescence in the ALF group, which was significantly weakened after treatment with tFNA and NP; however, the degree of attenuation in the tFNA group was significantly lower than that in the NP group. The expression of Bcl-2 was the opposite. In summary, the inhibitory effect of NP on ALF originated from the synergistic inhibition of intracellular oxidative stress and apoptosis. NP activated the Keap1 / Nrf2 signaling pathway and blocked hepatocyte apoptosis by regulating the mitochondrial apoptosis pathway, exerting its efficient ROS scavenging activity.

[0123] Experimental Example 4 The therapeutic effect of NP on acute kidney injury (AKI) in mice

[0124] 1. Experimental Methods

[0125] 1. AKI mouse model: The AKI mouse model was induced by ischemia-reperfusion (I / R). The experimental protocol is as follows Fig.30As shown in A. After the mice were anesthetized, a small incision was made on the bilateral dorsal skin of the Balb / C mice, and then the kidneys were separated and exposed. The bilateral renal pedicles were clamped with non-traumatic vascular clamps for 30 minutes. The vascular clamps were removed to start reperfusion, and then normal saline, tFNA or NP (100 μL) was given intraperitoneally every 12 hours after the incision was closed. The mice were killed on the third day after surgery, and serum and kidneys were collected.

[0126] 2. Experimental Grouping

[0127] Ctrl Group: no I / R surgery;

[0128] AKI Group: underwent I / R surgery and no follow-up treatment;

[0129] tFNAGroup: I / R surgery combined with tFNA treatment;

[0130] NP Group: I / R surgery combined with NP treatment;

[0131] In the experimental results graph, "1" represents the Ctrl Group, "2" represents the AKI Group, "3" represents the tFNA Group, and "4" represents the NP Group.

[0132] 3. HE staining: Perform histopathological analysis on kidney sections.

[0133] 4. PAS staining: Perform histopathological analysis on kidney sections.

[0134] 5. Immunohistochemistry: Expression of CD68, KIM-1, and p53 in the liver.

[0135] 6. Western blotting: analyze the expression of KIM-1, p53, Bax, Bcl-2, Caspase-3, Cyclin B1 and PCNA proteins.

[0136] 7. Flow cytometry: evaluate the effect of NP on cell apoptosis.

[0137] 8. TUNEL staining: evaluate the anti-apoptotic ability of NP.

[0138] 2. Experimental Results

[0139] Serum urea and creatinine (CREA) levels were measured in each group to determine renal function ( Fig.30C, E). Bilateral I / R injury caused a significant increase in urea and CREA, indicating severe renal function. After tFNA and NP treatment, these serum biochemical indicators were significantly weakened, and the urea and CREA levels in the NP group were the lowest, indicating that NP is more conducive to the recovery of renal excretion function than tFNA. HE and PAS staining more intuitively show the histopathological damage of the kidney ( Fig.30 B and D). Compared with the control group, the AKI group showed severe tubular injury with disorganized arrangement, manifested by numerous areas of coagulative necrosis, formation of hyaline casts, loss of brush borders, and mucous exudates. After tFNA, coagulative necrosis was reduced, and a large number of hyaline-like materials and mucous exudates were observed in the damaged tubules. NP treatment significantly alleviated tubular injury in I / r-induced AKI, with a significant reduction in tubular necrosis and hyaline casts, and preservation of brush borders. Semiquantitative analysis of the extent of tubular injury by HE and PAS staining supported our histopathological observations ( Fig.30 F and H), and the kidneys of each group were scored from 0 to 4 points according to the percentage of tubular damage. The results showed that the tubular necrosis score was the highest in the AKI group. After tFNA and NP treatment, the tubular necrosis score decreased, with the most obvious decrease in the NP group.

[0140] I / R injury often leads to renal mitochondrial damage and dysfunction, which in turn leads to various pathological states of AKI. To determine the protective effect of NP on mitochondria, transmission electron microscopy was used to observe the mitochondrial morphology of renal tubular epithelial cells. Representative parts are shown in Figure 2. Fig.30 G. I / R injury leads to extensive mitochondrial damage, characterized by swelling, disorganization, or loss of cristae, and fragmentation with extravasation of matrix material. Mitochondria in the tFNA-treated group showed mild rupture, but swelling persisted. However, after NP treatment, mitochondria were dense and elongated, with well-organized cristae and normal morphology, suggesting that NP can significantly reverse AKI-induced mitochondrial damage in renal tubular cells. The expression of CD68 in macrophages was monitored by immunohistochemistry ( Fig.30 I and K). The results showed that NP significantly alleviated the tubulointerstitial infiltration of inflammatory cells (including macrophages) induced by AKI, as shown by a significant decrease in CD68 expression. To evaluate the therapeutic effect of NP on ischemic AKI, immunohistochemistry ( Fig.30 J and M) and western blotting ( Fig.30L and N) detected the expression of kidney injury molecule-1 (KIM-1), a key biomarker of early renal injury. According to immunohistochemistry results, I / R injury caused a significant upregulation of KIM-1 expression in renal tubules, which was almost restored to the level of control mice after NP treatment. Western blotting further supported this result; NP significantly reversed the increase in KIM-1 expression caused by ischemic injury. Overall, the I / R-induced AKI mouse model was successfully established, and NP showed improvements in pathology, serology, microstructure, and protein expression, which can effectively treat renal injury.

[0141] P53 is a nuclear phosphorylated protein encoded by the tumor suppressor gene P53, which is closely involved in important biological functions such as cell cycle regulation, DNA repair, and cell apoptosis. Fig.31 As shown in the literature, I / R injury can lead to cellular stress in renal tubular epithelial cells (RTECs), including ROS accumulation, hypoxia, and DNA damage, which can activate p53 expression. Dysregulation of p53 has been shown to be a key factor leading to apoptosis and G2 / M phase arrest through different signaling pathways, which is a potential mechanism of cell or tissue necrosis during AKI. On the one hand, p53 upregulation can cause mitochondrial damage and activate its subordinate apoptotic pathways, thereby inducing apoptosis of RTEC cells. On the other hand, nuclear p53 transcriptionally inhibits the binding of cyclin-dependent kinase 1 (CDK1) to cyclin B1 and the expression of proliferating cell nuclear antigen (PCNA), leading to G2 / M phase arrest of RTECs and subsequently inhibiting cell proliferation. Studies have found that MiRNA-125 can interact with p53 and downregulate its expression, thereby coordinating the transcription of downstream apoptotic proteins and cycle-related proteins, inhibiting apoptosis and G2 / M phase arrest.

[0142] Therefore, in order to investigate the mechanism by which NP improves I / R-induced AKI in mice, we first performed western blotting ( Fig.32 ) and immunohistochemical staining ( Fig.33 ) demonstrated that NP could normalize AKI-induced upregulated p53 levels, while tFNA alone had minimal effect on p53 expression. Flow cytometry confirmed the effect of NP on cell apoptosis. The percentage of apoptotic cells ( Fig.34 ) confirmed that NP has a good inhibitory effect on I / r-induced RTEC cell apoptosis. Kidney TUNEL staining further confirmed the above findings ( Fig.35 ). The number of apoptotic cells in the AKI model increased significantly, and the green fluorescence intensity was significantly enhanced. The fluorescence weakened after tFNA and NP intervention, and the fluorescence intensity of the NP group was weaker, suggesting that NP is more conducive to reversing I / R-induced cell apoptosis than tFNA. Therefore, western blotting was used to further study the expression levels of proteins related to the mitochondrial apoptosis pathway. Fig.36 As shown in the figure, the levels of pro-apoptotic proteins Bax and Caspase-3 were the highest in the AKI group and the lowest in the NP group. The expression of anti-apoptotic protein Bcl-2 was the opposite, with the lowest expression level in the AKI group. Similar conclusions were drawn after Bax immunohistochemical staining ( Fig.37 These results indicate that NP significantly enhanced the inhibitory effect of miRNA-125 on I / R-induced cell apoptosis by activating p53 to regulate the downstream proteins of the mitochondrial apoptosis pathway, such as Bax, Bcl-2, and Caspase-3.

[0143] p53 is a cross-target between apoptosis and cell cycle arrest. Western blotting was used to detect the expression levels of Cyclin B1 and PCNA in the renal tissues of each group. Fig.38 ). Compared with the control group, I / R surgery significantly suppressed the protein levels of Cyclin B1 and PCNA, which is consistent with the biological characteristics of I / R injury. TFNA restored the levels of these proteins to a certain extent; however, the enhancement was much lower than that of NP treatment. Fig.39 The PCNA immunohistochemical staining shown also showed similar results; in the AKI group, the brown staining symbolizing PCNA expression was significantly lighter, but recovered to a similar depth as the control group after NP intervention. Therefore, the identification of the expression levels of cell cycle-related proteins showed that NP effectively eliminated the reduction in Cyclin B1 and PCNA expression caused by AKI, thereby inhibiting the G2 / M phase arrest of cells, promoting cell repair and proliferation, and ultimately accelerating injury repair.

[0144] In summary, the NP prepared by the present invention can improve the delivery efficiency of miRNA, has high stability and good biological compatibility, effectively inhibits liver necrosis, reduces inflammation, maintains ROS homeostasis, blocks hepatocyte apoptosis, and can effectively treat APAP-induced liver injury; at the same time, NP can significantly reverse I / r-induced cell apoptosis, effectively treat renal injury, and open up a new way for the innovative treatment of liver and kidney related diseases.

Claims

1. A functionalized tetrahedral framework nucleic acid, characterized in that: It is a tetrahedral framework nucleic acid embedded with miRNA; the tetrahedral framework nucleic acid is assembled by 3 DNA single strands and 3 miRNA strands through base complementary pairing; the miRNA strand includes miRNA and 2-6 ribonucleotides at the end; there are 4-8bp ribonucleic acid on the miRNA that does not participate in the formation of the tetrahedral framework nucleic acid; the 2-6 deoxyribonucleotides at the end of the DNA single strand are complementary to the 2-6 ribonucleotides at the end of the miRNA strand, and the part of the DNA single strand that is complementary to the miRNA is a ribonucleotide.

2. The functionalized tetrahedral framework nucleic acid according to claim 1, characterized in that: In the miRNA chain, the length of the miRNA is 22-24 bp, and the number of terminal ribonucleotides is 2-6; Preferably, in the miRNA chain, the length of the miRNA is 22 bp and the number of terminal ribonucleotides is 4.

3. The functionalized tetrahedral framework nucleic acid according to claim 2, characterized in that: In the miRNA chain, the miRNA is selected from miRNA125; and / or the terminal ribonucleic acid is cuua.

4. The functionalized tetrahedral framework nucleic acid according to any one of claims 1 to 3, characterized in that: The nucleotide sequence of the miRNA chain is shown in SEQ ID NO.

4.

5. The functionalized tetrahedral framework nucleic acid according to claim 1, characterized in that: In the miRNA chain, the 4-8 bp ribonucleotides starting from the 8th to the 11th ribonucleotides of the miRNA do not participate in forming the tetrahedral framework nucleic acid, and the remaining ribonucleotides complement the ribonucleotides on the DNA single strand to form the tetrahedral framework nucleic acid; Preferably, in the miRNA chain, the 6 bp ribonucleic acid starting from the 9th ribonucleotide of miRNA does not participate in the formation of tetrahedral framework nucleic acid, and the 1st to 8th ribonucleotides and the 15th to 22nd ribonucleotides of miRNA complement the ribonucleotides on the DNA single strand to form a tetrahedral framework nucleic acid.

6. The functionalized tetrahedral framework nucleic acid according to claim 1, characterized in that: The structure of the single-stranded DNA is RNA 1-deoxyribonucleic acid 2-RNA 3-deoxyribonucleic acid 4, wherein RNA 1 and RNA 3 are complementary to miRNA, and deoxyribonucleic acid 4 is complementary to the ribonucleotide at the end of miRNA; the lengths of RNA 1 and RNA 3 are (7-10) bp respectively, the length of deoxyribonucleic acid 4 is (2-6) bp, and the total length of the single-stranded DNA is 39-111 bp; Preferably, the lengths of RNA 1 and RNA 3 are 8 bp respectively, the length of deoxyribonucleic acid 4 is 4 bp, and the total length of the DNA single strand is 63 bp.

7. The functionalized tetrahedral framework nucleic acid according to claim 1, characterized in that: The nucleotide sequences of the three DNA single strands are shown as SEQ ID NO.1 to SEQ ID NO.

3.

8. The method for preparing a functionalized tetrahedral framework nucleic acid according to any one of claims 1 to 7, characterized in that: The method comprises the following steps: mixing three single-stranded DNAs and three miRNA chains, treating and incubating with a thermal cycler, and obtaining the obtained product; the molar ratio of the three single-stranded DNAs is 1:1:1, and the molar ratio of any one single-stranded DNA to any one miRNA chain is 1:

1.

9. Use of the functionalized tetrahedral framework nucleic acid according to any one of claims 1 to 7 in the preparation of a medicament for treating acute liver injury and / or acute kidney injury.

10. A drug for treating acute liver injury and / or acute kidney injury, characterized in that: The invention is prepared by taking the functionalized tetrahedral framework nucleic acid described in any one of claims 1 to 7 as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

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