Nucleic acid self-assembled medicine as well as preparation method and application thereof

Abstract

The invention belongs to the field of nano medicine, and particularly relates to a nucleic acid self-assembled medicine and a preparation method and application thereof.The nucleic acid self-assembled medicine is an assembled medicine with an addressable double-strand DNA tetrahedral structure as a carrier and a flavonoid compound loaded into the carrier through embedding, the nucleic acid self-assembled medicine is applied to preparation of liver-targeted flavone nano-medicines and prevention and / or treatment of Wilson's disease. According to the invention, the problem that the drug is specifically enriched into the focus is solved, the flavonoid drug can better reach the focus so as to exert an excellent treatment effect, a new technical scheme and a new treatment strategy are provided for treatment of Wilson diseases, and a new angle and a breakthrough are provided for development of human medical undertaking.

Description

Technical Field

[0001] The present invention belongs to the field of nanomedicine, and in particular relates to a nucleic acid self-assembly drug and a preparation method and application thereof. Background Art

[0002] As a strong oxidant, copper can activate free radicals, thereby promoting the production of intracellular reactive oxygen species (ROS). Intracellular copper overload, and the subsequent oxidative stress it induces, is a common feature and mechanism of pathogenesis in most neurodegenerative diseases. The ROS generated by oxidative stress damage the physiological functions of various macromolecules through various pathways and attack neurons in specific brain tissue regions, leading to cell apoptosis and ultimately the onset of disease.

[0003] Wilson disease (WD), also known as hepatolenticular degeneration (HLD), is characterized by abnormal copper metabolism caused by mutations in the ATP7B gene. This leads to excessive copper accumulation in the body, primarily affecting the liver, brain, kidneys, and cornea, causing liver damage, basal ganglia degeneration, renal damage, and corneal pigment rings (KF rings). It can even cause severe central nervous system damage, which can be life-threatening. The liver is a key organ involved in copper metabolism and one of the most vulnerable organs to copper accumulation. Treating WD requires not only reducing copper accumulation but also addressing and protecting the liver. The most commonly used treatments for WD are oral copper chelators and zinc supplements. However, these treatments have drawbacks, including numerous adverse reactions, poor efficacy, the need for lifelong treatment, and poor patient compliance.

[0004] Flavonoids, originally a general term for compounds derived from 2-phenylchromone as a skeleton, are widely found in plants. Medicinal compounds within the flavonoid class can be used to prevent and treat cardiovascular and cerebrovascular diseases, including hypertension in the elderly, cerebral hemorrhage, coronary heart disease, and angina pectoris, as well as to dilate coronary vessels and increase coronary flow. Many flavonoids possess antitussive, expectorant, and antiasthmatic properties, as well as antibacterial and liver-protecting properties, including detoxification, antifungal, and therapeutic effects in acute and chronic hepatitis and cirrhosis, and as anti-free radical and antioxidant agents.

[0005] While flavonoids have the potential to treat WD, the route of administration plays a key role in drug efficacy. Lack of a suitable delivery method can lead to ineffective drug treatment. Efficient drug delivery to the liver via carriers presents technical challenges, and modification of targeting groups can directly impact delivery efficacy.

[0006] Nucleic acid tetrahedrons (NATs) are three-dimensional nanostructures formed by the self-assembly of nucleic acid molecules. These structures are typically composed of oligonucleotide chains interconnected by base pairing and annealing to form a tetrahedral framework. NATs have broad potential applications in nanotechnology, biomaterials, and biomedicine. NATs can be used as drug delivery systems, encapsulating molecules such as drugs, proteins, and RNA within their internal cavities or adsorbing them onto their surfaces. Due to the inherent biocompatibility of nucleic acid molecules, NATs exhibit low immunogenicity when used in vivo.

[0007] Drug nanocarriers based on nucleic acid self-assembly materials, such as DNA tetrahedrons, exhibit excellent biocompatibility, high drug loading capacity and multi-drug synergistic delivery. DNA tetrahedrons are formed by spontaneously assembling single strands of DNA (or RNA single strands) of a specific sequence through annealing assembly to form a tetrahedral structure, and the size of the structure is at the nanometer level. As a type of nanomaterial with good biosafety, DNA tetrahedrons have been proven to be effectively taken up by mammalian cells. DNA tetrahedrons have therapeutic prospects for a variety of diseases, including tumors, infectious diseases, inflammation, etc. Through rational design, different types of drugs can be precisely positioned and assembled on tetrahedrons through complementary base pairing, which can simultaneously produce targeted delivery and synergistic effects, solving the treatment problems of some stubborn diseases. Based on the principle of DNA base pairing, therapeutic elements, chemical reactors and targeting ligands composed of DNA single strands can be effectively loaded through base pair hybridization, which is simple to operate and has high loading efficiency.

[0008] In summary, existing technologies indicate that treatment options for Wilson's disease primarily focus on traditional Chinese medicine preparations such as Gandoutang (Gandou Tang) and diagnostic probes, as well as gene-editing sequences. There are no reports of flavonoid compounds being used to treat Wilson's disease. Currently, most nucleic acid tetrahedrons used in drug delivery systems are single-stranded DNA tetrahedrons, with each arm consisting of a DNA double helix. However, there are no reports of double-stranded DNA tetrahedrons being used as drug delivery systems for Wilson's disease (there are no reports in Chinese, but we have published some in English). Modifying small nucleic acid drug RNAs to promote drug accumulation in the liver is a common strategy, but modifying the self-assembled structure of double-stranded DNA tetrahedrons with multiple GalNAc residues of fixed numbers and distributions to address the challenge of specific drug accumulation in lesions has not yet been studied. Summary of the Invention

[0009] The purpose of the present invention is to provide a nucleic acid self-assembly drug and a preparation method and application thereof.

[0010] The present invention provides a nucleic acid self-assembly drug. The nucleic acid self-assembly drug is an assembly drug which uses an addressable double-stranded DNA tetrahedron structure as a carrier and loads a flavonoid compound into the carrier by insertion.

[0011] Each arm of the addressable double-stranded DNA tetrahedron is composed of two DNA double helix chains. Each arm of the addressable double-stranded DNA tetrahedron is composed of 8 DNA helices (with an average length of 10.5 base pairs (bp) and a distance between base pair planes of 0.34 nm), with a length of approximately 28.5 nm (10.5 × 8 × 0.34 = 28.5 nm). This tetrahedral structure is addressable, meaning that any position within the DNA tetrahedron can be modified at a specific site. The addressable double-stranded DNA tetrahedron has 80 modifiable sites, including 12 GalNAc modification sites. The particle diameter of the addressable double-stranded DNA tetrahedron is approximately 20 nm.

[0012] The addressable double-stranded DNA tetrahedron is self-assembled by DNA single strands through multiple annealing procedures based on the base pairing principle. The sequence of the DNA single strands is shown in SEQ ID NO: 1-38.

[0013] The addressable double-stranded DNA tetrahedron is modified with an N-acetylgalactose group.

[0014] The single-stranded DNA sequences modified with N-acetylgalactosamine groups are SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 22 and SEQ ID NO: 31.

[0015] The present invention provides a method for preparing a nucleic acid self-assembly drug, characterized in that the method comprises the following steps:

[0016] 1) mixing single-stranded DNA sequences as shown in SEQ ID NOs: 1-11 to form a first mixture, mixing single-stranded DNA sequences as shown in SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NOs: 12-20 to form a second mixture, mixing single-stranded DNA sequences as shown in SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NOs: 21-29 to form a third mixture, and mixing single-stranded DNA sequences as shown in SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NOs: 30-38 to form a fourth mixture.

[0017] 2) Annealing and assembling the first, second, third, and fourth mixtures to obtain the four corner portions GV1, GV2, GV3, and GV4 that constitute the addressable double-stranded DNA tetrahedron structure. Specifically, the annealing and assembly procedures are: 95°C for 5 minutes; 65°C for 15 minutes; 55°C for 30 minutes; and 36°C for 30 minutes.

[0018] 3) The four corner components GV1, GV2, GV3, and GV4 are mixed and annealed to form an addressable double-stranded DNA tetrahedron. Specifically, the four components are mixed in a ratio of 1:1:1:1. The annealing process is as follows: hold at 37°C for 5 minutes, then slowly cool to 25°C over 12 hours in a gradient thermal cycler.

[0019] 4) The flavonoid compound is loaded into the above-mentioned addressable double-stranded DNA tetrahedron by intercalation to obtain a nucleic acid self-assembled drug.

[0020] The present invention provides an application of a nucleic acid self-assembly drug in preparing a drug for preventing and / or treating Wilson's disease.

[0021] In some embodiments, the flavonoid compound can be replaced with curcumin and other compounds such as polyphenols having multiple benzene rings, and loaded into the addressable double-stranded DNA tetrahedron by insertion to obtain a drug for treating Wilson's disease with the effect of chelating copper ions.

[0022] In some embodiments, the above applications include targeting liver cells, improving cell viability without producing cytotoxicity, reducing copper content in copper-overloaded cells, inhibiting cell apoptosis, reducing the level of intracellular reactive oxygen species, reducing liver copper levels, and promoting liver copper metabolism.

[0023] In some embodiments, the Wilson's disease drug is a liver-targeted flavonoid nanomedicine.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] The nucleic acid self-assembly drug provided by the present invention uses a double-stranded DNA tetrahedron as a carrier. Compared with a DNA tetrahedron with only one double-stranded DNA in each arm, the double-stranded DNA tetrahedron has good structural stability, a large drug loading capacity, and many modification sites. The nucleic acid self-assembly drug provided by the present invention modifies multiple GalNAcs of a fixed number and distribution position on its double-stranded DNA tetrahedron carrier, which solves the problem of specific enrichment of drugs in lesions, allowing flavonoids to better reach the lesions and thus exert excellent therapeutic effects. The nucleic acid self-assembly drug provided by the present invention achieves the effect of treating Wilson's disease by utilizing flavonoids, provides a new technical solution and treatment strategy for the prevention and treatment of Wilson's disease, and provides a new perspective and breakthrough for the development of human medical care. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the structure of the nucleic acid self-assembly drug of the present invention.

[0027] Figure 2The atomic force microscopy observation results of the nucleic acid self-assembly drug of the present invention are shown (scale bar: 50 nm).

[0028] Figure 3 This is a statistical graph showing the dynamic light scattering (DLS) particle size of the nucleic acid self-assembly drug of the present invention.

[0029] Figure 4 The cell viability assay results of the present invention at different concentrations of nucleic acid self-assembly drugs.

[0030] Figure 5 This is the therapeutic effect of the nucleic acid self-assembly drug of the present invention on copper-overloaded cells.

[0031] Figure 6 This is the inhibitory effect of the nucleic acid self-assembly drug of the present invention on cell apoptosis.

[0032] Figure 7 This is the Western blot analysis result of apoptosis-related signaling proteins of the present invention.

[0033] Figure 8 This is a fluorescence microscope image of intracellular reactive oxygen species according to the present invention.

[0034] Figure 9 This is the HE staining result of the rat liver tissue of the present invention.

[0035] Figure 10 This is the test result of copper content in rat liver of the present invention. DETAILED DESCRIPTION

[0036] The following is a further explanation of the technical solutions of the nucleic acid self-assembly drug and its preparation method and application through specific examples.

[0037] Example 1 Preparation of Nucleic Acid Self-Assembly Drugs

[0038] The DNA sequences used to assemble the 20-nanometer DNA tetrahedron structure are shown in Table 1. The sequences used to assemble the 20-nanometer DNA tetrahedron in this example were designed by our team and reported for the first time.

[0039] Table 1 DNA single-strand sequences used to assemble nucleic acid self-assembly drugs

[0040]

[0041]

[0042] First, purchased DNA strands were purified on denaturing agarose gels. For the single strands to be GalNAc-modified (G2, G-1-2, G-2-2, G-3-2, and G-4-2), dibenzocyclooctyne (DBCO)-modified DNA strands (DBCO2, DBCO-1-2, DBCO-2-2, DBCO-3-2, and DBCO-4-2) were purchased. These five strands were then reacted with N-azidoacetylgalactosamine tetraacylation reagent using click chemistry. The DBCO-modified single strands were dispersed in phosphate buffer at a ratio of 1:5 with N-azidoacetylgalactosamine tetraacylation reagent. A 1,3-dipolar cycloaddition reaction between DBCO and azide resulted in the covalent linkage of the DBCO-modified DNA strands to the azide-modified mannose. After reacting at room temperature for 12 hours, the mixture was ultrafiltered (5 kDa) to remove impurities. Quantification was then performed by measuring OD260 absorbance.

[0043] Purchased and synthesized DNA single strands were dissolved in ddH2O, separated by denaturing polyacrylamide gel electrophoresis, eluted, and purified by ethanol precipitation. The concentration of the purified DNA single strands was calculated by measuring the UV absorbance at OD260. The purified DNA single strands were assembled according to the following top angles:

[0044] Vertex 1GV1: YL, 1-1, G-2, G-1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9;

[0045] Top corner 2GV2: YL, 2-1, G-2, G-2-2, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9;

[0046] Top corner 3GV3: YL, 3-1, G-2, G-3-2, 3-3, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9;

[0047] Top corner 4GV4: YL, 4-1, G-2, G-4-2, 4-3, 4-4, 4-5, 4-6, 4-7, 4-8, 4-9.

[0048] After combination, dispersed in 1×TAE / Mg 2+ Buffer (40mM Tris, 20mM acetic acid, 2mM EDTA, 12.5mM magnesium acetate, pH = 8.3) was added and mixed and assembled at a final concentration of 100nM for each chain to obtain four single-chain mixtures (first mixture, second mixture, third mixture, and fourth mixture). Pay attention to the accuracy of the quantification.

[0049] The four single-stranded mixtures were mixed and placed in a gradient thermal cycler with a cooling program. The first assembly step, annealing, was as follows: 95°C for 5 minutes, then 68°C for 15 minutes, then 55°C for 30 minutes, then 36°C for 30 minutes. The resulting vertex-shaped assembly products were GV1, GV2, GV3, and GV4. The four vertex-shaped structures were then mixed in a 1:1:1:1 ratio. The second assembly step, annealing, was as follows: the mixed solution was heated to 37°C for 5 minutes, then slowly cooled to 25°C over 12 hours in a gradient thermal cycler. Upon completion of the program, a 20-nm-diameter DNA double-stranded tetrahedron structure with four distinct vertex structures was obtained. The assembled DNA double-stranded tetrahedrons were concentrated by ultrafiltration through a 5 kDa ultrafiltration cup, and the buffer was exchanged with saline or PBS.

[0050] The prepared DNA double-stranded tetrahedron is mixed with a flavonoid solution (galangin is selected here), incubated at room temperature for 6h-12h (or incubated at 37°C), and then ultrafiltration (3KDa) is performed to remove impurities to obtain a nucleic acid self-assembly drug. The schematic diagram of the nucleic acid self-assembly drug structure of the present invention is shown in FIG. Figure 1 shown.

[0051] The results of this example show that the loading capacity of galangin is an average of 4.1 galangin molecules per base pair. Therefore, the higher the base pair content and density, the higher the drug loading capacity of the DNA tetrahedron. The solubility of galangin is: soluble in ethanol and ether, easily soluble in chloroform and benzene, insoluble in water, and very poorly soluble in aqueous solutions, which limits its biological application. The design of this project solves the problem of biological application of poorly soluble flavonoid drugs such as galangin.

[0052] Example 2 Synthesis, Verification and Characterization of Nucleic Acid Self-Assembly Drugs

[0053] (1) Atomic force microscopy (AFM characterization)

[0054] Take the newly prepared DNA tetrahedron structure and disperse it in 1×TAE / Mg 2+ Buffer was added to a final concentration of 1 nM. 25 μL of the sample was dropped onto a freshly treated mica sheet and allowed to settle for 10 minutes. After deposition, the sample was removed, and the mica sheet was rinsed with ultrapure water and dried with nitrogen. The prepared sample was imaged using ScanAsyst-Fluid mode.

[0055] Atomic force microscopy observation results of nucleic acid self-assembly drugs are as follows Figure 2 As shown (scale bar: 50 nm), a regular, monodisperse, and uniform structure can be seen, and the size of the DNA tetrahedron structure is about 20 nm, indicating that the prepared nucleic acid self-assembled drug has a uniform, monodisperse morphology.

[0056] (2) Dynamic light scattering (DLS) detection

[0057] The freshly prepared nucleic acid self-assembly drug was dispersed in PBS buffer (sample concentration 100 nM) and DLS measurement was performed at 25°C.

[0058] Dynamic light scattering (DLS) particle size characterization statistics of nucleic acid self-assembled drugs are shown in the figure below: Figure 3 As shown, the hydrated particle size of the nucleic acid self-assembled drug is about 25 nm, which is consistent with the expected size range.

[0059] Example 3 Safety Testing of Nucleic Acid Self-Assembly Drugs

[0060] To evaluate the biosafety of nucleic acid self-assembly drugs, the CCK-8 assay was used to analyze cell viability. Human hepatocyte Huh-7 cells were seeded at a density of 4×103 cells per well in 96-well plates and cultured until adherent. Medium containing various concentrations of nucleic acid self-assembly drugs was added to the cell culture plates, and the cells were incubated at 37°C for 48 hours. The medium was then removed, and the cells were washed three times with cold PBS. Serum-free medium containing CCK-8 solution was added, and the cells were incubated in a culture incubator for 1 hour. After incubation, absorbance at 450 nm was measured using a Bio-Rad 680 microplate reader.

[0061] The results of cell viability assays under different concentrations of nucleic acid self-assembly drugs are as follows: Figure 4 The results show that nucleic acid self-assembly drugs not only do not produce cytotoxicity, but also have a certain promoting effect on cell proliferation. This result is consistent with the properties of nucleic acid tetrahedral structure and flavonoids that have a certain effect on promoting cell viability.

[0062] Example 4 Nucleic Acid Self-Assembly Drug Reduces Copper Content in Copper-Overloaded Cells

[0063] A cellular copper overload model was constructed by adding CuSO4 to the culture medium. Cells were transferred to a medium containing a 200 μM CuSO4 solution and incubated. PBS or a nucleic acid self-assembly drug (10 nM nucleic acid self-assembly drug, calculated as DNA tetrahedrons; free flavonoids were determined based on the amount of free flavonoids loaded by the 10 nM nucleic acid self-assembly drug) was added. A control group was set up. After 24 hours of culture, equal numbers of cells were counted and lysed in phosphate buffer (PBS) using an ultrasonic disruptor. The copper content in the cell lysate was determined according to the instructions of the Copper Quantification Detection Kit (BioAssay Systems, USA). The copper concentration was calculated based on the absorbance at 359 nm and a standard curve.

[0064] The therapeutic effect of nucleic acid self-assembly drugs on copper-overloaded cells Figure 5 As shown in the results, based on changes in intracellular copper levels, the experimental results indicate that the nucleic acid self-assembly drug is more effective at promoting copper ion efflux than the free flavonoid drug. Cells treated with the nucleic acid self-assembly drug showed a significant decrease in copper levels, approaching the copper levels in control cells. In a copper-overload cell model, the nucleic acid self-assembly drug was able to significantly reduce copper levels in copper-overloaded cells. Furthermore, since flavonoids themselves have a certain ability to chelate copper ions, they also have a certain cytoprotective effect. In this experiment, the free flavonoid drug (galangin) was used as a control.

[0065] Example 5 Nucleic acid self-assembly drugs reduce the effect of copper flux-induced cell apoptosis

[0066] Huh-7 hepatocytes were seeded at a density of 2.5×10⁴ cells per well in 24-well plates and cultured until adherent. Serum-containing medium containing 200 μM CuSO⁴ solution was added to the plates. Different experimental groups were set up. The treatment group was treated with 10 nM of the nucleic acid self-assembly drug, and the cells were cultured at 37°C for 24 hours. After incubation, the cells were washed twice with cold PBS and stained with Annexin V-FITC / PI according to the manufacturer's instructions. Samples were then analyzed by fluorescence microscopy and flow cytometry.

[0067] The inhibitory effect of nucleic acid self-assembly drugs on cell apoptosis Figure 6 As shown. Copper overload can induce a series of apoptosis-inducing effects in cells, including oxidative stress. Apoptosis in copper-overloaded cells can be detected using an apoptosis kit. Experimental results show that nucleic acid self-assembly drugs can significantly reduce copper overload-induced apoptosis.

[0068] Example 6 Analysis of signaling proteins related to the inhibition of apoptosis by nucleic acid self-assembly drugs

[0069] Huh-7 hepatocytes were seeded at a density of 2.5×10⁴ cells per well in 24-well plates and cultured until adherent. Serum-containing medium containing 200 μM CuSO⁴ solution was added to the plates to determine the experimental groups. The treatment group was treated with 10 nM of the nucleic acid self-assembly drug, and the cells were cultured at 37°C for 24 hours. After incubation, the cells were harvested, trypsinized, and washed three times with cold PBS. RIPA lysis buffer was then added to the cells and shaken to fully lyse them. Following lysis, the samples were centrifuged at 15,000 g for 10 minutes at 4°C, and the supernatant was used for subsequent experiments. The extracted total protein was quantified using the BCA assay according to the manufacturer's instructions. 20 μg of total protein was subjected to SDS-PAGE, and the separated proteins were transferred to a PVDF membrane. After transfer, the membrane was blocked and washed, and then incubated with the corresponding antibody solution, using β-actin as a loading control. After washing, the secondary antibody was added for incubation, and after washing again, luminescence detection was performed using an enhanced ECL-Western detection kit (Thermo-Fisher Scientific, USA).

[0070] Western blot analysis results of apoptosis-related signaling proteins are shown in Figure 2. Figure 7 As shown. Western blot analysis of apoptosis-related signaling proteins showed that, using Actin as the internal reference, the expression level of the pro-apoptotic factor Bax in the Wilson disease model induced by excess copper ions was significantly increased. Compared with the control group without the addition of excess copper ions for induction, Bax was at a high level. However, after treatment with nucleic acid self-assembly drugs, the level of crude apoptotic factors was significantly reduced. Because overexpression of Bax can antagonize the protective effect of Bcl-2, cells tend to die. As a marker for inhibiting cell apoptosis, the expression level of Bcl-2 can reflect the anti-apoptotic effect of therapeutic drugs. The experimental results showed that after treatment with nucleic acid self-assembly drugs, the expression level of Bcl-2 increased, indicating that nucleic acid self-assembly drugs have excellent therapeutic effects in inhibiting cell apoptosis induced by excess copper ions.

[0071] Example 7 Analysis of Antioxidant Damage of Nucleic Acid Self-Assembly Drugs

[0072] In this part of the experiment, 400 μM CuSO4 was added to Huh-7 cells to induce a cell oxidative aging model. After the cells were cultured and adhered, DMEM culture medium containing different drug components was added (the concentration of the nucleic acid self-assembly drug was 10 nM). After 48 hours of culture, the culture medium was removed, the cells were washed three times with cold PBS, and fresh DMEM culture medium was added. The level of intracellular reactive oxygen species was detected using a reactive oxygen species detection kit (Beyotime Biotechnology). After the treated cells were washed with cold PBS, DMEM culture medium containing DCFH-DA (10 μM) was added and incubated at room temperature for 10 minutes. After incubation, the culture medium was removed and the cells were washed with cold PBS, followed by fluorescence fiber microscopy observation.

[0073] Fluorescence microscopy imaging of intracellular reactive oxygen species Figure 8 As shown in the figure, the results of the anti-oxidative damage experiment showed that the accumulation of copper ions in cells produces a large amount of reactive oxygen species, which causes oxidative damage to cells. However, after treatment with nucleic acid self-assembly drugs, the level of reactive oxygen species in cells was significantly reduced.

[0074] Example 8 Copper Accumulation Rat Model of Nucleic Acid Self-Assembly Drugs - In Vivo Therapeutic Effect Analysis

[0075] SPF-grade healthy male SD rats were fed ad libitum for 12 consecutive weeks. The model and treatment groups were fed a copper-loaded diet (1 g / kg) and water (0.185%) for 12 consecutive weeks to replicate the rat model. Starting from the ninth week of modeling, rats in the treatment group were injected with 100 nM / 500 μL of the nucleic acid self-assembly drug via the tail vein (administered every other day for seven consecutive doses) until the end of treatment. After treatment, fresh liver tissue was removed, fixed in 10% neutral buffered formalin for 24 hours, and embedded in paraffin. Hematoxylin and eosin staining was performed and microscopic imaging was performed.

[0076] HE staining was used to observe the pathological changes of rat liver tissue. The results of HE staining of rat liver tissue were as follows: Figure 9 As shown, in the left control group, healthy rats showed intact hepatic lobule morphology and structure, with hepatocyte cords arranged radially around the central vein, cells of essentially uniform size, and no cell degeneration or necrosis. In the middle model group, severe hepatic lobule structure was observed, with disorganized, non-radial arrangement of hepatocyte cords, localized hepatocyte nuclei effaced or pyknotic, and cells necrotic, dissolved, or disappeared. In the right group treated with the nucleic acid self-assembly drug, stem cells were arranged more neatly than in the model group, with normal cell morphology. These results demonstrate the excellent therapeutic efficacy of nucleic acid self-assembly drugs.

[0077] Example 9 Copper Accumulation Rat Model of Nucleic Acid Self-Assembly Drugs - Metabolic Analysis

[0078] The rat liver copper level was detected using a kit. After treatment, fresh liver tissue was obtained, washed with physiological saline, 0.5 g was weighed, homogenized, and centrifuged at 4°C and 2000 r / min for 10 min. The supernatant was collected. Alternatively, 24-hour urine was collected and added to a 96-well plate. The plate was incubated at 37°C in the dark, and the photometric value was measured at a wavelength of 600 nm using an enzyme reader.

[0079] The results of the copper content test in rat liver are as follows Figure 10 The experimental results showed that after treatment, the nucleic acid self-assembly drug was able to significantly reduce the copper level in the liver of the rat copper accumulation model, indicating that the nucleic acid self-assembly drug can promote copper metabolism in the liver of the rat Wilson disease model, and further indicating that the nucleic acid self-assembly drug can treat diseases caused by high copper.

[0080] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A nucleic acid self-assembly drug, characterized in that: The nucleic acid self-assembly drug is an assembled drug that uses an addressable double-stranded DNA tetrahedron structure as a carrier, and loads flavonoid compounds into the carrier through insertion.

2. The nucleic acid self-assembly drug according to claim 1, characterized in that Each arm of the addressable double-stranded DNA tetrahedron consists of two DNA double helix chains.

3. The nucleic acid self-assembly drug according to claim 1, characterized in that The addressable double-stranded DNA tetrahedron is self-assembled by DNA single strands based on the base pairing principle through multiple annealing procedures, and the sequence of the DNA single strand is shown in SEQ ID NO: 1-38.

4. The nucleic acid self-assembly drug according to claim 1, characterized in that The addressable double-stranded DNA tetrahedron is modified with an N-acetylgalactose group.

5. The nucleic acid self-assembly drug according to claim 4, characterized in that The single-stranded DNA sequences modified with N-acetylgalactose groups are SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 22 and SEQ ID NO:

31.

6. A method for preparing a nucleic acid self-assembly drug, characterized in that: The method comprises the following steps: 1) mixing single-stranded DNAs with sequences such as SEQ ID NOs: 1-11 to form a first mixture, mixing single-stranded DNAs with sequences such as SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NOs: 12-20 to form a second mixture, mixing single-stranded DNAs with sequences such as SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NOs: 21-29 to form a third mixture, and mixing single-stranded DNAs with sequences such as SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NOs: 30-38 to form a fourth mixture; 2) annealing and assembling the first mixture, the second mixture, the third mixture, and the fourth mixture, respectively, to obtain four corner portions GV1, GV2, GV3, and GV4 constituting an addressable double-stranded DNA tetrahedron structure; 3) The four corner portions GV1, GV2, GV3, and GV4 are mixed and annealed to assemble to obtain an addressable double-stranded DNA tetrahedron; 4) The flavonoid compound is loaded into the above-mentioned addressable double-stranded DNA tetrahedron by intercalation to obtain a nucleic acid self-assembled drug.

7. Use of any one of the nucleic acid self-assembly drugs according to claims 1 to 5 in the preparation of drugs for preventing and / or treating Wilson's disease.

8. The use according to claim 7, characterized in that The applications include targeting liver cells, improving cell viability without producing cytotoxicity, reducing copper content in copper-overloaded cells, inhibiting cell apoptosis, reducing the level of intracellular reactive oxygen species, reducing liver copper levels, and promoting liver copper metabolism.

9. The use according to claim 7, characterized in that The Wilson disease drug is a liver-targeted flavonoid nanomedicine.

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