Cholesterol modified DNA tetrahedral structure nano-drug carrier

Abstract

The invention provides a DNA tetrahedral nano device with cholesterol molecule space accurate arrangement and a preparation method and application thereof. Specifically, the invention provides a nucleic acid tetrahedral nanostructure which comprises a tetrahedral composite structure formed by assembling a plurality of nucleic acid monomers, and the tetrahedral composite structure is coupled with cholesterol molecules. According to the invention, the addressability and programmability of the DNA nanotechnology are utilized, and the spatial position of cholesterol molecules in the DNA nanostructure can be accurately controlled, so that the advantages of greatly improving the biological stability, improving the pharmacokinetic and pharmacodynamic properties, enhancing the stability, improving the in-vivo distribution of drugs and the like can be achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of materials and biomedicine, and specifically relates to a cholesterol-modified DNA tetrahedral structure nano drug carrier. Background Art

[0002] The presence of cholesterol is considered to be essential for regulating functional lipid structures and interactions between proteins and lipids. However, the functional relevance of cholesterol at the interface of biological structures other than cell membranes has not been fully studied. A study showed that lipid layers containing cholesterol counteract bioadhesion through an entropic repulsion mechanism, thereby reducing the adhesion of proteins, bacteria, etc. to them. Modifying drug molecules with cholesterol has the advantages of improving pharmacokinetic and pharmacodynamic properties, enhancing stability, and improving drug distribution in vivo.

[0003] However, the current cholesterol modification sites are limited, and it is difficult to obtain cholesterol-modified products with precise spatial arrangement of cholesterol molecules, which cannot effectively improve the function of drug molecules.

[0004] Therefore, there is an urgent need in the art to develop cholesterol modification methods and products with precise spatial arrangement of cholesterol molecules. Summary of the invention

[0005] The purpose of the present invention is to provide a cholesterol modification method and product with precise spatial arrangement of cholesterol molecules.

[0006] In a first aspect of the present invention, a nucleic acid tetrahedron nanostructure is provided, wherein the nucleic acid tetrahedron nanostructure comprises a tetrahedron composite structure formed by assembling a plurality of nucleic acid monomers, and the tetrahedron composite structure is coupled to a cholesterol molecule.

[0007] In another preferred embodiment, the nucleic acid monomer is selected from the following group: DNA, RNA, locked nucleic acid, peptide nucleic acid, or a combination thereof.

[0008] In another preferred embodiment, the nucleic acid tetrahedron nanostructure is a DNA nucleic acid tetrahedron nanostructure.

[0009] In another preferred embodiment, among the nucleic acid single chains (or nucleic acid monomers) constituting the nucleic acid tetrahedral nanostructure, 2, 3, or 4 nucleic acid monomers are modified with cholesterol.

[0010] In another preferred embodiment, the number of sites modified by cholesterol on a nucleic acid monomer is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, preferably 1-12.

[0011] In another preferred embodiment, the nucleic acid monomer is coupled to a cholesterol molecule at the end (5' end and / or 3' end) and / or non-end.

[0012] In another preferred embodiment, the nucleic acid monomer is coupled to a cholesterol molecule at least at the non-terminal end (other nucleic acids other than the 5' end and the 3' end).

[0013] In another preferred embodiment, the nucleic acid monomer is coupled with a cholesterol molecule only at the non-terminal portion (other nucleic acids other than the 5' end and the 3' end).

[0014] In another preferred embodiment, the size of the nucleic acid tetrahedral nanostructure is about 5-30 nanometers, preferably 7-20 nanometers.

[0015] In another preferred embodiment, the nucleic acid tetrahedral nanostructure has a structure of formula A:

[0016] Z-CHOL (A)

[0017] In the formula,

[0018] Z is a nucleic acid tetrahedral nanostructure formed by the assembly of nucleic acid monomers;

[0019] CHOL represents a cholesterol molecule coupled to the nucleic acid tetrahedral nanostructure.

[0020] In another preferred embodiment, the nucleic acid tetrahedral nanostructure is a complex of formula I:

[0021] (Z-CHOL)-Dm (I)

[0022] In the formula,

[0023] Z is a nucleic acid tetrahedral nanostructure formed by the assembly of nucleic acid monomers;

[0024] CHOL represents a cholesterol molecule coupled to the nucleic acid tetrahedral nanostructure;

[0025] “(Z-CHOL)” represents a nucleic acid tetrahedral nanostructure formed by Z and CHOL with the surface modified by cholesterol molecules;

[0026] D represents a drug molecule, which is coupled to the nucleic acid tetrahedral nanostructure modified with a cholesterol molecule;

[0027] m is a positive integer ≥1.

[0028] In another preferred embodiment, the D is coupled to or connected to the nucleic acid monomer.

[0029] In a second aspect of the present invention, a method for preparing the nucleic acid tetrahedral nanostructure according to the first aspect of the present invention is provided, comprising the steps of:

[0030] (a) providing nucleic acid monomers, wherein at least one nucleic acid monomer is coupled to a cholesterol molecule;

[0031] (b) mixing and annealing the nucleic acid monomers to assemble and form the nucleic acid tetrahedral nanostructure according to the first aspect of the present invention.

[0032] In another preferred embodiment, among the nucleic acid monomers constituting the nucleic acid tetrahedral nanostructure, ≥50%, preferably ≥80%, more preferably ≥90%, and most preferably ≥95% or 100% of the nucleic acid monomers are coupled with cholesterol molecules.

[0033] In the third aspect of the present invention, there is provided a use of the nucleic acid tetrahedral nanostructure described in the first aspect of the present invention, which is used to prepare cholesterol-modified drugs.

[0034] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The assembly of a DNA tetrahedral nanomachine is shown.

[0036] Figure 2 The atomic force microscopy characterization of a DNA tetrahedron with a spatial conformation of cholesterol molecules is shown, in which cholesterol-modified DNA bases are evenly distributed in the single-stranded DNA. The length of the dimension bar in the small image in the atomic force microscopy characterization image is 5 nm.

[0037] Figure 3 The size representation of the DNA tetrahedron with the spatial conformation of the cholesterol molecule is shown.

[0038] Figure 4 The polyacrylamide characterization results of cholesterol-modified DNA nanomachines are shown. Sample No. 1 is a tetrahedral DNA nanomachine without cholesterol labeling; Sample No. 2 is a tetrahedral DNA nanomachine modified with 3×4 cholesterol molecules; Sample No. 3 is a tetrahedral DNA nanomachine modified with 6×4 cholesterol molecules; Lane 4 is a 20bp DNA standard, and the DNA fragments from top to bottom are 500bp, 400bp, 300bp, 200bp, 180bp, 160bp, 140bp, 120bp, 100bp, 80bp, 60bp, 40bp, and 20bp. DETAILED DESCRIPTION

[0039] After extensive and in-depth research, the inventors have provided a unique nucleic acid tetrahedron nanostructure with a precise spatial arrangement of cholesterol molecules for the first time. The inventors have designed and prepared nucleic acid tetrahedron nanostructures (such as DNA tetrahedron nanostructures) of different conformations, thereby achieving precise control of the spatial position of cholesterol molecules in the DNA nanostructure. A nanomachine that can carry drugs constructed by the nucleic acid tetrahedron nanostructure of the present invention greatly improves the biological stability and safety of the loaded drugs and improves the renal clearance rate of the loaded drugs. The present invention has been completed on this basis.

[0040] Cholesterol molecules

[0041] In the present invention, there is a hydrophobic surface formed by cholesterol molecules on the outer surface of the DNA nanostructure. In the present invention, since cholesterol molecules have hydrophobicity and assembly properties, the surface of the DNA nanostructure can be easily modified to form a hydrophobic layer.

[0042] Cholesterol molecules suitable for use in the present invention include dehydrocholesterol, thiol-modified cholesterol and palmitic acid cholesterol.

[0043] In the present invention, the cholesterol molecules can be directly or indirectly linked to the DNA nanostructure or its constituent monomers (nucleic acid monomers), for example, through covalent bonds, linking groups or other similar means.

[0044] In the present invention, a preferred cholesterol is a cholesterol molecule with modified hydroxyl groups, and the structural formula is shown below.

[0045]

[0046] In another preferred embodiment, one or more identical or different cholesterol molecules may be coupled to the nucleic acid tetrahedral nanostructure of the present invention.

[0047] Nucleic acid tetrahedral nanostructure of the present invention

[0048] As used herein, the terms "the nucleic acid tetrahedral nanostructure of the present invention", "the nanostructure of the present invention", "the cholesterol-modified nucleic acid tetrahedral nanostructure" and "the nucleic acid tetrahedral nanostructure with precise spatial arrangement of cholesterol molecules" are used interchangeably and refer to the nucleic acid tetrahedral nanostructure described in the first aspect of the present invention, which has a tetrahedral structure formed by the assembly of single-stranded nucleic acids, and the nucleic acid tetrahedral nanostructure is also coupled with cholesterol molecules in a predetermined space of the nucleic acid tetrahedral nanostructure (or a predetermined position of a nucleic acid monomer).

[0049] It should be understood that the nucleic acid monomers in the nucleic acid tetrahedral nanostructure of the present invention may include (but are not limited to): DNA, RNA, locked nucleic acid, peptide nucleic acid, or a combination thereof.

[0050] In a preferred embodiment, the nucleic acid tetrahedral nanostructure of the present invention is a DNA nucleic acid tetrahedral nanostructure.

[0051] In the present invention, the multiple nucleic acid monomers constituting the nucleic acid tetrahedral nanostructure are independent of each other, but can interact with each other through base pairing and the like to form a tetrahedral composite structure.

[0052] In the present invention, although the nucleic acid tetrahedral nanostructure is modified by at least one or more cholesterol molecules, each nucleic acid monomer can be independently modified by a cholesterol molecule or not. In other words, in the present invention, at least one nucleic acid monomer is coupled to a cholesterol molecule or modified by a cholesterol molecule.

[0053] Preferably, in the present invention, in the nucleic acid single strands (or nucleic acid monomers) constituting the nucleic acid tetrahedral nanostructure, 2, 3, or 4 nucleic acid monomers are modified with cholesterol. More preferably, all nucleic acid monomers are modified with cholesterol.

[0054] For each nucleic acid monomer modified by cholesterol, its modified site can be one or more. Typically, on a nucleic acid monomer, the number of sites modified by cholesterol is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, preferably 1-12.

[0055] In the present invention, the length of each nucleic acid monomer is generally 21-63 nt, preferably 57-63 nt.

[0056] Preferably, in the nucleic acid tetrahedral nanostructure of the present invention, the lengths of the nucleic acid monomers are the same or substantially the same (eg, the lengths differ by ≤10%, or ≤5 nt).

[0057] In a preferred embodiment, the size of the nucleic acid tetrahedral nanostructure of the present invention can be controlled to be between 5 and 30 nanometers, preferably between 7 and 20 nanometers, by setting the length of the nucleic acid monomer sequence.

[0058] In a preferred embodiment, taking a nucleic acid tetrahedral nanostructure in which each tetrahedral DNA chain is 63 nt in length as an example, the distribution of representative cholesterol molecules may include (but is not limited to) the following distribution:

[0059] 1. Each chain has one cholesterol molecule, which can be distributed at base positions 1-5, 30-35, or 60-65.

[0060] 2. Each chain has 3 cholesterol molecules, which can be distributed at base positions 15, 36, and 57. The average interval is 20 bases. The position can be designed as needed.

[0061] 3. Each chain has 6 cholesterol molecules, which can be distributed at base positions 5, 15, 26, 36, 47, and 57. The average interval is 10 bases.

[0062] It should be understood that for nucleic acid monomers of different lengths and different application scenarios, the connection position and / or number of cholesterol molecules can be adjusted as needed.

[0063] application

[0064] The present invention also provides applications of the nucleic acid tetrahedron nanostructure in various occasions.

[0065] Typically, the nucleic acid tetrahedral nanostructure of the present invention can be used as a carrier for loading drugs. It should be understood that the loaded drugs are small molecule drugs, peptide drugs, or protein drugs.

[0066] The DNA tetrahedral nanostructure of the present invention has a nanometer-level size, and the spatial position of the coupled cholesterol molecules can be precisely controlled. Therefore, a highly uniform nanomachine (or nanocarrier) for drug delivery can be prepared, thereby greatly improving the biological stability, half-life and safety of the drug.

[0067] In another preferred embodiment, a drug formed by covalently coupling the nucleic acid tetrahedral nanostructure of the present invention with the small molecule compound deferoxamine is constructed. The drug has an improved renal clearance rate and can be applied to blood purification, especially the efficient treatment of iron overload, and thus used for the treatment of related diseases such as hemosiderosis.

[0068] It should be understood that in the art, any active pharmaceutical ingredient that can be treated with cholesterol can be covalently coupled to the cholesterol on the nucleic acid tetrahedral nanostructure of the present invention to obtain a complex of formula I:

[0069] Z-CHOL-D (I)

[0070] In the formula,

[0071] Z is a nucleic acid tetrahedral nanostructure formed by the assembly of nucleic acid monomers;

[0072] CHOL represents a cholesterol molecule coupled to the nucleic acid tetrahedral nanostructure;

[0073] D stands for drug.

[0074] Preferably, the drug-carrying nucleic acid tetrahedral nanostructure has a structure of Formula II:

[0075] Z-CHOLn-Dm (II)

[0076] In the formula,

[0077] Z is a nucleic acid tetrahedral nanostructure formed by the assembly of nucleic acid monomers;

[0078] CHOL represents a cholesterol molecule coupled to the nucleic acid tetrahedral nanostructure;

[0079] n is the number of cholesterol molecules coupled to a nucleic acid tetrahedral nanostructure, which is 1-100, preferably 2-50, and more preferably 3-30;

[0080] D stands for drug;

[0081] m is the number of drugs covalently coupled to a nucleic acid tetrahedral nanostructure via a cholesterol molecule, and m is ≥1.

[0082] In another preferred embodiment, m≤n.

[0083] In another preferred embodiment, m is 1-100, preferably 2-50, more preferably 3-30.

[0084] In another preferred embodiment, the ratio of m / n is 10-100%, preferably 20-90%, and more preferably 50-80%.

[0085] Preparation method

[0086] The present invention also provides a method for preparing the nucleic acid tetrahedron nanostructure of the present invention, comprising the steps of:

[0087] (a) providing nucleic acid monomers, wherein at least one nucleic acid monomer is coupled to a cholesterol molecule;

[0088] (b) mixing and annealing the nucleic acid monomers to assemble and form the nucleic acid tetrahedral nanostructure of the present invention.

[0089] Typically, in the present invention, a predetermined number of cholesterol molecules are first synthesized into the phosphate backbone of DNA nucleotides by covalent linkage, thereby obtaining a conjugate of DNA nucleotide monomers and cholesterol molecules; then, these conjugates of nucleotide monomers and cholesterol are added one by one on a solid phase and reacted to synthesize a long DNA chain, thereby achieving the coupling of a predetermined number (such as 2-15) of cholesterol molecules on a single DNA strand; thirdly, through complementary base pairing of DNA, the assembly of DNA chains is achieved to form a three-dimensional nucleic acid tetrahedral nanostructure, thereby precisely spatially arranging and regulating the cholesterol molecules according to the requirements of three-dimensional distribution.

[0090] When it is necessary to prepare a nucleic acid tetrahedron nanostructure loaded with a drug, nucleic acid monomers pre-coupled with drug molecules can be directly used to assemble the nucleic acid tetrahedron nanostructure.

[0091] In addition, when it is necessary to prepare a drug-loaded nucleic acid tetrahedral nanostructure, the method of the present invention further comprises step (c): covalently coupling the nucleic acid tetrahedral nanostructure obtained in step (b) with the drug to obtain the drug-loaded nucleic acid tetrahedral nanostructure.

[0092] The main advantages of the present invention include:

[0093] (a) The drug platform of the present invention is based on DNA nanotechnology, has a simple preparation process, and has uniform structure and properties.

[0094] (b) The DNA nanostructure of the present invention is highly designable and its morphological characteristics can be precisely controlled.

[0095] (c) The yield of the DNA nanostructure prepared by the present invention is high, which can reach more than 90%.

[0096] (d) The present invention utilizes the addressability of DNA nanotechnology to precisely control the spatial position of cholesterol molecules in the DNA nanostructure to achieve a conformation with the highest spatial coverage, which can greatly improve its biological stability.

[0097] (f) The present invention uses the programmability of DNA nanotechnology to construct a DNA molecular machine with precisely controllable size, which can quickly pass through the glomerular filtration membrane of the kidney (less than 6 nanometers) in the body, thereby controlling its biological distribution in the body, achieving rapid clearance and excretion of the drug, and increasing its circulation time while greatly improving its biosafety.

[0098] (g) The DNA molecular machine provided by the present invention can be used as a universal drug delivery platform to target ions, small molecules, nucleic acid macromolecules, proteins and peptides in the body to achieve the function of targeted and directional removal in the body. It has very good promotion and universality.

[0099] (h) The hydrophobic surface forms a thicker hydration film, which prevents the occurrence of coagulation and precipitation, thereby increasing the stability of the structure. This flexible modification brings a greater steric hindrance effect, which protects the modified substance from the attack of proteases and increases the stability of the modified substance.

[0100] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are weight percentages and weight parts.

[0101] Example 1

[0102] Preparation of Tetrahedral Nucleic Acid Monomers

[0103] The sequences of a set of nucleic acid monomers used to construct nucleic acid tetrahedral nanostructures are shown in Tables 1 and 2.

[0104] The nucleic acid monomers shown in Table 1 and Table 2 were synthesized using conventional artificial synthesis methods.

[0105] Table 1 Normal tetrahedral sequence without cholesterol modification

[0106]

[0107] Table 2 24 tetrahedral sequences modified by cholesterol molecules, where the underlined bases are cholesterol-modified bases

[0108]

[0109] For the nucleic acid monomers shown in Table 2, a predetermined number of cholesterol molecules are first synthesized into the backbone of DNA nucleotides by covalent linkage, and a predetermined number (here 24) of cholesterol molecules are coupled to each edge of the tetrahedron using DNA solid phase synthesis.

[0110] Example 2

[0111] Assembly of DNA nanomachines

[0112] In the example, nucleic acid monomers shown in Table 2 prepared in Example 1 were used to prepare nucleic acid tetrahedral nanostructures.

[0113] The method is as follows: 4 oligonucleotides (nucleic acid monomer chains) of 63 bases are annealed to assemble, wherein each chain extends along the three sides of one face of a tetrahedron, and each single strand of DNA is structurally split into three edge regions, each edge being composed of two complementary DNA double strands. Therefore, each position of the tetrahedron is formed by double-stranded complementary hybridization with base sequence specificity. The sequences of the 4 nucleic acid monomer chains are described in Tables 1 and 2.

[0114] All single-stranded DNA oligonucleotides were in 1×TAE-Mg 2+ (12.5 mM magnesium acetate, 2 mM EDTA, 20 mM acetic acid and 40 mM Tris base, pH 8.0). The four oligonucleotides were then mixed and the mixture was annealed from 90°C to 4°C using the temperature gradient shown in Table 3 below.

[0115] Table 3

[0116]

[0117] During the annealing process, DNA chains are assembled through complementary base pairing to form a three-dimensional DNA nanostructure, thereby precisely spatially arranging and regulating cholesterol molecules according to the requirements of three-dimensional distribution.

[0118] The prepared nucleic acid tetrahedral nanostructures were characterized by atomic force microscopy and polyacrylamide electrophoresis using conventional instruments and methods.

[0119] result

[0120] The results are as follows Figure 2 , Figure 3 and Figure 4 shown.

[0121] Figure 2 Atomic force microscopy characterization of DNA tetrahedron with the spatial conformation of cholesterol molecules is shown, indicating that the assembly forms a tetrahedral structure.

[0122] Figure 3 The size and electrical characterization results of the DNA tetrahedron with the spatial conformation of cholesterol molecules are shown, indicating that its size is 5-30nm.

[0123] Figure 4 The results of structural characterization of the DNA nanomachine synthesized by the present invention are shown, wherein the molecular weight of the cholesterol-containing nucleic acid tetrahedron nanostructure is larger, which is consistent with the expected value.

[0124] Example 3

[0125] Preparation of drug-loaded nucleic acid tetrahedral nanostructures

[0126] In this example, the nucleic acid tetrahedron nanostructure prepared in Example 2 is covalently coupled with drug molecules to obtain a drug-loaded nucleic acid tetrahedron nanostructure. The method is as follows:

[0127] Deferoxamine is a very specific Fe ion (Fe 3+ ) chelator has a very strong affinity for iron ions. Through click chemistry, deferoxamine molecules can be coupled to the ends of DNA chains to form stable DNA modifications. By assembling different numbers (4-20) of deferoxamine nanochelators on tetrahedral DNA nanomachines, Fe ions can be chelated in the blood. 3+ effective removal.

[0128] The cholesterol nucleic acid tetrahedral structure loaded with deferoxamine is identical to the tetrahedral conformation characterized by the original design, and the structure is consistent. Because the small molecule deferoxamine is small in size, the size of the tetrahedral structure has not changed significantly, and its edge length is also 5-10nm.

[0129] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A nucleic acid tetrahedral nanostructure, characterized in that: The nucleic acid tetrahedron nanostructure comprises a tetrahedron composite structure formed by assembling a plurality of nucleic acid monomers, and the tetrahedron composite structure is coupled with a cholesterol molecule.

2. The nucleic acid tetrahedral nanostructure according to claim 1, characterized in that: The nucleic acid monomer is selected from the group consisting of DNA, RNA, locked nucleic acid, peptide nucleic acid, or a combination thereof.

3. The nucleic acid tetrahedral nanostructure according to claim 1, characterized in that: The nucleic acid tetrahedron nanostructure is a DNA nucleic acid tetrahedron nanostructure.

4. The nucleic acid tetrahedral nanostructure according to claim 1, characterized in that: In the nucleic acid single strands (or nucleic acid monomers) constituting the nucleic acid tetrahedral nanostructure, 2, 3, or 4 nucleic acid monomers are modified with cholesterol.

5. The nucleic acid tetrahedral nanostructure according to claim 1, characterized in that: On a nucleic acid monomer, the number of sites modified by cholesterol is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more, preferably 1-12; In another preferred embodiment, the nucleic acid monomer is coupled with a cholesterol molecule at the end (5' end and / or 3' end) and / or non-end; In another preferred embodiment, the nucleic acid monomer is coupled with a cholesterol molecule at least at the non-terminal end (other nucleic acids other than the 5' end and the 3' end); In another preferred embodiment, the nucleic acid monomer is coupled with a cholesterol molecule only at the non-terminal portion (other nucleic acids other than the 5' end and the 3' end).

6. The nucleic acid tetrahedral nanostructure according to claim 1, characterized in that: The size of the nucleic acid tetrahedral nanostructure is about 5-30 nanometers, preferably 7-20 nanometers.

7. The nucleic acid tetrahedral nanostructure according to claim 1, characterized in that: The nucleic acid tetrahedral nanostructure has a structure of formula A: Z-CHOL(A) In the formula, Z is a nucleic acid tetrahedral nanostructure formed by the assembly of nucleic acid monomers; CHOL represents a cholesterol molecule coupled to the nucleic acid tetrahedral nanostructure.

8. The nucleic acid tetrahedral nanostructure according to claim 1, characterized in that: The nucleic acid tetrahedral nanostructure is a complex of formula I: (Z-CHOL)-D m (I) In the formula, Z is a nucleic acid tetrahedral nanostructure formed by the assembly of nucleic acid monomers; CHOL represents a cholesterol molecule coupled to the nucleic acid tetrahedral nanostructure; "(Z-CHOL)" represents a nucleic acid tetrahedral nanostructure formed by Z and CHOL with the surface modified by cholesterol molecules; D represents a drug molecule, which is coupled to the nucleic acid tetrahedral nanostructure modified with a cholesterol molecule; m is a positive integer ≥ 1; In another preferred embodiment, the D is coupled to or connected to the nucleic acid monomer.

9. The method for preparing nucleic acid tetrahedral nanostructures according to claim 1, characterized in that: Includes steps: (a) providing nucleic acid monomers, wherein at least one nucleic acid monomer is coupled to a cholesterol molecule; (b) mixing and annealing the nucleic acid monomers to assemble and form the nucleic acid tetrahedral nanostructure according to claim 1; In another preferred embodiment, among the nucleic acid monomers constituting the nucleic acid tetrahedral nanostructure, ≥50%, preferably ≥80%, more preferably ≥90%, and most preferably ≥95% or 100% of the nucleic acid monomers are coupled with cholesterol molecules.

10. The use of the nucleic acid tetrahedral nanostructure according to claim 1, characterized in that: Used to prepare cholesterol-modified drugs.

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