Liposome-nucleic acid tetrahedron nano-composite structure as well as preparation method and application thereof

Abstract

The invention provides a lipid-coated DNA tetrahedral nanostructure as well as a preparation method and application thereof. Specifically, the invention provides a liposome-nucleic acid tetrahedron nano-composite structure, which comprises a tetrahedron composite structure formed by a plurality of nucleic acids and a liposome, and the liposome is coated on the outer surface of the tetrahedron composite structure. According to the invention, the DNA structure is used as a skeleton, and lipid is coated outside the DNA structure through charge action, so that the size and shape can be flexibly regulated and controlled, the stability, biological stability and tissue penetrability of a nano device can be improved, and cells can be effectively transfected.

Description

Technical Field

[0001] The invention belongs to the technical field of materials and biomedicine, and specifically relates to a liposome-nucleic acid tetrahedron nanocomposite structure and a preparation method and application thereof. Background Art

[0002] Lipid nanoparticles (LNP) are nanocarriers that deliver nucleic acid substances to cells. They are composed of ionizable cationic lipids, neutral auxiliary phospholipids, cholesterol, and polyethylene glycol (PEG) lipids. Currently, nucleic acid drugs using LNP have been approved, such as the siRNA drug Onpattro approved by Alnylam. LNP is a relatively mature technology platform used to deliver RNA drugs, vaccines or gene editing tools. Compared with other types of nucleic acid drug delivery systems, LNP has many advantages, such as high nucleic acid encapsulation rate and effective cell transfection, strong tissue penetration, low cytotoxicity and immunogenicity, and more conducive to drug delivery. These advantages make LNP an excellent nucleic acid delivery system.

[0003] However, based on the preparation principle, LNP can only qualitatively control the size but not the shape, and its non-liver targeting is poor, only 10%-20%. In addition, the LNP delivery system has poor stability and has high requirements for storage and transportation, which greatly increases the cost.

[0004] The DNA nanostructures obtained by DNA self-assembly technology can programmatically control the size and shape to achieve a high degree of organ targeting, among which tetrahedral DNA of a specific size can achieve 80-90% kidney targeting. Such DNA structures can easily carry nucleic acid drugs and deliver them to the targeted sites through base pairing, but they have poor biological stability and poor tissue penetration, and often have difficulty entering cells to exert their effects.

[0005] Therefore, there is an urgent need in this field to develop nano-drug delivery devices with flexible shape control and good stability. Summary of the invention

[0006] The purpose of the present invention is to provide a nano drug delivery device with flexible shape control and good stability. Specifically, the present invention provides a liposome-nucleic acid tetrahedron nanocomposite structure and its preparation method and application.

[0007] In a first aspect of the present invention, a liposome-nucleic acid tetrahedron nanocomposite structure is provided, wherein the liposome-nucleic acid tetrahedron nanocomposite structure comprises:

[0008] (a) a nucleic acid tetrahedral nanostructure, wherein the nucleic acid tetrahedral nanostructure comprises a tetrahedral composite structure composed of a plurality of nucleic acids; and

[0009] (b) liposomes, wherein the liposomes are coated on the outer surface of the tetrahedral composite structure to form a liposome-nucleic acid tetrahedral nanocomposite structure;

[0010] In another preferred embodiment, the liposome covers ≥50% (preferably ≥90% or 100%) of the outer surface of the tetrahedral complex structure.

[0011] In another preferred embodiment, in the nucleic acid tetrahedral nanostructure, the nucleic acid monomer is selected from the following group: DNA, RNA, locked nucleic acid, peptide nucleic acid, or a combination thereof.

[0012] In another preferred embodiment, the nucleic acid tetrahedron nanostructure is a DNA nucleic acid tetrahedron nanostructure.

[0013] In another preferred embodiment, the nucleic acid tetrahedral nanostructure has a size of about 5-30 nanometers, preferably 7-20 nanometers.

[0014] In another preferred embodiment, in the nucleic acid tetrahedral nanostructure, the lipid comprises: ionizable cationic lipid, neutral auxiliary phospholipid, cholesterol, polyethylene glycol (PEG), or a combination thereof.

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

[0016] Z-LNP(A)

[0017] In the formula,

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

[0019] LNP refers to lipid molecules coated on the surface of the nucleic acid tetrahedral nanostructure.

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

[0021] (ZD)-LNP(I)

[0022] In the formula,

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

[0024] D represents a drug, and the drug is connected to the nucleic acid tetrahedral nanostructure;

[0025] LNP refers to lipid molecules coated on the surface of the nucleic acid tetrahedral nanostructure.

[0026] 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:

[0027] (a) providing nucleic acid monomers;

[0028] (b) mixing and annealing the nucleic acid monomers to assemble into nucleic acid tetrahedral nanostructures;

[0029] (c) mixing the nucleic acid tetrahedral nanostructure with lipid molecules, and coating the lipid molecules on the outer surface of the nucleic acid tetrahedral nanostructure through charge action.

[0030] 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 for preparing nanomolecules for delivering nucleic acid drugs.

[0031] 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

[0032] Figure 1 A schematic diagram of the liposome-nucleic acid tetrahedron nanocomposite structure is shown, where the blue area is the DNA tetrahedron and the yellow area is the lipid.

[0033] Figure 2 Atomic force microscopy characterization of liposome-nucleic acid tetrahedron nanocomposite structures is shown.

[0034] Figure 3 The device for preparing liposome-nucleic acid tetrahedron nanocomposite structure is shown. DETAILED DESCRIPTION

[0035] After extensive and in-depth research, the inventors combined LNP and DNA nanostructure technology, used DNA structure as the skeleton, and coated the main components of LNP (ionizable cationic lipids, neutral auxiliary phospholipids, cholesterol, PEG lipids) on the outside of the DNA structure through charge to form a lipid bilayer, which can not only regulate the specific size and shape of LNP, but also improve the stability, biological stability and tissue penetration of nanodevices, and can effectively transfect cells. On this basis, the present invention was completed.

[0036] The lipids in the present invention

[0037] In the present invention, "lipid", "lipid molecule", "lipid nanoparticle" and "LNP" are used interchangeably, referring to the lipids described in the first aspect of the present invention, including: ionizable cationic lipids, neutral auxiliary phospholipids, cholesterol, polyethylene glycol (PEG). Among them, the cationic lipid can promote the autonomous aggregation of nucleic acid drugs (negatively charged) and lipids into particles of the same size as the virus (~100nm) through charge action, and can help the release of nucleic acid drugs in the cytoplasm; the PEG can prolong the blood half-life of the complex; the cholesterol, as a stabilizer, can improve the stability of the complex; the phospholipid supports the formation of a lipid bilayer structure.

[0038] In the present invention, preferred lipid molecules and their structural formulas are shown in Table 1:

[0039] Table 1 Structural formula of lipids used

[0040]

[0041] Nucleic acid tetrahedral nanostructure of the present invention

[0042] As used herein, the terms "nucleic acid tetrahedral nanostructure of the present invention", "nanostructure of the present invention", "nanodevice of the present invention", "lipid-coated nucleic acid tetrahedral nanostructure" and "LNP-coated nucleic acid tetrahedral nanostructure" are used interchangeably and refer to the lipid-coated 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 outer surface of the nucleic acid tetrahedral nanostructure is coated with lipid molecules.

[0043] 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.

[0044] In a preferred embodiment, the nucleic acid tetrahedron nanostructure of the present invention is a DNA nucleic acid tetrahedron nanostructure.

[0045] In the present invention, the schematic diagram of the nucleic acid tetrahedron nanostructure is as follows Figure 1 As shown, the blue area is the DNA tetrahedron and the yellow area is the lipid.

[0046] 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.

[0047] In the present invention, the length of each nucleic acid monomer is generally 21-63 nt, preferably 57-63 nt.

[0048] Preferably, in the nucleic acid tetrahedral nanostructure of the present invention, the length of each nucleic acid monomer is the same or substantially the same (eg, the length difference is ≤10%, or ≤5 nt).

[0049] 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.

[0050] Preparation method

[0051] The present invention also provides a method for preparing the nucleic acid tetrahedron nanostructure of the present invention, comprising the steps of:

[0052] (a) providing nucleic acid monomers;

[0053] (b) mixing and annealing the nucleic acid monomers to assemble and form the nucleic acid tetrahedral nanostructure of the present invention.

[0054] (c) mixing the nucleic acid tetrahedral nanostructure with lipid molecules, and coating the lipid molecules on the surface of the nucleic acid tetrahedral nanostructure through charge action.

[0055] When it is necessary to prepare a drug-loaded nucleic acid tetrahedral nanostructure, the method of the present invention comprises the steps of:

[0056] (a) providing nucleic acid monomers;

[0057] (b) mixing and annealing the nucleic acid monomers to assemble and form the nucleic acid tetrahedral nanostructure of the present invention.

[0058] (c) hybridizing the nucleic acid tetrahedron nanostructure with a nucleic acid drug to obtain a drug-loaded nucleic acid tetrahedron nanostructure.

[0059] (d) mixing the drug-loaded nucleic acid tetrahedral nanostructure with lipid molecules, and coating the lipid molecules on the surface of the nucleic acid tetrahedral nanostructure through charge action.

[0060] application

[0061] The present invention also provides applications of the nucleic acid tetrahedron nanostructure in various occasions.

[0062] Typically, the lipid-coated nucleic acid tetrahedron 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 or peptide drugs or protein drugs.

[0063] In a preferred example, a drug formed by coupling the nucleic acid tetrahedral nanostructure of the present invention with doxorubicin is constructed. As a type of chemotherapy drug, the drug can effectively kill cancer cells. The drug here can also be a small nucleic acid drug, which can bind to the target molecule RNA to inhibit its translation or regulation, thereby achieving gene therapy and editing.

[0064] 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.

[0065] The main advantages of the present invention include:

[0066] (a) The drug platform of the present invention is based on DNA nanotechnology, has a simple preparation process, and has uniform structure and properties.

[0067] (b) The yield of the DNA nanostructure prepared by the present invention is high, which can reach more than 90%.

[0068] (c) The lipid-coated DNA nanostructure of the present invention is highly designable and can flexibly regulate the specific size and shape of LNP.

[0069] (d) 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.

[0070] (f) The lipid-coated DNA nanostructure of the present invention improves non-liver targeting (up to 80-90%), and has good biological stability, tissue penetration and kidney targeting, greatly increasing the probability of effective cell transfection.

[0071] (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.

[0072] 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.

[0073] Example 1

[0074] Preparation of Tetrahedral Nucleic Acid Monomers

[0075] The sequences of a set of nucleic acid monomers used to construct nucleic acid tetrahedral nanostructures are shown in Table 2.

[0076] The nucleic acid monomers shown in Table 2 were synthesized using conventional artificial synthesis methods.

[0077] Table 2 Sequences of nucleic acid monomers in tetrahedrons without lipid coating

[0078]

[0079] Example 2

[0080] Assembly of DNA nanomachines

[0081] In this example, nucleic acid tetrahedral nanostructures were prepared using the nucleic acid monomers shown in Table 2 prepared in Example 1.

[0082] 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 Table 2.

[0083] All single-stranded DNA oligonucleotides were purified 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.

[0084] Table 3

[0085]

[0086] During the annealing process, DNA chains are assembled through complementary base pairing to form a three-dimensional DNA nanostructure.

[0087] Example 3

[0088] Lipid molecule coating

[0089] Preparation of lipid-ethanol solution: ((4-hydroxybutyl)azepinediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315, ionizable cationic lipid), 2-[(polyethylene glycol)-2000]-N,N-tetracosylacetamide (ALC-0159, PEG lipid), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC, neutral auxiliary phospholipid) and cholesterol were mixed in a molar ratio of 50:1.5:10:38.5 to prepare an ethanol solution with a total concentration of 12 mM.

[0090] Preparation of nucleic acid buffer: Prepare the DNA tetrahedron obtained in Example 2 into a mother solution with a concentration of 50 mM and pH = 4. When used, dilute the mother solution to the required concentration, that is, the DNA concentration is 0.107 g / L calculated based on a nitrogen-phosphorus ratio of 6.

[0091] Preparation of nucleic acid lipid nanoparticles: Microfluidic mixing technology is used to prepare nucleic acid lipid nanoparticles. The prepared lipid-ethanol solution and nucleic acid buffer solution are injected into the two inlet channels of the preparation system, and the two-phase flow rate is adjusted to 1:3 (lipid-ethanol solution flow rate 3mL / min, nucleic acid buffer flow rate 9mL / min) to mix the two phases to complete the preparation of nucleic acid lipid nanoparticles.

[0092] Post-treatment: After the sample is collected, the mixed solution is diluted to reduce the solubility of lipids, which gradually precipitate and solidify to form lipid nanoparticles, while efficiently coating DNA tetrahedrons. The samples are then ultrafiltered through an ultrafiltration tube to neutralize the pH value of the buffer solution to obtain lipid-coated DNA tetrahedron nanodevices.

[0093] The prepared lipid-coated nucleic acid tetrahedral nanostructures were characterized by atomic force microscopy using conventional instruments and methods.

[0094] The results are as follows Figure 2 shown. Figure 2 Atomic force microscopy characterization of lipid-coated DNA tetrahedrons is shown, demonstrating the assembly into a tetrahedral structure.

[0095] discuss

[0096] LNP can only qualitatively regulate size, not shape, and has poor non-liver targeting. LNP delivery system has poor stability, high requirements for storage and transportation, and high preparation cost; while DNA nanostructures obtained through DNA self-assembly technology can programmatically regulate size and shape to achieve higher organ targeting, among which tetrahedral DNA of a specific size can achieve 80-90% kidney targeting. Such a DNA structure can easily carry nucleic acid drugs and deliver them to the targeted site through base pairing, but it has poor biological stability and poor tissue penetration, and it is often difficult to enter cells to exert its effect.

[0097] If LNP and DNA nanostructure technology are combined, with the DNA structure as the skeleton, the main components of LNP (ionizable cationic lipids, neutral auxiliary phospholipids, cholesterol, PEG lipids) are coated on the outside of the DNA structure through the action of charges to form a lipid bilayer. This can not only regulate the specific size and shape of LNP, but also improve the stability, biological stability and tissue penetration of the nanodevice, and can effectively transfect cells.

[0098] 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 liposome-nucleic acid tetrahedron nanocomposite structure, characterized in that: The liposome-nucleic acid tetrahedron nanocomposite structure includes: (a) a nucleic acid tetrahedral nanostructure, wherein the nucleic acid tetrahedral nanostructure comprises a tetrahedral composite structure composed of a plurality of nucleic acids; and (b) liposomes, wherein the liposomes are coated on the outer surface of the tetrahedral composite structure to form a liposome-nucleic acid tetrahedral nanocomposite structure; In another preferred embodiment, the liposome covers ≥50% (preferably ≥90% or 100%) of the outer surface of the tetrahedral complex structure.

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: The size of the nucleic acid tetrahedral nanostructure is about 5-30 nanometers, preferably 7-20 nanometers.

5. The nucleic acid tetrahedral nanostructure according to claim 1, characterized in that: The lipids include: ionizable cationic lipids, neutral auxiliary phospholipids, cholesterol, polyethylene glycol (PEG), or a combination thereof.

6. The nucleic acid tetrahedral nanostructure according to claim 1, characterized in that: The nucleic acid tetrahedral nanostructure has a structure of formula A: Z-LNP(A) In the formula, Z is a nucleic acid tetrahedral nanostructure formed by the assembly of nucleic acid monomers; LNP refers to lipid molecules coated on the surface of the nucleic acid tetrahedral nanostructure.

7. The nucleic acid tetrahedral nanostructure according to claim 1, characterized in that: The nucleic acid tetrahedral nanostructure is a complex of formula I: (ZD)-LNP(I) In the formula, Z is a nucleic acid tetrahedral nanostructure formed by the assembly of nucleic acid monomers; D represents a drug, and the drug is connected to the nucleic acid tetrahedral nanostructure; LNP refers to lipid molecules coated on the surface of the nucleic acid tetrahedral nanostructure.

8. The method for preparing nucleic acid tetrahedral nanostructures according to claim 1, characterized in that: Includes steps: (a) providing nucleic acid monomers; (b) mixing and annealing the nucleic acid monomers to assemble into nucleic acid tetrahedral nanostructures; (c) mixing the nucleic acid tetrahedral nanostructure with lipid molecules, and coating the lipid molecules on the outer surface of the nucleic acid tetrahedral nanostructure through charge action.

9. The use of the nucleic acid tetrahedral nanostructure according to claim 1, characterized in that: Used to prepare nanomolecules for delivering nucleic acid drugs.

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