Flaky DNA nano-composite structure for treating acute kidney injury and preparation method of flaky DNA nano-composite structure

Abstract

The invention provides a flaky nucleic acid nano-composite structure for treating acute kidney injury and a preparation method of the flaky nucleic acid nano-composite structure. Specifically, the invention provides a flaky nucleic acid nano-composite structure, and a capture chain on the surface of the flaky nucleic acid nano-composite structure is coupled with cholesterol molecules. The cholesterol-modified flaky nucleic acid nano structure can greatly improve the stability of the cholesterol-modified flaky nucleic acid nano structure in a living body. The cholesterol-modified flaky nucleic acid nano-structure can be used as a nano-drug for removing active oxygen in a body, so that a new way for treating acute kidney injury is provided.

Description

Technical Field

[0001] The invention belongs to the technical field of materials and biomedicine, and particularly relates to a sheet-like DNA nanocomposite structure for treating acute kidney injury and a preparation method thereof. Background Art

[0002] Acute kidney injury (AKI) is a disease that seriously threatens human health and has a high morbidity and mortality rate. Therefore, the treatment of AKI is an important issue that needs to be solved urgently. Oxidative stress is one of the main pathogenesis of AKI and is closely related to the severity of the disease and the prognosis of patients.

[0003] Oxidative stress refers to the sharp increase in reactive oxygen species (ROS) levels when mitochondria are stimulated by external factors or have dysfunction, causing serious damage to cell structures and tissues and organs of the body. Reactive oxygen species broadly refers to free radicals and non-free radicals derived from oxygen, including superoxide anions (O2-), hydrogen peroxide (H2O2), hydroxyl radicals (OH-), ozone (O3) and singlet oxygen ( 1 O2), because they contain unpaired electrons, they are highly chemically reactive and react with biological molecules to destroy their normal functions.

[0004] To date, a variety of therapies have been used to treat AKI targeting antioxidant-induced inflammatory responses. For example, the reactivity of acetylcysteine ​​(NAC) with ROS can be used to clear ROS, or natural drugs can be used to inhibit inflammatory responses and reduce kidney damage caused by AKI. However, most drug treatments have limited effects and lack sustained and lasting intervention.

[0005] ROS can react with DNA to generate harmless metabolites, and the latest progress in DNA nanotechnology has enabled DNA nanodevices to be innovatively used for disease treatment. DNA origami technology refers to the technology of constructing the required graphic structure by co-folding long and short DNA chains. Self-assembled DNA nanostructures provide a precisely controllable addressing platform for the design of nanodevices and nanomedicines. This provides ideas for DNA nanostructures to be used as nanomedicines to remove reactive oxygen species in the body.

[0006] However, DNA origami is susceptible to attack by proteases when circulating in the blood and has poor biological stability, so its properties often need to be improved through modification.

[0007] The interface obtained by cholesterol self-assembly has the property of dynamic adaptation in response to changes in environmental polarity, and the adsorption of any biological molecule requires the orientation (polarity) adaptation of its interface, which limits the orientation state of the cholesterol at the interface, thereby reducing the entropy of the system. It is an entropy reduction process and is thermodynamically unfavorable. Therefore, the cholesterol interface counteracts biological adhesion through an entropy repulsion mechanism, that is, it can inhibit the adsorption of proteins and the like. Cholesterol molecules are considered to have many advantages as modifiers, but the application of cholesterol modifiers in drug delivery and AKI treatment has yet to be studied.

[0008] Therefore, there is an urgent need to develop DNA nanodevices that can effectively treat AKI. Summary of the invention

[0009] The purpose of the present invention is to provide a DNA nanodevice that can effectively treat AKI.

[0010] In a first aspect of the present invention, a sheet-like nucleic acid nanocomposite structure is provided, wherein the composite sheet-like nucleic acid nanocomposite structure comprises:

[0011] (a) a sheet-like structure formed by assembling nucleic acid chains, wherein at least one major surface of the sheet-like structure is provided with a protruding capture chain;

[0012] (b) a single-stranded functional nucleic acid molecule coupled to a cholesterol molecule, wherein the single-stranded functional nucleic acid molecule is captured by the capture chain to form a composite structure of "sheet structure-capture chain-single-stranded functional nucleic acid molecule-cholesterol";

[0013] In another preferred embodiment, the capture strand is a single-stranded structure;

[0014] In another preferred embodiment, the capture chain has a complementary region, and the sequence of the complementary region is partially or completely complementary to the sequence of the single-stranded functional nucleic acid molecule, so that the single-stranded functional nucleic acid molecule is captured by the capture chain;

[0015] In another preferred embodiment, the single-stranded functional nucleic acid molecule forms a double-stranded complementary structure by base complementation with the capture strand;

[0016] In another preferred embodiment, the cholesterol molecules are exposed to the outside or outer surface of the sheet-like nucleic acid nanocomposite structure;

[0017] In another preferred embodiment, the cholesterol molecule is coupled to the 5' or 3' end of the single-stranded functional nucleic acid molecule;

[0018] In another preferred embodiment, the capture chain in the composite structure includes a protruding capture chain segment and a constituent segment that is a part of the sheet structure;

[0019] In another preferred embodiment, ≥50% (preferably ≥80%, more preferably ≥90%, and most preferably ≥95% or 100%) of the capture segments in the composite structure have the same sequence;

[0020] In another preferred embodiment, the sequence of the capture segment is polyA, polyG, polyT and / or polyC;

[0021] In another preferred embodiment, in the composite structure, the length of the capture segment is 10-25 nt.

[0022] In another preferred embodiment, in the sheet-like nucleic acid nanocomposite structure, the nucleic acid monomer is selected from the following group: DNA, RNA, locked nucleic acid, peptide nucleic acid, or a combination thereof.

[0023] In another preferred embodiment, the sheet-like nucleic acid nanocomposite structure is a sheet-like DNA nanostructure.

[0024] In another preferred embodiment, in the sheet-like nucleic acid nanocomposite structure, the cholesterol is covalently linked to a nucleotide sequence, and the nucleotide sequence is base-paired with the capture chain, so that the cholesterol molecule is coupled to the capture chain.

[0025] In another preferred embodiment, in the sheet-like nucleic acid nanocomposite structure, the number of the capture chains is n, where n is an integer ≥ 1, preferably, n ≥ 8;

[0026] In another preferred embodiment, n is 10-1000, preferably 12-300.

[0027] In another preferred embodiment, the sheet-like nucleic acid nanocomposite structure has a size of about 5-30 nanometers, preferably 7-20 nanometers.

[0028] In a second aspect of the present invention, a method for preparing the sheet-like nucleic acid nanocomposite structure is provided, comprising the steps of:

[0029] (a) providing nucleic acid monomers;

[0030] (b) mixing and annealing the nucleic acid monomers to assemble into a sheet-like structure with protruding capture chains;

[0031] (c) mixing the single-stranded functional nucleic acid molecule coupled with the cholesterol molecule with the sheet-like structure of the capture chain with protrusions in step (b) to obtain the sheet-like nucleic acid nanocomposite structure according to the first aspect of the present invention.

[0032] In the third aspect of the present invention, there is provided a use of the sheet-like nucleic acid nanocomposite structure for preparing a drug for treating acute kidney injury.

[0033] 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

[0034] Figure 1 The cholesterol-modified sheet-like DNA nanostructure of the present invention is shown, wherein the blue area is the sheet-like DNA structure, the yellow part is the capture strand, and the purple part is the cholesterol molecule (taking 16 modification sites as an example).

[0035] Figure 2 Atomic force microscopy characterization of the cholesterol-modified sheet-like DNA structure of the present invention is shown.

[0036] Figure 3 The polyacrylamide characterization of the cholesterol-modified sheet-like DNA structure of the present invention is shown, wherein lane 1 is a DNA standard, and the DNA fragments from top to bottom are 2000bp, 1000bp, 750bp, 500bp, 250bp, and 100bp respectively; lane 2 is a single-stranded M13DNA; and lane 3 is a rectangular DNA sheet structure with a capture chain. DETAILED DESCRIPTION

[0037] After extensive and in-depth research, the inventors provided a cholesterol molecule-modified sheet-like nucleic acid nanocomposite structure (such as a sheet-like DNA nanostructure) for the first time, and constructed a drug-carrying nanomachine, which can greatly improve its stability in the body, improve pharmacokinetic and pharmacodynamic properties, improve drug distribution in the body, and prolong the drug's in vivo treatment time. On this basis, the present invention was completed.

[0038] Nucleic acid nanostructures of the present invention

[0039] In the present invention, the terms "nucleic acid nanostructure of the present invention", "cholesterol-modified nucleic acid nanostructure of the present invention", or "cholesterol-modified sheet-like DNA nanostructure of the present invention" are used interchangeably to refer to the sheet-like nucleic acid nanocomposite structure described in the first aspect of the present invention, which has a sheet-like nucleic acid nanocomposite structure and a capture chain on the surface of the structure, and the end of the capture chain is connected to the modifier.

[0040] As used herein, the terms "sheet-like DNA nanostructure" or "DNA origami structure" are used interchangeably to refer to a sheet-like structure formed by assembling multiple nucleic acid chains. In addition to having a sheet-like main structure, the sheet-like DNA nanostructure of the present invention also has protrusions.

[0041] In a preferred embodiment, the sheet-like nucleic acid nanocomposite structure of the present invention is a sheet-like DNA nanostructure.

[0042] In a preferred embodiment, the sheet-like DNA structure of the present invention is a polygonal sheet-like DNA structure. In another preferred embodiment, the sheet-like DNA structure of the present invention is a rectangular DNA structure.

[0043] In the present invention, the schematic diagram of the cholesterol molecule modified sheet-like DNA nanostructure is as follows Figure 1 As shown, the blue area is a rectangular DNA sheet structure, the yellow part is the capture chain, and the purple part is the cholesterol molecule (taking 16 modification sites as an example).

[0044] In the present invention, the capture strand is a DNA strand protruding outward from a specific modification site of the sheet-like DNA nanoplanar structure.

[0045] Cholesterol molecules suitable for use in the present invention include (but are not limited to): dehydrocholesterol, thiol-modified cholesterol and palmitic acid cholesterol.

[0046] In the present invention, a preferred cholesterol is a cholesterol molecule with modified hydroxyl groups, and the structural formula is shown below.

[0047]

[0048] In the present invention, cholesterol molecules can be covalently linked to nucleotide chains that are complementary chains to the capture chains, so that the cholesterol molecules can be coupled to the sheet-like nucleic acid nanocomposite structure through complementary base pairing.

[0049] Preparation method

[0050] The present invention also provides a method for preparing a cholesterol-modified sheet-like nucleic acid nanocomposite structure, comprising the steps of:

[0051] (a) providing nucleic acid monomers, including a long chain and a plurality of short chains (staple chains);

[0052] (b) mixing and annealing the nucleic acid monomers to assemble into a sheet-like nucleic acid nanocomposite structure with a capture chain;

[0053] (c) Mixing the single-stranded functional nucleic acid molecule coupled with the cholesterol molecule with the sheet-like structure of the capture chain with protrusions in step (b) to obtain the sheet-like nucleic acid nanocomposite structure of the present invention.

[0054] The main advantages of the present invention include:

[0055] (1) The drug platform of the present invention is based on DNA nanotechnology, with a simple preparation process and uniform structure and properties. The DNA nanostructure is highly designable and its morphological characteristics can be precisely controlled.

[0056] (2) The present invention utilizes the addressability of DNA nanotechnology and can achieve self-assembly to form an interface by precisely controlling the spatial position of cholesterol molecules in the DNA nanostructure, thereby greatly improving its biological stability, thereby improving its pharmacokinetic and pharmacodynamic properties, improving the drug's distribution in the body, and prolonging the drug's in vivo treatment time.

[0057] (3) 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 in vivo, 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.

[0058] (4) The DNA nanomedicine provided by the present invention can continuously and efficiently remove reactive oxygen species in the body, and can be targeted to organs such as the kidney and liver in the body to achieve the function of targeted and directional removal in the body, and has very good scalability and universality.

[0059] 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.

[0060] Example 1: Preparation of DNA origami structures

[0061] DNA origami structures 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) using established methods. 20 nM circular m13 ssDNA was mixed with a five-fold excess of stapler strands (sequences shown in SEQ ID No: 1-226), and the mixture was annealed from 90°C to 4°C using the temperature gradient shown in Table 1. The mixture was filtered using a 100 kDa Amicon ultrafilter in 1× TAE-Mg 2+ Filter the annealing solution in buffer or phosphate buffered saline (PBS, pH 7.2-7.5). The concentration of the DNA sheet structure can be calculated by its absorbance at 260 nm, and the average extinction coefficient is 109119009M -1 cm -1 .

[0062] Table 1

[0063]

[0064]

[0065] Example 2: Modification of cholesterol molecules

[0066] Cholesterol molecules were coupled to the ends of single-stranded functional nucleic acid molecules by click chemistry, and the sequence of the single-stranded functional nucleic acid molecule was shown in SEQ ID No: 227. Capture chains were designed at 16 sites on the plane of the sheet DNA structure, and the capture chains were complementary to the DNA sequence of the cholesterol-DNA conjugate, and the sequence of the capture chains was shown in SEQ ID No: 228-243. The purified sheet DNA structure (20nM) and the coupling product (100μM, 5 times excess) were allowed to stand at room temperature for 1 hour for hybridization to achieve the modification of cholesterol molecules, and then the interface was formed by self-assembly of cholesterol molecules to achieve the inhibition of biomolecule adhesion, and the sheet DNA nanostructure was obtained.

[0067] Example 3: Characterization of cholesterol-modified sheet-like DNA nanostructures

[0068] Agarose gel electrophoresis (AGE): 1% agarose gel electrophoresis (90V, 4°C, 85min) was performed in 1×TAE / Mg2+ buffer (triacetic acid 40mM, pH 8.0, magnesium acetate 12.5mM, EDTA 1mM). The sample loading volume was 1nM, 10μL, and a 20bp DNA ladder (1μL) was selected as a marker. After the electrophoresis, the samples were stained with ethidium bromide for 5 minutes and further analyzed on a chemiluminescent imaging system.

[0069] Atomic force microscopy (AFM): DNA nanodevices (2 μL, 20 nm) were added to freshly cut mica sheets and kept adsorbed for 2-3 min, then 1×TAE-Mg2+ buffer (400 μL) was added thereto and the samples were scanned in liquid mode on a PicoPlus atomic force microscope.

[0070] The results are as follows Figure 2 , Figure 3 shown. Figure 2 Atomic force microscopy characterization of sheet-like DNA structures; Figure 3 Represents polyacrylamide characterization, where lane 3 is a sheet-like DNA nanostructure with a capture strand.

[0071] Example 4: Experimental verification of sheet-like DNA nanostructures in scavenging reactive oxygen species

[0072] Electron spin resonance spectroscopy (ESR) was used to examine the scavenging effect of the sheet-like DNA nanostructure on hydroxyl radicals. The steps are as follows:

[0073] The Fenton reaction (0.735 mMFeSO4, 20 μL and 0.315 mMH2O2, 30 μL) produced hydroxyl radicals (·OH). 5,5-Dimethyl-1-pyrroline-N-oxide (DMPO) was selected as a spin trapping agent (98%, 4 μL). In an electron spin resonance spectrometer (JEOL-FA200), the ESR spectrum changes of DMPO-OH· adducts showed that DNA nanostructures had the ability to scavenge·OH, confirming the scavenging effect of sheet-like DNA nanostructures on ROS.

[0074] Cell experiments verify the mitigation effect of DNA nanostructures on cell oxidative stress damage. The steps are as follows:

[0075] The renal proximal tubule HK-2 cell line was cultured at 37°C, 5% CO2, in DMEM medium supplemented with 10% FBS and 1% penicillin / streptomycin. The cells were plated in 96-well cell culture plates (cell number per well: 10,000) and then cultured for 24 hours at 37°C and 5% CO2. The test substance, i.e., sheet-like DNA nanostructures (20nM, 100μL), was added to the cells and cultured for 30 minutes. The cells were then cultured with H2O2 (final concentration in each well: 250μM) at 37°C and 5% CO2 for 24 hours. Colorimetric analysis was performed using an MTT assay kit (catalog number: ST316; Beyotime) to detect cell viability. The experiment was performed according to the protocol provided by the manufacturer.

[0076] The results showed that in the cell group containing DNA nanostructures, cell viability was not affected by H2O2 and decreased; while in the cell group without DNA nanostructures, cell viability was significantly affected by H2O2 and decreased significantly. This experiment shows that DNA nanostructures can effectively react with H2O2, remove excessive reactive oxygen at the cellular level, and play a role in protecting cells.

[0077] 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 sheet-like nucleic acid nanocomposite structure, characterized in that: The composite sheet-like nucleic acid nanocomposite structure comprises: (a) a sheet-like structure formed by assembling nucleic acid chains, wherein at least one major surface of the sheet-like structure is provided with a protruding capture chain; (b) a single-stranded functional nucleic acid molecule coupled to a cholesterol molecule, wherein the single-stranded functional nucleic acid molecule is captured by the capture chain to form a composite structure of "sheet structure-capture chain-single-stranded functional nucleic acid molecule-cholesterol"; In another preferred embodiment, the capture strand is a single-stranded structure; In another preferred embodiment, the capture chain has a complementary region, and the sequence of the complementary region is partially or completely complementary to the sequence of the single-stranded functional nucleic acid molecule, so that the single-stranded functional nucleic acid molecule is captured by the capture chain; In another preferred embodiment, the single-stranded functional nucleic acid molecule forms a double-stranded complementary structure by base complementation with the capture strand; In another preferred embodiment, the cholesterol molecules are exposed to the outside or outer surface of the sheet-like nucleic acid nanocomposite structure; In another preferred embodiment, the cholesterol molecule is coupled to the 5' or 3' end of the single-stranded functional nucleic acid molecule; In another preferred embodiment, in the composite structure, the capture chain comprises a protruding capture chain segment and a constituent segment that is a part of a sheet-like structure; In another preferred embodiment, in the composite structure, ≥50% (preferably ≥80%, more preferably ≥90%, and most preferably ≥95% or 100%) of the capture segments have the same sequence; In another preferred embodiment, the sequence of the capture segment is polyA, polyG, polyT and / or polyC; In another preferred embodiment, in the composite structure, the length of the capture segment is 10-25 nt.

2. The sheet-like nucleic acid nanocomposite structure 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 sheet-like nucleic acid nanocomposite structure according to claim 1, characterized in that: The sheet-like nucleic acid nanocomposite structure is a sheet-like DNA nanostructure.

4. The sheet-like nucleic acid nanocomposite structure according to claim 1, wherein: The cholesterol is covalently linked to a nucleotide sequence, and the nucleotide sequence performs base complementary pairing with the capture chain, so that the cholesterol molecule is coupled to the capture chain.

5. The sheet-like nucleic acid nanocomposite structure according to claim 1, wherein: The number of the capture chains is n, where n is an integer ≥ 1, preferably, n ≥ 8; In another preferred embodiment, n is 10-1000, preferably 12-300.

6. The sheet-like nucleic acid nanocomposite structure according to claim 1, wherein: The size of the sheet-like nucleic acid nanocomposite structure is about 5-30 nanometers, preferably 7-20 nanometers.

7. The method for preparing a sheet-like nucleic acid nanocomposite structure according to claim 1, wherein: Includes steps: (a) providing nucleic acid monomers; (b) mixing and annealing the nucleic acid monomers to assemble into a sheet-like structure with protruding capture chains; (c) mixing the single-stranded functional nucleic acid molecule coupled with the cholesterol molecule with the sheet-like structure of the capture chain with protrusions in step (b) to obtain the sheet-like nucleic acid nanocomposite structure as claimed in claim 1.

8. The use of the sheet-like nucleic acid nanocomposite structure according to claim 1, characterized in that: Used for preparing medicines for treating acute kidney injury.

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