Preparation method of DNA nanoframe-assisted monodisperse perfluorocarbon nanoparticles and its application in fluorine nuclear magnetic resonance imaging

Abstract

The present invention discloses a method for preparing monodisperse perfluorocarbon nanoparticles assisted by a DNA nanoframe and its application in fluorine nuclear magnetic resonance imaging. The preparation method of the present invention comprises: introducing a fluorocarbon chain modification inside the space of a DNA nanoframe structure by hybridization complementation; dispersing perfluorocarbon PFC in a surfactant to form a dispersed solution, co-incubating with the DNA nanoframe structure with the fluorocarbon chain modification, using the fluorocarbon chain inside the DNA nanoframe structure as a "seed" to capture the PFC particles entering the space, so that the PFC self-assembles inside the DNA nanoframe structure to form nanoparticles of uniform size, that is, to obtain a nano-fluorine probe. The present invention utilizes a variety of three-dimensional DNA nanoframes (DNF) with different structures to prepare PFC nanoparticles. The method has the advantages of design controllability and uniform size. The prepared fluorine probe nuclear magnetic resonance signal is more single and sharp, and has good application prospects.

Description

Technical Field

[0001] The invention relates to a method for preparing monodisperse perfluorocarbon nanoparticles assisted by a DNA nanoframe and application thereof in fluorine nuclear magnetic resonance imaging, belonging to the technical fields of DNA nanotechnology and material assembly and nuclear magnetic resonance imaging. Background Art

[0002] Magnetic resonance imaging (MRI) is a non-invasive, deep scanning, non-radioactive imaging method that has been widely used in the screening and diagnosis of major diseases such as cancer. The MRI method currently used in clinical and medical research is mainly based on hydrogen protons ( 1 However, due to the high water content in the body, the magnetic resonance imaging signals within the tissue and between different tissues are prone to mutual interference, causing high background noise and reducing the resolution of the image; and both normal tissues and lesions contain a large number of hydrogen protons, so sometimes 1 H-MRI cannot directly distinguish normal tissue from lesions.

[0003] The above problems have promoted the development of non-hydrogen nuclear magnetic resonance imaging technology, including 19 F-MRI has attracted widespread attention due to its characteristics. 19 The F nucleus has a spin of 1 / 2 and a natural abundance of 100%, making it as sensitive as a hydrogen nucleus. Most importantly, 19 The content of F in the human body is very small, and it is mainly fixed in the solid phase of bones and teeth, which makes its transverse relaxation time (T2) very short and the signal is far below the detection limit of MRI instruments. 19 F does not generate background signal during imaging. 19 As an exogenous molecular probe, F-MRI contrast agent can be imaged in vivo in the form of "hot spots" (only the contrast agent is developed and appears as a bright spot in the magnetic resonance image). Based on its many advantages, 19 F-MRI is regarded as a diagnostic technology with potential for clinical application and has been used for in vitro and in vivo cell tracing, angiography and other studies.

[0004] The preparation method and material state of fluorinated contrast agents affect 19 One of the most important factors in the effectiveness of F-MRI imaging is the development and improvement of 19 The preparation process of F contrast agent will be expanded 19 The application scope of F-MRI is to promote clinical transformation. 19F contrast agents are all prepared based on PFC, but PFC (Perfluorocarbons, PFC) materials are highly hydrophobic. When using PFC as a contrast agent, surfactants and advanced dispersion equipment are needed to disperse PFC in an aqueous solution to form an emulsion. The preparation process is complicated; the preservation of emulsion samples is difficult; the contrast effect is limited by the uneven size and instability of the emulsion droplets, such as flocculation, coalescence, and Oswald ripening of nanodroplets. Using the new method to assemble uniform, size-controlled, and stable 19 F contrast agents can better study the relaxation properties of contrast agents, enhance the intensity of nuclear magnetic resonance signals, reduce the storage cost of contrast agents, and accelerate the development of 19 Clinical translation of F-MRI.

[0005] DNA nanotechnology has experienced 40 years of vigorous development since its creation by Nadrian Seeman in 1982, and has extremely wide applications in biology, medicine, materials, chemistry and other fields. The basic principle of DNA origami is based on the principle of complementary base pairing, using hundreds of short DNA chains called staple chains to fold a long DNA chain called a scaffold chain into pre-designed shapes, such as squares, rectangles, triangles, stars and smiley faces. In the following decade of development, DNA origami has made continuous breakthroughs in structural dimensions, structural curvature, topological weaving, dynamic control and modular super-assembly, achieving the assembly of finer, larger and more complex structures.

[0006] Using DNA nanostructures as framework templates to guide the assembly of functional materials, thereby giving rise to materials with new properties and functions, has become one of the effective means of exploring the world of nanomaterials.

[0007] There is no report on the use of DNA nanotechnology to guide the preparation of PFC monodisperse particles and their application in 19 Related reports on F-MRI angiography. Summary of the invention

[0008] The purpose of the present invention is to address the deficiencies of the prior art and provide a method for preparing monodisperse perfluorocarbon nanoparticles assisted by a DNA nanoframework (DNF) and its application in fluorine nuclear magnetic resonance imaging. The present invention captures PFC particles free in the solution by binding fluorocarbon chain seeds in the DNA nanoframework and performing fusion growth in a confined space, thereby obtaining monodisperse PFC particles with uniform size and controllable shape and size, which can be used for 19 F-NMR / MRI imaging.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] The first aspect of the present invention provides a method for preparing monodisperse perfluorocarbon nanoparticles assisted by a DNA nanoframe, comprising: introducing a fluorocarbon chain modification into the space of a DNA nanoframe structure by hybridization complementation; dispersing a perfluorocarbon PFC in a surfactant to form a PFC dispersion solution, and co-incubating the solution with the DNA nanoframe structure with the fluorocarbon chain modification introduced therein, so that the PFC enters the space of the DNA nanoframe structure under the action of the fluorocarbon chain (i.e., the fluorocarbon chain inside the DNA nanoframe structure is used as a "seed" to capture the PFC particles entering the space), so that the PFC self-assembles inside the DNA nanoframe structure to form nanoparticles of uniform size, i.e., obtaining a nanofluorine probe, and obtaining a purified nanofluorine probe after removing the surfactant.

[0011] In some embodiments of the present invention, the fluorocarbon chain is derived from the fluorocarbon chain in the fluorocarbon surfactant, and the fluorocarbon chain can be 1H,1H,2H,2H-perfluoro-1-octyl and / or 1H,1H,2H,2H-perfluoro-1-decyl.

[0012] Preferably, the DNA nanoframe structure is a type of self-enclosed three-dimensional structure with internal and external topological characteristics, including but not limited to truncated icosahedron (soccer ball shape), regular icosahedron, regular dodecahedron structure, etc. Utilizing the high designability of the DNA nanoframe structure, all vertices and "staple chains" on the edges can be selected as modification sites, and they can extend an anchor sequence inside or outside the structure for functional modification. Taking the soccer ball-shaped structure as an example, it is composed of 12 regular pentagons and 20 regular hexagons, with a total of 60 vertices and 90 edges, and 90 anchor sequences and fluorocarbon chain-modified DNA (complementary chains) can be extended inside or outside the ball to hybridize with each other.

[0013] Preferably, the DNA nanoframe structure is constructed by DNA origami technology, and the construction method of the DNA nanoframe structure comprises the following steps:

[0014] Step 1: Use DNA nanostructure design software to construct the DNA nanoframe structure;

[0015] Step 2: Based on step 1, further construct variants of the DNA framework structure and establish internal and external modifiable sites;

[0016] Step 3: self-assembly and purification of the DNA framework structure constructed in step 2;

[0017] In the above method, the step 1 subsequently comprises obtaining the corresponding sequences of hundreds of staple chains by inputting the template chain sequence, and performing sequence synthesis; the present invention uses the long-chain ssDNA template p7560 in the M13 phage genome as the template chain sequence of the DNA framework structure;

[0018] In the above method, the structural self-assembly and purification of step 3 specifically include: annealing the template chain sequence and the synthesized staple chain sequence, base pairing self-assembly to obtain a DNA nanoframe and separation and purification to obtain a purified DNA framework; the purification includes using size exclusion high performance liquid chromatography (HPLC) and ultrafiltration concentration to obtain purified structural monomers, and removing unbound staple chains and impurities such as dimers and polymers.

[0019] Preferably, the fluorocarbon chain modification is achieved by the following steps:

[0020] Step 1: reacting a DNA single strand modified with maleimide at the 3' end with 1H,1H,2H,2H-perfluoro-1-octanethiol or 1H,1H,2H,2H-perfluoro-1-decanethiol, and then purifying it by reverse HPLC to obtain a DNA single strand modified with a fluorine carbon chain at the 3' end, i.e., a DNA complementary strand modified with a fluorine carbon chain at the 3' end; or phosphoramidating 1H,1H,2H,2H-perfluoro-1-decanol, coupling it to the 5' end of the DNA single strand (coupling with -OH at the 5' end of the DNA single strand) by a DNA synthesizer, and purifying it by reverse HPLC to obtain a DNA single strand modified with a fluorine carbon chain at the 5' end, i.e., a DNA complementary strand modified with a fluorine carbon chain at the 5' end;

[0021] Step 2: hybridize the DNA complementary chain modified with fluorocarbon chain at the 3' end or 5' end with the anchoring chain inside the DNA nanoframe structure through the principle of base complementary pairing to achieve the modification of fluorocarbon chain inside the DNA nanoframe structure.

[0022] In some embodiments of the present invention, the 5' end of the fluorocarbon chain-modified DNA complementary chain points to the interior of the DNA nanoframe structure, and the 3' end is close to the frame edge of the DNA nanoframe structure; the fluorocarbon chain acts as a seed to capture free PFC fluoro balls, and the size of the fluoro balls is regulated by regulating the spatial position of the fluorocarbon chain seeds.

[0023] In some embodiments of the present invention, the spatial arrangement of the fluorocarbon chain seeds on the DNA nanoframe structure is adjusted by the position of the fluorocarbon chain modification and the sequence length of the DNA complementary chain, which is specifically divided into the following two types: ① A 21 nt DNA complementary chain modified with a fluorocarbon chain at the 3' end: The fluorocarbon chain is closely attached to the internal edge of the frame of the DNA nanoframe structure through complementary hybridization; ② A 21 nt, 41 nt or 61 nt DNA complementary chain (three different sequence lengths of DNA) modified with a fluorocarbon chain at the 5' end, with the 5' end pointing to the inside of the DNA frame; thereby achieving four different spatial positions of the fluorocarbon chain arranged at the internal edge of the DNA nanoframe structure, 21 nt, 41 nt or 61 nt DNA length from the inside of the frame. The fluorocarbon chain seeds form a spatial distribution from the internal edge of the frame to the center, with an interval of 20 nt DNA length. Therefore, fluorine probes of different sizes can be assembled inside a DNA frame.

[0024] In some embodiments of the present invention, the sequence of the anchoring chain is as shown in SEQ ID NO: 1, and the sequence of the anchoring chain is a universal sequence, which is applicable to different DNA nanoframe structures: truncated icosahedron (football-shaped), icosahedron, dodecahedron structure, etc., and hybridization with DNA complementary chains of different lengths.

[0025] In some embodiments of the present invention, the position of the fluorocarbon chain modification and the sequence length of the DNA complementary chain may be different, and specifically may include the following: the sequence of the DNA complementary chain is as shown in SEQ ID NO: 2, with a length of 21nt, and the 3' end is modified with a fluorocarbon chain; the sequence of the DNA complementary chain is as shown in SEQ ID NO: 2, with a length of 21nt, and the 5' end is modified with a fluorocarbon chain; the sequence of the DNA complementary chain is as shown in SEQ ID NO: 3, with a length of 41nt, and the 5' end is modified with a fluorocarbon chain; the sequence of the DNA complementary chain is as shown in SEQ ID NO: 4, with a length of 61nt, and the 5' end is modified with a fluorocarbon chain.

[0026] Preferably, the perfluorocarbon PFC includes at least one of 1,1,1-tris(perfluoro-tert-butoxymethyl)ethane (TPFBME), perfluoro-15-crown-5 ether (PFCE), linear perfluoropolyether (PFPE) and perfluorooctane bromide (PFOB).

[0027] Preferably, the preparation method of the PFC dispersion solution comprises: preparing a dispersion containing Mg 2+ The buffer solution containing the surfactant n-Octyl-β-D-glucopyranoside (OGP) was mixed with perfluorocarbon and dispersed by ultrasonication.

[0028] In some embodiments of the present invention, the buffer is 1xTE buffer (pH 7.0).

[0029] Preferably, dialysis is performed simultaneously with the incubation.

[0030] Preferably, the step of ultrafiltration concentration is further included after the dialysis is completed.

[0031] In some embodiments of the present invention, 110 μL of PFC dispersion solution and 2 pmol of internally fluorinated soccer-shaped DNA framework are mixed and dialyzed overnight. The dialysate is directly subjected to NMR characterization or ultrafiltration and concentration followed by cryo-EM characterization.

[0032] The DNA nanoframes used in the present invention include but are not limited to the above-mentioned truncated icosahedron (soccer ball shape, diameter of about 74 nm), two icosahedrons (diameters of 86 nm and 43 nm, respectively), and dodecahedron structures (diameter of 121 nm). According to the diameter of the inscribed circle of each structure, the internal space volume is calculated, so as to perform dialysis incubation with PFC dispersion solutions of different volumes and concentrations. Because the size of each DNA nanoframe is different, fluorine nanoprobes of different sizes can be assembled.

[0033] The second aspect of the present invention provides a nano-fluorine probe prepared by the preparation method of the first aspect as 19 Application of F contrast agents in magnetic resonance imaging.

[0034] The third aspect of the present invention provides the use of the nano-fluorine probe prepared by the preparation method described in the first aspect in the preparation of diagnostic reagents or kits.

[0035] The present invention adopts the advantages of DNA nanoframes, such as controllable shape and size, uniform size, precise internal and external modification, and the natural negative charge of DNA, and proposes a new 19 F contrast agent assembly method solves 19 The problems of F contrast agents being uneven in size, easy to agglomerate and difficult to preserve can be solved by precisely controlling the size of the fluorine balls at the nanoscale through the morphology and size of the nanoframe and the spatial arrangement of the internal fluorocarbon chain seeds.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The present invention utilizes a variety of three-dimensional DNA nanoframeworks (DNA nanoframeworks, DNF) with different structures to prepare PFC nanoparticles. The present invention utilizes the advantages of the designability of DNA nanoframeworks, the controllable uniform size and morphology, and the natural negative charge of DNA can prevent the aggregation of fluorine nanoparticles; the size of the fluorine balls can be precisely controlled at the nanometer level through the design of the morphology and size of the DNA nanoframework and the spatial arrangement of the internal fluorocarbon chain seeds; compared with the existing technology, the PFC nanoparticles that can be assembled by the DNA nanoframe have the great advantage of uniform and controllable size, and can be used to study the nuclear magnetic properties of PFC nanoparticles of different sizes; in addition, the DNA structure has the advantages of internal and external distinguishability, modifiability, low toxicity, and low immunogenicity, and the anchor points outside the structure can be programmably modified, and in the future, positioning and quantitative modifications can be performed, thereby achieving 19 Precise imaging and intelligent imaging of contrast agents for F-MRI; the present invention is 19 The development and utilization of F-MRI probes provide a new class of general methods and new ideas. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is a schematic diagram of the structure of a DNA nanoframe structure and AFM or TEM characterization results in an embodiment of the present invention;

[0039] Figure 2 1 is a schematic diagram of a process for preparing a fluorine probe using a soccer ball-shaped DNA nanostructure in an embodiment of the present invention;

[0040] Figure 3 The present invention is a schematic diagram of the dispersed assembly of three perfluorocarbon materials, PFCE, TPFBME and PFOB, taking the soccer ball-shaped DNA framework structure as an example, as well as cryo-electron microscopy characterization and 19 F-NMR / MRI signals;

[0041] Figure 4 Schematic diagram of assembling fluorine probes with four different morphological sizes of DNA framework structures and cryo-electron microscopy characterization and 19 F-NMR / MRI signals;

[0042] Figure 5 It is a schematic diagram and cryo-electron microscopy characterization of the nano-fluorine probes modified and assembled with fluorine-carbon chains arranged in different spaces inside the icosahedral DNA framework in an embodiment of the present invention;

[0043] Figure 6 In the embodiment of the present invention, the DNA nanoframe assembly method is used to fluorine probe 19 Effect of F-NMR characteristic peaks (A) and relaxation parameters of nanofluorine probes (B) and in vitro and in vivo 19Relationship between F-MRI imaging signal intensity and probe concentration (CD);

[0044] Figure 7 This is the imaging result of the nano-fluorine probe in mice according to the present invention. DETAILED DESCRIPTION

[0045] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0046] The present invention relates to using DNA nanoframes to assemble uniform and size-controllable nanofluorine particles for 19 F-NMR / MRI imaging. For reference by those skilled in the art.

[0047] The present invention is further described in detail by the following examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product instructions; the materials, reagents, etc. used, unless otherwise specified, are conventional commercially available products.

[0048] Example 1: Construction of DNA nanoframe structure

[0049] This embodiment provides a method for constructing a type of DNA nanoframe structure, wherein the DNA nanoframe is a self-enclosed three-dimensional structure with internal and external topological characteristics, including a regular icosahedron, a regular dodecahedron, a truncated regular icosahedron (soccer ball-shaped) structure, etc., which specifically includes the following steps:

[0050] Step 1: Construct a DNA framework structure such as a regular dodecahedron, a regular icosahedron, or a soccer ball. This step can use 2D / 3D images to present the schematic diagram of the structure, and then use DNA nanostructure professional design software Tiamat and caDNAno to design the framework DNA. Step 1 also includes the following steps: input

[0051] The M13 phage genome sequence was input, and the stapled chain sequence generated by the software was exported for sequence synthesis.

[0052] Step 2: Based on step 1, further construct variants of the DNA framework structure and establish internal and external modifiable sites;

[0053] Step 3: Assemble and purify the DNA framework structure constructed in step 2, obtain structural monomers by size exclusion chromatography and ultrafiltration concentration, and remove unbound staple chains and impurities such as dimers and polymers;

[0054] After the DNA framework is assembled and the icosahedron is synthesized, agarose gel electrophoresis can be used to preliminarily identify the structure. Transmission electron microscopy and atomic force microscopy are used to evaluate the size, integrity and compliance of the structure with the design.

[0055] Among them, size exclusion chromatography is to separate particles in a chromatographic column according to their different sizes. The retention time in the chromatogram is inversely proportional to the size of the molecule. The polymer of the DNA nanostructure has the shortest retention time, the free single strand has the longest retention time, and the target nanostructure has a medium retention time.

[0056] Ultrafiltration is a membrane separation method that uses the microporous structure of a semipermeable membrane to achieve selective separation and recovery of substances. The diameter of ultrafiltration retained particles is between 0.002 and 0.1 μm, the cut-off molecular weight of the ultrafiltration membrane pore size is between 1,000 and 500,000, and the ultrafiltration operating pressure is generally between 0.1 and 0.6 MPa. When a solution flows through the surface of an ultrafiltration membrane with a certain pore size at a certain flow rate, under the action of external pressure, ultrafiltration allows small molecules, inorganic salts and solutes smaller than the cut-off molecular weight to pass through, forming a filtrate (referred to as filtrate), while intercepting substances such as colloids, proteins, microorganisms and macromolecular organic matter larger than the membrane cut-off molecular weight to form a concentrated solution, thereby achieving the purpose of a concentrated structure.

[0057] Agarose gel electrophoresis is the most basic technology in genetic engineering. It is simple and rapid to operate and is now widely used in nucleic acid research. DNA molecules carry negative charges in a pH solution above the isoelectric point and move toward the positive electrode in an electric field. Due to the structural repetitive nature of the sugar-phosphate backbone, the same amount of double-stranded DNA has almost the same net charge, so they can move toward the positive electrode at the same rate.

[0058] A transmission electron microscope (TEM) transmits a beam of electrons through a sample to produce an image. As the electrons pass through the sample, some are scattered by the heavier atoms in the sample, forming a projected image. By rapidly freezing the sample, it becomes vitrified and glassy, ​​without forming ice crystals. In this state, the sample deteriorates much more slowly, making it easier to study biological materials.

[0059] Atomic force microscopy (AFM), also known as scanning force microscopy, is a nanoscale high-resolution scanning probe microscope that is 1000 times better than the optical diffraction limit.

[0060] Figure 12 is a schematic diagram of the structure of the four DNA nanoframe structures constructed in this embodiment and the AFM or TEM characterization results, from left to right are the AFM characterization results of the 121 nm dodecahedron, the 86 nm icosahedron, the 74 nm truncated icosahedron (soccer ball shape) structure and the TEM characterization results of the 43 nm icosahedron.

[0061] Example 2: DNA nanoframe assembly of nanofluorine probes

[0062] This embodiment provides a method for assembling a fluorine nanoprobe using the above-mentioned DNA nanoframe, and the specific steps are as follows:

[0063] Step 1: Modify the internal fluorocarbon chain of the DNA nanoframe. First, modify the fluorocarbon chain to the complementary chain of the anchor sequence through maleimide-thiol reaction or phosphoramidite chemistry. Use the staple chain at the specific position of the origami structure to extend the anchor sequence, and then modify it at the specific site of the structure through molecular hybridization.

[0064] In this step, the modification of the fluorocarbon chain can be carried out in two specific ways: ① using the DNA single strand modified with maleimide at the 3' end to react with 1H,1H,2H,2H-perfluoro-1-octanethiol or 1H,1H,2H,2H-perfluoro-1-decanethiol, and then purifying it by reverse HPLC to obtain DNA with fluorocarbon chain modified at the 3' end; ② 1H,1H,2H,2H-perfluoro-1-decanol is phosphoramidated and coupled to the 5' end of the DNA single strand by a DNA synthesizer (coupled with -OH at the 5' end of the DNA single strand), and purified by reverse HPLC to obtain a DNA single strand modified with fluorocarbon chain at the 5' end (i.e., a complementary strand modified with fluorocarbon chain).

[0065] In this step, the anchor sequence and complementary sequence of the four DNA nanoframe structures are universal, the anchor chain sequence: 5'-CTTCACACCACACTCCATCTA-3' (SEQ ID NO: 1); the complementary chain sequence: 5'-TAGATGGAGTGTGGTGTGAAG-3' (SEQ ID NO: 2).

[0066] Step 2: Taking perfluoro-15-crown-5 ether (PFCE) as an example: prepare 2 mL of 10 mM Mg 2+ The cells were then mixed with 4 μL of PFCE in 1xTE buffer (pH 7.0) containing 2% octyl-β-D-pyranoglucoside (OG) and dispersed by ultrasonication.

[0067] Step 3: Take the soccer ball-shaped DNA nanoframe as an example: Take 110 μL of the dispersion solution and 2 pmol of the internally fluorinated soccer ball-shaped DNA framework and dialyze overnight (this process is actually dialysis during incubation). Take the dialysate and directly perform NMR characterization or ultrafiltration concentration and then perform cryo-EM characterization.

[0068] Figure 2 This is a schematic diagram of assembling a nanofluorine probe using a soccer ball-shaped DNA nanoframe in one embodiment of the present invention.

[0069] Figure 3 The present invention is a schematic diagram of the process of assembling a nanofluorine probe using a soccer ball-shaped DNA nanoframe and three PFC materials, 1,1,1-tris(perfluorotert-butoxymethyl)ethane (TPFBME), perfluoro-15-crown-5 ether (PFCE) and perfluorooctyl bromide (PFOB), as well as cryo-electron microscopy characterization and 19 F-NMR / MRI results characterization. Cryo-electron microscopy results showed that uniform-sized nano-fluorine spheres grew inside the DNA nanostructure. 19 The characteristic peaks of the three PFC materials can be clearly observed in the F-NMR spectrum. The above results prove that DNA nanostructures can be used for 19 The feasibility of assembling F probes. Because the NMR characteristic peak signals of PFCE and TPFBME are symmetrical and single, and the displacement difference is large, we also use 19 F-MRI performed multi-color imaging of the two types of nanofluorine spheres.

[0070] Figure 4 The present invention is a schematic diagram of the process of assembling nanofluorine probes using four DNA nanoframes and PFCE in one embodiment, as well as cryo-electron microscopy characterization and 19 F-NMR results characterize the four DNA nanoframes, including a dodecahedron with a size of 121 nm, an icosahedron with a size of 86 nm and 43 nm, and a soccer ball-shaped structure with a size of 74 nm. After incubation and dialysis with PFCE, cryo-electron microscopy results confirmed that DNA nanostructures with different morphologies can be assembled into nanostructures of different sizes. 19 F probe. DNA nanostructures assembled with the same amount of material 19 F probe 19 The signal intensity of F-NMR is linearly related to the internal volume of the structure. The above results prove that the assembly method of the present invention can be used for DNA nanostructures and PFC materials of different shapes and sizes, and has strong versatility.

[0071] Figure 5It is a schematic diagram and cryo-electron microscopy characterization results of regulating the size of nano-fluorine balls by spatial arrangement of fluorocarbon chain seeds in one embodiment of the present invention. By modifying the fluorocarbon chains at different ends of DNA and using anchor chain complementary sequences of different lengths, we can arrange the fluorocarbon chains at the inner edge of the DNA nanoframe, 21 nt, 41 nt and 61 nt DNA lengths from the inner part of the framework, wherein the anchor chain sequences of the fluorocarbon chains arranged at the four different spatial positions are the same, the sequence is 5'-CTTCACACCACACTCCATCTA-3' (SEQ ID NO: 1); the lengths of the complementary chains or the modification positions of the fluorocarbon chains are inconsistent, respectively: 5'-TAGATGGAGTGTGGTGTGAAG-3' (SEQ ID NO: 2), the 3' end is modified with a fluorocarbon chain; 5'-TAGATGGAGTGTGGTGTGAAG-3' (SEQ ID NO: 2), the 5' end is modified with a fluorocarbon chain; 5'-TTTTTTTTTTTTTTTTTTTTTAGATGGAGTGTGGTGTGAAG-3' (SEQ ID NO: 3), the 5' end is modified with a fluorocarbon chain; 5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAGATGGAGTGTGGTGTGAAG-3' (SEQ ID NO: 4), the 5' end is modified with a fluorocarbon chain. Taking the icosahedral structure with a diameter of 83 nm as an example, after incubation with PFCE dialysis, the cryo-electron microscopy results showed that there were obvious differences in the diameters of the internally grown fluorospheres, which were 67.6 ± 1.7 nm, 63.0 ± 1.7 nm, 52.1 ± 2.6 nm and 42.1 ± 2.5 nm, respectively. The above results prove that the present invention can accurately control the size of the nanofluorospheres by adjusting the length of the fluorocarbon chain-modified DNA within the same DNA nanostructure, reflecting that the DNA nanostructure has the advantages of controllable shape and size and uniform size in the assembly of fluoro probes.

[0072] Example 3: Performance Characterization of Nanofluorine Probes

[0073] This example detects the nanofluorine probe obtained by the DNA nanoframe assembly method in Example 2. 19 F-NMR characteristic peaks, relaxation parameters and in vitro and in vivo 19 F-MRI imaging effect

[0074] Figure 6A in the figure represents the comparison between the fluorine probe assembled by DNA nanostructure and the liquid PFCE and the PFCE dispersed by surfactant. The results show that the shim cannot be adjusted during the NMR measurement due to the lack of deuterated reagent that can dissolve PFCE. The peak of liquid PFCE is wide and not single. The PFCE emulsion dispersed by ultrasonic crushing and surfactant also shows several chemical shift peaks. The nanofluorine probe assembled by the football-shaped DNA nanoframe shows a sharp and single NMR signal, proving that the nanofluorine probe has a great advantage in the sensitivity of fluorine nuclear magnetic signal detection. This example further measures the relaxation times T1 and T2 of the nanofluorine probe, which are 767 ms and 643 ms, respectively, and the ratio of T2 / T1 is 0.84 ( Figure 6 In B). This embodiment also utilizes 19 F-MRI measured the signal of nano-fluorine probes with different concentrations and found that the signal intensity was proportional to the concentration of the probe, indicating that within the concentration range measured by MRI, the fluorine probes would not aggregate ( Figure 6 Medium CD).

[0075] At the same time, this embodiment conducted a live detection experiment by injecting nanofluorine probes subcutaneously in mice and performing 19 When the measurement time is 4 minutes, a clear MRI signal can be observed, indicating that the nano-fluorine probe prepared by the present invention can be used for imaging in vivo ( Figure 7 ).

[0076] The above description is only a preferred embodiment of the present invention and is not any formal or substantial limitation of the present invention. It should be pointed out that a person skilled in the art can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing monodisperse perfluorocarbon nanoparticles assisted by DNA nanoframes, characterized in that: include: Fluorocarbon chain modification was introduced into the interior of the DNA nanoframe structure through hybridization complementation; Dispersing perfluorocarbon PFC in a surfactant to form a PFC dispersion solution, and co-incubating with the DNA nanoframe structure modified with the fluorocarbon chain, so that the PFC enters the space inside the DNA nanoframe structure under the action of the fluorocarbon chain, so that the PFC self-assembles inside the DNA nanoframe structure to form nanoparticles of uniform size, that is, obtaining a nanofluorine probe, and removing the surfactant to obtain a purified nanofluorine probe; The fluorocarbon chain modification is specifically achieved by the following steps: Step 1: reacting a DNA single strand modified with maleimide at the 3' end with 1H,1H,2H,2H-perfluoro-1-octanethiol or 1H,1H,2H,2H-perfluoro-1-decanethiol, and then purifying it by reverse HPLC to obtain a DNA single strand modified with a fluorine carbon chain at the 3' end, i.e., a DNA complementary strand modified with a fluorine carbon chain at the 3' end; or phosphoramidating 1H,1H,2H,2H-perfluoro-1-decanol, coupling it to the 5' end of the DNA single strand by a DNA synthesizer, and purifying it by reverse HPLC to obtain a DNA single strand modified with a fluorine carbon chain at the 5' end, i.e., a DNA complementary strand modified with a fluorine carbon chain at the 5' end; Step 2: hybridizing the complementary DNA chain with fluorocarbon chain modification at the 3' end or 5' end with the anchoring chain inside the DNA nanoframe structure through the base complementary pairing principle to achieve the modification of the fluorocarbon chain inside the DNA nanoframe structure; The preparation method of the PFC dispersion solution comprises: preparing a dispersion containing Mg 2+ The buffer solution containing octyl-β-D-pyranoglucoside, a surfactant, is mixed with perfluorocarbon and dispersed by ultrasonication.

2. The preparation method according to claim 1, characterized in that The DNA nanoframe structure is a type of self-enclosed three-dimensional structure with internal and external topological characteristics, including truncated icosahedron, regular icosahedron, and regular dodecahedron structures.

3. The preparation method according to claim 1, characterized in that: The DNA nanoframe structure is constructed by DNA origami technology, and the construction method of the DNA nanoframe structure comprises the following steps: Step 1: Use DNA nanostructure design software to construct the DNA nanoframe structure; Step 2: Based on step 1, further construct variants of the DNA framework structure and establish internal and external modifiable sites; Step 3: The DNA framework structure constructed in step 2 is self-assembled and purified.

4. The preparation method according to claim 1, characterized in that: The 5' end of the fluorocarbon chain-modified DNA complementary chain points to the interior of the DNA nanoframe structure, and the 3' end is close to the frame edge of the DNA nanoframe structure; the fluorocarbon chain acts as a seed to capture free PFC fluorine balls, and the size of the fluorine balls is regulated by regulating the spatial position of the fluorocarbon chain seeds.

5. The preparation method according to claim 4, characterized in that: The spatial arrangement of fluorocarbon chain seeds on the DNA nanoframe structure is regulated by the position of fluorocarbon chain modification and the sequence length of the DNA complementary chain, which is specifically divided into the following two types: ① A 21nt DNA complementary chain with a fluorocarbon chain modification at the 3' end: through complementary hybridization, the fluorocarbon chain is close to the internal edge of the DNA nanoframe structure; ② A 21nt, 41nt or 61nt DNA complementary chain with a fluorocarbon chain modification at the 5' end, with the 5' end pointing to the inside of the DNA frame; thereby achieving four different spatial positions where the fluorocarbon chains are arranged at the internal edge of the DNA nanoframe structure, and at a distance of 21nt, 41nt or 61nt DNA length from the inside of the frame.

6. The preparation method according to claim 1, characterized in that: The perfluorocarbon PFC includes at least one of 1,1,1-tris(perfluoro-tert-butoxymethyl)ethane, perfluoro-15-crown-5 ether, linear perfluoropolyether and brominated perfluorooctane.

7. The nano-fluorine probe prepared by the preparation method according to any one of claims 1 to 6 is used in the preparation 19 Application of F contrast agents in magnetic resonance imaging.

8. Use of the nano-fluorine probe prepared by the preparation method according to any one of claims 1 to 6 in the preparation of diagnostic reagents or kits.

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