DNA tetrahedral framework and nanocomposite and application of DNA tetrahedral framework and nanocomposite in treatment of spinal cord injury

Abstract

The invention discloses a DNA tetrahedral framework, a nano-composite and application of the nano-composite in treatment of spinal cord injury, and relates to the field of biological medicine, the DNA tetrahedral framework capable of loading ginger-derived exosome-like nanoparticles GELN is researched and developed, the DNA tetrahedral framework comprises four DNA single chains, nucleotide sequences of the four DNA single chains have at least 80% identity with sequences shown in SEQ ID NO: 1-4 respectively, and the DNA single chains are used for loading ginger-derived exosome-like nanoparticles GELN. The novel nano-composite obtained by compounding the DNA tetrahedral framework and GELN can target and enduringly act on microglial cells, and a way is provided for development of drugs for spinal cord injury and research of related pathological mechanisms.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a DNA tetrahedral framework and a nanocomposite and applications thereof in treating spinal cord injury. Background Art

[0002] Spinal cord injury is a damage to the spinal cord function caused by direct or indirect external factors. It is the "number one killer" of death and disability in young and middle-aged people in modern society. After spinal cord injury occurs, microglia polarize to M1 or M2 in response to continuous signal stimulation from the microenvironment, exerting pro-inflammatory or anti-inflammatory repair effects, and are the key hub for secondary spinal cord injury and good prognosis. Regulating microglial M2 polarization is a key link in reducing the immune inflammatory response to spinal cord injury and promoting spinal cord repair, but the regulatory mechanism is still unclear.

[0003] Currently, the market lacks drug carriers that can target and persistently act on microglia and precisely regulate their phenotype.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a DNA tetrahedral framework (tFNA) and a nanocomplex (GELN-tFNA) and applications thereof in treating spinal cord injury.

[0006] The present invention is achieved in that:

[0007] In the first aspect, an embodiment of the present invention provides a DNA tetrahedral framework, which includes four DNA single strands: S1 strand, S2 strand, S3 strand and S4 strand; wherein the nucleotide sequences of the S1 strand to the S4 strand are at least 80% identical to the sequences shown in SEQ ID NO: 1 to 4, respectively.

[0008] In a second aspect, an embodiment of the present invention provides a method for preparing a DNA tetrahedral framework as described in the aforementioned embodiment, which comprises the following steps: mixing the S1 to S4 chains described in the aforementioned embodiment in a buffer solution.

[0009] In a third aspect, embodiments of the present invention provide use of the DNA tetrahedral framework described in the preceding embodiments or the DNA tetrahedral framework prepared by the preparation method described in the preceding embodiments in the preparation of drugs.

[0010] In a fourth aspect, an embodiment of the present invention provides a nanocomplex comprising: ginger-derived exosome-like nanoparticles (GELN) and the DNA tetrahedral framework described in the preceding embodiment or the DNA tetrahedral framework prepared by the preparation method described in the preceding embodiment.

[0011] In a fifth aspect, an embodiment of the present invention provides a method for preparing a nanocomplex as described in any of the preceding embodiments, comprising the following steps: mixing ginger-derived exosome-like nanoparticles with the DNA tetrahedral framework described in the preceding embodiment or the DNA tetrahedral framework prepared by the preparation method described in the preceding embodiment.

[0012] In a sixth aspect, embodiments of the present invention provide use of the nanocomposite as described in the preceding embodiments or the nanocomposite prepared by the preparation method as described in the preceding embodiments in preparing a product for preventing, treating or assisting in the treatment of spinal cord injury.

[0013] In a seventh aspect, an embodiment of the present invention provides a product for preventing, treating or assisting in the treatment of spinal cord injury, wherein the active ingredient includes the nanocomposite described in the preceding embodiment or the nanocomposite prepared by the preparation method described in the preceding embodiment.

[0014] The present invention has the following beneficial effects:

[0015] The present invention has developed a DNA tetrahedral framework that can load ginger-derived exosome-like nanoparticles. The novel nanocomplex obtained by combining the DNA tetrahedral framework with ginger-derived exosome-like nanoparticles can target and persistently act on microglia, providing a way for the development of spinal cord injury drugs and the study of related pathological mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0017] Figure 1 The DNA tetrahedral framework in Example 1;

[0018] Figure 2 The cholesterol-modified DNA chain in Example 1;

[0019] Figure 3 The robot arm 1 in Example 1;

[0020] Figure 4 The robot arm 2 in Example 1;

[0021] Figure 5 is the TMEM119 aptamer in Example 1;

[0022] Figure 6The research and development ideas of the GELN-tFNA nanocomplex of Example 1 and the key mechanism for regulating the polarization of injury-activated spinal microglia M2;

[0023] Figure 7 Schematic diagram of the DNA tetrahedron framework of Examples 1 to 5. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0025] Ginger is a typical plant-based traditional Chinese medicine that is both medicinal and edible. Ginger-derived exosome-like nanoparticles are nanoscale vesicles secreted by ginger cells, with a particle size of 80 to 120 nm (average 100 nm), rich in gingerol, microRNA (miRNA) and a small amount of protein. They have the characteristics of wide sources, non-toxicity, no pollution, low immunogenicity, high biocompatibility and strong transformability. They participate in intercellular communication and mediate "cross-border intervention". Ginger-derived exosome-like nanoparticles are surrounded by a lipid bilayer, mainly phosphatidic acid, followed by digalactosyldiacylglycerol and monogalactosyldiacylglycerol, and are inlaid with several plant aquaporins (AQPs). The inventors of this application used high performance liquid chromatography to measure that the gingerol in ginger-derived exosome-like nanoparticles is mainly 6-gingerol, and its content is 10 times that of ginger; existing researchers have identified 116 conserved miRNAs in ginger-derived exosome-like nanoparticles. Compared with ginger, the upregulated ath-miR159a and ath-miR319a are highly enriched in ginger-derived exosome-like nanoparticles, and participate in regulating human immunity and cancer-related signaling pathways. It can be seen that ginger-derived exosome-like nanoparticles are nano delivery carriers with highly aggregated active ingredients of ginger.

[0026] However, ginger-derived exosome-like nanoparticles are still two shortcomings in becoming an ideal carrier for precisely regulating the polarization of spinal microglia M2: ① Ginger-derived exosome-like nanoparticles lack membrane proteins that specifically recognize spinal microglia, cannot migrate and aggregate in spinal microglia, and are easily nonspecifically adsorbed by surrounding tissue cells, reducing the efficacy; ② Ginger-derived exosome-like nanoparticles are easily degraded in an acidic environment and release the encapsulated active pharmaceutical ingredients instantaneously, with a short duration of action. Therefore, the inventors of the present invention intend to develop intelligent bionic nanomaterials to load ginger-derived exosome-like nanoparticles to construct a new nanocomposite that has the functions of targeting spinal microglia and pH-responsive controlled release.

[0027] On the one hand, an embodiment of the present invention provides a DNA tetrahedron framework, wherein the DNA tetrahedron framework comprises four DNA single strands: an S1 strand, an S2 strand, an S3 strand, and an S4 strand;

[0028] The nucleotide sequences of the S1 to S4 chains are at least 80% identical to the sequences shown in SEQ ID NOs: 1 to 4, respectively.

[0029] Four single DNA strands can self-assemble into a basic model for embedded loading of ginger-derived exosome-like nanoparticles. Compared with other DNA tetrahedral frameworks, the sequence sizes of the S1 chain, S2 chain, S3 chain and S4 chain provided by the present invention are more suitable for loading ginger-derived exosome-like nanoparticles.

[0030] Without further limitation, "specifically at least 80%" herein refers to any one of 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98%, 100% or a range between any two of them.

[0031] In some embodiments, the DNA tetrahedron framework further comprises: any one or more of a TMEM119 aptamer and a DNA robotic arm for capturing a target substance loaded by the DNA tetrahedron framework. The target substance may be ginger-derived exosome-like nanoparticles or other substances with a size ≤ ginger-derived exosome-like nanoparticles.

[0032] In some embodiments, the DNA robot is a single strand of DNA.

[0033] In some embodiments, more than 50% of the bases in the sequence of the DNA robot are modified with cholesterol. The more than 50% can be any one of 50%, 60%, 70%, 80%, 90%, 100%, or a range between any two. Cholesterol is similar and compatible with the lipid bilayer. By modifying the phosphate group of the DNA chain through a phosphate bond, the DNA robot can be made lipophilic, and can quickly and efficiently adsorb lipid membranes, or directly insert into lipid bilayers to surround membrane proteins. The more bases modified with cholesterol, the higher the lipophilicity.

[0034] In some embodiments, the nucleotide sequence of the DNA robot can form a palindrome structure. The palindrome structure can make the DNA single strand self-assemble into a double strand due to base pairing, prevent the two DNA robot arms from being entangled with each other due to base pairing, and also increase the contact area between the DNA robot arm and the target substance, so as to realize the automatic loading of the target substance in the DNA tetrahedron framework and form a new type of nanocomplex.

[0035] In some embodiments, the nucleotide sequence of the DNA robot contains one or more DNA loops, and the DNA loop includes (TGC)n, that is, n TGC repeating units form a DNA loop, n is a positive integer and is selected from 1 to 10, and n can be any one of 1, 2, 3, 4, 5, 6, 8, 10 or a range between any two of them.

[0036] The DNA tetrahedron framework can protect the target substances it carries, such as ginger-derived exosome-like nanoparticles, from instantaneous drug release caused by degradation in an acidic environment. Therefore, program initiation and control of drug release from ginger-derived exosome-like nanoparticles are the key points of the DNA tetrahedron framework design. There are 5 to 6 plant AQPs distributed on the lipid membrane of ginger-derived exosome-like nanoparticles, which belong to the γ subclass of intrinsic protein of the vacuole membrane and are the "transportation hub" for controlling the output of active substances from ginger-derived exosome-like nanoparticles. The plant AQP family has highly conserved structural characteristics. It is composed of four subunits as a tetramer, each of which is composed of three transmembrane α helices. Its primary structure is highly homologous, that is, the three transmembrane α helices of the four subunits all contain a conserved amino acid sequence: asparagine-proline-alanine (Asn-Pro-Ala, NPA). The four subunits surround the pore, and under physiological conditions, NPA folds to make the pore narrow and closed in an "hourglass shape". TGC can bind to the amino acid sequence of NPA through hydrogen bonds. The DNA ring formed by TGC repeating units can spontaneously couple to the NAP domain and regulate the opening and closing of AQP.

[0037] In some embodiments, the nucleotide sequence of the DNA robot contains at least one DNA buckle, the DNA buckle includes poly A and poly T, and the DNA buckle and the DNA ring together constitute a DNA buckle ring; the structure of the DNA buckle ring includes: poly A-DNA ring-poly T; the sequence length of the poly A and the poly T is 3 to 10 nt.

[0038] After ischemia and hypoxia, the pH value of the spinal microglial cytoplasm is an acidic environment of 4 to 5. However, the AQP of ginger-derived exosome-like nanoparticles is insensitive to changes in pH value. After being taken up by microglia, AQP cannot be opened, and the active ingredients of ginger-derived exosome-like nanoparticles are released in an orderly manner. The adenine-rich sequence (poly A) base pairs with the complementary chain (poly T) at pH = 7.4, but is protonated at pH = 5.0, transforming into a single-strand cross-linked A-motif, loosening the tension of the original double-stranded coiling. Poly A and poly T are used as DNA buckles to form a DNA buckle with the DNA ring, which can respond to changes in environmental pH, regulate the "DNA loop" conformation of the TGC repeat sequence, and then link the NAP domain to regulate the opening and closing state of AQP.

[0039] In some embodiments, the sequence length of the poly A or the poly T can be any one of 3, 4, 5, 6, 7, 8, 9, 10 nt or a range between any two of them.

[0040] In some embodiments, the DNA buckle further includes a first palindromic sequence and a second palindromic sequence capable of forming a palindromic structure with the first palindromic sequence; the first palindromic sequence and the second palindromic sequence are respectively located on the side of the poly A and the poly T away from the DNA loop.

[0041] In some embodiments, the number of DNA circles can be any one of 1, 2, 3, 4, 5, or a range between any two.

[0042] In order to reduce the impact of conformational changes of the DNA clasp on the structure of the DNA manipulator, and also to increase the range of activity of the functional units of the DNA clasp in capturing and contacting AQP in the lipid bilayer, according to the number of AQPs distributed on the ginger-derived exosome-like nanoparticles, the three DNA clasp were each connected to the two sides and the top (or near the top) of the DNA manipulator through a cholesterol-modified DNA double-strand, so that the DNA manipulator evolved into a "three-finger shape", which has the functions of fixation and pH-responsive control of the slow release of ginger-derived exosome-like nanoparticles.

[0043] In some embodiments, the DNA robot comprises any one or more of robot 1 and robot 2; the nucleotide sequences of robot 1 and robot 2 are at least 80% identical to the sequences shown in SEQ ID NOs: 5 to 6, respectively, and more than 50% of the bases based on the shown sequences have cholesterol modification.

[0044] In some embodiments, the nucleotide sequences of the robot arm 1 and the robot arm 2 are shown in SEQ ID NOs: 5 to 6. In some embodiments, the robot arm 1 is connected to any one or more of the S1 to S4 chains; the robot arm 2 is connected to any one or more of the S1 to S4 chains.

[0045] In some embodiments, the robotic arm 1 is connected to the 5' end of any one or more of the S1 to S4 chains, and the 5' end of the single-stranded DNA connected to the robotic arm 1 is a sticky end; the robotic arm 2 is connected to the 5' end of any one or more of the S1 to S4 chains, and the 5' end of the single-stranded DNA connected to the robotic arm 2 is a sticky end.

[0046] In some embodiments, the single-stranded DNA connected to the robotic arm 1 and the single-stranded DNA connected to the robotic arm 2 are opposite edges in the DNA tetrahedron framework.

[0047] In some embodiments, the sticky ends of the single-stranded DNA connected to the robotic arm 1 and the sticky ends of the single-stranded DNA connected to the robotic arm 2 are respectively located in the central area of ​​the skew edges, and the central area is the area extending 10 to 15 nt from the midpoint of the edge to both ends.

[0048] In some embodiments, the 5' end of the DNA single strand connected to the robot arm 1 and the 5' end of the DNA single strand connected to the robot arm 2 are the midpoints of the non-planar edges of the DNA tetrahedral framework.

[0049] TMEM119 is an intrinsic transmembrane protein specifically expressed by microglia. It does not change with the change of polarization state. It is specifically recognized by exogenous ligands, thereby activating mesh protein-mediated endocytosis and intracellular transport. Nucleic acid aptamers are single-stranded oligonucleotides that can be folded into a specific structure and bind to the target protein with high specificity. As a new type of molecular recognition tool, it shows broad application prospects in the field of disease diagnosis and treatment. The embodiment of the present invention screened out TMEM119-specific DNA aptamers (referred to as TMEM119 aptamers), which can guide the DNA tetrahedral framework and its nanocomplexes to deliver ginger-derived exosome-like nanoparticles to spinal microglia.

[0050] In some embodiments, the TMEM119 aptamer is connected to any one or more of the S1 to S4 chains.

[0051] In some embodiments, the TMEM119 aptamer is located at a vertex of the DNA tetrahedron framework. The DNA tetrahedron framework has four vertices, and the TMEM119 aptamer can be disposed at any one or more of the vertices.

[0052] In some embodiments, the TMEM119 aptamer is connected to the 5' end of any one or more of the S1 to S4 chains, and the 5' end of the DNA single strand connected to the TMEM119 aptamer is a sticky end, which is located at the vertex of the DNA tetrahedral framework after the S1 to S4 chains are assembled. In some embodiments, the TMEM119 aptamer is connected to the 5' end of the S1 chain, and the 5' end of the S1 chain is a sticky end, which is located at the vertex of the DNA tetrahedral framework after assembly. Driven by aptamer-ligand recognition, the DNA tetrahedral framework migrates and aggregates toward spinal microglia in a "corner attack" posture, and is then ingested.

[0053] In some embodiments, the nucleotide sequence of the TMEM119 aptamer is at least 80% identical to the sequence shown in SEQ ID NO:7.

[0054] In some embodiments, the nucleotide sequence of the TMEM119 aptamer is shown in SEQ ID NO:7.

[0055] On the other hand, an embodiment of the present invention provides a method for preparing a DNA tetrahedral framework as described in any of the above embodiments, comprising the following steps: mixing the S1 to S4 chains described in any of the above embodiments in a buffer.

[0056] In some embodiments, the preparation method further comprises: heating the mixed product and then cooling it to room temperature to obtain a DNA tetrahedral framework skeleton (tFNA skeleton).

[0057] In some embodiments, the molar ratio of the S1 to S4 chains is 1 to 3: 1 to 3: 1 to 3: 1 to 3. The molar ratio may be 1: 1: 1: 1, and the molar ratio between any two chains may be any one of 1: 1, 1: 2, 1: 3, or a range between any two of them.

[0058] In some embodiments, the heating conditions include: 85-105° C., 1-20 min. The heating temperature can be any one of 85, 86, 88, 90, 92, 94, 95, 96, 98, 100, 102, 104, 105° C. or a range between any two thereof. The heating time can be any one of 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 min or a range between any two thereof.

[0059] In some embodiments, when the DNA tetrahedral framework also includes the TMEM119 aptamer described in any of the preceding embodiments and / or the DNA robotic arm described in any of the preceding embodiments, the preparation method further includes: mixing the DNA tetrahedral framework skeleton with the TMEM119 aptamer and / or the DNA robotic arm, adding DNA ligase, and obtaining the final DNA tetrahedral framework.

[0060] In some embodiments, the molar ratio of the DNA tetrahedral framework, the TMEM119 aptamer and the DNA robot is 1 to 3:1 to 3:1 to 6. The molar ratio can be any one of 1:1:1, 2:1:1, 1:2:1, 1:1:2, 3:1:1, 1:3:1, 1:1:4, 2:1:2, 1:2:2, or any two thereof.

[0061] When the DNA robot includes robot 1 and robot 2, the mixing molar ratio of the DNA tetrahedral framework skeleton, the TMEM119 aptamer, robot 1 and robot 2 is 1-3:1-3:1-3:1-3, and the molar ratio between any two of them can be any one of 1:1, 1:2, 1:3 or a range between any two of them.

[0062] On the other hand, an embodiment of the present invention provides the use of the DNA tetrahedral framework described in any of the preceding embodiments or the DNA tetrahedral framework prepared by the preparation method described in any of the preceding embodiments in the preparation of drugs.

[0063] In some embodiments, the drug includes: a drug for preventing, treating or assisting in the treatment of spinal cord injury.

[0064] On the other hand, an embodiment of the present invention provides a nanocomplex comprising: ginger-derived exosome-like nanoparticles and the DNA tetrahedral framework described in any of the foregoing embodiments or the DNA tetrahedral framework prepared by the preparation method described in any of the foregoing embodiments.

[0065] In some embodiments, the molar ratio of the DNA tetrahedral framework to the ginger-derived exosome-like nanoparticles is 1-3:1-3.

[0066] On the other hand, an embodiment of the present invention further provides a method for preparing the nanocomposite as described in any of the above embodiments, comprising the following steps:

[0067] The ginger-derived exosome-like nanoparticles are mixed with the DNA tetrahedral framework described in any of the preceding embodiments or the DNA tetrahedral framework prepared by the preparation method described in any of the preceding embodiments.

[0068] In some embodiments, the mixing conditions include: 30-45°C, 3-9h. The mixing temperature can be any one of 30, 32, 34, 36, 38, 40, 42, 44, 45°C or a range between any two thereof. The mixing time can be any one of 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9h or a range between any two thereof.

[0069] In some embodiments, the preparation method further comprises preparing ginger-derived exosome-like nanoparticles.

[0070] In some embodiments, ginger juice is subjected to a first centrifugation and a second centrifugation to remove the precipitate to obtain a supernatant; the supernatant is mixed and incubated with a polyethylene glycol solution, the incubated product is centrifuged for a third time, and the precipitate is collected to obtain ginger-derived exosome-like nanoparticles.

[0071] In some embodiments, the conditions of the first centrifugation or the third centrifugation are: 2000-4000g, 10-30min. The centrifugal gravitational acceleration can be any one of 2000, 2500, 3000, 3500, 4000g or a range between any two of them. The centrifugal time can be any one of 10, 15, 20, 25, 30min or a range between any two of them.

[0072] In some embodiments, the second centrifugation condition is: 8000-12000g, 30-50min. The centrifugal gravitational acceleration can be any one of 8000, 9000, 10000, 11000, 12000g or any two thereof; the centrifugal time can be any one of 30, 35, 40, 45, 50min or any two thereof.

[0073] In some embodiments, the volume percentage of polyethylene glycol in the polyethylene glycol solution is 5% to 20%. The volume percentage can be any one of 5%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, or any two thereof. The polyethylene glycol solution is prepared by mixing polyethylene glycol and water.

[0074] In some embodiments, the molecular weight of the polyethylene glycol is 5000-7000, and specifically can be any one of 5000, 5500, 6000, 6500 and 7000, or a range between any two of them.

[0075] On the other hand, embodiments of the present invention further provide use of the nanocomposite as described in any of the foregoing embodiments or the nanocomposite prepared by the preparation method as described in any of the foregoing embodiments in preparing a product for preventing, treating or assisting in treating spinal cord injury.

[0076] In addition, an embodiment of the present invention further provides a product for preventing, treating or assisting in treating spinal cord injury, wherein the active ingredient includes the nanocomposite described in any of the foregoing embodiments or the nanocomposite prepared by the preparation method described in any of the foregoing embodiments.

[0077] As used herein, "treating" includes preventing or alleviating a condition, reducing the rate at which a condition develops or develops, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or stopping symptoms associated with a condition, producing complete or partial reversal of a condition, curing a condition, or a combination of the above.

[0078] For cancer, "treatment" can refer to inhibiting or slowing the growth, reproduction, or metastasis of tumors or malignant cells, or some combination of the above. For tumors, "treatment" includes eliminating all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of the tumor, or some combination of the above.

[0079] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0080] The steps of the animal model and some related detection methods used in the subsequent examples are as follows:

[0081] (1) Establishment of rat spinal cord injury model: modified Allen impact method, removal of T 10 The spinal cord was exposed through the vertebral lamina, and a 1 mm thick small gasket was attached to the dura mater. A 30 g Kirschner wire was dropped vertically from a height of 3.0 cm along the cannula to hit the gasket, causing spinal cord injury. The rat's whole body quickly retracted and shook, indicating that the model was successful.

[0082] (2) In vivo fluorescence imaging to detect the aggregation of GELN-tFNA in spinal cord injuries: After anesthesia, the rat was placed on the sample stage of the imager to ensure that it was in the appropriate focal length and position. The fluorescence in vivo imager was started and imaging was performed according to the preset parameters to obtain fluorescence images for data analysis.

[0083] (3) Immunofluorescence staining to detect the aggregation of GELN-tFNA in spinal cord microglia: The spinal cord tissue was fixed with 4% paraformaldehyde, frozen and sectioned, penetrated, blocked, incubated with Iba-1 primary antibody, then incubated with fluorescent secondary antibody, stained with DAPI, and observed under a fluorescence microscope.

[0084] (4) Detection of 6-gingerol concentration by HPLC: The supernatant of acidic PBS or spinal cord homogenate at different time points was filtered through a 0.45 μm microporous filter membrane and then subjected to chromatographic analysis. Chromatographically pure methanol and purified water were mixed in a ratio of 25:75 to prepare the mobile phase. The peak area was recorded and the results were calculated based on the standard curve.

[0085] (5) Fluorescence quantitative PCR detection of miRNA levels: miRNA was extracted from acidic PBS or spinal cord homogenate supernatant at different time points, and stem-loop reverse transcription primers were designed to obtain cDNA under the action of reverse transcriptase. Downstream and downstream primers were added for PCR reaction, and the results were calculated based on the Ct value and standard curve.

[0086] (6) BBB motor function score, angle of inclined board test, gait analysis and motor evoked potential of rats: BBB motor function score: rat hind limb movement is divided into 22 levels, including the number and range of joint movements, load-bearing degree, coordination of front and rear limbs, and the movement of front and rear paws and tail. Complete paralysis of the hind limbs is 0 points, and completely normal is 21 points; Inclined board test angle: the rat's body longitudinal axis is parallel to the longitudinal axis of the inclined board, with its head facing the elevated side of the inclined board. Starting from 0°, the maximum angle at which the rat can stay on the inclined board for 5 seconds is observed by raising it 5° each time. The average value is obtained after 5 measurements; Gait analysis: a piece of white paper with the same length and width is placed on a narrow board, and the dorsum of the rat's hind limb is smeared with red ink. The sole of the foot was smeared with blue ink, and the rat was immediately placed on one side of a white paper and walked toward the dark box. The white paper was put away after the rat entered the dark box, and the dorsum of the hind limbs was dragged on the ground (red footprint pixel value / red footprint pixel value+blue footprint pixel value), step length (the distance between the two footprints on the same side) and step width (the distance between the left and right footprints) were measured; Motor evoked potential: a needle stimulation electrode was implanted 2mm in front of the coronal suture and 2mm beside the sagittal suture (cerebral cortical motor area), with a stimulation intensity of 40mA, a stimulation wave width of 0.1ms, a stimulation frequency of 1Hz, a superposition number of 300-500 times, a scanning speed of 5ms / D, a sensitivity of 5mV / D, and the latency and amplitude were recorded.

[0087] (7) Spinal cord injury detection: The spinal cord injury area was measured using small animal magnetic resonance imaging. The spinal cord was fixed with 4% paraformaldehyde and T was taken. 11 ~L2 spinal cord tissue was embedded in paraffin and sectioned, and the number of normal neurons in the anterior horn, intermediate zone, and posterior horn of the spinal cord was counted using Nissl staining.

[0088] (8) ELISA detection of M1 and M2 microglial cell marker levels: Total protein was extracted from the supernatant of spinal cord homogenate and quantitatively analyzed in an ELISA instrument using the corresponding ELISA kit.

[0089] Example 1

[0090] This example provides the design and synthesis of GELN-tFNA nanocomposites. Figure 6 , specifically including the following steps.

[0091] 1. Design of tFNA.

[0092] (1) tFNA backbone: Based on the tetrahedral inscribed sphere model and the principle of base complementary pairing, four DNA single strands were designed, and the 5' ends (sticky ends) of the S1 strand, S2 strand, and S4 strand were located at the A vertex, the AD edge midpoint, and the BC edge midpoint, respectively. Specifically, the nucleotide sequences of the S1 strand, S2 strand, S3 strand, and S4 strand are shown in SEQ ID NOs: 1 to 4, respectively.

[0093] (2) DNA robot (three-finger robot): Two cholesterol-modified (all bases have cholesterol modification) DNA single strands with palindromic sequences are folded into a "three-finger robot" (robot arm 1 and robot arm 2), each connected to the midpoint of the skew edges (AD edge and BC edge); a pH-responsive DNA clamp composed of poly A complementary double strand and TGC repeat sequence is designed and coupled to the three "finger tips" of the "three-finger robot" on both sides. Specifically, the nucleotide sequences of robot arm 1 and robot arm 2 are shown in SEQ ID NOs: 5-6.

[0094] (3) “Navigator”: The TMEM119 aptamer was screened using the SELEX technique and connected to the 5′ end (sticky end) of the S1 chain at the A vertex.

[0095] The SELEX technology screening process includes: ①TMEM119 protein expression and purification: construct plasmid pET-32a-TMEM119 to transform DH5α Escherichia coli competent cells, take a single colony that has been successfully transformed and inoculate LB medium to induce TMEM119 protein expression, and then purify TMEM119 protein by affinity chromatography column. ②ssDNA initial library construction: design an 81bp oligonucleotide ssDNA library with 18bp fixed sequences at both ends and 45bp random sequences in the middle, and design upstream and downstream primers. ③ssDNA initial library screening: TMB magnetic beads are incubated with TMEM119 solution at 4℃ overnight to obtain TMB-TMEM119 complex, which is then mixed with 1OD of ssDNA, incubated at 4℃ for 2h, separated by magnetic stand to obtain TMB-TMEM119-ssDNA complex, eluted ssDNA on the TMB-TMEM119-ssDNA complex with TE buffer at 95℃ water bath for 15min, and amplified by PCR to obtain ssDNA secondary library. ④ ssDNA secondary library screening: Perform 10 rounds of repeated screening according to the steps described in ③. When the screening reaches the 4th round, perform a counter-screening, that is, incubate the screened ssDNA library with TMB magnetic beads without TMEM119 for 2 hours, remove the magnetic beads through the magnetic frame, and obtain ssDNA that is not bound to TMB magnetic beads. At the end of each round of screening, the ELISA method is used to determine the binding ability of the screened ssDNA with TMEM119, and a counter-screening is performed every 4 rounds. ⑤ Cloning and sequencing: After the 10th round of screening, the obtained ssDNA is amplified by PCR and transfected into DH5α competent cells. Single colonies with good growth conditions are selected by agarose gel electrophoresis for positive clones of about 230bp, and DNA sequencing is performed.

[0096] The nucleotide sequence of the screened TMEM119 aptamer is shown in SEQ ID NO:7.

[0097] The nucleotide sequences of the tFNA backbone (S1 chain to S4 chain), robotic arm 1, robotic arm 2, and TMEM119 aptamer are shown in Table 1 , and the schematic diagram is shown in Figures 1 to 5 .

[0098] Table 1 Sequence information

[0099]

[0100] 2. Synthesis of tFNA.

[0101] The obtained S1-S4 chains were mixed with TAE-Mg 2+ The mixture was mixed in a buffer solution, heated at 95°C for 5 min, and then slowly cooled to room temperature to synthesize the tFNA backbone.

[0102] Synthesize robot 1, robot 2, and TMEM119 aptamers using DNA synthesis technology.

[0103] The tFNA backbone, robotic arm 1, robotic arm 2, and TMEM119 aptamer were mixed at a molar ratio of 1:1:1:1 and incubated with E. coli DNA ligase at 37°C for 6 h to obtain tFNA.

[0104] 3. Isolate and purify GELN.

[0105] Ginger was crushed into juice, and the precipitate was removed by centrifugation at low speed (3000g, 20min) and high speed (10000g, 40min). The supernatant was added with 10% by volume polyethylene glycol 6000 and incubated overnight. The precipitate was collected by centrifugation at low speed (3000g, 20min) to obtain GELN.

[0106] 4. Investigation of the synthesis efficiency and characteristics of GELN-tFNA nanocomplexes.

[0107] (1) Synthesis of GELN-tFNA nanocomplex: tFNA and isolated and purified GELN were incubated at 37° C. for 6 h in an equimolar ratio to synthesize the GELN-tFNA nanocomplex.

[0108] Determination of synthesis efficiency: pre-modify the front and middle bases of robot arm 1 with Cy3 fluorescent groups, and pre-modify the front and middle bases of robot arm 2 with Cy5 fluorescent groups; when the two robot arms surround and fix GELN in the internal space of tFNA, the front and middle sections of the two robot arms will approach each other. According to the principle of fluorescence resonance energy transfer, when Cy3 is excited, part or all of the energy will be transferred to Cy5 to induce the emission of red fluorescence. The synthesis efficiency is calculated as Cy5 red fluorescence intensity / (Cy3 green fluorescence intensity+Cy5 red fluorescence intensity).

[0109] (2) Characterization: The particle size of the GELN-tFNA nanocomposite was analyzed using a particle size analyzer, and its appearance was observed using a transmission electron microscope.

[0110] Analysis of the particle size of the GELN-tFNA nanocomplex: GELN-tFNA was mixed with PBS to prepare a 100 nM suspension, 50 μl of the suspension was added to a cuvette, and measured in a laser particle size analyzer.

[0111] Observe the appearance of GELN-tFNA nanocomplex: aspirate 10 μl GELN-tFNA suspension and drop it on the copper mesh, aspirate 1% glutaraldehyde solution and drop it on the copper mesh to fix for 5 minutes, then wash and add 10 μl uranyl acetate to stain in the dark for 30 minutes, wash the copper mesh again, and observe it under a transmission electron microscope after drying.

[0112] (3) Stability study: GELN-tFNA was placed in acidic PBS (pH = 5.0) and its stability in an acidic environment was studied by agarose gel electrophoresis to observe the stability of DNA molecular weight at 6, 12, 24 h and 3, 5, and 7 days. The method was as follows: TAE-Mg was added to agarose 2+ 1% agarose gel was prepared by dissolving in buffer solution, heated in a microwave oven until clear and transparent, cooled to 60°C and introduced into a gel casting apparatus. After solidification for 30 minutes, the sample was loaded. The sample concentration was 300 nM and separated at a voltage of 100 V for 2 hours for staining and imaging.

[0113] (4) Biocompatibility study: GELN-tFNA was injected into rats via the tail vein. Serum myocardial enzymes (CK, CK-MB), liver function (ALT, AST, TBil, DBil), renal function (BUN, Cr) and arterial blood gas (pH, PaO2, PaCO2) indicators were measured at 24 hours, confirming its good biocompatibility.

[0114] Example 2

[0115] This embodiment provides a GELN-tFNA nanocomplex and a synthesis method thereof, which is substantially the same as that of Embodiment 1, except that the number of DNA rings of the DNA tetrahedral framework is different, and the number of DNA rings of both robot arm 1 and robot arm 2 is 5 (five-finger robot arm).

[0116] Example 3

[0117] This embodiment provides a GELN-tFNA nanocomplex and a synthesis method thereof, which are substantially the same as those of Embodiment 1, except that the number of DNA rings of the DNA tetrahedral framework is different, and the number of DNA rings of both robotic arm 1 and robotic arm 2 is 0.

[0118] Example 4

[0119] This embodiment provides a GELN-tFNA nanocomplex and a synthesis method thereof, which is substantially the same as that of Embodiment 1, except that the DNA tetrahedral framework of this embodiment does not have a DNA mechanical arm.

[0120] Example 5

[0121] This example provides a GELN-tFNA nanocomplex and a synthesis method thereof, which is substantially the same as Example 1, except that the DNA tetrahedron framework of this example does not have a DNA robotic arm and a TMEM119 aptamer.

[0122] The schematic diagrams of the DNA tetrahedral frameworks of Examples 1 to 5 can be found in Figure 7 .

[0123] Example 6

[0124] The synthesis efficiency and characteristics of the GELN-tFNA nanocomposites synthesized in Examples 1 to 5 were investigated.

[0125] (1) Determination of synthesis efficiency: The front and middle bases of robot arm 1 are modified with Cy3 fluorescent groups, and the front and middle bases of robot arm 2 are modified with Cy5 fluorescent groups. When the two robot arms surround and fix GELN in the internal space of tFNA, the front and middle sections of the two robot arms will approach each other. According to the principle of fluorescence resonance energy transfer, when Cy3 is excited, part or all of the energy will be transferred to Cy5, inducing the emission of red fluorescence. The synthesis efficiency is calculated as Cy5 red fluorescence intensity / (Cy3 green fluorescence intensity+Cy5 red fluorescence intensity).

[0126] The results are shown in the following table.

[0127] Table 2 Synthesis efficiency

[0128]

[0129] The results confirmed that Examples 1 to 3 can efficiently synthesize GELN-tFNA nanocomposites.

[0130] (2) Characterization: The particle size of the GELN-tFNA nanocomposite was analyzed using a particle size analyzer, and its appearance was observed using a transmission electron microscope.

[0131] Analysis of the particle size of the GELN-tFNA nanocomplex: GELN-tFNA was mixed with PBS to prepare a 100 nM suspension, 50 μl of the suspension was added to a cuvette, and measured in a laser particle size analyzer.

[0132] Observe the appearance of GELN-tFNA nanocomplex: aspirate 10 μl GELN-tFNA suspension and drop it on the copper mesh, aspirate 1% glutaraldehyde solution and drop it on the copper mesh to fix for 5 minutes, then wash and add 10 μl uranyl acetate to stain in the dark for 30 minutes, wash the copper mesh again, and observe it under a transmission electron microscope after drying.

[0133] The results confirmed that the particle sizes of the GELN-tFNA nanocomposites synthesized in Examples 1 to 5 fluctuated between 160 and 200 nm, and all of them were regular tetrahedrons in appearance.

[0134] (3) Stability study: GELN-tFNA was placed in acidic PBS (pH = 5.0), and agarose gel electrophoresis was used to observe the changes in DNA molecular weight at 6, 12, 24 hours and 3, 5, and 7 days: TAE-Mg was added to agarose 2+ 1% agarose gel was prepared by dissolving in buffer solution, heated in a microwave oven until clear and transparent, cooled to 60°C and introduced into a gel casting apparatus. After solidification for 30 minutes, the sample was loaded. The sample concentration was 300 nM and separated at a voltage of 100 V for 2 hours for staining and imaging.

[0135] The results confirmed that the DNA molecular weight of the GELN-tFNA nanocomplexes synthesized in Examples 1 to 5 remained constant at 6, 12, 24 h and 3, 5, 7 d, confirming their stability in an acidic environment.

[0136] (4) Biocompatibility study: GELN-tFNA was injected into rats via the tail vein, and serum myocardial enzymes (CK, CK-MB), liver function (ALT, AST, TBil, DBil), renal function (BUN, Cr) and arterial blood gas (pH, PaO2, PaCO2) indicators were measured at 24 hours.

[0137] The results are as follows.

[0138] Table 3 Serum myocardial enzymes, liver function, renal function and arterial blood gas indexes

[0139]

[0140]

[0141] The results showed that the above indicators were all within the normal range, confirming its good biocompatibility.

[0142] Example 7: pH-responsive controlled release function of GELN-tFNA nanocomplex.

[0143] The same dose of GELN and tFNA-GELN nanocomplexes (Examples 1 to 5) were placed in acidic PBS (pH=5.0), and the cumulative release amounts of 6-gingerol, ath-miR159a and ath-miR319a at 6, 12, 24 h and 3, 5, 7 d were calculated.

[0144] The results are shown in the following table.

[0145] Table 4 Cumulative release of 6-gingerol, ath-miR159a and ath-miR319a

[0146]

[0147]

[0148] The results confirmed that the GELN-tFNA nanocomplex synthesized in Examples 1 and 2 was a pH-responsive slow-release drug.

[0149] Example 8: GELN-tFNA nanocomplex targets spinal cord injury and microglia function.

[0150] The lipid membrane of GELN and GELN-tFNA nanocomplexes (Examples 1 to 5) was labeled with DiR lipophilic red fluorescent probe. The same dose of GELN and GELN-tFNA was injected into the tail vein of rats 0.5 h after spinal cord injury. In vivo fluorescence imaging and immunofluorescence staining were used to observe the aggregation of GELN and GELN-tFNA in the spinal cord injury and microglia at 6 h. The levels of 6-gingerol, ath-miR159a and ath-miR319a in the supernatant of spinal cord homogenate were measured at 6, 12, 24 h and 3, 5, and 7 d.

[0151] The results are shown in the following table.

[0152] Table 5 GELN and GELN-tFNA nanocomplexes in spinal cord injury and microglial aggregation and release of 6-gingerol, ath-miR159a and ath-miR319a

[0153]

[0154]

[0155] The results confirmed that the GELN-tFNA nanocomplexes synthesized in Examples 1 to 3 can target spinal cord injury and microglia, while Examples 1 to 2 can continuously release active ingredients.

[0156] Example 9: The advantage of GELN-tFNA nanocomplex in promoting microglial M2 polarization in protecting spinal cord neurons.

[0157] The rats were injected with the same dose of GELN and GELN-tFNA nanocomplexes (Examples 1 to 5) via the tail vein at 0.5 h after spinal cord injury, and the BBB motor function scores and the angles of the inclined board test were measured at 6, 12, 24 h and 3, 5, and 7 d; the gait and motor evoked potentials were analyzed at 7 d, the spinal cord injury area was measured by small animal magnetic resonance imaging, the number of normal neurons in the anterior horn, intermediate zone and posterior horn of the spinal cord was counted by Nissl staining, and the expression levels of M1 microglial markers iNOS, CD86, TNF-α, IL-1β, IL-6 and M2 microglial markers Arg-1, CD206, IL-10, IL-4, and TGF-β were determined by ELISA.

[0158] The results are as follows.

[0159] Table 6 BBB motor function score, inclined board test angle, gait and motor evoked potential

[0160]

[0161]

[0162] Table 7 Spinal cord injury area and number of normal neurons

[0163]

[0164] Table 8 M1 and M2 microglial cell markers

[0165]

[0166] The results show that the GELN-tFNA nanocomplex has the advantage of promoting microglial M2 polarization to protect spinal neurons. Its mechanism of action can be referred to Figure 6 .

[0167] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A DNA tetrahedral framework, characterized in that: The DNA tetrahedral framework includes four DNA single strands: S1 strand, S2 strand, S3 strand and S4 strand; The nucleotide sequences of the S1 to S4 chains are at least 80% identical to the sequences shown in SEQ ID NOs: 1 to 4, respectively.

2. The DNA tetrahedral framework according to claim 1, characterized in that The DNA tetrahedron framework further includes any one or more of a TMEM119 aptamer and a DNA mechanical arm for capturing a target substance loaded on the DNA tetrahedron framework.

3. The DNA tetrahedral framework according to claim 2, characterized in that: The DNA robotic arm is a single-stranded DNA; Optionally, more than 50% of the bases in the sequence of the DNA robot have cholesterol modification; Optionally, the nucleotide sequence of the DNA robot may form a palindrome; Optionally, the nucleotide sequence of the DNA robot contains one or more DNA loops, and the DNA loops include (TGC)n, where n is a positive integer selected from 1 to 10; Optionally, the nucleotide sequence of the DNA robot contains at least one DNA buckle, the DNA buckle includes polyA and poly T, and the DNA buckle and the DNA ring together form a DNA buckle ring; the structure of the DNA buckle ring includes: poly A-DNA ring-poly T; the sequence lengths of the poly A and the poly T are 3 to 10 nt; Optionally, the DNA buckle further comprises a first palindromic sequence and a second palindromic sequence capable of forming a palindromic structure with the first palindromic sequence; the first palindromic sequence and the second palindromic sequence are respectively located on a side of the poly A and the poly T away from the DNA loop; Optionally, the DNA robot comprises any one or more of robot arm 1 and robot arm 2; the nucleotide sequences of robot arm 1 and robot arm 2 are at least 80% identical to the sequences shown in SEQ ID NOs: 5 to 6, and more than 50% of the bases on the basis of the sequences have cholesterol modification; Optionally, the robot arm 1 is connected to any one or more of the S1 chain to the S4 chain; the robot arm 2 is connected to any one or more of the S1 chain to the S4 chain; Optionally, the robot arm 1 is connected to the 5' end of any one or more of the S1 to S4 chains, and the 5' end of the DNA single strand connected to the robot arm 1 is a sticky end; the robot arm 2 is connected to the 5' end of any one or more of the S1 to S4 chains, and the 5' end of the DNA single strand connected to the robot arm 2 is a sticky end; Optionally, the DNA single strand connected to the robot arm 1 and the DNA single strand connected to the robot arm 2 are skew edges; Optionally, the sticky end of the DNA single strand connected to the robot arm 1 and the sticky end of the DNA single strand connected to the robot arm 2 are respectively located in the central region of the skew edge, and the central region is a region extending 10 to 15 nt from the midpoint of the edge to both ends; Optionally, the 5' end of the DNA single strand connected to the robot arm 1 and the 5' end of the DNA single strand connected to the robot arm 2 are the midpoints of the skew edges. Optionally, the TMEM119 aptamer is connected to any one or more of the S1 to S4 chains; Optionally, the TMEM119 aptamer is located at the vertices of the DNA tetrahedral framework; Optionally, the TMEM119 aptamer is connected to the 5' end of any one or more of the S1 to S4 chains, and the 5' end of the DNA single strand connected to the TMEM119 aptamer is a sticky end; Optionally, the nucleotide sequence of the TMEM119 aptamer has at least 80% identity with the sequence shown in SEQ ID NO: 7; Optionally, the nucleotide sequence of the TMEM119 aptamer is shown in SEQ ID NO:

7.

4. The method for preparing a DNA tetrahedral framework according to any one of claims 1 to 3, characterized in that: It includes the following steps: Mixing the S1 to S4 chains described in claim 1 in a buffer; Optionally, the preparation method further comprises: heating the mixed product and then cooling it to room temperature to obtain a DNA tetrahedral framework skeleton; Optionally, the mixing molar ratio of S1 chain to S4 chain is 1-3:1-3:1-3:1-3; Optionally, the heating conditions include: 85-105° C., 1-20 min; Optionally, when the DNA tetrahedral framework further comprises the TMEM119 aptamer described in claim 2 or 3 and / or the DNA robotic arm described in claim 2 or 3, the preparation method further comprises: mixing the DNA tetrahedral framework skeleton with the TMEM119 aptamer and / or the DNA robotic arm, and adding DNA ligase to obtain the final DNA tetrahedral framework; Optionally, the mixing molar ratio of the DNA tetrahedral framework skeleton, the TMEM119 aptamer and the DNA robotic arm is 1-3:1-3:1-6.

5. Use of the DNA tetrahedral framework according to any one of claims 1 to 3 or the DNA tetrahedral framework prepared by the preparation method according to claim 4 in the preparation of drugs; Optionally, the drug includes: Drugs used to prevent, treat or assist in the treatment of spinal cord injuries.

6. A nanocomposite, characterized in that: The invention comprises: ginger-derived exosome-like nanoparticles and the DNA tetrahedron framework described in any one of claims 1 to 3 or the DNA tetrahedron framework prepared by the preparation method described in claim 4.

7. The nanocomposite according to claim 6, characterized in that The molar ratio of the DNA tetrahedral framework to the ginger-derived exosome-like nanoparticles is 1-3:1-3.

8. The method for preparing a nanocomposite according to claim 6 or 7, characterized in that: It includes the following steps: Mixing ginger-derived exosome-like nanoparticles with the DNA tetrahedral framework described in any one of claims 1 to 3 or the DNA tetrahedral framework prepared by the preparation method described in claim 4; Optionally, the mixing conditions include: 30-45° C., 3-9 h; Optionally, the preparation method further comprises the preparation of ginger-derived exosome-like nanoparticles; Optionally, the ginger juice is subjected to a first centrifugation and a second centrifugation to remove the precipitate to obtain a supernatant; the supernatant is mixed with a polyethylene glycol solution and incubated, the incubated product is subjected to a third centrifugation, and the precipitate is collected to obtain ginger-derived exosome-like nanoparticles; Optionally, the conditions of the first centrifugation are: 2000-4000g, 10-30min; Optionally, the conditions for the second centrifugation are: 8000-12000 g, 30-50 min; Optionally, the conditions for the third centrifugation are: 2000-4000g, 10-30min; Optionally, the volume percentage of polyethylene glycol in the polyethylene glycol solution is 5% to 20%; Optionally, the molecular weight of the polyethylene glycol is 5000-7000.

9. Use of the nanocomposite according to claim 6 or 7 or the nanocomposite prepared by the preparation method according to claim 8 in preparing a product for preventing, treating or assisting in treating spinal cord injury.

10. A product for preventing, treating or assisting in the treatment of spinal cord injury, characterized in that: The active ingredient comprises the nanocomposite according to claim 6 or 7 or the nanocomposite prepared by the preparation method according to claim 8.

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