Tetrahedral framework nucleic acid as well as preparation method and application thereof

Abstract

The invention relates to the field of gene therapy, in particular to tetrahedral framework nucleic acid as well as a preparation method and application thereof. And one of four oligonucleotide chains of the tetrahedral framework nucleic acid comprises the sequence of the miR-26b-5p, so that the stable bearing of the miR-26b-5p is realized, and the miR-26b-5p can be completely and stably transferred into a target cell. After miR-26b-5p enters a human body, the degradation of cartilage is inhibited by adjusting the expression condition of the marker, so that the inhibition and improvement of osteoarthritis are realized.

Description

Technical Field

[0001] The present invention relates to the field of gene therapy, and in particular to a tetrahedral framework nucleic acid and a preparation method and application thereof. Background Art

[0002] There are a variety of existing treatment options for osteoarthritis, but each has its limitations. Surgery is currently the most effective treatment, with total joint replacement being highly effective and capable of completely resolving joint problems. However, it carries risks, high costs, and postoperative complications. Injections, such as sodium hyaluronate injections, can lubricate joints and reduce friction, but their effectiveness is short-lived, and some patients respond poorly. This may be due to individual differences in drug response and the irreversible nature of disease progression. Physical therapies, such as heat therapy, acupuncture, and rehabilitation training, can alleviate symptoms and strengthen muscles to a certain extent, but they often fail to improve joint damage. Their effectiveness is limited and depends on patient compliance. Medication is currently the most common treatment option. Nonsteroidal anti-inflammatory drugs (NSAIDs) can reduce pain and inflammation, but they may cause side effects such as gastrointestinal discomfort and cardiovascular risks. This is because these drugs, while suppressing inflammation, can also affect normal tissues. Chondroitin sulfate and other cartilage protectants have uncertain effects and a slow onset of action. This may be due to the complex nature of cartilage metabolism and the difficulty of drugs effectively reaching cartilage tissue.

[0003] To improve treatment efficacy and reduce the potential health risks associated with treatment, it is necessary to develop more effective medications. However, the pathogenesis of osteoarthritis remains unclear, making it difficult to fundamentally curb the onset of disease and reduce damage. Furthermore, significant individual variability among patients makes it difficult to uniformly evaluate the therapeutic effects of related drugs during clinical trials, complicating the optimization of treatment plans.

[0004] Based on the above problems, those skilled in the art have considered developing a biological agent that can inhibit and improve the body damage caused by osteoarthritis by regulating the expression of related influencing factors. Summary of the Invention

[0005] The purpose of the present invention is to provide a tetrahedral framework nucleic acid for treating osteoarthritis, and a preparation method and application thereof.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] A tetrahedral framework nucleic acid comprises a first chain, a second chain, a third chain and a fourth chain interconnected by self-assembly, wherein the first chain, the second chain, the third chain and the fourth chain are all oligonucleotide chains, and the sequence of the fourth chain includes the sequence of miR-26b-5p.

[0008] Optionally, the tetrahedral framework nucleic acid further comprises WYRGRL modified on the surface.

[0009] Optionally, the sequence of the first chain is SEQ ID NO: 1;

[0010] The sequence of the second chain is SEQ ID NO: 2;

[0011] The sequence of the third chain is SEQ ID NO: 3;

[0012] The sequence of the fourth chain is SEQ ID NO: 4.

[0013] In a second aspect, the present invention further provides a method for preparing the above-mentioned tetrahedral framework nucleic acid, comprising:

[0014] Obtain the first chain, the second chain, the third chain, and the fourth chain;

[0015] The first chain, the second chain, the third chain and the fourth chain are dissolved in a buffer solution and mixed, heated to a first temperature and annealed for a first time, and then quickly cooled to a second temperature and maintained for a second time to self-assemble into a tetrahedral structure.

[0016] Optionally, the preparation method further comprises:

[0017] The self-assembled tetrahedral structure is mixed with WYRGRL, and the mixture is allowed to stand to allow the WYRGRL to be adsorbed on the surface of the tetrahedral structure, thereby obtaining the tetrahedral framework nucleic acid.

[0018] Optionally, the molar ratio of the first chain, the second chain, the third chain and the fourth chain is (0.9-1.1): (0.9-1.1): (0.9-1.1);

[0019] The first temperature is any value between 90°C and 95°C;

[0020] The first time is any value between 5 minutes and 10 minutes;

[0021] The second temperature is any value between 2°C and 6°C;

[0022] The second time is any value between 20 minutes and 60 minutes.

[0023] In a third aspect, the present invention also provides the use of the above-mentioned tetrahedral framework nucleic acid in the preparation of anti-osteoarthritis drugs.

[0024] Optionally, the drug is administered by injection into the affected area.

[0025] Optionally, the tetrahedral framework nucleic acid is used to improve osteophyte formation, cartilage degradation and bone microstructure damage caused by osteoarthritis.

[0026] Optionally, the tetrahedral framework nucleic acid enhances the expression of cartilage matrix markers SOX9 and COL 2, and inhibits the expression of cartilage degradation marker MMP13.

[0027] The beneficial effects of the present invention are: by binding miR-26b-5p to the mRNA of its target gene, it helps to regulate the immune response, fight inflammation and promote cartilage repair. By using tetrahedral framework nucleic acids (tFNAs) as carriers of miRNAs, it helps to stably transfer structurally unstable single-stranded miRNA into the body and transfect it into cells, thereby exerting its biological effects. Because tFNAs have high stability, good biocompatibility and adjustable physicochemical properties, they help to improve the delivery efficiency of miR-26b-5p and prevent miR-26b-5p from being rapidly degraded by nucleases, thereby improving its therapeutic effect. The shape and structure of tFNAs can be adjusted through synthetic methods and surface modification, and have high development potential, so they are more practical.

[0028] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a flow chart of the method for preparing a tetrahedral framework nucleic acid as shown in Example 1 of the present invention;

[0030] Figure 2 Schematic diagram of the preparation process of the tetrahedral framework nucleic acid shown in Example 1 of the present invention;

[0031] Figure 3 1 is a micro-computed tomography image of the knee joint samples of each group of mice in Example 1 of the present invention;

[0032] Figure 4 Statistical graphs of microstructural parameters of knee joint samples of mice in each group in Example 1 of the present invention;

[0033] Figure 5 This is a tissue staining image of the knee joint samples of each group of mice in Example 1 of the present invention;

[0034] Figure 6 These are immunohistochemical staining images of the knee joint samples of each group of mice in Example 1 of the present invention;

[0035] Figure 7 This is an analysis chart of the expression of multiple mRNAs in the knee joint samples of each group of mice in Example 1 of the present invention. DETAILED DESCRIPTION

[0036] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0037] In the description of the present invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings and are intended solely to facilitate and simplify the description of the present invention. They are not intended to indicate or imply that the devices or components referred to must have, be constructed, or operate in a specific orientation, and therefore should not be construed as limitations on the present invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0038] In the description of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "mounted," "connected," and "connected" should be understood in a broad sense. For example, they may refer to fixed, detachable, or integral connections; mechanical or electrical connections; direct or indirect connections through an intermediate medium; and internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on the specific circumstances.

[0039] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0040] Macrophages are an important component of the immune system and are generally divided into two subtypes: M1 macrophages and M2 macrophages. M1 macrophages primarily participate in proinflammatory responses, secreting large amounts of proinflammatory cytokines such as TNF-α, IL-1β, and IL-6, and play a crucial role in infection, tumor progression, and acute inflammation. Unlike M1 macrophages, M2 macrophages typically exhibit anti-inflammatory properties and participate in tissue repair and reparative immune responses. By secreting anti-inflammatory factors such as IL-10 and TGF-β, they promote tissue repair, reduce immune responses, and contribute to the resolution of inflammation. M2 macrophages play a crucial role in the recovery process of chronic diseases and trauma by regulating the immune environment.

[0041] While studying the mechanism of osteoarthritis, the inventors found that miR-26b-5p was enriched in M2 macrophage-derived exosomes compared with M1 macrophage-derived exosomes, and the expression of miR-26b-5p was significantly different, suggesting that miR-26b-5p may play an important role in regulating immune response, anti-inflammation, and cartilage repair.

[0042] In response to the above inference, the inventors conducted verification experiments and studied its mechanism.

[0043] The present invention applies to protect a tetrahedral framework nucleic acid, comprising a first chain, a second chain, a third chain and a fourth chain interconnected by self-assembly, wherein the first chain, the second chain, the third chain and the fourth chain are all oligonucleotide chains, and the sequence of the fourth chain includes the sequence of miR-26b-5p.

[0044] By binding miR-26b-5p to the mRNA of its target genes, it helps regulate immune responses, combat inflammation, and promote cartilage repair. Using tetrahedral framework nucleic acids (tFNAs) as carriers for miR-26b-5p, tFNAs containing miR-26b-5p, known as tFNAs-miR-26b-5p, facilitate the stable delivery of the unstable single-stranded miR-26b-5p into the body and transfection into cells, thereby exerting its biological effects. Due to their high stability, good biocompatibility, and tunable physicochemical properties, tFNAs help improve the delivery efficiency of miR-26b-5p and protect it from rapid degradation by nucleases, thereby enhancing its therapeutic efficacy. The shape and structure of tFNAs can be regulated through synthetic methods and surface modification, offering high development potential and therefore high practicality.

[0045] In some embodiments, the tetrahedral framework nucleic acid further comprises a WYRGRL modified on the surface.

[0046] WYRGRL is a cartilage-targeting peptide sequence that imparts targeting properties to the tetrahedral framework nucleic acid, thereby enhancing the efficiency of miR-26b-5p transfection into chondrocytes and, consequently, improving therapeutic efficacy. WYRGRL has a high affinity for tFNAs-miR-26b-5p and binds to the surface of tFNAs-miR-26b-5p simply through electrostatic adsorption to form WYRGRL-tFNAs-miR-26b-5p (WT@26). This allows for a low-cost preparation without affecting the structure and biological effects of miR-26b-5p.

[0047] In some embodiments, the sequence of the first strand is SEQ ID NO: 1;

[0048] The sequence of the second chain is SEQ ID NO: 2;

[0049] The sequence of the third strand is SEQ ID NO: 3;

[0050] The sequence of the fourth chain is SEQ ID NO:4.

[0051] Through sequence design, miR-26b-5p is carried in tFNAs, making tFNAs easy to synthesize and able to effectively retain the original structure and biological effects of miR-26b-5p.

[0052] Second, see Figure 1 The present invention also provides a method for preparing the above-mentioned tetrahedral framework nucleic acid, comprising:

[0053] S1. Obtain the first chain, the second chain, the third chain, and the fourth chain.

[0054] S2. Dissolve the first chain, the second chain, the third chain and the fourth chain in a buffer solution, mix them, heat them to a first temperature and anneal them for a first time, then quickly cool them to a second temperature and maintain them for a second time to self-assemble into a tetrahedral structure.

[0055] The synthesis of tFNAs by direct heating annealing method helps to improve the preparation efficiency and the reaction conditions are easy to control.

[0056] In some embodiments, see Figure 2 , the preparation method further comprises:

[0057] S3. Mixing the self-assembled tetrahedral structure with WYRGRL, and allowing the WYRGRL to be adsorbed on the surface of the tetrahedral structure to obtain a tetrahedral framework nucleic acid.

[0058] Through simple mixing, WYRGRL is modified on the surface of tFNAs by electrostatic adsorption, which is highly efficient and simple.

[0059] In some embodiments, the molar ratio of the first chain, the second chain, the third chain, and the fourth chain is (0.9-1.1): (0.9-1.1): (0.9-1.1), for example, any one of (1:1:1:1), (0.9:1:1.1:1), (1:1.1:1:0.95), (1.1:0.95:0.9:1.05), and (1:0.9:0.9:0.9);

[0060] The first temperature is any value between 90°C and 95°C, for example, any value between 90°C, 91°C, 92°C, 93°C, 94°C and 95°C;

[0061] The first time is any value between 5 minutes and 10 minutes, for example, it can be any value between 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes and 10 minutes;

[0062] The second temperature is any value between 2°C and 6°C, for example, any value between 2°C, 3°C, 4°C, 5°C and 6°C;

[0063] The second time period is any value between 20 minutes and 60 minutes, for example, it may be any value between 20 minutes, 30 minutes, 40 minutes, 50 minutes and 60 minutes.

[0064] In a third aspect, the present invention also provides the use of the above-mentioned tetrahedral framework nucleic acid in the preparation of anti-osteoarthritis drugs.

[0065] In some embodiments, the drug is administered by injection into the affected area.

[0066] In some embodiments, tetrahedral framework nucleic acids are used to improve osteophyte formation, cartilage degradation, and bone microarchitecture damage caused by osteoarthritis.

[0067] In some embodiments, the tetrahedral framework nucleic acid enhances the expression of the cartilage matrix markers SOX9 and COL 2, and inhibits the expression of the cartilage degradation marker MMP13.

[0068] Please refer to the following examples for details.

[0069] Example 1:

[0070] See Figure 1 The method for preparing a tetrahedral framework nucleic acid as shown in a preferred embodiment of the present application includes:

[0071] S1. Obtain the first chain, the second chain, the third chain, and the fourth chain.

[0072] S2. Dissolve the first chain, the second chain, the third chain and the fourth chain in a buffer solution, mix them, heat them to a first temperature and anneal them for a first time, then quickly cool them to a second temperature and maintain them for a second time to self-assemble into a tetrahedral structure.

[0073] In step S1, the sequences of the first, second, third, and fourth strands were designed based on the sequence of miR-26b-5p, i.e., SEQ ID NO:5. The first strand sequence is SEQ ID NO:1, the second strand sequence is SEQ ID NO:2, the third strand sequence is SEQ ID NO:3, and the fourth strand sequence is SEQ ID NO:4, including the sequence of miR-26b-5p. In this example, the first, second, third, and fourth strands were custom-made at Sangon Biotechnology, China. The specific sequences are shown in Table 1 below.

[0074] Table 1:

[0075]

[0076] Among them, the first chain, the second chain and the third chain are all DNA chains, and the fourth chain is a hybrid chain formed by inserting the miR-26b-5p chain segment into the DNA chain, which contains both "U" and "T" bases.

[0077] In step S2, the buffer solution includes 10 mmol / L tris-HCl and 50 mmol / L MgCl2, and the pH is adjusted to 8.0. 100 μL of the buffer solution is added to a 200 μL centrifuge tube, and equimolar amounts of the first, second, third, and fourth chains are added and dissolved. In this embodiment, the first temperature is 95°C, the first time is 10 minutes, and the second temperature is 4°C, and the second time is 20 minutes to obtain tFNAs-miR-26b-5p.

[0078] Store part of tFNAs-miR-26b-5p at 4°C and process part of tFNAs-miR-26b-5p through step S3. Figure 2 , step S3 operates as follows.

[0079] S3. Mixing the self-assembled tetrahedral structure with WYRGRL, and allowing the WYRGRL to be adsorbed on the surface of the tetrahedral structure to obtain a tetrahedral framework nucleic acid.

[0080] In step S3, WYRGRL was purchased from Sangon Biotechnology, China, mixed with tFNAs-miR-26b-5p and allowed to stand at room temperature for 30 min to obtain the tetrahedral framework nucleic acid WT@26.

[0081] According to the sequence of miR-26b-5p, it was customized at Sangon Biotechnology, China to obtain miR-26b-5p. After deleting the sequence of miR-26b-5p in the fourth chain, step S2 was performed to obtain tFNA.

[0082] The miR-26b-5p, tFNA, tFNAs-miR-26b-5p, and WT@26 were dissolved in the above buffer solution to form a pharmaceutical composition. The therapeutic effect of the pharmaceutical composition obtained in this example on osteoarthritis was verified by animal experiments.

[0083] Take 24 healthy C57BL / J6 mice, all of which are male, weighing between 19 and 22 g, aged 8 to 10 weeks, and all are specific pathogen-free (SPF) grade experimental mice. The experimental mice in this example were provided by the Animal Experiment Center of Soochow University. The feeding conditions were 6 per cage, room temperature of 18 to 20 ° C, humidity of 50 to 60%, good ventilation, and free access to food and water. All procedures in this example were carried out in accordance with the Animal Care Committee and the Guide for the Care and Use of Animals of the National Institutes of Health.

[0084] The experimental mice were randomly divided into 4 groups: Ctrl, PBS, miR, tFNA, TM, and WT@26. The mice in the PBS, miR, tFNA, TM, and WT@26 groups were induced to form osteoarthritis models. The modeling methods included:

[0085] After fasting for 12 hours, the experimental mice were anesthetized by intraperitoneal injection of 10% chloral hydrate. The injection volume was calculated based on the weight of the mice at 500 mg / kg. The mice were fixed on the operating table in a supine position to ensure that the knee joint was completely exposed, and the right knee joint area of ​​the anesthetized mice was disinfected with 75% alcohol. A longitudinal incision of about 1 cm was made in the skin in front of the right knee joint, and the subcutaneous tissue and fascia were separated until the knee joint was completely exposed. The anterior cruciate ligament (ACL) was then carefully separated and transected. After hemostasis, the fascia and skin were sutured in sequence with absorbable sutures, and the sutures were disinfected again.

[0086] In the Ctrl group, the skin was incised and the knee joint was exposed, and then the anterior cruciate ligament was sutured directly without transection.

[0087] After surgery, mice in each group were placed in a warm, dry environment for recovery and given appropriate analgesics and antibiotics. Eight volumes of phosphate-buffered saline (PBS) solution were injected into the knee joints of mice in the PBS group. The same volume of miR-26b-5p at a concentration of 250 nmol / L was injected into the same area of ​​the miR group, the same volume of tFNA at a concentration of 250 nmol / L was injected into the same area of ​​the tFNA group, the same volume of tFNAs-miR-26b-5p at a concentration of 250 nmol / L was injected into the same area of ​​the TM group, and the same volume of WT@26 at a concentration of 250 nmol / L was injected into the same area of ​​the WT@26 group. The drugs were administered once weekly for four weeks. After that, each mouse was anesthetized with an intraperitoneal injection of 10% chloral hydrate. The knee joints and surrounding tissues were quickly removed for specimen preparation. After sampling, the mice were sacrificed.

[0088] All animals in each group regained consciousness within 30 to 60 minutes after surgery, moving freely within their cages and eating normally, with no significant changes in their mental state. There was no inflammatory reaction, including redness, swelling, or exudate, and all wounds healed by primary intent. No animals died during the experiment.

[0089] One knee joint sample from each mouse was fixed in 4% paraformaldehyde for 24 hours and then scanned with micro-computed tomography (μCT). The 2D and 3D reconstruction images of the axial plane scan (AP) and lateral plane scan (LAT) as well as the subchondral bone images are shown in the table. Figure 3 . The scanning parameters were: resolution 18 μm, voltage 80 kV; current 100 μA; exposure time 100 ms each time; 0.9° / 8 images. The Wedemeyer C method (see Wedemeyer C, et al. Particle-induced osteolysis in three-dimensional micro-computed tomography. Calcif Tissue Int. 2007; 81(5): 394-402.) was used to select a cylindrical region of interest (ROI). The diameter of the ROI was 3 mm and the height was 1 mm. Micro-CT image analysis software was used to perform 3D analysis on the images and record the bone volume (Bone volume, BV, mm) of the ROI area. 3 ), combined with the total volume of the ROI area (total bone volume, TV, mm 3 ) was used to calculate the bone volume fraction (the trabecular bone volume to total bone volume ratio, BV / TV,%), and record Tb.Th (trabecular thickness) and Tb.Sp (trabecular space) in the ROI area. The statistical results are shown in Figure 3 .

[0090] See Figure 3 and Figure 4 , it can be seen that the PBS group mice had significant osteophyte formation in the joints compared with the Ctrl group mice, accompanied by severe cartilage degradation and bone microstructure damage. This included decreased BV / TV and Tb.Th, and increased Tb.Sp. After treatment with miR-26b-5p, WYRGRL, tFNA-miR-26b-5p and WT@26, the BV / TV of each group of samples was effectively improved, and the WT@26 group had the most obvious treatment effect, while Tb.Th was only effectively improved in the TM group and the WT@26 group, indicating that the pharmaceutical composition in this embodiment exhibited an excellent joint structure protective effect.

[0091] After μCT testing, each sample was decalcified with 10% ethylenediaminetetraacetic acid (EDTA) for 3 weeks, embedded in paraffin, and then sectioned for histological examination.

[0092] The paraffin sections were stained with hematoxylin-eosin (HE) and Safranin O-Fast Green (SO / FG). The staining methods are known in the art and will not be described in detail here.

[0093] The morphology of the knee joints of mice in each group was observed under a light microscope, and the degree of synovial inflammation was assessed by examining the thickening of the synovial layer, cell infiltration, and vascular dilation. Figure 5 ,The results of HE staining and SO / FG staining showed that the cartilage was severely lost and the matrix degradation was obvious in the PBS group, while the cartilage integrity was maintained after WT@26 treatment.

[0094] The paraffin sections of each group were subjected to immunohistochemical staining to detect the levels of SOX9, COL 2, ACAN and MMP13 in the samples.

[0095] Paraffin sections were soaked in xylene for 5 minutes, repeated twice, and then soaked in anhydrous ethanol for 2 minutes, 95% ethanol for 1 minute, 80% ethanol for 1 minute, and 75% ethanol for 1 minute. The sections were then rinsed in distilled water for 2 minutes. After rinsing, the sections were incubated in 3% H2O2 at room temperature for 5-10 minutes to eliminate endogenous peroxidase activity. The sections were then rinsed in distilled water and soaked in phosphate-buffered saline (PBS) for approximately 5 minutes to obtain pretreated samples.

[0096] Place the pretreated sample in a container filled with citrate buffer and heat in a microwave oven to maintain the liquid temperature between 92°C and 98°C for 10-15 minutes. Remove from the container and cool at room temperature for 10-20 minutes before performing antigen retrieval. Rinse with PBS to obtain the repaired sample.

[0097] Nonspecific binding sites on the repaired samples were blocked with 5% to 10% normal goat serum diluted in PBS and incubated at room temperature for 10 minutes. Primary antibodies containing affinity-purified rabbit antibodies against SOX9, COL 2, and MMP13 were added at a working titer of 1:400. After incubation at 37°C for 1 hour, the samples were rinsed three times with PBS for 5 minutes each. HRP-conjugated IG antibody, a secondary antibody, was then added at a working titer of 1:500. The samples were incubated in a humidified chamber at room temperature for 60 minutes. After incubation, the samples were developed with diaminobenzidine (DAB) as a colorimetric reagent. After staining at room temperature for 5 to 30 minutes, the samples were rinsed with distilled water and dehydrated with ethanol and xylene until transparent. Neutral resin was added to the coverslips, and the labeled samples were mounted.

[0098] Select 5 consecutive labeled samples, observe them under a 20x light microscope, and count the positive cells with brown-yellow granules in the cytoplasm. Figure 6 It can be seen that the levels of SOX9, COL 2, ACAN and MMP13 in the PBS group decreased significantly compared with the Ctrl group, while the levels increased significantly after treatment with WT@26. The transcription factor SOX9 plays an important regulatory role in the development of chondrocytes and matrix synthesis. The decreased expression of SOX9 may lead to chondrocyte matrix degradation and cartilage degeneration. COL 2 (type II collagen) is the main component of the cartilage matrix and is essential for maintaining the elasticity and toughness of cartilage. A decrease in COL 2 content usually indicates cartilage tissue damage or degeneration. ACAN (aggregant proteoglycan) is another important component of the cartilage matrix that helps maintain the compressive resistance and smoothness of cartilage. A decrease in ACAN content may mean cartilage degeneration. MMP13 (matrix metalloproteinase 13) is a key collagenase responsible for degrading type II collagen. An increase in MMP13 content usually marks the degradation process of the cartilage matrix, which may be caused by factors such as inflammation or mechanical stress. The changes in the content of the above factors can prove that WT@26 has a therapeutic effect on cartilage damage and inflammatory damage in the joints.

[0099] In this example, the expression of SOX9, COL2, AGC and MMP13 was also detected by quantitative RT-PCR.

[0100] Immediately after killing the mice, the knee joints were removed under sterile conditions, soft tissue removed, and cryopreserved in liquid nitrogen. Approximately 100 mg of the sample was placed in a liquid nitrogen-chilled mortar and ground into a powder under liquid nitrogen. The powder was transferred to a 15 ml centrifuge tube and 8 ml of TRIzol (a cell lysing agent, nuclease inhibitor, and ion protectant) was added. The tubes were homogenized using a high-speed tissue homogenizer for 5 minutes and allowed to stand at room temperature for 30 minutes. After addition of 1.6 ml of chloroform, the tubes were shaken vigorously for 15 seconds and allowed to stand at room temperature for approximately 5 minutes. The tubes were centrifuged at 12,000 g / min at 4°C for 15 minutes. The supernatant was aspirated and 4.0 ml of isopropanol was added. The tubes were gently inverted to mix five times and allowed to stand at room temperature for 10 minutes. Centrifuge at 12,000 g / min for 10 minutes at 4°C, discard the supernatant, and add 3 ml of 75% ethanol to the pellet, mix thoroughly, and wash once. Centrifuge again at 10,000 g / min for 10 minutes, and discard the supernatant. The resulting pellet represents the total RNA extracted from the sample. Invert the pellet to air dry, then dissolve it in 200 μl of diethylpyrocarbonate (DEPC)-treated water and transfer it to a 1.5 ml centrifuge tube. Measure the OD260 / 280, identify it by electrophoresis, and store it at -80°C.

[0101] Purchase a reverse transcription kit from Promega and perform reverse transcription according to the instructions.

[0102] The cDNA obtained by reverse transcription was subjected to fluorescent real-time quantitative PCR. The reaction system included 2.0 μl of cDNA, 0.5 μl of upstream primer at a concentration of 10 μmol / L, 0.5 μl of downstream primer at a concentration of 10 μmol / L, 0.5 μl of ROX Reference Dye, 12.5 μl of SYBR Premix Ex Taq, and deionized water to a total volume of 25 μl. The reaction system was added to a real-time fluorescent quantitative PCR (qPCR) instrument, denatured at 95°C for 10 minutes, and amplified for 40 cycles. In each cycle, the primers for SOX9, COL 2, AGC, and MMP13 were denatured at 94°C for 15 seconds and annealed at 60°C for 1 minute. The primers for SOX9, COL 2, AGC, and MMP13 in this example were purchased from Sangon Biotechnology, China. Four sets of primers were used to amplify β-actin, SOX9, COL 2, AGC, and MMP13 in cDNA, respectively.

[0103] Set the detection parameters of the qPCR instrument. The fluorescence background signal is set to the fluorescence value of the first 12 cycles, and the threshold is set to 10 times the standard deviation of the fluorescence signal from the 4th to the 12th cycle. Using β-actin as the internal reference gene, calibrate the Ct value of each sample, that is, the number of cycles experienced when the fluorescence signal in each reaction tube reaches the set threshold, and compare the expression differences of each gene in different samples based on the Ct value. Each pair of primers is repeated 3 times in each template. The average Ct value of each target gene is subtracted from the average Ct value of the internal reference gene of the corresponding template to obtain ΔCt, and calculate 2 -ΔCt As the average relative content of each target gene in cDNA, a higher value indicates a higher expression level of the target gene in the template.

[0104] The results were analyzed using SPSS 11.0 statistical software and the data were expressed as mean ± standard deviation. For comparisons of multiple groups, one-way ANOVA was used. For pairwise comparisons, LSD and Dunnett-t methods were used under the condition of homogeneity of the overall variance. Differences were considered statistically significant when p < 0.05, marked with ns, and the number of "*" marks the degree of significance of the difference. Figure 7, it can be seen that after joint degeneration, the expression of multiple mRNAs in the knee joint changes, including downregulation of SOX9, COL 2, and AGC, and upregulation of MMP13. After treatment, these changes were improved. In particular, after treatment with WT@26, the mRNA expression of SOX9, COL 2, and MMP13 was no longer significantly different from that of the Ctrl group, which did not undergo anterior cruciate ligament transection. AGC mRNA expression was also significantly improved.

[0105] Combined with the results of the above-mentioned validation experiments, it can be seen that miR-26b-5p has the effect of improving osteoarthritis. Using tFNA as a carrier of miR-26b-5p can improve the stability of miR-26b-5p in the body and transfected into cells, thereby improving the therapeutic effect. Attaching WYRGRL on the surface can improve the efficiency of miR-26b-5p transfection into chondrocytes by incubating cartilage targeting, thereby improving the therapeutic effect, and the modification method is simple. In other words, the tetrahedral framework nucleic acid applied for protection in the present invention has a significant therapeutic effect on osteoarthritis and is easy to modify and optimize.

[0106] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0107] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A tetrahedral framework nucleic acid, characterized in that The invention comprises a first chain, a second chain, a third chain and a fourth chain connected to each other by self-assembly, wherein the first chain, the second chain, the third chain and the fourth chain are all oligonucleotide chains, and the sequence of the fourth chain includes the sequence of miR-26b-5p.

2. The tetrahedral framework nucleic acid according to claim 1, wherein Also included are surface-modified WYRGRLs.

3. The tetrahedral framework nucleic acid according to claim 1, wherein The sequence of the first strand is SEQ ID NO: 1; The sequence of the second chain is SEQ ID NO: 2; The sequence of the third chain is SEQ ID NO: 3; The sequence of the fourth chain is SEQ ID NO:

4.

4. The method for preparing a tetrahedral framework nucleic acid according to any one of claims 1 to 3, wherein: include: Obtain the first chain, the second chain, the third chain, and the fourth chain; The first chain, the second chain, the third chain and the fourth chain are dissolved in a buffer solution and mixed, heated to a first temperature and annealed for a first time, and then quickly cooled to a second temperature and maintained for a second time to self-assemble into a tetrahedral structure.

5. The preparation method according to claim 4, wherein Also includes: The self-assembled tetrahedral structure is mixed with WYRGRL, and the mixture is allowed to stand to allow the WYRGRL to be adsorbed on the surface of the tetrahedral structure, thereby obtaining the tetrahedral framework nucleic acid.

6. The preparation method according to claim 4, wherein The molar ratio of the first chain, the second chain, the third chain and the fourth chain is (0.9-1.1): (0.9-1.1): (0.9-1.1); The first temperature is any value between 90°C and 95°C; The first time is any value between 5 minutes and 10 minutes; The second temperature is any value between 2°C and 6°C; The second time is any value between 20 minutes and 60 minutes.

7. Use of the tetrahedral framework nucleic acid according to any one of claims 1 to 3 in the preparation of anti-osteoarthritis drugs.

8. The use according to claim 7, characterized in that The drug is administered by injection into the affected area.

9. The use according to claim 7, characterized in that The tetrahedral framework nucleic acid is used to improve osteophyte formation, cartilage degradation and bone microstructure damage caused by osteoarthritis.

10. The use according to claim 7, characterized in that The tetrahedral framework nucleic acid enhances the expression of cartilage matrix markers SOX9 and COL 2, and inhibits the expression of cartilage degradation marker MMP13.

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