Tetrahedral framework nucleic acid carrying small molecule drug and preparation method and application of tetrahedral framework nucleic acid

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a tetrahedral framework nucleic acid carrying a small molecule drug as well as a preparation method and application of the tetrahedral framework nucleic acid. According to the invention, good cell entry performance, editability, anti-inflammatory and anti-oxidation capability of tetrahedral framework nucleic acid and anti-inflammatory and osteogenesis promoting effects of a JAK inhibitor are combined, and the JAK inhibitor Upatinib (UPA) is successfully loaded onto the tetrahedral framework nucleic acid (tFNA) to obtain a tFNA / UPA compound (TUC); the TUC can significantly inhibit inflammatory reaction by inhibiting a JAK / STAT pathway on the premise of reducing the drug dosage, enhances a WNT signal pathway, promotes an osteogenesis function, more effectively treats rheumatoid arthritis, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a tetrahedral framework nucleic acid carrying a small molecule drug, a preparation method thereof and uses thereof. Background Art

[0002] At present, the first-line drug for the treatment of rheumatoid arthritis (RA) is methotrexate, but only about 30% of monotherapy has achieved the effect of reducing disease activity. Although non-steroidal anti-inflammatory drugs (NSAIDs) can relieve pain and stiffness and improve physical function, they do not interfere with joint damage and thus do not change the disease. Glucocorticoids have rapid symptomatic and disease-improving effects, but have serious long-term side effects. Disease-modifying antirheumatic drugs (DMARDs) can target inflammation. As a targeted synthetic DMARD, JAK inhibitors can target the downstream JAK signal transduction pathway involved in RA, and thus have faster efficacy than other therapeutic drugs. At the same time, compared with most biosynthetic DMARDs, the first-generation Jaknib tofacitinib has better clinical efficacy than methotrexate. Most importantly, Jakinibs show the possibility of effectively controlling inflammation while maintaining stable immune function. So far, jakinibs including tofacitinib, baricitinib and UPA have been successively approved by the FDA for the treatment of RA patients. The second-generation JAK inhibitor upadacitinib (UPA) has higher targeting than the first-generation JAK inhibitor and thus fewer side effects. The third-generation targeted synthetic Disease-modifying antirheumatic drugs (DMARDs) of Jakinib can achieve the dual effects of anti-inflammatory and promoting osteogenesis by inhibiting the JAK / STAT pathway, and are emerging personalized therapeutic drugs for RA patients at present, but their bioavailability is low, and they are accompanied by serious liver and kidney damage and other side effects.

[0003] As a representative of functional nucleic acids, Tetrahedral framework nucleic acids (tFNAs) are an efficient drug delivery carrier. It is composed of four specific base sequences and has a unique three-dimensional structure synthesized through an annealing process. It has good cell entry performance, editability, anti-inflammatory and antioxidant properties, and is an ideal drug carrier.

[0004] Due to the poor solubility and short half-life of upadacitinib (UPA), the bioavailability of UPA is severely hindered. The in vivo administration dose of UPA is usually very high, which is extremely likely to cause side effects such as liver and kidney damage and immunosuppression, and at the same time brings a greater economic burden to patients. Therefore, there is an urgent need to develop a drug delivery system to increase the bioavailability of the drug, reduce systemic side effects, and avoid frequent drug administration, so as to treat rheumatoid arthritis more safely and effectively. Summary of the Invention

[0005] In view of the problems of the prior art, the present invention provides a tetrahedral framework nucleic acid carrying a small molecule drug, and its preparation method and use.

[0006] A functionalized tetrahedral framework nucleic acid, wherein the functionalized tetrahedral framework nucleic acid is modified with a JAK inhibitor.

[0007] Preferably, it is assembled by embedding a JAK inhibitor into the helical groove on the surface of the DNA double helix of the tetrahedral framework nucleic acid; the tetrahedral framework nucleic acid is formed by base complementary pairing of 4 single-stranded DNAs.

[0008] Preferably, the nucleotide sequences of the 4 single-stranded DNAs are as shown in SEQ ID NO.1-SEQ ID NO.4.

[0009] Preferably, the JAK inhibitor is selected from upadacitinib.

[0010] The present invention also provides a preparation method of the above functionalized tetrahedral framework nucleic acid, comprising the following steps:

[0011] Step 1: Mix 4 single-stranded DNAs, incubate, and anneal to obtain a tetrahedral framework nucleic acid;

[0012] Step 2: Mix the tetrahedral framework nucleic acid with a JAK inhibitor solution, and incubate with shaking to obtain the product.

[0013] Preferably, in Step 2, the concentration ratio of the tetrahedral framework nucleic acid to the JAK inhibitor solution is 1:(10-60);

[0014] And / or, the temperature of the incubation with shaking is 20-25°C, and the time is 5-7h.

[0015] The present invention also provides the use of the above functionalized tetrahedral framework nucleic acid in a drug for treating rheumatoid arthritis.

[0016] Preferably, the drug is used to inhibit the inflammatory response.

[0017] Preferably, the drug is used to promote osteogenic differentiation.

[0018] The present invention also provides a drug for treating rheumatoid arthritis, which is prepared by using the above-mentioned functionalized tetrahedral framework nucleic acid as an active ingredient and adding pharmaceutically acceptable excipients.

[0019] The DNA double helix is formed by two anti-parallel polynucleotide chains winding around each other to form a right-handed double helix structure. The bases of the DNA double helix are located inside the double helix, while the phosphates and sugar groups are on the outside, connected by phosphodiester bonds to form the backbone of the nucleic acid. Among them, the DNA double helix refers to the outer surface of the DNA double strand, which is composed of phosphate groups and deoxyribose.

[0020] The helical groove refers to the concave part on the surface of the DNA double helix structure, which is divided into the major groove and the minor groove. The wider groove is called the major groove, and the narrower groove is called the minor groove. Both the major groove and the minor groove are caused by base pair stacking and the twist of the sugar-phosphate backbone.

[0021] The present invention successfully loaded the JAK inhibitor upadacitinib (UPA) onto the tetrahedral framework nucleic acid (tFNA) to obtain the tFNA / UPA complex (TUC). TUC has good biocompatibility, stability, and endocytosis performance. While significantly reducing the drug dosage, TUC can inhibit the inflammatory response by inhibiting the JAK-STAT pathway and enhance the osteogenic differentiation by promoting the WNT signaling pathway, providing a new approach for the clinical treatment of rheumatoid arthritis.

[0022] Obviously, based on the above content of the present invention, according to the common general technical knowledge and customary means in the art, without departing from the above basic technical idea of the present invention, various other forms of modifications, substitutions, or changes can be made.

[0023] The following is a further detailed description of the above content of the present invention through specific embodiments in the form of examples. However, this should not be understood as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. Description of the Drawings

[0024] Figure 1For the synthesis, characteristics, and cellular uptake of TUC. (A) Schematic illustration of the fabrication of TUC. (B) The bands became lighter with the increasing TDN:UPA ratio. (C) Successful synthesis of TDN and TUC was confirmed by PAGE. (D) Successful synthesis of TDN and TUC was confirmed by high-performance capillary electrophoresis. (E) Chemical structure of UPA. (F, G) Zeta potential and hydrodynamic size of UPA, TDN, and TUC measured by DLS. (H) Encapsulation efficiency of UPA on TDN at different ratios of TDN to UPA (TDN:UPA = 1:10 - 1:50). (J) TEM image showing that TUC has a triangular structure (scale bars: 100 and 50 nm). (j) AFM image showing the successful synthesis of TUC (scale bars: 50 nm and 50 nm). (K - L) Serum stability and quantitative analysis of TUC at 5% and 10% FBS, 37 °C.

[0025] Figure 2 For TUC to be taken up by BMSCs through the JAK / STAT pathway and reduce LPS-induced inflammation. (A) Immunofluorescence images of BMSCs cells taking up Cy5-labeled TDN and TUC (Cy5: red; cytoskeleton: green; nucleus: blue) (scale bar: 50 μm). (B) Statistical analysis of the immunofluorescence results. (C) Cell viability results of BMSCs treated with different concentrations of TUC. (D, E) Immunofluorescence images of the expression of JAK2 and STAT1 in BMSCs after different treatments for 48 h. (JAK2, STAT1: red; cytoskeleton: green; nucleus: blue; 3D thermal imaging: reconstruction of the fluorescence intensity of JAK2 and STAT1) (scale bar: 200 μm). (F) Western blot detection of the expression of STAT1, P-STAT1, STAT3, P-STAT3, JAK2, and P-JAK2. (G - K) Quantitative analysis of the Western blot results.

[0026] Figure 3 For TUC to promote osteogenesis of LPS-treated BMSCs through the WNT pathway. (A) Osteogenic differentiation of BMSCs was observed by ALP staining on day 7. (B) Quantitative analysis of the ALP staining results. (C) Formation of calcium nodules was monitored by alizarin red S staining on day 14. (D) Quantitative analysis of the alizarin red S staining results. (E) Western blot detection of the expression results of WNT1 and SOCS2. (F) Quantitative analysis of the expression levels of the above proteins. (G, H) Immunofluorescence images of the expression of WNT1 and SOCS2 in BMSCs after different treatments for 7 days. (WNT1 and SOCS2: red; cytoskeleton: green; nucleus: blue; 3D thermal imaging: fluorescence reconstruction).

[0027] Figure 4TUC can alleviate the clinical symptoms of the RA rat model. (A) Schematic diagram of the method and time points of in vivo drug injection (the RA model is RA rats). (B) General conditions of RA rats after treatment with CON, RA, TDN, UPA, and TUC (n = 5). (C) Quantitative analysis of the hind paw thickness after treatment with CON, RA, TDN, UPA, and TUC (n = 5). (D) Images of gait experiments. (E, F) Quantitative analysis of the average heel swing speed (milliseconds) and average hind paw area (square centimeters). (G, H) Quantitative analysis detected by Elisa. (I) TUNEL staining of BMSCs in different groups. (TUNEL: green; three-dimensional thermal imaging: reconstruction of TUNEL fluorescence intensity) (scale bar: 200 μm). (J) Quantitative analysis of the levels of serum neutrophils, lymphocytes, and white blood cells.

[0028] Figure 5 TUC can restore bone loss in the RA rat model. (A, B) Micro-CT images of joint sections of different treatment groups. (C - F) Statistical analysis of BV / TV, Tb.N, Tb.Sp, and Tb.Th under the ROI according to the micro-CT results (n = 5). (G, H) Quantitative analysis of immunofluorescence results. (I, J) Immunofluorescence images of JAK2 / STAT1 and TNF-α expression (STAT1 and TNF-α: red; JAK2: green; cell nucleus: blue) (scale bar: 200 μm).

[0029] Figure 6 TUC has a bone protection effect in vivo. (A) Images of the ankle stained with Goldner, TB, safranine O, and Masson (scale bar: 200 μm). (B) Bone area ratio. (C) Quantitative analysis of CVF in different treatment groups. (D, E) IHC images of WNT1 and STAT3 expression (scale bars: 200 μm and 100 μm). (F, G) Quantitative analysis of IHC images. Detailed implementation methods

[0030] In the following examples and experimental examples, the reagents and raw materials not specifically described are all commercially available products.

[0031] Example 1 Preparation of tetrahedral framework nucleic acid (TUC) modified with JAK inhibitor upadacitinib In this example, the JAK inhibitor upadacitinib (UPA) was successfully loaded onto the tetrahedral framework nucleic acid (tFNA) to obtain the tFNA / UPA complex (TUC); specifically, first, 4 pre-designed single-stranded DNAs (S1, S2, S3, S4) were synthesized into the tetrahedral framework nucleic acid through base complementary pairing, and then upadacitinib was embedded in the helical groove on the surface of the DNA double helix of the tetrahedral framework nucleic acid and assembled.

[0032] Among them, the nucleotide sequence of single-stranded DNA S1 is shown in SEQ ID NO.1, the nucleotide sequence of single-stranded DNA S2 is shown in SEQ ID NO.2, the nucleotide sequence of single-stranded DNA S3 is shown in SEQ ID NO.3, and the nucleotide sequence of single-stranded DNA S4 is shown in SEQ ID NO.4. The specific nucleotide sequences are shown in Table 1.

[0033] Table 1

[0034]

[0035] The structural formula of upadacitinib is:

[0036] The preparation method is as follows: Add equimolar amounts of four single-stranded DNAs (S1, S2, S3, S4) to TM buffer (10 mM Tris base, 50 mM MgCl2, pH 8.0). After vortexing and centrifuging, heat to 95 °C for 10 minutes in a thermal cycler, and then cool to 4 °C for 20 minutes. Self-assemble according to the base complementary pairing principle to obtain tetrahedral framework nucleic acid (tFNA); Incubate the UPA solution (10 μM) with the tetrahedral framework nucleic acid at room temperature (25 °C) with shaking for 6 h to allow UPA to embed into the helical groove of the DNA double strand of the tetrahedral framework nucleic acid, where the concentration ratio of tFNA:UPA is 1:40, so that UPA is successfully loaded onto tFNA to prepare TUC( Figure 1 A).

[0037] This comparative example provides the control samples used in the experiment.

[0038] Comparative Example 1 Tetrahedral framework nucleic acid (tFNA)

[0039] This sample is assembled from 4 pre-designed single-stranded DNAs (S1, S2, S3, S4) based on base complementarity. Its preparation method is the same as that of tFNA in Example 1. The sequences of single-stranded DNAs (S1, S2, S3, S4) are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4 respectively.

[0040] The technical solution of the present invention will be further described below through experiments.

[0041] Experimental Example 1 Characterization of the TUC Structure

[0042] This experimental example characterizes the TUC structure prepared in Example 1.

[0043] I. Experimental Method

[0044] Agarose gel electrophoresis: Observe the fluorescent bands in the gel imaging system and analyze the binding of tFNA and UPA at different ratios.

[0045] Polyacrylamide gel electrophoresis (PAGE): Prove the successful synthesis of tFNA and TUC.

[0046] Capillary electrophoresis: Detect the molecular weight, particle size, and potential of TUC and tFNA.

[0047] Microscopic characterization: Use atomic force microscopy (AFM) and transmission electron microscopy (TEM) to detect the morphology of tFNA and TUC.

[0048] II. Experimental Results

[0049] The results of agarose gel electrophoresis showed that as the ratio of tFNA:UPA increased continuously (the concentration ratios of tFNA:UPA were 1:10, 1:20, 1:40, 1:60) ( Figure 1 B), the bands gradually became lighter until they disappeared. When the ratio of tFNA to UPA was 1:40, the bands completely disappeared, indicating that the encapsulation efficiency reached the highest at this time, and UPA completely occupied the original binding position of Gel-red and tFNA.

[0050] Polyacrylamide gel electrophoresis (PAGE) proved the successful synthesis of tFNA and TUC ( Figure 1 C). At the same time, the results of capillary electrophoresis showed that the molecular weight of TUC was slightly larger than that of tFNA ( Figure 1 D). Upadacitinib is a small molecule monomer drug ( Figure 1 E) with a molecular weight of 380.37. In addition, the particle sizes of tFNA and TUC were 8.72 nm and 15.69 nm respectively, and the potentials were -9.29 and -6.26 mV respectively ( Figure 1 F, G). UPA itself is positively charged, so in addition to the physical embedding effect between UPA and tFNA, the electrostatic attraction between positive and negative charges also plays a very important role. TUC has a larger particle size than tFNA, which once again proves the successful loading of UPA. To investigate the maximum efficiency of tFNA to load UPA, the encapsulation efficiency of 250 nM tFNA at different UPA concentrations (10 - 60 μM) was measured ( Figure 1 H). As the UPA concentration increased continuously, the encapsulation efficiency first increased and then decreased. Therefore, the ratio of UPA to tFNA of 1:40 was selected as the drug concentration for subsequent cell experiments and animal experiments. In addition, AFM and TEM showed the morphology of TUC ( Figure 1 I, J). Because UPA is embedded between the DNA double strands, TUC is not triangular like tFNA, but more blunt, which proves the successful synthesis of TUC.

[0051] Experimental Example 2: Stability and Cellular Uptake Performance of TUC

[0052] I. Experimental Methods

[0053] Agarose gel electrophoresis: To verify the stability of TUC in the cell culture environment.

[0054] Flow cytometry and confocal microscopy: To observe the uptake of Cy5-labeled TUC and tFNA by LPS-induced BMSCs.

[0055] II. Experimental Results

[0056] Agarose gel electrophoresis verified that TUC showed good stability in both 5% and 10% FBS ( Figure 1 K, L), especially within 6 h, further demonstrating the structural stability of TUC, with good biocompatibility and stability.

[0057] The uptake of Cy5-labeled TUC and tFNA (TDN group) by LPS-induced BMSCs was observed by flow cytometry and confocal microscopy ( Figure 2 A). The results showed that both Cy5-labeled tFNA and TUC were widely distributed in the cytoplasm, and the percentage of cells with detectable fluorescence in both the TUC group and the TDN group showed good cellular uptake efficiency ( Figure 2 B), and there was no statistical difference between tFNA and TUC. The above results indicate that TUC has good biocompatibility, structural stability, and cellular uptake performance, and the loading of UPA does not affect the cellular uptake ability of tFNA.

[0058] Experimental Example 3: TUC Inhibits LPS-Induced Inflammation by Inhibiting the JAK-STAT Pathway

[0059] I. Experimental Methods

[0060] Cell culture: Bone marrow mesenchymal stem cells (BMSCs), with the culture medium consisting of DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin.

[0061] Cell model: A BMSC model induced by LPS (1 μg / ml) was used to simulate the situation in the joint cavity of RA patients. Experimental groups: (1) Control group; (2) LPS group (LPS 1 μg / ml); (3) TDN group (LPS 1 μg / ml + tFNA 250 nM): also known as the "tFNA group"; (4) UPA group (LPS 1 μg / ml + UPA 10 μM); (5) TUC group (LPS 1 μg / ml + TUC).

[0062] CCK-8: Detect the effect of tFNA loaded with different concentrations of UPA on the cell viability of LPS-induced BMSCs.

[0063] ALP detection: Observe the effect of tFNA loaded with different concentrations of UPA on the osteogenic differentiation of LPS-induced BMSCs.

[0064] Western blotting (WB): Detect the expression of JAK- and STAT-related proteins.

[0065] II. Experimental Results

[0066] Previous experiments have demonstrated that the optimal concentration of tetrahedral framework nucleic acid is 250 nM. Six different ratios from low to high were set according to the ratio of 1:40 between tFNA and UPA ((TDN / UPA ratio of 1:10, 1:20, 1:40, 1:60)) Figure 2 C) CCK-8 assay was performed, and the results showed that cell viability of LPS-induced BMSCs could be promoted at all concentrations. Meanwhile, for the effect of TUC on the osteogenic differentiation of LPS-induced BMSCs, the same six concentration gradients were set, and the differences in the osteogenic differentiation of LPS-induced BMSCs were observed by ALP. Finally, the experimental results of CCK-8 and ALP both showed that the optimal concentration of UPA was 10 μM (tFNA = 250 nM), which was used for subsequent in vivo and in vitro experiments.

[0067] Janus family kinases (JAKs), namely JAK1, JAK2, JAK3, and TYK2, are non-receptor protein tyrosine kinases. The abnormal activation of the JAK / STAT signaling pathway through JAK mutations or constitutive TYK2 signaling has been proven to be the key to inducing abnormal development of hematopoietic stem cells, blood malignancies, autoimmunity, and certain immunodeficiency syndromes. At the same time, various pro-inflammatory cytokines such as IL-6, TNF-α, IL-1β, etc. all exert their effects through the JAK / STAT pathway. To verify the anti-inflammatory ability of TUC, LPS was used to induce BMSCs to simulate the situation in the inflamed joints of RA patients. First, all six concentration gradients of TUC could promote cell viability Figure 2 C), demonstrating that TUC has good biocompatibility. As the concentration of TUC increased continuously, cell viability increased continuously. Especially when the concentration of TUC was 10 μM, the cell viability was the best. Subsequently, to verify the effect of TUC on the JAK / STAT signaling pathway, LPS-induced BMSCs were treated with tFNA, UPA, and TUC respectively. Under the action of LPS, the expression of JAK2- and STAT1-related inflammatory factors in BMSCs was significantly increased Figure 2D, E), the results of WB also showed that tFNA (TDN group) could only slightly reduce the expression of JAK and STAT related proteins, while both the UPA and TUC groups showed good effects in inhibiting the expression of related proteins, and the inhibitory effect of the TUC group was better ( Figure 2 F - G). In addition, the expressions of P - STAT3, P - JAK2, and P - STAT1 were significantly inhibited compared with those of STAT3, JAK2, and STAT1 (Figure 2H - K), and the JAK / STAT pathway was effectively inhibited. The results indicated that tFNA was an ideal drug carrier, which could significantly reduce the drug dosage. At the same time, TUC could inhibit the LPS - induced inflammatory response by inhibiting the JAK - STAT pathway.

[0068] Experimental Example 4 TUC promotes the osteogenic differentiation of BMSCs by enhancing the WNT signaling pathway

[0069] I. Experimental methods

[0070] 1. Experimental grouping:

[0071] (1) Control group; (2) LPS group (LPS 1 μg / ml); (3) TDN group (LPS 1 μg / ml + tFNA 250 nM): also known as the "tFNA group"; (4) UPA group (LPS 1 μg / ml + UPA 10 μM); (5) TUC group (LPS 1 μg / ml + TUC 250 nM).

[0072] 2. ALP staining

[0073] 3. Alizarin red S staining: To evaluate the ability of cells to differentiate into osteoblasts.

[0074] 4. Western blotting (WB): To detect the expression of WNT - related pathway proteins.

[0075] 5. Immunofluorescence: To detect the expression of WNT signaling pathway - related proteins.

[0076] II. Experimental results

[0077] One of the important clinical features of RA patients is severe bone loss. The JAK / STAT pathway can be induced by different inflammatory cytokines in osteoblasts. To compare the osteogenic ability of TUC and UPA under inflammatory conditions, ALP staining was used to verify the osteogenic effects of the five groups. The results showed that the TDN group had a certain anti - inflammatory effect, so its osteogenic ability was slightly better than that of the LPS group. The UPA group and the TUC group showed better osteogenic effects compared with the LPS group ( Figure 3 A, B). The results of Alizarin red S staining were consistent with those of ALP staining ( Figure 3C, D), the calcium nodules in the tFNA group were slightly more than those in the LPS group, while the calcium nodules in the UPA group and the TUC group were significantly more than those in the LPS group, and there was no significant statistical difference between the UPA group and the TUC group. The Western blotting results showed ( Figure 3 E, F), the UPA group and the TUC group could effectively promote the expression of WNT-related pathway proteins, thus promoting the osteogenesis of BMSCs. Suppressor of cytokine signaling 2 (SOCS2), as a negative regulator of β-catenin, was significantly inhibited in the UPA and TUC groups. Immunofluorescence (Figure 3G, H) once again confirmed that TUC could effectively promote the expression of WNT signaling pathway-related proteins, thus promoting the osteogenic ability of BMSCs under inflammatory conditions. The above results indicate that TUC promotes the osteogenic differentiation of BMSCs by enhancing the WNT signaling pathway.

[0078] Experimental Example 5 Anti-inflammatory and bone protection effects of TUC in in vivo experiments

[0079] I. Experimental methods

[0080] 1. Animal model: Select 6-8-week-old SD rats (Jiangsu Jicui Yakang Biotechnology Co., Ltd., 6 rats in each group, 30 rats in total). After completely mixing Bovine typeⅡ (chondex, 5 ml, 2 mg / ml) and incomplete Freund's adjuvant (chondex, 5 ml) using an emulsifier, the SD rats were immunized for the first time (inserted parallel to the tail 2 cm from the bottom of the tail until the tip of the needle was 0.5 cm from the bottom, and the whole needle should be subcutaneous, injecting 0.2 ml subcutaneously at the bottom of the tail) and the second immunization (injecting 0.1 ml subcutaneously at the tail) on the first day and the seventh day respectively. After 19 days, a collagen-induced arthritis model (CIA model) was successfully established ( Figure 4 A).

[0081] 2. Experimental grouping: The rats were randomly divided into the following 5 groups:

[0082] control group: SD rats, without any treatment.

[0083] CIA group: CIA rats with successful modeling;

[0084] TDN group: Also known as the "tFNA group"; injecting 100 μL, 250 nM tFNA via the tail vein;

[0085] UPA group: Injecting 2.5 μL, 10 μM UPA via the tail vein;

[0086] TUC group: Injecting 100 μL TUC via the tail vein.

[0087] Record the paw pad thickness and clinical index of each group every day. Administer drugs via the tail vein injection every other day. Sacrifice all CIA rats on the 31st day, and collect the heart, liver, spleen, lungs, kidneys, joint lavage fluid, blood, and the right ankle joint.

[0088] 3. Elisa assay

[0089] 4. micro-CT scan: Evaluate the trabecular bone parameters of the ankle joint.

[0090] 5. Immunofluorescence

[0091] 6. Goldner trichrome staining: Reflect the expression level of new bone.

[0092] 7. Toluidine Blue (TB) staining: Stain the cartilage tissue.

[0093] 8. Safranine O staining: Stain the cartilage and bone tissues.

[0094] 9. Masson staining: Observe the fibrosis process and the distribution of collagen fibers.

[0095] II. Experimental results

[0096] In the inflamed joints of RA patients, the joints often show swelling due to the infiltration of various inflammatory factors. To verify the anti-inflammatory and bone-protective effects of TUC in vivo, a CIA rat model was established to simulate the in vivo situation of RA patients. After 2 weeks of treatment, both macroscopically and in terms of the measured values of paw pad thickness, the TUC and UPA groups showed significant improvement compared with the CIA group ( Figure 4 B, C), and at the same time, the therapeutic effect of the drugs on CIA rats was further verified from the behavioral aspect ( Figure 4 D). The swing speed of the TUC group and the UPA group was significantly increased compared with the CIA group ( Figure 4 E), and the paw area was significantly increased ( Figure 4 F), indicating that TUC has a good in vivo therapeutic effect on the joint swelling symptoms of RA patients and can significantly improve the behavioral and appearance conditions caused by swelling. Elisa assays were performed on the sera and joint lavage fluids of the five groups of rats collected after 2 weeks of treatment ( Figure 4 G, H). Pro-inflammatory cytokines such as IL-6, IL-8, TNF-α, and IL-1β were all significantly inhibited, and the blood routine results also confirmed the above results. The neutrophils, white blood cells, and lymphocytes in the blood of the rats in the TUC group and the UPA group were all reduced, and the reduction in the TUC group was more obvious ( Figure 4 J).

[0097] In the joints of RA patients, due to cytokine dysregulation, the osteoclast-mediated bone resorption and osteoblast-mediated bone formation, which are in balance under physiological homeostasis, are disrupted. Bone resorption increases while bone formation decreases, leading to bone loss. The Janus kinase (JAK) / signal transducers and activators of transcription (STAT) system is a central pathway that mediates the cell's response to various cytokines and growth factors. Previous studies have shown that the JAK / STAT system also plays a role in bone biology, having a bone-protective effect while exerting an anti-inflammatory effect. Micro-CT was used to evaluate the trabecular bone parameters of the ankle joint. The results of the three-dimensional reconstruction of trabecular bone ( Figure 5 A, B) showed that compared with the control group, the CIA group exhibited obvious bone loss and reduced bone density. The tFNA group had a slight improvement. It was initially inferred that this might be because the anti-inflammatory ability of tFNAs improved the osteogenic ability of BMSCs. The bone density of the UPA group and the TUC group increased significantly, and there was no significant statistical difference between the two, indicating that TUC could still have an efficient anti-inflammatory and osteogenic effect while significantly reducing the dosage of UPA. Combining the corresponding statistical analysis of trabecular thickness (Tb.Th) and trabecular separation (Tb.Sp) ( Figure 5 C, D) could show the changes in the trabecular bone structure. The CIA group had the highest Tb.Sp and the lowest bone density. The UPA group and the TUC group showed higher Tb.Th. Further processing the results by quantitative analysis ( Figure 5 E, F), compared with other treatment groups, the TUC group showed higher bone volume / total volume (BV / TV), bone mineral density (BMD), and trabecular number (Tb.N), proving that TUC has good osteogenic ability in vivo, can effectively improve bone loss in RA patients, and has a good bone-protective effect. Immunofluorescence and statistical analysis of tissue sections also confirmed that TUC has good JAK / STAT inhibitory and anti-inflammatory effects ( Figure 5 G-J).

[0098] The role of TUC in RA was further investigated by histological analysis of tissues stained with Goldner, toluidine blue (TB), Safranine O, and Masson. Goldner staining mainly reflects the expression level of new bone. After staining, mineralized bone appears green, while new bone (osteoid) appears orange or red. The orange or red staining area in the UPA group and TUC group was significantly larger than that in other treatment groups. The area fraction of the orange or red region in the figure was calculated. Toluidine blue staining makes cartilage stain blue, while the missing cartilage remains colorless. Masson staining shows that fibrous tissue is red and cartilage collagen is blue, and the loss of collagen in the blue region is reduced. In addition, Safranine O staining was used to stain glycosaminoglycans in cartilage. When cartilage is damaged, glycosaminoglycans decrease. Safranine O staining clearly shows cartilage damage in CIA rats, demonstrating that TUC can reduce cartilage damage by anti-inflammation ( Figure 6 A), and the red staining area in the UPA group and TUC group was significantly larger than that in the CIA group and TDN group ( Figure 6 B), and the quantitative analysis of collagen volume fraction (CVF) ( Figure 6 C) further confirmed this. Subsequently, the immunohistochemistry and statistical results of ankle bone tissue sections ( Figure 6 D-G) also showed that the expression of WNT1 in the UPA group and TUC group increased significantly, while the expression of STAT3 decreased significantly, once again confirming the previous conclusion that TUC can promote the WNT signaling pathway while inhibiting inflammation through the JAK / STAT pathway, and synergistically treat rheumatoid arthritis by anti-inflammation and osteogenesis.

[0099] In summary, the present invention constructs a nano-drug delivery system TUC, which has good biocompatibility, stability, and endocytosis performance. While significantly reducing the drug dosage, TUC can inhibit the inflammatory response by inhibiting the JAK-STAT pathway, enhance the WNT signaling pathway to promote osteogenic differentiation, providing a new approach for the clinical treatment of rheumatoid arthritis.

Claims

1. A functionalized tetrahedral framework nucleic acid, characterized in that: The functionalized tetrahedral framework nucleic acid is modified with a JAK inhibitor.

2. The functionalized tetrahedral framework nucleic acid according to claim 1, characterized in that: It is assembled by embedding a JAK inhibitor into a spiral groove on the surface of a DNA double helix of a tetrahedral framework nucleic acid; the tetrahedral framework nucleic acid is formed by four single-stranded DNAs through complementary base pairing.

3. The functionalized tetrahedral framework nucleic acid according to claim 2, characterized in that: The 4 single-stranded DNA nucleotide sequences are shown as SEQ ID NO.1-SEQ ID NO.

4.

4. The functionalized tetrahedral framework nucleic acid according to claim 2, characterized in that: The JAK inhibitor is selected from upadacitinib.

5. The method for preparing the functionalized tetrahedral framework nucleic acid according to any one of claims 1 to 4, characterized in that: The steps include: Step 1: Mix, incubate and anneal four single-stranded DNAs to obtain a tetrahedral framework nucleic acid; Step 2: Mix the tetrahedral framework nucleic acid and the JAK inhibitor solution, and incubate by shaking to obtain the product.

6. The preparation method according to claim 5, characterized in that: In step 2, the concentration ratio of the tetrahedral framework nucleic acid to the JAK inhibitor solution is 1:(10-60); And / or, the shaking incubation temperature is 20-25° C. and the time is 5-7 h.

7. Use of the functionalized tetrahedral framework nucleic acid according to any one of claims 1 to 4 in a drug for treating rheumatoid arthritis.

8. The use according to claim 7, characterized in that: The drug is used to suppress inflammatory responses.

9. The use according to claim 7, characterized in that: The drug is used for promoting osteogenic differentiation.

10. A drug for treating rheumatoid arthritis, characterized in that: The invention is prepared by taking the functionalized tetrahedral framework nucleic acid as any one of claims 1 to 4 as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

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