Tetrahedral framework nucleic acid nano compound carrying miR-23b as well as preparation method and application of tetrahedral framework nucleic acid nano compound

Abstract

The invention provides a miR-23b-carrying tetrahedral framework nucleic acid nano-composite as well as a preparation method and application thereof, and belongs to the field of biological medicines. The compound disclosed by the invention is formed by four single-stranded DNA (Deoxyribose Nucleic Acid) through complementary base pairing; the nucleotide sequences of the four single-stranded DNAs are respectively as shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 5. The compound T-23b can prevent miR-23b from being rapidly degraded in vivo, can inhibit infiltration of inflammatory cells and inhibit synovial hyperplasia to relieve synovial inflammation in treatment of rheumatoid arthritis, and can also prevent pathological angiogenesis, cartilage matrix degradation and osteoclast generation to protect bone joint structures. Results show that T-23b plays a synergistic role in treating rheumatoid arthritis, is a safe and effective choice for clinically treating rheumatoid arthritis, and has a good application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a tetrahedral framework nucleic acid nanocomplex carrying miR-23b, a preparation method and a use thereof. Background Art

[0002] Rheumatoid arthritis is an inflammatory autoimmune disease characterized by chronic inflammation of the joints, resulting in joint swelling, pain, and dysfunction. It is a systemic disease that affects not only the joints but also other parts of the body, such as the skin, eyes, heart, lungs, and blood vessels. Rheumatoid arthritis is a clinical syndrome that spans several disease subgroups. These different subgroups include several inflammatory cascades that all lead to a final common pathway that results in persistent synovial inflammation and associated damage to the articular cartilage and underlying bone.

[0003] Simple miR-23b has the effect of treating rheumatoid arthritis, but the single-stranded structure of pure miRNA is unstable and easily degraded, which makes it difficult for it to exert its function alone. As an efficient and safe drug delivery system, tFNAs have high structural stability and good biocompatibility, which may protect miRNA from degradation by nucleases, enhance its stability, and enable efficient delivery and release of miRNA in biological systems. However, the presence of tFNAs may also affect the efficiency of miRNA in cells. Therefore, it is necessary to explore how to provide a drug delivery system that carries miR-23b without affecting its effects. Summary of the invention

[0004] The purpose of the present invention is to provide a tetrahedral framework nucleic acid nanocomplex carrying miR-23b, as well as a preparation method and use thereof.

[0005] The present invention provides a tetrahedral framework nucleic acid nanocomplex carrying miR-23b, which is formed by four single-stranded DNAs through base complementary pairing; the nucleotide sequences of the four single-stranded DNAs are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.5 respectively.

[0006] Furthermore, the aforementioned tetrahedral framework nucleic acid nanocomplex carrying miR-23b is prepared by the following method:

[0007] Place the four single-stranded DNAs at a temperature sufficient to denature them for 10 to 15 minutes, then lower the temperature to 2 to 8°C and maintain it for more than 20 minutes.

[0008] Furthermore, the aforementioned tetrahedral framework nucleic acid nanocomplex carrying miR-23b is prepared by the following method:

[0009] Place the four single-stranded DNAs at 95°C for 10 minutes, then lower the temperature to 4°C and maintain for more than 20 minutes.

[0010] Furthermore, the molar ratio of the four single-stranded DNAs is 1:1:1:1.

[0011] Furthermore, the four single-stranded DNAs are first dissolved in a solvent and then denatured.

[0012] Furthermore, the solvent is TM buffer, wherein the concentration of Tris-HCl is 10 mM, the concentration of MgCl2 is 50 mM, and the pH is 8.0.

[0013] The present invention also provides a method for preparing the aforementioned tetrahedral framework nucleic acid nanocomplex carrying miR-23b, which comprises the following steps:

[0014] Place the four single-stranded DNAs at a temperature sufficient to denature them for 10 to 15 minutes, then lower the temperature to 2 to 8°C and maintain it for more than 20 minutes.

[0015] Furthermore, the aforementioned method comprises the following steps:

[0016] Place the four single-stranded DNAs at 95°C for 10 minutes, then lower the temperature to 4°C and maintain for more than 20 minutes.

[0017] Further,

[0018] The molar ratio of the four single-stranded DNAs is 1:1:1:1;

[0019] and / or, the four single-stranded DNAs are first dissolved in a solvent and then denatured;

[0020] Preferably, the solvent is TM buffer, wherein the concentration of Tris-HCl is 10 mM, the concentration of MgCl2 is 50 mM, and the pH is 8.0.

[0021] The present invention also provides use of the aforementioned tetrahedral framework nucleic acid nanocomplex carrying miR-23b in preparing a drug for treating rheumatoid arthritis.

[0022] In the present invention, for the convenience of description, the single-stranded hybrid chain formed by DNA and RNA (such as SEQ ID NO. 5) is also referred to as "single-stranded DNA".

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] tFNAs can be used as a carrier and miR-23b to successfully prepare a nanocomposite T-23b. The nanocomposite obtained by the present invention can prevent the rapid degradation of miR-23b in the body, and can inhibit the infiltration of inflammatory cells and synovial hyperplasia to relieve synovial inflammation in the treatment of rheumatoid arthritis, and can also prevent pathological angiogenesis, cartilage matrix degradation and osteoclast formation to protect bone joint structure. The results show that T-23b plays a synergistic role in the treatment of rheumatoid arthritis, is a safe and effective choice for the clinical treatment of rheumatoid arthritis, and has a good application prospect.

[0025] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0026] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the PAGE electrophoresis diagram of T-23b and its ssDNA single chain.

[0028] Figure 2 This is the particle size diagram of T-23b.

[0029] Figure 3 This is the Zeta potential diagram of T-23b.

[0030] Figure 4 This is a transmission electron microscope image of T-23b.

[0031] Figure 5 This is a diagram of T-23b cell occlusion.

[0032] Figure 6 Schematic diagram of the animal experimental treatment process and the clinical scores of mice at different time points.

[0033] Figure 7 The following are the gross morphological images of mouse limbs at different time points.

[0034] Figure 8 These are the ELISA test results for the levels of inflammatory factors in mouse plasma.

[0035] Fig. 9 These are the results of HE staining and Safranin O staining of mouse knee joint and limb tissue sections.

[0036] Fig.10These are the results of TRAP staining and immunofluorescence staining of mouse knee joint tissue sections.

[0037] Fig.11 These are the results of PCR detection of multiple genes related to inflammation and osteoclastosis in mouse knee joint tissues. DETAILED DESCRIPTION

[0038] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0039] Example 1. Preparation of tetrahedral framework nucleic acid nanocomplex (T-23b) carrying miR-23b

[0040] The four ssDNA single strands (S1, S2, S3, S4-miR23b) were dissolved in TM buffer (10mM Tris-HCl, 50mM MgCl2, pH = 8.0), and the final concentration of the four ssDNA single strands was 1μΜ. They were fully mixed, quickly heated to 95°C and maintained for 10 minutes, and then quickly cooled to 4°C and maintained for more than 20 minutes to obtain T-23b (tFNAs-miR23b).

[0041] The four ssDNA single strands (S1, S2, S3, S4) were dissolved in TM buffer (10 mM Tris-HCl, 50 mM MgCl2, pH = 8.0) with a final concentration of 1 μM. The mixture was fully mixed and rapidly heated to 95°C for 10 minutes, then rapidly cooled to 4°C and maintained for more than 20 minutes to obtain tetrahedral framework nucleic acids (tFNAs).

[0042] The nucleotide sequence of the ssDNA single strand is shown in Table 1.

[0043] Table 1. Nucleotide sequences

[0044]

[0045]

[0046] The beneficial effects of the present invention are demonstrated below through specific test examples.

[0047] Test Example 1: Physical Property Characterization of T-23b

[0048] 1. Experimental methods

[0049] (1) Polyacrylamide gel electrophoresis (PAGE) was used to verify whether T-23b was successfully synthesized.

[0050] (2) Dynamic light scattering was used to measure the particle size and zeta potential of T-23b.

[0051] (3) The morphological characteristics of T-23b were detected using transmission electron microscopy.

[0052] (4) Immunofluorescence detection of T-23b entry into cells:

[0053] (4.1) Extraction and culture of synovial fibroblasts (FLS)

[0054] The mouse knee joint was soaked in PBS containing 3% penicillin-streptomycin and then in 0.25% trypsin cell digestion solution. The mixture was shaken in a 37°C water bath for 30 min and centrifuged at 1200 r / min for 3 min. The supernatant was discarded and an appropriate amount of complete culture medium (DMEM / F12+10% FBS+1% penicillin-streptomycin) was added to terminate the digestion. The mixture was inoculated into 75 cm 2 Cells will adhere to the culture flask and grow into P0 cells. After that, the medium will be changed every 3 days until the cells are about 80% confluent.

[0055] (4.2) Cell immunofluorescence staining

[0056] Cy5 was connected to the S1 chain, and tFNAs-Cy5 was prepared by using S1 connected with Cy5 and S2, S3 and S4 according to the method described in Example 1. Cy3 was connected to miR23b to obtain miR23b-Cy3. tFNAs were prepared by using S1 connected with Cy5 and S4-miR23b connected with Cy3 and S2 and S3 according to the method described in Example 1. Cy5 -miR23b Cy3 .

[0057] After the cells reached about 80% confluency, tFNAs-Cy5, miR23b-Cy3 and tFNAs were added at a concentration of 62.5 nM. Cy5 -miR23b Cy3 (Solvent is culture medium), after incubation for 24 hours, the cells were fixed with 4% cold paraformaldehyde, permeabilized with 0.5% Triton X-100, and blocked with 5% goat serum. After washing with PBS, the samples were incubated with primary antibodies (anti-TNF-α, 1:200; anti-IL-1β, 1:200; anti-MMP-3, 1:200) at 4°C overnight. After re-washing on the second day, secondary antibodies (1:1000) were added and stained with phalloidin and DAPI. Immunofluorescence images were obtained by confocal laser microscopy (N-SIM S, Nikon, Japan).

[0058] 2. Experimental results

[0059] Depend on Figure 1 It can be seen that T-23b and tFNAs were successfully synthesized. Figure 1In the figure, tS1 is the band of single-chain S1 on PAGE gel electrophoresis, tS2 is the band of single-chain S2 on PAGE gel electrophoresis, tS3 is the band of single-chain S3 on PAGE gel electrophoresis, and tS4 is the band of single-chain S4-mi-R23b on PAGE gel electrophoresis.

[0060] Depend on Figure 2 and Figure 4 It can be seen that the particle size of T-23b is approximately 12.91±5.54nm.

[0061] Depend on Figure 3 It can be seen that T-23b has a negative charge and the potential is about -0.02±1.51mV.

[0062] Figure 5 This is a diagram of T-23b entering cells. About 80% of T-23b enters cells in about 6 hours, indicating that T-23b has good ability to enter cells.

[0063] Experimental Example 2: The therapeutic effect of T-23b on rheumatoid arthritis model

[0064] 1. Experimental methods

[0065] (1) Adult female BALB / c mice (20-25 g body weight, 4-6 weeks old) were fed with standard chow and sterile water and housed under laboratory conditions for 1 week for adaptive feeding.

[0066] (2) In order to verify the therapeutic effect of T-23b prepared in Example 1 on rheumatoid arthritis, the present invention established a collagen-induced rheumatoid arthritis (RA) mouse model. Female BALB / c mice aged 4-6 weeks were used, and Freund's complete adjuvant was injected intraperitoneally 3 times (experimental day 0, day 7, and day 21), and the drug was administered according to the weight (g) of the mouse, with a dose of 2.5 μl / g, and dissolved in PBS to prepare a 200ul injection solution. The modeling was evaluated in the 4th week of the experiment (experimental day 28), and the mice with successful modeling could clearly observe redness and swelling of the limb joints. From the 4th week of the experiment (experimental day 28), mice were treated with miR-23b, tFNAs and T-23b at a concentration of 250nM, respectively, with each injection dose of 200μl (PBS dissolved), and treated once every two days, for a total of 4 weeks (treatment until the 56th day of the experiment).

[0067] (3) Samples were collected and analyzed at the beginning of the 4th week (28th day of the experiment), the 6th week (42nd day of the experiment), the 8th week (56th day of the experiment), and the 10th week (70th day of the experiment).

[0068] (3.1) The RA clinical score was used to score the clinical symptoms of each group of mice at different time points.

[0069] The ACR / EULAR 2010 criteria are as follows:

[0070] I. Joint involvement (0-5 points)

[0071] One medium to large joint (0 points)

[0072] Two to ten medium to large joints (1 point)

[0073] One to three small joints (excluding large joints) (2 points)

[0074] Four to ten small joints (excluding large joints) (3 points)

[0075] More than ten joints (at least one small joint) (5 points)

[0076] II. Serology (0-3 points)

[0077] Negative rheumatoid factor and negative anti-citrullinated antigen antibodies (0 points)

[0078] Low rheumatoid factor or low anti-citrullinated antigen antibodies (2 points)

[0079] High rheumatoid factor or high anti-citrullinated antigen antibodies (3 points)

[0080] III. Acute phase reactants (0-1 points)

[0081] Normal C-reactive protein and normal erythrocyte sedimentation rate (0 points)

[0082] Abnormal C-reactive protein or abnormal erythrocyte sedimentation rate (1 point)

[0083] IV. Duration of symptoms (0-1)

[0084] Less than 6 weeks (0 points)

[0085] 6 weeks or longer (1 point).

[0086] (3.2) Use gross images to observe the swelling of the limb joints of each group of mice.

[0087] (3.3) ELISA was used to detect the levels of inflammatory factors in mouse plasma on days 14, 28, 42, and 56 of the experiment.

[0088] (3.4) Samples were collected on the 70th day of the experiment, and tissue sections of the mouse knee joints and limbs were observed and analyzed using HE staining and Safranin O staining.

[0089] (3.5) Samples were collected on the 70th day of the experiment, and osteoclasts were observed using TRAP staining. Immunofluorescence was used to detect the expression of inflammation and osteoclast-related proteins in joint tissues.

[0090] (3.6) Samples were collected on the 70th day of the experiment, and PCR was used to detect the expression of inflammation and osteoclast-related genes.

[0091] 2. Experimental results

[0092] Figure 6 This indicates that T-23b treatment significantly reduced the clinical scores of RA mice.

[0093] Figure 7 It shows that T-23b treatment significantly reduced the swelling of the limb joints of RA mice.

[0094] Figure 8 It shows that T-23b reduces the serum levels of inflammatory factors, and the effect is better than using tFNAs and miR-23b alone.

[0095] Fig. 9 It shows that after T-23b treatment, the joint surface of mice became smoother, the capsule-bone interface was clear and complete, the vascular proliferation and inflammatory cell infiltration were reduced, the synovial proliferation was less, and the synovial thickness was significantly reduced. The red staining area of ​​articular cartilage increased, the proteoglycan content increased, and the number of chondrocytes and the volume of cartilage cavities also decreased.

[0096] Fig.10 The results showed that after T-23b treatment, the number and volume of osteoclasts in the knee joints of mice decreased. T-23b treatment can also reduce the production of inflammatory factors and osteoclast-related proteins in the joints.

[0097] Fig.11 It shows that T-23b treatment can also downregulate the expression of inflammation-related genes at the transcriptional level, thereby inhibiting the inflammatory response and protecting the cartilage matrix in the joints. T-23b treatment inhibits the formation of osteoclasts by downregulating Csf2 and RANK and upregulating the expression of the OPG gene at the transcriptional level. T-23b also inhibits osteoclast-mediated bone resorption by downregulating the expression of Ctsk and MMP-3 genes at the transcriptional level. T-23b treatment can inhibit the infiltration of inflammatory cells and the production of inflammatory cytokines, inhibit synovial hyperplasia and angiogenesis; at the same time, it inhibits the formation of osteoclasts and avoids excessive degradation of the cartilage matrix, thereby protecting the bone joint structure.

[0098] Compared with the use of miR-23b and tFNAs alone, the anti-inflammatory and therapeutic effects of T-23b (tFNAs-miR23b) of the present invention on rheumatoid arthritis are significantly improved, and a synergistic effect is exerted.

[0099] In summary, tFNAs as carriers and miR-23b can be successfully prepared into nanocomplex T-23b. The nanocomplex obtained by the present invention can prevent the rapid degradation of miR-23b in the body, inhibit the infiltration of inflammatory cells and inhibit synovial hyperplasia to relieve synovial inflammation in the treatment of rheumatoid arthritis, and can also prevent pathological angiogenesis, cartilage matrix degradation and osteoclastogenesis to protect bone joint structure. The results show that T-23b plays a synergistic role in the treatment of rheumatoid arthritis, is a safe and effective choice for the clinical treatment of rheumatoid arthritis, and has a good application prospect.

Claims

1. A tetrahedral framework nucleic acid nanocomplex carrying miR-23b, characterized in that: It is formed by four single-stranded DNAs through complementary base pairing; the nucleotide sequences of the four single-stranded DNAs are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.5 respectively.

2. The tetrahedral framework nucleic acid nanocomplex carrying miR-23b according to claim 1, characterized in that: It is prepared by the following method: Place the four single-stranded DNAs at a temperature sufficient to denature them for 10 to 15 minutes, then lower the temperature to 2 to 8°C and maintain it for more than 20 minutes.

3. The tetrahedral framework nucleic acid nanocomplex carrying miR-23b according to claim 2, characterized in that: It is prepared by the following method: Place the four single-stranded DNAs at 95°C for 10 minutes, then lower the temperature to 4°C and maintain for more than 20 minutes.

4. The tetrahedral framework nucleic acid nanocomplex carrying miR-23b according to claim 2 or 3, characterized in that: The molar ratio of the four single-stranded DNAs is 1:1:1:

1.

5. The tetrahedral framework nucleic acid nanocomplex carrying miR-23b according to claim 2 or 3, characterized in that: The four single-stranded DNAs are first dissolved in a solvent and then denatured.

6. The tetrahedral framework nucleic acid nanocomplex carrying miR-23b according to claim 6, characterized in that: The solvent is TM buffer, wherein the concentration of Tris-HCl is 10 mM, the concentration of MgCl2 is 50 mM, and the pH is 8.

0.

7. A method for preparing the tetrahedral framework nucleic acid nanocomplex carrying miR-23b according to any one of claims 1 to 6, characterized in that: It includes the following steps: Place the four single-stranded DNAs at a temperature sufficient to denature them for 10 to 15 minutes, then lower the temperature to 2 to 8°C and maintain it for more than 20 minutes.

8. The method according to claim 7, characterized in that: It includes the following steps: Place the four single-stranded DNAs at 95°C for 10 minutes, then lower the temperature to 4°C and maintain for more than 20 minutes.

9. The method according to claim 7 or 8, characterized in that: The molar ratio of the four single-stranded DNAs is 1:1:1:1; and / or, the four single-stranded DNAs are first dissolved in a solvent and then denatured; Preferably, the solvent is TM buffer, wherein the concentration of Tris-HCl is 10 mM, the concentration of MgCl2 is 50 mM, and the pH is 8.

0.

10. Use of the tetrahedral framework nucleic acid nanocomplex carrying miR-23b according to any one of claims 1 to 6 in the preparation of a drug for treating rheumatoid arthritis.

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