CN-121975798-A - DNA tetrahedron nano-framework compound with miRNA integrated at vertex, and preparation method and application thereof

Abstract

The invention provides a DNA tetrahedron nano-frame compound with miRNA integrated at the vertex, a preparation method and application thereof, and belongs to the technical field of biological medicine. The DNA single-stranded DNA is a compound with a DNA tetrahedron nano-framework structure formed by self-assembly of DNA single-stranded S1, S2 and S3 and miRNA, the sequences of the DNA single-stranded S1, S2 and S3 are respectively shown as SEQ ID NO.5-7, the miRNA comprises an active fragment and a connecting fragment, and the connecting fragment is complementary with the terminal base of the DNA single-stranded. The compound has longer residence time in the OA joint, excellent serum stability, storage stability and systemic biosafety, can be efficiently absorbed by chondrocytes, can be more intensively acted on a lesion part, has remarkable anti-apoptosis and antioxidant capacity, can remarkably inhibit the damage of cartilage, promote the maintenance and synthesis of a healthy matrix, has excellent tissue repair capacity and cartilage protection effect, and provides a more effective treatment strategy for inflammatory or degenerative joint diseases.

Inventors

• LUO DELUN
• CHEN XINGYU

Assignees

• 成都云海四面体生物科技有限公司

Dates

Publication Date

20260505

Application Date

20251219

Claims (10)

1. 1. The DNA tetrahedral nano-framework compound with miRNA integrated at the vertex is characterized in that the compound is formed by DNA single chains S1, S2, S3 and miRNA in a self-assembly way and has a DNA tetrahedral nano-framework structure; The sequences of the DNA single strands S1, S2 and S3 are respectively shown in SEQ ID NO. 5-7; the miRNA comprises an active fragment and a connecting fragment, wherein the connecting fragment is complementary with the terminal base of the DNA single strand.
2. 2. The complex of claim 1, wherein the active fragment comprises miR-143, miR-332, miR-140, miR-17, miR-20a, miR-146a, or miR-155.
3. 3. The compound according to any one of claims 1-2, wherein the sequence of the miRNA is shown in SEQ ID No. 8.
4. 4. The complex of claim 1, wherein the molar ratio of DNA single strands S1, S2, S3 and miRNA is 1:1:1:3.
5. 5. The method for preparing the complex according to any one of claims 1 to 4, wherein the method comprises the steps of mixing DNA single strands S1, S2, S3 and miRNA in a buffer solution to obtain a mixed solution, swirling and centrifuging the mixed solution, placing the mixed solution in a PCR instrument, and annealing the mixed solution to self-assemble the mixed solution into the complex with the DNA tetrahedral nano-framework structure.
6. 6. The preparation method according to claim 5, wherein the final concentration of the DNA single strands S1, S2 and S3 in the mixed solution is 1. Mu.M, and the annealing procedure is to incubate at 90-100 ℃ for 5-20 minutes, then rapidly cool to 0-10 ℃ and hold for 10-30 minutes.
7. 7. Use of a complex according to any one of claims 1 to 4 in the manufacture of a medicament for the prevention and/or treatment of inflammation, inflammatory joint disease or degenerative joint disease.
8. 8. The use according to claim 7, wherein the inflammatory condition comprises inflammatory bowel disease, rheumatoid arthritis, encephalomyelitis, pneumonia, or hepatitis, the inflammatory joint disease comprises rheumatoid arthritis, ankylosing spondylitis, gout, pseudogout, psoriatic arthritis, infectious arthritis, or systemic lupus erythematosus-associated arthritis, and the degenerative joint disease comprises primary osteoarthritis, secondary osteoarthritis, spinal degenerative joint disease, or degenerative interphalangeal joint disease.
9. 9. The use according to claim 8, wherein the primary osteoarthritis comprises knee osteoarthritis, hip osteoarthritis, ankle osteoarthritis, hand osteoarthritis or foot osteoarthritis, the degenerative joint disease of the spine comprises cervical facet arthritis, lumbar facet arthritis, degenerative disc disease, degenerative lumbar spondylolisthesis or degenerative scoliosis, and the secondary osteoarthritis comprises post-traumatic joint disease, joint disease secondary to dysplasia or anatomic abnormalities, joint disease secondary to osteonecrosis, neurogenic joint disease or heavy physical load acceleration joint disease.
10. 10. A pharmaceutical composition, characterized in that it is a preparation prepared by taking the compound of any one of claims 1-4 as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

Description

DNA tetrahedron nano-framework compound with miRNA integrated at vertex, and preparation method and application thereof Technical Field The invention belongs to the technical field of biological medicine, and particularly relates to a DNA tetrahedron nano-frame compound with miRNA integrated at vertexes, a preparation method and application thereof. Background Degenerative joint disease is a chronic osteoarthropathy characterized by degeneration of articular cartilage, proliferation of subchondral bone and inflammation of synovium, which seriously affects joint function and quality of life of patients, wherein Osteoarthritis (OA) is the most common degenerative joint disease worldwide, and is also the most leading cause of disability in the elderly. OA is a complex pathological condition of the whole joint characterized by chronic inflammation, progressive cartilage degradation and subchondral bone remodeling, the pathogenesis of which is multifactorial and heterogeneous, driven mainly by an imbalance between mechanical load and biochemical signals. Abnormal mechanical stress (e.g., caused by obesity or injury) activates chondrocytes and other articular cells, resulting in elevated levels of pro-inflammatory cytokines, particularly interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha), which disrupt chondrocyte homeostasis, inhibit synthesis of essential extracellular matrix (ECM) components (type II collagen, aggrecan ACAN), and induce matrix degrading enzymes such as Matrix Metalloproteinases (MMPs) and thrombospondin motif-containing disintegrin-like metalloproteinases (ADAMTS), thereby accelerating ECM degradation, a vicious circle initiated by mechanical stress and amplified by inflammatory mediators, ultimately resulting in irreversible cartilage damage. The pathological complexity of OA involves multiple tissues (cartilage, bone, synovium) and cross-pathways (e.g. inflammation, catabolism, apoptosis) such that single-target therapies tend to be poorly effective, which motivates research into multi-molecular pathway therapies, particularly through epigenetic regulatory factors such as micrornas (micrornas, mirnas). These non-coding RNAs 19-25 nucleotides in length serve as key molecular switches that can simultaneously regulate multiple genes involved in basic biological processes such as proliferation, differentiation, apoptosis, and stress responses. In fact, mirnas are critical for normal skeletal development and joint homeostasis, e.g., mouse chondrocyte-specific knockout of the Dicer gene results in severe skeletal growth defects and reduced chondrocyte proliferation, clearly demonstrating the fundamental role of mirnas in cartilage biology. However, endogenous mirnas suffer from multiple limitations of poor stability, potential off-target effects, and low efficiency of delivery to target cells. Therefore, the development of a nanocarrier system capable of stably and efficiently delivering mirnas is critical to fully exploiting its therapeutic potential. Various nanoscale vectors, such as liposomes, viral vectors, polymers, and inorganic nanoparticles, are widely used for miRNA delivery. However, inherent limitations, including poor immunogenicity, cytotoxicity, and batch reproducibility, limit their use. In recent years, DNA has become a promising alternative as a genetic template for RNA and protein synthesis due to its ease of preparation, excellent natural biocompatibility and unprecedented site-specific editability. Among them, the tetrahedral DNA nano-framework (TETRAHEDRAL DNA Nanostructure, TDN) is an ideal nano-carrier due to its simple structure, good monodispersity and excellent cell/tissue penetration ability, exhibiting high efficiency in nucleic acid delivery. However, conventional TDN loading strategies typically attach cargo to the apex via a sticky end or chemical linker, which may expose miRNA, alter particle geometry, TDN enters the cell primarily via a "corn attack" mechanism, which may impair cell penetration, and furthermore, conventional loading approaches lack a controllable intracellular release mechanism, which may prematurely release or insufficiently release miRNA before reaching the site of action, and the residence time of free miRNA in the joint cavity is extremely short, rapidly cleared, and does not maintain effective therapeutic concentrations, requiring frequent dosing. Therefore, a drug system for stably, controllably releasing and efficiently delivering miRNA is developed, a more effective treatment strategy is provided for degenerative joint diseases, and the drug system has important clinical application value. Disclosure of Invention The invention aims to provide a DNA tetrahedral nano-framework compound with miRNA integrated at the vertex, a preparation method and application thereof. The invention provides a DNA tetrahedron nanometer frame compound with miRNA integrated at the vertex, which is a compound with a D