DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system, and preparation method and application thereof

Abstract

A DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system, its preparation method, and its application are described. The delivery system includes: a triangular DNA origami carrier, formed by the self-assembly of a single-stranded DNA backbone and stapled strands, with sides A, B, and C; an SOX9 plasmid bound to side A; a type II collagen-targeting peptide grafted to side B; a stem cell-targeting aptamer modified to side C; and an anti-inflammatory and antioxidant small molecule drug loaded inside the DNA origami carrier. The preparation method includes: S1: The backbone and auxiliary strands are annealed to complete self-assembly, obtaining a triangular DNA origami carrier WA; S2: The DNA origami carrier WA and the SOX9 plasmid are annealed to obtain a triangular DNA origami carrier WAS with the SOX9 plasmid bound to side A; the DNA origami carrier WAS is mixed and incubated with the anti-inflammatory and antioxidant small molecule drug, and purified to obtain the delivery system; the delivery system is applied in the preparation of drugs for treating osteoarthritis; this application has multifunctional integrated features, realizing targeted and efficient treatment of osteoarthritis.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system, its preparation method, and its application. Background Technology

[0002] Osteoarthritis (OA) is a common chronic degenerative musculoskeletal disease. Due to the lack of nerves and blood vessels, the articular cartilage has a very weak proprioceptive ability to feel pain, resulting in severely impaired self-repair mechanisms after injury. Cartilage tissue faces a deterioration of its microenvironment in the early stages of injury, including chronic low-grade inflammation and oxidative stress, which further weakens its regenerative potential.

[0003] Mesenchymal stem cells (MSCs) possess multipotent differentiation, immunomodulatory, and tissue repair potential, making them ideal seed cells for cartilage injury repair. However, exogenous infusion strategies have significant limitations. Intravenous injection results in extremely low stem cell migration to the target site, with most cells remaining in the lungs and only about one percent reaching the joint cavity. While direct intra-articular injection allows for local administration, the downregulation of chemokine receptor expression in the injury microenvironment weakens stem cell homing ability and makes them susceptible to aging-related secretory phenotypes, posing a risk of rapid aging. Furthermore, injected stem cells tend to aggregate in the synovial tissue, making it difficult to effectively target low-inflammation cartilage defect areas, leading to poor repair outcomes.

[0004] To circumvent the drawbacks of exogenous stem cell transplantation, the industry is gradually shifting towards the therapeutic approach of in-situ recruitment of endogenous stem cells. If these cells can be efficiently recruited to the cartilage defect area and precisely induced to differentiate into hyaline chondrocytes, while simultaneously improving the local inflammatory microenvironment, in-situ cartilage repair can be achieved. However, although endogenous mesenchymal stem cells exist in periarticular tissues, such as synovium, bone marrow, and adipose-derived cells, their natural recruitment process is limited by factors such as long physiological distances and weak migration ability, resulting in a scarcity that cannot meet the repair needs. Developing biomaterials with highly efficient "siphoning" functions to directionally recruit endogenous stem cells to the injury site would provide a new approach to cartilage repair. However, simply increasing the number of stem cells is insufficient; the key lies in promoting their precise differentiation into hyaline chondrocytes rather than fibrocartilage. The expression regulation of the early cartilage-specific marker gene SOX9 becomes a core element. Exogenous introduction of SOX9 gene fragments may activate the cartilage differentiation pathway, but a safe and efficient delivery vector is lacking.

[0005] Meanwhile, inflammatory factors and oxidative stress in the regenerative microenvironment easily weaken stem cell stemness. Although resveratrol has the potential for antioxidant, anti-inflammatory, and stem cell proliferation-maintaining effects, it is difficult to exert a stabilizing effect due to low bioavailability and the risk of local accumulation. While aptamer technology can provide highly specific targeting capabilities, such as the recognition of mesenchymal stem cells by the Apt19S aptamer, single functional components cannot synergistically address the multiple challenges of recruitment, differentiation induction, and microenvironment regulation. Existing delivery systems mostly focus on exogenous cell therapy or simple drug loading, failing to integrate the multimodal functions of targeting peptides, gene plasmids, and protective small molecules, resulting in unsatisfactory recruitment efficiency, directed differentiation capacity, and long-term survival rate of endogenous stem cells.

[0006] Therefore, there is an urgent need to propose an endogenous stem cell recruitment and cartilage repair delivery system that can simultaneously achieve cartilage targeting, stem cell recruitment, chondrogenic gene activation, and microenvironment optimization, in order to overcome the technical bottleneck of cartilage damage repair.

[0007] To address the aforementioned issues, existing technologies urgently need improvement. Summary of the Invention

[0008] One objective of this invention is to provide a DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system, which integrates multiple functions including cartilage targeting, endogenous stem cell recruitment, chondrogenic differentiation induction, and microenvironment regulation. This system maintains extracellular matrix homeostasis and significantly delays the progression of osteoarthritis, thus achieving targeted and efficient treatment of osteoarthritis and solving at least one of the aforementioned technical problems.

[0009] One objective of this invention is to provide a method for preparing an endogenous stem cell recruitment and cartilage repair delivery system based on DNA origami, which has the advantages of simple preparation technology, strong operability, stable process, fast cycle, easy mass production and wide application.

[0010] Another objective of this invention is to provide an application of a DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system in the preparation of a drug for treating osteoarthritis.

[0011] To achieve the above objectives, in one aspect, according to embodiments of the present invention, a DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system includes: The triangular DNA origami carrier is formed by the self-assembly of a single-stranded DNA backbone and multiple staple chains. The three sides of the DNA origami carrier are respectively divided into side A, side B, and side C. The SOX9 plasmid binds to side A of the DNA origami vector; Type II collagen-targeting peptides are grafted onto side B of the DNA origami carrier; A stem cell targeting aptamer is modified on the C-side of the DNA origami vector; Anti-inflammatory and antioxidant small molecule drugs are loaded inside the DNA origami carrier.

[0012] The DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system according to the above embodiments of the present invention may also have the following additional technical features: According to one embodiment of the present invention, the backbone chain comprises M13mp18 single-stranded DNA; The staple chain includes: As shown in SEQ ID NO:1~65, the A-group staple chain is formed by self-assembly with the backbone chain through base complementary pairing to form the A-side; As shown in SEQ ID NO:66~130, the B-group staple chain is formed by self-assembly with the backbone chain through base complementary pairing to form the B-side; As shown in SEQ ID NO:131 to SEQ ID NO:195, the C-group staple chain forms the C-side by self-assembling with the backbone chain through complementary base pairing.

[0013] Furthermore, according to an embodiment of the present invention, the group A staple chain includes a plasmid trapping sub-chain as shown in SEQ ID NO:23 and SEQ ID NO:53, wherein the 5' end of the plasmid trapping sub-chain is modified with streptavidin; The 3' end of the SOX9 plasmid is biotin-labeled and binds to the A side of the DNA origami vector through the affinity of biotin for streptavidin.

[0014] Furthermore, according to one embodiment of the present invention, the biotin comprises Biotin-dUTP biotin.

[0015] Furthermore, according to an embodiment of the present invention, the group B staple chain includes a peptide capture sub-chain as shown in SEQ ID NO:88 and SEQ ID NO:118, wherein the 5' end of the peptide capture sub-chain is modified with maleimide; The type II collagen-targeting peptide is a thiolized WYRGRL-targeting peptide, which is grafted onto the B side of the DNA origami carrier through the covalent cross-linking property of maleimide and thiol.

[0016] Furthermore, according to one embodiment of the present invention, the C-group staple chain includes aptamer-modified sub-chains as shown in SEQ ID NO:153 and SEQ ID NO:183; The stem cell targeting aptamer includes the Apt19S aptamer, wherein the Apt19S aptamer is modified at the 5' end of the aptamer modified subchain to form an aptamer chain as shown in SEQ ID NO:209 and SEQ ID NO:210.

[0017] Furthermore, according to one embodiment of the present invention, the anti-inflammatory and antioxidant small molecule drug includes resveratrol.

[0018] Furthermore, according to one embodiment of the present invention, the Apt19S aptamer is modified with a Cy5 fluorescent group.

[0019] To achieve the above objectives, on the one hand, according to the embodiments of the present invention, a method for preparing a DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system as described in any of the foregoing claims is provided, the preparation method comprising the following steps: S1: The backbone chain and the auxiliary chain containing the modified staple chain, type II collagen targeting peptide, and stem cell targeting aptamer are placed in a buffer solution, annealed and cooled to room temperature to complete self-assembly, and after removing the excess auxiliary chain, a triangular DNA origami vector WA with type II collagen targeting peptide grafted on side B and stem cell targeting aptamer modified on side C is obtained. S2: The DNA origami vector WA and the SOX9 plasmid containing the free complementary sequence are placed in a buffer solution, annealed and cooled to room temperature to obtain a triangular DNA origami vector WAS with the SOX9 plasmid bound to side A; S3: The DNA origami carrier WAS is mixed and incubated with an anti-inflammatory and antioxidant small molecule drug, and after purification, the DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system is obtained.

[0020] Further, according to an embodiment of the present invention, in step S1, the backbone chain is M13mp18 single-stranded DNA, which is mixed with the auxiliary chain in a molar ratio of 1:(8-12) and added to the buffer solution. The annealing conditions are slow cooling from 95°C to room temperature, and the entire annealing process is not less than 12 h.

[0021] Further, according to an embodiment of the present invention, in step S1 and / or step S2, the buffer solution comprises Tris at a concentration of 30-50 mmol / L, acetic acid at a concentration of 18-22 mmol / L, EDTA at a concentration of 1-3 mmol / L, and magnesium acetate at a concentration of 10-15 mmol / L, and the pH value of the buffer solution is 8.0-8.5.

[0022] Further, according to an embodiment of the present invention, in step S1, the concentration of the M13mp18 single-stranded DNA is 3 to 7 nmol / L; and / or, the concentration of the auxiliary strand is 24 to 84 nmol / L.

[0023] Further, according to an embodiment of the present invention, in step S2, the DNA origami vector WA and the SOX9 plasmid are mixed in a molar ratio of 1:(1-3) and added to the buffer solution, and the annealing conditions are slow cooling from 37°C to room temperature, and the entire annealing process is not less than 4 hours.

[0024] Furthermore, according to an embodiment of the present invention, in step S1 and / or step S2, the buffer solution comprises a magnesium-containing TAE buffer solution, which is prepared by adding magnesium chloride to the TAE buffer solution, with 0.099 to 0.139 mg of magnesium chloride added per 100 μL of TAE buffer solution.

[0025] Furthermore, according to an embodiment of the present invention, in step S3, the incubation conditions for the DNA origami carrier WAS and the anti-inflammatory and antioxidant small molecule drug are shaking at 1-10°C for 6-10 h; The purification was performed using ultrafiltration centrifugation under the following conditions: molecular weight cutoff of 3–100 kDa, centrifuged at 8000–15000 g for 15–25 min.

[0026] Furthermore, according to an embodiment of the present invention, in step S3, the concentration of the anti-inflammatory and antioxidant small molecule drug is 3 to 7 μmol / L, and the DNA origami carrier WAS is mixed and incubated with the anti-inflammatory and antioxidant small molecule drug at a molar ratio of 1:(18 to 22).

[0027] To achieve the above objectives, on the other hand, the application of the DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system as described in any of the foregoing embodiments of the present invention in the preparation of a medicament for treating osteoarthritis is also discussed.

[0028] To address the shortcomings of existing technologies, this invention constructs a multifunctional, integrated DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system. It uses a triangular DNA origami as the core nanocarrier, leveraging the precise modifiability of DNA structure to precisely distribute multiple functional units at different locations within the carrier. Each component works synergistically: the type II collagen-targeting peptide distributed on side B specifically recognizes type II collagen in cartilage tissue, allowing the carrier to target and enrich cartilage at the site of injury and prolong its residence time within the joint cavity; the stem cell-targeting aptamer distributed on side C specifically recognizes and recruits endogenous mesenchymal stem cells (MSCs) around the joint, achieving in situ stem cell recruitment; the SOX9 plasmid distributed on side A, after being taken up by recruited stem cells, can express cartilage-related genes, inducing the directed differentiation of stem cells into hyaline chondrocytes; and the anti-inflammatory and antioxidant small molecule drugs loaded within the carrier can be delivered along with the triangular DNA... The origami carrier framework structure degrades and releases nutrients gradually, exerting antioxidant and anti-inflammatory effects, improving the microenvironment of chronic low-grade inflammation in joints, while maintaining stem cell activity and inhibiting stem cell aging.

[0029] Based on the above technical solution, the DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system proposed in this invention achieves efficient repair of cartilage damage and effectively delays the progression of osteoarthritis through a complete pathway of "targeted enrichment—in situ recruitment of stem cells—directed differentiation into chondrocytes—regulation of the inflammatory microenvironment." Its beneficial effects are manifested in at least the following aspects: (1) This invention integrates type II collagen-targeting peptides, stem cell-targeting aptamers, SOX9 plasmids, and anti-inflammatory and antioxidant small molecule drugs into a triangular DNA origami carrier. It has the characteristics of promoting hyaline cartilage differentiation and maintaining stem cell proliferation. It can realize the in situ recruitment of endogenous stem cells and the sustained release of anti-inflammatory small molecule drugs (such as resveratrol). It breaks through the limitations of traditional single-function carriers, realizes the synergistic effect of multiple components, and has outstanding comprehensive efficacy.

[0030] (2) Based on the complementary base pairing characteristics between the single-stranded DNA backbone strand and the staple strand, the present invention realizes the self-assembly of the triangular DNA origami carrier. The carrier structure has a certain rigidity and good stability, and can realize the fixed-point modification and fixation of each functional unit on the three sides of the carrier, which significantly improves the loading efficiency of target molecules and anti-inflammatory drugs and the targeted therapeutic effect of cartilage damage.

[0031] (3) The delivery system proposed in this invention has excellent biocompatibility, no obvious organ toxicity or immunogenicity, is safe for in vivo application, and can significantly delay cartilage damage and degeneration.

[0032] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures / processes / experimental results shown in these drawings without creative effort.

[0034] Appendix Figure 1 This is a schematic diagram of the framework structure of a triangular DNA origami carrier in an endogenous stem cell recruitment and cartilage repair delivery system based on DNA origami, according to an embodiment of the present invention. Appendix Figure 2 This is a schematic diagram of the synthetic route and characterization results of a DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system proposed in one embodiment of the present invention. A is a schematic diagram of the synthesis of the DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system; B is a schematic diagram of the product obtained by reacting the peptide capture strand on side B of the triangular DNA origami carrier with a thiolized WYRGRL targeting peptide, followed by polyacrylamide gel electrophoresis detection; C is a schematic diagram of the product obtained by modifying the 5' end of the aptamer-modified strand on side C of the triangular DNA origami carrier with an Apt19s aptamer, followed by polyacrylamide gel electrophoresis detection; D is a schematic diagram of the molecular weight of the final product after ligation of the synthesized DNA origami carrier WA with different amounts of SOX9 plasmid (1:1, 1:2, 1:3), as determined by agarose gel electrophoresis; E is a schematic diagram of the morphological detection results of the obtained DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system using AFM (scale bar: 120). (nm); F is a schematic diagram of the zeta potential detection results of triangular DNA origami products with different drug loading; G is the cumulative release of resveratrol in the supernatant after incubation for 48 h under different pH conditions; Appendix Figure 3This diagram illustrates the targeting properties and in vivo retention effects of triangular DNA origami products with different drug loading methods. A represents the triangular DNA origami vector, and the diagram shows the uptake efficiency of the product obtained after modifying the 5' end of the aptamer strand on side C with Apt19s in different cells (scale bar: 40 µm). B represents the triangular DNA origami vector, and the diagram shows the imaging results of the product obtained before and after grafting a type II collagen-targeting peptide onto side B in the joint cavity of OA mice, illustrating its retention effect. C represents the triangular DNA origami vector, and the diagram shows the imaging results of the product obtained before and after grafting a type II collagen-targeting peptide onto side B in different organs of OA mice, illustrating its retention and metabolism. Appendix Figure 4 This diagram illustrates the effects of different drug-loaded triangular DNA origami products on chondrogenic differentiation of mesenchymal stem cells (MSCs). A shows the results of alicin blue staining analysis after MSCs underwent chondrogenic differentiation using triangular DNA origami products loaded with different drugs; B shows the results of toluidine blue staining analysis after MSCs underwent chondrogenic differentiation using triangular DNA origami products loaded with different drugs (scale bar: 200 µm); C shows the results of analyzing the expression of different chondrogenic marker genes (Col II and SOX9) after MSCs underwent chondroid differentiation induced by triangular DNA origami products loaded with different drugs (scale bar: 50 µm). Appendix Figure 5 This diagram illustrates the effects of different drug-loaded triangular DNA origami products on mesenchymal stem cell proliferation, migration, and in vivo stem cell recruitment. A shows the results of alicin blue staining analysis of mesenchymal stem cells after chondrogenic differentiation by different drug-loaded triangular DNA origami products (scale bar: 100 nm); B shows the results of toluidine blue staining analysis of mesenchymal stem cells after chondrogenic differentiation by different drug-loaded triangular DNA origami products (scale bar: 500 nm); C shows the results of expression analysis of different chondrogenic marker genes (ColII and SOX9) after induction of chondroid differentiation by different drug-loaded triangular DNA origami products (scale bar: 100 nm (left); 50 nm (right)). Appendix Figure 6This diagram illustrates the effects of triangular DNA origami products loaded with different drugs on cartilage degradation and synovitis in an OA mouse model. A represents the observation period and detection indicators in the in vivo experiment; B shows the cartilage wear in OA mice after intervention with triangular DNA origami products loaded with different drugs, detected by Safranin-Fix Green staining (scale bar: 100 µm (top); 50 µm (bottom)); C shows the cartilage wear in OA mice after intervention with triangular DNA origami products loaded with different drugs, detected by toluidine blue staining (scale bar: 100 µm (top); 50 µm (bottom)); D shows the synovial hyperplasia in OA mice after intervention with triangular DNA origami products loaded with different drugs, detected by H&E staining (scale bar: 50 µm). Appendix Figure 7 This diagram illustrates the effects of triangular DNA origami products loaded with different drugs on cartilage matrix homeostasis, osteophyte formation, and subchondral bone sclerosis in an OA mouse model. A shows the expression of anabolism (Col II, SOX9) and catabolism (MMP13) in cartilage after intervention with triangular DNA origami products loaded with different drugs, as detected by immunofluorescence (scale bar: 100 µm (top); 25 µm (middle, bottom)). B shows the osteophyte formation in OA mice after intervention with triangular DNA origami products loaded with different drugs, as detected by MicroCT. C shows the subchondral bone sclerosis in OA mice after intervention with triangular DNA origami products loaded with different drugs, as detected by MicroCT. The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0036] The following description, in conjunction with the accompanying drawings and embodiments, further illustrates the DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system, its preparation method, and its applications proposed in this invention: I. Explanation 1. Raw materials and sources The raw materials used in this invention for assembling the target delivery system, namely the aforementioned DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system (hereinafter referred to as tDON-WARS or DNR), are all commercially available products: The backbone chain in this application embodiment is the M13mp18 single-stranded DNA commonly used in the art, which has a fixed publicly available reference sequence (see GenBank: X02513.1 for the specific sequence), and is typically 7249 bp in length. It was purchased from NEB (New England Biolabs), catalog number: N4040S.

[0037] All staple chains that form drug-free triangular DNA origami vectors (hereinafter referred to as tDONs) through base complementary pairing and self-assembly of the backbone chain include group A staple chains as shown in SEQ ID NO:1-65 in the sequence listing, group B staple chains as shown in SEQ ID NO:66-130 in the sequence listing, group C staple chains as shown in SEQ ID NO:131-195 in the sequence listing, linker chains as shown in SEQ ID NO:196-207 in the sequence listing, circular chains as shown in SEQ ID NO:208 in the sequence listing, as well as streptavidin-modified DNA single strands, maleimide-modified DNA single strands, etc. All of the above oligonucleotide sequences were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0038] The SOX9 plasmid, Apt19S aptamer, thiolized WYRGRL targeting peptide, resveratrol, and Tris, acetic acid, EDTA, magnesium acetate, and magnesium chloride in the TAE buffer used in this application can all be commercially available products. Specifically, the biotin-dUTP modified SOX9 plasmid was purchased from Guangzhou Aiji Biotechnology Co., Ltd., batch number: AJX0915-2202; resveratrol was purchased from Med Chem Express (USA), catalog number: No. HY-115595; Tris, acetic acid, and EDTA were purchased from Solarbio Life Sciences, catalog number: T1060; and magnesium chloride was purchased from Maclean Biotechnology Co., Ltd., catalog number: 7786-30-3.

[0039] 2. Sample Naming Rules For ease of differentiation, the following uses a unified name for DNA origami products loaded with different drugs: tDONs: These are triangular DNA origami carriers, with no modified or grafted functional units on their three sides and no loaded with any active pharmaceutical ingredients. They constitute the main framework of the delivery system of this application. tDONs-W: namely, triangular DNA origami vector W, is a monofunctional DNA origami product in which a WYRGRL targeting peptide is grafted only onto a specific sequence on the B side of tDONs; tDONs-A: namely, triangular DNA origami vector A, is a single-function DNA origami product in which the Apt19S aptamer is modified only on a specific sequence of the C side of tDONs; tDONs-WA: or triangular DNA origami vector WA, is a bifunctional DNA origami product in which a WYRGRL targeting peptide is grafted onto a specific sequence on the B side of tDONs and an Apt19S aptamer is modified onto a specific sequence on the C side of tDONs. tDONs-WAR: also known as triangular DNA origami vector WAR, is a trifunctional DNA origami product based on tDONs-WA and loaded with resveratrol. tDONs-WAS: also known as triangular DNA origami vector WAS, is a trifunctional DNA origami product based on tDONs-WA, with a specific sequence on the A side of tDONs containing the SOX9 plasmid. tDON-WARS (DNR): a DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system, is a multifunctional DNA nanorobot based on tDONs-WAS and internally loaded with resveratrol. It is the target DNA origami product of this application.

[0040] 3. Control group design This application employs a univariate progressive control principle, progressively setting up groups from tDONs without functional units—tDONs-W and tDONs-A with single functional units—tDONs-WA with dual functional units—tDONs-WAR and tDONs-WAS with three functional units—to the fully integrated tDONs-WARS (DNR). Combining in vitro cell experiments, in vivo animal experiments, and physicochemical characterization experiments, the independent functions of each functional unit of the delivery system and the synergistic effects of multiple components were verified one by one. Except for the experimental variable, all experimental conditions, such as sample concentration, operating parameters, culture / incubation conditions, and drug administration duration, were kept consistent across all groups. Two baseline controls were included: Blank control (Ctrl): Normal cells / normal laboratory mice, without modeling or vector intervention; Model control group (Saline): An inflammation model was constructed in vitro using 1 ng / mL IL-1β, and an osteoarthritis model was constructed in vivo using the medial meniscus instability (DMM) method. Saline was administered only, without any carrier intervention. Specific Implementation

[0041] In conventional osteoarthritis treatment, exogenous mesenchymal stem cells (MSCs) are administered via intravenous infusion or intra-articular injection. However, due to the low expression of the cell surface chemokine receptor CXCR4, their homing efficiency to cartilage defect sites is reduced. Simultaneously, the chronic low-grade inflammatory microenvironment within the joint cavity is enriched with aging-related secreted phenotypic factors, which limits the differentiation potential of stem cells at the injury site and increases the risk of premature aging. Furthermore, the insufficient natural recruitment capacity of endogenous MSCs makes it difficult to achieve in-situ repair of cartilage defects, thus affecting the overall effect of cartilage tissue regeneration.

[0042] To address the aforementioned technical problems, one aspect of this application proposes a DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system (i.e., the aforementioned tDONs-WARS (DNR), or simply a delivery system, which will not be elaborated further below). This delivery system includes: The triangular DNA origami vector (i.e., the tDONs mentioned above, which will not be described again below) is formed by the self-assembly of a single-stranded DNA backbone and multiple staple chains. The three sides of the DNA origami vector are divided into side A, side B, and side C in sequence. SOX9 plasmid binds to side A of the DNA origami vector; Type II collagen-targeting peptides are grafted onto the B side of the DNA origami vector. Stem cell targeting aptamers are modified on the C-side of a DNA origami vector. Anti-inflammatory and antioxidant small molecule drugs (such as resveratrol) are loaded inside DNA origami vectors.

[0043] For ease of understanding, the following explains some key terms in this embodiment: DNA origami is a technique that utilizes the self-assembly of DNA molecules to form pre-defined nanostructures. By designing sequences of long single-stranded DNA backbones and multiple short staple chains (typically 20–60 bp in length), these strands are precisely folded into two-dimensional or three-dimensional nanostructures of specific shapes and sizes through complementary base pairing. This technique provides highly addressable surfaces that can be precisely anchored and modified with various functional units.

[0044] "Endogenous stem cell recruitment" refers to the process by which the body's own stem cells (such as mesenchymal stem cells) are attracted to migrate to the site of injury through specific biological signals or physical structures. This differs from exogenous stem cell transplantation, which aims to utilize the body's own repair potential to improve treatment efficiency.

[0045] The "Cartilage Repair Delivery System" is a carrier system designed to efficiently and specifically deliver active substances with cartilage repair functions (such as genes, drugs, and cytokines) to damaged cartilage tissue to promote cartilage regeneration and repair. It overcomes the problems of low drug bioavailability and poor targeting in traditional treatment methods.

[0046] The triangular DNA origami vector, or tDONs, is the core framework structure of the delivery system in this application. Its overall structure is triangular, as shown below. Figure 1As shown, in counter-clockwise order, its three sides are named side A (base of the triangle), side B (right side of the triangle), and side C (left side of the triangle). They are designed with different chemical modification sites for binding or grafting different functional units, thereby achieving synergistic effects of multifunctional units. Furthermore, this specific shape design contributes to its stability in the biological environment and the spatial arrangement of functional units. As one implementation, the single-stranded DNA backbone can be selected from single-stranded DNA commonly used in DNA origami, such as M13 phage genomic DNA or other single-stranded DNA with similar properties. The staple chain can be composed of chemically synthesized short DNA oligonucleotides, whose sequences are designed to be complementary to specific regions on the backbone chain to ensure precise folding. By controlling the annealing conditions of PCR, such as slow cooling in a buffer containing an appropriate ion concentration, efficient self-assembly of the backbone chain and staple chain can be achieved, forming a stable triangular nanostructure.

[0047] "Self-assembly" refers to the process by which the backbone chain and auxiliary chains, including staple chains and type II collagen targeting peptides, spontaneously form an ordered structure through intermolecular interactions (such as base pairing, hydrogen bonds, and van der Waals forces) without external intervention. In DNA origami, the backbone chain and staple chains achieve precise self-assembly through base pairing in a buffer system.

[0048] The SOX9 plasmid is a circular DNA molecule containing the SOX9 gene. SOX9 is a key transcription factor in chondrogenesis; its expression induces mesenchymal stem cells to differentiate into chondrocytes and promotes cartilage matrix synthesis. The SOX9 plasmid is bound to side A of the DNA origami vector to provide recruited mesenchymal stem cells with a gene-induced signal for chondrogenic differentiation. As one implementation, the SOX9 plasmid can be bound to side A via various non-covalent or covalent mechanisms. For example, side A can be pre-modified with molecules of specific affinity, such as biotin or streptavidin, and then the SOX9 plasmid can be anchored to side A through biotin-streptavidin specific binding. Alternatively, a specific DNA sequence can be designed on side A to bind to a complementary sequence on the SOX9 plasmid via base pairing.

[0049] The type II collagen-targeting peptide is a short peptide sequence that specifically recognizes and binds to type II collagen. Type II collagen (Col II) is the main extracellular matrix component of cartilage tissue; therefore, this peptide can guide the delivery system to precisely target cartilage injury sites. The type II collagen-targeting peptide is grafted onto the B-side of the DNA origami carrier to guide the delivery system to specifically target cartilage injury areas rich in type II collagen. Endogenous mesenchymal stem cells are recruited to the cartilage surface using a stem cell-targeting aptamer (e.g., the Apt19S aptamer), thereby prolonging the residence time of the delivery system within the joint and increasing its local concentration and efficacy. As one implementation, the type II collagen-targeting peptide can be grafted onto the B-side via a chemical coupling reaction. For example, the B-side can be pre-modified with reactive groups, such as maleimide or N-hydroxysuccinimide (NHS), and then the type II collagen-targeting peptide with thiol or amino groups can be covalently linked to the B-side. Alternatively, the DNA sequence on the B side can be designed to link with a peptide with a specific DNA sequence through DNA hybridization.

[0050] A stem cell targeting aptamer is a single-stranded DNA or RNA molecule capable of binding to specific stem cell surface receptors with high specificity and high affinity. Through this aptamer, the delivery system can actively recognize and capture target stem cells, achieving efficient cell recruitment. The stem cell targeting aptamer is modified to the C-side of the DNA origami vector to specifically recognize and capture endogenous mesenchymal stem cells within the joint cavity, thereby achieving efficient stem cell recruitment. The recruited endogenous stem cells can efficiently take up the delivery system loaded with the SOX9 plasmid, thereby directly promoting cartilage formation. As one implementation method, the stem cell targeting aptamer can be modified to the C-side in various ways. For example, a DNA sequence can be pre-designed on the C-side to form a stable link with an aptamer carrying a complementary sequence through base pairing. Alternatively, a chemically active group can be pre-modified on the C-side, and then the aptamer can be covalently linked to the C-side.

[0051] Resveratrol (Res), an anti-inflammatory and antioxidant small molecule drug, is a natural polyphenol compound widely reported to possess various biological activities, including antioxidant, anti-inflammatory, anti-aging, and maintenance of stem cell stemness. Recent studies have shown that resveratrol (Res) can maintain the stemness of mesenchymal stem cells (MSCs) and delay their aging or premature differentiation through multiple mechanisms: on the one hand, by activating stemness-related signaling pathways, such as effectively activating Sirt1, and further inhibiting premature differentiation of MSCs, thereby prolonging their proliferative capacity; on the other hand, by regulating Wnt signaling to promote the self-renewal of MSCs while inhibiting their excessive adipogenic differentiation. After the DNA origami carrier framework of the delivery system is gradually degraded, the slowly released Res can enhance the proliferation and migration capacity of stem cells in a low-inflammatory microenvironment. Loading Res into the delivery system aims to achieve sustained release of active substances, improve the damaged microenvironment, and enhance the survival and differentiation potential of stem cells recruited by the delivery system. As one implementation method, resveratrol can be loaded through physical adsorption, encapsulation, or non-covalent binding to specific sites within a DNA origami carrier. For example, the hydrophobic regions or cavity structures within the DNA origami carrier can be utilized to allow resveratrol molecules to diffuse and stably bind within the carrier through simple mixing and incubation. Alternatively, specific DNA sequences can be designed within the carrier to form weak interactions with resveratrol molecules, thereby achieving loading. In other embodiments, anti-inflammatory and antioxidant small molecule drugs can also be selected from small molecule drug components with bone and joint repair effects, such as curcumin, icariin, tea polyphenols, and ginkgo biloba extract; this application does not specifically limit this.

[0052] In summary, the delivery system provided in this application integrates multiple functions such as precise targeting, efficient recruitment, directed differentiation induction, and microenvironment regulation into a single nanocarrier platform, forming a synergistic overall solution. This integrated and multifunctional design effectively solves the bottleneck problems in existing technologies, such as low efficiency of exogenous stem cell transplantation, weak recruitment capacity of endogenous stem cells, and insufficient chondrogenic differentiation potential of recruited stem cells in the inflammatory microenvironment, making it difficult to achieve efficient in-situ cartilage repair. This provides an innovative and efficient strategy for the treatment of cartilage injury diseases such as osteoarthritis.

[0053] Furthermore, in some embodiments, the backbone chain comprises M13mp18 single-stranded DNA.

[0054] Please refer to the sequence list of this application. The staple chain includes: As shown in SEQ ID NO:1~65, the A-group staple chains are self-assembled with the backbone chain through base complementary pairing to form the A-side.

[0055] As shown in SEQ ID NO:66~130, the B-group staple chain forms the B-side by self-assembling with the backbone chain through complementary base pairing.

[0056] As shown in SEQ ID NO:131 to SEQ ID NO:195, the C-group staple chain forms the C-side by self-assembling with the backbone chain through complementary base pairing.

[0057] Specifically, the M13mp18 single-stranded DNA is a long-chain DNA molecule widely used in DNA nanotechnology, with a known sequence and good biocompatibility. As the core supporting structure of the DNA origami carrier, the M13mp18 single-stranded DNA provides sufficient length and a specific base sequence to achieve precise base complementarity pairing with multiple staple chains, thereby forming a pre-defined triangular DNA origami carrier structure.

[0058] Groups A through C are a carefully designed series of short-chain DNA molecules whose sequences are complementary to specific regions of the M13mp18 single-stranded DNA backbone. These staple chains, through precise binding to the backbone, fold and fix the backbone into side A of a triangular DNA origami vector. They not only define the physical boundaries and shapes of sides A, B, and C, but also provide specific anchoring regions for subsequent binding to the SOX9 plasmid, ensuring precise spatial positioning of functional molecules; specific sites for grafting type II collagen-targeting peptides, ensuring accurate presentation of the target peptides on the vector; and specific sites for modifying stem cell-targeting aptamers, ensuring accurate presentation of the aptamers on the vector, thereby achieving effective recruitment of stem cells. The sequence design of the staple chains can be adjusted according to the desired DNA origami structure shape and binding sites of functional molecules to accommodate different backbones or achieve different structural features. For example, specific chemical modification groups can be introduced onto the staple chains to facilitate subsequent coupling of functional molecules.

[0059] This application's approach introduces M13mp18 single-stranded DNA as the core backbone, leveraging its long-chain characteristics to provide a stable physical basis for constructing the DNA origami structure. By designing three specific sets of staple chain sequences, modular programming and precise self-assembly of the three structural sides of the triangular DNA origami vector were achieved. Set A staple chains, through specific base complementarity with the backbone chain, spatially define the structure of side A, providing a dedicated anchor point for subsequent binding of the SOX9 plasmid. Set B staple chains, through specific sequence arrangements, construct side B, providing spatial support for the grafting of type II collagen-targeting peptides. Set C staple chains, through precise pairing with the backbone chain, form side C, providing a positioning region for the modification of stem cell-targeting aptamers. This sequence-programmed self-assembly method not only ensures the integrity of the triangular vector structure but, more importantly, achieves spatial decoupling of functional partitions. This allows plasmids, targeting peptides, and aptamers to be precisely placed on different borders of the vector, thus avoiding spatial interference between functional components and providing structural assurance for the subsequent efficient recruitment and directed differentiation of endogenous stem cells.

[0060] Furthermore, in some embodiments, please refer to the sequence listing of this application, the group A staple chain includes a plasmid capture sub-chain as shown in SEQ ID NO:23 and SEQ ID NO:53, wherein the 5' end of the plasmid capture sub-chain is modified with streptavidin (SA).

[0061] Correspondingly, the 3' end of the SOX9 plasmid is biotin-labeled and binds to the A side of the DNA origami vector through the affinity properties of biotin and streptavidin.

[0062] This application's scheme involves introducing a specially designed plasmid capture strand into the A side of a triangular DNA origami vector, modifying its 5' end with streptavidin, and simultaneously labeling the 3' end of the SOX9 plasmid with biotin. This leverages the extremely high affinity between biotin and streptavidin to achieve precise and stable binding of the SOX9 plasmid to the A side of the DNA origami vector. Specifically, during the self-assembly of the DNA origami vector, the plasmid capture strand from the A group of staples is integrated into the A side of the DNA origami vector, with its exposed streptavidin at the 5' end providing multiple high-affinity binding sites. Subsequently, the biotin-labeled SOX9 plasmid specifically binds to these streptavidin sites through the biotin at its 3' end. This binding mechanism utilizes the ultra-high affinity of the biotin-streptavidin interaction, ensuring that the SOX9 plasmid can be firmly anchored to the DNA origami vector in a predetermined direction and position, greatly improving the specificity and stability of the plasmid loading. This precise anchoring method not only avoids random shedding or degradation of the plasmid during delivery but also ensures that the SOX9 plasmid maintains its biological activity after reaching the target endogenous stem cells, thereby effectively exerting its gene transduction function and promoting the differentiation of stem cells into hyaline cartilage. This binding method, combined with the precise nanostructure of the DNA origami vector itself, allows the SOX9 plasmid to be presented in a highly ordered manner, which is crucial for its delivery efficiency in vivo and the gene transduction effect on targeted cells.

[0063] In one specific implementation, when preparing the DNA origami vector, a plasmid capture strand containing sequences as shown in SEQ ID NO:23 and SEQ ID NO:53 can be synthesized in advance, and streptavidin can be chemically coupled to its 5' end. The SOX9 plasmid can be obtained by polymerase chain reaction (PCR) amplification or enzyme digestion and ligation, and biotinylated deoxynucleotides (e.g., Biotin-dUTP) can be added to the 3' hydroxyl end of the plasmid via a terminal deoxynucleotidyl transferase (TdT) catalytic reaction to achieve biotin labeling. After the DNA origami vector self-assembles, the streptavidin-modified DNA origami vector and the biotinylated SOX9 plasmid are mixed and incubated in an appropriate buffer (e.g., TAE buffer containing magnesium ions). During incubation, the biotinylated SOX9 plasmid spontaneously and specifically binds to the streptavidin site on the A side of the DNA origami vector. After binding is complete, the product can be purified by methods such as ultrafiltration and centrifugation to remove unbound SOX9 plasmids, thereby obtaining DNA origami products loaded with the functional unit of SOX9 plasmid.

[0064] Furthermore, in some embodiments, the biotin includes Biotin-dUTP biotin. Biotin-dUTP biotin is a nucleotide derivative formed by combining biotin with deoxyuridine triphosphate. Biotin is known for its extremely high affinity for streptavidin, one of the strongest non-covalent interactions in nature. Deoxyuridine triphosphate is a substrate for DNA synthesis and can be incorporated into the DNA chain by DNA polymerase. Therefore, Biotin-dUTP biotin can be efficiently and specifically incorporated into the 3' end or interior of DNA molecules (such as SOX9 plasmids) via enzymatic reactions, thereby achieving biotinylation labeling of DNA molecules. This labeling method is site-controllable, avoiding random modification of functional regions of DNA molecules and ensuring that the labeled DNA molecules retain their biological activity. In addition, the introduction of Biotin-dUTP biotin does not significantly alter the physicochemical properties of DNA molecules, thus ensuring good biocompatibility.

[0065] Furthermore, in some embodiments, please refer to the sequence listing of this application, the group B staple chain includes a peptide capture sub-chain as shown in SEQ ID NO:88 and SEQ ID NO:118, wherein the 5' end of the peptide capture sub-chain is modified with maleimide.

[0066] Correspondingly, the type II collagen-targeting peptide is a thiolized WYRGRL-targeting peptide (i.e., WYRGRL-SH), which is grafted onto the B side of the DNA origami carrier through the covalent cross-linking property of maleimide and thiol.

[0067] Specifically, the peptide capture strands are carefully designed DNA oligonucleotide chains whose main function is to provide a specific anchoring point on the B-side of the DNA origami carrier. These strands, through base complementarity pairing with the backbone chain and other staple chains, can be precisely integrated into the three-dimensional structure of the DNA origami carrier, thereby ensuring that the subsequently grafted target peptides can be located on the B-side of the DNA origami carrier.

[0068] The 5' end modification of the peptide trapping chain with maleimide refers to the covalent attachment of a maleimide group to the 5' end of the peptide trapping chain. Maleimide is a highly reactive chemical group that can undergo a specific Michael addition reaction with molecules containing a thiol group (-SH) to form a stable thioether bond. This modification ensures high chemoselectivity and stability for the subsequent attachment of the targeting peptide. In practice, the maleimide group can be introduced during oligonucleotide synthesis by introducing a maleimide-modified phosphoramide monomer, or introduced after oligonucleotide synthesis by reacting with a crosslinking agent containing a maleimide group.

[0069] The type II collagen-targeting peptide is a thiolized WYRGRL targeting peptide, a peptide with a specific amino acid sequence that has been shown to specifically recognize and bind to type II collagen. Type II collagen is a major component of articular cartilage, thus this peptide endows the delivery system with the ability to target cartilage tissue. Simultaneously, this peptide is thiolized, meaning a thiol group (-SH) is introduced into its molecular structure. This thiol group is a key reaction site for covalent cross-linking with maleimide groups, ensuring that the targeting peptide can firmly attach to the DNA origami carrier. Thiolization can be achieved by introducing cysteine ​​residues during peptide synthesis or by chemically modifying the thiol group into non-cysteine ​​peptides.

[0070] This application's approach precisely integrates maleimide-modified peptide capture strands into the B-side of a DNA origami carrier, allowing maleimide groups to be arranged in a predetermined spatial pattern on the DNA origami carrier surface. Based on this, a highly specific covalent cross-linking reaction is used between a thiolized WYRGRL targeting peptide (i.e., WYRGRL-SH) and these maleimide groups to firmly graft the targeting peptide onto the B-side of the DNA origami carrier. This design ensures that the type II collagen targeting peptide is not only positioned on the B-side of the DNA origami carrier but also, through stable covalent bonding, is not easily detached in the complex joint cavity microenvironment, thus maintaining the delivery system's continuous targeting capability to cartilage tissue. This precise positioning and stable connection mechanism effectively solves the technical challenges of unstable connection, low efficiency, and poor biocompatibility between the targeting peptide and the DNA carrier, significantly improving the targeting efficiency and stability of the delivery system in vivo.

[0071] Through the above technical solution, this application achieves stable, efficient, and biocompatible grafting of type II collagen-targeting peptides onto DNA origami carriers. This covalent cross-linking method ensures that the targeting peptides are not easily detached or inactivated in the complex joint cavity microenvironment, thereby significantly enhancing the delivery system's ability to target and bind to cartilage tissue. This is of great significance for achieving precise recruitment of endogenous stem cells to cartilage injury sites and promoting their in situ repair, effectively overcoming the limitations of low targeting efficiency and poor carrier stability in traditional delivery methods.

[0072] Furthermore, in some embodiments, please refer to the sequence listing of this application, the C group staple chain includes aptamer-modified sub-chains as shown in SEQ ID NO:153 and SEQ ID NO:183.

[0073] Correspondingly, the stem cell targeting aptamer includes the Apt19S aptamer, wherein the Apt19S aptamer is modified at the 5' end of the aptamer modified subchain to form an aptamer chain as shown in SEQ ID NO:209 and SEQ ID NO:210.

[0074] Specifically, the aptamer-modified aptamer is a special functional staple chain within the C-group of staple chains, primarily serving as a linker for stem cell targeting aptamers. This aptamer is specially designed so that its sequence can perform base-complementary pairing with the backbone chain of the DNA origami carrier, thus stably integrating itself onto the C-side of the DNA origami carrier. Simultaneously, this aptamer also possesses specific modification sites to connect the stem cell targeting aptamer to the DNA origami carrier, achieving specific recognition and binding to target cells. For example, the aptamer can be designed to contain a chemical modification group at a specific position, or its sequence itself can serve as a binding site for the aptamer. A stem cell targeting aptamer is a nucleic acid molecule, typically single-stranded DNA or RNA, capable of specifically recognizing and binding to specific molecules (such as receptors and antigens) on the surface of stem cells. Its function is similar to that of an antibody, but it offers advantages such as higher stability, lower immunogenicity, and easier synthesis. Stem cell targeting aptamers achieve precise capture and recruitment of stem cells through high-affinity binding to markers on the stem cell surface.

[0075] The Apt19S aptamer is a known single-stranded DNA aptamer with high specificity and affinity for mesenchymal stem cells (MSCs). Its unique sequence and three-dimensional structure enable it to precisely recognize and bind to specific receptors on the surface of MSCs, thereby achieving effective targeting and recruitment of MSCs. This aptamer acts as a "chemical antibody," playing a crucial role in cell recognition and capture in biological delivery systems. The 5' end of the aptamer modification strand is the specific site where the stem cell-targeting aptamer attaches to the aptamer modification strand. Choosing to modify the 5' end is typically to ensure that the aptamer maintains its correct spatial conformation and biological activity after ligation, thus effectively binding to the target stem cells. Furthermore, 5' end modification facilitates stable ligation of the aptamer to the aptamer modification strand through various methods such as chemical coupling or enzymatic ligation. Besides the 5' end, modifications can also be considered at the 3' end or internal bases, but it must be ensured that the targeting function of the aptamer and the self-assembly stability of the DNA origami vector are not affected. An aptamer chain is a complex chain formed by modifying the 5' end of an aptamer modified daughter chain with a stem cell-targeting aptamer (such as the Apt19S aptamer). This complex chain is the direct executor of the DNA origami vector's function of targeted recruitment of stem cells. It combines the targeting ability of the aptamer with the structural stability of the DNA origami vector, ensuring that the aptamer is presented on the surface of the DNA origami vector in the correct orientation and density, thereby efficiently recognizing and capturing target stem cells.

[0076] Through the above technical solution, this application effectively solves the problem of low recruitment efficiency of endogenous stem cells in traditional methods. By precisely modifying the C-side of the DNA origami vector with the Apt19S aptamer, this delivery system achieves high specificity and high affinity targeting of intra-articular mesenchymal stem cells. This "chemical antibody"-like recruitment mechanism enables the delivery system to actively and efficiently capture free mesenchymal stem cells and enrich them at the cartilage injury site. This not only significantly increases the number of stem cells reaching the injury site, providing sufficient "seed cells" for cartilage repair, but also avoids interference with non-target cells due to the specificity of the recruitment process, improving the precision of treatment. Therefore, this approach greatly improves the efficiency and success rate of in-situ repair of cartilage defects using endogenous stem cells, providing a more effective approach for the treatment of cartilage injury diseases such as osteoarthritis.

[0077] Furthermore, in some embodiments, this application further proposes that the Apt19S aptamer be modified with a Cy5 fluorescent group.

[0078] Cy5 fluorophores are commonly used near-infrared fluorescent dyes, primarily functioning to provide optical tracer signals, enabling labeled molecules to fluoresce under specific excitation light for visual detection. This application modifies the Apt19S aptamer with a Cy5 fluorophore, imbuing the aforementioned DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system with fluorescence tracer functionality. Specifically, when the delivery system is introduced into the body, its C-side modified Apt19S aptamer specifically recognizes and binds to target endogenous stem cells. Because the Apt19S aptamer is linked to a Cy5 fluorophore, once the aptamer successfully binds to stem cells, the Cy5 fluorophore accumulates on or near the stem cell surface. Under external excitation, the Cy5 fluorophore emits a detectable fluorescence signal, allowing researchers to observe and track the distribution, migration, and binding of the delivery system to stem cells in real time using fluorescence imaging technology. This visualization capability not only verified the targeted recruitment efficiency of the Apt19S aptamer, but also provided an intuitive basis for assessing the enrichment of the entire delivery system at the cartilage defect site, ensuring the effective delivery of subsequent therapeutic components.

[0079] The following is a specific example illustrating a particular implementation method. The Apt19S aptamer can be pre-modified with an amino group at its 5' or 3' end. Subsequently, activated Cy5-NHS ester (N-hydroxysuccinimide ester) is reacted with the amino-containing Apt19S aptamer in a suitable buffer, covalently linking the Cy5 fluorescent group to the Apt19S aptamer through the formation of a stable amide bond. After the reaction, the Apt19S-Cy5 modified aptamer is purified by methods such as gel electrophoresis or high-performance liquid chromatography. This modified Apt19S-Cy5 aptamer can then be modified at the 5' end of the aptamer modifying strand in the manner described above to form the aptamer strand, and finally integrated into the C-side of a triangular DNA origami carrier through a DNA origami self-assembly process. In practical applications, such as in animal models, the joint sites injected with the delivery system can be scanned using an in vivo fluorescence imaging system. By utilizing the excitation and emission wavelength characteristics of Cy5, the enrichment of the delivery system in the articular cartilage defect area and its binding efficiency with endogenous stem cells can be monitored in real time.

[0080] Through the aforementioned technical solution, this delivery system achieves real-time visual tracking capabilities, effectively addressing the technical challenges of monitoring stem cell aptamer binding and assessing the distribution and enrichment efficiency of the delivery system in complex biological environments. This allows researchers to precisely track the stem cell recruitment process and intuitively evaluate the targeting efficiency and enrichment level of the delivery system at cartilage defect sites, thus providing important experimental evidence and monitoring methods for optimizing treatment plans and improving cartilage repair outcomes.

[0081] One aspect of this application discloses a method for preparing an endogenous stem cell recruitment and cartilage repair delivery system based on DNA origami, for use in preparing the aforementioned delivery system, namely tDONs-WARS or DNR. The preparation method includes the following steps: S1: The backbone chain and the auxiliary chain containing the modified staple chain, type II collagen targeting peptide, and stem cell targeting aptamer are placed in a buffer solution, annealed and cooled to room temperature to complete self-assembly. After removing the excess auxiliary chain, a triangular DNA origami vector WA with type II collagen targeting peptide grafted on side B and stem cell targeting aptamer modified on side C is obtained.

[0082] S2: Place the DNA origami vector WA and the SOX9 plasmid containing the free complementary sequence in a buffer solution, anneal and cool to room temperature to obtain a triangular DNA origami vector WAS with the SOX9 plasmid bound to side A.

[0083] S3: The DNA origami carrier WAS was mixed with anti-inflammatory and antioxidant small molecule drugs and incubated. After purification, an endogenous stem cell recruitment and cartilage repair delivery system based on DNA origami was prepared.

[0084] The preparation method provided in this application integrates type II collagen-targeting peptides, stem cell-targeting aptamers, SOX9 plasmids, and anti-inflammatory and antioxidant small molecule drugs (such as resveratrol) onto specific sides of a triangular DNA origami carrier. This achieves spatial isolation and synergistic effects of functional molecules, forming an intelligent delivery system that integrates precise targeting, efficient recruitment, directional induction, and microenvironment improvement. Specifically, in step S1, the backbone chain and the modified staple chain are annealed and self-assembled in a magnesium-containing TAE buffer. This method can precisely control the geometry of the DNA origami carrier, ensuring that the type II collagen-targeting peptides and stem cell-targeting aptamers are site-specifically modified on sides B and C of the carrier. This lays the structural foundation for subsequent precise localization of cartilage injury sites and efficient recruitment of endogenous stem cells. Since type II collagen is the main extracellular matrix component of cartilage tissue, this targeting peptide can guide the delivery system to specifically bind to the damaged cartilage area; while the stem cell targeting aptamer can recognize and bind to the surface receptors of endogenous mesenchymal stem cells with high affinity, realize the active recruitment of stem cells, and significantly increase the local concentration of stem cells in the damaged area.

[0085] In step S2, the vector WA and the SOX9 plasmid are annealed together using complementary sequences. This base-pairing-based ligation method is highly specific and stable, ensuring that the SOX9 plasmid can be stably loaded onto the A side of the vector. As a key transcription factor in chondrogenesis, SOX9 plasmid can effectively activate the chondrogenesis pathway through gene transduction after stem cells are recruited to the injury site. This promotes the directed differentiation of stem cells towards hyaline cartilage, preventing the formation of fibrocartilage or hypertrophic cartilage, thereby ensuring the quality of cartilage repair.

[0086] In step S3, the WAS carrier is mixed and incubated with an anti-inflammatory and antioxidant small molecule drug (such as resveratrol), and the resulting product is purified to obtain the delivery system. This loading method utilizes the internal spatial structure of the DNA origami carrier to protect resveratrol from environmental degradation and ensure effective drug loading. Resveratrol, as a small molecule compound with antioxidant, anti-inflammatory, and stem cell-maintaining properties, can improve the chronic inflammatory microenvironment within the joint cavity, reduce oxidative stress, and enhance the survival rate and chondrogenic differentiation potential of recruited stem cells, thereby providing sustained impetus for cartilage repair.

[0087] Through the above technical solution, the embodiments of this application achieve in situ, efficient recruitment, directed differentiation, and continuous repair of endogenous stem cells at the site of cartilage injury. Compared with traditional methods, this system eliminates the shortcomings of exogenous stem cell infusion and instead utilizes the body's own endogenous stem cells. Through the synergistic effects of precise targeting, efficient recruitment, directed induction, and microenvironment regulation, it significantly improves the efficiency and quality of cartilage repair, providing an innovative and efficient strategy for the treatment of cartilage injury diseases such as osteoarthritis.

[0088] Furthermore, in some embodiments, in step S1 of the preparation method, the backbone chain is M13mp18 single-stranded DNA, which is mixed with the auxiliary chain at a molar ratio of 1:(8-12) and added to the buffer solution. The annealing conditions are slow cooling from 95°C to room temperature, and the entire annealing process is not less than 12 h.

[0089] Specifically, the molar ratio of the backbone strand to the auxiliary strands is one of the key parameters for successful DNA origami self-assembly. The number of auxiliary strands (including staple chains from groups A to C as shown in SEQ ID NO:1–195, at least 12 linker strands as shown in SEQ ID NO:196–207, and at least one loop strand as shown in SEQ ID NO:208) needs to be sufficient to completely cover the backbone strand and form the desired three-dimensional structure. A molar ratio of 1:(8–12) indicates that there is a sufficient amount of auxiliary strands relative to the backbone strand, which helps improve the efficiency of self-assembly and the integrity of the structure, reducing structural defects caused by insufficient auxiliary strands. In practice, this molar ratio can be adjusted according to the length of the backbone strand, the design of the auxiliary strands, and the complexity of the target origami structure. For example, a molar ratio of 1:8, 1:10, or 1:12 can be used to optimize the assembly effect, with 1:10 being the preferred ratio.

[0090] Annealing is performed by slowly cooling from 95°C to room temperature. Annealing is a core step in the PCR process of DNA origami self-assembly, where the DNA strands undergo denaturation, annealing, and extension through temperature control within the PCR program. Heating the system to 95°C aims to completely unwind the DNA backbone and auxiliary strands, eliminating any pre-existing secondary structures. Subsequently, slow cooling to room temperature provides sufficient time for base pairing and structural rearrangement of the DNA strands, resulting in the thermodynamically most stable origami structure. The cooling rate can also be precisely controlled using a programmable thermal cycler, for example, decreasing by 0.1°C or 0.5°C per minute.

[0091] Furthermore, the duration of the annealing process is crucial to the assembly quality of the DNA origami. Sufficiently long annealing times allow the DNA strands to fully explore the conformational space, perform necessary error corrections and rearrangements, thereby overcoming kinetic traps and forming highly uniform and stable DNA origami structures. Insufficient annealing time may lead to misfolding or incomplete assembly. In other embodiments, the annealing time can also be adjusted according to the specific DNA sequence, the complexity of the origami structure, and the desired assembly efficiency; for example, it can be extended to 16 hours or 24 hours to further improve assembly accuracy.

[0092] Through the above technical solution, this application ensures that the auxiliary strand fully covers the backbone strand by precisely controlling the molar ratio of M13mp18 single-stranded DNA to the helper strand at 1:(8-12), thereby forming a well-structured and defect-free DNA origami backbone. Simultaneously, the annealing process, employing slow cooling from 95°C to room temperature for at least 12 hours, allows sufficient time for precise base pairing and structural rearrangement of the DNA strand, significantly improving the assembly uniformity and structural stability of the DNA origami vector. This optimized self-assembly process provides a solid foundation for the stable binding of the subsequent SOX9 plasmid, type II collagen-targeting peptide, and stem cell-targeting aptamer, thus ensuring that the entire delivery system can efficiently and stably recruit endogenous stem cells and promote cartilage repair.

[0093] Furthermore, in some embodiments, this application further proposes that in step S1 and / or step S2, the buffer solution comprises Tris at a concentration of 30–50 mmol / L, acetic acid at a concentration of 18–22 mmol / L, EDTA at a concentration of 1–3 mmol / L, and magnesium acetate at a concentration of 10–15 mmol / L, and the pH value of the buffer solution is 8.0–8.5.

[0094] Specifically, Tris, or tris(hydroxymethyl)aminomethane, is a commonly used biological buffer agent with good buffering capacity in aqueous solutions, effectively maintaining the pH stability of the solution. In this application, a Tris concentration of 30–50 mmol / L is used as the main component of the buffer system, providing a stable pH environment for the self-assembly of DNA origami and preventing DNA structural instability or degradation due to pH fluctuations. In practice, the Tris concentration can be 30 mmol / L, 35 mmol / L, 40 mmol / L, 45 mmol / L, 50 mmol / L, etc., with 40 mmol / L being preferred.

[0095] Acetic acid, a weak acid, forms the Tris-acetic acid buffer system together with Tris. In this application, acetic acid at a concentration of 18–22 mmol / L synergistically enhances the buffering capacity of the buffer solution near pH 8.3, ensuring the precise maintenance of the weakly alkaline environment required for DNA origami self-assembly. Specifically, the acetic acid concentration can be 18 mmol / L, 19 mmol / L, 20 mmol / L, 21 mmol / L, 22 mmol / L, etc., with 20 mmol / L being preferred.

[0096] EDTA, or ethylenediaminetetraacetic acid, is a potent metal ion chelating agent. In this application, EDTA at a concentration of 1–3 mmol / L can effectively chelate divalent or polyvalent metal ion impurities, such as copper and iron ions, that may be present in the solution. These ions may catalyze the oxidative degradation of DNA or interfere with the interactions between DNA strands. Through chelation, EDTA protects the integrity of DNA and reduces potential negative impacts on the self-assembly process. Specifically, the EDTA concentration can be 1 mmol / L, 2 mmol / L, 3 mmol / L, etc., with 2 mmol / L being preferred.

[0097] Magnesium acetate, as a source of magnesium ions, plays a crucial role in DNA origami technology. In this application, a magnesium acetate concentration of 10–15 mmol / L provides an appropriate amount of magnesium ions, which effectively neutralize the electrostatic repulsion between negatively charged phosphate groups on the DNA backbone. By shielding these electrostatic repulsions, the magnesium ions allow the DNA strands to fold more tightly and promote precise self-assembly between multiple staple strands and the backbone strand according to the base complementary pairing principle, thereby ensuring the regularity and stability of the triangular DNA origami carrier structure. Specifically, the magnesium acetate concentration can be, for example, 10 mmol / L, 10.5 mmol / L, 11 mmol / L, 12.5 mmol / L, 13 mmol / L, 14.5 mmol / L, 15 mmol / L, etc., with 12.5 mmol / L being preferred.

[0098] The magnesium-containing TAE buffer solution has a pH of 8.0–8.5. This slightly alkaline environment is crucial for the stability and self-assembly efficiency of DNA origami. Under this pH condition, the DNA phosphate backbone maintains a stable negative charge, while avoiding DNA hydrolysis or denaturation that may occur in excessively acidic or alkaline environments, providing an ideal chemical environment for precise folding of DNA origami. In specific implementations, the pH of the magnesium-containing TAE buffer solution can be, for example, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5, with 8.3 being the preferred value.

[0099] Through the aforementioned technical solution, this application significantly improves the self-assembly efficiency and structural stability of the DNA origami carrier by precisely controlling the chemical environment of the magnesium-containing TAE buffer during the preparation of the DNA origami delivery system. This optimized buffer system effectively avoids problems such as DNA folding errors and low yield, ensuring that functional molecules (such as SOX9 plasmids, type II collagen-targeting peptides, and stem cell-targeting aptamers) can bind efficiently and stably to the DNA origami carrier, thereby maintaining the structural integrity and bioactivity of the delivery system in subsequent applications. This provides a reliable carrier basis for achieving efficient endogenous stem cell recruitment and cartilage repair, thereby enhancing the repair efficacy of the entire delivery system in treating osteoarthritis.

[0100] Furthermore, in some embodiments, this application further proposes that in step S1 of the preparation method, the concentration of the M13mp18 single-stranded DNA is 3-7 nmol / L; and / or, the concentration of the auxiliary strand is 24-84 nmol / L.

[0101] The concentration of M13mp18 single-stranded DNA, as the backbone strand in DNA origami structures, along with its auxiliary strands, has a decisive impact on the efficiency of the self-assembly process and the quality of the product. This application sets the concentration of M13mp18 single-stranded DNA at 3–7 nmol / L to ensure an appropriate template density of the backbone strand in the reaction system. This avoids non-specific intermolecular aggregation due to excessive concentration or slow self-assembly kinetics due to insufficient concentration, thus affecting the formation of the final product. In practice, the concentration can be set to, for example, 3 nmol / L, 4 nmol / L, 5 nmol / L, 6 nmol / L, or 7 nmol / L, depending on the complexity of the DNA origami structure and the desired yield, to adapt to different experimental conditions and objectives. 5 nmol / L is preferred.

[0102] The concentration of the auxiliary chain is set to 24–84 nmol / L, so that the molar ratio of the auxiliary chain to the backbone chain is maintained at a specific level (i.e., satisfying the aforementioned limitation of 1:(8–12)). This excess auxiliary chain design can drive the base complementary pairing reaction in the direction of generating a complete origami structure, ensuring that each staple chain can fully bind to the corresponding position of the backbone chain. In practice, the concentration of auxiliary chains can be set to, for example, 24 nmol / L, 27 nmol / L, 30 nmol / L, 32 nmol / L, 33 nmol / L, 36 nmol / L, 40 nmol / L, 44 nmol / L, 45 nmol / L, 48 nmol / L, 50 nmol / L, 54 nmol / L, 56 nmol / L, 60 nmol / L, 63 nmol / L, 70 nmol / L, 77 nmol / L, 84 nmol / L, etc., according to the concentration of the backbone chain and the required stability of the origami structure, in order to optimize the self-assembly efficiency and product quality. Among these, 50 nmol / L is preferred.

[0103] Through the above technical solutions, the preparation method proposed in this application significantly improves the efficiency and product quality of DNA origami vector self-assembly by precisely controlling the concentration of M13mp18 single-stranded DNA to 3–7 nmol / L and / or the concentration of the auxiliary strand to 24–84 nmol / L. This effectively solves the problems of low self-assembly efficiency, incomplete structure, or insufficient yield caused by improper concentration ratios. The DNA origami vector WA obtained in this way has higher structural uniformity and stability, thus providing a solid foundation for subsequent binding of SOX9 plasmid, grafting of type II collagen-targeting peptides, and modification of stem cell-targeting aptamers, ensuring the effective loading and delivery performance of functional molecules. Ultimately, this enables the prepared DNA origami-based delivery system to more efficiently recruit endogenous stem cells and promote cartilage repair, thereby improving the overall therapeutic effect.

[0104] Furthermore, in some embodiments, this application further proposes that in the preparation of the above-mentioned delivery system, in step S2, the DNA origami vector WA and the SOX9 plasmid are mixed and added to the buffer solution at a molar ratio of 1:(1-3), and the annealing conditions are slow cooling from 37°C to room temperature, and the entire annealing process is not less than 4 hours.

[0105] The DNA origami vector WA, also known as tDONs-WA, is a bifunctional triangular DNA origami product. Its B side is grafted with a type II collagen-targeting peptide, and its C side is modified with a stem cell-targeting aptamer. This product has a specific trap strand designed on its A side for binding to the SOX9 plasmid. The SOX9 plasmid is a circular DNA molecule carrying the SOX9 gene, containing a sequence complementary to the trap strand on the A side of the DNA origami vector WA. This plasmid is designed to induce endogenous stem cells to differentiate into cartilage cells via gene transfection. The DNA origami vector WA and the SOX9 plasmid are mixed at a molar ratio of 1:(1–3). This ratio ensures that the SOX9 plasmid binds sufficiently to the DNA origami vector WA while avoiding excessive free plasmid. In practice, depending on the actual binding efficiency and the characteristics of the plasmid, the molar ratio of the DNA origami vector WA to the SOX9 plasmid can be used, such as 1:1, 1:1.6, 1:2, 1:2.5, or 1:3, to optimize the binding effect, with 1:2 being the preferred ratio. Adding the mixture to the buffer solution means dissolving or dispersing the reactants in a specific solution environment to provide a stable chemical environment that promotes the binding reaction between the DNA origami vector WA and the SOX9 plasmid.

[0106] Annealing, characterized by slow cooling from 37°C to room temperature, refers to the process of promoting specific binding between DNA molecules by controlling temperature changes. Slow cooling from 37°C provides a suitable binding temperature for the capture strand on the DNA origami vector WA to bind with the complementary sequence on the SOX9 plasmid, ensuring efficient and specific base pairing. The initial annealing temperature can be fine-tuned based on the Tm value of the DNA sequence, for example, starting cooling from 40°C or 35°C; the cooling rate can be more precisely controlled through the PCR program, for example, decreasing by 0.1°C per minute. The entire annealing process should be at least 4 hours, referring to the time from 37°C to room temperature and maintaining this temperature. This duration ensures sufficient time for the DNA origami vector WA and the SOX9 plasmid to bind adequately and reach equilibrium, thereby improving binding efficiency and stability. In addition to 4 hours, the annealing time can be extended to 6 or 8 hours, depending on experimental results and binding efficiency requirements, to further optimize the binding effect.

[0107] The above technical solution effectively improves the binding efficiency and stability of SOX9 plasmid and DNA origami vector WA, avoiding the impact on subsequent function due to insufficient binding or plasmid damage. The prepared DNA origami vector WAS has a more stable structure and higher functional activity, thus enabling more effective delivery of SOX9 plasmid to target endogenous stem cells, promoting their chondrogenic differentiation, and thereby enhancing the application effect of the entire delivery system in cartilage repair.

[0108] Furthermore, in some embodiments, this application further proposes that, in preparing the above-mentioned delivery system, the buffer solution includes a magnesium-containing TAE buffer, prepared by adding magnesium chloride to the TAE buffer, with 0.099–0.139 mg of magnesium chloride added per 100 μL of TAE buffer. The magnesium-containing TAE buffer is a commonly used electrophoresis buffer, whose base buffer consists of Tris base, acetic acid, and EDTA, and its main function is to maintain DNA stability and provide conductivity. In this application, it serves as the reaction medium for DNA origami self-assembly, and the introduction of magnesium ions (Mg²⁺) is crucial. Magnesium ions can neutralize the negatively charged phosphate groups on the DNA backbone, reducing electrostatic repulsion between DNA chains, thereby promoting base pairing between the single-stranded DNA backbone chain and multiple staple chains, ensuring that the DNA origami structure can fold efficiently and accurately into the desired three-dimensional conformation. This buffer solution can be prepared in advance as a TAE stock solution, then diluted and magnesium salts added as needed, or commercially available TAE buffer can be used directly. TAE buffer is prepared by adding magnesium chloride, a common salt that provides magnesium ions. By adding magnesium chloride to TAE buffer, the concentration of magnesium ions in the solution can be precisely controlled.

[0109] The addition of 0.099–0.139 mg of magnesium chloride per 100 μL of TAE buffer precisely defines the range of magnesium chloride addition. This precise ratio ensures optimal magnesium ion concentration during DNA origami self-assembly. In practice, this can be achieved by accurately weighing solid magnesium chloride and dissolving it in a known volume of TAE buffer, or by preparing a high-concentration magnesium chloride stock solution and then precisely transferring it to the TAE buffer. This precise ratio is crucial for DNA origami formation, preventing incomplete DNA folding or structural instability due to insufficient magnesium ion concentration, and avoiding non-specific DNA aggregation or precipitation that may occur due to excessive magnesium ion concentration. In practice, magnesium chloride can be added to every 100 μL of TAE buffer at concentrations such as 0.099 mg, 0.105 mg, 0.108 mg, 0.11 mg, 0.116 mg, 0.119 mg, 0.12 mg, 0.122 mg, 0.125 mg, 0.13 mg, 0.132 mg, 0.136 mg, and 0.139 mg, with 0.119 mg of magnesium chloride per 100 μL of TAE buffer being preferred.

[0110] Through the above technical solution, the preparation method provided in this application precisely controls the concentration of magnesium ions during the DNA origami self-assembly process, effectively solving the problems of low folding efficiency, poor stability, and unsatisfactory subsequent functional molecule loading effects caused by improper magnesium ion concentration ratio. Specifically, the precise ratio of 0.099–0.139 mg magnesium chloride added to every 100 μL of TAE buffer provides the optimal ionic environment for DNA origami self-assembly, significantly improving the folding efficiency and structural stability of the DNA origami carrier. This not only ensures that the triangular DNA origami carrier WA and the DNA origami carrier WAS can form a regular and stable three-dimensional structure, but also provides a solid structural foundation for its A-side binding of SOX9 plasmid, B-side grafting of type II collagen-targeting peptides, C-side modification of stem cell-targeting aptamers, and internal loading of anti-inflammatory and antioxidant small molecule drugs such as resveratrol. Ultimately, this ensured the overall structural integrity and bioactivity of the DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system, thereby improving its delivery efficiency and therapeutic efficacy in recruiting endogenous stem cells and promoting cartilage repair.

[0111] Furthermore, in some embodiments, this application further proposes that in the preparation of the above-mentioned delivery system, in step S3, the DNA origami carrier WAS and the anti-inflammatory and antioxidant small molecule drug (such as resveratrol) are incubated at 1-10°C with shaking for 6-10 h; purification is carried out by ultrafiltration centrifugation, with centrifugation conditions of: molecular weight cutoff of 3-100 kDa, centrifuged at 8000-15000 g for 15-25 min.

[0112] The incubation conditions are designed to optimize the loading process of anti-inflammatory and antioxidant small molecule drugs (such as resveratrol) within the DNA origami carrier WAS. A low-temperature environment of 1–10°C effectively maintains the structural integrity and stability of the DNA origami carrier WAS, preventing disintegration or conformational changes at higher temperatures, thus ensuring that resveratrol can stably enter and bind within the carrier. Specifically, incubation can be performed at temperatures such as 1°C, 3°C, 4°C, 6°C, 8°C, and 10°C to maintain DNA structural stability, with 4°C being the preferred temperature. Shaking for 6–10 hours provides sufficient mixing and time to promote adequate contact and binding of resveratrol molecules with the internal space of the DNA origami carrier WAS, improving drug loading efficiency and uniformity. Besides continuous shaking, intermittent shaking or magnetic stirring can also be used, as long as it ensures thorough mixing of resveratrol and the carrier. In practice, the incubation time can be adjusted according to the actual loading efficiency and carrier characteristics, for example, 6 hours, 8 hours, or 10 hours, with 8 hours being the preferred setting. Purification was performed using ultrafiltration centrifugation, a highly efficient physical separation technique. The principle is to use centrifugal force to drive the solution through a filter membrane with a specific molecular weight cutoff, thereby separating substances of different molecular weights. The purpose of this method is to efficiently remove unbound free resveratrol and other small molecule impurities, ensuring the purity of the delivery system and avoiding potential non-specific toxic side effects from free drugs. Specifically, the selection of a filter membrane with a molecular weight cutoff of 3–100 kDa is based on the significant difference in molecular weight between the DNA origami carrier WAS and resveratrol. This ensures that the larger molecular weight DNA origami carrier WAS is effectively retained, while the smaller molecular weight resveratrol can pass through the filter membrane and be discharged with the filtrate. A centrifugal force of 8000–15000 g provides sufficient driving force to rapidly pass the solution through the filter membrane, thereby improving purification efficiency. A centrifugation time of 15–25 min ensures thorough separation and purification. The molecular weight cutoff can be adjusted according to the actual molecular weight of the DNA origami carrier WAS and the molecular weight of resveratrol, for example, selecting filter membranes of 30 kDa, 50 kDa, or 100 kDa. The centrifugal force can be adjusted according to equipment conditions and purification requirements, such as 8000 g, 12000 g, 15000 g, etc., with 12000 g being the preferred option. The centrifugation time can also be adjusted according to actual conditions, such as 15 minutes, 20 minutes, 25 minutes, etc., with 20 minutes being the preferred option.

[0113] Through the above technical solutions, the preparation method provided in this application, by precisely controlling the incubation conditions of the DNA origami carrier WAS and resveratrol (i.e., shaking at 1–10°C for 6–10 h), effectively maintains the structural stability of the DNA origami carrier WAS and promotes the efficient and uniform loading of resveratrol. Simultaneously, ultrafiltration centrifugation, with a molecular weight cutoff of 3–100 kDa and centrifugation conditions of 8000–15000 g for 15–25 min, efficiently and thoroughly removes unbound free resveratrol, thereby significantly improving the purity of the delivery system. This series of refined preparation and purification steps ensures that the final DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system possesses stable biological activity and excellent safety, avoiding potential toxic side effects from free drugs. Therefore, this delivery system can more effectively leverage the role of resveratrol in enhancing the chondrogenic differentiation potential of mesenchymal stem cells and maintaining stemness, thus providing a solid guarantee for achieving precise recruitment and efficient cartilage repair of endogenous stem cells.

[0114] Furthermore, in some embodiments, this application further proposes that in preparing the above-mentioned delivery system, in step S3, the concentration of resveratrol is set to 3-7 μmol / L, and the DNA origami carrier WAS and resveratrol are mixed and incubated at a molar ratio of 1:(18-22).

[0115] This scheme achieves standardized control of the drug loading process by precisely limiting the concentration of resveratrol and its molar ratio with the DNA origami carrier WAS, thereby ensuring the stability and effectiveness of the delivery system in cartilage repair applications. In the preparation of the DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system, step S3 involves mixing and incubating the DNA origami carrier WAS with resveratrol to achieve resveratrol loading. By precisely controlling the concentration of resveratrol at 3–7 μmol / L and mixing and incubating the DNA origami carrier WAS with resveratrol at a molar ratio of 1:(18–22), the encapsulation efficiency and uniformity of resveratrol within the DNA origami carrier WAS can be optimized. This precise condition control allows resveratrol to be stably loaded into the DNA origami carrier WAS, avoiding aggregation, precipitation, or uneven distribution of the drug during loading due to improper concentration or ratio. Ultimately, the purified delivery system, with its internally loaded resveratrol, exerts its effects at the desired dosage and distribution, effectively enhancing the chondrogenic differentiation potential of endogenous stem cells and maintaining their stemness, thus providing continuous pharmacological support for cartilage repair. Specifically, the concentration of resveratrol can be 3 μmol / L, 4 μmol / L, 5 μmol / L, 6 μmol / L, 7 μmol / L, etc., with 5 μmol / L being preferred. The molar ratio of the DNA origami carrier WAS to resveratrol can be set to 1:18, 1:19, 1:20, 1:21, 1:22, etc., with 1:20 being preferred.

[0116] By precisely controlling the concentration of resveratrol and its mixing ratio with the DNA origami carrier WAS through the above technical solution, the loading efficiency and distribution uniformity of resveratrol within the carrier can be significantly improved. This effectively avoids potential cytotoxicity caused by excessively high drug concentrations and reduces drug waste. This ensures that the delivery system can stably and efficiently deliver resveratrol to the target site, thereby maximizing the bioactivity of resveratrol in maintaining stem cell stemness, anti-inflammation, and promoting chondrogenic differentiation. Ultimately, this enhances the therapeutic efficacy and bioavailability of the entire delivery system in endogenous stem cell recruitment and cartilage repair.

[0117] The technical solution of this application will be further described below through a specific embodiment 1: The preparation method proposed in this application further includes step S0: auxiliary chain modification, which involves targeted chemical modification of specific sequences of each group of staple chains to prepare for the subsequent coupling of functional units, specifically including: The plasmid capture strands in group S01 and A (as shown in SEQ ID NO:23 and SEQ ID NO:53) are modified with streptavidin at their 5' end, allowing the modified plasmid capture strands to connect to the 3' end of the SOX9 plasmid labeled with biotin-dUTP. The reaction system was a liquid medium (enzyme-free water) incubated on a horizontal shaker at 150 rpm at room temperature for 30 minutes. The binding effect of the SOX9 plasmid to the A side of the DNA origami vector was verified by 1% agarose gel electrophoresis after the reaction.

[0118] The 5' end of the peptide capture strand in group S02 and B staple chains (as shown in SEQ ID NO:88 and SEQ ID NO:118) is modified with maleimide, which allows the modified peptide capture strand to link with the thiolized WYRGRL targeting peptide (hereinafter referred to as WY). The reaction system was placed in an oscillator and oscillated at 500 times / min for 3 hours. After the reaction, the effectiveness of grafting the WYRGRL targeting peptide onto the B side of the DNA origami vector was verified by 6% polyacrylamide gel electrophoresis.

[0119] The aptamer-modified daughter chains in the S03 and C group staple chains (as shown in SEQ ID NO:153 and SEQ ID NO:183) are modified at their 5' ends with an Apt19S aptamer carrying a Cy5 fluorescent group, thereby achieving in situ stem cell recruitment and tracking effects, and thus constructing aptamer chains as shown in SEQ ID NO:209 and SEQ ID NO:210. The synthesis effect of the aptamer chains can be verified by 6% PAGE gel electrophoresis after the reaction.

[0120] Furthermore, the preparation method of the delivery system of this application also includes: S1. Mix M13mp18 single-stranded DNA (5 nmol / L) with the modified auxiliary strand (50 nmol / L) at a molar ratio of 1:10, and add 1×TAE / Mg²⁺ buffer (components: 40 mmol / L Tris, 20 mmol / L acetic acid, 2 mmol / L EDTA, 12.5 mmol / L magnesium acetate, pH=8.3).

[0121] The system was slowly cooled from 95℃ to room temperature using a PCR program, with an annealing time of no less than 12 h, to complete self-assembly. Then, the free auxiliary strand was removed using a 100 kDa ultrafiltration centrifuge tube (repeated 3 times) to obtain a DNA origami vector WA with WYRGRL targeting peptide grafted on side B and Apt 19S aptamer modified on side C, namely tDONs-WA.

[0122] S2. The obtained tDONs-WA and the SOX9 plasmid containing the free complementary sequence are mixed in a molar ratio of 1:2 in a magnesium-containing TAE buffer (0.119 mg of magnesium chloride is added per 100 μL of TAE buffer); the system is slowly cooled from 37℃ to room temperature for at least 4 h to obtain a triangular DNA origami vector WAS with the SOX9 plasmid bound to side A, namely tDONs-WAS.

[0123] S3: Prepare a 5 μmol / L resveratrol solution, mix the obtained tDONs-WAS with the resveratrol solution and incubate at 4°C for 8 hours with shaking; then pass the resulting mixture through a 50 kDa ultrafiltration tube and centrifuge at 12000g for 20 minutes to remove residual single-stranded DNA (ssDNA) and free Res, to obtain a delivery system tDONs-WARS / DNR that integrates four factors or modifications, namely WYRGRL targeting peptide, Apt19S aptamer, Res and SOX9 plasmid.

[0124] Another aspect of this application proposes the application of a DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system in the preparation of drugs for treating osteoarthritis.

[0125] The DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system proposed in this application integrates multiple therapeutic functions into a nanoscale carrier and, through pharmaceutical formulation, enables it to function as a drug for treating osteoarthritis. This delivery system uses a triangular DNA origami carrier as its core scaffold, formed through a precise self-assembly process, and pre-loaded or grafted with various bioactive molecules at specific locations. Specifically, the SOX9 plasmid binds to side A of the DNA origami carrier, serving as a key gene for inducing chondrogenic differentiation; a type II collagen-targeting peptide is grafted onto side B, ensuring the delivery system can specifically recognize and bind to damaged cartilage areas; a stem cell-targeting aptamer is modified on side C, responsible for efficiently recruiting endogenous stem cells within the joint cavity; simultaneously, resveratrol is loaded inside the DNA origami carrier to improve the local microenvironment and enhance stem cell activity. When this delivery system is formulated into a drug and administered intra-articularly to osteoarthritis patients, the type II collagen-targeting peptide first guides the system to precisely locate the cartilage injury site. Subsequently, the stem cell targeting aptamer exerts a "siphoning" effect, efficiently attracting and enriching surrounding endogenous mesenchymal stem cells to the damaged area. Simultaneously with stem cell recruitment, the SOX9 plasmid is delivered into these stem cells, activating their specific differentiation towards hyaline cartilage. Furthermore, the internally loaded resveratrol is continuously released, optimizing the pathological microenvironment within the joint cavity through its anti-inflammatory, antioxidant, and stem cell-protective effects, providing favorable conditions for the recruited stem cells to continuously differentiate and repair. The entire system, through the precise spatial arrangement of DNA origami carriers, achieves integrated functions of targeted localization, stem cell recruitment, differentiation induction, and microenvironment regulation, thereby promoting in-situ regeneration and repair of damaged cartilage.

[0126] This application presents a drug for treating osteoarthritis, developed using a DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system. This effectively addresses the clinical translational challenges of traditional exogenous stem cell transplantation, such as low efficiency, poor homing ability, and limited recruitment and differentiation of endogenous stem cells. The drug achieves precise targeting of damaged cartilage, significantly improving the enrichment efficiency of endogenous stem cells at the lesion site. Simultaneously, delivery via the SOX9 plasmid efficiently induces recruited stem cells to specialize towards hyaline cartilage, preventing the formation of fibrocartilage or hypertrophic cartilage. Furthermore, the sustained release of resveratrol improves the inflammatory and oxidative stress microenvironment within the joint cavity, providing favorable conditions for stem cell survival, proliferation, and continuous differentiation, thereby promoting effective cartilage regeneration and repair. This multifunctional integrated drug formulation offers a highly efficient, precise, and practically valuable solution for the clinical treatment of osteoarthritis.

[0127] III. Characterization Experiment This application verifies the synthesis, microstructure, surface potential, and detects in vitro drug release of all the DNA origami products obtained in Example 1 above. The experimental samples include: tDONs, tDONs-W, tDONs-A, tDONs-WA, tDONs-WAR, tDONs-WAS, and DNR.

[0128] 3.1 Electrophoresis verification: PAGE electrophoresis (e.g.) Figure 2 (As shown in B and 2C): The electrophoretic bands of the peptide conjugation product on side B and the aptamer conjugation product on side C both lagged behind the original single strand, proving that the target peptide and aptamer were successfully grafted. Agarose gel electrophoresis (e.g.) Figure 2 As shown in D): When tDONs-WA and SOX9 plasmid are mixed in ratios of 1:1, 1:2, and 1:3, the molecular weight of the product gradually increases with the increase of the proportion of plasmid, proving that the plasmid binding efficiency is good.

[0129] 3.2 Microscopic morphology detection (e.g.) Figure 2 As shown in E): The morphology of DNR was observed using atomic force microscopy (AFM). The results showed that the sample had a regular triangular structure, and the binding functional units were visible around the vector. The DNA origami framework was fully assembled.

[0130] 3.3 Zeta potential detection (as shown in Figure 2F): As the number of functional units on the support surface increases, the negative potential of the sample gradually increases, proving that each functional molecule is successfully modified on the support surface.

[0131] 3.4 In vitro drug release experiment (as shown in Figure 2G) Take 1 mL of 1 mmol / L DNR sample and place it into a dialysis bag with a molecular weight cutoff of 3000 Da. Place it in buffer solutions at pH 5.0 (acidic inflammatory environment) and pH 7.4 (normal physiological environment) and shake at 37℃ and 100 r / min. Take samples at 0, 3, 6, 12, 24, 36 and 48 h and detect the drug release using the UV characteristic absorption peak of resveratrol at 306 nm.

[0132] Results: Resveratrol release rate was significantly faster under acidic conditions, with a cumulative release of over 30% in 6 hours, which can meet the medication needs of the inflammatory microenvironment of joints.

[0133] IV. In vitro cell experiments 4.1 Cellular uptake and target specificity detection (as shown in Figure 3A) Experimental cells: mouse bone marrow macrophages, synovial fibroblasts, chondrocytes, and bone marrow mesenchymal stem cells (MSCs); Sample: tDONs-A (carrying Cy5 fluorescent label), concentration 10 nmol / L.

[0134] The samples were co-incubated with cells for 12 h, washed with PBS, fixed, and transmembrane-treated. The nuclei were then stained with DAPI, and the fluorescence signal was observed using a laser confocal microscope.

[0135] Results: Strong fluorescence signals were detected only in the MSCs group, while the fluorescence of the other cells was extremely weak, proving that the Apt19S aptamer can specifically target mesenchymal stem cells.

[0136] 4.2 Joint cavity retention and in vivo distribution detection (see attached) Figure 3 (As shown in B and 3C) Experimental animals: OA model mice; groups: tDONs group (no targeting peptide), tDONs-W group (grafted WYRGRL targeting peptide), sample concentration 1 μM, intra-articular injection dose 10 μL.

[0137] Duration of stay (as attached) Figure 3 (As shown in B): Joint fluorescence was monitored continuously for 7 days using a small animal in vivo imaging system (IVIS). Significant differences were observed between the two groups on day 3 post-injection. By day 5, the fluorescence of the blank carrier tDONs had essentially disappeared, while tDONs-W still retained strong fluorescence, demonstrating that the targeting peptide could significantly prolong the carrier's residence time in the joint cavity.

[0138] Distribution of organs (as shown in the appendix) Figure 3 (As shown in C): Mice were dissected on day 3 after injection, and the heart, liver, spleen, lungs, kidneys, and lower limb joints were harvested for fluorescence detection. tDONs are mainly metabolized by the kidneys, with weak signals at the joints; tDONs-W are mainly enriched in the knee joint, with little distribution in non-target organs, and excellent cartilage targeting.

[0139] 4.3 Detection of stem cell proliferation and migration capacity (as shown in Figures 5A and 5B) Modeling conditions: 1 ng / mL IL-1β induced MSCs to establish an inflammatory injury model; experimental groups: blank control group (Ctrl), model group (IL-1β), tDONs-WA group, tDONs-WAR group, tDONs-WAS group, DNR group, and the carrier concentration was 10 nmol / L.

[0140] Cell proliferation assay: Cell proliferation activity was detected using an EDU kit 24 h after treatment in each group. Results: IL-1β significantly inhibited MSC proliferation, and this damage could be reversed in all DNA origami samples, with DNR showing the best protective effect on stem cell proliferation.

[0141] Cell scratch migration assay: Cells were pretreated with samples for 6 h before scratch treatment. Scratch morphology was recorded at 0 h and 24 h, and cell migration rate was calculated. Results: DNR can maximize the migration ability of MSCs under inflammatory conditions.

[0142] 4.4 In vitro chondrogenic differentiation experiment (as attached) Figure 4 ) (1) Two-dimensional cell differentiation staining Groups: simple cartilage induction medium group (Ctrl), tDONs-WA group, tDONs-WAR group, tDONs-WAS group, DNR group, carrier concentration 10 nmol / L, continuous culture for 7 days, medium changed every 2 days.

[0143] Alcian blue staining was used respectively (e.g. Figure 4 As shown in A), toluidine blue staining (as shown in A) Figure 4 (As shown in B) Detect the amount of cartilage matrix secretion.

[0144] Results: All functional carriers could promote the differentiation of MSCs into chondrocytes, with DNR showing the most significant effect in chondrocyte formation.

[0145] (2) Three-dimensional cartilage ball induction experiment MSCs were prepared into cell spheroids and added to cartilage induction medium containing the samples according to the above grouping. They were cultured continuously for 14 days, with the medium changed every 3 days. After fixing, embedding, and sectioning the cartilage spheroids, the expression of cartilage marker proteins Col II and SOX9 was detected by immunofluorescence (see attached). Figure 4 (As shown in C); Western blotting was used to detect the expression levels of Col II and SOX9 in the total protein of chondrocyte spheroids (as shown in the attached image). Figure 4 (as shown in DF) Results: DNR significantly upregulated the expression of Col II and SOX9, confirming at the molecular level its efficient induction of MSCs to chondrogenic differentiation.

[0146] V. In vivo animal experiments 5.1 Construction of an animal model of osteoarthritis A C57BL / 6J mouse OA model was established using medial meniscus instability surgery (DMM): After anesthesia, the right knee joint was prepared and disinfected, the tissue was cut layer by layer, the anterior cruciate ligament was severed, the wound was sutured, and the mice were fed normally.

[0147] 5.2 In vivo stem cell recruitment experiment (as attached) Figure 5 (as shown in C) Samples: Cy5 fluorescently labeled tDONs-W, tDONs-WA, tDONs-WAS, and DNR, at a concentration of 100 nmol / L, administered intra-articularly at a dose of 10 μL per animal, for 4 consecutive weeks.

[0148] After the drug administration was completed, knee joint tissue was taken, fixed, decalcified, embedded, and sectioned. Immunofluorescence co-staining was performed using MSC markers CD73 and CD90.

[0149] Results: Vectors modified with the Apt19S aptamer could recruit endogenous MSCs; the DNR loaded with SOX9 plasmid and resveratrol achieved the highest stem cell recruitment efficiency.

[0150] 5.3 In vivo pharmacodynamic evaluation (as shown in Figures 6 and 7) (1) Experimental grouping and dosing regimen The experiment consisted of 6 groups, with 10 mice in each group. Drug administration began in the first week after DMM modeling, with intra-articular injections (100 nmol / L, 10 μL) once weekly for 10 consecutive weeks. Sham surgery + saline solution group (Ctrl); DMM model + saline group (Saline); DMM model + tDONs-WA group; DMM model + tDONs-WAR group; DMM model + tDONs-WAS group; DMM model + DNR group.

[0151] (2) Histological staining examination (as shown in Figure 6) Ten weeks after drug administration, tissue samples were collected, and knee joint sections were stained and analyzed. Safranin-Fix Green and Toluidine Blue staining (as shown in Figures 6B and 6C): The DMM model group showed severe cartilage wear; all functional carriers could delay cartilage degeneration, with DNR showing the best protective effect.

[0152] H&E staining (as shown in Figure 6D): The synovium in the model group was thickened and inflammatory cell infiltration was obvious; the synovium morphology in the DNR group was close to that of normal tissue and the anti-inflammatory effect was significant.

[0153] (3) MicroCT and immunofluorescence detection (as shown in Figure 7) MicroCT scans (as shown in Figures 7B and 7C): The model group showed extensive osteophyte formation and severe subchondral bone sclerosis; all intervention groups showed inhibition of osteophyte formation and bone sclerosis, with DNR showing the strongest effect.

[0154] Immunofluorescence assay (as shown in Figure 7A): Detection of anabolism indicators (Col II, SOX9) and catabolism indicators (MMP13).

[0155] Results: DNR can upregulate cartilage synthesis-related proteins and downregulate degradation proteins, thus maintaining cartilage matrix homeostasis.

[0156] VI. Comprehensive Experimental Conclusions The triangular DNA origami delivery system (DNR) constructed in this invention achieves multifunctional unit partitioning modification and synergistic effects: B-side WYRGRL targeting peptides can specifically bind to type II collagen in articular cartilage, prolonging the carrier's joint residence time and achieving targeted enrichment; C-side Apt19S aptamers efficiently recruit endogenous mesenchymal stem cells around the joint; After the SOX9 plasmid is taken up by stem cells, it induces them to differentiate into hyaline chondrocytes. Resveratrol loaded inside the vector is slowly released as the DNA structure degrades, exerting anti-inflammatory and antioxidant effects, maintaining stem cell activity, and inhibiting aging.

[0157] This integrated delivery system can significantly improve the inflammatory microenvironment of joints, promote cartilage regeneration, and inhibit the progression of osteoarthritis, making it extremely valuable for the preparation of drugs for the treatment of osteoarthritis.

[0158] The above description is merely an embodiment of this application and is not intended to limit the scope of protection of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system, characterized in that, include: The triangular DNA origami carrier is formed by the self-assembly of a single-stranded DNA backbone and multiple staple chains. The three sides of the DNA origami carrier are respectively divided into side A, side B, and side C. The SOX9 plasmid binds to side A of the DNA origami vector; Type II collagen-targeting peptides are grafted onto side B of the DNA origami carrier; A stem cell targeting aptamer is modified on the C-side of the DNA origami vector; Anti-inflammatory and antioxidant small molecule drugs are loaded inside the DNA origami carrier.

2. The DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system according to claim 1, characterized in that, The backbone chain includes M13mp18 single-stranded DNA. The staple chain includes: As shown in SEQ ID NO:1~65, the A-group staple chain is formed by self-assembly with the backbone chain through base complementary pairing to form the A-side; As shown in SEQ ID NO:66~130, the B-group staple chain is formed by self-assembly with the backbone chain through base complementary pairing to form the B-side; As shown in SEQ ID NO:131 to SEQ ID NO:195, the C-group staple chain forms the C-side by self-assembling with the backbone chain through complementary base pairing.

3. The DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system according to claim 2, characterized in that, The group A staple chain includes plasmid capture sub-chains as shown in SEQ ID NO:23 and SEQ ID NO:53, wherein the 5' end of the plasmid capture sub-chain is modified with streptavidin; The 3' end of the SOX9 plasmid is biotin-labeled and binds to the A side of the DNA origami vector through the affinity of biotin for streptavidin.

4. The DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system according to claim 3, characterized in that, The biotin includes Biotin-dUTP biotin.

5. The DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system according to claim 2, characterized in that, The group B staple chain includes a peptide capture sub-chain as shown in SEQ ID NO:88 and SEQ ID NO:118, wherein the 5' end of the peptide capture sub-chain is modified with maleimide; The type II collagen-targeting peptide is a thiolized WYRGRL-targeting peptide, which is grafted onto the B side of the DNA origami carrier through the covalent cross-linking property of maleimide and thiol.

6. The DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system according to claim 2, characterized in that, The C-group staple chain includes aptamer-modified sub-chains as shown in SEQ ID NO:153 and SEQ ID NO:183; The stem cell targeting aptamer includes the Apt19S aptamer, wherein the Apt19S aptamer is modified at the 5' end of the aptamer modified subchain to form an aptamer chain as shown in SEQ ID NO:209 and SEQ ID NO:

210.

7. The DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system according to claim 6, characterized in that, The Apt19S aptamer is modified with a Cy5 fluorescent group.

8. The DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system according to claim 1, characterized in that, The anti-inflammatory and antioxidant small molecule drugs include resveratrol.

9. A method for preparing an endogenous stem cell recruitment and cartilage repair delivery system based on DNA origami as described in any one of claims 1 to 8, characterized in that, Includes the following steps: S1: The backbone chain and the auxiliary chain containing the modified staple chain, type II collagen targeting peptide, and stem cell targeting aptamer are placed in a buffer solution, annealed and cooled to room temperature to complete self-assembly, and after removing the excess auxiliary chain, a triangular DNA origami vector WA with type II collagen targeting peptide grafted on side B and stem cell targeting aptamer modified on side C is obtained. S2: The DNA origami vector WA and the SOX9 plasmid containing the free complementary sequence are placed in a buffer solution, annealed and cooled to room temperature to obtain a triangular DNA origami vector WAS with the SOX9 plasmid bound to side A. S3: The DNA origami carrier WAS is mixed and incubated with an anti-inflammatory and antioxidant small molecule drug, and after purification, the DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system is obtained.

10. The preparation method according to claim 9, characterized in that, In step S1, the backbone strand is M13mp18 single-stranded DNA, which is mixed with the auxiliary strand at a molar ratio of 1:(8-112) and added to the buffer solution. The annealing conditions are slow cooling from 95°C to room temperature, and the entire annealing process is not less than 12 h.

11. The preparation method according to claim 9, characterized in that, In step S1 and / or step S2, the buffer solution contains Tris at a concentration of 30–50 mmol / L, acetic acid at a concentration of 18–22 mmol / L, EDTA at a concentration of 1–3 mmol / L, and magnesium acetate at a concentration of 10–15 mmol / L, and the pH value of the buffer solution is 8.0–8.

5.

12. The preparation method according to claim 9, characterized in that, In step S1, the concentration of the M13mp18 single-stranded DNA is 3–7 nmol / L; And / or, the concentration of the auxiliary chain is 24–84 nmol / L.

13. The preparation method according to claim 9, characterized in that, In step S2, the DNA origami vector WA and the SOX9 plasmid are mixed in a molar ratio of 1:(1-3) and added to the buffer solution. The annealing conditions are slow cooling from 37°C to room temperature, and the entire annealing process is not less than 4 hours.

14. The preparation method according to claim 9, characterized in that, In step S1 and / or step S2, the buffer solution includes a magnesium-containing TAE buffer solution, which is prepared by adding magnesium chloride to TAE buffer solution, with 0.099 to 0.139 mg of magnesium chloride added per 100 μL of TAE buffer solution.

15. The preparation method according to claim 9, characterized in that, In step S3, the incubation conditions for the DNA origami carrier WAS and the anti-inflammatory and antioxidant small molecule drug are shaking at 1-10°C for 6-10 h. The purification was performed using ultrafiltration centrifugation under the following conditions: molecular weight cutoff of 3–100 kDa, centrifuged at 8000–15000 g for 15–25 min.

16. The preparation method according to claim 9, characterized in that, In step S3, the concentration of the anti-inflammatory and antioxidant small molecule drug is 3-7 μmol / L, and the DNA origami carrier WAS is mixed and incubated with the anti-inflammatory and antioxidant small molecule drug at a molar ratio of 1:(18-22).

17. The use of the DNA origami-based endogenous stem cell recruitment and cartilage repair delivery system according to any one of claims 1 to 8 in the preparation of a medicament for treating osteoarthritis.

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