DNA (deoxyribonucleic acid) nano patch for enhancing MSC (mesenchymal stem cell) bone targeting capability as well as preparation method and application of DNA nano patch

Abstract

The invention belongs to the technical field of cell membrane surface engineering, and particularly relates to a DNA nano patch for enhancing MSC bone targeting ability and a preparation method and application thereof, and the preparation method comprises the following steps: mixing an annular scaffold chain and a staple chain to obtain rectangular DNA origami; the preparation method comprises the following steps: modifying a malinimide group at the 3'end of a bone targeting peptide to obtain a malinimide group modified bone targeting peptide, and mixing the malinimide group modified bone targeting peptide with a 5 '-thiol modified short chain to obtain a bone targeting peptide-short chain; modifying cholesterol on the DNA short chain to obtain a cholesterol-short chain; and mixing the rectangular DNA origami, the bone targeting peptide-short chain and the cholesterol-short chain to obtain the DNA nano patch for enhancing the bone targeting ability of the MSC. The DNA nano patch can be used for strengthening MSC (mesenchymal stem cells) so as to prepare drugs for treating degenerative diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of cell membrane surface engineering, and specifically relates to a DNA nanopatch for enhancing the bone targeting ability of MSCs, and a preparation method and application thereof. Background Art

[0002] Osteoporosis is caused by an unbalanced interaction between osteoblastic bone formation and osteoclast bone resorption. Current treatments focus on anti-resorptive and anabolic drugs, lacking treatments that can exert multiple effects simultaneously. It is worth noting that the extracellular microenvironment, including reactive oxygen species (ROS), inorganic components, immune cells, and cytokines, also significantly affects the progression of osteoporosis. Recent studies have shown that ROS bone metabolism is harmful, and it can inhibit osteoblast differentiation while stimulating osteoclast differentiation. Therefore, new treatment strategies must simultaneously address the problems caused by bone remodeling and the osteoporotic microenvironment.

[0003] As the precursor of osteoblasts, mesenchymal stem cell (MSC) transplantation can promote osteogenic differentiation, enhance bone mineral density, and slow the progression of osteoporosis. However, current MSC therapies lack targeting capabilities and microenvironmental regulation. Engineered MSCs focusing on gene editing have been developed to address these issues, but they also increase the risk of immunogenic reactions and other complications associated with genetic engineering. Non-gene-edited cell surface engineering endows MSCs with enhanced homing, adhesion, and migration capabilities, as well as attenuated immunogenicity and avoidance of genetic risks. Functionalized DNA is known for its ease of synthesis and biocompatibility, making it an ideal choice for such engineering. Framework nucleic acids (FNAs), especially DNA origami (DON), have superior stability, programmability, and their phosphate backbone can chelate with bone calcium, giving them certain bone targeting capabilities, but their potential in anchoring MSCs for the treatment of osteoporosis remains unexplored. Summary of the invention

[0004] In order to solve the above technical problems, the present invention develops a DNA nanopatch for enhancing the bone targeting ability of MSCs and a preparation method and application thereof, which can be used to strengthen MSCs and thus prepare drugs for degenerative diseases.

[0005] The technical solution provided by the present invention is as follows:

[0006] A method for preparing a DNA nanopatch for enhancing the bone targeting ability of MSCs comprises the following steps: mixing a circular scaffold chain and a staple chain to obtain a rectangular DNA origami; 6)'s 3' end is modified with a maleimide group to obtain a bone-targeting peptide modified with a maleimide group, and the bone-targeting peptide modified with a maleimide group is mixed with a 5'-thiol-modified short chain to obtain a bone-targeting peptide-short chain; cholesterol is modified on the DNA short chain to obtain a cholesterol-short chain; and a rectangular DNA origami, a bone-targeting peptide-short chain and a cholesterol-short chain are mixed to obtain a DNA nanopatch that enhances the bone-targeting ability of MSCs.

[0007] Furthermore, the molar ratio of the rectangular DNA origami, the bone targeting peptide-short chain and the cholesterol-short chain is 1:5~10:10~20.

[0008] Furthermore, after the rectangular DNA origami, bone-targeting peptide-short chain and cholesterol-short chain were mixed, the mixture was heated to 40-50°C and cooled to 20-30°C at a rate of 0.15-0.25°C / min to obtain a DNA nanopatch with enhanced MSC bone targeting ability.

[0009] Furthermore, the preparation method of the bone-targeted peptide-short chain includes: adding 6-maleimidocaproic acid to a dimethylformamide solution containing o-benzotriazole-N,N,N' and diisopropylethylamine to react with the bone-targeted peptide, removing the peptide protecting group with trifluoroacetic acid, incubating at room temperature, and purifying to obtain a bone-targeted peptide modified with a maleimide group; reacting the bone-targeted peptide modified with a maleimide group with a 5'-thiol-modified short chain to generate a bone-targeted peptide-short chain.

[0010] Furthermore, the preparation method of the cholesterol-short chain includes: adding the DNA short chain to dichloromethane containing trichloroacetic acid to obtain a deprotected DNA short chain; adding the deprotected DNA short chain to a mixture of 5-cholesterol CE and tetrazole to obtain an intermediate; adding the intermediate to an acetonitrile solution containing acetic anhydride and 1-methylimidazole, and then oxidizing it with iodine to obtain a cholesterol-short chain.

[0011] The present invention provides a DNA nanopatch, which is prepared by the above-mentioned preparation method.

[0012] The present invention also provides the use of the DNA nanopatch in preparing osteoporosis medicine.

[0013] Furthermore, the method for preparing the osteoporosis drug comprises: anchoring the above-mentioned DNA nanopatch on the surface of mesenchymal stem cells to obtain DNA nanopatch-mesenchymal stem cells, and using the DNA nanopatch-mesenchymal stem cells to prepare the osteoporosis drug.

[0014] Furthermore, the preparation method of the DNA nanopatch-mesenchymal stem cells includes: adding the above-mentioned DNA nanopatch to a mesenchymal stem cell suspension so that the final concentration of the DNA nanopatch is 4~6 nM, and after incubation in an oscillating metal bath, anchoring the DNA nanopatch on the surface of the mesenchymal stem cells to obtain DNA nanopatch-mesenchymal stem cells.

[0015] Furthermore, the osteoporosis drug is used to increase the bone density of postmenopausal mice and improve the oxidative stress level in the body.

[0016] Beneficial Effects

[0017] Rectangular DNA origami (rDON) and bone targeting peptide (DSS 6 ) dual targeting effect, MSCs with therapeutic potential selectively accumulate in bone tissue after intravenous injection, directly increasing bone mass. Rectangular DNA origami, as a ROS scavenger, can not only promote bone differentiation but also inhibit the formation of osteoclasts by alleviating oxidative stress in the osteoporotic microenvironment.

[0018] DNA nanopatch (DNP, also abbreviated as D / CH rDON is derived from rDON and loaded with cholesterol (CH) and bone targeting peptide (DSS) 6 ). DNP is anchored to the MSC cell membrane through the hydrophobic effect of cholesterol to form DNP-engineered MSC (DNP-MSC). After intravenous injection, these engineered MSCs preferentially accumulate in bone tissue driven by the bone-targeting properties of DNP. In bone tissue, MSCs home to their target tissues, differentiate into osteoblasts, secrete cytokines, and help increase bone mass. At the same time, DNP in the bone microenvironment promotes osteogenic differentiation and inhibits the occurrence of osteoclasts by scavenging ROS prevalent in the osteoporotic environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the mechanism of using DNA nanopatch (DNP) to engineer mesenchymal stem cells (MSC) to treat osteoporosis;

[0020] Figure 2 Schematic diagram of the basic structure of unfunctionalized rectangular DNA origami (rDON); the green lines represent staple chains, and the red lines represent scaffold chains (M13mp18);

[0021] Figure 3 Representative atomic force microscopy images of unfunctionalized rectangular DNA origami (rDON) morphology; scale bar: 100 nm;

[0022] Figure 4The diagram is a schematic diagram of the synthesis of cholesterol-short chain (CH-ssDNA) by the amidophosphorus method and its mass spectrometry characterization; a is a schematic diagram of the synthesis of cholesterol-short chain by the amidophosphorus method; b is the mass spectrometry characterization;

[0023] Figure 5 Bone targeting peptide-short chain (DSS 6 -ssDNA) synthesis schematic diagram and mass spectrometry characterization; a is the synthesis schematic diagram of bone-targeting peptide-short chain; b is the mass spectrometry characterization;

[0024] Figure 6 Schematic diagram of the main nanostructures and coupling sites of DNA nanopatch (DNP). The red circles indicate the 27 sites for covalent attachment of cholesterol molecules, and the purple triangles indicate the sites for bone targeting peptide (DSS 6 ) The 10 sites for covalent attachment; the green rectangles indicate the locations of the FAM fluorescent dye within the DNP for easy tracking and imaging.

[0025] Figure 7 Agarose gel electrophoresis analysis and atomic force microscopy images of different nanostructures, where a is agarose gel electrophoresis analysis of different nanostructures, and b is a representative atomic force microscopy image, scale bar: 100 nm;

[0026] Figure 8 This is a confocal microscopic image of DNA nanopatch-engineered MSCs. The green part of the outer circle of the cell is the DNA nanopatch labeled with FAM fluorescence;

[0027] Fig. 9 Representative small animal in vivo optical imaging (IVIS) images of isolated mouse hind limbs after administration of DNA nanopatch engineered MSCs (DNP-MSCs); mice were euthanized 48 hours after injection, and hind limbs were immediately harvested for IVIS imaging ( CH rDON-MSCs are rectangular DNA origami-engineered MSCs modified with only cholesterol).

[0028] Fig.10 Micro-CT scans were performed on the lower femurs of mice in each group of the experiment to reconstruct three-dimensional images and quantitative results of bone density ( D rDON is a rectangular DNA origami modified with only the bone-targeting peptide);

[0029] Fig.11are the test results of mouse serum, where a is the level of type 1 procollagen amino terminal peptide (P1NP) in serum of each group of mice detected by enzyme-linked immunosorbent assay, b is the level of type 1 collagen carboxyl terminal peptide (β-CTX) in serum of each group of mice detected by enzyme-linked immunosorbent assay, c is the level of superoxide dismutase (SOD) in serum of each group of mice detected by kit, d is the level of malondialdehyde (MDA) in serum of each group of mice detected by kit (OVX is bilateral ovariectomy group, D rDON is a rectangular DNA origami modified with only the bone targeting peptide). DETAILED DESCRIPTION

[0030] Experimental Materials

[0031] 1. Experimental Animals

[0032] C57 / B6J mice, 50 females, 6-8 weeks old, weighing 21-37 g, SPF grade, were provided by Changzhou Cavens Experimental Animal Co., Ltd. During the experiment, the temperature in the breeding room was 22±3.0℃, the relative humidity was 60-70%, and the mice were free to eat and drink.

[0033] Animal experiments were in compliance with relevant laws and approved by the Institutional Animal Care and Use Committee of Soochow University (approval number: ecsu-2019000198).

[0034] 2. Reagents

[0035] M13mp18 DNA (scaffold chain), 10ug / tube, provided by Japan Baori Medical Biotechnology (Beijing) Co., Ltd., stored in a -20℃ refrigerator.

[0036] ssDNA short chain (staple chain), 5OD / tube, provided by China Sangon Biotechnology (Shanghai) Co., Ltd., stored in a -20℃ refrigerator.

[0037] Bone-targeted peptide DSS 6 , 100 mg / tube, synthesized and provided by Shanghai Qiangyao Biotechnology Co., Ltd., China, and stored in a -80℃ refrigerator.

[0038] β-CTX ELISA kit was provided by Shanghai ELISA Biotechnology Co., Ltd., China, with product number YJ002251.

[0039] P1NP ELISA kit was provided by Shanghai ELISA Biotechnology Co., Ltd., China, with the product number YJ063063.

[0040] The MDA kit was provided by Shanghai ELISA Biotechnology Co., Ltd., China, with the product number YJ016824.

[0041] The SOD kit was provided by Shanghai ELISA Biotechnology Co., Ltd., China, with the product number YJ643059.

[0042] 3. Instruments

[0043] The micro-computed tomography scanner (micro-CT, brand: Skyscan), manufactured in Belgium, was provided by the School of Radiation Medicine and Protection of SU.

[0044] The ELISA reader (multi-function microplate reader, brand: bio-tek) is produced in the United States and provided by the Institute of Functional Nanomaterials and Soft Matter of Soochow University.

[0045] Atomic force microscope (AFM, brand: Bruke), produced in Germany, was provided by the School of Radiation Medicine and Protection of SU.

[0046] Confocal microscope (brand: ZEISS), made in Germany, provided by the Institute of Functional Nanomaterials and Soft Matter of Soochow University.

[0047] Small animal in vivo fluorescence imaging system (brand: perkinelmer), produced in the United States, provided by the Institute of Functional Nanomaterials and Soft Matter of Soochow University.

[0048] Example 1

[0049] Synthesis and characterization of DNA nanopatch (DNP)

[0050] A 7,249-nucleotide circular scaffold chain (M13mp18, 10 nM) and a staple chain (20-60 nucleotides, the staple chain is based on the "Supplementary Information" in the document "Xiangbowen Jin. et al. DNA Nanopatch-Specific Modification of Probiotics for Ultrasound-Triggered Inflammatory BowelDisease Therapy. Journal of the American Chemical Society. 2024.", specifically, the sequences 1-216 and loops 1-10 in Table S1 disclosed in the above document, and the staple chain used in the present invention has some site sequences replaced based on the sequences in Table S1 above, and the replaced sequences are detailed in Tables 1 and 2 below) were mixed in 1×TAE-Mg 2+ rDON was formed in a buffer (20 mM acetic acid, 40 mM Tris base, 2 mM EDTA, 12.5 mM magnesium acetate, pH 8.0). The mixture was heated to 65 °C, cooled to 25 °C at a rate of 0.1 °C / min, and ultrafiltered (3500 g, 15 min) through a 100 kDa centrifugal filter (Amicon) to remove excess short chains, obtaining rectangular DNA origami for eliminating ROS.

[0051] Bone targeting peptide (DSS 6 , amino acid sequence: DSSDSSDSSDSSDSSDSS, SEQ ID NO.1) was synthesized by Shanghai Qiangyao Company, and Shanghai Shenggong synthesized a 5'-thiol-modified short chain (SH-ssDNA, 100 μM) (sequence: SH-TAAACTCTTTGCGCAC, SEQ ID NO.2). 6 The 3' end of the molecule was modified with a maleimide group (6-MAL) by adding a 3-fold excess of 6-maleimidocaproic acid to a dimethylformamide (DMF) solution containing a 3-fold excess of o-benzotriazole-N,N,N' (HBTU) and a 10-fold excess of diisopropylethylamine (DIEA) with DSS. 6 The peptide protecting groups were removed by trifluoroacetic acid (TFA), and after incubation at room temperature for 45 min, the final product, maleimide-modified bone-targeting peptide (MAL-DSS) was purified by high performance liquid chromatography (HPLC). 6 Then, SH-ssDNA was treated with 10 mM tris(2-carboxyethyl)phosphine (TCEP) aqueous solution for 30 min, and the excess TCEP was removed by ultrafiltration. 6The bone-targeting peptide-short chain (DSS-ssDNA) was reacted with SH-ssDNA in Dulbecco's phosphate buffer (DPBS) at a molar ratio of 5:1 at room temperature for 1 h. 6 -ssDNA). Purified by HPLC and characterized by mass spectrometry.

[0052] Cholesterol-short chain (CH-ssDNA) for anchoring on the surface of MSC membrane was synthesized by phosphoramidite method. 5-Cholesterol CE phosphoramidite (5-Cholesterol CE phosphoramidite) and DNA short chain (sequence: CGCATTCAGGATTCTCA, SEQ ID NO.3) were purchased from Shanghai Bioengineering. The DNA short chain was added to dichloromethane (DCM) containing 3% trichloroacetic acid (TCA) to block the protective group and obtain a free 5′-hydroxyl end. Next, the deprotected DNA short chain was added to a mixture of 0.1 M 5-Cholesterol CE (5-Cholesterol CE) and 0.5 M tetrazolium to obtain an intermediate. The intermediate was added to an acetonitrile solution containing acetic anhydride and 17.6% 1-methylimidazole, and then oxidized with 0.015 M iodine. The final product, cholesterol-short chain (CH-ssDNA), was purified by high performance liquid chromatography (HPLC) and determined by mass spectrometry.

[0053] Finally, 10-fold excess molar ratio of CH-ssDNA and 5-fold excess molar ratio of DSS were added. 6 -ssDNA was mixed with rDON. The mixture was heated to 45°C and cooled to 25°C at a rate of 0.2°C / min to obtain DNA nanopatch (DNP). Excess short chains were removed using a 100 kDa centrifugal filter.

[0054] Table 1. Staple chain replacement chain sequences for capturing cholesterol-short chains (5'-3' from left to right)

[0055]

[0056] Table 2. Staple chain replacement chain sequences for capturing bone targeting peptide-short chains (left to right 5'-3').

[0057]

[0058] Atomic force microscopy: 2 μl of 10 nM rDON or DNP was deposited on freshly cleaved mica cells and allowed to adsorb for 2-3 min. Then, 1×TAE / Mg was added to the liquid cell. 2+ The samples were scanned in an atomic force microscope (MultiMode 8 AFM (Bruker) ScanAsyst imaging system).

[0059] A 90 × 60 nm rectangular DNA origami (rDON) was successfully constructed by assembling scaffold chains and staple chains ( Figure 2 ), atomic force microscopy (AFM) images ( Figure 3 ) confirmed the successful assembly. Next, cholesterol-short chain (CH-ssDNA) was synthesized by the amidophosphorus method ( Figure 4 a), mass spectrometry showed that the synthesis was successful ( Figure 4 b). To further improve the bone targeting specificity of the nanostructures, we used the bone targeting peptide DSS 6 Modification of rDON,DSS 6 It is composed of 6 Asp-Ser-Ser repeating sequences and has a high affinity for low-crystalline hydroxyapatite and calcium phosphate in bone tissue. 6 The coupling with SH-ssDNA is based on DSS 6 The reaction between 6-MAL at one end and the thiol group of SH-ssDNA ( Figure 5 a). DSS was confirmed by mass spectrometry analysis 6 -ssDNA successful coupling ( Figure 5 b). We will then 6 -ssDNA and CH-ssDNA were simultaneously loaded onto rDON to form a DNA nanopatch (DNP / D / CH rDON)( Figure 6 ). Agarose gel electrophoresis showed that all nanostructures had clear main bands. As the number of loaded short chains increased, the migration speed became slower, confirming that the assembly was successful ( Figure 7 a). AFM images further confirmed the successful formation of DNPs ( Figure 7 b). Finally, MSCs and DNPs with green fluorescence were incubated at room temperature for 20 minutes. Confocal images showed that DNPs were successfully anchored on the MSC cell membrane ( Figure 8 ).

[0060] Example 2

[0061] Construction of DNA nanopatch-mesenchymal stem cells:

[0062] 10 6 MSCs were resuspended in 1 mL PBS, centrifuged at 300 × g for 3 min, and the supernatant was removed. The washed cells were resuspended in 200 µL PBS. DNP was added to the cell suspension to a final concentration of 5 nM, and the cells were incubated in a 500 rpm oscillating metal bath for 20 min to anchor DNP on the surface of MSCs to obtain DNA nanopatch-mesenchymal stem cells (DNP-MSCs). After the reaction, the cells were washed twice with 1 mL PBS, and finally resuspended in 200 µL PBS and subjected to fluorescence confocal microscopy imaging.

[0063] Mouse modeling and drug administration:

[0064] The groups were as follows: sham operation group (control), bilateral ovariectomy group (OVX), OVX+MSC treatment group, OVX+ D rDON( D There were 6 mice in each group.

[0065] Control group: The mice were 6-8 weeks old normal C57 mice, which were only subjected to sham surgery for control.

[0066] OVX group: 6-8 weeks old bilaterally ovariectomized mice were used for type I (postmenopausal) osteoporosis model.

[0067] OVX+MSC group: The mice were bilaterally ovariectomized at 6-8 weeks old. Four weeks after surgery, 200 μL PBS (containing 10 6 MSCs), once a week for 4 weeks.

[0068] OVX+ D rDON group: 6-8 weeks old bilaterally ovariectomized mice were injected with 200 μL PBS (containing 5 nM D rDON), once a week for 4 weeks.

[0069] OVX+ DNP-MSC group: The mice were bilaterally ovariectomized at 6-8 weeks of age. Four weeks after surgery, 200 μL PBS (10 μL modified with 5 nM DNP) was injected into the tail vein. 6 MSCs), once a week for 4 weeks.

[0070] The mouse samples of each group were as follows:

[0071] Collection of serum samples: Orbital blood sampling method was used. The mouse was pressed on the mouse cage with the left hand. The mouse's head was fixed with the thumb and index finger. The neck was tightly grasped and the two sides of the neck were compressed to make the eyeball protrude and the orbital venous plexus congested. The capillary was held in the right hand and inserted from the inner canthus of the eye at a 45° angle to the mouse's face. The mouse's body was fixed, the hind limbs were pressed, and the fingers were relaxed to adjust the capillaries so that the blood flowed out smoothly. After the blood was collected, the neck pressure was immediately removed to pull out the blood collector, and compression was immediately applied to stop the bleeding. The blood sample tube was gently shaken and placed on ice for 30 minutes. After that, it was centrifuged at 4°C and 3000rpm for 20 minutes. The supernatant was taken and centrifuged again at 4°C and 4000rpm for 10 minutes. The supernatant was a relatively pure serum sample for the detection of P1NP, β-CTX, SOD, and MDA levels.

[0072] Collection of mouse femur specimens: The mice were killed by cervical dislocation, the skin and muscles at the proximal end of the mouse lower limbs were cut to fully expose the femur and hip joint, the femur was carefully separated, the femur was taken out intact, and the muscles and other soft tissues were removed. The left femur was fixed with 4% PFA for 24-48 hours, and then replaced with 70% alcohol for micro-CT detection.

[0073] Mesenchymal stem cells were labeled with red fluorescent dye (Vybrant-DiD) and DNP-engineered MSCs (DNP-MSCs) were injected into mice via the tail vein. IVIS imaging was used for 48 hours to observe the targeting of MSCs to bone tissue. CH Compared with rDON-MSC, DNP-MSC had a significantly increased homing to bone tissue after administration. Further quantification using flow cytometry revealed that the fluorescence intensity of the femur was higher than that of the CH The rDON-MSC group was 5.25 times stronger than the natural MSC group and 7.97 times stronger ( Fig. 9 ). This indicates that the bone tissue targeting ability of DNP-engineered MSCs (DNP-MSCs) is significantly improved.

[0074] like Fig.10 As shown in the figure, the use of a micro-computed tomography (Micro-CT) scanner to scan the lower end of the femur of the above experimental mice and perform three-dimensional reconstruction and quantitative analysis can accurately describe the bone density and bone microstructure, thereby determining the degree of osteoporosis. The three-dimensional image reconstruction and quantitative results showed that compared with the control group, the number of trabeculae in the ovariectomized group of mice was significantly reduced, thinner, and the bone density decreased. After DNP-MSC intervention, the bone density of mice increased from 0.08 g / cm to 1.3 g / cm. 3 Increased to 0.13 g / cm 3 Compared with the natural MSC group, bone density was further increased by 30%.

[0075] Enzyme-linked immunosorbent assay (ELISA) was further used to detect the levels of serum bone metabolism indicators (β-CTX, P1NP) and oxidative stress-related factors (SOD, MDA) in mice of each experimental group. The levels of β-CTX and P1NP in serum can be used as markers of bone formation and bone resorption, respectively. SOD and MDA are the two most commonly used indicators in the evaluation of oxidative stress. SOD is an important antioxidant enzyme that removes superoxide anion free radicals in the body and can protect cells from damage by oxygen free radicals. MDA is one of the products formed by the reaction of lipids and oxygen free radicals, and its content represents the degree of lipid peroxidation. Fig.11As shown, the P1NP level in the DNP-MSC group was significantly increased (P<0.0001), and the β-CTX level was significantly decreased (P<0.0001). In addition, we also found that the SOD level of the OVX group (bilateral ovariectomy) mice was significantly decreased (P<0.0001), and the MDA level was increased (P<0.0001), while the DNP-MSC group could better restore the SOD and MDA levels of bilateral ovariectomized mice, and the effect was significantly better than that of the natural MSC group. This shows that DNP-MSC can further restore the bone mass loss and oxidative stress level in mice after bilateral ovariectomy compared with natural MSC.

Claims

1. A method for preparing a DNA nanopatch for enhancing MSC bone targeting ability, characterized in that: The method comprises the following steps: mixing a circular scaffold chain and a staple chain to obtain a rectangular DNA origami; modifying the 3' end of the bone targeting peptide with a maleimide group to obtain a bone targeting peptide modified with a maleimide group; mixing the bone targeting peptide modified with a maleimide group with a 5'-thiol modified short chain to obtain a bone targeting peptide-short chain; modifying cholesterol on the DNA short chain to obtain a cholesterol-short chain; Rectangular DNA origami, bone-targeting peptide-short chains and cholesterol-short chains were mixed to obtain a DNA nanopatch with enhanced MSC bone-targeting ability.

2. The method for preparing a DNA nanopatch for enhancing MSC bone targeting ability according to claim 1, characterized in that: The molar ratio of rectangular DNA origami, bone targeting peptide-short chain and cholesterol-short chain is 1:5~10:10~20.

3. The method for preparing a DNA nanopatch for enhancing MSC bone targeting ability according to claim 1, characterized in that: After the rectangular DNA origami, bone-targeting peptide-short chain and cholesterol-short chain were mixed, the mixture was heated to 40-50°C and cooled to 20-30°C at a rate of 0.15-0.25°C / min to obtain a DNA nanopatch with enhanced MSC bone targeting ability.

4. The method for preparing a DNA nanopatch for enhancing MSC bone targeting ability according to claim 1, characterized in that: The preparation method of the bone-targeting peptide-short chain comprises: adding a maleimide group to a dimethylformamide solution containing o-benzotriazole-N,N,N' and diisopropylethylamine to react with a bone-targeting peptide, removing the peptide protecting group with trifluoroacetic acid, incubating at room temperature, and purifying to obtain a bone-targeting peptide modified with a maleimide group; and reacting the bone-targeting peptide modified with a maleimide group with a 5'-thiol-modified short chain to generate a bone-targeting peptide-short chain.

5. The method for preparing a DNA nanopatch for enhancing MSC bone targeting ability according to claim 1, characterized in that: The preparation method of the cholesterol-short chain comprises: adding the DNA short chain into dichloromethane containing trichloroacetic acid to obtain a deprotected DNA short chain; adding the deprotected DNA short chain into a mixture of 5-cholesterol CE and tetrazole to obtain an intermediate; adding the intermediate into an acetonitrile solution containing acetic anhydride and 1-methylimidazole, and then oxidizing with iodine to obtain the cholesterol-short chain.

6. A DNA nanopatch, characterized in that: Prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the DNA nanopatch according to claim 6 in the preparation of osteoporosis drugs.

8. The use according to claim 7, characterized in that: The method for preparing the osteoporosis drug comprises: anchoring the DNA nanopatch according to claim 6 on the surface of mesenchymal stem cells to obtain DNA nanopatch-mesenchymal stem cells, and using the DNA nanopatch-mesenchymal stem cells to prepare the osteoporosis drug.

9. The use according to claim 8, characterized in that: The preparation method of the DNA nanopatch-mesenchymal stem cells comprises: adding the DNA nanopatch described in claim 6 to a mesenchymal stem cell suspension so that the final concentration of the DNA nanopatch is 4-6 nM, and after incubation in an oscillating metal bath, anchoring the DNA nanopatch on the surface of the mesenchymal stem cells to obtain the DNA nanopatch-mesenchymal stem cells.

10. The use according to claim 7, characterized in that: The osteoporosis drug is used for increasing the bone density of postmenopausal mice and improving the oxidative stress level in the body.

Images