A DNA nanosheet for enhancing the bone targeting ability of MSCs, its preparation method and application

Abstract

The present invention belongs to the technical field of cell membrane surface engineering, and specifically relates to a DNA nanosheet for enhancing the bone targeting ability of MSCs, its preparation method and application. The preparation method includes the following steps: mixing a circular scaffold strand and staple strands to obtain a rectangular DNA origami; modifying a maleimide group at the 3'-end of a bone targeting peptide to obtain a maleimide group-modified bone targeting peptide, and mixing the maleimide group-modified bone targeting peptide with a 5'-thiol-modified short chain to obtain a bone targeting peptide-short chain; modifying cholesterol on a DNA short chain to obtain a cholesterol-short chain; mixing the rectangular DNA origami, the bone targeting peptide-short chain and the cholesterol-short chain to obtain a DNA nanosheet for enhancing the bone targeting ability of MSCs. The DNA nanosheet can be used to strengthen MSCs, thereby preparing drugs for degenerative diseases.

Description

Technical Field

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

[0002] Osteoporosis is caused by the imbalance between osteoblast bone formation and osteoclast bone resorption. Current treatment methods mainly focus on anti-resorptive drugs and anabolic drugs, lacking treatment methods 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, etc., will also significantly affect the progression of osteoporosis. Recent studies have shown that ROS bone metabolism is harmful, which can inhibit osteogenic differentiation while stimulating osteoclast differentiation. Therefore, new treatment strategies must simultaneously address the problems brought by bone remodeling and the osteoporosis microenvironment.

[0003] As a precursor of osteoblasts, mesenchymal stem cell (MSC) transplantation can promote osteogenic differentiation, enhance bone mineral density, and slow down the progression of osteoporosis. However, current MSC therapies lack targeting ability and microenvironment regulation. Engineered MSCs focusing on gene editing have been developed to solve these problems, but they also increase the risk of immunogenic reactions and other complications related to genetic engineering. Non-gene-edited cell surface engineering endows MSCs with enhanced homing, adhesion, and migration abilities, as well as weakened immunogenicity and avoidance of genetic risks. Functionalized DNA is well-known for its easy synthesis and biocompatibility, and is an ideal choice for this kind of engineering. Framework nucleic acids (FNAs), especially DNA origami (DON), have superior stability and programmability. In addition, its phosphate backbone can chelate with osteocalcin, making it have a certain bone targeting ability, but their potential in anchoring MSCs for treating osteoporosis has not been explored yet. Summary of the Invention

[0004] To solve the above technical problems, the present invention develops a DNA nanosheet for enhancing the bone targeting ability of MSCs, a preparation method thereof, and an application thereof, which can be used to strengthen MSCs, thereby preparing drugs for degenerative diseases.

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

[0006] A preparation method of a DNA nanosheet for enhancing the bone targeting ability of MSCs, comprising the following steps: mixing a circular scaffold strand and staple strands to obtain a rectangular DNA origami; in a bone targeting peptide (DSS 6) The 3'-end of the peptide was modified with a maleimide group to obtain a bone-targeting peptide modified with a maleimide group. The bone-targeting peptide modified with a maleimide group was mixed with a 5'-thiol-modified short chain to obtain a bone-targeting peptide-short chain; cholesterol was modified on the DNA short chain to obtain a cholesterol-short chain; the rectangular DNA origami, the bone-targeting peptide-short chain, and the cholesterol-short chain were mixed to obtain a DNA nanosheet 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 mixing the rectangular DNA origami, the bone-targeting peptide-short chain, and the cholesterol-short chain, 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 nanosheet that enhances the bone-targeting ability of MSCs.

[0009] Furthermore, the preparation method of the bone-targeting 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-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; 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.

[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 with iodine to obtain a cholesterol-short chain.

[0011] The present invention provides a DNA nanosheet prepared by the above preparation method.

[0012] The present invention also provides the application of the above DNA nanosheet in the preparation of osteoporosis drugs.

[0013] Furthermore, the preparation method of the osteoporosis drug includes: anchoring the above DNA nanosheet on the surface of mesenchymal stem cells to obtain a DNA nanosheet-mesenchymal stem cell, and using the DNA nanosheet-mesenchymal stem cell to prepare an osteoporosis drug.

[0014] Furthermore, the preparation method of the DNA nanosheet-mesenchymal stem cell includes: adding the above DNA nanosheet to the mesenchymal stem cell suspension so that the final concentration of the DNA nanosheet is 4-6 nM, incubating in a shaking metal bath, and then anchoring the DNA nanosheet on the surface of the mesenchymal stem cell to obtain the DNA nanosheet-mesenchymal stem cell.

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

[0016] Beneficial effects

[0017] The dual targeting effects of rectangular DNA origami (rDON) and bone-targeting peptide (DSS 6 ) enable the therapeutically-potential MSCs to selectively accumulate in bone tissues after intravenous injection and directly play a role in increasing bone mass. As a ROS scavenger, rectangular DNA origami can not only promote osteogenic differentiation but also inhibit osteoclast formation by alleviating oxidative stress in the osteoporosis microenvironment.

[0018] The DNA nanosheet (DNP, which can also be abbreviated as D / CH rDON) is derived from rDON and is synthesized by loading cholesterol (CH) and bone-targeting peptide (DSS 6 ) on rDON. The DNP is anchored on the MSC cell membrane through the hydrophobic effect of cholesterol to form DNP-engineered MSCs (DNP-MSCs). After intravenous injection, these engineered MSCs preferentially accumulate in bone tissues driven by the bone-targeting properties of DNP. In bone tissues, MSCs home to their target tissues, differentiate into osteoblasts, secrete cytokines, and contribute to increasing bone mass. Meanwhile, DNP in the bone microenvironment promotes osteogenic differentiation and inhibits osteoclastogenesis by scavenging ROS prevalent in the osteoporosis environment. Description of the drawings

[0019] Figure 1 It is a schematic diagram of the mechanism for treating osteoporosis by engineering mesenchymal stem cells (MSCs) with DNA nanosheets (DNPs);

[0020] Figure 2 It is a schematic diagram of the basic structure of unfunctionalized rectangular DNA origami (rDON); the green lines represent staple strands, and the red lines represent the scaffold strand (M13mp18);

[0021] Figure 3 It is a representative atomic force microscopy image characterization of the morphology of unfunctionalized rectangular DNA origami (rDON); scale bar: 100 nm;

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

[0023] Figure 5 For the synthesis schematic diagram and mass spectrometry characterization of bone-targeting peptide-short chain (DSS 6 -ssDNA); where a is the schematic diagram of the synthesis 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 nanoplaster (DNP). The red circles represent 27 sites for covalent attachment of cholesterol molecules, and the purple triangles represent 10 sites for covalent attachment of bone-targeting peptide (DSS 6 ); The green rectangles represent the positions of FAM fluorescent dyes 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 the agarose gel electrophoresis analysis of different nanostructures, b is the representative atomic force microscopy image, scale bar: 100 nm;

[0026] Figure 8 Confocal microscopy image of DNA nanoplaster-engineered MSCs, and the green part outside the cells is the FAM fluorescent-labeled DNA nanoplaster;

[0027] Figure 9 Representative small animal in vivo optical imaging (IVIS) images of the lower limbs of mice after administration of DNA nanoplaster-engineered MSCs (DNP-MSCs); The mice were euthanized 48 hours after injection, and then the hind limbs were harvested immediately for IVIS imaging ( CH rDON-MSC is a rectangular DNA origami-engineered MSC modified only with cholesterol).

[0028] Figure 10 Micro-CT scan of the lower femurs of mice in each group and reconstruction of three-dimensional images and bone density quantification results ( D rDON is a rectangular DNA origami modified only with bone-targeting peptide);

[0029] Figure 11Results of mouse serum tests. Among them, a is the level of type 1 procollagen amino terminal peptide (P1NP) in the serum of mice in each experimental group detected by enzyme-linked immunosorbent assay (ELISA), b is the level of type 1 collagen carboxyl terminal peptide (β-CTX) in the serum of mice in each experimental group detected by ELISA, c is the level of superoxide dismutase (SOD) in the serum of mice in each experimental group detected by a kit, and d is the level of malondialdehyde (MDA) in the serum of mice in each experimental group detected by a kit (OVX is the bilateral ovariectomy group, D rDON is a rectangular DNA origami modified only with a bone-targeting peptide). Specific implementation methods

[0030] Experimental materials

[0031] 1. Experimental animals

[0032] 50 C57 / B6J female mice, 6 - 8 weeks old, weighing 21 - 37 g, SPF grade, provided by Changzhou Cavens Experimental Animal Co., Ltd. During the experiment, the temperature in the breeding room was 22 ± 3.0 °C, and the relative humidity was 60 - 70%. They had free access to food and water.

[0033] The animal experiment complied with relevant laws and was approved by the Institutional Animal Care and Use Committee of Soochow University (Approval No.: ecsu - 2019000198).

[0034] 2. Reagents

[0035] M13mp18 DNA (scaffold strand), 10 μg / tube, provided by Takara Bio Inc. (Beijing, China), stored in a -20 °C refrigerator.

[0036] ssDNA short chains (staple strands), 5 OD / tube, provided by Sangon Biotech (Shanghai) Co., Ltd., stored in a -20 °C refrigerator.

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

[0038] β-CTX ELISA kit, provided by Shanghai Enzyme-linked Biotechnology Co., Ltd., China, product catalog number is YJ002251.

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

[0040] MDA kit, provided by Shanghai Enzyme-linked Biotechnology Co., Ltd., China, with the product number YJ016824.

[0041] SOD kit, provided by Shanghai Enzyme-linked Biotechnology Co., Ltd., China, with the product number YJ643059.

[0042] 3. Instruments

[0043] Micro-computed tomography scanner (micro-CT, brand: Skyscan), produced in Belgium, provided by Soochow University School of Radiation Medicine and Protection.

[0044] Microplate reader (multifunctional microplate detector, brand: bio-tek), produced in the United States, provided by Soochow University Institute of Functional Nano and Soft Materials.

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

[0046] Confocal microscope (brand: ZEISS), produced in Germany, provided by Soochow University Institute of Functional Nano and Soft Materials.

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

[0048] Example 1

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

[0050] Mix the circular scaffold strand (M13mp18, 10 nM) of 7,249 nucleotides and staple strands (20 - 60 nucleotides, the staple strands are based on the "Supplementary Information" in the literature "Xiangbowen Jin. et al. DNA Nanopatch-Specific Modification of Probiotics for Ultrasound-Triggered Inflammatory Bowel Disease Therapy. Journal of the American Chemical Society. 2024.", specifically, the sequences 1 - 216 and loops 1 - 10 in Table S1 disclosed in the above literature, and some site sequences of the staple strands used in the present invention have been replaced on the basis of the sequences in Table S1 above, and the replaced sequences are shown in Table 1 and Table 2 below) in 1×TAE-Mg 2+ buffer (20 mM acetic acid, 40 mM Tris base, 2 mM EDTA, 12.5 mM magnesium acetate, pH 8.0) to form rDON. Heat the mixture to 65 °C and cool it to 25 °C at a rate of 0.1 °C / min. Ultrafilter (3500 g, 15 min) through a 100 kDa centrifugal filter (Amicon) to remove the excess short strands, obtaining a rectangular DNA origami for ROS elimination.

[0051] Bone-targeting peptide (DSS 6 , amino acid sequence: DSSDSSDSSDSSDSSDSS, SEQ ID NO.1) was synthesized by Shanghai Qiangyao Co., Ltd., and Shanghai Sangon synthesized the 5'-thiol-modified short strand (SH-ssDNA, 100 μM) (sequence: SH-TAAACTCTTTGCGCAC, SEQ ID NO.2). To modify the maleimide group (6-MAL) at the 3' end of DSS 6 , add 3-fold excess of 6-maleimidocaproic acid to a dimethylformamide (DMF) solution containing 3-fold excess of o-benzotriazole-N,N,N' (HBTU) and 10-fold excess of diisopropylethylamine (DIEA) and react with DSS 6 . Remove the peptide protecting group with trifluoroacetic acid (TFA). After incubating at room temperature for 45 min, purify by high performance liquid chromatography (HPLC) to obtain the final product maleimide group-modified bone-targeting peptide (MAL-DSS 6 ). Then, treat SH-ssDNA with 10 mM aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) for 30 min and ultrafilter to remove the excess TCEP. Then MAL-DSS 6React with SH-ssDNA at a molar ratio of 5:1 in Dulbecco's phosphate buffered saline (DPBS) at room temperature for 1 h to generate bone-targeting peptide-short chain (DSS 6 -ssDNA). Purify by HPLC and characterize by mass spectrometry.

[0052] Synthesize cholesterol-short chain (CH-ssDNA) for anchoring on the MSC membrane surface using the phosphoramidite method. 5-Cholesterol CE phosphoramidite and DNA short chain (sequence: CGCATTCAGGATTCTCA, SEQ ID NO.3) were both purchased from Sangon Biotech (Shanghai). Add the DNA short chain to dichloromethane (DCM) containing 3% trichloroacetic acid (TCA) to block the protecting group, obtaining a free 5'-hydroxyl end. Next, add the deprotected DNA short chain to a mixture of 0.1 M 5-Cholesterol CE and 0.5 M tetrazole to obtain an intermediate. Add the intermediate to an acetonitrile solution containing acetic anhydride and 17.6% 1-methylimidazole, and then oxidize 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, mix 10-fold excess molar ratio of CH-ssDNA and 5-fold excess molar ratio of DSS 6 -ssDNA with rDON. Heat the mixture to 45 °C and cool it to 25 °C at a rate of 0.2 °C / min to finally obtain DNA nanoplaster (DNP). Remove the excess short chain using a 100 kDa centrifugal filter.

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

[0055]

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

[0057]

[0058] Atomic force microscopy imaging: Deposit 2 μL of 10 nM rDON or DNP on a freshly cleaved mica cell and allow it to adsorb for 2 - 3 minutes. Then, add 1×TAE / Mg 2+ buffer in the liquid cell. Place the sample in an atomic force microscope (MultiMode 8 AFM (Bruker) ScanAsyst imaging system) for scanning.

[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 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] Harvesting of mouse femoral specimens: The mice were sacrificed by cervical dislocation. The skin and muscles of the proximal part of the lower limbs of the mice were incised to fully expose the femur and hip joint. The femur was carefully separated, taken out intact, and the muscles and other soft tissues were removed. The left femur was fixed with 4% PFA and stored for 24 - 48 hours, and then replaced with 70% alcohol for storage, for detection by micro-computed tomography (micro-CT).

[0073] Mesenchymal stem cells were labeled with a 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. Compared with natural MSCs and simple rectangular origami-engineered MSCs ( CH rDON-MSCs), the homing ability of DNP-MSCs to bone tissue increased significantly after administration. Further quantification using flow cytometry found that the fluorescence intensity of the femur was 5.25 times stronger than that of the CH rDON-MSC group and 7.97 times stronger than that of the natural MSC group ( Figure 9 ). It shows that the bone tissue targeting ability of DNP-engineered MSCs (DNP-MSCs) is significantly improved.

[0074] As Figure 10 shown, the lower end of the femur of the above experimental mice was scanned using a micro-computed tomography scanner (Micro-CT) and three-dimensional reconstruction and quantitative analysis were performed, which can accurately describe bone density and the microscopic structure of bones, thereby determining the degree of osteoporosis. The results of three-dimensional image reconstruction and quantification showed that: compared with the control group, the number of trabecular bones in the ovariectomized group of mice decreased significantly, became thinner, and bone density decreased. After intervention with DNP-MSCs, the bone density of the mice increased from 0.08 g / cm 3 to 0.13 g / cm 3 compared with the ovariectomized group, and further increased by 30% compared with the natural MSC group.

[0075] Furthermore, enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of serum bone metabolism indexes (β-CTX, P1NP) and oxidative stress-related factors (SOD, MDA) in mice of each experimental group. The contents 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 indexes in evaluating the oxidative stress process. SOD is an important antioxidant enzyme that scavenges superoxide anion free radicals in vivo and can protect cells from oxygen free radical damage. MDA is one of the products formed by the reaction of lipids with oxygen free radicals, and its content represents the degree of lipid peroxidation. As Figure 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 in the OVX group (bilateral ovariectomy) mice was significantly decreased (P<0.0001), and the MDA level was increased (P<0.0001). The DNP-MSC group could better restore the SOD and MDA levels in bilateral ovariectomy mice, and the effect was significantly better than that of the natural MSC group. This indicates that DNP-MSC can further restore the bone mass loss and the in vivo oxidative stress level in mice after bilateral ovariectomy compared with natural MSC.

Claims

1. A method for preparing a DNA nanopatch for enhancing the bone targeting ability of MSCs, 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; The rectangular DNA origami, bone-targeting peptide-short chain and cholesterol-short chain were mixed to obtain a DNA nanopatch that enhances the bone-targeting ability of MSCs; the annular scaffold chain was M13mp18; the staple chain was based on the literature "Xiangbowen Jin. et al. DNANanopatch-Specific Modification of Probiotics for Ultrasound-TriggeredInflammatory Bowel Disease The invention relates to a method for treating a bone-targeted peptide comprising: preparing a bone-targeted peptide comprising: a peptide having an amino acid sequence as shown in SEQ ID NO. 1, a 5'-thiol-modified short chain sequence as shown in SEQ ID NO. 2, and a DNA short chain sequence as shown in SEQ ID NO.

3.

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 HBTU 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.

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