Bone-targeted functionalized tetrahedral framework nucleic acid nano system as well as preparation method and application thereof

Abstract

The invention discloses a bone-targeted functionalized tetrahedral framework nucleic acid nano system as well as a preparation method and application thereof, the bone-targeted functionalized tetrahedral framework nucleic acid nano system comprises tetrahedral framework nucleic acid tFNA-Sa, polypeptide SDSSD with bone cell specific targeting and an osteoporosis treatment drug, the bone targeting tetrahedral framework nucleic acid tFNA-Sa is used for modifying bone targeting polypeptide to obtain the bone targeting tetrahedral framework nucleic acid tFNAs-SDSSD, the editability and the biocompatibility of the nucleic acid tetrahedron are utilized, the SDSSD bone targeting peptide is modified on the surface of the tetrahedron, the bone targeting ability can be given to the bone targeting peptide, and then the purposes of efficient aggregation of the medicine in bone tissue, high-efficiency drug delivery and the like are achieved. The distribution of the medicine in non-target tissues is reduced. The targeted delivery system not only can improve the treatment effect of the medicine, but also can greatly reduce the side effect of the medicine, and brings a new scheme for targeted therapy for postmenopausal osteoporosis.

Description

Technical Field

[0001] This invention relates to the field of medicine, specifically to a bone-targeting functionalized tetrahedral framework nucleic acid nanosystem, its preparation method, and its applications. Background Technology

[0002] Postmenopausal osteoporosis (PMOP) is a common metabolic bone disease in postmenopausal women. PMOP is the main type of osteoporosis in women. The mouse embryonic osteoblast precursor cell line (MC3T3-E1), as a cell line with a clearly defined osteogenic lineage and directional differentiation ability, plays a crucial role in the study of bone formation and development, exhibiting unique biological characteristics and has been proven to be a good model for in vitro osteogenic studies. Traditional treatments for osteoporosis mainly involve basic nutritional support such as calcium and vitamin D supplementation, and the use of anti-resorption drugs such as bisphosphonates, denosumab, and hormone replacement therapy. However, these methods have many drawbacks. For example, long-term use of bisphosphonates may lead to increased bone fragility and gastrointestinal problems; denosumab may cause weakened immunity; and hormone replacement therapy may increase the risk of breast cancer and cardiovascular disease.

[0003] In recent years, the use of single-component traditional Chinese medicines (TCM) in the treatment of osteoporosis has gradually gained public attention. Gentian root, a plant-derived estrogen, has a chemical structure similar to mammalian estrogens. It can regulate bone metabolism, reduce bone resorption, and promote osteoblast proliferation, differentiation, and bone tissue formation by binding to estrogen receptors in the body, significantly improving bone mineral density and bone quality in postmenopausal osteoporosis patients. For example, a randomized controlled trial confirmed that 24 months of treatment with genistein had a positive effect on bone mineral density in postmenopausal women with osteoporosis; a Cochrane systematic review indicated that soy isoflavones (containing genistein) may slow down bone mineral density loss in postmenopausal women. Although genistein shows promising potential in the treatment of postmenopausal osteoporosis, its clinical application still faces some challenges, such as low bioavailability and poor targeting. In the development of modern pharmaceutical technology, nanocarrier drug delivery systems have shown significant application prospects, particularly in improving drug stability, targeting, bioavailability, and reducing side effects. However, existing nanodelivery systems still have some shortcomings and challenges: for example, liposomes have a retention effect, resulting in uneven drug distribution; polymer nanoparticles have certain cytotoxicity at high concentrations; and lipid nanoparticles have limited delivery efficiency, usually requiring higher doses. These shortcomings limit their widespread application in the clinical field.

[0004] Tetrahedral framework nucleic acids (tFNAs), as an emerging type of functional nanomaterial, exhibit excellent biocompatibility and low immunogenicity in the biomedical field. This unique material is a three-dimensional nanostructure formed by the self-assembly of four single-stranded DNA (ssDNA) strands through a precise base-complementary pairing mechanism.

[0005] Furthermore, tFNAs possess unique editability. This characteristic allows tFNAs to achieve diverse functional applications through a series of simple chemical modifications, thus playing an important role in gene regulation and targeted delivery. For example, Cai et al. linked tFNAs to si-RAGE via sticky ends, effectively and continuously downregulating RAGE expression, thereby inhibiting inflammatory responses by blocking the NF-κB pathway and exhibiting antioxidant function. Liao et al. developed a tFNA-based bioswitchable siRNA delivery system (BiRDS) to carry CKIP-1 (a negative regulator of bone formation) siRNA, which significantly promoted osteogenic differentiation and bone regeneration in rat mandibular defects. SDSSD peptide is a short peptide composed of five amino acids: serine, aspartate, serine, serine, aspartate. It specifically recognizes and binds to osteoblasts in bone tissue and has been widely studied for the development of bone-targeted drug delivery systems. Researchers have combined SDSSD peptide with the cationic polymer polyethyleneamine (PVAm) to construct a bone-targeted delivery system for delivering therapeutic RNA (such as anti-miR-138-5p). This system has demonstrated good bone-targeting ability and osteoblast differentiation-promoting capacity in both in vitro and in vivo experiments. Other researchers have developed SDSSD-modified polyurethane (PU) nanomicelles encapsulating siRNA / microRNA, which can deliver anti-miR-214 to osteoblasts. Although both of these delivery systems have solved the problem of targeted delivery, they both exhibit certain toxicity at high concentrations, limiting their application. Summary of the Invention

[0006] The purpose of this invention is to provide a bone-targeting functionalized tetrahedral framework nucleic acid nanosystem, its preparation method, and its applications.

[0007] The objective of this invention is achieved as follows:

[0008] A bone-targeting functionalized tetrahedral framework nucleic acid nanosystem includes tetrahedral framework nucleic acid tFNA-Sa, a peptide SDSSD with specific targeting of bone cells, and an osteoporosis treatment drug. The molar ratio of tetrahedral framework nucleic acid to peptide SDSSD is 1:1. Tetrahedral framework nucleic acid tFNA-Sa is modified with peptide SDSSD to obtain bone-targeting tetrahedral framework nucleic acid tFNAs-SDSSD. The molar ratio of bone-targeting tetrahedral framework nucleic acid tFNAs-SDSSD to osteoporosis treatment drug is 1:40.

[0009] The tetrahedral framework nucleic acid tFNA-Sa is obtained by base complementary pairing of single-stranded deoxyribonucleic acid S3, single-stranded deoxyribonucleic acid S1, single-stranded deoxyribonucleic acid S2 and single-stranded deoxyribonucleic acid S4 carrying sticky ends with special sequences. The single-stranded deoxyribonucleic acid S3 carrying sticky ends with special sequences is single-stranded deoxyribonucleic acid S3-Sa2.

[0010] The amino acid sequence of the bone cell-specific targeting peptide SDSSD is: Ser-Asp-Ser-Ser-Asp, Ser-Asp-Ser-Ser-As, which are respectively serine, aspartate, serine, serine, and aspartate. After chemical modification, it is combined with Fmoc-L-propargylglycine Fmoc-Pra-OH to synthesize propargylglycine-modified bone-targeting peptide Pra-SDSSD.

[0011] The osteoporosis treatment drug is genistein.

[0012] A method for preparing a bone-targeting functionalized tetrahedral framework nucleic acid nanosystem includes the following steps:

[0013] Step 1: Single-stranded deoxyribonucleic acid S1, single-stranded deoxyribonucleic acid S2, single-stranded deoxyribonucleic acid S3-Sa2 and single-stranded deoxyribonucleic acid S4 are synthesized into tetrahedral framework nucleic acid tFNA-Sa2 through nucleic acid self-assembly;

[0014] Step 2: By using a click chemical reaction, the azide-modified sticky terminal Sa2*Sa2*-N3 is combined with Pra-SDSSD to synthesize the bone-targeting peptide Sa2*-SDSSD modified with the Sa2* sticky terminal.

[0015] Step 3: Then, the tFNA-Sa2 obtained in Step 1 and the Sa2*-SDSSD obtained in Step 2 are combined through the sticky end Sa2 and Sa2* base complementary pairing to obtain bone-targeting tetrahedral framework nucleic acid tFNAs-SDSSD;

[0016] Step 4: After mixing bone-targeting tetrahedral framework nucleic acids tFNAs-SDSSD with genistein, the mixture is reacted by shaking at room temperature to obtain a bone-targeting functionalized tetrahedral framework nucleic acid nanosystem.

[0017] The reaction conditions for the synthesis of tFNA-Sa2 were as follows: first, reaction at 95℃ for 10 min; then reaction at 4℃ for 20 min; and finally reaction at 4℃ for 60 min. The synthesis reactions of Sa2*-SDSSD and tFNAs-SDSSD were both at room temperature, with reaction times of 6 h and 3 h, respectively. The reaction of bone-targeting tetrahedral framework nucleic acid tFNAs-SDSSD with genistein was carried out at room temperature for 3 h.

[0018] Applications of a bone-targeting functionalized tetrahedral framework nucleic acid nanosystem: its application in the preparation of specific targeting and enrichment drugs for mouse embryonic osteoblast precursor cell lines and mouse femoral, tibial, and vertebral bone tissues; and its application in the preparation of drugs for osteoporosis-related diseases.

[0019] The osteoporosis-related disease mentioned is postmenopausal osteoporosis.

[0020] The drug is a drug that promotes the bone regeneration capacity of osteoblasts.

[0021] The beneficial effects of this invention are as follows: By utilizing the editability and biocompatibility of nucleic acid tetrahedra, this invention modifies the surface of SDSSD bone-targeting peptides, endowing them with bone-targeting capabilities. This allows for efficient drug aggregation in bone tissue and reduces drug distribution in non-target tissues. This targeted delivery system not only improves the therapeutic effect of drugs but also significantly reduces drug side effects, providing a new approach for targeted therapy of postmenopausal osteoporosis. Functionalized tetrahedral framework nucleic acid nanomedicines loaded with genistein possess osteoblast targeting ability, the biological advantages of tFNAs, and the ability of Gen to promote osteoblast formation in osteoporotic cells, thereby achieving the goal of high-quality osteoporosis treatment. Attached Figure Description

[0022] Figure 1 This is a schematic diagram illustrating the preparation of the bone-targeting functionalized tetrahedral framework nucleic acid nanosystem (hereinafter referred to as tFNAs-SDSSD&Gen) of the present invention;

[0023] Figure 2 Image showing the PAGE detection results of Cy5-tFNAs-SDSSD-FITC;

[0024] Figure 3 The graph shows the particle size and potential detection results for tFNAs-SDSSD and tFNAs-SDSSD / Gen.

[0025] Figure 4 The images show the AFM and TEM detection results for tFNAs-SDSSD and tFNAs-SDSSD / Gen.

[0026] Figure 5 Image showing the encapsulation rate test results for tFNAs-SDSSD equipped with Gen;

[0027] Figure 6 Figure A shows the immunofluorescence (IF) detection results of tFNAs-SDSSD / Gen-Cy5 and Gen-Cy5 entering cells, and Figure B shows the flow cytometry detection results.

[0028] Figure 7 A graph showing the ability of Cy5-tFNAs-SDSSD-FITC to target osteoblasts (MC3T3) in vitro was obtained by co-culturing Cy5-tFNAs-SDSSD-FITC and Cy5-tFNAs-Sa2 with mouse osteoblasts (MC3T3) and detecting cell IF at different time points.

[0029] Figure 8 Figure 1 shows the IF assay results of different cells (MC3T3, RAW264.7 and BMSCs) treated with Cy5-tFNAs-SDSSD-FITC, Cy5-tFNAs-Sa2 and Sa2*-SDSSD-FITC.

[0030] Figure 9 Fluorescence imaging of bone tissue at different time points after tail vein injection of Cy5-tFNAs-Sa2 and Cy5-tFNAs-SDSSD-FITC in mice. Figure A shows in vitro fluorescence imaging of the hind limbs of mice in each group; Figure B shows quantitative analysis of the fluorescence signal in each hind limb in Figure A; Figure C shows in vitro fluorescence imaging of the spine of mice in each group; Figure D shows quantitative analysis of the fluorescence signal in each spine sample in Figure C.

[0031] Figure 10 Serial in vivo fluorescence imaging of small animals after tail vein administration of tFNAs-SDSSD / Gen-Cy5, tFNAs / Gen-Cy5, and Gen-Cy5. Figure A shows the ex vivo fluorescence imaging of the hind limbs of mice in each group; Figure B shows the quantitative analysis of the fluorescence signal in each hind limb in Figure A; Figure C shows the ex vivo fluorescence imaging of the spine of mice in each group; Figure D shows the quantitative analysis of the fluorescence signal in each spine sample in Figure C.

[0032] Figure 11 ALP and Alizarin Red staining images of IL-1β-pretreated MC3T3 cells with tFNAs-SDSSD, Gen, and tFNAs-SDSSD / Gen after drug treatment;

[0033] Figure 12 The results of ALP (Figure A) and OSX (Figure B) cellular IF detection for each group of cells are shown in the figure.

[0034] Figure 13 The graph shows the Western blot (WB) results and statistical analysis results of osteogenic-related proteins in each group of cells; the data are presented in the figure.

[0035] Mean ± standard deviation (n=3); Statistical analysis: *p<0.05, **p<0.01, **p<0.001;

[0036] Figure 14 Schematic diagram of tFNAs-SDSSD, Gen, and tFNAs-SDSSD / Gen in vivo treatment of osteoporosis in mice;

[0037] Figure 15 The results of Micro-CT scans of the femur and related indicators of mice in each group after treatment are presented as quantitative analysis graphs; data are expressed as mean ± standard deviation (n=3); statistical analysis: *p<0.05, **p<0.01, **p<0.001;

[0038] Figure 16 The results of Micro-CT scans of the tibia and related indicators of mice in each group after treatment are presented as quantitative analysis graphs; data are expressed as mean ± standard deviation (n=3); statistical analysis: *p<0.05, **p<0.01, **p<0.001;

[0039] Figure 17 The images show the H&E and Masson staining results of femoral and tibial pathological sections of mice in each group after treatment.

[0040] Figure 18 The images show the H&E staining results of the heart, liver, spleen, lungs, and kidneys of mice in each group after treatment. Detailed Implementation

[0041] In the following examples and experimental cases, reagents and materials not specifically described are all commercially available products. "Room temperature" as used in this invention refers to 25±10℃, and "overnight" refers to 12±5h.

[0042] Example 1. Synthesis and identification of tFNAs-SDSSD and tFNAs-SDSSD / Gen complex

[0043] I. Experimental Methods: 1. Synthesis of tFNAs-SDSSD: 1.1 Synthesis of Sa2*-SDSSD

[0044] Powdered Sa2*-N3 and Pra-SDSSD were dissolved in enzyme-free water under aseptic conditions to prepare stock solutions of 200 μM and 600 μM, respectively. After vortexing and centrifugation, the solutions were obtained. Their sequences are shown in Table 2.

[0045] Table 2: Single-stranded sequences of Sa2*-SDSSD

[0046]

[0047] 1.2. Synthesis of tFNAs-Sa2: Four single-stranded deoxyribonucleic acid (ssDNA) samples were used in this experiment. Three of them, S1, S2, and S4, were powdered single-stranded deoxyribonucleic acid (ssDNA: 8.5 nmol), and the other was ssDNA with sticky ends (S3-Sa2). Under aseptic conditions, these samples were dissolved in enzyme-free water to prepare a 100 μM stock solution, vortexed, and centrifuged. Their ssDNA sequences are shown in Table 1:

[0048] Table 1: Single-stranded sequences of Sa2-tFNAs

[0049]

[0050] Prepare a TM buffer solution consisting of 50 mM Tris-HCl and 8 mM MgSO4, adjusting the pH to 8.0. Add 96 μL of TM buffer to an eight-tube bundle, then add 1 μL each of the dissolved S1, S2, S3-Sa2, and S4 solutions to the bundle, bringing the total volume of the mixture in the bundle to 100 μL. Shake the bundle to ensure thorough mixing, then centrifuge to ensure the stability of the solution. Place the eight-tube bundle containing single-stranded DNA (ssDNA) into a thermal cycling apparatus and program it for a light-protected reaction. The specific program is as follows: maintain the temperature at 95°C for 10 minutes, then adjust the temperature to 4°C and maintain it for 20 minutes. When the reaction continues for 30 minutes, tFNAs-Sa2 synthesis is complete, with a concentration of 1000 nM. The synthesized tFNAs-Sa2 was stored in a refrigerator at 4°C for long-term preservation. Unless otherwise specified, all subsequent experiments using tFNAs-Sa2 were synthesized according to the detailed steps and conditions described above.

[0051] In a clean bench, 29.86 μL of dimethyl sulfoxide (DMSO) was first added to a 1 mL EP tube. Next, 10 μL of 10 mM copper sulfate (CuSO4) and 3.54 μL of 28.3 μM tris(benzyltriazolylmethyl)amine (TBTA) were added sequentially to the EP tube. After the additions were complete, the EP tube was gently shaken to ensure thorough mixing. A complexation reaction was then carried out at room temperature for 5 minutes to chelate divalent copper ions. After the reaction was complete, 10 μL of vitamin C (Ascorbic Acid) was added to the EP tube, and the mixture was shaken again and centrifuged to ensure sufficient contact between the vitamin C and the solution. The reaction was carried out at room temperature for 5 minutes to reduce divalent copper ions to monovalent copper ions. Finally, 10 μL of Sa2*-N3 (200 μM) and 6.6 μL of Pra-SDSSD (600 μM) were added sequentially, the mixture was shaken and centrifuged, and the solution was allowed to react overnight at room temperature to complete the synthesis of Sa2*-SDSSD. The synthesized Sa2*-SDSSD was purified on the second day. The specific steps were as follows: 175 μL of ice-cold anhydrous ethanol was mixed with 7 μL of ice-cold 3M sodium acetate solution. This mixture was then added to the above reaction system. After shaking and mixing, the mixture was centrifuged at 4°C and 14000 RCF for 15 minutes. A yellow-green precipitate was observed to form. After centrifugation, the supernatant was carefully discarded, and an equal volume of 70% ethanol was added to resuspend the precipitate. The precipitate was washed and centrifuged again at 4°C and 14000 RCF for 15 minutes. The supernatant was discarded, the EP tube cap was opened, and the precipitate was allowed to air dry at room temperature. 20 μL of enzyme-free water was added to dissolve the precipitate, and the purified Sa2*-SDSSD with a concentration of 100 μM was obtained.

[0052] 1.3 Synthesis of tFNAs-SDSSD: In a clean bench, open an eight-tube containing 100 μL of tFNA-Sa2, add 1 μL of Sa2*-SDSSD, shake to mix and centrifuge, incubate at room temperature for 1 hour, and synthesize tFNAs-SDSSD by the principle of complementary base pairing.

[0053] 1.4. Synthesis of tFNAs-SDSSD / Gen: Powdered Gen (Selleck, S2301) was dissolved in dimethyl sulfoxide (DMSO) to prepare a 40 mM stock solution. The stock solution was aliquoted and placed in light-proof bags, then stored at -80°C. The synthesized tFNAs-SDSSD and Gen stock solutions were precisely mixed at a ratio of 1:40, i.e., 1 μL of Gen (40 μM) was added to 1000 μL of tFNAs-SDSSD (1000 nM). Under light-proof conditions, the mixture of tFNAs-SDSSD and Gen was continuously shaken at room temperature for 3 hours to synthesize the tFNAs-SDSSD / Gen complex.

[0054] 2. Particle Size and Potential Detection: Following the steps described above, synthesize 1 mL each of tFNAs-SDSSD and tFNAs-SDSSD / Gen. Add each to the inner tube of an ultrafiltration centrifuge tube. Centrifuge the samples at 5000g for 5 minutes in a dark environment, carefully discarding the waste liquid in the outer tube. Next, invert the inner tube centrifuge column and centrifuge again at the same force (5000g) for 5 minutes. Collect the resulting bottom liquid and store it on ice to maintain sample stability. Strictly follow the standard operating procedures to turn on the equipment power, start the Zetasizer software, and connect it to the nanoparticle size analyzer (Malvern, Nano ZS, UK). During connection, ensure a stable connection to guarantee the accuracy of subsequent detections. Place the tFNAs-SDSSD and tFNAs-SDSSD / Gen samples into the nanoparticle size analyzer column and use the nanoparticle size analyzer to detect the particle size and charge of the samples. Each sample group was set up with 3 replicates. The results of each sample group were recorded in detail and statistically analyzed. Based on the results of the statistical analysis, corresponding charts were drawn to show relevant information such as particle size and charge of the samples.

[0055] 3. Atomic Force Microscopy (AFM) Detection: Take 10 μL each of the synthesized and ultrafiltered tFNAs-SDSSD and tFNAs-SDSSD / Gen, and dilute the sample solution to be tested at a volume ratio of 1:10. Add an appropriate amount of the diluted solution evenly to the surface of the mica sheet, ensuring the droplets completely cover the surface. Place the mica sheet loaded with the sample in a well-ventilated environment at room temperature to dry naturally. After the surface liquid has completely evaporated, use sterile micro-tweezers to transfer the dried mica sheet to the AFM detection system (Shimadzu, SPM-9700, Japan). Following standardized laboratory management procedures, the detection process was performed by qualified professional technicians. The pretreated mica sheet was fixed on the AFM detection platform, the sample was tested, and the surface morphology and dimensional parameters of tFNAs-SDSSD and tFNAs-SDSSD / Gen were recorded.

[0056] 4. Transmission Electron Microscopy (TEM) Detection: The synthesized and ultrafiltered tFNAs-SDSSD and tFNAs-SDSSD / Gen were stored on ice in the dark. Approximately 20 μL of the sample was evenly dropped onto a clean sample plate. A copper mesh was gently placed upside down on the surface of the droplet, ensuring full contact and wetting of the front side of the mesh. After standing for 3 minutes, the mesh was uprighted using tweezers, and the sample-loaded mesh was allowed to air dry at room temperature, maintaining a clean environment to avoid contamination. Subsequently, 2% phosphotungstic acid (pH 7.0) was used as a staining agent. The dried copper mesh was again inverted and placed over the staining solution, maintaining the staining time for 2 minutes. After staining, the edges of the mesh were held with tweezers, and excess staining solution was absorbed with filter paper. The treated copper mesh was then transferred to the TEM detection well. According to laboratory management regulations, this testing operation was performed by professional technicians who were qualified to operate a transmission electron microscope (TEM, JEM-1400FLASH, Japan), and the surface dimensions and morphology of tFNAs-SDSSD and tFNAs-SDSSD / Gen were recorded during the testing process.

[0057] 5. Polyacrylamide Gel Electrophoresis Detection: 5.1. Preparation of Polyacrylamide Gel and Electrophoresis Buffer: Add double-distilled water (ddH2O), 40% acrylamide (Acr), 10x TAE buffer (tris(hydroxymethyl)aminomethane, acetic acid, and ethylenediaminetetraacetic acid), 10% ammonium persulfate (APS), and tetramethylethylenediamine (TEMED) to a 50mL centrifuge tube according to the ratio of 4.2mL:1.2mL:0.6mL:60μL:6μL. Gently shake to thoroughly mix all components. Then pour the mixture into a clean glass plate. Insert a 1mm thick comb from left to right between the two glass plates to avoid air bubbles. Allow it to solidify completely. Once a uniform and transparent colloidal structure has formed, the polyacrylamide gel meeting the experimental requirements is obtained. (Gels, PAGE), and all polyacrylamide gels required for subsequent experiments were prepared according to this standardized procedure. Next, 20 mL of 50x TAE was taken to prepare 1000 mL of 1x TAE buffer.

[0058] 5.2. Polyacrylamide gel electrophoresis detection: Take an eight-tube and add 2 μL of 6x DNA dilution buffer. Take 10 μL of 1000 nM Cy5-S1, S2, S3, S4, Cy5-tFNAs-Sa2, and Cy5-tFNAs-2SDSSD-FITC and add them to the eight-tube. Mix them thoroughly with the 6x DNA to dilute it to 1x. The polyacrylamide gel (PAGE) was immersed in 1xTAE buffer. The comb was carefully removed vertically from the PAGE gel. Following a pre-set experimental sequence, 6 μL of DNA markers, 10 μL of 1000 nM Cy5-S1, S2, S3, S4, Cy5-tFNAs-Sa2, and Cy5-tFNAs-2SDSSD-FITC were added sequentially to the corresponding positions on the gel. Electrophoresis was performed on the polyacrylamide gel (PAGE) at a constant voltage of 80 V for 1 hour. After electrophoresis, nucleic acid staining solution (Gel stain red:ddH2O = 1:10000 = 10 μL:100 mL) was prepared. The gel was carefully removed and incubated in the developing solution with shaking for 15 minutes in the dark. The gel was then placed on an imaging system (Thermo Fisher Scientific, iBright CL1500, USA), and imaging was performed according to the preset parameters and procedures.

[0059] In another eight-tube strip, add 2 μL of 6x DNA dilution buffer. Then, add 10 μL of 1000 nM Cy5-tFNAs-Sa2, Cy5-tFNAs-SDSSD-FITC, Cy5-tFNAs-2SDSSD-FITC, and Cy5-tFNAs-3SDSSD-FITC to the eight-tube strip and mix thoroughly with the 6x DNA. After soaking the PAGE gel in 1x TAE solution, the comb was removed vertically, and 6 μL of DNA marker, 10 μL of 1000 nM Cy5-tFNAs-Sa2, Cy5-tFNAs-SDSSD-FITC, Cy5-tFNAs-2SDSSD-FITC, Cy5-tFNAs-3SDSSD-FITC, and 6 μL of DNA marker were added sequentially. Electrophoresis was performed on the polyacrylamide gel (PAGE) at a constant voltage of 80 V for 1 hour. After electrophoresis, the gel was carefully removed and placed in a dark environment for incubation with nucleic acid staining solution for 15 minutes with shaking. The gel was then placed in an imaging system for imaging.

[0060] 6. Drug Encapsulation Efficiency Assay: Following the above-described method for synthesizing tFNAs-SDSSD / Gen, tFNAs-SDSSD (1000 nM, 200 μL) was mixed with Gen (40 mM) at ratios of 1:10, 1:20, 1:40, 1:80, 1:160, and 1:320. The mixture was shaken at room temperature for 3 hours to achieve final Gen concentrations of 10 μM, 20 μM, 40 μM, 80 μM, 160 μM, and 320 μM. The samples were ultrafiltered at 5000 g for 5 minutes, and the precipitate was collected and balanced to 200 μL. Gen was diluted with 200 μL of DMSO to create different concentration gradients: 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM, and 80 μM. Using dimethyl sulfoxide (DMSO) as a blank control (Blank group), the UV-Vis absorbance analyzer was initialized and calibrated. Gen was diluted to a concentration of 1 μM, placed on the detection stage, and detected using the calibrated UV-Vis absorbance analyzer to determine the characteristic absorption peak value of Gen. Based on the measured characteristic absorption peak value of Gen, the absorption intensity of Gen solutions at different concentrations was measured, and a standard curve was plotted. Then, the absorption peak values ​​of tFNAs-SDSSD / Gen precipitates at different concentration gradients were detected sequentially. By comparing with the standard curve, the concentration of Gen that did not complex with tFNAs-SDSSD was calculated. This concentration was compared with the initial Gen concentration to obtain the proportion of tFNAs-SDSSD without Gen and the efficiency of tFNAs-SDSSD encapsulating Gen. The experiment was repeated three times.

[0061] 7. Detection of tFNAs-SDSSD / Gen cell penetration ability: 7.1. Cell IF detection: MC3T3-E1 single-cell suspension was seeded in confocal microplates, approximately 10⁴ cells per well. Three experimental groups were set up: blank control group (CON), Gen-Cy5 group, and tFNAs-SDSSD / Gen-Cy5 group. Genistein was labeled with the Cy5 fluorescent group (excitation light: 650 nm), and the complex tFNAs-SDSSD / Gen-Cy5 was synthesized according to the above method. When the cells grew to 80% confluence, the culture medium (2% FBS, 1% penicillin antibiotics) containing TM buffer, Gen-Cy5, and tFNAs-SDSSD / Gen-Cy5 was replaced. After culturing for 8 hours in a cell culture incubator at 37°C and 5% CO₂, the culture medium in the confocal microplates was gently aspirated under light-protected conditions, and the cells were washed three times with PBS for 10 minutes each time. Then, 1 mL of 4% paraformaldehyde was added to each confocal microplate to fix the cells. The microplates were placed in a refrigerator at 4°C for 30 minutes to fix the cells. The cells were washed three times with PBS for 10 minutes each time. 500 μL of freshly prepared 0.5% phalloidin (FITC) (excitation light: 488 nm) was added, and the microplates were incubated at 37°C for 30 minutes. The cells were washed three times with PBS for 10 minutes each time. 0.1% 4',6-diamidinyl-2-phenylindole (DAPI, excitation light: 350 nm) needs to be freshly prepared and used immediately. Before use, it should be thoroughly mixed. 500 μL of the prepared DAPI solution was added to the confocal microplates and incubated at room temperature for 15 minutes. The cells were washed three times with PBS for 10 minutes each time. Finally, the cells were kept moist by immersing them in 10% glycerol solution. The cells were observed and fluorescent images were captured using a fluorescence microscope (Olympus, FV3000, Japan). The distribution of the drug within the cells was analyzed by observing fluorescence images. The experiment was repeated three times.

[0062] 7.2 Flow cytometry (FC) assay: MC3T3-E1 single-cell suspensions were seeded into six-well plates, approximately 10 cells per well. 5Three experimental groups were set up using 100% confluence of cells: a blank control group (CON), a Gen-Cy5 group, and a tFNAs-SDSSD / Gen-Cy5 group. Once the cells reached 100% confluence, the culture medium (2% FBS, 1% penicillin antibody) was replaced with TM buffer, Gen-Cy5, and tFNAs-SDSSD / Gen-Cy5, respectively. The cells were then cultured at 37°C in a 5% CO2 incubator for 8 hours. After 8 hours of culture, the cells were washed three times with PBS buffer. EDTA-free trypsin was preheated by adding 1 mL of trypsin to each well of the cultured cells for digestion, and the cells were digested at 37°C for 2 minutes. During digestion, cell morphology was closely observed under a microscope. When cells appeared shrunken and bright, but had not yet floated, trypsin was carefully removed using a pipette. Digestion was immediately terminated with 2 mL of PBS containing 2% FBS, and the cells were resuspended. The digested cell suspension was collected into three separate 15 mL centrifuge tubes and centrifuged at 1000 rpm for 5 min. After centrifugation, the supernatant was carefully discarded using a pipette, retaining the cell pellet at the bottom of the tube. Then, 1 mL of PBS was added to the centrifuge tube to resuspend the cells, ensuring they were evenly dispersed in the buffer. The cell suspension was centrifuged again under the same conditions (1000 rpm, 5 min), the supernatant was discarded, and the cell pellet was resuspended in 500 μL of PBS to prepare a single-cell suspension. Single-cell suspensions were placed on the flow cytometer stage. The voltage parameters of the flow cytometer were adjusted using blank control samples to ensure accurate identification and detection of cell signals. Simultaneously, based on the signal characteristics observed in the blank control samples, the target cell population was selected. The flow cytometer parameters were adjusted, and an appropriate excitation wavelength was selected. The number of positive cells in the Gen-Cy5 and tFNAs-SDSSD / Gen-Cy5 groups was recorded. All experiments were conducted under dark conditions. The experiments were repeated three times. Statistical analysis was then performed, and graphs were generated.

[0063] II. Experimental Results: According to the aforementioned ( Figure 1The following steps were performed using PAGE gel electrophoresis. The molecular weights of Cy5-tFNAs-Sa2 were approximately 230 bp, Cy5-tFNAs-SDSSD-FITC approximately 280 bp, Cy5-tFNAs-2SDSSD-FITC approximately 340 bp, and Cy5-tFNAs-3SDSSD-FITC approximately 400 bp. The molecular weight increased with the increase in the number of SDSSD peptides attached to the tFNAs, confirming the successful synthesis of tFNAs-SDSSD. Furthermore, the co-localization of Cy5 fluorescence on the S1 chain and FITC fluorescence on the SDSSD peptide further confirms that the SDSSD peptide successfully modified the tFNAs, indicating the successful synthesis of tFNAs-SDSSD. Figure 2 Serum stability assays showed that both Cy5-tFNAs-Sa2 and Cy5-tFNAs-SDSSD-FITC exhibited only minor degradation after 6 hours of incubation. After 12 hours of incubation, both materials showed low degradation rates and remained relatively stable. The tetrahedral structure did not exhibit decreased stability due to modification with bone-targeting peptides, demonstrating that tFNAs-SDSSD is a relatively stable nanonucleic acid material. Figure 3 Particle size analysis showed that the particle size of tFNAs-SDSSD was approximately 21.98 nm, and the particle size of tFNAs-SDSSD / Gen was approximately 32.57 nm. Potential analysis showed that the potential of pure tFNAs was approximately -9.20 mV, the SDSSD peptide was positively charged, while Gen was neutral. After SDSSD modification, the potential of tFNAs-SDSSD was approximately -2.87 mV, and the potential of tFNAs-SDSSD / Gen was approximately -2.86 mV. Figure 6 The message indicates successful synthesis of tFNAs-SDSSD / Gen. Figure 5 AFM detection results showed that the diameter of tFNAs-SDSSD was approximately 20 nm, and the diameter of tFNAs-SDSSD / Gen was approximately 40 nm. TEM detection results showed that the diameter of tFNAs-SDSSD was approximately 20 nm, and the diameter of tFNAs-SDSSD / Gen was approximately 40 nm, and both exhibited a three-dimensional structure, proving the successful synthesis of tFNAs-SDSSD and tFNAs-SDSSD / Gen. Figure 8 The results of UV-Vis spectrophotometer analysis indicate that a molar mass ratio of tFNAs-SDSSD to Gen of 1:40 can ensure both the loading efficiency of tFNAs-SDSSD and the concentration of Gen. Therefore, a tFNAs-SDSSD to Gen loading ratio of 1:40 is optimal. Figure 9After treating MC3T3-E1 cells with tFNAs-SDSSD / Gen-Cy5 and Gen-Cy5 for 8 hours, confocal microscopy was used to detect their entry into the cells. The results after 8 hours showed that the Cy5 fluorescence in MC3T3-E1 cells in the tFNAs-SDSSD / Gen-Cy5 group was significantly stronger than that in the Gen-Cy5 group. These results demonstrate that tFNAs-SDSSD can facilitate the entry of more Gen into cells, significantly improving Gen bioavailability. Figure 6 A). After treating MC3T3-E1 cells with tFNAs-SDSSD / Gen-Cy5 and Gen-Cy5 for 8 hours, the cell entry was quantitatively detected by flow cytometry. The results showed that the positive percentage of MC3T3-E1 cells in the Gen-Cy5 group was 10.5%, while the positive rate in the tFNAs-SDSSD / Gen-Cy5 group was 99.3%, which was significantly higher than that in the Gen-Cy5 group, and the difference was statistically significant. The results demonstrate that tFNAs-SDSSD can significantly improve the efficiency of Gen entry into cells, increasing the efficiency by approximately 9 times. Figure 6 B). *P<0.05, **P<0.01, ***P<0.001, ns= No significance.

[0064] Example 2. tFNAs-SDSSD / Gen targeting detection

[0065] I. Experimental Methods: 1. Detection of tFNAs-SDSSD's ability to penetrate MC3T3-E1 cells: Experimental groups were set up: blank control group, Cy5-tFNAs-Sa2, and Cy5-tFNAs-SDSSD-FITC. SDSSD peptides were labeled using fluorescently labeled FITC (excitation light: 488nm), and Cy5-tFNAs-Sa2 and Cy5-tFNAs-SDSSD-FITC were synthesized according to the above method. MC3T3-E1 cells were seeded in confocal dishes, with approximately 1 x 10-1 cells per well. 4Cells were cultured at 37°C in a 5% CO2 incubator. When the cells reached 80% confluence, they were treated with medium containing TM buffer, Cy5-tFNAs-Sa2, and Cy5-tFNAs-SDSSD-FITC (2% FBS, 1% penicillin antibody). After 2, 4, and 8 hours, under dark conditions, the medium was removed from the confocal dish, and the cells were washed three times with PBS for 10 minutes each time. Then, 4% paraformaldehyde was added for cell fixation at 4°C for 30 minutes. The cells were washed three times with PBS for 10 minutes each time, and then 500 μL of freshly prepared 0.5% red fluorescent phalloidin (FITC) (excitation light: 550 nm) was added. The cells were stained at 37°C for 30 minutes, and then washed three times with PBS for 10 minutes each time. DAPI needs to be prepared and used immediately. Before use, it should be thoroughly mixed. Add 500 μL of the prepared DAPI solution to a confocal dish and incubate at room temperature for 15 minutes. Wash the cells with PBS three times for 10 minutes each time. Finally, immerse the cells in 10% glycerol solution. Adjust the fluorescence microscope parameters, selecting an appropriate laser wavelength and scanning depth based on the excitation spectrum characteristics of the target fluorescent dye. Use a 60x oil immersion microscope to scan and image the fluorescence of the cells, and detect the targeting ability of tFNAs-SDSSD to enter MC3T3-E1 cells at different time points. The experiment was repeated three times.

[0066] 2. Detection of tFNAs-SDSSD targeting of MC3T3-E1 cells: MC3T3-E1 cells were seeded in confocal microplates, with approximately 1 x 10-1 cells per well. 4 BMSCs and RAW264.7 cells were cultured in a 37℃, 5% CO2 incubator. Cells were seeded in the same manner and used when they reached 80% confluence. Cy5-tFNAs-Sa2, Cy5-tFNAs-SDSSD-FITC, and Sa2*-SDSSD-FITC were synthesized according to the above method. Experimental groups were set up for each cell type: Sa2*-SDSSD-FITC, Cy5-tFNAs-Sa2, and Cy5-tFNAs-SDSSD-FITC. The synthesized drugs were added to each of the three cell types, and the cells were cultured for another 4 hours. Cell nuclei and cytoplasm were stained as described above, and cell images were acquired under a fluorescence microscope to observe the targeting of tFNAs-SDSSD to different cell types. The experiment was repeated three times.

[0067] 3. Small Animal In vivo Imaging Detection: 3.1. Detection of bone targeting ability of tFNAs-SDSSD in vivo: Experimental groups were set up: Cy5-tFNAs-Sa2 group and Cy5-tFNAs-SDSSD-FITC group. 15 female C57BL / 6 mice were in each group, with 5 time points (1h, 2h, 3h, 4h, 6h), and 3 mice in each group at each time point. The synthesized Cy5-tFNAs-Sa2 (1μM) and Cy5-tFNAs-SDSSD-FITC (1μM) were administered via tail vein injection, 100μL / mouse. The specific procedure is as follows: Mice were fixed in a cage, and their tails were wiped with 75% alcohol swabs to fully dilate blood vessels. The tails were then illuminated with a tail vein ring lamp to fully expose the tail veins. The lateral tail vein was selected for injection, with the needle inserted parallel to the tail at an angle of approximately 10°. The syringe was gently pushed, and injection continued when there was no resistance. After injection, the needle was withdrawn, and hemostasis was achieved by applying pressure with a dry cotton ball. Mice were euthanized by intraperitoneal injection of sodium pentobarbital solution at 1, 2, 3, 4, and 6 hours later, followed by tissue sampling. The spine, bilateral femurs, and tibias of the mice were removed under light-protected conditions. The mouse bone tissue was placed in the dark chamber of a small animal in vivo imaging system (IVIS, Lumina III, USA). The imaging parameters were adjusted, and a suitable excitation wavelength (650 nm for Cy5) was selected for imaging. The imaging results were analyzed to observe the enrichment of Cy5-tFNAs-Sa2 and Cy5-tFNAs-SDSSD-FITC in the mouse bone tissue.

[0068] 3.2. Detection of in vivo bone targeting ability of tFNAs-SDSSD / Gen: Three experimental groups were set up: Gen-Cy5, tFNAs-Sa2 / Gen-Cy5, and tFNAs-SDSSD / Gen-Cy5. Eighteen female C57BL / 6 mice were used in each group, with three mice per group at six time points (1h, 2h, 3h, 4h, 6h, and 8h). Drug administration was performed via tail vein injection. Bone tissue samples were collected from the mice at 1, 2, 3, 4, 6, and 8 hours. Subsequently, bone tissue images of these mice were created using a small animal in vivo imaging system. The drug accumulation in bone tissue was compared among the three experimental groups by analyzing the imaging results.

[0069] II. Experimental Results: After treating MC3T3-E1 cells with TM buffer, Cy5-tFNAs-Sa2, and Cy5-tFNAs-SDSSD-FITC at 2, 4, and 8 hours respectively, confocal microscopy was used to detect drug targeting and cell entry. The results showed that at 4 hours, the fluorescence of the Cy5-tFNAs-SDSSD-FITC group was significantly stronger than that of Cy5-tFNAs-Sa2, and the two fluorescence types co-localized in the Cy5-tFNAs-SDSSD-FITC group, indicating that Cy5-tFNAs-SDSSD-FITC is relatively stable, can be taken up by cells, and its targeting and cell entry efficiency is significantly higher than that of Cy5-tFNAs-Sa2. At 8 hours, both groups showed strong fluorescence, with Cy5-tFNAs-SDSSD-FITC showing stronger fluorescence. The results above suggest that the SDSSD peptide can target MC3T3-E1 cells. After modification with tFNAs, tFNAs-SDSSD possesses both targeting and intracellular penetration capabilities. Its targeting effect allows it to recognize MC3T3-E1 cells more quickly and accurately, improving the efficiency of tFNAs entering MC3T3-E1 cells (Figure 7). Four hours after treating MC3T3-E1, BMSCs, and RAW264.7 cells with Cy5-tFNAs-Sa2, Cy5-tFNAs-SDSSD-FITC, and Sa2*-SDSSD-FITC materials, respectively, confocal microscopy was used to detect drug targeting and intracellular penetration. The results showed that after treatment with Sa2*-SDSSD-FITC, the MC3T3-E1 group exhibited significantly higher fluorescence compared to the BMSCs and RAW264.7 groups, while the BMSCs and RAW264.7 groups showed no significant fluorescence. This indicates that Sa2*-SDSSD-FITC only targets MC3T3-E1 cells, and has no targeting effect on BMSCs and RAW264.7 cells. Cy5-tFNAs-Sa2 and Cy5-tFNAs-SDSSD-FITC all showed some cell entry ability in the MC3T3-E1, BMSCs, and RAW264.7 cell groups, but Cy5-tFNAs-SDSSD-FITC showed the strongest targeting and entry ability and the strongest fluorescence in MC3T3-E1 cells compared to the other two cell types. This suggests that modification with bone-targeting peptides improved the entry efficiency of tFNAs into MC3T3-E1 cells, while having no such effect on the other two cell types. Figure 8Bone tissue samples were collected from mice at 1h, 2h, 3h, 4h, and 6h after Cy5-tFNAs-Sa2 and Cy5-tFNAs-SDSSD-FITC were administered via tail vein injection. Imaging results showed that Cy5-tFNAs-SDSSD-FITC was highly enriched in bone tissues such as the femur, tibia, and spine, and had a longer retention time compared to Cy5-tFNAs-Sa2, indicating that Cy5-tFNAs-SDSSD-FITC has good bone targeting properties. Imaging of mouse bone tissue at 1h, 2h, 3h, 4h, 6h, and 8h after administration of tFNAs-SDSSD / Gen-Cy5, tFNAs / Gen-Cy5, and Gen-Cy5 via tail vein injection showed that, compared to the tFNAs / Gen-Cy5 and Gen-Cy5 groups, tFNAs-SDSSD / Gen-Cy5 was significantly enriched in bone tissues such as the femur, tibia, and spine, and had a longer retention time. There was no significant difference between the tFNAs / Gen-Cy5 and Gen-Cy5 groups, indicating that tFNAs-SDSSD / Gen-Cy5 can effectively deliver therapeutic drugs to bone tissue.

[0070] Example 3. Effect of tFNAs-SDSSD / Gen on osteogenic differentiation capacity of MC3T3-E1 cells in vitro.

[0071] I. Experimental Methods: 1. Alkaline Phosphatase Detection: 1.1. Effect of Alkaline Phosphatase Detection on the Osteogenic Differentiation Capacity of MC3T3-E1 Cells: Recombinant Murine IL-1β lyophilized powder was dissolved in sterile water to a concentration of 10000 ng / mL, aliquoted, and stored at -20℃ protected from light. MC3T3-E1 cells were digested and centrifuged to prepare cells with a density of 102... 51 mL of cell suspension was evenly seeded into one well of a 12-well plate, with 6 groups (0 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, 40 ng / mL). Cells were used when they reached 100% adherence. IL-1β was added to 1 mL of osteogenic induction medium to prepare IL-1β suspensions at concentrations of 0 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, and 40 ng / mL. These suspensions were then added to the corresponding wells of the plate. The IL-1β-containing osteogenic induction medium was replaced every 2 days until day 7 of induction. The IL-1β-containing osteogenic induction medium was then removed. Cells were washed three times with PBS for 10 min each time. 4% paraformaldehyde was added, and cells were fixed at 4°C for 30 min. Cells were then washed three times with PBS for 10 min each time. Following the instructions for alkaline phosphatase (ALP) (Beyotime, C3206, China), prepare 6 mL of ALP staining working solution under light-protected conditions and mix thoroughly. Add 1 mL of the prepared ALP staining working solution to each of the six groups. Incubate the 12-well plates at 37°C for 30 minutes. Carefully aspirate the ALP staining solution with a pipette, wash three times with PBS to ensure complete removal of residual staining, and photograph the samples using a stereomicroscope. Repeat the experiment three times.

[0072] 1.2. Effect of alkaline phosphatase on the osteogenic differentiation capacity of MC3T3-E1 cells: Experimental groups were set up as follows: MC3T3-E1 group, MC3T3-E1+IL-1β group, MC3T3-E1+IL-1β+tFNAs-SDSSD group, MC3T3-E1+IL-1β+Gen group, and MC3T3-E1+IL-1β+tFNAs-SDSSD / Gen group. 250 μL of tFNAs-SDSSD (1000 nM), Gen (40 μM), and tFNAs-SDSSD / Gen (1000 nM:40 μM) were synthesized according to the aforementioned method. The synthesized materials were added to 750 μL of α-MEM complete medium containing 2% FBS and mixed thoroughly. MC3T3-E1 cells were digested and centrifuged to prepare cells with a density of 102. 51 mL of cell suspension was evenly seeded into one well of a 12-well plate. When MC3T3-E1 cells reached 100% confluence, the original culture medium was aspirated, and 1 mL of the pre-prepared mixture was added to the corresponding well. After addition, the plate was returned to the incubator and cultured for another 8 hours. The mixture in the well was carefully aspirated, and the cells were gently washed twice with pre-warmed PBS. Subsequently, 1 mL of osteogenic induction solution containing IL-1β (10 ng / mL) was added to each well for osteogenic induction. The drug administration and osteogenic induction were repeated every 2 days until day 7 of induction. Cells were fixed with 4% paraformaldehyde at 4°C, and 1 mL of ALP staining solution was added to each well under light-protected conditions. The plate was then placed in a 37°C incubator for staining for 30 minutes. The ALP staining solution was carefully aspirated with a pipette, and the cells were washed three times with PBS to ensure thorough removal of residual staining solution. The samples were photographed using a stereomicroscope. The experiment was repeated three times.

[0073] 2. Alizarin Red Staining Detection of the Effect of tFNAs-SDSSD / Gen on the Osteogenic Differentiation Capacity of MC3T3-E1 Cells: Experimental groups were set up as follows: MC3T3-E1 group, MC3T3-E1+IL-1β group, MC3T3-E1+IL-1β+tFNAs-SDSSD group, MC3T3-E1+IL-1β+Gen group, and MC3T3-E1+IL-1β+tFNAs-SDSSD / Gen group. Drug synthesis, drug addition, and osteogenic induction were performed according to the above steps. Drug addition and osteogenic induction were repeated every 2 days. Induction was stopped after 28 days when calcium nodules were observed under a microscope. Aspirate the osteogenic induction solution containing IL-1β, wash cells with PBS, add 1 mL of fixative from the osteoblast mineralization nodule staining kit (Alizarin Red S method, Beyotime, C0148S), fix cells for 20 minutes, aspirate the fixative, wash three times with PBS, then add 1 mL of Alizarin Red S staining solution to each well, gently agitate to ensure even staining, stain at room temperature for 30 minutes, observe for successful staining, aspirate the staining solution, and finally wash thoroughly with distilled water to remove residual staining solution. Observe and photograph under a microscope. Repeat the experiment three times.

[0074] 3. Cellular IF assay to detect the effect of tFNAs-SDSSD / Gen on the expression of osteogenic-related proteins in MC3T3-E1 cells: Experimental groups were set up as follows: MC3T3-E1 group, MC3T3-E1+IL-1β group, MC3T3-E1+IL-1β+tFNAs-SDSSD group, MC3T3-E1+IL-1β+Gen group, and MC3T3-E1+IL-1β+tFNAs-SDSSD / Gen group. MC3T3-E1 cells were seeded in confocal microarrays according to the aforementioned steps. Drug synthesis, drug addition, and osteogenic induction were performed according to the above steps. Drug addition and osteogenic induction were repeated every 2 days. After 7 days of osteogenic induction, the osteogenic induction solution was aspirated with a pipette, and the cells were washed three times with PBS for 10 min each time. 4% paraformaldehyde was added to the cells, and the confocal microarrays were placed in a refrigerator at 4°C for 30 min to fix the cells. The cells were then washed three more times with PBS. Prepare 0.5% Triton-100 perforation buffer (Aladdin, 9002-93-1, China) at a ratio of PBS:Triton-100 = 200:1, add 1 mL to each well, and perforate for 20 minutes to facilitate antibody entry. Wash cells three times with PBS for 10 minutes each time, then add goat serum (PBS:goat serum = 20:1) (Soleb, SL038, China) to block the cells and incubate at 37°C for 20 minutes. After removing the goat serum, add 300 μL of primary antibody (1:200, diluted in PBS) to each well, evenly covering the cells (Table 3), and incubate overnight at 4°C. The next day, remove the cells from the low-temperature environment and allow them to warm to room temperature for 30 minutes. Carefully aspirate the primary antibody solution from the confocal dish using a pipette. Wash the cells three times with PBS for 10 minutes each time to thoroughly remove residual primary antibody. Add 500 μL of secondary antibody solution (excitation light: 594 nm, goat anti-rabbit) diluted 1:200 with PBS to each well. After adding the secondary antibody, place the confocal dish on a shaker and incubate at room temperature for 1 hour. Wash three times with PBS, 10 min each time. Then, following the previous steps, stain MC3T3-E1 cells with 500 μL of phalloidin (excitation light: 488 nm) and 500 μL of DAPI (excitation light: 350 nm), respectively. Finally, add 10% glycerol solution to the cells to fully infiltrate them, and observe and image the cells using a fluorescence microscope at 60x magnification. All experiments involving cellular immunofluorescence (IF) detection in this study were strictly performed according to this procedure.

[0075] Table 3: Antibody Sources and Dilution Ratios

[0076]

[0077] 4. Immunoblot analysis of the effect of tFNAs-SDSSD / Gen on the expression of osteogenic-related proteins in MC3T3-E1 cells: Experimental groups were set up as follows: MC3T3-E1 group, MC3T3-E1+IL-1β group, MC3T3-E1+IL-1β+tFNAs-SDSSD group, MC3T3-E1+IL-1β+Gen group, and MC3T3-E1+IL-1β+tFNAs-SDSSD / Gen group. Cells were seeded in 6-well plates according to the above steps, and drugs were synthesized, added, and osteogenic induction was performed. On day 7 of osteogenic induction, the osteogenic induction solution was gently aspirated with a pipette. Cell lysis buffer was prepared according to the instructions of the total protein extraction kit (KGI, KGP250, China), and the lysis buffer was placed on ice after preparation. After washing cells three times with pre-chilled PBS, 300 μL of cell lysis buffer was added to each well of a 6-well plate, ensuring the buffer completely covered the cell surface. The plate was then transferred to ice and lysed by shaking for 15 minutes. Once fully lysed, cells were scraped from the wells using a sterile cell scraper. The collected cell lysates were transferred to 1.5 mL centrifuge tubes and sonicated three times at low temperature. Subsequently, the protein concentration of each group of cell lysates was determined using a BCA protein quantification kit (KGPBCA, China). After adjusting the protein sample concentrations of all groups to be consistent, 5x protein loading buffer (Beyotime, P0015, China) was added. After thorough mixing, the protein samples were heated at 100°C for 30 minutes to complete the protein denaturation process. Prepare a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel according to the instructions. Place the gel in the electrophoresis tank, add electrophoresis buffer to cover the gel, remove the comb, and add 10 μL of protein marker and total protein for each group sequentially. After electrophoresis, cut the gel, transfer the membrane, block with rapid blocking buffer for 15 min, add specific primary antibody solution diluted 1:1000, and incubate overnight in a shaker at 4°C. The next day, remove the bands from the 4°C environment and allow them to warm to room temperature for 30 min. Recover the primary antibody, transfer the membrane to a culture dish containing Tris buffered saline-Tween (TBST), and wash three times with low-speed shaking at room temperature for 10 min each time. Then incubate with secondary antibody (1:3000) for 1 hour. The TBST washed the strip three times again, and the developer solution was prepared according to the instructions (Affinity, KF8001, China). The strip was immersed in the developer solution and incubated for 1 minute. Finally, it was placed in the imaging system (Thermofisher, USA) for development and imaging.To ensure the reliability and accuracy of the experimental results, the above experiment was repeated three times. After all three experiments were completed, the detection results obtained from each experiment were systematically collected. Using ImageJ professional image processing software, quantitative statistical analysis of the grayscale values ​​of the collected detection results was performed, and corresponding charts were generated. All detection experiments involving protein immunoblotting (WB) in this study were strictly performed according to the above steps.

[0078] 5. Statistical Analysis: In this study, for cytological experiments, the number of replicates was three to ensure the reproducibility and accuracy of the results. All experimental data in this study are presented as mean ± standard deviation. The data provided in this paper have undergone rigorous screening and validation, are highly representative, and can truly reflect the actual experimental situation. Statistical analysis in this study was performed using GraphPad Prism 9 software (GraphPad, USA). Student's t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. Where: * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, and ns represents No significance.

[0079] II. Experimental Results: Following the aforementioned administration and osteogenic induction methods, ALP and Alizarin Red staining were performed 7 and 28 days after osteogenic induction. Results showed that the IL-1β group had significantly reduced ALP secretion and calcium nodule formation compared to the CON group, indicating successful IL-1β modeling. The IL-1β+tFNAs-SDSSD / Gen group had significantly more ALP secretion and calcium nodule formation compared to the IL-1β+Gen and IL-1β+tFNAs-SDSSD groups. Figure 11 The results demonstrated that tFNAs-SDSSD / Gen promoted ALP secretion, calcium salt secretion, and calcium nodule formation in MC3T3-E1 cells under IL-1β conditions. MC3T3-E1 cells treated with the drug were induced to osteogenic processes for 7 days with an osteogenic induction solution containing IL-1β (10 ng / mL). Immunofluorescence staining of osteogenic-related proteins (alkaline phosphatase (ALP) and osteoblast-specific transcription factor (Osterix, OSX)) was performed according to the aforementioned steps. The results showed that IL-1β treatment significantly reduced the expression of osteogenic-related proteins ALP and OSX. In contrast, IL-1β + tFNAs-SDSSD / Gen treatment significantly increased the expression of osteogenic-related proteins ALP and OSX compared to the IL-1β + Gen and IL-1β + tFNAs-SDSSD groups. Figure 12This further demonstrates that tFNAs-SDSSD / Gen promotes the expression of osteogenic-related proteins in MC3T3-E1 cells under IL-1β conditions. Whole protein extraction and Western blotting were performed on the cells in each group, revealing osteogenic-related proteins: Runt-related transcription factor 2 (RUNX2), ALP, OSX, and Osteopontin (OPN). The results showed that after treatment with IL-1β+Gen, IL-1β+tFNAs-SDSSD, and IL-1β+tFNAs-SDSSD / Gen, compared to the IL-1β group, the levels of osteogenic-related proteins RUNX2, ALP, OSX, and OPN were all increased. However, the increase was most significant in the IL-1β+tFNAs-SDSSD / Gen group, indicating that tFNAs-SDSSD / Gen has a more pronounced osteogenic effect. The statistical analysis of the WB band gray values ​​was significant. Figure 13 The results showed that tFNAs-SDSSD / Gen promotes the expression of early and late osteogenic-related proteins in MC3T3-E1 cells in the IL-1β environment.

[0080] Example 4. Therapeutic effect of tFNAs-SDSSD / Gen on OVX mice

[0081] 1. Animal Experiment Grouping and Drug Administration: Osteoporosis-prone mice (OVX mice) were created through castration and divided into 5 groups: CON+TM buffer group, OVX+TM buffer group, OVX+tFNAs-SDSSD group, OVX+TM buffer+Gen group, and OVX+tFNAs-SDSSD / Gen group, with 6 mice in each group. After successful OVX modeling, the mice were routinely fed for one month, and then injected with 100 μL of the drug via the tail vein every two days. The drug dosages were tFNAs-SDSSD (1000 nM), Gen (40 μM), and tFNAs-SDSSD / Gen (1000 nM: 40 μM). After two months of treatment, the mice were euthanized, and tissues such as the heart, liver, spleen, lung, kidney, femur, and tibia were collected. During the collection process, care was taken to avoid damaging the tissues and ensure their integrity. The collected tissues were promptly tested.

[0082] 2. Micro-computed tomography (MCT): Mice that were previously grouped (n=6 per group) were anesthetized and euthanized. Both femurs and tibias were collected intact using surgical instruments. The surface muscles of both femurs and tibias were carefully removed using ophthalmic scissors. Bone tissue samples were fixed in a 4% paraformaldehyde solution at room temperature for 72 hours. Micro-CT scans were then performed on the bone tissue samples to obtain three-dimensional reconstructed images and bone-related parameters. The acquired bone parameters were statistically analyzed using statistical analysis software, and corresponding charts were generated.

[0083] 3. Hematoxylin-eosin staining: Femoral and tibial samples fixed in 4% paraformaldehyde for 72 hours were decalcified until the bone tissue became pliable. The bone tissue was then dehydrated, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Finally, the samples were observed under a microscope, and the acquired images were recorded.

[0084] 4. Masson staining: Femoral and tibial samples fixed in 4% paraformaldehyde for 72 hours were decalcified until the bone tissue became pliable. The bone tissue was then dehydrated, embedded in paraffin, sectioned, and stained with Masson's stain. Finally, the samples were observed and images recorded under a microscope.

[0085] 5. Biosafety Testing: After two months of drug treatment in mice, vital internal organs were removed, fixed, embedded, and sectioned. After sectioning, H&E staining was performed. The sections were then placed under a microscope for careful observation of their tissue structure and photographic recording.

[0086] 6. Statistical Analysis: In this study, the sample size for each animal group was 6 animals. All experimental data in this study are presented as mean ± standard deviation. The data provided in this paper have undergone rigorous screening and validation, are highly representative, and can accurately reflect the actual experimental situation. Statistical analysis in this study was performed using GraphPad Prism 9 software (GraphPad, USA). Student's t-test was used for comparisons between two groups, and one-way ANOVA was used for comparisons among multiple groups. *P<0.05, **P<0.01, ***P<0.001, and ns represent No significance.

[0087] II. Experimental Results: Based on the aforementioned steps ( Figure 14Micro-CT scans of the bilateral femurs and tibias of mice in each group showed that, compared with the sham-operated group, the castration group had a significantly reduced number of trabeculae in the femur and tibia, and a significantly increased intertrabecular spacing. Treatment with tFNAs-SDSSD / Gen had a preventive effect against PMOP formation, with more trabeculae than the castration group, tFNAs-SDSSD, and Gen, and a smaller intertrabecular spacing. Thermographic results for Tb. Th and Tb. Sp were consistent with this. Statistical analysis of bone-related parameters of the ROI indicated that tFNAs-SDSSD / Gen had a significant preventive effect against osteoporosis after castration, and its effect was stronger than that of Gen alone. The results demonstrate that tFNAs-SDSSD / Gen has a preventive effect against osteoporosis in castrated mice in vivo. Figure 15 , 16 H&E and Masson staining of femoral tissue sections from each group of mice showed that, compared with the simple tFNAs-SDSSD and Gen groups, the tFNAs-SDSSD / Gen group had an increased number of bone fibers, tighter connections between the bone fibers, and a more orderly arrangement, resulting in a more significant therapeutic effect. This demonstrates that tFNAs-SDSSD / Gen can preserve the quantity and quality of trabeculae in the medullary cavity of castrated mice and can effectively prevent and treat postmenopausal osteoporosis. Figure 17 H&E staining of heart, liver, spleen, lung, and kidney samples from mice in each treatment group revealed that tFNAs-SDSSD / Gen, tFNAs-SDSSD, and Gen alone did not show significant damaging effects on these critical internal organs under long-term use. Figure 18 This indicates that tFNAs-SDSSD / Gen has long-term biological safety and no obvious toxic side effects occurred during use.

Claims

1. A bone-targeting functionalized tetrahedral framework nucleic acid nanosystem, characterized in that, The formulation includes tetrahedral framework nucleic acid tFNA-Sa, a peptide SDSSD with specific targeting of bone cells, and an osteoporosis treatment drug. The molar ratio of tetrahedral framework nucleic acid to peptide SDSSD is 1:

1. Tetrahedral framework nucleic acid tFNA-Sa modifies peptide SDSSD to obtain bone-targeting tetrahedral framework nucleic acid tFNAs-SDSSD. The molar ratio of bone-targeting tetrahedral framework nucleic acid tFNAs-SDSSD to osteoporosis treatment drug is 1:

40.

2. The bone-targeting functionalized tetrahedral framework nucleic acid nanosystem according to claim 1, characterized in that, The tetrahedral framework nucleic acid tFNA-Sa is obtained by base complementary pairing of single-stranded deoxyribonucleic acid S3, single-stranded deoxyribonucleic acid S1, single-stranded deoxyribonucleic acid S2 and single-stranded deoxyribonucleic acid S4 carrying sticky ends with special sequences. The single-stranded deoxyribonucleic acid S3 carrying sticky ends with special sequences is single-stranded deoxyribonucleic acid S3-Sa2.

3. The bone-targeting functionalized tetrahedral framework nucleic acid nanosystem according to claim 1, characterized in that, The amino acid sequence of the bone cell-specific targeting peptide SDSSD is: Ser-Asp-Ser-Ser-Asp, Ser-Asp-Ser-Ser-As, which are respectively serine, aspartate, serine, serine, and aspartate. After chemical modification, it is combined with Fmoc-L-propargylglycine Fmoc-Pra-OH to synthesize propargylglycine-modified bone-targeting peptide Pra-SDSSD.

4. The bone-targeting functionalized tetrahedral framework nucleic acid nanosystem according to claim 1, characterized in that, The osteoporosis treatment drug is genistein.

5. A method for preparing a bone-targeting functionalized tetrahedral framework nucleic acid nanosystem, characterized in that, Includes the following steps: Step 1: Single-stranded deoxyribonucleic acid S1, single-stranded deoxyribonucleic acid S2, single-stranded deoxyribonucleic acid S3-Sa2 and single-stranded deoxyribonucleic acid S4 are synthesized into tetrahedral framework nucleic acid tFNA-Sa2 through nucleic acid self-assembly; Step 2: By using a click chemical reaction, the azide-modified sticky terminal Sa2*-N3 is combined with Pra-SDSSD to synthesize the bone-targeting peptide Sa2*-SDSSD modified with the Sa2* sticky terminal. Step 3: Then, the tFNA-Sa2 obtained in Step 1 and the Sa2*-SDSSD obtained in Step 2 are combined through the sticky end Sa2 and Sa2* base complementary pairing to obtain bone-targeting tetrahedral framework nucleic acid tFNAs-SDSSD; Step 4: After mixing bone-targeting tetrahedral framework nucleic acids tFNAs-SDSSD with genistein, the mixture is reacted by shaking at room temperature to obtain a bone-targeting functionalized tetrahedral framework nucleic acid nanosystem.

6. The method for preparing the bone-targeting functionalized tetrahedral framework nucleic acid nanosystem according to claim 5, characterized in that, The reaction conditions for the synthesis of tFNA-Sa2 were as follows: first, reaction at 95℃ for 10 min; then reaction at 4℃ for 20 min; and finally reaction at 4℃ for 60 min. The synthesis reactions of Sa2*-SDSSD and tFNAs-SDSSD were both at room temperature, with reaction times of 6 h and 3 h, respectively. The reaction of bone-targeting tetrahedral framework nucleic acid tFNAs-SDSSD with genistein was carried out at room temperature for 3 h.

7. An application of a bone-targeting functionalized tetrahedral framework nucleic acid nanosystem, characterized in that, Applications in the preparation of mouse embryonic osteoblast precursor cell lines and specific targeted and enriched drugs for mouse femoral, tibial and vertebral bone tissues; applications in the preparation of drugs for osteoporosis-related diseases.

8. The application of the bone-targeting functionalized tetrahedral framework nucleic acid nanosystem according to claim 7, characterized in that, The osteoporosis-related disease mentioned is postmenopausal osteoporosis.

9. The application of the bone-targeting functionalized tetrahedral framework nucleic acid nanosystem according to claim 7, characterized in that, The drug is a drug that promotes the bone regeneration capacity of osteoblasts.

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