Bisphosphonate-nucleic acid nanoparticles, methods of making and using the same

Abstract

This invention discloses a bisphosphonate-nucleic acid nanoparticle, its preparation method, and its applications. The preparation method involves adding divalent metal ions to a rolling circle amplification (RoBA) reaction solution, followed by the addition of bisphosphonate, to perform a RoBA reaction to obtain the bisphosphonate-nucleic acid nanoparticles. The RoBA reaction solution contains a circular DNA template. The preparation method of this invention has the advantages of high speed, precise synthesis, and simplicity, realizing multiple reactions—RoBA, ion coordination, crystallization, and co-assembly of DNA and bisphosphonate—in the same reaction system, greatly simplifying the reaction process and improving reaction efficiency. The obtained bisphosphonate-nucleic acid nanoparticles exhibit dual-pathway therapeutic effects and excellent ribozyme stability, showing promising application prospects.

Description

Technical Field

[0001] This invention relates to the fields of polymer materials and biomedical technology, and specifically to a bisphosphonate-nucleic acid nanoparticle, its preparation method, and its applications. Background Technology

[0002] With the increasing aging of the global population, osteoporosis has become a systemic disease affecting millions of people worldwide. Currently, strategies to inhibit bone resorption are widely used in the treatment of osteoporosis, and alendronate is a commonly used first-line drug in this strategy. However, long-term or high-dose use of alendronate often inhibits bone turnover, interferes with the natural mechanisms and processes of bone repair, and increases the risk of osteonecrosis of the jaw and atypical fractures.

[0003] Since osteoporosis is caused by an imbalance between bone resorption and bone formation, another important strategy for treating osteoporosis is to promote bone formation. Studies have found that sclerosingin, a glycoprotein mainly secreted by osteocytes, can specifically block the Wnt / β-Catenin signaling pathway by binding to low-density lipoprotein receptor-associated proteins (LRP5 / 6), thereby inhibiting osteoblast formation and differentiation and exhibiting a significant negative regulatory effect on bone formation.

[0004] In recent years, DNA-inorganic hybrid nanomaterials have attracted widespread attention in the biomedical field due to their simultaneous inheritance of the excellent properties of both DNA molecules and inorganic materials. Rolling circle amplification (RoBA) is a common method for the large-scale preparation of DNA. Using RoBA, primers are extended on circular template DNA to generate long-chain DNA, which then self-assembles with inorganic magnesium pyrophosphate generated during the reaction, forming magnesium pyrophosphate@DNA nanoparticles that possess the properties of both DNA and inorganic pyrophosphate. Studies have shown that nanomaterials prepared by RoBA contain a large number of pre-defined aptamer repeat units that encode the base sequence of the template DNA, enabling efficient recognition of corresponding protein-like biomolecules. Inorganic pyrophosphate not only plays a crucial "nucleation" role in the nanoassembly process but also significantly improves the ribozyme tolerance and in vivo circulation time of DNA molecules.

[0005] It is worth noting that although pyrophosphate possesses the biological activity of inhibiting bone resorption, it is ineffective when taken orally, and is rapidly inactivated by enzymatic hydrolysis when administered by injection. As an analogue of pyrophosphate, alendronate exhibits excellent chemical stability, acid tolerance, and hydrolytic enzyme resistance due to the replacement of the POP group in pyrophosphate with a PCP group. However, there is currently no existing technology for preparing alendronate@DNA nanoparticles.

[0006] Therefore, developing a method for preparing alendronate@DNA nanoparticles is of great practical significance. Summary of the Invention

[0007] Due to the above-mentioned deficiencies in the existing technology, the present invention provides a method for preparing alendronate@DNA nanoparticles. Specifically, it is based on rolling circle amplification reaction to co-assemble bisphosphonates and DNA nucleic acid aptamers to prepare bisphosphonate@DNA nanoparticles with good biocompatibility and high ribozyme stability. These bisphosphonate@DNA nanoparticles have both the functions of inhibiting bone resorption and promoting bone formation, and have broad application prospects in the field of osteoporosis treatment.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] A method for preparing bisphosphonate-nucleic acid nanoparticles involves adding divalent metal ions to a rolling circle amplification reaction solution, followed by the addition of bisphosphonate, and then performing a rolling circle amplification reaction to obtain bisphosphonate-nucleic acid nanoparticles. The rolling circle amplification reaction solution contains a circular DNA template.

[0010] As a preferred technical solution:

[0011] The circular DNA template is prepared using the method described above, and the preparation method is as follows:

[0012] (1) Mix the specific DNA sequence template and its primers in DNA ligase buffer (10-100mM Tris-HCl, 5-100mM MgCl2, 1-20mM DTT, 5-20mM adenosine triphosphate, pH 6-8) and anneal.

[0013] (2) The annealing product was co-incubated with DNA ligase, and then the DNA ligase was inactivated.

[0014] (3) The product obtained in step (2) is purified to remove excess primer DNA from the reaction system and obtain a circular DNA template.

[0015] In the preparation method described above, in step (1), the specific DNA sequence template is a osteoschizoprotein aptamer complementary sequence or other sequences;

[0016] The concentration of a specific DNA sequence template in DNA ligase buffer is 0.1–1 μM, and the length of the template DNA is 20–150 bp.

[0017] The concentration of the primer in the DNA ligase buffer is 0.1–1 μM, and the length of the primer DNA is 20–60 bp.

[0018] Annealing refers to heating at 50–120°C for 5–15 minutes, followed by gradual cooling to room temperature over 1–5 hours.

[0019] In the preparation method described above, in step (2), the DNA ligase is T4 DNA ligase;

[0020] The concentration of the DNA ligase in the co-incubation system is 5000–50000 U / mL, and the co-incubation refers to culturing at room temperature for 10–30 hours.

[0021] The inactivation process involves heating to 50–100°C and maintaining the temperature for 10–30 minutes.

[0022] As described above, step (3) specifically involves placing the product obtained in step (2) in an EXO I enzyme reaction buffer (10-100 mM Glycine-KOH, 5-10 mM MgCl2, 0.5-5 mM β-mercapteothanol), then adding the EXO I enzyme for reaction, and then inactivating the EXO I enzyme.

[0023] In the preparation method described above, the concentration of the EXO I enzyme in the system is 200–2000 U / mL;

[0024] The reaction involving the addition of EXO I enzyme was carried out at a temperature of 37°C for 0.5–2 hours.

[0025] The EXO I enzyme is inactivated by maintaining it at 50–120°C for 5–30 minutes.

[0026] As described above, the rolling circle amplification reaction solution is obtained by uniformly mixing a circular DNA template, deoxyribonucleoside triphosphates (dNTPs), bovine serum albumin (BSA), inorganic pyrophosphatase, phi29 buffer (10–100 mM Tris-HCl, 5–50 mM (NH4)2SO4, 5–50 mM MgCl2, 1–10 mM DTT), and phi29 DNA polymerase.

[0027] The rolling circle amplification reaction refers to incubation at 30°C for 20–72 hours;

[0028] After the rolling circle amplification reaction, the phi29 DNA polymerase was inactivated by heating at 60–120 °C for 10–20 minutes.

[0029] The concentrations of each dNTP in the rolling circle amplification reaction system were 0.5–5 mM, the concentration of bisphosphonates was 0.05–10 mM, the concentration of inorganic pyrophosphatase was 1–10 U / mL, the concentration of bovine serum albumin (BSA) was 0.5–5 mg / mL, the concentration of phi29 DNA polymerase was 1000–5000 U / mL, the concentration of circular DNA template was 0.1–2 μM, and the concentration of divalent metal ions was 5–50 mM.

[0030] The divalent metal ions are magnesium ions, calcium ions, cobalt ions, or strontium ions;

[0031] The bisphosphonate is one or more of etidronate, clodronate, pamidronate, tiludronate, alendronate, neridronate, opaldronate, risedronate, ibandronate, and zoledronic acid.

[0032] The bisphosphonate-nucleic acid nanoparticles have a particle size of 40–1000 nm.

[0033] As described above, after inactivating the phi29 DNA polymerase, the product is thoroughly mixed in ultrapure water and centrifuged at 5000–50000 r / min for 5–30 minutes, and this process is repeated several times.

[0034] The specific DNA sequence template and its primers in this application are pre-designed, programmed, and synthesized. After annealing hybridization, the template DNA and primer DNA form a stable circular DNA template under the action of T4 DNA ligase. After purification, the length of the primer DNA is extended under the action of PhI29 DNA polymerase, and then co-assembled with bisphosphonate crystals in the reaction system to form bisphosphonate@DNA nanoparticles. Specifically, a bisphosphonate@DNA nanoparticle containing an anti-osteointe protein aptamer sequence can be prepared accordingly. On the one hand, it can improve the ribozyme tolerance of the DNA aptamer, prolong its in vivo half-life, and enhance the osteogenic potential of osteoblasts. On the other hand, by targeting cancellous bone, it can release alendronate at the cancellous bone site, thereby inhibiting the osteoclast activity of osteoclasts and reducing the side effects of the drug. This nanoparticle has the ability to promote bone formation and inhibit bone resorption, providing a new approach for the treatment of osteoporosis.

[0035] This invention also provides bisphosphonate-nucleic acid nanoparticles prepared by the method described above. These bisphosphonate-nucleic acid nanoparticles possess both bone resorption inhibition and bone formation promotion properties. This invention utilizes a synergistic effect to address the problems of single bone resorption inhibition strategies, providing a combination therapy that integrates bisphosphonates with anti-sclerosing acid aptamers. This helps to avoid the drawbacks of both and achieve complementary advantages, improving the treatment effect of osteoporosis. In particular, its application in the treatment of osteoporosis and bone defects has an irreplaceable role compared to other carriers.

[0036] Furthermore, the present invention also provides the application of the bisphosphonate-nucleic acid nanoparticles described above in drug carriers or pharmaceutical compositions for bone-related diseases, which can be used for multi-pathway treatment of orthopedic diseases such as osteoporosis.

[0037] The above technical solution is only one feasible technical solution of the present invention. The scope of protection of the present invention is not limited thereto. Those skilled in the art can reasonably adjust the specific design according to actual needs.

[0038] The above invention has the following advantages or beneficial effects:

[0039] (1) The method of the present invention uses rolling circle amplification for preparation, which has the advantages of fast speed, accurate synthesis and simple and easy operation. It utilizes programmed temperature-controlled enzymatic reaction, and by changing the types and concentrations of reactants in the reaction process, controlling the reaction time and temperature, etc., multiple reactions such as rolling circle amplification, ion coordination, crystallization and DNA co-assembly with bisphosphonates are realized in the same reaction system, thereby greatly simplifying the reaction process and improving the reaction efficiency.

[0040] (2) The bisphosphonate-nucleic acid nanoparticles of the present invention have dual therapeutic effects, wherein bisphosphonates can inhibit the proliferation of osteoclasts, thereby reducing bone resorption, while DNA aptamers promote the proliferation and differentiation of osteoblasts by binding to bone proteins, thereby promoting bone mineralization and formation.

[0041] (3) The bisphosphonate-nucleic acid nanoparticles of the present invention have excellent ribozyme stability. DNA aptamers can bind to characteristic proteins efficiently, but they are often easily degraded by ribozymes in vivo. After nano-sizing, the ribozyme tolerance and in vivo half-life of the aptamers are greatly improved.

[0042] (4) The bisphosphonate-nucleic acid nanoparticles of the present invention have bone targeting and pH responsiveness. The bisphosphonates in the nanoparticles can coordinate with calcium ions on the bone surface, showing good bone targeting. In addition, the microenvironment of osteoporosis and other lesions is acidic, and the decomposition rate of the nanoparticles is faster than that under normal physiological conditions. Therefore, the specific aggregation and responsive release of the nanoparticles at the osteoporosis site greatly improves the utilization efficiency of the drug and achieves precise treatment.

[0043] (5) The bisphosphonate-nucleic acid nanoparticles of the present invention have few toxic side effects. Bisphosphonates are the most commonly used drugs in the clinical treatment of osteoporosis. Long-term and high-dose use of bisphosphonates can lead to serious side effects such as osteonecrosis of the jaw. After self-assembly, the bisphosphonates in the nanoparticles are slowly released, which reduces their side effects on cells and the body. Attached Figure Description

[0044] The invention, its features, shape, and advantages will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings. Like reference numerals denote like parts throughout the drawings. The drawings are not drawn to scale; the emphasis is on illustrating the gist of the invention.

[0045] Figure 1 A schematic diagram of the synthesis route of bisphosphonate-nucleic acid nanoparticles (a) and a schematic diagram of their in vivo action (b);

[0046] Figure 2 A schematic diagram of single-crystal XRD analysis of magnesium alendronate crystals;

[0047] Figure 3 This is a gel electrophoresis image of the synthesis process of bisphosphonate-nucleic acid nanoparticles;

[0048] Figure 4 SEM image of bisphosphonate-nucleic acid nanoparticles;

[0049] Figure 5 TEM image of bisphosphonate-nucleic acid nanoparticles;

[0050] Figure 6 This is a particle size distribution diagram of bisphosphonate-nucleic acid nanoparticles;

[0051] Figure 7 The fluorescence distribution of nucleic acid and bisphosphonate-nucleic acid nanoparticles in various organs of mice 4 hours after in vivo;

[0052] Figure 8 The fluorescence distribution of nucleic acid and bisphosphonate-nucleic acid nanoparticles in various organs of mice 12 hours after in vivo;

[0053] Figure 9 The graph shows the concentration of P1NP (osteoblast-related protein) in rats after different drug treatment groups.

[0054] Figure 10 The graph shows the concentration of CTX1 (an osteoclast-associated protein) in rats after different drug treatment groups.

[0055] Figure 11 Micro-CT images of three-dimensional reconstruction of femoral trabeculae in rats after treatment with different groups of drugs;

[0056] Figure 12 This is a density map of trabecular bone in the femur of rats after different treatment methods. Detailed Implementation

[0057] The present invention will be further described below with reference to specific embodiments, but these are not intended to limit the invention.

[0058] Example 1

[0059] A method for preparing bisphosphonate-nucleic acid nanoparticles includes the following steps (Note: all concentrations below refer to the concentrations in the final reaction system):

[0060] (1) Annealing:

[0061] DNA template T1 (containing the apocrine protein aptamer complementary sequence) or T2 (without the apocrine protein aptamer complementary sequence) (5 μM) and its corresponding primer P (10 μM) were mixed in T4 ligase buffer (50 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT, 1 mM adenosine triphosphate, pH 7.5). The reaction mixture was heated at 95 °C for 10 minutes, and then the sample was gradually cooled to room temperature over 3 hours.

[0062] (2) Connection:

[0063] The product from step (1) was incubated with T4 DNA ligase (20000 U / mL) at room temperature for 16 hours. This allowed phosphodiester bonds to form at both ends of the DNA template, resulting in a circular DNA template. Subsequently, the ligation product was heated to 65°C and held for 10 minutes to inactivate the T4 DNA ligase.

[0064] (3) Template purification:

[0065] The product from step (2) was added to EXO I enzyme reaction buffer (67mM Glycine-KOH, 6.7mM MgCl2, 10mM β-mercapteothanol), and EXO I enzyme (480U / mL) was added. The reaction was carried out at 37°C for 1.5 hours. Excess primer DNA in the reaction system was removed by passing the EXO I enzyme reaction system. Then, the reaction was kept at 80°C for 20 minutes to inactivate the EXO I enzyme.

[0066] (4) Rolling circle amplification:

[0067] The reaction product from step (3) was mixed with dNTPs (1 mM each), rolling circle amplification reaction buffer (50 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM MgCl2, 4 mM DTT), phi29 DNA polymerase (200 U / mL), sodium alendronate (1.5 mM), BSA (1 mg / mL), and inorganic pyrophosphatase (2 U / mL). The mixture was incubated at 30 °C for 24 hours, and then heated to 90 °C and held for 10 minutes to inactivate the phi29 DNA polymerase, thus obtaining magnesium alendronate-nucleic acid nanoparticles.

[0068] (5) Purification of alendronate-nucleic acid nanoparticles:

[0069] Add 200 μL of deionized water to the 25 μL of rolling circle amplification product, mix well with a pipette, then centrifuge at 14000 r / min for 15 min, remove 200 μL of supernatant, and repeat the operation three times to obtain 25 μL of magnesium alendronate-nucleic acid nanoparticles.

[0070] The schematic diagrams of the synthesis route and the in vivo action route in this embodiment are as follows: Figure 1 As shown, the single-crystal XRD structure of the magnesium alendronate coordination compound obtained during the synthesis process is as follows: Figure 2 As shown, the gel electrophoresis imaging of each component during the synthesis process is as follows: Figure 3 As shown, the SEM image of alendronate-nucleic acid nanoparticles is as follows: Figure 4 As shown, the TEM image of the nanoparticle is as follows. Figure 5 As shown, the particle size distribution of the nanoparticles is as follows. Figure 6 As shown.

[0071] In this embodiment, the nanoparticles endow the aptamer with excellent ribozyme tolerance and bone targeting, such as Figure 7 and Figure 8 As shown, 4 and 12 hours after drug injection into mice, in the absence of significant differences in fluorescence intensity in other organs, the fluorescence intensity of nanoparticles in bone was much higher than that of nucleic acids (p < 0.05). At the same time, the sustained release process of alendronate reduced its toxic side effects and effectively improved its biosafety.

[0072] The bone-promoting effect of alendronate-nucleic acid nanoparticles in this embodiment is as follows: Figure 9 As shown, it effectively promotes the production of osteogenic-associated protein P1NP and inhibits bone resorption. Figure 10 As shown, the production of the osteoclast-associated protein CTX1 was effectively inhibited. The final three-dimensional micro-CT reconstruction images of the trabecular bone in each group after treatment are shown below. Figure 11 As shown, bone density is as follows Figure 12 As shown, alendronate-nucleic acid nanoparticles achieve a dual-pathway treatment for osteoporosis.

[0073] Example 2

[0074] A method for preparing bisphosphonate-nucleic acid nanoparticles is basically the same as that in Example 1, except that the concentration of sodium alendronate in the system is 1 mM.

[0075] Example 3

[0076] A method for preparing bisphosphonate-nucleic acid nanoparticles is basically the same as that in Example 1, except that the concentration of sodium alendronate in the system is 2 mM.

[0077] Example 4

[0078] A method for preparing bisphosphonate-nucleic acid nanoparticles is basically the same as that in Example 1, except that the concentration of sodium alendronate in the system is 3 mM.

[0079] Example 5

[0080] A method for preparing bisphosphonate-nucleic acid nanoparticles is basically the same as that in Example 1, except that the reaction time for rolling circle amplification is 20 hours.

[0081] Example 6

[0082] A method for preparing bisphosphonate-nucleic acid nanoparticles is basically the same as that in Example 1, except that the reaction time for rolling circle amplification is 48 hours.

[0083] Example 7

[0084] A method for preparing bisphosphonate-nucleic acid nanoparticles is basically the same as that in Example 1, except that the reaction time for rolling circle amplification is 72 hours.

[0085] After measuring the particle size of the bisphosphonate-nucleic acid nanoparticles prepared in Examples 1 to 7, it was found that the particle size of the nanoparticles gradually decreased as the concentration of alendronate increased, while the particle size of the nanoparticles gradually increased as the reaction time increased.

[0086] Example 8

[0087] A method for preparing bisphosphonate-nucleic acid nanoparticles is basically the same as that in Example 1, except that sodium alendronate is replaced with sodium etidronate.

[0088] Example 9

[0089] A method for preparing bisphosphonate-nucleic acid nanoparticles is basically the same as that in Example 1, except that sodium alendronate is replaced with sodium zoledronic acid.

[0090] Example 10

[0091] A method for preparing bisphosphonate-nucleic acid nanoparticles is basically the same as that in Example 1, except that sodium alendronate is replaced with sodium pamidronate.

[0092] Example 11

[0093] A method for preparing bisphosphonate-nucleic acid nanoparticles is basically the same as that in Example 1, except that sodium alendronate is replaced with sodium risedronate.

[0094] In fact, other bisphosphonates such as chlorophosphonate, tiluphosphonate, neridonate, opalphosphonate, and ibandronate also possess the PCP bisphosphonate structure, and therefore can also self-assemble with DNA during rolling circle amplification to form nanoparticles.

[0095] Example 12

[0096] A method for preparing bisphosphonate-nucleic acid nanoparticles includes the following steps (Note: all concentrations below refer to the concentrations in the final reaction system):

[0097] (1) Annealing:

[0098] The DNA template (Taptscl56 (osteoprotein aptamer sequence) or T2 (apopaenzyme sequence), 5 μM) and its primers (10 μM) were mixed in T4 ligase buffer (50 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT, 1 mM adenosine triphosphate, pH 7.5). The reaction mixture was heated at 95 °C for 10 min, and then the sample was gradually cooled to room temperature over 3 hours.

[0099] (2) Connection:

[0100] The product from step (1) was incubated with T4 DNA ligase (20000 U / mL) at room temperature for 16 h. This allowed phosphodiester bonds to form at both ends of the DNA template, resulting in a circular DNA template. Subsequently, the ligation product was heated to 65°C and held for 10 minutes to inactivate the T4 DNA ligase.

[0101] (3) Template purification:

[0102] The product from step (2) was added to EXO I enzyme reaction buffer (67mM Glycine-KOH, 6.7mM MgCl2, 10mM β-mercapteothanol), and EXO I enzyme (480U / mL) was added. The reaction was carried out at 37°C for 1.5h. Excess primer DNA in the reaction system was removed by passing the EXO I enzyme reaction system. Then, the reaction was kept at 80°C for 20 minutes to inactivate the EXO I enzyme.

[0103] (4) Rolling circle amplification:

[0104] The reaction product from step (3) was mixed with dNTPs (1 mM each), rolling circle amplification reaction buffer (50 mM Tris-HCl, 10 mM (NH4)2SO4, 10 mM MgCl2, 4 mM DTT), and phi29 DNA polymerase (1000 U / mL). The mixture was incubated at 30 °C for 20 hours, and then heated to 90 °C and held for 10 minutes to inactivate the phi29 DNA polymerase.

[0105] (5) DNA purification:

[0106] Add inorganic pyrophosphatase to the reaction product in step (4), mix it with a pipette, and react it at 37°C for 24 hours. Then, incubate it at 65°C for 10 minutes to inactivate the inorganic pyrophosphatase. Then, add 200 μL of ultrapure water, mix it, and centrifuge it at 14000 r / min for 2 hours in a 10K ultrafiltration centrifuge tube to obtain long-chain aptamer DNA.

[0107] (6) Synthesis of alendronate-nucleic acid nanoparticles:

[0108] The purified DNA obtained in step (5) was added to MgCl2 (12.5 mM), mixed well, and incubated at 37°C for 24 hours. Then, alendronate sodium (1.5 mM) solution was added, mixed well, and incubated at 37°C for 20 hours to obtain alendronate-nucleic acid nanoparticles.

[0109] (7) Purification of alendronate-nucleic acid nanoparticles:

[0110] Add 200 μL of deionized water to the product obtained in step (6), mix with a pipette, then centrifuge at 14000 r / min for 15 minutes, remove 200 μL of supernatant, repeat the operation three times to obtain 25 μL of alendronate-nucleic acid nanoparticles.

[0111] Example 13

[0112] A method for preparing bisphosphonate-nucleic acid nanoparticles is basically the same as that in Example 12, except that MgCl2 in step (5) is replaced with CaCl2.

[0113] Example 14

[0114] A method for preparing bisphosphonate-nucleic acid nanoparticles is basically the same as that in Example 12, except that MgCl2 in step (5) is replaced with SrCl2.

[0115] Those skilled in the art should understand that variations can be implemented by combining existing technology with the above embodiments, which will not be elaborated here. Such variations do not affect the essence of the present invention, and will not be elaborated here either.

[0116] The preferred embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and the devices and structures not described in detail should be understood as being implemented in a conventional manner in the art. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the scope of the present invention. This does not affect the essential content of the present invention. Therefore, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the present invention's technical solutions still fall within the protection scope of the present invention.

Claims

1. A method for preparing bisphosphonate-nucleic acid nanoparticles, characterized in that: Divalent metal ions were added to the rolling circle amplification reaction solution, followed by the addition of bisphosphonate, and the rolling circle amplification reaction was carried out to obtain phosphonate-nucleic acid nanoparticles. The rolling circle amplification reaction solution contained a circular DNA template. The rolling circle amplification reaction solution is obtained by uniformly mixing a circular DNA template, deoxyribonucleoside triphosphate, bovine serum albumin, inorganic pyrophosphatase, phi29 buffer, and phi29 DNA polymerase. The rolling circle amplification reaction refers to incubation at 30°C for 20-72 hours; After the rolling circle amplification reaction, the phi29 DNA polymerase was inactivated by heating at 60-120℃ for 10-20 minutes. The concentrations of each dNTP in the rolling circle amplification reaction system were 0.5–5 mM, the concentration of bisphosphonates was 0.05–10 mM, the concentration of inorganic pyrophosphatase was 1–10 U / mL, the concentration of bovine serum albumin (BSA) was 0.5–5 mg / mL, the concentration of phi29 DNA polymerase was 1000–5000 U / mL, the concentration of circular DNA template was 0.1–2 μM, and the concentration of divalent metal ions was 5–50 mM. The divalent metal ions are magnesium ions, calcium ions, cobalt ions, or strontium ions; The bisphosphonate is one or more of etidronate, clodronate, pamidronate, tiludronate, alendronate, neridronate, opaldronate, risedronate, ibandronate, and zoledronic acid. The bisphosphonate-nucleic acid nanoparticles have a particle size of 40~1000 nm.

2. The preparation method according to claim 1, characterized in that, The method for preparing the circular DNA template is as follows: (1) Mix the specific DNA sequence template and its primers in DNA ligase buffer and then anneal; (2) The annealing product was co-incubated with DNA ligase, and then the DNA ligase was inactivated; (3) The product obtained in step (2) is purified to obtain a circular DNA template.

3. The preparation method according to claim 2, characterized in that, In step (1), the specific DNA sequence template is the osteoschizoprotein aptamer complementary sequence or other sequences; The concentration of a specific DNA sequence template in DNA ligase buffer is 0.1~1μM, and the length of the template DNA is 20~150bp; The concentration of the primer in the DNA ligase buffer is 0.1~1μM, and the length of the primer DNA is 20~60 bp; Annealing refers to heating at 50~120℃ for 5~15 minutes and then gradually cooling to room temperature over 1~5 hours.

4. The preparation method according to claim 2, characterized in that, In step (2), the DNA ligase is T4 DNA ligase; The concentration of the DNA ligase in the co-incubation system is 5000~50000 U / mL, and the co-incubation refers to culturing at room temperature for 10~30 hours; The inactivation process involves heating to 50-100°C and maintaining the temperature for 10-30 minutes.

5. The preparation method according to claim 2, characterized in that, Step (3) specifically involves placing the product obtained in step (2) in an EXO I enzyme reaction buffer, adding EXO I enzyme for reaction, and then inactivating the EXO I enzyme.

6. The preparation method according to claim 5, characterized in that, The concentration of the EXO I enzyme in the system is 200~2000 U / mL; The reaction involving the addition of EXO I enzyme was carried out at a temperature of 37°C for 0.5 to 2 hours. The EXO I enzyme is inactivated by maintaining it at 50-120℃ for 5-30 minutes.

7. The preparation method according to claim 1, characterized in that, After inactivating the phi29 DNA polymerase, the product was thoroughly mixed in ultrapure water and centrifuged at 5000~50000 r / min for 5~30 minutes, and the process was repeated several times.

8. Bisphosphonate-nucleic acid nanoparticles prepared by the preparation method according to any one of claims 1 to 7.

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