A kind of double phosphate and adenine nucleoside triphosphate synergistic modified calcium phosphate nanoparticles and its preparation method and application

Abstract

The application discloses a kind of double phosphate and adenine nucleoside triphosphate synergic modification's calcium phosphate nanoparticles and preparation method and application thereof, belong to the field of biomedicine, including the following steps: calcium chloride solution, Tris buffer solution is dissolved in water, to obtain first solution;HEPES buffer solution containing disodium hydrogen phosphate is added to water, then double phosphate, adenine nucleoside triphosphate is added, and stirred uniformly, to obtain second solution;Second solution is dropped into first solution, and stirred uniformly, to obtain mixed solution, and the mixed solution is centrifuged, washed, dried, to obtain double phosphate and adenine nucleoside triphosphate synergic modification's calcium phosphate nanoparticles;The application improves the size of calcium phosphate nanoparticles by the synergistic effect of double phosphate and adenine nucleoside triphosphate, and adjusts its Zeta potential, to improve its circulation time in vivo and delivery efficiency and stability, improve the delivery efficiency of nucleic acid vaccine, and then enhance the immune effect of vaccine.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a calcium phosphate nanoparticle synergistically modified with bisphosphate and adenine nucleoside triphosphate, its preparation method, and its application. Background Technology

[0002] Calcium phosphate nanoparticles (CP) are a biocompatible inorganic nanocarrier that exhibits unique advantages in the field of nucleic acid vaccine delivery. Their composition is similar to natural bone minerals, possessing low toxicity, high nucleic acid loading capacity, and pH-responsive release properties. They can rapidly dissolve and release plasmid DNA (pDNA) or mRNA in acidic intracellular environments (pH 5.0–6.5), avoiding nucleic acid degradation by lysosomal enzymes. However, traditional CP particles suffer from severe stability defects in physiological solutions: the particles easily aggregate due to the hydroxyapatite crystallization phase transition, resulting in sizes exceeding the 20–200 nm range preferred by immune cells (average sizes often reach 600–700 nm or larger), significantly reducing cellular uptake efficiency. Simultaneously, their high surface zeta potential (+10 to +40 mV) easily triggers nonspecific protein adsorption and reticuloendothelial system clearance, limiting in vivo delivery efficacy. These issues necessitate high doses (e.g., 300 μg DNA / dose) of CP nanoparticles in nucleic acid vaccine applications to induce an immune response, increasing potential toxicity risks.

[0003] Bisphosphonates (BP) are a class of small-molecule drugs commonly used to treat osteoporosis and exhibit good affinity for CP materials. Based on this, researchers have attempted to modify CP nanoparticles, developing nanoparticles (BCP) that use bisphosphonates to partially or completely replace the phosphate (P) in CP nanoparticles. However, BCP nanoparticles are not without their flaws. While BP modification improves particle stability, in practical applications, the control over particle size and surface charge remains unsatisfactory, leaving room for optimization. Excessive surface charge can lead to decreased nanoparticle stability in vivo, affecting their cycle retention time and potentially impacting their delivery efficiency and immune activation. Furthermore, excessive BP can also produce certain toxicities.

[0004] Therefore, this application is hereby submitted. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide calcium phosphate nanoparticles synergistically modified with bisphosphonate (BP) and adenine nucleoside triphosphate (ATP), as well as their preparation method and applications. Through the synergistic effect of bisphosphonate and adenine nucleoside triphosphate, the size of calcium phosphate nanoparticles can be regulated and improved, and their zeta potential can be adjusted, thereby increasing their circulation time, delivery efficiency and stability in vivo, effectively increasing their chance of contact with target cells, improving the delivery efficiency of nucleic acid vaccines, and thus enhancing the immune effect of vaccines.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preparing calcium phosphate nanoparticles synergistically modified with bisphosphate and adenine nucleoside triphosphate includes the following steps: The first solution is obtained by dissolving calcium chloride solution and Tris (abbreviation for tris(hydroxymethyl)aminomethane) buffer in water; Dissolve HEPES (abbreviation for 4-hydroxyethylpiperazine ethanesulfonic acid) buffer containing disodium hydrogen phosphate in water, add bisphosphate and adenine nucleoside triphosphate, stir well to obtain the second solution; The second solution was added dropwise to the first solution and stirred until homogeneous to obtain a mixture. The mixture was then centrifuged, washed, and dried to obtain calcium phosphate nanoparticles co-modified by diphosphate and adenine nucleoside triphosphate. The amount of the bisphosphate added is 20-50% of the molecular weight of phosphate in the second solution; The ratio of adenine triphosphate to HEPES buffer containing disodium hydrogen phosphate is (0.15~1.125) mg : (0.4~0.5) mL. This invention, by adding bisphosphate and adenine triphosphate, can regulate and improve the size of calcium phosphate nanoparticles. BP modification can, to a certain extent, stabilize the structure of CP nanoparticles and enhance particle dispersibility and growth. The addition of ATP allows for more precise control of nanoparticle size, achieving a size of approximately 100 nm, which meets the optimal uptake size for immune cells. BP increases the amino groups on the nanoparticle surface, raising the zeta potential. Meanwhile, the phosphate groups in the ATP molecule interact with the nanoparticle surface, adjusting the zeta potential and effectively improving the stability and dispersibility of calcium phosphate nanoparticles. This allows the zeta potential to remain stable between -4 and +4 mV. This potential state enables the nanoparticles to maintain good dispersion in the physiological environment, reducing aggregation and adsorption, thereby increasing their circulation time, delivery efficiency, and stability in vivo. It also effectively increases their contact with target cells (such as antigen-presenting cells), improving the delivery efficiency of nucleic acid vaccines and thus enhancing the immune effect of the vaccine.

[0007] As an embodiment of this application, the bisphosphate includes sodium alendronate.

[0008] As an embodiment of this application, the ratio of adenine nucleoside triphosphate to HEPES buffer containing disodium hydrogen phosphate is (0.75~1.05) mg: 0.43 mL.

[0009] The inventors of this invention have discovered that the amount of adenine triphosphate added has a significant impact on the technical effect. By controlling the amount of adenine triphosphate added to 50-70% of the amount of calcium phosphate nanoparticles precipitated, the size of the calcium phosphate nanoparticles can be adjusted to around 100 nm. At the same time, the Zeta potential is between -4 and +4 mV, which is stable and suppresses the generation of excessive negative charge without the risk of sudden charge drop or excessive aggregation, thus providing an optimal carrier basis for subsequent vaccine delivery.

[0010] In one embodiment of this application, the concentration of the calcium chloride solution is 0.5~5M; The pH of the Tris buffer solution is 9-10.

[0011] As an embodiment of this application, the volume ratio of the calcium chloride solution, Tris buffer, and water is 1:(1~4):(4~10).

[0012] As an embodiment of this application, the HEPES buffer solution further includes sodium chloride; The concentration of disodium hydrogen phosphate in the HEPES buffer is 5~20mM; The concentration of sodium chloride in the HEPES buffer is 200~350mM; The concentration of HEPES in the HEPES buffer solution is 20~80mM.

[0013] As an embodiment of this application, the volume ratio of HEPES buffer containing disodium hydrogen phosphate to water is 1:(0.5~2).

[0014] As an embodiment of this application, the volume ratio of the first solution to the HEPES buffer is (5~10):1.

[0015] As an embodiment of this application, serum albumin is also added to the mixture.

[0016] As an embodiment of this application, the final concentration of serum albumin is 0.04~0.8 μg / mL.

[0017] The present invention also provides calcium phosphate nanoparticles synergistically modified with bisphosphate and adenine nucleoside triphosphate, which are prepared by the preparation method described above.

[0018] This invention also provides the application of calcium phosphate nanoparticles synergistically modified with bisphosphate and adenine nucleoside triphosphate in nucleic acid vaccine delivery.

[0019] The beneficial effects of this invention are as follows: By adding bisphosphate and adenine nucleoside triphosphate, the size of calcium phosphate nanoparticles can be regulated and improved through their synergistic effect. BP modification can stabilize the structure of calcium phosphate nanoparticles to a certain extent, enhancing their dispersibility and growth. The addition of ATP can further precisely control the size of the nanoparticles to meet the optimal uptake size of immune cells. BP increases the amino groups on the nanoparticle surface, raising the zeta potential; while the phosphate groups in ATP molecules interact with the nanoparticle surface, adjusting the zeta potential and effectively improving the stability and dispersibility of calcium phosphate nanoparticles. This allows the zeta potential to remain stable between -4 and +4 mV. This potential state enables the nanoparticles to maintain a good dispersed state in the physiological environment, reducing aggregation and adsorption, thereby improving their circulation time, delivery efficiency, and stability in vivo. This effectively increases their contact with target cells, improving the delivery efficiency of nucleic acid vaccines and thus enhancing the immune effect of the vaccine. Attached Figure Description

[0020] Figure 1 The figure shows the effect of the amount of adenine triphosphate added on the size of calcium phosphate nanoparticles co-modified by bisphosphate and adenine triphosphate.

[0021] Figure 2 The figure shows the effect of the amount of adenine nucleoside triphosphate added on the zeta potential of calcium phosphate nanoparticles co-modified by bisphosphate and adenine nucleoside triphosphate.

[0022] Figure 3 The figure shows the effect of the amount of adenine triphosphate added on the size of calcium phosphate nanoparticles modified with adenine triphosphate.

[0023] Figure 4 This is a transmission electron microscope image of calcium phosphate nanoparticles synergistically modified with bisphosphate and adenine nucleoside triphosphate as described in Example 1.

[0024] Figure 5 This is a transmission electron microscope image of calcium phosphate nanoparticles from Comparative Example 1.

[0025] Figure 6 Transmission electron microscopy image of ATP-modified calcium phosphate nanoparticles.

[0026] Figure 7 Transmission electron microscopy image of bisphosphate-modified calcium phosphate nanoparticles.

[0027] Figure 8The graph shows the expression of eGFP in Examples 3 and Comparative Examples 4-6. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0029] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0030] In this application, numerical ranges are referred to as continuous unless otherwise specified, and include the minimum and maximum values ​​of the range, as well as every value between the minimum and maximum values. Furthermore, when the range refers to integers, it includes every integer between the minimum and maximum values ​​of the range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges may be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges to which they are incorporated.

[0031] In this application, there are no particular restrictions on the specific dispersion and mixing methods.

[0032] Unless otherwise specified, all components, raw materials, or instruments used in the embodiments and comparative examples of this invention are commercially available, and the same type of components and raw materials are used in each parallel experiment.

[0033] The following embodiments are provided to facilitate understanding of the invention. These embodiments are not intended to limit the scope of the claims.

[0034] Example 1 A method for preparing calcium phosphate nanoparticles synergistically modified with bisphosphate and adenine nucleoside triphosphate includes the following steps: (1) Dissolve 0.42 mL of 2M calcium chloride solution and 0.84 mL of pH 10 Tris buffer (10 mM) in 2 mL of water to obtain the first solution; (2) Prepare HEPES buffer containing disodium hydrogen phosphate. The HEPES buffer contains 280 mM NaCl, 7.5 mM Na2HPO4 and 50 mM HEPES (the remainder is water).

[0035] Add 0.43 mL of HEPES buffer containing disodium hydrogen phosphate to 0.3 mL of water, then add alendronate sodium and adenine nucleoside triphosphate, stir well to obtain the second solution; (3) The second solution above is added dropwise to the first solution above, and stirred at 500 rpm for 30 min to obtain a mixture. The mixture is centrifuged at 15000 g for 10 min, washed with water, and dried to obtain calcium phosphate nanoparticles (BACP) co-modified by bisphosphate and adenine nucleoside triphosphate.

[0036] The amount of the bisphosphate added is 50% of the molecular weight of phosphate in the second solution; The amount of adenine triphosphate added is 10-75% of the amount of BCP nanoparticle precipitation (1.5 mg) (i.e., 0.15-1.125 mg), and the corresponding ratio of adenine triphosphate to HEPES buffer containing disodium hydrogen phosphate is (0.15-1.125) mg: 0.43 mL.

[0037] When the amount of adenosine triphosphate added ranges from 10% to 75% of the BCP nanoparticle precipitation (i.e., 0.15 to 1.125 mg), the particle size variation of the nanoparticles (BACP) is as follows: Figure 1 As shown, from Figure 1 As can be seen, from 10% to 60% (0.15 to 0.9 mg) of adenine triphosphate (ATP), the average particle size gradually decreases with increasing ATP mass ratio, reaching its minimum at 60% ATP. From 60% to 75% (0.9 to 1.125 mg) of ATP, the size increases further with increasing ATP content. At 75% ATP, the particles gradually become larger, losing control over particle growth. Experimental data show that the average size of BACP nanoparticles is smallest at 60% ATP content, proving that ATP has the best inhibitory effect on particle growth at this ratio. When the ATP content is 10% to 40% (0.15 to 0.6 mg), the particle size is too large, resulting in poor stability. Furthermore, excessively large particle size significantly reduces cellular uptake efficiency.

[0038] like Figure 2 As shown, the zeta potential changes of calcium phosphate nanoparticles (BACP) when the amount of adenine nucleoside triphosphate added ranges from 10% to 70% (0.15 to 1.05 mg) are as follows: Figure 2 As shown, for 10–70% (0.15–1.05 mg) ATP, the Zeta potential gradually decreased from a weakly positive charge (+4 mV) to a moderately negative charge (-4 mV), without a sudden drop (bisphosphate buffered the charge shock of ATP). This demonstrates that dual modification inhibits the generation of excessive negative charge.

[0039] The nanoparticle size and zeta potential were measured using a Malvern Zetasizer Nano microscope, and the samples were examined simultaneously using transmission electron microscopy (TEM). The TEM used was a Hitachi 7700 electron microscope.

[0040] Based on the data in Figures 1 and 2, the optimal ratio of dual-modified particles is when the amount of adenosine triphosphate added is 50-70% (0.75-1.05 mg) of the calcium phosphate nanoparticle (BCP) precipitation, especially at 60%, where the ratio of dual-modified particles is optimal: at this point, the BACP nanoparticles have the smallest size (adapted to cellular uptake), a moderately negative Zeta potential (-2 mV, balancing dispersibility and membrane interaction), and no risk of sudden charge drop or excessive aggregation, providing the optimal carrier basis for subsequent vaccine delivery.

[0041] Example 2 A method for preparing calcium phosphate nanoparticles synergistically modified with bisphosphate and adenine nucleoside triphosphate includes the following steps: (1) Dissolve 0.42 mL of 2M calcium chloride solution and 0.84 mL of Tris buffer solution with pH 10 in 2 mL of water to obtain the first solution; (2) Prepare HEPES buffer containing disodium hydrogen phosphate. The HEPES buffer contains 280 mM NaCl, 7.5 mM Na2HPO4 and 50 mM HEPES (the remainder is water).

[0042] Add 0.43 mL of HEPES buffer containing disodium hydrogen phosphate to 0.3 mL of water, then add alendronate sodium and adenine nucleoside triphosphate, stir well to obtain the second solution; (3) The second solution above is added dropwise to the first solution above, and stirred at 500 rpm for 30 min to obtain a mixture. Human serum gamma globulin is added to the mixture at a final concentration of 0.04~0.8 μg / mL and stirred evenly to complete the protein encapsulation of BACP nanoparticles. This step can further increase the stability of the particles in vivo. Centrifuge at 15000g for 10 min, wash with water, and dry to obtain calcium phosphate nanoparticles co-modified by bisphosphate and adenine nucleoside triphosphate.

[0043] The amount of the bisphosphate added is 50% of the molecular weight of phosphate in the second solution; The amount of adenine nucleoside triphosphate added is 60% of the amount of calcium phosphate nanoparticles (BCP) precipitate (0.9 mg).

[0044] Example 3 The application of calcium phosphate nanoparticles co-modified with bisphosphate and adenine nucleoside triphosphate for loading eGFP includes the following steps: (1) Dissolve 0.42 mL of 2M calcium chloride solution and 0.84 mL of Tris buffer solution with pH 10 in 2 mL of water to obtain the first solution; (2) Prepare HEPES buffer containing disodium hydrogen phosphate. The HEPES buffer contains 280mM NaCl, 7.5mM Na2HPO4 and 50mM HEPES. Then add 10µg eGFP plasmid (the remainder is water).

[0045] Add 0.43 mL of HEPES buffer containing disodium hydrogen phosphate to 0.3 mL of water, then add alendronate sodium and adenine nucleoside triphosphate, stir well to obtain the second solution; (3) The second solution above is added dropwise to the first solution above, and stirred at 500 rpm for 30 min to obtain a mixture. The mixture is centrifuged at 15000 g for 10 min, washed with water, and dried to obtain calcium phosphate nanoparticles (BACP-eGFP) loaded with eGFP and co-modified by adenine nucleoside triphosphate.

[0046] The amount of the bisphosphate added is 50% of the molecular weight of phosphate in the second solution; The amount of adenine nucleoside triphosphate added is 60% of the amount of calcium phosphate nanoparticles (BCP) precipitate (0.9 mg).

[0047] Comparative Example 1 A method for preparing calcium phosphate nanoparticles includes the following steps: (1) Dissolve 0.42 mL of 2M calcium chloride solution and 0.84 mL of Tris buffer solution with pH 10 in 2 mL of water to obtain the first solution; (2) Prepare HEPES buffer containing disodium hydrogen phosphate. The HEPES buffer contains 280 mM NaCl, 15 mM Na2HPO4 and 50 mM HEPES (the remainder is water).

[0048] Add 0.43 mL of HEPES buffer containing disodium hydrogen phosphate to 0.3 mL of water and stir until homogeneous to obtain the second solution; (3) The second solution above is added dropwise to the first solution above, and stirred at 500 rpm for 30 min to obtain a mixture. The mixture is centrifuged at 15000 g for 10 min, washed with water, and dried to obtain calcium phosphate nanoparticles (CP).

[0049] Comparative Example 2 A method for preparing adenosine triphosphate modified calcium phosphate nanoparticles includes the following steps: (1) Dissolve 0.42 mL of 2M calcium chloride solution and 0.84 mL of Tris buffer solution with pH 10 in 2 mL of water to obtain the first solution; (2) Prepare HEPES buffer containing disodium hydrogen phosphate. The HEPES buffer contains 280 mM NaCl, 15 mM Na2HPO4 and 50 mM HEPES (the remainder is water).

[0050] Add 0.43 mL of HEPES buffer containing disodium hydrogen phosphate to 0.3 mL of water, add adenine nucleoside triphosphate, stir well to obtain the second solution; (3) The second solution above is added dropwise to the first solution above, and the mixture is stirred at 500 rpm for 30 min to obtain a mixture. The mixture is centrifuged at 15000 g for 10 min, washed with water, and dried to obtain ATP-modified calcium phosphate nanoparticles (ACP).

[0051] The amount of adenine nucleoside triphosphate added is 10-70% (0.1mg-0.7mg) of the amount of calcium phosphate nanoparticles (CP) precipitated (1mg).

[0052] Among them, the Zeta potential of ATP-modified calcium phosphate nanoparticles (ACP) in Comparative Example 2 is as follows: Figure 3 As shown.

[0053] like Figure 3 As shown, when the amount of adenine nucleoside triphosphate added is 10-20% of the amount of calcium phosphate nanoparticle precipitation, the Zeta potential drops sharply from a strong positive charge (+10mV) to near neutral (-1mV). The charge neutralization effect is enhanced with the increase of ATP. At 20-30%, the Zeta potential drops sharply (-1mV→-6mV). At 40-70%, the potential is relatively stable (-7mV→-10mV), but the potential fluctuation is large.

[0054] As can be seen, the present invention, through double modification with bisphosphate and adenine nucleoside triphosphate, can effectively improve the Zeta potential, making the Zeta potential stable between -4 and +4 mV. This potential state allows the nanoparticles to maintain a good dispersion state in the physiological environment, reducing the occurrence of aggregation and adsorption phenomena, while eliminating the risk of sudden charge drop or excessive aggregation. This improves the circulation time, delivery efficiency, and stability in vivo, effectively increasing the chance of contact with target cells, improving the delivery efficiency of nucleic acid vaccines, and thus enhancing the immune effect of vaccines.

[0055] Comparative Example 3 A method for preparing bisphosphate-modified calcium phosphate nanoparticles includes the following steps: (1) Dissolve 0.42 mL of 2M calcium chloride solution and 0.84 mL of Tris buffer solution with pH 10 in 2 mL of water to obtain the first solution; (2) Prepare HEPES buffer containing disodium hydrogen phosphate. The HEPES buffer contains 280 mM NaCl, 7.5 mM Na2HPO4 and 50 mM HEPES (the remainder is water).

[0056] Add 0.43 mL of HEPES buffer containing disodium hydrogen phosphate to 0.3 mL of water, add sodium alendronate, stir well to obtain the second solution; (3) The second solution above is added dropwise to the first solution above, and stirred at 500 rpm for 30 min to obtain a mixture. The mixture is centrifuged at 15000 g for 10 min, washed with water, and dried to obtain bisphosphate modified calcium phosphate nanoparticles (BCP).

[0057] The amount of bisphosphate added is 50% of the molecular weight of phosphate in the second solution.

[0058] Comparative Example 4 An application of calcium phosphate nanoparticles for loading eGFP includes the following steps: (1) Dissolve 0.42 mL of 2M calcium chloride solution and 0.84 mL of Tris buffer solution with pH 10 in 2 mL of water to obtain the first solution; (2) Prepare HEPES buffer containing disodium hydrogen phosphate. The HEPES buffer contains 280mM NaCl, 15mM Na2HPO4 and 50mM HEPES. Add 10µg eGFP plasmid (the remainder is water).

[0059] Add 0.43 mL of HEPES buffer containing disodium hydrogen phosphate to 0.3 mL of water and stir until homogeneous to obtain the second solution; (3) The second solution above is added dropwise to the first solution above, and stirred at 500 rpm for 30 min to obtain a mixture. The mixture is centrifuged at 15000 g for 10 min, washed with water, and dried to obtain calcium phosphate nanoparticles loaded with eGFP (CP-eGFP).

[0060] Comparative Example 5 The application of adenosine triphosphate modified calcium phosphate nanoparticles for loading eGFP includes the following steps: (1) Dissolve 0.42 mL of 2M calcium chloride solution and 0.84 mL of Tris buffer solution with pH 10 in 2 mL of water to obtain the first solution; (2) Prepare HEPES buffer containing disodium hydrogen phosphate. The HEPES buffer contains 280mM NaCl, 15mM Na2HPO4 and 50mM HEPES. Add 10µg eGFP plasmid (the remainder is water).

[0061] Add 0.43 mL of HEPES buffer containing disodium hydrogen phosphate to 0.3 mL of water, add adenine nucleoside triphosphate, stir well to obtain the second solution; (3) The second solution above is added dropwise to the first solution above, and stirred at 500 rpm for 30 min to obtain a mixture. The mixture is centrifuged at 15000 g for 10 min, washed with water, and dried to obtain adenosine triphosphate modified calcium phosphate nanoparticles loaded with eGFP (ACP-eGFP).

[0062] The amount of adenine nucleoside triphosphate added is 60% (0.6 mg) of the amount of calcium phosphate nanoparticles (CP) precipitate.

[0063] Comparative Example 6 The application of a bisphosphate-modified calcium phosphate nanoparticle for loading eGFP includes the following steps: (1) Dissolve 0.42 mL of 2M calcium chloride solution and 0.84 mL of Tris buffer solution with pH 10 in 2 mL of water to obtain the first solution; (2) Prepare HEPES buffer containing disodium hydrogen phosphate. The HEPES buffer contains 280mM NaCl, 7.5mM Na2HPO4 and 50mM HEPES. Then add 10µg eGFP plasmid (the remainder is water).

[0064] Add 0.43 mL of HEPES buffer containing disodium hydrogen phosphate to 0.3 mL of water, add sodium alendronate, stir well to obtain the second solution; (3) The second solution above is added dropwise to the first solution above, and the mixture is stirred at 500 rpm for 30 min to obtain a mixture. The mixture is centrifuged at 15000 g for 10 min, washed with water, and dried to obtain calcium phosphate nanoparticles loaded with eGFP and modified with bisphosphate (BCP-eGFP).

[0065] The amount of bisphosphate added is 50% of the molecular weight of phosphate in the second solution.

[0066] 1. Particle size detection: The transmission electron microscopy (TEM) images of Example 1 (BACP), Comparative Example 1 (CP), Comparative Example 2 (ACP, with ATP content of 60%), and Comparative Example 3 (BCP) are shown below. Figures 4-7 As shown, Figure 4 The results show that the particles in Example 1 (BACP) have a size of 90 nm, are rounded, and have good dispersibility. Comparative Example 1 (CP) exhibits severe aggregation and poor dispersibility. Comparative Example 2 (ACP) shows significant particle aggregation and poor dispersibility, with a size of 180 nm. Comparative Example 3 (BCP) has uniformly dispersed particles with blurred edges, and a size of 140 nm. Experimental data show that the bisphosphate and adenine triphosphate have a significant synergistic effect in regulating the size of calcium phosphate, effectively improving the size of calcium phosphate nanoparticles and adjusting their Zeta potential. This improves their circulation time, delivery efficiency, and stability in vivo, thereby enhancing the delivery efficiency of nucleic acid vaccines and ultimately strengthening their immune efficacy.

[0067] 2. Detection of eGFP expression: RAW264.7 cells were passaged in DMEM medium containing 10% fetal bovine serum to induce logarithmic growth. One day before transfection, cells were cultured at a rate of 5 × 10⁶ cells / mL. 4 Cells / wells were seeded into 24-well plates and incubated at 37°C in a 5% CO2 humidifier for 24 hours.

[0068] Add 100 µL of eGFP-containing nanoparticle solution (containing 100 ng of eGFP plasmid, nanoparticles synthesized in Examples 3 and Comparative Examples 4-6, respectively) to each well. Add 100 µL of eGFP-containing PBS solution (containing 100 ng of eGFP plasmid) to the control group and mix gently. Incubate at 37°C in a 5% CO2 incubator for 48 hours.

[0069] 48 hours after transfection, carefully collect the cells. If the cells are adherent, they need to be digested with trypsin or other digestive solutions to detach them from the culture vessel surface; if they are suspension cells, the cell culture medium can be directly transferred to centrifuge tubes.

[0070] The collected cells were precipitated by centrifugation (1500, 5 min), and the supernatant was discarded. The cells were washed three times with PBS. Then, the cells were resuspended in 200 μL of 4% paraformaldehyde (PFA) solution and fixed at room temperature for 15 min.

[0071] The fixed cells were centrifuged again (1500g, 5min), the fixative was discarded, and the cells were washed three times with PBS buffer to remove residual fixative and impurities. The washed cells were resuspended in 500µL PBS buffer and transferred to flow cytometry tubes. Green fluorescence (excitation 488nm, emission 525nm) was detected using a flow cytometer (e.g., CytoFLEX) to assess eGFP expression. Figure 8 As shown.

[0072] Comparison of eGFP expression (mean fluorescence intensity, MFI) among the different nanoparticles (including CP, ACP, BCP, and BACP). The results show that BACP significantly outperforms the other nanoparticles, and BCP also outperforms ACP.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing calcium phosphate nanoparticles synergistically modified with bisphosphate and adenine nucleoside triphosphate, characterized in that, Includes the following steps: Dissolve calcium chloride solution and Tris buffer in water to obtain the first solution; Add HEPES buffer containing disodium hydrogen phosphate to water, then add bisphosphate and adenine nucleoside triphosphate, stir well to obtain the second solution; The second solution was added dropwise to the first solution and stirred until homogeneous to obtain a mixture. The mixture was then centrifuged, washed, and dried to obtain calcium phosphate nanoparticles co-modified by diphosphate and adenine nucleoside triphosphate. The amount of the bisphosphate added is 20-50% of the molecular weight of phosphate in the second solution; The volume ratio of adenosine triphosphate to HEPES buffer containing disodium hydrogen phosphate is (0.9~1.05) mg: 0.43 mL; The bisphosphate includes sodium alendronate; The volume ratio of the calcium chloride solution, Tris buffer, and water is 1:(1~4):(4~10). The volume ratio of the first solution to the HEPES buffer is (5~10):

1.

2. The method for preparing calcium phosphate nanoparticles synergistically modified with bisphosphate and adenine nucleoside triphosphate according to claim 1, characterized in that, The concentration of the calcium chloride solution is 0.5~5M; The pH of the Tris buffer solution is 9-10.

3. The method for preparing calcium phosphate nanoparticles synergistically modified with bisphosphate and adenine nucleoside triphosphate according to claim 1, characterized in that, The HEPES buffer also includes sodium chloride; The concentration of disodium hydrogen phosphate in the HEPES buffer is 5~20mM; The concentration of sodium chloride in the HEPES buffer solution is 200~350mM; The concentration of HEPES in the HEPES buffer solution is 20~80mM.

4. The calcium phosphate nanoparticles synergistically modified with bisphosphate and adenine nucleoside triphosphate according to claim 1, characterized in that, This also includes adding serum albumin to the mixture.

5. The calcium phosphate nanoparticles synergistically modified with bisphosphate and adenine nucleoside triphosphate according to claim 4, characterized in that, The final concentration of serum albumin is 0.04~0.8 μg / mL.

6. A calcium phosphate nanoparticle synergistically modified with bisphosphate and adenine nucleoside triphosphate, characterized in that, It is prepared by any one of the preparation methods described in claims 1 to 5.

7. The application of the calcium phosphate nanoparticles synergistically modified with bisphosphate and adenine nucleoside triphosphate as described in claim 6 in the preparation of nucleic acid vaccines.

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