Calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid, preparation method of calcium phosphate nanoparticles and application of calcium phosphate nanoparticles in hard tissue repair products

Abstract

The invention provides calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid, a preparation method of the calcium phosphate nanoparticles and application of the calcium phosphate nanoparticles in hard tissue repair products, the diameter of the calcium phosphate nanoparticles is 1 nm-10 microns, and the mass fraction of the polyphosphate or the polyphosphoric acid in the calcium phosphate nanoparticles is 10-80%; the calcium-phosphorus composite material is obtained by mixing a calcium-containing solution and a phosphorus-containing solution, the phosphorus-containing solution comprises polyphosphate or polyphosphoric acid and phosphate or phosphoric acid, and the concentration of polyphosphate or polyphosphoric acid in the phosphorus-containing solution is 0.1-500 mg / mL. The calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid have the advantages of small size, controllable particle size and high biological safety, the preparation method is simple to operate and low in cost, and large-scale preparation of the calcium phosphate nanoparticles can be realized; the collagen fibers can be effectively mineralized, the hard tissues such as bones and enamel can be repaired, and the method has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of nano inorganic materials, and in particular relates to calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid, a preparation method thereof, and application of the nanoparticles in hard tissue repair products. Background Art

[0002] Calcium phosphate particles are cheap and easy to manufacture. Due to their unusual functional pleiotropy, they are abundant and naturally available nanomaterials that can be used as a means for various technological purposes. They are used in many industries, including biomedicine. However, there are few stable preparation methods for inorganic nano calcium phosphates, so it is of great significance to develop a new and easy-to-operate preparation method for calcium phosphate nanoparticles and their application.

[0003] Polyphosphates, also known as condensed phosphates, are phosphate polymers linked by phosphoanhydride bonds. Polyphosphates have many biological roles, including storage of phosphate, energy source in anoxic environments, and sequestration of multivalent cations. Polyphosphates have only recently been identified in a wide range of life forms, including bacteria, plants, animals, yeast, and fungi. The biochemical roles of polyphosphates continue to be discovered; enzymes responsible for polyphosphate condensation and hydrolytic degradation continue to be identified. Three important properties of polyphosphates make them a useful resource for apatite biomineralization, namely, their affinity for calcium, their ability to sequester local high concentrations of orthophosphate while maintaining low orthophosphate activity, and their ability to be synthesized and degraded by enzymes. A large number of studies have shown that polyphosphates play an important role in the mineralization and repair of hard tissues in vivo (Role and mechanism of polyphosphate in bone tissue regeneration [J]. Chinese Journal of Tissue Engineering Research, 2023, 27 (21): 3375-3381.).

[0004] At present, the preparation method of calcium phosphate nanoparticles using aqueous solution as the reaction system has good biosafety. However, due to the lack of stabilizers and the easy aggregation of small-sized nanoparticles, the size of the calcium phosphate obtained is often submicron to micron. Small-sized calcium phosphate has higher mineralization and biological activity, promotes the repair of hard tissues such as teeth and bones, and effectively prepares bulk materials of continuous structure. However, it is still difficult to achieve the stable preparation of ultra-small-sized calcium phosphate nanoparticles and apply them to the repair of hard tissues such as bones and enamel. Summary of the invention

[0005] The purpose of the present invention is to provide a calcium phosphate nanoparticle containing polyphosphate or phosphate, a preparation method thereof and application in hard tissue repair products; the nanoparticles provided by the present invention have the advantage of controllable size, so that they can effectively repair hard tissues; and the preparation method is simple and can achieve large-scale preparation of calcium phosphate nanoparticles.

[0006] The present invention provides the following technical solutions:

[0007] A calcium phosphate nanoparticle containing polyphosphate or polyphosphoric acid, wherein the diameter of the calcium phosphate nanoparticle is 1nm-10μm; the mass percentage of the polyphosphate or polyphosphoric acid in the calcium phosphate nanoparticle is 10-80%.

[0008] The diameter of the calcium phosphate nanoparticles is 1-100 nm.

[0009] The present invention also provides a method for preparing calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid, comprising the following steps:

[0010] (1) dissolving an inorganic calcium salt in a solvent and stirring until the inorganic calcium salt solid is completely dissolved to prepare a calcium-containing solution;

[0011] (2) adding polyphosphate or polyphosphoric acid to a solvent, stirring until completely dissolved, and then adding phosphate or phosphoric acid thereto, stirring until completely dissolved, to prepare a phosphorus-containing solution; the concentration of the polyphosphate or polyphosphoric acid in the phosphorus-containing solution is 0.1 to 500 mg / mL;

[0012] (3) Slowly adding the phosphorus-containing solution into the calcium-containing solution and adjusting the pH to 5.5-8.5, the reaction obtains calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid.

[0013] In the present invention, polyphosphate or polyphosphoric acid competitively binds to calcium ions with phosphate groups. If the concentration is lower than the polyphosphate or polyphosphoric acid concentration range, severe precipitation will occur, and the calcium phosphate particles will be unstable and agglomerated. If the concentration is higher than the polyphosphate or polyphosphoric acid concentration range, calcium phosphate nanoparticles cannot be formed. The particle size of the calcium phosphate nanoparticles is related to the amount of polyphosphate added. The higher the amount of polyphosphate added, the smaller the particle size.

[0014] Preferably, in step (1), the solvent is deionized water, ethanol, methanol or N-methylpyrrolidone.

[0015] Preferably, in step (1), the inorganic calcium salt comprises one or both of calcium chloride and calcium nitrate.

[0016] Preferably, in step (1), the concentration of calcium ions in the calcium-containing solution is in the range of 0.001 to 2 mol / mL.

[0017] Preferably, in step (2), the concentration of phosphate in the phosphorus-containing solution is in the range of 0.001 to 2 mol / mL.

[0018] Preferably, in step (2), the phosphate includes one or more of dihydrogen phosphate, hydrogen phosphate and phosphate; the dihydrogen phosphate includes one or more of potassium dihydrogen phosphate (KH2PO4), sodium dihydrogen phosphate (NaH2PO4) and ammonium dihydrogen phosphate (NH4H2PO4); the hydrogen phosphate includes one or more of dipotassium hydrogen phosphate (K2HPO4), disodium hydrogen phosphate (Na2HPO4) and diammonium hydrogen phosphate ((NH4)2HPO4); the phosphate includes one or more of tripotassium phosphate (K3PO4) and trisodium phosphate (Na3PO4·12H2O).

[0019] Preferably, in step (2), the degree of polymerization of the polyphosphate or polyphosphoric acid is ≥2.

[0020] Preferably, in step (2), the polyphosphate includes one or more of ammonium polyphosphate, sodium polyphosphate and potassium polyphosphate.

[0021] Preferably, in step (3), the molar ratio of calcium ions in the calcium-containing solution to phosphate or phosphoric acid in the phosphorus-containing solution is 0.1-2.

[0022] The calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid are in an amorphous state.

[0023] The preparation method provided by the invention is simple to operate and low in cost, and can realize large-scale preparation of calcium phosphate nanoparticles containing polyphosphate.

[0024] The present invention also provides an application of the calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid in the mineralization of collagen fibers.

[0025] In the present invention, the self-assembled collagen fibers are immersed in the solution of calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid and incubated at 37° C. for a period of time, and the calcium phosphate nanoparticles containing polyphosphate penetrate into the bone to increase the mineral content.

[0026] The present invention also provides an application of the above-mentioned calcium phosphate nanoparticles containing polyphosphate in a product for repairing hard tissue; the hard tissue includes bone and tooth damage in orthopedics, plastic surgery, craniomaxillofacial surgery, brain surgery or stomatology.

[0027] In the present invention, enamel is immersed in the solution of calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid and incubated at 37°C for a period of time. The calcium phosphate nanoparticles containing polyphosphate are adsorbed on the surface of the enamel to form a repair layer, which is a continuous structure with the natural enamel crystals.

[0028] In the present invention, the solution of calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid is injected into the femur of C57 mice modeled with osteoporosis to perform penetration repair on the osteoporotic femur and increase its bone content to treat osteoporosis.

[0029] In the present invention, calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid are freeze-dried and then filled into the femoral defect of the SD mouse thigh modeled with osteoporosis to perform filling-type repair on the osteoporotic femoral defect.

[0030] Compared with the prior art, the calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid provided by the present invention have the advantages of controllable particle size and strong stability, the preparation method is simple to operate, low cost, and can achieve large-scale preparation of calcium phosphate nanoparticles containing polyphosphate; and can effectively repair hard tissues, such as bone matrix and enamel repair, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a transmission electron microscope characterization image of the polyphosphate-containing calcium phosphate nanoparticles prepared in Example 1 of the present invention.

[0032] Figure 2 This is an X-ray diffraction characterization diagram of the calcium phosphate nanoparticles containing polyphosphate prepared in Example 1 of the present invention.

[0033] Figure 3 This is the Tyndall phenomenon of the calcium phosphate nanoparticle solution containing polyphosphate prepared in Example 1 of the present invention, wherein a is a fresh sample and b is a sample left for 24 hours.

[0034] Figure 4 Transmission electron microscope characterization image (a) and particle size distribution image (b) of the calcium phosphate nanoparticles containing polyphosphate prepared in Example 2 of the present invention.

[0035] Figure 5 This is a transmission electron microscopy characterization image of collagen fibers mineralized using calcium phosphate nanoparticles containing polyphosphate in Example 1 of the present invention.

[0036] Figure 6 This is a scanning electron microscope image of enamel repair using polyphosphate-containing calcium phosphate nanoparticles in Example 1 of the present invention.

[0037] Figure 7 This is a Micro-CT image of Example 1 of the present invention after the femur of osteoporotic C57 mice was repaired by injection of calcium phosphate nanoparticles containing polyphosphate; wherein a is a picture of the osteoporotic femur, and b is a picture of the osteoporosis repair effect.

[0038] Figure 8This is the morphology of the calcium phosphate nanoparticles containing polyphosphate after freeze-drying in Example 1 of the present invention.

[0039] Fig. 9 This is a Micro-CT image of Example 1 of the present invention, in which calcium phosphate nanoparticles containing polyphosphate are used to fill and repair the femoral defect of osteoporotic SD rats after freeze-drying; wherein a is a picture of the femoral defect, and b is a picture of the defect repair effect.

[0040] Specific implementation method

[0041] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0042] Example 1

[0043] 1. Weigh 0.111 g of calcium chloride dihydrate and add it to 10 mL of deionized water. Stir until the calcium chloride particles are completely dissolved to prepare a calcium solution.

[0044] 2. Weigh 0.2 g of sodium polyphosphate (degree of polymerization is about 250) and add it to 10 mL of deionized water, stirring thoroughly until the sodium polyphosphate is dissolved. Continue to add 0.142 g of disodium hydrogen phosphate and stir until the disodium hydrogen phosphate particles are completely dissolved to prepare a phosphorus-containing solution.

[0045] 3. Use a pipette to slowly add the calcium-containing solution to the phosphorus-containing solution, control the magnetic stirring speed to 500 rpm, and use 0.1 M hydrochloric acid solution to adjust the pH to 7.4.

[0046] 4. When the calcium-containing solution is slowly added to the phosphorus-containing solution, sodium polyphosphate, disodium hydrogen phosphate and calcium ions react, and the solution gradually changes from clear and transparent to pale white, thus obtaining a calcium phosphate nanoparticle solution containing polyphosphate (25 mg / mL). The transmission electron microscopy characterization of calcium phosphate nanoparticles containing polyphosphate (sodium polyphosphate) is shown in the figure below: Figure 1 As shown, the particle size is about 2nm, and the X-ray diffraction characterization diagram is as follows Figure 2 As shown, it is shown that the calcium phosphate nanoparticles containing polyphosphate are in an amorphous state, such as Figure 3 As shown, the Tyndall phenomenon still occurs in the calcium phosphate nanoparticle solution containing polyphosphate after being placed for 24 hours.

[0047] Example 2

[0048] 1. Weigh 0.111 g of calcium chloride dihydrate and add it to 10 mL of deionized water. Stir until the calcium chloride particles are completely dissolved to prepare a calcium solution.

[0049] 2. Weigh 0.2 g of sodium polyphosphate (degree of polymerization is about 50) and add it to 10 mL of deionized water, stirring thoroughly until the sodium polyphosphate is dissolved. Continue to add 0.142 g of disodium hydrogen phosphate and stir until the disodium hydrogen phosphate particles are completely dissolved to prepare a phosphorus-containing solution.

[0050] 3. Use a pipette to slowly add the calcium-containing solution to the phosphorus-containing solution, control the magnetic stirring speed to 500 rpm, and use 0.1 M hydrochloric acid solution to adjust the pH to 7.4.

[0051] 4. When the calcium-containing solution is slowly added to the phosphorus-containing solution, sodium polyphosphate, disodium hydrogen phosphate and calcium ions react, and the solution gradually changes from clear and transparent to pale white, thus obtaining a calcium phosphate nanoparticle solution containing polyphosphate (25 mg / mL). The transmission electron microscopy characterization and particle size distribution diagram of calcium phosphate nanoparticles containing polyphosphate (sodium polyphosphate) are shown in Figure 2. Figure 4 As shown, the particle size is about 30 nm.

[0052] Example 3

[0053] Collagen fiber self-assembly and collagen mineralization: 3 mg mL -1 The pH value of type I collagen was adjusted to 9 with NaOH solution, and then rinsed with deionized water after incubation for 30 minutes. Then, the collagen fibers were further cross-linked with 0.05% glutaraldehyde for 4 hours. The self-assembled collagen fibers were immersed in 100 mL of calcium phosphate nanoparticle solution containing polyphosphate (25 mg / mL), incubated at 37°C for 30 minutes, and the mineralization of the collagen fibers was observed by transmission electron microscopy after drying ( Figure 5 ), the darker electron-dense areas indicate that the collagen fibers have been severely mineralized.

[0054] Example 4

[0055] The restoration of enamel was simulated as an early caries lesion model. The enamel was soaked in 37wt% phosphoric acid solution for 30 seconds, then ultrasonically treated in deionized water for 20 minutes to ensure the removal of any residual contaminants and air-dried. The enamel was soaked in 100mL of calcium phosphate nanoparticle solution containing polyphosphate (25mg / mL) and incubated at 37℃ for 72 hours. The restoration was observed by scanning electron microscopy ( Figure 6 ). The enamel repair area is significantly higher than the unrepaired area, indicating the formation of new enamel columns and their epitaxial growth along the direction of the original enamel columns.

[0056] Example 5

[0057] The osteoporosis repair experiment used C57 mice (female) as a model. 48 mice weighing 0.2±0.02kg (4-5 months old) were randomly divided into 4 groups (n=12). Osteoporosis modeling surgery: The mice were anesthetized and fixed on the surgical board in a prone position. The hair on the back was removed and wiped with iodine and 75% alcohol for disinfection. An incision was made at 1 / 3 below the midline on both sides of the back. The skin and muscle were bluntly separated, and the muscle layer could be seen from the incision. The psoas major muscle was cut under the ribs 1 cm away from the spine. The fat tissue wrapped around the ovary and the uterine horn closely connected to the ovary could be immediately seen. The fat mass was gently clamped with small tweezers, pulled out from the incision, and then separated from the fat mass. The fallopian tube under the ovary was ligated with silk thread, and the ovary was removed. After the operation, the uterine horn was smoothly returned to the abdominal cavity. After suturing the incision, the other ovary was removed in the same way. The successful modeling was confirmed by animal in vivo imaging 4 weeks after the operation. The calcium phosphate nanoparticle solution containing polyphosphate was injected into the femur of osteoporotic C57 mice. The experimental period was 4 weeks and 12 weeks, at which time the animals were killed by overdose of anesthesia, and the osteoporosis repair effect in the thigh femur was observed by Micro-CT. Figure 7 This is a Micro-CT photo. The picture shows that calcium phosphate nanoparticles containing polyphosphate significantly promote bone growth and have obvious therapeutic and restorative effects on osteoporosis.

[0058] Example 6

[0059] The osteoporotic bone defect repair experiment was conducted using SD rats (female) as a model. 48 mice weighing 2.5±0.2 kg (4-5 months old) were randomly divided into 4 groups (n=12). The osteoporosis modeling surgery was performed as described in Example 4, and then the implantation surgery was performed. During the implantation surgery, 3% sodium pentobarbital (1 mL kg -1 ) anesthetized rats. The thigh was cut open to expose the femur. A defect area was created at the femur using a dental drill. Then, a powder (morphology as shown) obtained by freeze-drying calcium phosphate nanoparticles containing polyphosphate was implanted. Figure 8 The muscle and skin were sutured in layers. The implantation experiment period was 4 weeks and 12 weeks, at which time the animals were killed by overdose of anesthesia, and the defect repair effect was observed by Micro-CT. Fig. 9 This is a Micro-CT photo, which shows that the site filled with polyphosphate-containing calcium phosphate nanoparticles has been completely healed, indicating that polyphosphate-containing calcium phosphate nanoparticles can degrade and promote new bone growth in the defect site, even under osteoporotic conditions.

[0060] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiment. All technical solutions belonging to the principle of the present invention belong to the protection scope of the present invention. For those skilled in the art, some improvements and modifications made without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A calcium phosphate nanoparticle containing polyphosphate or polyphosphoric acid, characterized in that: The diameter of the calcium phosphate nanoparticles is 1 nm to 10 μm, and the mass fraction of polyphosphate or polyphosphoric acid in the calcium phosphate nanoparticles is 10 to 80%.

2. A method for preparing calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid according to claim 1, characterized in that: The steps include: (1) dissolving an inorganic calcium salt in a solvent and stirring until the inorganic calcium salt solid is completely dissolved to prepare a calcium-containing solution; (2) adding polyphosphate or polyphosphoric acid to a solvent, stirring until completely dissolved, and then adding phosphate or phosphoric acid thereto, stirring until completely dissolved, to prepare a phosphorus-containing solution; the concentration of the polyphosphate or polyphosphoric acid in the phosphorus-containing solution is 0.1 to 500 mg / mL; (3) Slowly adding the phosphorus-containing solution into the calcium-containing solution and adjusting the pH to 5.5-8.5, and reacting to obtain calcium phosphate nanoparticles containing polyphosphate.

3. The method for preparing calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid as claimed in claim 1, characterized in that: In step (1), the concentration of calcium ions in the calcium-containing solution is in the range of 0.001 to 2 mol / mL.

4. The method for preparing calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid as claimed in claim 1, characterized in that: In step (2), the concentration of phosphate or phosphoric acid in the phosphorus-containing solution is in the range of 0.001 to 2 mol / mL.

5. The method for preparing calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid as claimed in claim 1, characterized in that: In step (2), the phosphate includes one or more of dihydrogen phosphate, hydrogen phosphate and phosphate.

6. The method for preparing calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid as claimed in claim 1, characterized in that: In step (2), the degree of polymerization of the polyphosphate is ≥2.

7. The method for preparing calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid as claimed in claim 1 or 6, characterized in that: In step (2), the polyphosphate includes one or more of ammonium polyphosphate, sodium polyphosphate and potassium polyphosphate.

8. The method for preparing calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid as claimed in claim 1, characterized in that: In step (3), the molar ratio of calcium ions in the calcium-containing solution to phosphate or phosphoric acid in the phosphorus-containing solution is 0.1-2.

9. Use of the calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid according to claim 1 in the mineralization of collagen fibers.

10. Use of the calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid according to claim 1 in a product for repairing hard tissue.

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