Calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid, methods of making and use in hard tissue repair products

Abstract

The application provides a kind of polyphosphate or polyphosphoric acid containing calcium phosphate nanoparticles and its preparation method and application in hard tissue repair products, the diameter of the calcium phosphate nanoparticles is 1nm-10μm, the mass fraction of polyphosphate or polyphosphoric acid in calcium phosphate nanoparticles is 10-80%; it is obtained by mixing calcium-containing solution and phosphorus-containing solution, the phosphorus-containing solution includes polyphosphate or polyphosphoric acid and phosphate or phosphoric acid, the concentration of polyphosphate or polyphosphoric acid in the phosphorus-containing solution is 0.1-500mg / mL.The polyphosphate or polyphosphoric acid containing calcium phosphate nanoparticles obtained by the application has the advantages of small size, controllable particle size and high biological safety, the preparation method is simple to operate, low in cost, and can realize large-scale preparation of calcium phosphate nanoparticles;And can effectively mineralize collagen fibers, and repair hard tissues such as bone and enamel repair, has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of nano-inorganic materials technology, specifically relating to a calcium phosphate nanoparticle containing polyphosphate or polyphosphoric acid, its preparation method, and its application in products for repairing hard tissues. Background Technology

[0002] Calcium phosphate particles are inexpensive and easy to manufacture. Due to their unusual versatility, they are abundant and naturally available nanomaterials that can be used for various technical purposes. They are used in many industries, including biomedicine. However, stable preparation methods for inorganic calcium phosphate nanoparticles are still scarce. Therefore, developing a new and simple method for preparing calcium phosphate nanoparticles and its applications is of great significance.

[0003] Polyphosphates, also known as condensed phosphates, are phosphate polymers linked by phosphate anhydride bonds. Polyphosphates have numerous biological functions, including phosphate storage, energy generation in anaerobic environments, and the sequestration of polyvalent cations. Polyphosphates have only recently been identified in a wide range of life forms, including bacteria, plants, animals, yeast, and fungi. The biochemical functions of polyphosphates are constantly being discovered; enzymes responsible for the condensation and hydrolytic degradation of polyphosphates are also being identified. Three important properties of polyphosphates make them a useful resource for apatite biomineralization: their affinity for calcium, their ability to insulate localized high concentrations of orthophosphate while maintaining low orthophosphate activity, and their ability to be synthesized and degraded by enzymes. Numerous studies have shown that polyphosphates play a crucial 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] Currently, aqueous solutions are considered to have good biocompatibility in the preparation of calcium phosphate nanoparticles. However, due to the lack of stabilizers and the tendency of small nanoparticles to aggregate, the resulting calcium phosphate particles are often submicron to micron in size. Small-sized calcium phosphate exhibits higher mineralization and bioactivity, promoting the repair of hard tissues such as teeth and bone, and is effective in preparing continuous bulk materials. However, it remains difficult to achieve the stable preparation of ultra-small calcium phosphate nanoparticles and apply them to the repair of hard tissues such as bone and tooth enamel. Summary of the Invention

[0005] The purpose of this invention is to provide calcium phosphate nanoparticles containing polyphosphate or phosphate, their preparation method, and their application in products for repairing hard tissues. The nanoparticles provided by this invention have the advantage of controllable size, which enables them to effectively repair hard tissues. Moreover, the preparation method is simple and can realize the large-scale preparation of calcium phosphate nanoparticles.

[0006] This invention provides the following technical solution:

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

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

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

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

[0011] (2) Add polyphosphate or polyphosphoric acid to a solvent and stir until completely dissolved. Then add phosphate or phosphoric acid and stir until completely dissolved to prepare a phosphorus-containing solution. The concentration of polyphosphate or polyphosphoric acid in the phosphorus-containing solution is 0.1-500 mg / mL.

[0012] (3) Slowly add the phosphorus-containing solution to the calcium-containing solution and adjust the pH to 5.5-8.5 to obtain calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid.

[0013] In this invention, polyphosphate or polyphosphoric acid competitively binds to calcium ions with phosphate groups. Below the specified concentration range of polyphosphate or polyphosphoric acid, severe precipitation occurs, and the unstable calcium phosphate particles lead to aggregation. Above the specified concentration range of polyphosphate or polyphosphoric acid, calcium phosphate nanoparticles cannot be formed. Furthermore, 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 includes 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); and 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 to 2.

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

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

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

[0025] In this invention, self-assembled collagen fibers are immersed in a 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 penetrate into the bone interior, increasing the mineral content.

[0026] The present invention also provides the application of the above-mentioned polyphosphate-containing calcium phosphate nanoparticles in products for repairing hard tissues; the hard tissues include bone and tooth damage in orthopedics, plastic surgery, craniofacial surgery, neurosurgery, or dentistry.

[0027] In this invention, the glaze is immersed in a 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 glaze to form a repair layer, which has a continuous structure with the natural glaze crystals.

[0028] In this invention, the solution of calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid is injected into the femur of C57 mice modeling osteoporosis, thereby penetrating and repairing the osteoporotic femur and increasing its bone content to treat osteoporosis.

[0029] In this invention, calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid are freeze-dried and then filled into the femoral bone defects of SD mice modeled for osteoporosis, thereby performing a filling-type repair of osteoporotic femoral defects.

[0030] Compared with existing technologies, the calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid provided by this invention have the advantages of controllable particle size and strong stability. The preparation method is simple to operate and low in cost, and it can realize the large-scale preparation of calcium phosphate nanoparticles containing polyphosphate. Moreover, it can effectively repair hard tissues, such as bone matrix and enamel, and has broad application prospects. Attached Figure Description

[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 The image shows the X-ray diffraction characterization of the polyphosphate-containing calcium phosphate nanoparticles prepared in Example 1 of this invention.

[0033] Figure 3 The Tyndall effect is observed in the calcium phosphate nanoparticle solution containing polyphosphate prepared in Example 1 of this invention, where a is a fresh sample and b is a sample placed for 24 hours.

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

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

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

[0037] Figure 7 This is a Micro-CT image of osteoporotic C57 mice after femoral repair using polyphosphate-containing calcium phosphate nanoparticles via injection, as described in Example 1 of this invention; where a is an image of the osteoporotic femur and b is an image of the osteoporosis repair effect.

[0038] Figure 8The image shows the morphology of calcium phosphate nanoparticles containing polyphosphate after freeze-drying in Example 1 of this invention.

[0039] Figure 9 The image shown is a Micro-CT image of calcium phosphate nanoparticles containing polyphosphate, which were freeze-dried and used to fill and repair femoral defects in osteoporotic SD rats in Example 1 of this invention; where a is the femoral defect image and b is the defect repair effect image.

[0040] Specific implementation methods

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

[0042] Example 1

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

[0044] 2. Weigh 0.2g of sodium polyphosphate (degree of polymerization approximately 250) and add it to 10mL of deionized water. Stir thoroughly until the sodium polyphosphate dissolves. Then add 0.142g 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, and control the magnetic stirring speed to 500 rpm. Adjust the pH to 7.4 using 0.1M hydrochloric acid solution.

[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 microscope characterization image of the calcium phosphate nanoparticles containing polyphosphate (sodium polyphosphate) is shown below. Figure 1 As shown, the particle size is approximately 2 nm, and the X-ray diffraction characterization pattern is as follows. Figure 2 As shown, this indicates that the calcium phosphate nanoparticles containing polyphosphate are in an amorphous state, such as... Figure 3 As shown, the Tyndall effect still exists after the above-mentioned calcium phosphate nanoparticle solution containing polyphosphate is left for 24 hours.

[0047] Example 2

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

[0049] 2. Weigh 0.2g of sodium polyphosphate (degree of polymerization approximately 50) and add it to 10mL of deionized water. Stir thoroughly until the sodium polyphosphate dissolves. Then add 0.142g 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, and control the magnetic stirring speed to 500 rpm. Adjust the pH to 7.4 using 0.1M hydrochloric acid solution.

[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). Transmission electron microscopy characterization and particle size distribution diagrams of the calcium phosphate nanoparticles containing polyphosphate (sodium polyphosphate) are shown below. Figure 4 As shown, the particle size is approximately 30 nm.

[0052] Example 3

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

[0054] Example 4

[0055] Enamel restoration was modeled using early caries lesions. The enamel was soaked in a 37wt% phosphoric acid solution for 30 seconds, followed by ultrasonic treatment in deionized water for 20 minutes to ensure removal of any residual contaminants, and then air-dried. The enamel was then immersed in 100 mL of a calcium phosphate nanoparticle solution containing polyphosphate (25 mg / mL) and incubated at 37°C for 72 hours. The restoration was observed using a scanning electron microscope. Figure 6 The enamel-repaired area is significantly higher than the unrepaired area, indicating the formation of new enamel pillars and their outward extension along the direction of the original enamel pillars.

[0056] Example 5

[0057] Osteoporosis repair experiments were conducted using female C57 mice as a model. Forty-eight mice weighing 0.2 ± 0.02 kg (4-5 months old) were randomly divided into four groups (n = 12). Osteoporosis modeling surgery: Mice were anesthetized and fixed prone on a surgical board. Hair on their backs was removed, and the area was disinfected with iodine and 75% alcohol. Incisions were made on both sides of the back at the lower third of the back. The skin and muscle were bluntly dissected, revealing the muscle layer. The psoas major muscle was incised 1 cm below the ribs, immediately exposing the adipose tissue surrounding the ovary and the uterine horn closely connected to it. The adipose tissue was gently grasped with small forceps, pulled out through the incision, and separated from the adipose tissue. The fallopian tubes below the ovary were ligated with silk sutures, and the ovary was removed. The uterine horn was successfully returned to the abdominal cavity postoperatively. After suturing the incision, the other ovary was removed using the same method. Successful modeling was confirmed by in vivo imaging four weeks postoperatively. A solution of calcium phosphate nanoparticles containing polyphosphate was injected into the femur of osteoporotic C57 mice. The experiments were conducted for 4 and 12 weeks, at which time the animals were euthanized due to overdose of anesthesia, and the osteoporosis repair effect in the femur was observed by micro-CT. Figure 7 The image is a Micro-CT scan showing that calcium phosphate nanoparticles containing polyphosphate significantly promote bone growth and have a marked therapeutic and restorative effect on osteoporosis.

[0058] Example 6

[0059] An osteoporotic bone defect repair experiment was conducted using female SD rats as a model. Forty-eight mice weighing 2.5 ± 0.2 kg (4-5 months old) were randomly divided into four groups (n = 12). Osteoporosis modeling surgery was performed as described in Example 4, followed by implantation surgery. During the implantation surgery, 3% sodium pentobarbital (1 mL / kg) was used. -1 Anesthetize rats. Make an incision on the lateral thigh to expose the femur. Create a defect area in the femur using a dental drill. Then implant calcium phosphate nanoparticles containing polyphosphate, which are then lyophilized to obtain a powder (morphology as shown in the image). Figure 8 (As shown), the muscle and skin were sutured in layers. The implantation experiments were conducted for 4 weeks and 12 weeks, at which time the animals were euthanized due to overdose of anesthesia, and the defect repair effect was observed by Micro-CT. Figure 9 The image is a micro-CT scan showing that the area filled with polyphosphate-containing calcium phosphate nanoparticles has completely healed, indicating that polyphosphate-containing calcium phosphate nanoparticles can degrade and promote new bone growth at the defect site, even under osteoporotic conditions.

[0060] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. All technical solutions that fall within the principles of the present invention are within the scope of protection of the present invention. For those skilled in the art, any improvements and modifications made without departing from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing calcium phosphate nanoparticles 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%. The method for preparing the calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid includes the following steps: (1) Dissolve the inorganic calcium salt in a solvent and stir until the inorganic calcium salt solid is completely dissolved to prepare a calcium-containing solution; (2) Add polyphosphate or polyphosphoric acid to a solvent and stir until completely dissolved. Then add phosphate or phosphoric acid and stir until completely dissolved to prepare a phosphorus-containing solution. The concentration of polyphosphate or polyphosphoric acid in the phosphorus-containing solution is 0.1~500 mg / mL. (3) The phosphorus-containing solution is slowly added to the calcium-containing solution, and the pH is adjusted to 5.5~8.

5. The reaction yields calcium phosphate nanoparticles containing polyphosphate. In step (2), the phosphate includes one or more of dihydrogen phosphate, hydrogen phosphate, and phosphate; in step (2), the degree of polymerization of the polyphosphate is ≥2.

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

3. The method for preparing calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid as described 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 to 2.

4. The application of calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid obtained by the method of claim 1 in the mineralization of collagen fibers.

5. The application of calcium phosphate nanoparticles containing polyphosphate or polyphosphoric acid obtained by the method of claim 1 in products for repairing hard tissue.

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