Preparation method and application of hexagonal prism assembled hydroxyapatite porous microspheres

Abstract

The invention belongs to the field of preparation of hydroxyapatite, and particularly relates to a preparation method and application of hexagonal prism assembled hydroxyapatite porous microspheres. The preparation method of the hexagonal prism assembled hydroxyapatite porous microspheres comprises the following steps: carrying out hydrothermal reaction on water-soluble calcium salt, dihydric pyrophosphate and urea at 150-200 DEG C; the molar ratio of the dihydric pyrophosphate to Ca in the water-soluble calcium salt to urea is 1: (3-5): (10-15). The preparation method comprises the following steps: taking water-soluble calcium salt, dihydric pyrophosphate and urea as raw materials to prepare hexagonal prism assembled hydroxyapatite porous microspheres; according to the method, additives such as an organic active agent and a template agent are not used, and the obtained hydroxyapatite porous microspheres are high in roundness, loose and porous in surface, beneficial to high adsorption and high loading of a target object and wide in application prospect in the fields of drugs, catalyst carriers and ion adsorption materials.

Description

Technical Field

[0001] The invention belongs to the field of hydroxyapatite preparation, and in particular relates to a preparation method and application of hydroxyapatite porous microspheres assembled in hexagonal prisms. Background Art

[0002] Hydroxyapatite (Ca 10 (PO4)6(OH)2; HA) is the main inorganic component of vertebrate bones and teeth, showing excellent biocompatibility and bioactivity, and is one of the most widely used calcium phosphate materials. At present, the reported morphological structures of HA are mainly plate-like, fibrous, columnar, spherical, etc. Among them, HA porous microspheres have broad application prospects in the fields of drugs and catalyst carriers, ion adsorption materials, etc. due to their good fluidity, high specific surface area and unique pore structure.

[0003] In the prior art, the preparation methods of HA porous microspheres are mostly template method, hydrothermal method, chemical precipitation method and microemulsion method, etc. A Chinese invention patent with the authorization announcement date of April 24, 2018 and the authorization announcement number of CN 106115642 B discloses a large-sized hydroxyapatite porous microsphere material and a preparation method thereof. The method adopts a hydrothermal homogeneous precipitation method, and needs to add glutamic acid, an essential amino acid for the human body, to the reactants as a regulator of microsphere growth, and utilizes the adsorption and chelation of glutamic acid to effectively regulate the growth of HA and make it grow into microspheres.

[0004] The porous microsphere structure obtained by adding a template as an additive inevitably has additive residues, and the production process is relatively complicated, and the cost of raw materials is also increased accordingly. In addition, there is also a method of adding an organic active agent as an additive to prepare HA porous microspheres, but the method is environmentally friendly and has adverse effects on the biocompatibility and bioactivity of HA materials. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing hexagonal prism-assembled hydroxyapatite porous microspheres, so as to solve the problems of complicated production process, high raw material cost, and environmental unfriendliness in the existing solution using additives.

[0006] The second object of the present invention is to provide the application of the hydroxyapatite porous microspheres obtained by the above preparation method in drug carriers.

[0007] In order to achieve the above first purpose, the technical solution adopted by the present invention is:

[0008] A method for preparing hexagonal prism-assembled hydroxyapatite porous microspheres comprises the following steps: subjecting water-soluble calcium salt, dihydrogen pyrophosphate and urea to a hydrothermal reaction at 150-200° C.; the molar ratio of Ca to urea in the dihydrogen pyrophosphate and water-soluble calcium salt is 1:(3-5):(10-15).

[0009] The present invention is a pioneering invention, which uses water-soluble calcium salt, dihydrogen pyrophosphate and urea as raw materials to prepare hexagonal prism-assembled hydroxyapatite porous microspheres; the method does not use additives such as organic surfactants and template agents, and the obtained hydroxyapatite porous microspheres have high roundness and a loose and porous surface, which is conducive to high adsorption and high loading of the target object, and has broad application prospects in the fields of drug and catalyst carriers and ion adsorption materials.

[0010] Preferably, the concentration of urea in the hydrothermal reaction system is 0.10-0.25 mol / L.

[0011] Preferably, the hydrothermal reaction time is 6 to 15 hours.

[0012] Further preferably, the dihydrogen pyrophosphate is disodium dihydrogen pyrophosphate.

[0013] Further preferably, the water-soluble calcium salt is calcium nitrate or calcium chloride.

[0014] Preferably, the particle size of the hexagonal prism-assembled hydroxyapatite porous microspheres is 30 to 50 μm.

[0015] Preferably, the length of the hexagonal prism assembly unit of the hexagonal prism assembled hydroxyapatite porous microspheres is 0.5 to 3 μm, and the cross-sectional width is 0.1 to 1.5 μm.

[0016] In order to achieve the above second purpose, the technical solution adopted by the present invention is:

[0017] An application of the hydroxyapatite porous microspheres obtained by the above preparation method in drug carriers.

[0018] When the above-mentioned hydroxyapatite porous microspheres are used in drug carriers, the special surface structure constructed based on the hexagonal assembly units can effectively increase the loading amount of BSA, which has been proven by experiments and has good application prospects in high-efficiency drug carriers. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The X-ray diffraction pattern of the hexagonal prism-assembled HA porous microspheres prepared in Example 1 of the present invention;

[0020] Figure 2 This is a field emission scanning electron microscope image of the hexagonal prism-assembled HA porous microspheres prepared in Example 1 of the present invention;

[0021] Figure 3 for Figure 2 A partial magnified view of the surface;

[0022] Figure 4 This is a field emission scanning electron microscope image of the hexagonal prism-assembled HA porous microspheres prepared in Example 2 of the present invention;

[0023] Figure 5 This is a field emission scanning electron microscope image of hydroxyapatite prepared in Comparative Example 1;

[0024] Figure 6 This is a field emission scanning electron micrograph of the calcium phosphate agglomerates prepared in Comparative Example 2. DETAILED DESCRIPTION

[0025] The technical concept of the present invention is to prepare HA porous microspheres by using a non-additive hydrothermal homogeneous precipitation method; during the reaction, urea is gradually hydrolyzed to release OH - , providing driving force and alkaline environment for the subsequent nucleation and growth of crystals. The dihydrogen pyrophosphate ions decompose into orthophosphate ions in an alkaline environment and combine with calcium ions to serve as nucleation sites. At the same time, the dihydrogen pyrophosphate ions can be adsorbed on the HA crystal surface, complexed with calcium ions, and regulate the rate of crystal nucleation and crystal surface growth to form hydroxyapatite hexagonal prisms. The final result is hexagonal prism-assembled HA porous microspheres.

[0026] During the hydrothermal reaction, water-soluble calcium salt, dihydrogen pyrophosphate, and urea are preferably mixed in the form of a solution. For example, the water-soluble calcium salt can be made into an aqueous solution of 0.06 to 0.15 mol / L, the dihydrogen pyrophosphate can be made into an aqueous solution of 0.02 to 0.05 mol / L, and the urea can be made into an aqueous solution of 0.6 to 1.2 mol / L. The three aqueous solutions can be directly mixed to obtain a reaction system suitable for the hydrothermal reaction. After the hydrothermal reaction, the final product is obtained by solid-liquid separation, washing, and freeze-drying. The method is simpler as a whole, easy to operate, and the raw materials used are cheap and easy to obtain, and the cost is low. In addition, no toxic reagents are used in the preparation process, which is green and environmentally friendly.

[0027] Further, by controlling the concentration of each raw material in the hydrothermal reaction system, the reaction efficiency and stability can be improved.

[0028] The porous microspheres are self-assembled from hexagonal prisms and have a loose porous structure, which is conducive to high adsorption and high loading of the target and has good application prospects in terms of efficient drug carriers.

[0029] The present invention is further described below in conjunction with specific examples, but the present invention is not limited to the following examples. The methods used in the examples are conventional methods unless otherwise specified, and the reagents used are commercially available unless otherwise specified.

[0030] 1. Specific embodiment of the method for preparing the porous hydroxyapatite microspheres assembled by hexagonal prisms of the present invention

[0031] Example 1

[0032] The preparation method of the hexagonal prism-assembled hydroxyapatite porous microspheres of the present embodiment comprises the following steps: 100 mL of 0.15 mol / L calcium chloride, 100 mL of 0.05 mol / L disodium dihydrogen pyrophosphate and 50 mL of 1 mol / L urea aqueous solution are mixed in a beaker, stirred evenly and then transferred to a high-pressure reactor (the filling degree is 80%, and the final urea concentration obtained by directly adding the volumes of the three solutions is 0.2 mol / L), the reaction temperature is set to 180° C., the reaction time is 10 h, and finally the obtained white precipitate is centrifuged and washed, washed twice with deionized water, washed once with anhydrous ethanol, and then freeze-dried to obtain hexagonal prism-assembled HA porous microspheres.

[0033] Example 2

[0034] The preparation method of the hexagonal prism-assembled hydroxyapatite porous microspheres of the present embodiment comprises the following steps: 100 mL of 0.06 mol / L calcium nitrate, 100 mL of 0.02 mol / L disodium dihydrogen pyrophosphate and 50 mL of 0.6 mol / L urea aqueous solution are mixed in a beaker, stirred evenly and then transferred to a high-pressure reactor (the filling degree is 80%, and the final urea concentration obtained by directly adding the volumes of the three solutions is 0.12 mol / L), the reaction temperature is set to 150° C., the reaction time is 6 h, and finally the obtained white precipitate is centrifuged and washed, washed twice with deionized water, washed once with anhydrous ethanol, and then freeze-dried to obtain hexagonal prism-assembled HA porous microspheres.

[0035] Example 3

[0036] The preparation method of the hexagonal prism-assembled hydroxyapatite porous microspheres of the present embodiment comprises the following steps: 100 mL of 0.15 mol / L calcium chloride, 100 mL of 0.05 mol / L dipotassium dihydrogen pyrophosphate and 50 mL of 1.2 mol / L urea aqueous solution are mixed in a beaker, and after magnetic stirring for 0.5 h, the mixture is transferred to a high-pressure reactor (the filling degree is 80%, and the final urea concentration obtained by directly adding the volumes of the three solutions is 0.24 mol / L), the reaction temperature is set to 200° C., the reaction time is 15 h, and finally the obtained white precipitate is centrifuged and washed, washed twice with deionized water, washed once with anhydrous ethanol, and then freeze-dried to obtain hexagonal prism-assembled HA porous microspheres.

[0037] 2. Comparison

[0038] Comparative Example 1

[0039] This comparative example uses disodium hydrogen phosphate as a phosphorus source to prepare hydroxyapatite, and the specific operation is as follows:

[0040] 100 mL of 0.15 mol / L calcium chloride, 100 mL of 0.05 mol / L disodium hydrogen phosphate and 50 mL of 1 mol / L urea aqueous solution were mixed in a beaker, stirred evenly and then transferred to a high-pressure reactor (filling degree was 80%), the reaction temperature was set to 180°C, the reaction time was 10 h, and finally the obtained white precipitate was centrifuged and washed, washed twice with deionized water and once with anhydrous ethanol, and then freeze-dried to obtain flaky hydroxyapatite.

[0041] Comparative Example 2

[0042] In this comparative example, urea is not added during the preparation of hydroxyapatite, and the specific operation is as follows:

[0043] 100 mL of 0.15 mol / L calcium chloride and 100 mL of 0.05 mol / L sodium dihydrogen pyrophosphate aqueous solution were mixed in a beaker, stirred evenly and then transferred to a high-pressure reactor (filling degree was 80%), the reaction temperature was set to 180° C., the reaction time was 10 h, and finally the obtained white precipitate was centrifuged and washed, washed twice with deionized water and once with anhydrous ethanol, and then freeze-dried to obtain calcium phosphate aggregates.

[0044] 3. Experimental Example 1

[0045] Experimental Example 1 XRD Analysis

[0046] The product obtained in Example 1 was subjected to XRD analysis, and its X-ray diffraction pattern was as follows: Figure 1 As shown. Figure 1 It can be seen that the phase of the product is hydroxyapatite HA (Ca 10 (PO4)6(OH)2).

[0047] Experimental Example 2 Field Emission Scanning Electron Microscope Analysis

[0048] The field emission scanning electron microscope image of the product obtained in Example 1 is as follows Figure 2 As shown by Figure 2 It can be seen that the microspheres are composed of hexagonal prisms and have a loose and porous surface. Figure 3 As shown, its hexagonal assembly unit structure can be clearly seen. The particle size of the porous microspheres obtained in Example 1 is 30-50 μm, the length of its hexagonal assembly unit is 0.5-3 μm, and the cross-sectional width is 0.1-1.5 μm.

[0049] The field emission scanning electron microscope image of the product obtained in Example 2 is as follows Figure 4As shown, it shows structural features that are basically consistent with those of Example 1. The particle size, length and cross-sectional width of the porous microspheres obtained in Example 2 and Example 3 are basically the same as those in Example 1.

[0050] Comparative Example 1: Under the same conditions as in Example 1, when the phosphorus source was replaced with another common phosphorus source, disodium hydrogen phosphate, a flaky hydroxyapatite HA ( Figure 5 ), without microsphere structure. This indicates that dihydrogen pyrophosphate plays an important role in the formation of hexagonal prism-assembled HA porous microspheres.

[0051] Comparative Example 2: Under the same conditions as in Example 1, when no urea was added, irregular agglomerates ( Figure 6 ), without microsphere structure. This indicates that urea also plays an important role in the formation of hydroxyapatite microsphere structure, and it and dihydrogen pyrophosphate jointly regulate the formation of hexagonal self-assembled microsphere structure.

[0052] Experimental Example 3 Application in drug carrier

[0053] The hydroxyapatite HA obtained in each embodiment and comparative example is used as a carrier to load bovine serum albumin (BSA), as described below:

[0054] Weigh 0.2 g of the hexagonal HA porous microspheres prepared in Example 1, the HA flakes prepared in Comparative Example 1, and the calcium phosphate aggregates prepared in Comparative Example 2, respectively, and soak them in 10 mL of 10 mg / mL bovine serum albumin (BSA) solution, and shake them in a constant temperature water bath at 37 ° C for 24 hours. After soaking, the BSA solution containing the sample was centrifuged at 3000 rpm for 3 minutes. Take the supernatant, measure the absorbance of the remaining BSA therein, and obtain the corresponding BSA concentration C.

[0055] BSA loading amount DLA (m BSA / m HA ) is calculated by the following formula:

[0056]

[0057] In the formula, m BSA / m HA represents the loading amount of BSA; C0 represents the BSA concentration in the solution at the beginning of the reaction; C represents the BSA concentration in the supernatant after the reaction is completed; V represents the volume of the BSA solution at the beginning of the reaction; M represents the sample mass at the beginning of the reaction.

[0058] Finally, the BSA loading amounts of the three samples were 102.5 mg / g, 31.6 mg / g, and 11.2 mg / g, respectively.

[0059] It can be seen from the above experiments that the hexagonal self-assembled HA porous microspheres constructed by the method of the present invention have a loose porous structure on the surface, which significantly improves the loading capacity of BSA and is a highly efficient drug carrier.

[0060] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing hexagonal prism-assembled hydroxyapatite porous microspheres, characterized in that: The following steps are involved: A water-soluble calcium salt, dihydrogen pyrophosphate and urea are subjected to a hydrothermal reaction at 150-200° C. The molar ratio of Ca to urea in the dihydrogen pyrophosphate and water-soluble calcium salt is 1:(3-5):(10-15).

2. The method for preparing hexagonal prism-assembled hydroxyapatite porous microspheres according to claim 1, characterized in that: The concentration of urea in the hydrothermal reaction system is 0.10-0.25 mol / L.

3. The method for preparing hexagonal prism-assembled hydroxyapatite porous microspheres according to claim 1, characterized in that: The hydrothermal reaction time is 6 to 15 hours.

4. The method for preparing hexagonal prism-assembled hydroxyapatite porous microspheres according to claim 1, 2 or 3, characterized in that: The dihydrogen pyrophosphate is disodium dihydrogen pyrophosphate.

5. The method for preparing hexagonal prism-assembled hydroxyapatite porous microspheres according to claim 1, 2 or 3, characterized in that: The water-soluble calcium salt is calcium nitrate or calcium chloride.

6. The method for preparing hexagonal prism-assembled hydroxyapatite porous microspheres according to claim 1, characterized in that: The particle size of the hexagonal prism-assembled hydroxyapatite porous microspheres is 30 to 50 μm.

7. The method for preparing hexagonal prism-assembled hydroxyapatite porous microspheres according to claim 1, characterized in that: The length of the hexagonal prism assembly unit of the hexagonal prism assembly hydroxyapatite porous microsphere is 0.5-3 μm, and the cross-sectional width is 0.1-1.5 μm.

8. Use of the hydroxyapatite porous microspheres obtained by the preparation method according to any one of claims 1 to 7 as a drug carrier.

Images