Hydroxyapatite microsphere injection with good water dispersibility and preparation method

Abstract

The application belongs to the technical field of material preparation, and particularly relates to a hydroxyapatite microsphere injection with good water dispersibility and a preparation method. The application first provides a hydroxyapatite microsphere injection with good water dispersibility, which comprises a solvent and double-group modified hydroxyapatite microspheres; the surface of the double-group modified hydroxyapatite microspheres comprises amino-carboxyl groups connected through electrostatic adsorption. The double-group modified hydroxyapatite microspheres can form a system with good dispersibility in an aqueous solution, and can keep the characteristics of uniform dispersion and non-sedimentation for a long time.

Description

[0001] Divisional application

[0002] This application is a divisional application based on the Chinese invention patent application with application number CN202410733307.6, application date June 6, 2024, and invention name “Preparation and Application of Hydroxyapatite Microsphere Composite Injection”. Technical Field

[0003] The invention belongs to the technical field of material preparation, and particularly relates to a hydroxyapatite microsphere injection with good water dispersibility and a preparation method thereof. Background Art

[0004] Hydroxyapatite (HAp), also known as hydroxyapatite, is a natural apatite mineral with the molecular formula of Ca5(PO4)3(OH). 10 (PO4)6(OH)2 represents a crystal structure consisting of two molecules. HAp is currently widely used in medical aesthetics due to its high biocompatibility, low risk of allergic reactions after injection, and ability to stimulate collagen regeneration after subcutaneous injection. However, HAp itself has some physical and chemical performance deficiencies when prepared as an injectable formulation, necessitating modification or engineering.

[0005] Most current mainstream hydroxyapatite injection products utilize modified or modified hydroxyapatite. A common example is the combination of sodium carboxymethylcellulose (CMC-Na) gel and hydroxyapatite microspheres. The CMC-Na gel disperses the hydroxyapatite microspheres, increasing the viscosity of the injection solution, improving their sedimentation, and preventing large particle aggregation. However, most hydroxyapatite microspheres are unevenly dispersed within the gel matrix. Following injection, they may aggregate and migrate subcutaneously or within the body, resulting in symptoms such as a foreign body sensation or granulomas at the injection site. Patents CN104853742A and CN104703582A disclose homogenizing hyaluronic acid, a biomacromolecule, with HAp microspheres to produce a viscoelastic mixed injectable gel. However, this technique involves simply physically mixing the hydroxyapatite microspheres with the gel, making it difficult to achieve a truly uniform distribution of the hydroxyapatite microspheres within the gel matrix. There are also some schemes to directly modify single chemical groups on the surface of hydroxyapatite microspheres. However, the modified hydroxyapatite prepared by this strategy is difficult to maintain long-term stability and dispersibility in aqueous solution.

[0006] In summary, it is necessary to propose new improvement strategies to address the shortcomings of existing technologies. Summary of the Invention

[0007] The object of the present invention is to provide a hydroxyapatite microsphere injection with good water dispersibility and a preparation method, so as to partially solve or alleviate the above-mentioned deficiencies in the prior art. The present invention specifically adopts the following technical solutions.

[0008] The first aspect of the present invention is to provide a hydroxyapatite microsphere injection with good water dispersibility.

[0009] A hydroxyapatite microsphere injection with good water dispersibility comprises a solvent and double-group-modified hydroxyapatite microspheres; the surfaces of the double-group-modified hydroxyapatite microspheres contain amino-carboxyl groups (carboxyl groups connected to amino groups) connected by electrostatic adsorption; the double-group-modified hydroxyapatite microspheres are obtained by sequentially performing amino modification and carboxyl modification on a hydroxyapatite microsphere matrix with a particle size range of 10-60 μm; the hydroxyapatite microsphere matrix reacts with a silane coupling agent providing amino groups at a mass ratio of 10:1 to obtain first modified hydroxyapatite microspheres; and the first modified hydroxyapatite microspheres react with sodium carboxymethyl cellulose providing carboxyl groups at a mass ratio of 5:1 or 6:1 to obtain the double-group-modified hydroxyapatite microspheres.

[0010] Preferably, the particle size of the hydroxyapatite microsphere matrix is ​​in the range of 30-50 μm.

[0011] Furthermore, the silane coupling agent providing amino groups includes γ-aminopropyltrimethoxysilane, γ-aminopropyltriethoxysilane or γ-aminopropylmethyldiethoxysilane.

[0012] Optionally, the silane coupling agent providing amino groups may further include N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane or γ-methacryloxypropyltrimethoxysilane.

[0013] It can be understood that the present invention can also provide hydroxyapatite microspheres with good water dispersibility, wherein the hydroxyapatite microspheres are double-group modified hydroxyapatite microspheres; the surfaces of the double-group modified hydroxyapatite microspheres contain amino-carboxyl groups connected by electrostatic adsorption (carboxyl groups connected to amino groups); the double-group modified hydroxyapatite microspheres are amino-modified and carboxyl-modified in sequence on a hydroxyapatite microsphere matrix with a particle size range of 10-60 μm; the hydroxyapatite microsphere matrix reacts with a silane coupling agent providing an amino group in a mass ratio of 10:1 to obtain a first modified hydroxyapatite microsphere; the first modified hydroxyapatite microspheres react with sodium carboxymethyl cellulose providing a carboxyl group in a mass ratio of 5:1 or 6:1 to obtain the double-group modified hydroxyapatite microspheres.

[0014] The hydroxyapatite microspheres with good water dispersibility are used in the preparation of medical injections. The injections are aqueous dispersions.

[0015] Another aspect of the present invention is to provide a method for preparing hydroxyapatite microsphere injection with good water dispersibility.

[0016] The preparation method of the hydroxyapatite microsphere injection comprises the following steps:

[0017] S01: weighing a calcium salt and adding it to an aqueous solution containing a morphology-forming agent, stirring and dissolving the morphology-forming agent to obtain a calcium-containing reaction solution, wherein the morphology-forming agent includes citric acid, polyaspartic acid, sodium polyacrylate or polyethylene glycol;

[0018] S02: preparing a phosphate solution and mixing it with the calcium-containing reaction solution prepared in S01, adjusting the pH of the reaction system to be greater than 7.0, and the molar ratio of calcium to phosphate in the reaction system to be 1.5-1.7:1 (preferably 1.68±0.2:1);

[0019] S03: placing the reaction system of S02 in a reactor and reacting at 120-160°C for 8-15 hours (or at 100-180°C for 5-24 hours), centrifuging, and collecting the precipitate after centrifugation to obtain hydroxyapatite microspheres;

[0020] S04: adding the hydroxyapatite microspheres obtained in S03 to an acidic solution with a pH of 5.5 to fully dissolve them, then adding an ethanol solution of a silane coupling agent, wherein the mass ratio of the hydroxyapatite microspheres to the ethanol solution of the silane coupling agent is 10:1, adjusting the pH of the reaction system to a pH of 9.0, stirring to fully react, centrifuging, and collecting the precipitated material after centrifugation to obtain amino-modified hydroxyapatite microspheres; the silane coupling agent includes γ-aminopropyltrimethoxysilane, γ-aminopropyltriethoxysilane, or γ-aminopropylmethyldiethoxysilane;

[0021] S05: dissolving and dispersing the amino-modified hydroxyapatite microspheres obtained in S04 in purified water, and then adding sodium carboxymethyl cellulose, wherein the mass ratio of the amino-modified hydroxyapatite microspheres to the sodium carboxymethyl cellulose is 5:1 or 6:1; adjusting the pH of the reaction system to 8.5, stirring at room temperature to fully react, and after the reaction is completed, dialyzing and freeze-drying for 48-72 hours to obtain the modified hydroxyapatite microspheres with double groups;

[0022] S06: adding a solvent to dissolve the modified hydroxyapatite microspheres with double groups to obtain the hydroxyapatite microsphere injection.

[0023] In some preferred embodiments, the reaction in the reactor in S03 is carried out at 120° C. for 15 h, 150° C. for 10 h, or 160° C. for 8 h.

[0024] In some embodiments, the solvent is an aqueous solution, but solvents commonly used in the art for preparing aqueous dispersants can also be used.

[0025] Furthermore, the calcium salt includes calcium carbonate, calcium chloride, calcium nitrate or calcium bicarbonate.

[0026] Furthermore, the phosphate includes potassium phosphate, sodium phosphate, disodium hydrogen phosphate or dipotassium hydrogen phosphate.

[0027] Furthermore, the acidic solution includes glacial acetic acid solution or hydrochloric acid solution.

[0028] Furthermore, the means for fully dissolving the hydroxyapatite microspheres in the acidic solution with a pH of 5.5 in S03 includes ultrasound and homogenization.

[0029] Furthermore, after the reaction is completed, the dialysis operation is performed using distilled water; specifically, the dialysis is performed multiple times using distilled water.

[0030] Furthermore, the pH of the reaction system was adjusted to 8.0 in S02.

[0031] The present invention can also provide a method for preparing digroup-modified hydroxyapatite microspheres, which is specifically as follows.

[0032] A method for preparing digroup-modified hydroxyapatite microspheres comprises the following steps:

[0033] S01: weighing a calcium salt and adding it to an aqueous solution containing a morphology-forming agent, stirring and dissolving the morphology-forming agent to obtain a calcium-containing reaction solution, wherein the morphology-forming agent includes citric acid, polyaspartic acid, sodium polyacrylate or polyethylene glycol;

[0034] S02: preparing a phosphate solution and mixing it with the calcium-containing reaction solution prepared in S01, adjusting the pH of the reaction system so that the pH value is greater than 7.0, and the molar ratio of calcium to phosphate in the reaction system is 1.5-1.7:1;

[0035] S03: placing the reaction system of S02 into a reactor and reacting at 120-160°C for 8-15 hours, centrifuging, and collecting the precipitated material to obtain hydroxyapatite microspheres;

[0036] S04: adding the hydroxyapatite microspheres obtained in S03 to an acidic solution with a pH of 5.5 to fully dissolve them, then adding an ethanol solution of a silane coupling agent, wherein the mass ratio of the hydroxyapatite microspheres to the ethanol solution of the silane coupling agent is 10:1, adjusting the pH of the reaction system to a pH of 9.0, stirring to fully react, centrifuging, and collecting the precipitated material after centrifugation to obtain amino-modified hydroxyapatite microspheres; the silane coupling agent includes γ-aminopropyltrimethoxysilane, γ-aminopropyltriethoxysilane, or γ-aminopropylmethyldiethoxysilane;

[0037] S05: dissolving and dispersing the amino-modified hydroxyapatite microspheres obtained in S04 with purified water, and then adding sodium carboxymethyl cellulose, wherein the mass ratio of the amino-modified hydroxyapatite microspheres to the sodium carboxymethyl cellulose is 5:1 or 6:1; adjusting the pH of the reaction system to 8.5, stirring at room temperature for sufficient reaction, and after the reaction is completed, dialyzing and freeze-drying for 48-72 hours to obtain the modified hydroxyapatite microspheres with double groups.

[0038] Beneficial technical effects:

[0039] The present invention first provides a hydroxyapatite microsphere injection with good water dispersibility, which is mainly composed of a double-group modified hydroxyapatite microsphere. The double-group modified hydroxyapatite microsphere can form a well-dispersible system in an aqueous solution and maintain the property of uniform dispersion for a long time without sedimentation. Experiments have shown that the double-group modified hydroxyapatite microspheres provided by the present invention only show a small amount of precipitation after being dispersed in an aqueous solution for 5 days, compared with the single-group modified hydroxyapatite microspheres, while the single-group modified hydroxyapatite microspheres show a large amount of precipitation. Figure 1 As can be seen in the figure, the surface of the double-group-modified hydroxyapatite microspheres has amino-carboxyl groups connected by electrostatic adsorption (i.e., the surface of the microspheres is connected to amino groups, and the amino groups are connected to carboxyl groups). Based on this, the highly water-dispersible hydroxyapatite microsphere injection provided by the present invention can reduce the nodule phenomenon caused by particle migration and aggregation after filling due to the high dispersibility of the double-group-modified microspheres.

[0040] The present invention also provides a corresponding preparation method. A morphology-forming agent (also called a morphology-controlling agent) is first used to induce a phosphate solution containing calcium ions to transform and crystallize toward a calcium-phosphate mineral, forming hydroxyapatite microspheres. The modified hydroxyapatite is then linked to donor groups via electrostatic adsorption to form composite microspheres. This method utilizes a hydrothermal process, with the vast majority of materials participating in the reaction in liquid form, and the reaction conditions are generally mild. This results in a simple, convenient preparation process that can be standardized and controlled, making it suitable for industrial scale-up. Without the introduction of toxic crosslinking agents, the electrostatic adsorption between groups is utilized to achieve uniform and prolonged dispersion of the hydroxyapatite microspheres in an aqueous solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the embodiments or the description of the prior art. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the various elements or parts are not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without inventive work.

[0042] Figure 1 A reaction mechanism diagram of one embodiment of the present invention;

[0043] Figure 2 This is a process flow chart of one embodiment of the present invention;

[0044] Figure 3 This is a particle size distribution diagram of modified hydroxyapatite with amino and carboxylic acid groups prepared in one embodiment of the present invention;

[0045] Figure 4 This is a scanning electron micrograph of modified hydroxyapatite with amino and carboxylic acid groups prepared in one embodiment of the present invention (scale bar: 30 μm);

[0046] Figure 5 This is a diagram showing the liquid dispersion effects of different samples tested in one embodiment of the present invention after 48 hours;

[0047] Figure 6 This is a diagram showing the liquid dispersion effects of different samples tested in one embodiment of the present invention after 5 days. DETAILED DESCRIPTION

[0048] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0049] As used herein, "and / or" includes any and all combinations of one or more of the associated listed items.

[0050] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0051] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0052] In this specification, certain embodiments may be disclosed in a format that is within a range. It should be understood that this description of "within a range" is merely for convenience and brevity and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within this range. For example, the description of a range of 1-6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5, and 6. Regardless of the breadth of the range, the above rules apply.

[0053] Example 1

[0054] This embodiment provides an example of a method for preparing digroup-modified hydroxyapatite composite microspheres.

[0055] Step 1: Preparation of hydroxyapatite microspheres (HA-MP).

[0056] S01: Weigh a certain amount of calcium salt and dissolve it in an aqueous solution containing a morphology-forming agent (such as citric acid, polyaspartic acid, sodium polyacrylate, and polyethylene glycol), stirring to dissolve it, to form a calcium-containing reaction solution. The calcium salt can be selected from calcium carbonate, calcium chloride, calcium nitrate, or calcium bicarbonate. The mass ratio of the calcium salt to the aqueous solution containing the morphology-forming agent is approximately 10:1 to 4:1.

[0057] S02: Weigh a certain amount of phosphate and dissolve it in purified water to prepare a phosphorus solution. The phosphate can be selected from potassium phosphate, sodium phosphate, disodium hydrogen phosphate, or dipotassium hydrogen phosphate.

[0058] S03: Mix the calcium-containing reaction solution and the phosphorus solution to adjust the calcium:phosphate molar ratio to 1.5-1.7:1 (optimal 1.68±0.2:1), and adjust the pH of the reaction system to be greater than or equal to 7.0.

[0059] S04: Stir the reaction solution until a white precipitate appears in the reaction system, pour the reaction system into a reactor, and react at 100-180° C. for 5-24 hours.

[0060] S05: After the reaction is completed, the reactor is taken out and cooled, and the white precipitate at the lower layer is collected by centrifugation and washed to obtain hydroxyapatite microspheres, which are the first reaction product.

[0061] Step 2: Preparation of the first modified hydroxyapatite microspheres (HA-MP-NH2).

[0062] S01: Dissolve a silane coupling agent in a 95% ethanol aqueous solution and stir at room temperature to dissolve. The silane coupling agent can be selected from γ-aminopropyltriethoxysilane (A-1100), γ-aminopropyltrimethoxysilane (A-1110), N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane (A-1120), γ-methacryloyloxypropyltrimethoxysilane (A-174), or γ-aminopropylmethyldiethoxysilane (A-2100).

[0063] S02: adding hydroxyapatite microsphere powder to an acid solution with a pH value of about 5.0-6.0, homogenizing and mixing thoroughly to obtain a hydroxyapatite microsphere solution. The acid solution can be selected from acetic acid, hydrochloric acid, etc.

[0064] S03: Pour the hydroxyapatite microsphere solution into the silane coupling agent solution so that the mass of the hydroxyapatite microspheres is 5-10 times the mass of the silane coupling agent.

[0065] S04: Adjust the pH value of the reaction solution to about 7.0-9.0, and stir the reaction for 3-5 hours.

[0066] S05: Collect the white precipitate in the lower layer by centrifugation, wash and dry it to obtain modified hydroxyapatite with amino groups, which is the second reaction product.

[0067] Step 3: Preparation of the second modified hydroxyapatite microspheres (HA-MP-NH2 / CMC).

[0068] S01: Dissolve the second reaction product in purified water and fully disperse it.

[0069] S02: Weigh a certain amount of sodium carboxymethyl cellulose (CMC-Na) and pour it into the aqueous solution of the second reaction product so that the mass ratio of the second reaction product to sodium carboxymethyl cellulose is 5:1-10:1, and mix thoroughly.

[0070] S03: Adjusting the pH of the reaction solution to make the pH value of the reaction solution approximately greater than or equal to 8.0.

[0071] S04: The reaction solution was further stirred at room temperature for 12-48 hours.

[0072] S05: After the reaction is completed, the alkali is removed by dialysis and the mixture is freeze-dried to obtain modified hydroxyapatite with amino and carboxylic acid groups, which is the final product of the reaction.

[0073] The reaction mechanism diagram of this embodiment is shown in Figure 1 .

[0074] Example 2

[0075] This embodiment provides an example of a specific preparation method of double-group modified hydroxyapatite composite microspheres.

[0076] Step 1: Preparation of hydroxyapatite microspheres (HA-MP).

[0077] Dissolve 1.67g of calcium carbonate in 20mL of purified water containing 150μg / mL citric acid and stir magnetically at 100rpm. Once fully dissolved, add 3.58g of sodium phosphate and stir to mix thoroughly. Adjust the pH of the reaction solution to approximately 8.0 with 0.1M NaOH until a white precipitate appears in the reaction vessel. Pour the resulting precipitate into a tetrafluoroethylene liner in a reactor, which is then heated at 150°C for 10 hours. After the reaction time is up, remove the reactor and cool it to room temperature. Collect the white precipitate in the liner by centrifugation at 2000rpm and wash it three times with purified water and three times with anhydrous ethanol. Finally, collect the white precipitate and dry it at 60°C for 12 hours to obtain hydroxyapatite microspheres, the first reaction product.

[0078] Step 2: Preparation of the first modified hydroxyapatite microspheres (HA-MP-NH2).

[0079] Weigh 0.2 g of the first reaction product and dissolve it in 40 mL of glacial acetic acid at pH 5.5. After 20 kHz sonication for 15 minutes, homogenize the mixture at 10,000 rpm for 2 minutes to fully dissolve the hydroxyapatite microspheres. Then, add a pre-prepared ethanol solution containing 0.02 g of γ-aminopropyltrimethoxysilane (A-1110). After thorough stirring, adjust the pH of the reaction solution to approximately 9.0 and allow the mixture to react for another 4 hours with further stirring. The reaction product is centrifuged, and the precipitate is collected and appropriately washed to yield amino-modified hydroxyapatite, the second reaction product.

[0080] Step 3: Preparation of the second modified hydroxyapatite microspheres (HA-MP-NH2 / CMC).

[0081] Weigh 0.5 g of the second reaction product and dissolve it in 40 mL of purified water. Use a homogenizer and ultrasound to evenly disperse it. Add 20 mL of an aqueous solution containing 0.1 g of sodium carboxymethylcellulose (CMC-Na) and stir to mix thoroughly. Adjust the pH of the reaction solution to approximately 8.5, and stir at room temperature for 24 hours. After completion of the reaction, dialyze against distilled water (using a 3500 Da dialysis bag) for 24 hours, changing the water four times. The reaction product is then freeze-dried in a vacuum for 48-72 hours to obtain modified hydroxyapatite with amino and carboxylic acid groups, the final product.

[0082] It should be understood that the specific preparation method shown in this embodiment is only an example and not a limitation.

[0083] The process flow chart of this embodiment is shown in Figure 2 The particle size distribution and scanning electron microscopy images of the modified hydroxyapatite with amino and carboxylic acid groups prepared in this example are shown in Figure 3 and Figure 4 . Figure 3 As can be seen, the particle size of the modified hydroxyapatite with amino and carboxylic acid groups prepared in this example ranges from approximately 10 to 60 μm. The present invention further prefers microspheres with a particle size range of 30 to 50 μm for use in the preparation of medical injections. This is because microspheres with a particle size range of 30 to 50 μm can better avoid being phagocytosed by immune cells in the body.

[0084] Comparative Example 1

[0085] This comparative example provides a modified hydroxyapatite microsphere with only carboxylic acid groups and an example of its preparation.

[0086] Weigh 0.5 g of the first reaction product from Example 2 and dissolve it in 40 mL of purified water. Use a homogenizer and ultrasound to disperse it evenly. Then, add 20 mL of an aqueous solution containing 0.1 g of CMC-Na and stir to mix thoroughly. Adjust the pH of the reaction solution to 8.5, and stir at room temperature for 24 hours. After the reaction is complete, dialyze the solution with distilled water for 24 hours (using a 3500 Da dialysis bag), changing the water four times. The reaction product is then freeze-dried in vacuo for 48-72 hours to obtain modified hydroxyapatite microspheres bearing only carboxylic acid groups.

[0087] Comparative Example 2

[0088] This comparative example provides a modified hydroxyapatite microsphere with only amino groups and an example of its preparation.

[0089] 0.2 g of the first reaction product from Example 2 was weighed and dissolved in 40 mL of glacial acetic acid solution at pH 5.5. After 20 kHz ultrasonication for 15 minutes, the mixture was homogenized at 10,000 rpm for 2 minutes to fully dissolve the hydroxyapatite microspheres. Subsequently, a previously prepared ethanol solution containing 0.02 g of γ-aminopropyltrimethoxysilane (A-1110) was added. After thorough stirring, the pH of the reaction solution was adjusted to approximately 9.0, and the mixture was stirred and reacted for another 4 hours. The reaction product was centrifuged, and the precipitate was collected and appropriately washed to obtain modified hydroxyapatite microspheres containing only amino groups.

[0090] Comparative Example 3

[0091] This comparative example provides an unmodified hydroxyapatite microsphere and a preparation example.

[0092] Dissolve 1.67g of calcium carbonate in 20mL of purified water containing 150μg / mL citric acid with magnetic stirring at 100rpm. Once fully dissolved, add 3.58g of sodium phosphate and stir to mix thoroughly. Adjust the pH of the reaction solution to approximately 8.0 with 0.1M NaOH until a white precipitate appears in the reaction vessel. Pour the resulting precipitate into a tetrafluoroethylene liner in a reactor, which is then heated at 150°C for 10 hours. After the reaction time is up, remove the reactor and cool it to room temperature. Collect the white precipitate in the liner by centrifugation at 2000rpm and wash it three times with purified water and three times with anhydrous ethanol. Finally, collect the white precipitate and dry it at 60°C for 12 hours to obtain unmodified hydroxyapatite microspheres.

[0093] Example 4

[0094] Weigh the samples prepared in Example 2 and Comparative Examples 1-3, dissolve them in 5 mL of aqueous solution, and observe the sedimentation of the samples in the liquid within 48 h and 5 days, as shown in Table 1 and Figure 5 、 Figure 6 . Figure 6 The results show that after 5 days, after a dispersion test compared to Comparative Example 1, the sample of Example 2 was still homogeneously dispersed in the solution containing CMC-Na, while the sample of Comparative Example 1 had some white precipitate aggregated at the bottom of the test tube. This indicates that the hydroxyapatite microspheres modified with two groups prepared by the preferred method of the present invention, when mixed with sodium carboxymethylcellulose, can effectively reduce the aggregation and precipitation of the microspheres in the solution. Therefore, after being prepared as an injection, it can effectively improve the nodular swelling and bulging phenomenon that occurs at the injection site of hydroxyapatite injection.

[0095] Table 1 Dispersion test of different sample solutions

[0096]

[0097] Conclusion: Unmodified hydroxyapatite microspheres and hydroxyapatite microspheres modified with only a single group began to precipitate after 6 hours, while the modified hydroxyapatite microspheres with amino and carboxylic acid groups prepared by the present invention only showed a small amount of precipitation after 5 days, indicating that these microspheres meet the requirements for further preparation as medical injections.

[0098] Example 5

[0099] This embodiment provides another example of a specific preparation method of double-group modified hydroxyapatite composite microspheres.

[0100] Step 1: Preparation of hydroxyapatite microspheres (HA-MP).

[0101] Dissolve 1.67g of calcium chloride in 20mL of purified water containing 150μg / mL polyaspartic acid (PA) with magnetic stirring at 100rpm. Once fully dissolved, add 3.58g of potassium phosphate and stir to mix thoroughly. Adjust the pH of the reaction solution to approximately 8.0 with 0.1M NaOH until a white precipitate appears in the reaction vessel. Pour the precipitated reaction solution into a tetrafluoroethylene liner in a reactor, and heat the entire reactor at 160°C for 8h. After the reaction time is up, remove the reactor and cool it to room temperature. Collect the white precipitate in the liner by centrifugation at 2000rpm and wash it three times with purified water and three times with anhydrous ethanol. Finally, collect the white precipitate and dry it at 65°C for 12h to obtain hydroxyapatite microspheres, the first reaction product.

[0102] Step 2: Preparation of the first modified hydroxyapatite microspheres (HA-MP-NH2).

[0103] Weigh 0.2 g of the first reaction product and dissolve it in 40 mL of hydrochloric acid solution at pH 5.5. After 10 minutes of ultrasonication at 40 kHz, homogenize the mixture at 10,000 rpm for 2 minutes to fully dissolve the hydroxyapatite microspheres. Then, add a pre-prepared ethanol solution containing 0.02 g of γ-aminopropyltriethoxysilane (A-1100). After thorough stirring, adjust the pH of the reaction solution to approximately 9.0 and allow the mixture to react for another 4 hours with further stirring. The reaction product is centrifuged, and the precipitate is collected and appropriately washed to yield amino-modified hydroxyapatite, the second reaction product.

[0104] Step 3: Preparation of the second modified hydroxyapatite microspheres (HA-MP-NH2 / CMC).

[0105] Weigh 0.6 g of the second reaction product and dissolve it in 40 mL of purified water. Use a homogenizer and ultrasound to evenly disperse it. Add 20 mL of an aqueous solution containing 0.1 g of sodium carboxymethylcellulose (CMC-Na) and stir to mix thoroughly. Adjust the pH of the reaction solution to approximately 8.5, and stir at room temperature for 24 hours. After completion of the reaction, dialyze against distilled water (using a 3500 Da dialysis bag) for 24 hours, changing the water four times. The reaction product is then freeze-dried in a vacuum for 48-72 hours to obtain modified hydroxyapatite with amino and carboxylic acid groups, the final product.

[0106] It should be understood that the specific preparation method shown in this embodiment is only an example and not a limitation.

[0107] Example 6

[0108] This embodiment provides another example of a specific preparation method of double-group modified hydroxyapatite composite microspheres.

[0109] Step 1: Preparation of hydroxyapatite microspheres (HA-MP).

[0110] Dissolve 1.67g of calcium bicarbonate in 20mL of purified water containing 150μg / mL polyethylene glycol with magnetic stirring at 100rpm. Once fully dissolved, add 3.58g of dipotassium hydrogen phosphate and stir to mix thoroughly. Adjust the pH of the reaction solution to approximately 8.0 with 0.1M NaOH until a white precipitate appears in the reaction vessel. Pour the precipitated reaction solution into a tetrafluoroethylene liner in a reactor, and heat the entire reactor at 120°C for 15h. After the reaction time is up, remove the reactor and cool it to room temperature. Collect the white precipitate in the liner by centrifugation at 2000rpm and wash it three times with purified water and three times with anhydrous ethanol. Finally, collect the white precipitate and dry it at 60°C for 12h to obtain hydroxyapatite microspheres, the first reaction product.

[0111] Step 2: Preparation of the first modified hydroxyapatite microspheres (HA-MP-NH2).

[0112] Weigh 0.2 g of the first reaction product and dissolve it in 40 mL of glacial acetic acid at pH 5.5. After 10 minutes of ultrasonication at 40 kHz, homogenize the mixture at 10,000 rpm for 2 minutes to fully dissolve the hydroxyapatite microspheres. Then, add a pre-prepared ethanol solution containing 0.02 g of γ-aminopropylmethyldiethoxysilane (A-2100). After thorough stirring, adjust the pH of the reaction solution to approximately 9.0 and allow the mixture to react for another 4 hours with further stirring. The reaction product is centrifuged, and the precipitate is collected and appropriately washed to yield amino-modified hydroxyapatite, the second reaction product.

[0113] Step 3: Preparation of the second modified hydroxyapatite microspheres (HA-MP-NH2 / CMC).

[0114] Weigh 0.5 g of the second reaction product and dissolve it in 40 mL of purified water. Use a homogenizer and ultrasound to evenly disperse it. Add 20 mL of an aqueous solution containing 0.1 g of sodium carboxymethylcellulose (CMC-Na) and stir to mix thoroughly. Adjust the pH of the reaction solution to approximately 8.5, and stir at room temperature for 24 hours. After completion of the reaction, dialyze against distilled water (using a 3500 Da dialysis bag) for 24 hours, changing the water four times. The reaction product is then freeze-dried in a vacuum for 48-72 hours to obtain modified hydroxyapatite with amino and carboxylic acid groups, the final product.

[0115] It should be noted that, in this document, the terms "comprises," "includes," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus comprising a series of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or apparatus comprising the element.

[0116] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are protected by the present invention.

Claims

1. A method for preparing hydroxyapatite microsphere injection with good water dispersibility, characterized in that: The following steps are involved: S01: weighing a calcium salt and adding it to an aqueous solution containing a morphology-forming agent, stirring and dissolving the morphology-forming agent to obtain a calcium-containing reaction solution, wherein the morphology-forming agent includes citric acid, polyaspartic acid, sodium polyacrylate or polyethylene glycol; S02: preparing a phosphate solution and mixing it with the calcium-containing reaction solution prepared in S01, adjusting the pH of the reaction system so that the pH value is greater than 7.0, and the molar ratio of calcium to phosphate in the reaction system is 1.5-1.7:1; S03: placing the reaction system of S02 into a reactor and reacting at 120-160°C for 8-15 hours, centrifuging, and collecting the precipitated material to obtain hydroxyapatite microspheres; S04: adding the hydroxyapatite microspheres obtained in S03 to an acidic solution of pH 5.5 to fully dissolve them, then adding an ethanol solution of a silane coupling agent, wherein the mass ratio of the hydroxyapatite microspheres to the ethanol solution of the silane coupling agent is 10:1, adjusting the pH of the reaction system to a pH value of 9.0, stirring to fully react, centrifuging, and collecting the precipitated material after centrifugation to obtain amino-modified hydroxyapatite microspheres; the silane coupling agent includes γ-aminopropyltrimethoxysilane, γ-aminopropyltriethoxysilane, or γ-aminopropylmethyldiethoxysilane; and the acidic solution includes glacial acetic acid solution or hydrochloric acid solution; S05: dissolving and dispersing the amino-modified hydroxyapatite microspheres obtained in S04 in purified water, and then adding sodium carboxymethyl cellulose, wherein the mass ratio of the amino-modified hydroxyapatite microspheres to the sodium carboxymethyl cellulose is 5:1 or 6:1; adjusting the pH of the reaction system to 8.5, stirring at room temperature to fully react, and after the reaction is completed, dialyzing and freeze-drying for 48-72 hours to obtain the modified hydroxyapatite microspheres with double groups; S06: adding a solvent to dissolve the modified hydroxyapatite microspheres with double groups to obtain the hydroxyapatite microsphere injection; the particle size of the hydroxyapatite microsphere matrix is ​​in the range of 30-50 μm.

2. The preparation method according to claim 1, wherein The calcium salt includes calcium carbonate, calcium chloride, calcium nitrate or calcium bicarbonate.

3. The preparation method according to claim 1, wherein The phosphate includes potassium phosphate, sodium phosphate, disodium hydrogen phosphate or dipotassium hydrogen phosphate.

4. The preparation method according to claim 1, wherein In S03, the means for fully dissolving the hydroxyapatite microspheres by adding them to the acidic solution with a pH of 5.5 include ultrasound and homogenization.

5. The preparation method according to claim 1, wherein After the reaction is completed, the dialysis operation is performed using distilled water.

6. The preparation method according to claim 1, wherein In S02, the pH of the reaction system was adjusted to 8.0.

Images