Hydroxyapatite microsphere injection with good water dispersibility and preparation method thereof

Abstract

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. The invention firstly provides a hydroxyapatite microsphere injection with good water dispersibility, wherein the hydroxyapatite microsphere injection comprises a solvent and a double-group modified hydroxyapatite microsphere; the surface of the double-group modified hydroxyapatite microsphere comprises an amino-carboxyl group which is connected through an electrostatic adsorption effect. The double-group modified hydroxyapatite microspheres can form a system with good dispersity in an aqueous solution, and the characteristic of uniform dispersion can be kept for a long time without sedimentation.

Description

[0001] Divisional application 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 Use of Hydroxyapatite Microsphere Composite Injection”. Technical Field

[0002] 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

[0003] Hydroxyapatite (HAp), also known as hydroxyapatite, is a natural apatite mineral with a molecular formula of Ca5(PO4)3(OH). 10 (PO4)6(OH)2 represents a crystal structure composed of two molecules. HAp is widely used in the field of medical cosmetology because it has a high level of biocompatibility, is not prone to allergic reactions after injection into the human body, and can stimulate collagen regeneration after subcutaneous injection. However, HAp itself has some physical and chemical properties deficiencies when prepared as an injection, so it often needs to be modified or transformed.

[0004] Most of the current mainstream hydroxyapatite injection products use modified or modified hydroxyapatite, such as the combination of sodium carboxymethylcellulose (CMC-Na) gel and hydroxyapatite microspheres. CMC-Na gel is used to disperse hydroxyapatite microspheres, increase the viscosity of the injection solution, improve the sedimentation of hydroxyapatite microspheres, and avoid the formation of large particle agglomeration. However, most hydroxyapatite microspheres are unevenly dispersed in the gel matrix, and may aggregate and migrate subcutaneously or in vivo after injection, causing patients to experience foreign body sensation or granulomas at the injection site. Patents with publication numbers CN104853742A and CN104703582A disclose homogenizing hyaluronic acid, a biomacromolecule, with HAp microspheres to obtain a viscoelastic mixed injection gel. However, this technology is only a simple physical uniform mixing of hydroxyapatite microspheres and gel, and it is difficult to truly achieve uniform distribution of hydroxyapatite microspheres in the gel matrix. There are also some schemes to directly modify a single chemical group 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.

[0005] In summary, it is necessary to propose new improvement strategies to address the shortcomings of existing technologies. Summary of the invention

[0006] 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.

[0007] The first aspect of the present invention is to provide a hydroxyapatite microsphere injection with good water dispersibility.

[0008] A hydroxyapatite microsphere injection with good water dispersibility, the hydroxyapatite microsphere injection comprising a solvent and a double-group modified hydroxyapatite microsphere; the surface of the double-group modified hydroxyapatite microsphere comprises an amino-carboxyl group connected by electrostatic adsorption (carboxyl group connected to the amino group); the double-group modified hydroxyapatite microsphere is prepared 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 an amino group at a mass ratio of 10:1 to obtain a first modified hydroxyapatite microsphere; the first modified hydroxyapatite microsphere reacts with sodium carboxymethyl cellulose providing a carboxyl group at a mass ratio of 5:1 or 6:1 to obtain the double-group modified hydroxyapatite microsphere.

[0009] Preferably, the particle size of the hydroxyapatite microsphere matrix is ​​in the range of 30-50 μm.

[0010] Furthermore, the silane coupling agent providing amino groups includes γ-aminopropyltrimethoxysilane, γ-aminopropyltriethoxysilane or γ-aminopropylmethyldiethoxysilane.

[0011] Optionally, the silane coupling agent providing amino groups may also include N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane or γ-methacryloxypropyltrimethoxysilane.

[0012] It can be understood that the present invention can also provide a hydroxyapatite microsphere with good water dispersibility, wherein the hydroxyapatite microsphere is a double-group modified hydroxyapatite microsphere; the surface of the double-group modified hydroxyapatite microsphere contains amino-carboxyl groups connected by electrostatic adsorption (carboxyl groups connected to amino groups); the double-group modified hydroxyapatite microsphere is a hydroxyapatite microsphere matrix with a particle size range of 10-60 μm, which is amino-modified and carboxyl-modified in sequence; the hydroxyapatite microsphere matrix is ​​reacted with a silane coupling agent providing an amino group at a mass ratio of 10:1 to obtain a first modified hydroxyapatite microsphere; the first modified hydroxyapatite microsphere is reacted with sodium carboxymethyl cellulose providing a carboxyl group at a mass ratio of 5:1 or 6:1 to obtain the double-group modified hydroxyapatite microsphere.

[0013] The hydroxyapatite microspheres with good water dispersibility are used in the preparation of medical injections. The injections are aqueous dispersions.

[0014] Another aspect of the present invention is to provide a method for preparing hydroxyapatite microsphere injection with good water dispersibility.

[0015] The preparation method of the hydroxyapatite microsphere injection comprises the following steps: S01: weighing a calcium salt and adding it to an aqueous solution containing a morphology-forming agent, stirring and dissolving the solution 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 value 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 (preferably 1.68±0.2:1); S03: placing the reaction system of S02 in a reactor and reacting at 120-160°C for 8-15h (or at 100-180°C for 5-24h), centrifuging, and collecting the precipitate after centrifugation to obtain hydroxyapatite microspheres; S04: adding the hydroxyapatite microspheres obtained in S03 to an acidic solution of pH 5.5 to fully dissolve, 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 value of the reaction system to 9.0, stirring to fully react, centrifuging, collecting the precipitated material after centrifugation, and obtaining amino-modified hydroxyapatite microspheres; the silane coupling agent includes γ-aminopropyltrimethoxysilane, γ-aminopropyltriethoxysilane or γ-aminopropylmethyldiethoxysilane; 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 value 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; S06: adding a solvent to dissolve the modified hydroxyapatite microspheres with double groups to obtain the hydroxyapatite microsphere injection.

[0016] 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.

[0017] In some embodiments, the solvent is an aqueous solution, but any solvent commonly used in the art for preparing aqueous dispersants can also be used.

[0018] Furthermore, the calcium salt includes calcium carbonate, calcium chloride, calcium nitrate or calcium bicarbonate.

[0019] Furthermore, the phosphate includes potassium phosphate, sodium phosphate, disodium hydrogen phosphate or dipotassium hydrogen phosphate.

[0020] Furthermore, the acidic solution includes glacial acetic acid solution or hydrochloric acid solution.

[0021] Furthermore, the means for adding the hydroxyapatite microspheres into the acidic solution of pH 5.5 to fully dissolve them in S03 includes ultrasound and homogenization.

[0022] Furthermore, after the reaction is completed, the dialysis operation is performed using distilled water; specifically, the dialysis is performed multiple times using distilled water.

[0023] Further, the pH in S02 is adjusted so that the pH value of the reaction system is 8.0.

[0024] The present invention can also provide a method for preparing double-group modified hydroxyapatite microspheres, which is specifically as follows.

[0025] A method for preparing bi-group modified hydroxyapatite microspheres comprises the following steps: S01: weighing a calcium salt and adding it to an aqueous solution containing a morphology-forming agent, stirring and dissolving the solution 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 value 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 in a reaction kettle and reacting at 120-160° C. for 8-15 hours, centrifuging, and collecting the precipitate after centrifugation to obtain hydroxyapatite microspheres; S04: adding the hydroxyapatite microspheres obtained in S03 to an acidic solution of pH 5.5 to fully dissolve, 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 value of the reaction system to 9.0, stirring to fully react, centrifuging, collecting the precipitated material after centrifugation, and obtaining amino-modified hydroxyapatite microspheres; the silane coupling agent includes γ-aminopropyltrimethoxysilane, γ-aminopropyltriethoxysilane or γ-aminopropylmethyldiethoxysilane; 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 value 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.

[0026] Beneficial technical effects: The present invention first provides a hydroxyapatite microsphere injection with good water dispersibility, and the injection is mainly composed of a double-group modified hydroxyapatite microsphere. The double-group modified hydroxyapatite microsphere can form a system with good dispersibility in an aqueous solution, and maintain the characteristics of uniform dispersion for a long time without sedimentation. Experiments have shown that the double-group modified hydroxyapatite microsphere provided by the present invention only shows a small amount of precipitation after being dispersed in an aqueous solution for 5 days, compared with the single-group modified hydroxyapatite microsphere, while the single-group modified hydroxyapatite microsphere shows a large amount of precipitation. Figure 1 It can be seen that the surface of the hydroxyapatite microspheres modified with double groups has amino-carboxyl groups connected by electrostatic adsorption (i.e., the surface of the microspheres is connected to the amino group, and the amino group is connected to the carboxyl group). Based on this, after the hydroxyapatite microsphere injection with good water dispersibility provided by the present invention is injected into the body as an actual product, due to the high dispersibility of the double-group modified microspheres, the nodule phenomenon caused by particle migration and aggregation after filling can be reduced.

[0027] In addition, the present invention also provides a corresponding preparation method, firstly using a morphology forming agent (also called a morphology control agent) to induce a phosphate solution containing calcium ions to transform and crystallize in the direction of calcium phosphate minerals to form hydroxyapatite microspheres, and then connecting the modified hydroxyapatite with a donor group by means of electrostatic adsorption to form composite microspheres. The method of the present invention adopts a hydrothermal method, most of the materials participate in the reaction in the form of liquid, and the reaction conditions are generally mild, so the preparation process is simple, convenient, and can be standardized and controlled, suitable for industrial promotion, and without introducing a toxic cross-linking agent, the electrostatic adsorption between groups is used to achieve uniform and long-term dispersion of hydroxyapatite microspheres in an aqueous solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is 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 paying creative labor.

[0029] Figure 1 A reaction mechanism diagram of one embodiment of the present invention; Figure 2 A process flow chart of one embodiment of the present invention; 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; Figure 4 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); 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; 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

[0030] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are 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 work are within the scope of protection of the present invention.

[0031] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0032] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0033] 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.

[0034] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges 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.

[0035] Example 1 This embodiment provides an example of a method for preparing double-group modified hydroxyapatite composite microspheres.

[0036] Step 1: Preparation of hydroxyapatite microspheres (HA-MP).

[0037] S01: Weigh a certain amount of calcium salt and dissolve it in an aqueous solution containing a morphology forming agent (citric acid, polyaspartic acid, sodium polyacrylate, polyethylene glycol, etc.), stir to dissolve it, and form a calcium-containing reaction solution. The calcium salt can be selected from calcium carbonate, calcium chloride, calcium nitrate or calcium bicarbonate, etc. The mass ratio of the calcium salt to the aqueous solution containing the morphology forming agent is about 10:1~4:1.

[0038] 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.

[0039] S03: Mix the calcium-containing reaction solution and the phosphorus solution so that the molar ratio of calcium to phosphate is 1.5-1.7:1 (optimum is 1.68±0.2:1), and adjust the pH so that the pH value of the reaction system is greater than or equal to 7.0.

[0040] S04: Stir the reaction solution until white precipitate appears in the reaction system, pour the reaction system into a reaction kettle, and react at 100-180° C. for 5-24 hours.

[0041] 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, i.e., the first reaction product.

[0042] Step 2: Preparation of the first modified hydroxyapatite microspheres (HA-MP-NH2).

[0043] S01: Dissolve the silane coupling agent in 95% ethanol aqueous solution and stir at room temperature to dissolve it. The silane coupling agent can be selected from γ-aminopropyl triethoxysilane (A-1100), γ-aminopropyl trimethoxysilane (A-1110), N-(β-aminoethyl)-γ-aminopropyl trimethoxysilane (A-1120), γ-methacryloxypropyl trimethoxysilane (A-174) or γ-aminopropyl methyl diethoxysilane (A-2100) and the like.

[0044] S02: adding hydroxyapatite microsphere powder to an acid solution with a pH value of about 5.0-6.0, and fully homogenizing and mixing to obtain a hydroxyapatite microsphere solution. The acid solution can be selected from acetic acid, hydrochloric acid, and the like.

[0045] S03: Pour the hydroxyapatite microsphere solution into the silane coupling agent solution so that the mass of the hydroxyapatite microsphere is 5-10 times the mass of the silane coupling agent.

[0046] S04: Adjust the pH value of the reaction solution to about 7.0-9.0, and stir the reaction for 3-5 hours.

[0047] S05: Collect the white precipitate at the lower layer by centrifugation, wash and dry it to obtain modified hydroxyapatite with amino groups, i.e., the second reaction product.

[0048] Step 3: Preparation of the second modified hydroxyapatite microspheres (HA-MP-NH2 / CMC).

[0049] S01: Dissolve the second reaction product in purified water and disperse it fully.

[0050] 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 them thoroughly.

[0051] S03: Adjusting the pH of the reaction solution so that the pH value of the reaction solution is approximately greater than or equal to 8.0.

[0052] S04: The reaction solution was further stirred at room temperature for 12-48 hours.

[0053] S05: After the reaction is completed, the alkali is removed by dialysis and freeze-dried to obtain modified hydroxyapatite with amino and carboxylic acid groups, which is the final product of the reaction.

[0054] The reaction mechanism diagram of this example is shown in Figure 1 .

[0055] Example 2 This embodiment provides an example of a specific preparation method of double-group modified hydroxyapatite composite microspheres.

[0056] Step 1: Preparation of hydroxyapatite microspheres (HA-MP).

[0057] Dissolve 1.67g of calcium carbonate in 20mL of purified water containing 150μg / mL citric acid and stir magnetically at 100rpm. After it is fully dissolved, add 3.58g of sodium phosphate and stir to mix evenly. Use 0.1M NaOH to adjust the pH and control the pH value of the reaction solution to be about 8.0 until a white precipitate appears in the reaction vessel. Further pour the precipitated reaction solution into the tetrafluoroethylene liner of the reactor, and then heat the entire reactor at 150℃ for 10h. After the reaction time is reached, take out the reactor and cool it to room temperature, collect the white precipitate in the liner by centrifugation at a rate of 2000rpm, and centrifuge and wash it 3 times with purified water and anhydrous ethanol respectively. Finally, collect the white precipitate and dry it at 60℃ for 12h to obtain hydroxyapatite microspheres, i.e., the first reaction product.

[0058] Step 2: Preparation of the first modified hydroxyapatite microspheres (HA-MP-NH2).

[0059] Weigh 0.2g of the first reaction product and dissolve it in 40mL of glacial acetic acid solution at pH 5.5. After using 20kHz ultrasound for 15min, use a homogenizer to homogenize at 10000rpm for 2min to fully dissolve the hydroxyapatite microspheres. Then pour the ethanol solution containing 0.02g of γ-aminopropyltrimethoxysilane (A-1110) prepared in advance into it, stir it well, adjust the pH value of the reaction solution to about 9.0, and further stir and react for 4 hours. Centrifuge the reaction product, collect the precipitate and wash the precipitate appropriately to obtain modified hydroxyapatite with amino groups, i.e., the second reaction product.

[0060] Step 3: Preparation of the second modified hydroxyapatite microspheres (HA-MP-NH2 / CMC).

[0061] Weigh 0.5g of the second reaction product and dissolve it in 40mL of purified water. Use a homogenizer and ultrasound to disperse it evenly. Add 20mL of aqueous solution containing 0.1g of sodium carboxymethyl cellulose (CMC-Na) and stir to mix it thoroughly. Adjust the pH value of the reaction solution to about 8.5, and stir the reaction at room temperature for 24 hours. After the reaction is completed, dialyze with distilled water for 24 hours (using a 3500Da dialysis bag), change the water 4 times during the process, and vacuum freeze-dry the reaction product for 48-72 hours to obtain modified hydroxyapatite with amino and carboxylic acid groups, which is the final product of the reaction.

[0062] It should be understood that the specific preparation method shown in this embodiment is only an example and not a limitation.

[0063] The process flow chart of this embodiment is shown in Figure 2The particle size distribution diagram and scanning electron microscope image of the modified hydroxyapatite with amino and carboxylic acid groups prepared in this example are shown in Figure 3 and Figure 4 . Figure 3 It can be seen that the particle size range of the modified hydroxyapatite with amino and carboxylic acid groups prepared in this embodiment is about 10-60 μm, and the present invention further preferably selects microspheres with a particle size range of 30-50 μm for preparing medical injections. This is because when the particle size range of the microspheres is 30-50 μm, they can better avoid being phagocytosed by immune cells in the body.

[0064] Comparative Example 1 This comparative example provides a modified hydroxyapatite microsphere having only carboxylic acid groups and an example of its preparation.

[0065] Weigh 0.5g of the first reaction product in Example 2 and dissolve it in 40mL of purified water. Use a homogenizer and ultrasound to disperse it evenly, then add 20mL of an aqueous solution containing 0.1g of CMC-Na and stir to mix it thoroughly. Adjust the pH of the reaction solution to 8.5, and stir at room temperature for 24h. After the reaction is completed, dialyze with distilled water for 24h (using a 3500Da dialysis bag), change the water 4 times, and vacuum freeze-dry the reaction product for 48-72h to obtain modified hydroxyapatite microspheres with only carboxylic acid groups.

[0066] Comparative Example 2 This comparative example provides a modified hydroxyapatite microsphere with only amino groups and an example of its preparation.

[0067] Weigh 0.2 g of the first reaction product in Example 2 and dissolve it in 40 mL of glacial acetic acid solution at pH 5.5. After using 20 kHz ultrasound for 15 minutes, use a homogenizer to homogenize at 10000 rpm for 2 minutes to fully dissolve the hydroxyapatite microspheres. Then pour the ethanol solution containing 0.02 g of γ-aminopropyltrimethoxysilane (A-1110) prepared in advance into it, stir it well, adjust the pH value of the reaction solution to about 9.0, and further stir and react for 4 hours. The reaction product is centrifuged, the precipitate is collected and the precipitate is properly washed to obtain modified hydroxyapatite microspheres with only amino groups.

[0068] Comparative Example 3 This comparative example provides an unmodified hydroxyapatite microsphere and a preparation example.

[0069] Dissolve 1.67g of calcium carbonate in 20mL of purified water containing 150μg / mL citric acid and stir magnetically at 100rpm. After it is fully dissolved, add 3.58g of sodium phosphate and stir to mix evenly. Use 0.1M NaOH to adjust the pH and control the pH value of the reaction solution to about 8.0 until a white precipitate appears in the reaction vessel. Further pour the precipitated reaction solution into the tetrafluoroethylene liner of the reactor, and then heat the entire reactor at 150℃ for 10h. After the reaction time is reached, take out the reactor and cool it to room temperature, collect the white precipitate in the liner by centrifugation at a rate of 2000rpm, and centrifuge and wash it 3 times with purified water and anhydrous ethanol respectively. Finally, collect the white precipitate and dry it at 60℃ for 12h to obtain unmodified hydroxyapatite microspheres.

[0070] Example 4 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 Tables 1 and Figure 5 , Figure 6 . Figure 6 It is shown that after 5 days, after the dispersion test with comparative example 1, the sample of Example 2 is still homogeneously dispersed in the solution containing CMC-Na, while the sample of comparative example 1 has some white precipitates gathered at the bottom of the test tube. This shows that after the hydroxyapatite microspheres with two group modifications prepared by the preferred method of the present invention are mixed with sodium carboxymethyl cellulose, the microsphere aggregation and precipitation phenomenon of the solution can be effectively reduced. Therefore, after being prepared as an injection, the nodular swelling and bulging phenomenon of the hydroxyapatite injection at the injection site can be effectively improved.

[0071] Table 1 Dispersion test of different sample solutions 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 such microspheres meet the requirements for further preparation as medical injections.

[0072] Example 5 This embodiment provides another specific preparation method example of double-group modified hydroxyapatite composite microspheres.

[0073] Step 1: Preparation of hydroxyapatite microspheres (HA-MP).

[0074] Dissolve 1.67g of calcium chloride in 20mL of purified water containing 150μg / mL polyaspartic acid, and stir magnetically at 100rpm. After it is fully dissolved, add 3.58g of potassium phosphate and stir to mix evenly. Use 0.1M NaOH to adjust the pH and control the pH value of the reaction solution to about 8.0 until a white precipitate appears in the reaction vessel. Further pour the precipitated reaction solution into the tetrafluoroethylene liner of the reactor, and then place the entire reactor at a high temperature of 160℃ and heat for 8h. After the reaction time is reached, take out the reactor and cool it to room temperature, collect the white precipitate in the liner by centrifugation at a rate of 2000rpm, and centrifuge and wash it 3 times with purified water and anhydrous ethanol respectively, finally collect the white precipitate, dry it at 65℃ for 12h, and obtain hydroxyapatite microspheres, i.e., the first reaction product.

[0075] Step 2: Preparation of the first modified hydroxyapatite microspheres (HA-MP-NH2).

[0076] Weigh 0.2g of the first reaction product and dissolve it in 40mL of hydrochloric acid solution with pH 5.5. After using 40kHz ultrasound for 10min, use a homogenizer at 10000rpm for 2min to fully dissolve the hydroxyapatite microspheres. Then pour the ethanol solution containing 0.02g of γ-aminopropyltriethoxysilane (A-1100) prepared in advance into it, stir it thoroughly, adjust the pH value of the reaction solution to about 9.0, and further stir and react for 4 hours. Centrifuge the reaction product, collect the precipitate and wash the precipitate appropriately to obtain modified hydroxyapatite with amino groups, i.e., the second reaction product.

[0077] Step 3: Preparation of the second modified hydroxyapatite microspheres (HA-MP-NH2 / CMC).

[0078] Weigh 0.6g of the second reaction product and dissolve it in 40mL of purified water. Use a homogenizer and ultrasound to disperse it evenly. Add 20mL of aqueous solution containing 0.1g of sodium carboxymethyl cellulose (CMC-Na) and stir to mix it thoroughly. Adjust the pH value of the reaction solution to about 8.5, and stir at room temperature for 24 hours. After the reaction is completed, dialyze with distilled water for 24 hours (using a 3500Da dialysis bag), change the water 4 times during the process, and vacuum freeze-dry the reaction product for 48-72 hours to obtain modified hydroxyapatite with amino and carboxylic acid groups, which is the final product of the reaction.

[0079] It should be understood that the specific preparation method shown in this embodiment is only an example and not a limitation.

[0080] Example 6 This embodiment provides another specific preparation method example of double-group modified hydroxyapatite composite microspheres.

[0081] Step 1: Preparation of hydroxyapatite microspheres (HA-MP).

[0082] Dissolve 1.67g of calcium bicarbonate in 20mL of purified water containing 150μg / mL polyethylene glycol and stir magnetically at 100rpm. After it is fully dissolved, add 3.58g of potassium dihydrogen phosphate and stir to mix evenly. Use 0.1MNaOH to adjust the pH and control the pH value of the reaction solution to about 8.0 until a white precipitate appears in the reaction vessel. Further pour the precipitated reaction solution into the tetrafluoroethylene liner of the reactor, and then heat the entire reactor at 120℃ for 15h. After the reaction time is reached, take out the reactor and cool it to room temperature, collect the white precipitate in the liner by centrifugation at a rate of 2000rpm, and centrifuge and wash it 3 times with purified water and anhydrous ethanol respectively. Finally, collect the white precipitate and dry it at 60℃ for 12h to obtain hydroxyapatite microspheres, i.e., the first reaction product.

[0083] Step 2: Preparation of the first modified hydroxyapatite microspheres (HA-MP-NH2).

[0084] Weigh 0.2g of the first reaction product and dissolve it in 40mL of glacial acetic acid solution at pH 5.5. After using 40kHz ultrasound for 10min, use a homogenizer at 10000rpm for 2min to fully dissolve the hydroxyapatite microspheres. Then pour the ethanol solution containing 0.02g of γ-aminopropylmethyldiethoxysilane (A-2100) prepared in advance into it, stir it thoroughly, adjust the pH value of the reaction solution to about 9.0, and further stir and react for 4 hours. Centrifuge the reaction product, collect the precipitate and wash the precipitate appropriately to obtain modified hydroxyapatite with amino groups, i.e., the second reaction product.

[0085] Step 3: Preparation of the second modified hydroxyapatite microspheres (HA-MP-NH2 / CMC).

[0086] Weigh 0.5g of the second reaction product and dissolve it in 40mL of purified water. Use a homogenizer and ultrasound to disperse it evenly. Add 20mL of aqueous solution containing 0.1g of sodium carboxymethyl cellulose (CMC-Na) and stir to mix it thoroughly. Adjust the pH value of the reaction solution to about 8.5, and stir the reaction at room temperature for 24 hours. After the reaction is completed, dialyze with distilled water for 24 hours (using a 3500Da dialysis bag), change the water 4 times during the process, and vacuum freeze-dry the reaction product for 48-72 hours to obtain modified hydroxyapatite with amino and carboxylic acid groups, which is the final product of the reaction.

[0087] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0088] 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 modes, which are merely illustrative rather than 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 within the protection of the present invention.

Claims

1. A hydroxyapatite microsphere injection with good water dispersibility, characterized in that the hydroxyapatite microsphere injection comprises a solvent and a double-group modified hydroxyapatite microsphere; the surface of the double-group modified hydroxyapatite microsphere contains an amino-carboxyl group connected by electrostatic adsorption; the double-group modified hydroxyapatite microsphere is a hydroxyapatite microsphere matrix with a particle size range of 10-60 μm, which is amino-modified and carboxyl-modified in sequence; the hydroxyapatite microsphere matrix is ​​reacted with a silane coupling agent providing an amino group at a mass ratio of 10:1 to obtain a first modified hydroxyapatite microsphere; the first modified hydroxyapatite microsphere is reacted with sodium carboxymethyl cellulose providing a carboxyl group at a mass ratio of 5:1 or 6:1 to obtain the double-group modified hydroxyapatite microsphere.

2. The hydroxyapatite microsphere injection according to claim 1, characterized in that: The particle size of the hydroxyapatite microsphere matrix is ​​in the range of 30-50 μm.

3. The hydroxyapatite microsphere injection according to claim 1, characterized in that: The silane coupling agent providing amino groups includes γ-aminopropyltrimethoxysilane, γ-aminopropyltriethoxysilane or γ-aminopropylmethyldiethoxysilane.

4. The method for preparing the hydroxyapatite microsphere injection according to any one of claims 1 to 3, 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 solution 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 value 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 in a reaction kettle and reacting at 120-160° C. for 8-15 hours, centrifuging, and collecting the precipitate after centrifugation to obtain hydroxyapatite microspheres; S04: adding the hydroxyapatite microspheres obtained in S03 to an acidic solution of pH 5.5 to fully dissolve, 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 value of the reaction system to 9.0, stirring to fully react, centrifuging, collecting the precipitated material after centrifugation, and obtaining amino-modified hydroxyapatite microspheres; the silane coupling agent includes γ-aminopropyltrimethoxysilane, γ-aminopropyltriethoxysilane or γ-aminopropylmethyldiethoxysilane; 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 value 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; S06: adding a solvent to dissolve the modified hydroxyapatite microspheres with double groups to obtain the hydroxyapatite microsphere injection.

5. The preparation method according to claim 4, characterized in that: The calcium salt includes calcium carbonate, calcium chloride, calcium nitrate or calcium bicarbonate.

6. The preparation method according to claim 4, characterized in that: The phosphate includes potassium phosphate, sodium phosphate, disodium hydrogen phosphate or dipotassium hydrogen phosphate.

7. The preparation method according to claim 4, characterized in that: The acidic solution includes glacial acetic acid solution or hydrochloric acid solution.

8. The preparation method according to claim 4, characterized in that: In S03, the means for adding the hydroxyapatite microspheres into the acidic solution of pH 5.5 to fully dissolve them include ultrasound and homogenization.

9. The preparation method according to claim 4, characterized in that: After the reaction is completed, the dialysis operation is performed using distilled water.

10. The preparation method according to claim 4, characterized in that: The pH of the reaction system was adjusted to 8.0.

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