Method for preparing chitosan-hydroxyapatite composite microparticles by high-speed shearing and synergistic double-inversion miniemulsion

Abstract

The application relates to a method for preparing chitosan-hydroxyapatite composite microparticles through high-speed shearing and synergic double-reverse-phase fine emulsion, which continuously acts on the emulsification and homogenization process of the emulsification mixing of a chitosan-containing calcium salt and phosphate precursor aqueous solution and an oil phase system and a triethanolamine precursor aqueous solution and an oil phase system (constituting double-reverse-phase fine emulsion droplets) and the subsequent collision-mixing-neutralization-reaction process of chitosan, calcium salt and phosphate in the dispersed phase emulsion droplets (water droplets) and triethanolamine in another dispersed phase emulsion droplet (water droplet), realizes the continuous operation of the high-speed shearing effect reinforced physical emulsification mixing process and in-situ precipitation chemical reaction process, and achieves the purpose of preparing CS-HAP composite microparticles which are good in dispersity, not prone to agglomeration, closely combined with each other, high in porosity, uniformly dispersed and nano-level dispersed. The method is simple in preparation process, low in equipment requirement and cost, mild in reaction condition, easy to control in preparation process and easy to enlarge.

Description

Technical Field

[0001] This invention relates to a method for preparing chitosan-hydroxyapatite composite microparticles, and particularly to a method for preparing chitosan-hydroxyapatite composite microparticles using a high-speed shear synergistic dual-phase reverse emulsion. Background Technology

[0002] Chitosan (CS) is a natural, weakly basic polysaccharide with excellent biocompatibility, biodegradability, and non-toxicity. However, its poor mechanical properties, lack of elasticity and flexibility limit its application in bone tissue engineering. Hydroxyapatite (HAP) is the main inorganic mineral component of bone tissue, possessing excellent biocompatibility, bioactivity, and osteoconductivity, but it is brittle and has low toughness. The nanocomposite material prepared by combining these two materials not only possesses the advantages of both but also exhibits excellent mechanical properties due to the synergistic effect between them, making it suitable for medical fields such as bone tissue repair, artificial bone scaffolds, and replacement materials. Therefore, the preparation of chitosan-hydroxyapatite (CS-HAP) composite materials has attracted considerable attention in the industry.

[0003] Currently, the main preparation methods for chitosan-hydroxyapatite composites include solution blending, electrochemical deposition, simulated body fluid mineralization, and co-precipitation. Solution blending involves directly mixing HAP particles and CS solution in a specific ratio, promoting uniform dispersion through stirring or ultrasound, then injecting the mixture into a mold and freeze-drying. This method is simple to operate, but it easily leads to uneven HAP dispersion and agglomeration in the CS-HAP composite. Furthermore, the weak interfacial interaction between CS and HAP can cause the mechanical properties to decrease with increasing HAP content. Electrochemical deposition utilizes an electric field to drive protonated chitosan to migrate towards the cathode, and under controlled electrochemical conditions, allows Ca2+ to be deposited on the cathode. 2+ and PO4 3- HAP can be co-deposited with chitosan at the electrode / solution interface. This method allows for easy control of HAP deposition on porous / irregular surfaces, but is limited by the requirement that the substrate material must be conductive. The simulated body fluid mineralization method involves immersing a chitosan membrane in simulated body fluid for approximately 7 days, allowing HAP particles to deposit on its surface under low ion concentration conditions. The particle size and morphology of HAP can be controlled by adjusting the immersion time and ion concentration, but this method has a long preparation cycle and poor reproducibility. The co-precipitation method involves mixing the HAP precursor solution with an acidic CS solution and adjusting the pH of the system. The increased pH causes HAP and CS to precipitate simultaneously. This method solves the dispersion problem of HAP in the CS matrix to some extent, but the process requires precise pH control, and HAP still exhibits varying degrees of aggregation.

[0004] Therefore, in order to effectively promote the interfacial bonding between HAP and CS, solve the problems of HAP particle agglomeration and uneven dispersion in CS-HAP composites, and improve their performance, it is necessary to study new process strengthening methods to prepare chitosan-hydroxyapatite composites to overcome the above-mentioned defects of existing methods. Summary of the Invention

[0005] The technical problem this invention aims to solve is to provide a method for preparing chitosan-hydroxyapatite (HAP) composite microparticles using a high-speed shear-assisted dual-phase reverse-phase microemulsion process. This method continuously applies a high-speed shear effect to the emulsification, mixing, and homogenization process of a chitosan-containing calcium salt and phosphate precursor aqueous solution with an oil phase system, and a triethanolamine-containing precursor aqueous solution with an oil phase system (forming dual-phase reverse-phase microemulsion droplets). Subsequently, the chitosan, calcium salt, and phosphate in the dispersed phase droplets (water droplets) undergo collision-mixing-neutralization-reaction processes with the triethanolamine in the other dispersed phase droplets (water droplets). This achieves continuous operation of the high-speed shear-enhanced physical emulsification and mixing process and the in-situ precipitation chemical reaction process, and achieves the goal of preparing CS-HAP composite microparticles with good dispersibility, low agglomeration, tight bonding between CS and HAP, high porosity, and uniform HAP dispersion at the nanoscale. This method has a simple preparation process, low equipment requirements, low cost, mild reaction conditions, easy process control, and is easy to scale up.

[0006] The technical solution to the above-mentioned technical problems is: a method for preparing chitosan-hydroxyapatite composite microparticles by high-speed shear synergistic dual-phase reverse emulsion, comprising the following steps: (1) Preparation of precursor aqueous solution I and precursor aqueous solution II: Dissolve appropriate amounts of calcium salt, phosphate and glacial acetic acid in the same deionized water to form an acidic solution containing calcium salt and phosphate with a pH of 2.5-4. Then dissolve an appropriate amount of chitosan in it to obtain an acidic solution with calcium salt and phosphate content of 0.03-1.5 mol / L and chitosan content of 5-20 g / L as precursor aqueous solution I; the calcium salt is one of calcium chloride, calcium nitrate and calcium acetate; the phosphate is one of soluble disodium hydrogen phosphate, sodium dihydrogen phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, sodium phosphate, potassium phosphate and ammonium phosphate. Dissolve an appropriate amount of triethanolamine in deionized water to form a triethanolamine solution with a concentration of 5.5–10.0 mol / L as precursor aqueous solution II; (2) Preparation of oil phase: A certain amount of surfactant is fully dissolved in vegetable oil to form the oil phase; the surfactant is Span-80 or Span-60; the amount of surfactant is 2.0 to 8.0% (v / v) of the volume of vegetable oil. (3) Preparation of reversed-phase precursor crude emulsion I and reversed-phase precursor crude emulsion II: Take an appropriate amount of precursor aqueous solution I from step (1) and add it to the oil phase of step (2) under stirring at 25-60 ℃ to form precursor crude emulsion I; take an appropriate amount of precursor aqueous solution II from step (1) and add it to the oil phase of step (2) under stirring at 25-60 ℃ to form precursor crude emulsion II; the calcium / phosphorus molar ratio in the precursor aqueous solution I and precursor aqueous solution II is 1.6-2.0; the volume ratio of the precursor aqueous solution I, precursor aqueous solution II and oil phase is 10-30% (v / v). (4) High-speed shear mixing preparation of reverse precursor fine emulsion I and reverse precursor coarse emulsion II: At the same temperature as the preparation of reverse precursor coarse emulsion I in step (3), reverse precursor coarse emulsion I and reverse precursor coarse emulsion II in step (3) are simultaneously treated by a high-speed shear homogenizer at a certain high shear rate for 12 to 30 min to obtain reverse precursor fine emulsion I and reverse precursor fine emulsion II with emulsion particle size in the nanometer range. (5) Preparation of chitosan-hydroxyapatite composite microparticles by high-speed shear mixing and synergistic two-phase reverse microemulsion: At the same temperature as the preparation of reverse precursor microemulsion I in step (4), reverse precursor microemulsion II in step (4) is added dropwise to reverse precursor microemulsion I under uninterrupted high-speed shear homogenization at a rate of 5-15 mL / min. The mixture is continuously high-speed shear mixing and homogenization is carried out at the same high shear rate as in step (4) for 60-150 min. The mixture is then rapidly cooled to 10-25℃ at a cooling rate of 8-12℃ / min. After centrifugation, washing and freeze drying, chitosan-hydroxyapatite composite microparticles are obtained.

[0007] Preferably, in step (1), the chitosan has a molecular weight of 20 to 1000 kDa and a degree of deacetylation of 55 to 95%.

[0008] Preferably, in step (2), the vegetable oil is palm oil, corn oil, soybean oil, rapeseed oil, olive oil, or sunflower oil.

[0009] Preferably, the high shear rate in steps (4) and (5) is 8000 to 16000 r / min.

[0010] High-speed shear mixing, as a high-energy emulsification and homogenization technology, relies on the shear chamber formed by the high-speed rotating rotor and stator to generate synergistic effects of turbulent inertial forces and viscous shear forces, achieving rapid homogenization and mixing between immiscible liquid materials. When the rotor rotates at high speed, it drives the liquid material to form strong turbulence. The turbulent inertial forces create dramatic velocity gradients and pressure fluctuations within the material, enhancing the mixing between immiscible liquids and causing large droplets to break down into smaller droplets. Simultaneously, the liquid material is compressed and impacted within the gap between the rotor and stator, and the viscous shear forces further tear and refine the droplet size, promoting thorough mixing between different phases of liquid material. Furthermore, the high-speed motion of the liquid material has a certain probability of generating cavitation effects. The resulting microbubbles burst instantaneously, generating localized high pressure, microjets, and intense turbulence, which further enhance the mixing and homogenization effect, ultimately achieving a uniform and stable dispersion state between the two phases of liquid material.

[0011] This invention discloses a novel method for preparing chitosan-hydroxyapatite composite microparticles using high-speed shear synergistic dual-phase reverse emulsion. The main technical principles include: (1) The pulsed cavitation jet effect (including turbulent inertial force and viscous shear stress, etc.) promotes the cutting and breaking of large-sized precursor calcium salts containing chitosan, phosphate salt (emulsion) droplets, and large-sized precursor water (emulsion) droplets containing triethanolamine suspended in the vegetable oil phase, so that the suspended water (emulsion droplets) in the vegetable oil phase are continuously refined, and a double reverse phase fine emulsion with a water (emulsion) droplet size of 100-500 nm can be prepared, forming an emulsion system with small droplet size, narrow distribution and high stability. This provides a nanoscale stable "template" reaction microsystem for the synchronous nucleation of chitosan and hydroxyapatite, and for obtaining chitosan-hydroxyapatite composite microparticles with good dispersibility, tight binding of chitosan and hydroxyapatite, and uniform dispersion of hydroxyapatite.

[0012] (2) Before the collision-contact reaction of the two-phase fine water (emulsion), under the action of the pulse cavitation jet effect, the aggregation (flocculation) between the precursor calcium salt and phosphate salt (emulsion) droplets I containing chitosan suspended in the oil phase and its breakup into fine water (emulsion) droplets will reach "approximate thermodynamic equilibrium"; similarly, the aggregation (flocculation) between the precursor emulsion droplets II containing triethanolamine suspended in the oil phase and its breakup into fine water (emulsion) droplets will also reach "approximate thermodynamic equilibrium". This provides a favorable microenvironment for the generation of ultrafine-sized, narrowly distributed chitosan-hydroxyapatite composite microparticles when the two collide, contact and react.

[0013] (3) During the collision-contact reaction of the two-phase fine water (emulsion), the space of the water (emulsion) droplets in the fine emulsion can be regarded as a series of micro-element reaction spaces. Therefore, under the combined action of high-speed shear effect, including turbulent inertial force and viscous shear force, and a certain probability of cavitation effect, the probability of contact and collision between the precursor calcium salt containing chitosan and the phosphate salt (emulsion) droplets I suspended in the oil phase and the precursor triethanolamine-containing emulsion droplets II is increased. This leads to the sequential neutralization reaction and in-situ precipitation reaction in the water (emulsion) droplet "microreactor". It promotes the simultaneous occurrence of chitosan desolvation and hydroxyapatite crystallization reaction on the same microscale, enhances the combination of chitosan and hydroxyapatite, and obtains CS-HAP composite microparticles dispersed in nano-sized hydroxyapatite.

[0014] (4) The generation of two-phase reverse water (emulsion) droplets is an important basis for preparing CS-HAP composite microparticles with good dispersibility, tight binding of chitosan and hydroxyapatite, high porosity, uniform HAP dispersion and nanoscale dispersion. The high-speed shear mixing effect is an important guarantee for generating water (emulsion) droplet size of 100-500 nm two-phase reverse water (emulsion) droplets. The cooperation and complementarity of the two are the key to obtaining CS-HAP composite microparticles with good dispersibility, low agglomeration, tight binding of chitosan and hydroxyapatite, high porosity, uniform HAP dispersion and nanoscale dispersion by this method.

[0015] The beneficial effects of this invention are: (1) The chitosan-hydroxyapatite composite microparticles provided by the present invention have good dispersibility, and their size is controllable in the range of 500 to 1300 nm, and their polydispersity index (PDI) is controllable in the range of 0.40 to 0.90.

[0016] (2) The porosity of the chitosan-hydroxyapatite composite microparticles provided by the present invention is controllable at 70-90%, which is significantly higher than that of conventional co-precipitation method, and is 1.1-2.0 times its porosity.

[0017] (3) The chitosan-hydroxyapatite composite microparticles provided by the present invention contain hydroxyapatite in which the hydroxyapatite is distributed in the composite microparticles with a finer grain size than that of conventional coprecipitation methods, and the grain size can reach 16 to 100 nm.

[0018] (4) Compared with conventional solution blending method, the interface between chitosan and hydroxyapatite in the composite microparticles obtained by the present invention is tightly bound, the nano-hydroxyapatite is evenly dispersed in the chitosan matrix, and it is not easy to agglomerate.

[0019] (5) The preparation process provided by the present invention realizes the preparation process of reverse emulsification physical mixing of precursor aqueous solutions I and II through high-speed shear mixing, as well as the continuous operation of subsequent neutralization reaction, in-situ precipitation chemical reaction granulation process, which simplifies the operation process.

[0020] (6) The high-speed shear synergistic dual reverse emulsion mixing mode adopted in this invention greatly avoids the problem of large-area agglomeration of chitosan-hydroxyapatite composite particles due to excessive local reaction during the pH control process in conventional coprecipitation method.

[0021] (7) The preparation process provided by the present invention can easily adjust the droplet size and distribution formed by the reverse precursor microemulsions I and II by controlling the high-speed shearing operation conditions and process parameters, thereby controlling the size and distribution of the generated chitosan-hydroxyapatite composite microparticles. This effectively avoids the problem of difficulty in controlling the size and distribution of chitosan / hydroxyapatite composite microparticles obtained in the conventional coprecipitation method.

[0022] (8) The preparation method provided by the present invention is simple to operate, efficient, and has good repeatability. The high-speed shear mixing homogenizer is inexpensive and easy to control, and has the advantage of large-scale industrial application. Attached Figure Description

[0023] Figure 1 Example 1 of this invention: A process flow diagram for preparing chitosan-hydroxyapatite composite microparticles using high-speed shear synergistic dual-phase microemulsion.

[0024] Figure 2 SEM image of the internal surface microstructure of the chitosan-hydroxyapatite composite microparticles prepared in Example 1 of this invention.

[0025] Figure 3 SEM image of the internal surface morphology of the chitosan-hydroxyapatite composite microparticles prepared in Example 2 of this invention. Detailed Implementation

[0026] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention. Example 1

[0027] A method for preparing chitosan-hydroxyapatite composite microparticles using high-speed shear synergistic dual-phase reverse emulsion is illustrated in the process flow diagram below. Figure 1 As shown, it includes the following steps: (1) Preparation of precursor aqueous solutions I and II: Dissolve 0.63 g anhydrous CaCl2, 0.44 g (NH4)2HPO4 and 1.2 mL glacial acetic acid in deionized water and dilute to a final volume in the same 100 mL volumetric flask. The pH of the mixed solution is 2.7. Then, dissolve 0.58 g chitosan (molecular weight 500 kDa, degree of deacetylation 85%) in the 100 mL acidic mixed solution at 50 °C with stirring. The above mixed solution is precursor aqueous solution I.

[0028] Dissolve 74.9 mL of triethanolamine solution in deionized water and bring the volume to 100 mL to obtain a 5.64 mol / L triethanolamine solution, which is the precursor aqueous solution II.

[0029] (2) Preparation of oil phases I and II: Dissolve 12.5 mL of Span-80 in 250 mL of palm oil to form the oil phase.

[0030] (3) Preparation of reversed-phase precursor crude emulsions I and II: Take 17.3 mL of precursor aqueous solution I from step (1) and add it to 100 mL of oil phase in step (2), and stir at 25 °C to form crude emulsion I; take 25.0 mL of precursor aqueous solution II from step (1) and add it to 144.5 mL of oil phase in step (2), and stir at 25 °C to form crude emulsion II.

[0031] (4) High-speed shear mixing preparation of reverse precursor microemulsions I and II: At 25 °C, the precursor crude emulsions I and II from step (3) were simultaneously treated by a high-speed shear homogenizer at 15000 r / min for 15 min to obtain reverse precursor microemulsions I and II with a particle size of nanometers.

[0032] (5) Preparation of chitosan-hydroxyapatite composite microparticles by high-speed shear mixing and synergistic two-phase reverse fine emulsion: Under the condition of 25 °C and uninterrupted high-speed shear homogenization, the precursor fine emulsion II from step (4) was added dropwise to the reverse fine precursor liquid I from step (4) at a rate of 10 mL / min. The mixture was then treated with a high-speed shear homogenizer at a speed of 15000 r / min for 60 min (timing started when the reverse precursor fine emulsion II was added). After centrifugation, washing with petroleum ether, anhydrous ethanol, and deionized water 9 times, and freeze-drying, chitosan / hydroxyapatite composite microparticles were obtained. The internal surface micromorphology is shown in the figure. Figure 2 .

[0033] The physicochemical properties and performance comparison of a novel method for preparing chitosan-hydroxyapatite (CS-HAP) composite microparticles using a high-speed shear-coordinated dual-phase reverse emulsion method of the present invention with those of CS-HAP composite microparticles prepared by the conventional coprecipitation method are shown in Table 1. The conventional coprecipitation method refers to the coprecipitation method described in Gao Haochen, Wang Pei, Cao Zhizhong, Ge Kuikui, Wang Yihan, and Lu Min's paper, "In Vitro Release and Antibacterial Properties of Minocycline-Loaded Nano-Hydroxyapatite / Chitosan Composites".

[0034]

[0035] From Table 1 and Figure 2 It can be seen that the CS-HAP composite microparticles prepared by high-speed shear synergistic dual-phase reverse emulsion have good dispersibility and rough surface. The HAP crystals are uniformly dispersed in the CS-HAP composite microparticles and have a nanometer-scale size (up to 22.3 nm). The size of the CS-HAP composite microparticles is 590.5 nm, the PDI is 0.554, and the porosity reaches 75.3%, indicating that the obtained microparticles are ultra-fine and have a narrow distribution. Compared with the CS-HAP composite material prepared by conventional coprecipitation method, which has a micrometer-scale particle size (>1000 nm) after passing through a 100-mesh sieve, the particle size is significantly coarser than that of the CS-HAP composite microparticles prepared by high-speed shear synergistic dual-phase reverse emulsion. In addition, the porosity of the CS-HAP composite particles prepared by conventional coprecipitation method after sieving is 43.68%, while the porosity of the CS-HAP composite microparticles prepared by high-speed shear synergistic dual-phase reverse emulsion is 1.72 times that of the conventional coprecipitation method. Moreover, in the CS-HAP composite particles prepared by conventional coprecipitation method, HAP tends to agglomerate and have an uneven distribution in the composite particles. This result shows that, compared with the conventional coprecipitation method, the CS-HAP composite microparticles prepared by high-speed shear mixing synergistic dual reverse phase fine emulsion are finer in size, narrower in distribution, better in dispersion, and higher in porosity. At the same time, HAP is uniformly dispersed in the CS-HAP composite microparticles and is dispersed at the nanoscale. Example 2

[0036] A novel method for preparing chitosan-hydroxyapatite composite microparticles using high-speed shear-coordinated dual-phase reverse emulsion includes the following steps: (1) Preparation of precursor aqueous solutions I and II: Dissolve 4.1 g of anhydrous Ca(NO3)2, 2.1 g of anhydrous Na2HPO4 and 1.0 mL of glacial acetic acid in deionized water and dilute to a final volume in the same 100 mL volumetric flask. The pH of the mixed solution is 2.75. Then, dissolve 1.0 g of chitosan (molecular weight 200 kDa, degree of deacetylation 90%) in the 100 mL acidic mixed solution at 50 °C with stirring. The above mixed solution is precursor aqueous solution I.

[0037] Dissolve 80.0 mL of triethanolamine solution in deionized water and bring the volume to 100 mL to obtain a 6.03 mol / L triethanolamine solution, which is the precursor aqueous solution II.

[0038] (2) Preparation of oil phases I and II: After melting an appropriate amount of surfactant Span-60, take 10.0 mL and dissolve it fully in 250 mL of corn oil to form the oil phase.

[0039] (3) Preparation of reversed-phase precursor crude emulsions I and II: Preparation of reversed-phase precursor crude emulsions I and II: Take 15.0 mL of precursor aqueous solution I from step (1) and add it to 100 mL of oil phase in step (2), and stir at 40 °C to form crude emulsion I; take 21.4 mL of precursor aqueous solution II from step (1) and add it to 142.9 mL of oil phase in step (2), and stir at 40 °C to form crude emulsion II.

[0040] (4) Preparation of reverse precursor fine emulsion I and II by high-speed shear mixing: At 40 °C, the precursor crude emulsion I and II from step (3) were simultaneously treated by a high-speed shear homogenizer at 12500 r / min for 20 min to obtain reverse precursor fine emulsion I and reverse precursor fine emulsion II with a particle size of nanometer.

[0041] (5) Preparation of chitosan-hydroxyapatite composite microparticles by high-speed shear mixing and synergistic two-phase reverse fine emulsion: Under the condition of 40 °C and uninterrupted high-speed shear homogenization, the precursor fine emulsion II from step (4) was added dropwise to the reverse fine precursor liquid I from step (4) at a rate of 12 mL / min. The mixture was then treated with a high-speed shear homogenizer at a speed of 12500 r / min for 90 min, and rapidly cooled to 15 °C at a cooling rate of 10 °C / min. After centrifugation, washing with petroleum ether, anhydrous ethanol, and deionized water 9 times, and freeze-drying, chitosan / hydroxyapatite composite microparticles were obtained. The internal surface micromorphology is shown in the figure. Figure 3 .

[0042] Table 2 compares the physicochemical properties and performance of a novel high-speed shear-coordinated dual-phase reverse emulsion method for preparing chitosan-hydroxyapatite (CS-HAP) composite microparticles with those prepared by the conventional coprecipitation method. The conventional coprecipitation method refers to the method described in Wang Jincheng's paper, "Research on Hydroxyapatite / Chitosan Composite Materials Carried with Lysocystis Bacillus Enzyme."

[0043]

[0044] From Table 2 and Figure 3It can be seen that the CS-HAP composite microparticles prepared by high-speed shear synergistic dual-phase reverse emulsion have good dispersibility and rough surface. The HAP crystals are uniformly dispersed in the CS-HAP composite microparticles and have a size of nanometer scale (up to 24.2 nm). The size of the CS-HAP composite microparticles is 630.7 nm, the PDI is 0.614, and the porosity reaches 74.4%, indicating that the obtained microparticles are ultra-fine and have a narrow distribution. Compared with the CS-HAP composite material prepared by conventional coprecipitation method, the particle size after grinding is micrometer scale (>1000 nm), which is significantly coarser than the size of the CS-HAP composite microparticles prepared by high-speed shear synergistic dual-phase reverse emulsion. In addition, the porosity of the CS-HAP composite particles prepared by conventional coprecipitation method after sieving is 64.1%, while the porosity of the CS-HAP composite microparticles prepared by high-speed shear synergistic dual-phase reverse emulsion is 1.16 times that of the conventional coprecipitation method. Moreover, in the CS-HAP composite particles prepared by conventional coprecipitation method, HAP tends to agglomerate and have uneven distribution in the composite particles. This result shows that, compared with the conventional coprecipitation method, the CS-HAP composite microparticles prepared by high-speed shear mixing synergistic dual reverse phase fine emulsion are finer in size, narrower in distribution, better in dispersion, and higher in porosity. At the same time, HAP is uniformly dispersed in the CS-HAP composite microparticles and is dispersed at the nanoscale.

Claims

1. A method for preparing chitosan-hydroxyapatite composite microparticles using high-speed shear synergistic dual-phase reverse emulsion, characterized in that, Includes the following steps: (1) Preparation of precursor aqueous solution I and precursor aqueous solution II: Dissolve appropriate amounts of calcium salt, phosphate and glacial acetic acid in the same deionized water to form an acidic solution containing calcium salt and phosphate with a pH of 2.5-4. Then dissolve an appropriate amount of chitosan in it to obtain an acidic solution with calcium salt and phosphate content of 0.03-1.5 mol / L and chitosan content of 5-20 g / L as precursor aqueous solution I; the calcium salt is one of calcium chloride, calcium nitrate and calcium acetate; the phosphate is one of soluble disodium hydrogen phosphate, sodium dihydrogen phosphate, ammonium dihydrogen phosphate, diammonium hydrogen phosphate, sodium phosphate, potassium phosphate and ammonium phosphate. Dissolve an appropriate amount of triethanolamine in deionized water to form a triethanolamine solution with a concentration of 5.5–10.0 mol / L as precursor aqueous solution II; (2) Preparation of oil phase: A certain amount of surfactant is fully dissolved in vegetable oil to form the oil phase; the surfactant is Span-80 or Span-60; the amount of surfactant is 2.0 to 8.0% (v / v) of the volume of vegetable oil. (3) Preparation of reversed-phase precursor crude emulsion I and reversed-phase precursor crude emulsion II: Take an appropriate amount of precursor aqueous solution I from step (1) and add it to the oil phase of step (2) under stirring at 25-60 ℃ to form precursor crude emulsion I; take an appropriate amount of precursor aqueous solution II from step (1) and add it to the oil phase of step (2) under stirring at 25-60 ℃ to form precursor crude emulsion II; the calcium / phosphorus molar ratio in the precursor aqueous solution I and precursor aqueous solution II is 1.6-2.0; the volume ratio of the precursor aqueous solution I, precursor aqueous solution II and oil phase is 10-30% (v / v). (4) High-speed shear mixing preparation of reverse precursor fine emulsion I and reverse precursor coarse emulsion II: At the same temperature as the preparation of reverse precursor coarse emulsion I in step (3), reverse precursor coarse emulsion I and reverse precursor coarse emulsion II in step (3) are simultaneously treated by a high-speed shear homogenizer at a certain high shear rate for 12 to 30 min to obtain reverse precursor fine emulsion I and reverse precursor fine emulsion II with emulsion particle size in the nanometer range. (5) Preparation of chitosan-hydroxyapatite composite microparticles by high-speed shear mixing and synergistic two-phase reverse microemulsion: At the same temperature as the preparation of reverse precursor microemulsion I in step (4), reverse precursor microemulsion II in step (4) is added dropwise to reverse precursor microemulsion I under uninterrupted high-speed shear homogenization at a rate of 5-15 mL / min. The mixture is continuously high-speed shear mixing and homogenization is carried out at the same high shear rate as in step (4) for 60-150 min. The mixture is then rapidly cooled to 10-25℃ at a cooling rate of 8-12℃ / min. After centrifugation, washing and freeze drying, chitosan-hydroxyapatite composite microparticles are obtained.

2. The method for preparing chitosan-hydroxyapatite composite microparticles by high-speed shear synergistic dual-phase reverse emulsion according to claim 1, characterized in that: In step (1), the chitosan has a molecular weight of 20-1000 kDa and a degree of deacetylation of 55-95%.

3. The method for preparing chitosan-hydroxyapatite composite microparticles by high-speed shear synergistic dual-phase reverse emulsion according to claim 1 or 2, characterized in that: In step (2), the vegetable oils are palm oil, corn oil, soybean oil, rapeseed oil, olive oil and sunflower oil.

4. The method for preparing chitosan-hydroxyapatite composite microparticles by high-speed shear synergistic dual-phase reverse emulsion according to claim 1 or 2, characterized in that: The high shear rate in steps (4) and (5) is 8000–16000 r / min.

Images