Raspberry-shaped degradable porous microspheres with high mesoporosity, and preparation method therefor and use thereof

Abstract

The present invention relates to the technical field of biomedical materials. Disclosed in the present invention are raspberry-shaped degradable porous microspheres with high mesoporosity, and a preparation method therefor and the use thereof. The raspberry-shaped degradable porous microspheres with high mesoporosity are nano-to micron-sized microspheres having a rough surface formed by means of the self-assembly of nano-sized hydroxyapatite particles driven by the thermodynamic behavior of excessive surface free energy reduction. Moreover, the raspberry-shaped degradable porous microspheres with high mesoporosity of the present invention have a rough surface and spherical morphology, and increased surface roughness and specific surface area, facilitating cell attachment, proliferation and the adsorption of drug molecules. The spherical structure thereof improves the mechanical microenvironment when in contact with surrounding tissues, and reduces the probability of inflammatory reactions occurring. The porous structure increases the contact area with body fluids, regulates the degradation rate and the release efficiency of calcium ions, promotes the repair of the skin barrier, and facilitates the loading of drugs and stem cells.

Description

A raspberry-like biodegradable porous microsphere with high mesopority, its preparation method and application Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a raspberry-like biodegradable porous microsphere with high mesoporous content, its preparation method, and its application. Background Technology

[0002] Hydroxyapatite (HAp) is one of the most common forms of the calcium phosphate family, with the chemical formula Ca. 10 (PO4)6(OH)2 is a major inorganic mineral component of human bones and teeth. Calcium hydroxyphosphate (CHP) is also widely found in nature, such as in the bones of vertebrates and the shells of mollusks. CHP possesses good biocompatibility and bioactivity, and as an inorganic bone substitute, it has been widely used in the medical field, demonstrating high application value in orthopedic surgery, maxillofacial surgery, and controlled drug release. However, because CHP belongs to the hexagonal crystal system, space group P63 / m, with crystallographic parameters a = 9.418 Å, c = 6.881 Å, β = 120°, the smallest structural unit of CHP prepared using traditional methods such as hydrothermal methods is usually needle-shaped. This needle-like structure can easily stimulate inflammatory reactions in surrounding tissues after implantation. Technical issues

[0003] Preparing calcium hydroxyphosphate into microspheres can improve the mechanical microenvironment when it comes into contact with surrounding tissues, reducing the probability of inflammatory responses. The porous structure can increase the contact area between calcium hydroxyphosphate and body fluids after injection, regulating its degradation rate to some extent, and also facilitating the loading of drugs and stem cells. Currently, microsphere preparation methods include hydrothermal methods, spray drying methods, sol-gel methods, and template methods.

[0004] For example, Chinese patent (patent application number 202210911941.5) discloses a hydrothermal preparation method for porous microspheres; however, its microstructure consists of clusters of needle-like and plate-like calcium hydroxyapatite, which may cause significant inflammatory reactions after injection. Chinese patent (patent application number 202210364112.X) discloses a spray-drying preparation method for hydroxyapatite microspheres, but it lacks a porous structure, which is detrimental to drug loading and degradation time control. Chinese patent (patent application number 202310547230.9) discloses a template-based preparation method for injectable high-purity medical hydroxyapatite microspheres, which also lacks a porous structure.

[0005] Therefore, there is an urgent need for a raspberry-like biodegradable porous microsphere with high mesoporous content, its preparation method, and its application to solve the above problems. Technical solutions

[0006] The purpose of this invention is to provide a raspberry-like biodegradable porous microsphere with high mesoporosity, its preparation method, and its application. This overcomes the shortcomings of existing microspheres, such as sharp microstructures, relatively poor biocompatibility, and lack of porous structure.

[0007] The solution of the present invention is:

[0008] A high-mesoporosity raspberry-shaped biodegradable porous microsphere is formed by the self-assembly of nano-sized calcium hydroxyphosphate particles under the thermodynamic behavior of reduced excess surface free energy, resulting in nano- to micro-sized microspheres with rough surfaces. The high-mesoporosity raspberry-shaped biodegradable porous microsphere is spherical or irregularly shaped and has a nano-sized pore structure, which has one or more combinations of cross-linked pores, through pores, and blind pores.

[0009] As a preferred technical solution, the surface roughness of the microspheres is 5–150 nm; the particle size of the microspheres is 300 nm–150 μm; the average pore size of the microspheres is 0.1 nm–150 nm; the porosity of the microspheres is 50%–95%; and the specific surface area of ​​the microspheres is 30–200 m². 2 / g.

[0010] As a preferred technical solution, the surface roughness of the microspheres is 50–90 nm; the particle size range of the microspheres is 600 nm–75 μm; the pore size range of the microspheres is 2–50 nm; the porosity of the microspheres is 65%–80%; and the specific surface area of ​​the microspheres is 60–150 m². 2 / g. A particle size range of 600 nm to 75 μm minimizes the body's reaction to foreign matter in high-mesoporous raspberry-like biodegradable porous microspheres. For high-mesoporous raspberry-like biodegradable porous microspheres, pore size, porosity, and specific surface area are interrelated. Within the range of pore size (2–50 nm), porosity (65%–85%), and specific surface area (60–100 m² / g), an optimal balance is achieved between the degradation time of calcium hydroxyphosphate, the calcium ion release rate, and the drug release rate. A surface roughness of 50–90 nm facilitates cell spread on the microsphere surface and enhances tissue compatibility.

[0011] This invention also discloses a method for preparing raspberry-like biodegradable porous microspheres with high mesoporosity, comprising the following steps:

[0012] By mineralizing the dextran microsphere template, mineralized dextran microsphere templates are obtained; after calcining the dextran microsphere templates, raspberry-like biodegradable porous microspheres with high mesopority are prepared.

[0013] As a preferred technical solution, the method for preparing the dextran microsphere template is as follows:

[0014] S1. Add the dextran solution with a concentration of 10-50% W / V to the alkaline solution, mix well, and obtain the aqueous phase;

[0015] S2. The aqueous phase is added to the organic phase, mixed and pre-emulsified to obtain an emulsion, wherein the organic phase is n-octane containing a surfactant;

[0016] S3. Add a crosslinking agent to the emulsion, react, filter and separate the crosslinked product;

[0017] S4. The crosslinked material is washed with n-octane and distilled water, then solvent exchanged with a volatile solvent, and then dried in a vacuum to obtain a dextran microsphere template.

[0018] As a preferred technical solution, a dual centrifugal stirrer is used in step S1 to ensure complete and uniform mixing;

[0019] The organic phase in S2 is injected into a three-necked glass reactor. The reactor is equipped with a heat-insulating jacket, a top stirrer and an arc-shaped blade stirring paddle, and is connected to an injection pump for adding reagents. The reaction temperature is controlled by a thermostatic bath. After the aqueous phase is added to the reactor, it is stirred and pre-emulsified to form a W / O type emulsion and to ensure that the surfactant is completely adsorbed on the surface of the droplets.

[0020] In step S3, the stirring speed is slowed down, and the crosslinking agent is added dropwise to the reaction vessel using a syringe pump. The reaction is stirred until the reaction is complete. The dextran microsphere template is separated from the reaction mixture by filtration through a filter membrane, and then washed twice with n-octane and distilled water in sequence. The dextran microsphere template is then solvent-exchanged with a volatile solvent and dried in a vacuum to completely remove the solvent.

[0021] The dried dextran microsphere template was placed in a chromatography column and the column was washed alternately with calcium salt aqueous solution and phosphate aqueous solution. After repeating this process several times, the dextran microsphere template was washed with purified water to obtain mineralized dextran microsphere template.

[0022] The mineralized dextran microsphere template was placed in a muffle furnace for calcination to remove the dextran microsphere template, thereby preparing raspberry-like biodegradable porous microspheres with high mesopority.

[0023] As a preferred technical solution, in step S1, the concentration of the dextran solution is 20–40% W / V; the weight-average molecular weight (Mw) of the dextran is 40,000–60,000, and its polymer dispersibility index (PDI) is <2.0; the alkaline solution is an aqueous NaOH solution with a concentration of 1–10 mol / L; the mixing in step S1 is performed by stirring at a speed of 2000–4000 rpm for 1–10 min.

[0024] As a preferred technical solution, the concentration of the NaOH aqueous solution is 2-4 mol / L.

[0025] As a preferred technical solution, the volume ratio of the aqueous phase to the organic phase in step S2 is 1 to 5:1;

[0026] The content of the surfactant in the organic phase is 0.1% to 2% W / V;

[0027] The surfactant is at least one selected from polyethylene glycol (200) monolaurate, glyceryl monolaurate, N,N-dimethylhexamethylene amide, polyglycerol fatty acid ester, polyethylene glycol (200) dilaurate, diglyceryl monolaurate, sorbitol monolaurate, nonylphenoxypolyethoxyethanol, and Span 80; the mixing in step S2 is performed by stirring at a speed of 1000-2000 rpm for 5-30 min;

[0028] In step S3, the crosslinking agent is at least one of diisocyanate, glutaraldehyde, epichlorohydrin, epibromopropane, ethylene glycol diglycidyl ether, and 1,4-butanediol diglycidyl ether.

[0029] The method for adding the crosslinking agent is to use a syringe pump to add it at a rate of 1 to 10 mL / h.

[0030] The molar ratio of dextran to the cross-linking agent is 1:0.5–40 kg / mol;

[0031] The reaction time is 12 to 48 hours; the reaction temperature is 20°C to 50°C; the reaction is stirred at a speed of 200 to 800 rpm.

[0032] In step S3, filtration is performed using a microporous membrane under reduced pressure, with the membrane having a pore size of 0.22 μm to 0.45 μm.

[0033] The volatile solvent in step S4 is at least one of ethanol or acetone.

[0034] As a preferred technical solution, the volume ratio of the aqueous phase to the organic phase in step S2 is 1 to 2:1;

[0035] The surfactant is at least one of polyethylene glycol (200) monolaurate, polyethylene glycol (200) dilaurate, and Span 80;

[0036] The mixing process involves stirring with a stirrer at a speed of 1000–1500 rpm for 10–20 minutes.

[0037] The crosslinking agent is epichlorohydrin;

[0038] The method for adding the crosslinking agent is to use a syringe pump to add it at a rate of 2 to 8 mL / h.

[0039] The molar ratio of the dextran to the cross-linking agent is 1:1.2–32 kg / mol;

[0040] The reaction time is 16–24 h; the reaction temperature is 35℃–45℃; and the stirring in the S3 reaction is performed by stirring with a stirrer at a speed of 300–500 rpm for 10–20 min.

[0041] As a preferred technical solution, the mineralization treatment method is as follows: the dextran microsphere template is alternately rinsed with calcium salt aqueous solution and phosphate aqueous solution, the number of alternating rinsing times is ≥2, and then rinsed with purified water.

[0042] As a preferred technical solution, the concentration of the phosphate aqueous solution is 0.1–2.0 mol / L; the flow rate of the phosphate aqueous solution during rinsing is 10–100 mL / h; and the phosphate aqueous solution is one of diammonium hydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, ammonium dihydrogen phosphate, sodium dihydrogen phosphate, and potassium dihydrogen phosphate.

[0043] The concentration of the calcium salt aqueous solution is 0.1–2.0 mol / L; the flow rate of the calcium salt aqueous solution during rinsing is 10–100 mL / h; the calcium salt aqueous solution is one of calcium chloride, calcium bromide, calcium nitrate, and dicalcium hydrogen phosphate.

[0044] As a preferred technical solution, the concentration of the phosphate aqueous solution is 0.3–0.5 mol / L; the phosphate aqueous solution is a diammonium hydrogen phosphate solution;

[0045] The concentration of the calcium salt aqueous solution is 0.3–0.5 mol / L; the calcium salt aqueous solution is a calcium nitrate solution.

[0046] As a preferred technical solution, the calcination temperature is 600–1200℃; the calcination time is 2–24 h.

[0047] As a preferred technical solution, the calcination temperature ranges from 800 to 1000°C, and the calcination time is from 15 to 18 hours.

[0048] As a preferred technical solution, the drying method is to dry in a vacuum oven at a temperature above 80°C until constant weight; the drying time is 2 hours to 24 hours; more preferably, the drying method is to dry at a temperature of 90°C and a pressure of 1.0 × 10⁻⁶. -3 Dry to constant weight in a vacuum oven at MPa.

[0049] The present invention also discloses the application of a raspberry-shaped biodegradable porous microsphere with high mesopority in drug sustained-release carriers or tissue engineering scaffolds.

[0050] As a preferred technical solution, raspberry-shaped biodegradable porous microspheres with high mesoporous ratio have good adsorption capacity and can be used as adsorbents for application in relevant fields.

[0051] The invention employs a high-mesoporous raspberry-like biodegradable porous microsphere, its preparation method, and its application. The high-mesoporous raspberry-like biodegradable porous microsphere is formed by the self-assembly of nano-sized calcium hydroxyphosphate particles under the thermodynamic behavior of reduced excess surface free energy, resulting in nano- to micro-sized microspheres with rough surfaces. The high-mesoporous raspberry-like biodegradable porous microsphere is spherical or irregularly shaped and has a nano-sized pore structure, which includes one or more combinations of cross-linked pores, through pores, and blind pores. Beneficial effects

[0052] Advantages of this invention:

[0053] The high mesoporous raspberry-shaped biodegradable porous microspheres of the present invention have a rough surface with high surface roughness, thereby providing more structural support points and bioactive sites, which helps cell attachment and proliferation, and is conducive to the adsorption of drug molecules; its spherical morphology can improve the mechanical microenvironment when it comes into contact with surrounding tissues, reducing the probability of inflammatory response.

[0054] Furthermore, specific surface area also affects the degradation rate of raspberry-shaped biodegradable porous microspheres with high mesoporousness. The high mesoporous raspberry-shaped biodegradable porous microspheres prepared in this invention also possess a porous structure, and the morphology and size parameters of the nanoscale calcium hydroxyphosphate particles can be adjusted through processing to obtain high mesoporous raspberry-shaped biodegradable porous microspheres with different pore sizes, porosities, and specific surface areas. The porous structure can increase the contact area between calcium hydroxyphosphate and body fluids after injection, thereby regulating its degradation rate to a certain extent and thus affecting the release efficiency of calcium ions. Calcium ions are an important element for the proliferation and differentiation of basal cells in the skin; regulating calcium ion concentration helps repair the skin barrier and is also beneficial for loading drugs and stem cells.

[0055] The preparation method used in this invention is simple, the production process has a high safety factor, and the resulting porous microspheres have a stable structure and various parameters can be adjusted according to requirements, making them suitable for industrial-scale production.

[0056] This invention enhances the stability of the emulsion system through the addition of surfactants; the addition of crosslinking agents, especially epichlorohydrin, adjusts the crosslinking density of the dextran template to regulate the subsequent mineralization efficiency; NaOH improves reactivity, accelerating the crosslinking rate compared to when it is not added. The crosslinking agent is added dropwise via an injection pump to prevent microspheres from agglomerating due to collisions, resulting in more uniform microsphere size compared to direct full addition. The use of calcium salts and phosphates in the biomimetic mineralization process alternately mineralizes and generates hydroxyapatite nanoparticles, which, compared to impregnation, improves mineralization efficiency while obtaining more structurally complete hydroxyapatite nanocrystals.

[0057] This invention uses cross-linked activated dextran microspheres as a template agent, which has a strong ability to induce the mineralization and deposition of calcium hydroxyphosphate. Compared with other methods for preparing calcium hydroxyphosphate, the biomimetic mineralization method not only has the advantages of being environmentally friendly, simple in process, low in cost, and able to effectively control the morphology and microstructure of crystals, but also the prepared composite microspheres have good interfacial compatibility between the calcium hydroxyphosphate and modified dextran microspheres due to the strong bonding between the two phases. The calcium hydroxyphosphate layer is not easy to fall off and can be dispersed uniformly. By adjusting the morphology and size parameters of the nano-sized calcium hydroxyphosphate particles prepared by the dextran template, raspberry-like biodegradable porous microspheres with different pore sizes, porosities, and specific surface areas can be obtained during sintering. The corresponding pore structure affects the degradation rate, which in turn affects the release efficiency of calcium ions. Calcium ions are an important element for the proliferation and differentiation of basal cells in the skin, and the regulation of calcium ion concentration helps to repair the skin barrier. In addition, due to their excellent adsorption capacity, raspberry-shaped biodegradable porous microspheres can also serve as tissue engineering scaffolds to load drugs and cells, and to sustainably repair tissue damage in the human body. Attached Figure Description

[0058] Figure 1 is a scanning electron microscope image of raspberry-like biodegradable porous microspheres with high mesopority obtained by alternating mineralization and calcination to remove the template using dextran microspheres as a soft template in Example 1 of the present invention.

[0059] Figure 2 is a scanning electron microscope image of the surface morphology of the raspberry-like biodegradable porous microspheres with high mesopority obtained by alternating mineralization and calcination to remove the template using dextran microspheres as a soft template in Example 1 of the present invention.

[0060] Figure 3 is a transmission electron microscope image of calcium hydroxyphosphate nanoparticles obtained by alternating mineralization and ultrasonic demolding using dextran microspheres as a soft template in Example 1 of the present invention. It shows the nanoscale calcium hydroxyphosphate particles enriched on the mineralized dextran microsphere template. The nanoscale calcium hydroxyphosphate particles will self-assemble into raspberry-like biodegradable porous microspheres with high mesopority during the subsequent calcination demolding process. The calcium hydroxyphosphate nanoparticles are spherical or irregular spherical particles with a particle size of 10-70 nm and an aspect ratio of 1-5.

[0061] Figure 4 is a BET nitrogen adsorption-desorption curve of the high mesoporous raspberry-shaped biodegradable porous microspheres of Example 1 of the present invention. Embodiments of the present invention

[0062] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below with reference to specific embodiments. Example 1:

[0063] The organic continuous phase consisted of 2.45 L of n-octane containing 1% w / v polyethylene glycol (200) monolaurate. The organic continuous phase was injected into a 10 L three-necked glass reactor equipped with a heat-insulating jacket, a top stirrer, and an arc-bladed impeller, and connected to a syringe pump for reagent addition. The reaction temperature was controlled at 40°C using a thermostat.

[0064] The aqueous phase was prepared by adding 1 L of 2 mol / L NaOH solution to 2 L of 20% w / v dextran solution, and then homogenizing at 3000 rpm for 5 min using a dual centrifugal stirrer until completely mixed. After adding the aqueous phase to the reactor, it was pre-emulsified by stirring at 1300 rpm for 15 min to form a w / o emulsion and ensure that the surfactant was completely adsorbed on the droplet surface.

[0065] The stirring speed was set to 400 rpm, and 1.6 mol of epichlorohydrin with a molar ratio of 200 to dextran was added dropwise to the reaction vessel at a rate of 4 mL / h using a syringe pump. The reaction was stirred for 18 h to ensure complete reaction.

[0066] Microspheres were separated from the reaction mixture by filtration through a 0.22 μm filter membrane and then washed twice, sequentially with n-octane and distilled water. The microspheres were then subjected to solvent exchange with acetone and / or ethanol and dried under vacuum to completely remove the solvent.

[0067] The dried microspheres were placed in a chromatography column and washed alternately with 0.5 mol / L calcium nitrate solution and 0.3 mol / L diammonium hydrogen phosphate solution at a flow rate of 60 mL / h. This process was repeated 10 times. After washing the microspheres with purified water, they were calcined in a muffle furnace at 1000℃ for 15 h to remove the dextran template, resulting in a microsphere with an average particle size of 22.3 μm, a porosity of 80.2%, an average pore size of 21.5 nm, and a specific surface area of ​​58.8 m². 2 / g, biomimetic biomineralized raspberry-like biodegradable porous microspheres with a surface roughness of 63 nm and a high mesoporous ratio. Example 2:

[0068] The organic continuous phase consisted of 2.45 L of n-octane containing 1% w / v polyethylene glycol (200) monolaurate. The organic continuous phase was injected into a 10 L three-necked glass reactor equipped with a heat-insulating jacket, a top stirrer, and an arc-bladed impeller, and connected to a syringe pump for reagent addition. The reaction temperature was controlled at 40°C using a thermostat.

[0069] The aqueous phase was prepared by adding 1 L of 4 mol / L NaOH solution to 2 L of 20% w / v dextran solution, and then homogenizing at 3000 rpm for 5 min using a dual centrifugal stirrer until completely mixed. After adding the aqueous phase to the reactor, it was pre-emulsified by stirring at 1300 rpm for 15 min to form a w / o emulsion and ensure that the surfactant was completely adsorbed on the droplet surface.

[0070] Set the stirring speed to 400 rpm and use a syringe pump to add 2.4 mol of ethylene glycol diglycidyl ether dropwise to the reaction vessel at a rate of 4 mL / h. Stir the reaction for 18 h to ensure complete reaction.

[0071] Microspheres were separated from the reaction mixture by filtration through a 0.22 μm filter membrane and then washed twice, sequentially with n-octane and distilled water. The microspheres were then subjected to solvent exchange with acetone and / or ethanol and dried under vacuum to completely remove the solvent.

[0072] The dried microspheres were placed in a chromatography column and washed alternately with 0.5 mol / L calcium nitrate solution and 0.3 mol / L diammonium hydrogen phosphate solution at a flow rate of 60 mL / h. This process was repeated 8 times. After washing the microspheres with purified water, they were calcined in a muffle furnace at 900 °C for 18 h to remove the dextran template, resulting in a microsphere with an average particle size of 35.8 μm, a porosity of 86.7%, an average pore size of 35.0 nm, and a specific surface area of ​​77.4 m². 2 / g, biomimetic biomineralized raspberry-like biodegradable porous microspheres with a surface roughness of 78 nm and a high mesoporous ratio. Example 3:

[0073] The organic continuous phase consisted of 2.45 L of n-octane containing 0.5% w / v Span 80. The organic continuous phase was injected into a 10 L three-necked glass reactor equipped with a heat-insulating jacket, a top stirrer, and an arc-bladed impeller, and connected to a syringe pump for reagent addition. The reaction temperature was controlled at 40°C using a thermostat.

[0074] The aqueous phase was prepared by adding 1 L of 2 mol / L NaOH solution to 2 L of 20% w / v dextran solution, and then homogenizing at 3000 rpm for 5 min using a dual centrifugal stirrer until completely mixed. After adding the aqueous phase to the reactor, it was pre-emulsified by stirring at 1300 rpm for 15 min to form a w / o emulsion and ensure that the surfactant was completely adsorbed on the droplet surface.

[0075] Set the stirring speed to 400 rpm and use a syringe pump to add 1.2 mol of ethylene glycol diglycidyl ether dropwise into the reaction vessel at a rate of 4 mL / h. Stir the reaction for 18 h to ensure complete reaction.

[0076] Microspheres were separated from the reaction mixture by filtration through a 0.22 μm filter membrane and then washed twice, sequentially with n-octane and distilled water. The microspheres were then subjected to solvent exchange with acetone and / or ethanol and dried under vacuum to completely remove the solvent.

[0077] The dried microspheres were placed in a chromatography column and washed alternately with 0.5 mol / L calcium nitrate solution and 0.3 mol / L diammonium hydrogen phosphate solution at a flow rate of 60 mL / h. This process was repeated 10 times. After washing the microspheres with purified water, they were calcined in a muffle furnace at 1000℃ for 15 h to remove the dextran template, resulting in a microsphere with an average particle size of 27.4 μm, a porosity of 82.3%, an average pore size of 31.2 nm, and a specific surface area of ​​70.9 m². 2 / g, biomimetic biomineralized raspberry-like biodegradable porous microspheres with a surface roughness of 69 nm and a high mesoporous ratio. Example 4:

[0078] The organic continuous phase consisted of 2.45 L of n-octane containing 1% w / v polyethylene glycol (200) dilaurate. The organic continuous phase was injected into a 10 L three-necked glass reactor equipped with a heat-insulating jacket, a top stirrer, and an arc-bladed impeller, and connected to a syringe pump for reagent addition. The reaction temperature was controlled at 40°C using a thermostat.

[0079] The aqueous phase was prepared by adding 1 L of 4 mol / L NaOH solution to 2 L of 40% w / v dextran solution, and then homogenizing at 3000 rpm for 5 min using a dual centrifugal stirrer until completely mixed. After adding the aqueous phase to the reactor, it was pre-emulsified by stirring at 1300 rpm for 15 min to form a w / o emulsion and ensure that the surfactant was completely adsorbed on the droplet surface.

[0080] The stirring speed was set to 400 rpm, and 2.4 mol of epichlorohydrin was added dropwise to the reaction vessel at a rate of 4 mL / h using a syringe pump. The reaction was stirred for 18 h to ensure complete reaction.

[0081] Microspheres were separated from the reaction mixture by filtration through a 0.22 μm filter membrane and then washed twice, sequentially with n-octane and distilled water. The microspheres were then subjected to solvent exchange with acetone and / or ethanol and dried under vacuum to completely remove the solvent.

[0082] The dried microspheres were placed in a chromatography column and washed alternately with 0.5 mol / L calcium nitrate solution and 0.3 mol / L diammonium hydrogen phosphate solution at a flow rate of 60 mL / h. This process was repeated 10 times. After washing the microspheres with purified water, they were calcined in a muffle furnace at 1000℃ for 15 h to remove the dextran template, resulting in a microsphere with an average particle size of 56.7 μm, a porosity of 90.1%, an average pore size of 43.0 nm, and a specific surface area of ​​85.8 m². 2 / g, biomimetic biomineralized raspberry-like biodegradable porous microspheres with a surface roughness of 107 nm and high mesopority. Example 5:

[0083] The organic continuous phase consisted of 2.45 L of n-octane containing 0.5% w / v polyethylene glycol (200) dilaurate. The organic continuous phase was injected into a 10 L three-necked glass reactor equipped with a heat-insulating jacket, a top stirrer, and an arc-bladed impeller, and connected to a syringe pump for reagent addition. The reaction temperature was controlled at 40°C using a thermostat.

[0084] The aqueous phase was prepared by adding 1 L of 2 mol / L NaOH solution to 2 L of 40% w / v dextran solution, and then homogenizing at 3000 rpm for 5 min using a dual centrifugal stirrer until completely mixed. After adding the aqueous phase to the reactor, it was pre-emulsified by stirring at 1300 rpm for 15 min to form a w / o emulsion and ensure that the surfactant was completely adsorbed on the droplet surface.

[0085] Set the stirring speed to 400 rpm and use a syringe pump to add 2.4 mol of epichlorohydrin dropwise to the reaction vessel at a rate of 4 mL / h. Stir the reaction for 18 h to ensure complete reaction.

[0086] Microspheres were separated from the reaction mixture by filtration through a 0.22 μm filter membrane and then washed twice, sequentially with n-octane and distilled water. The microspheres were then subjected to solvent exchange with acetone and / or ethanol and dried under vacuum to completely remove the solvent.

[0087] The dried microspheres were placed in a chromatography column and washed alternately with 0.5 mol / L calcium nitrate solution and 0.3 mol / L diammonium hydrogen phosphate solution at a flow rate of 60 mL / h. This process was repeated 10 times. After washing the microspheres with purified water, they were calcined in a muffle furnace at 1000℃ for 15 h to remove the dextran template, resulting in a microsphere with an average particle size of 46.9 μm, a porosity of 93.7%, an average pore size of 77.5 nm, and a specific surface area of ​​91.2 m². 2 / g, biomimetic biomineralized raspberry-like biodegradable porous microspheres with a surface roughness of 134 nm and a high mesoporous ratio. Example 6:

[0088] The organic continuous phase consisted of 2.45 L of n-octane containing 0.5% w / v polyethylene glycol (200) dilaurate. The organic continuous phase was injected into a 4 L three-necked glass reactor equipped with a heat-insulating jacket, a top stirrer, and an arc-bladed impeller, and connected to a syringe pump for reagent addition. The reaction temperature was controlled at 40 °C using a thermostat.

[0089] The aqueous phase was prepared by adding 1 L of 2 mol / L NaOH solution to 10 L of 20% w / v dextran solution, and then homogenizing at 3000 rpm for 5 min using a dual centrifugal stirrer until completely mixed. After adding the aqueous phase to the reactor, it was pre-emulsified by stirring at 1300 rpm for 15 min to form a w / o emulsion and ensure that the surfactant was completely adsorbed on the droplet surface.

[0090] Set the stirring speed to 400 rpm and use a syringe pump to add 0.6 mol of epichlorohydrin dropwise to the reaction vessel at a rate of 4 mL / h. Stir the reaction for 18 h to ensure complete reaction.

[0091] Microspheres were separated from the reaction mixture by filtration through a 0.22 μm filter membrane and then washed twice, sequentially with n-octane and distilled water. The microspheres were then subjected to solvent exchange with acetone and / or ethanol and dried under vacuum to completely remove the solvent.

[0092] The dried microspheres were placed in a chromatography column and washed alternately with 0.5 mol / L calcium nitrate solution and 0.3 mol / L diammonium hydrogen phosphate solution at a flow rate of 60 mL / h. This process was repeated 10 times. After washing the microspheres with purified water, they were calcined in a muffle furnace at 1000℃ for 15 h to remove the dextran template, resulting in a microsphere with an average particle size of 46.9 μm, a porosity of 93.7%, an average pore size of 38.1 nm, and a specific surface area of ​​96.2 m². 2 / g, biomimetic biomineralized raspberry-like biodegradable porous microspheres with a surface roughness of 72 nm and a high mesoporous ratio. Example 7:

[0093] The organic continuous phase consisted of 2.45 L of n-octane containing 0.5% w / v polyethylene glycol (200) dilaurate. The organic continuous phase was injected into a 10 L three-necked glass reactor equipped with a heat-insulating jacket, a top stirrer, and an arc-bladed impeller, and connected to a syringe pump for reagent addition. The reaction temperature was controlled at 40°C using a thermostat.

[0094] The aqueous phase was prepared by adding 1 L of 2 mol / L NaOH solution to 2 L of 40% w / v dextran solution, and then homogenizing at 3000 rpm for 5 min using a dual centrifugal stirrer until completely mixed. After adding the aqueous phase to the reactor, it was pre-emulsified by stirring at 1300 rpm for 15 min to form a w / o emulsion and ensure that the surfactant was completely adsorbed on the droplet surface.

[0095] Set the stirring speed to 400 rpm and use a syringe pump to add 3.2 mol of epichlorohydrin dropwise to the reaction vessel at a rate of 4 mL / h. Stir the reaction for 18 h to ensure complete reaction.

[0096] Microspheres were separated from the reaction mixture by filtration through a 0.22 μm filter membrane and then washed twice, sequentially with n-octane and distilled water. The microspheres were then subjected to solvent exchange with acetone and / or ethanol and dried under vacuum to completely remove the solvent.

[0097] The dried microspheres were placed in a chromatography column and washed alternately with 0.5 mol / L calcium nitrate solution and 0.3 mol / L diammonium hydrogen phosphate solution at a flow rate of 60 mL / h. This process was repeated 10 times. After washing the microspheres with purified water, they were calcined in a muffle furnace at 1000℃ for 15 h to remove the dextran template, resulting in a microsphere with an average particle size of 51.2 μm, a porosity of 94.8%, an average pore size of 114.5 nm, and a specific surface area of ​​133.1 m². 2 / g, biomimetic biomineralized raspberry-like biodegradable porous microspheres with a surface roughness of 145 nm and a high mesoporous ratio. Example 8:

[0098] The organic continuous phase consisted of 2.45 L of n-octane containing 0.5% w / v polyethylene glycol (200) dilaurate. The organic continuous phase was injected into a 10 L three-necked glass reactor equipped with a heat-insulating jacket, a top stirrer, and an arc-bladed impeller, and connected to a syringe pump for reagent addition. The reaction temperature was controlled at 40°C using a thermostat.

[0099] The aqueous phase was prepared by adding 1 L of 4 mol / L NaOH solution to 2 L of 20% w / v dextran solution, and then homogenizing at 3000 rpm for 5 min using a dual centrifugal stirrer until completely mixed. After adding the aqueous phase to the reactor, it was pre-emulsified by stirring at 1300 rpm for 15 min to form a w / o emulsion and ensure that the surfactant was completely adsorbed on the droplet surface.

[0100] Set the stirring speed to 400 rpm and use a syringe pump to add 1.6 mol of epichlorohydrin dropwise to the reaction vessel at a rate of 4 mL / h. Stir the reaction for 18 h to ensure complete reaction.

[0101] Microspheres were separated from the reaction mixture by filtration through a 0.22 μm filter membrane and then washed twice, sequentially with n-octane and distilled water. The microspheres were then subjected to solvent exchange with acetone and / or ethanol and dried under vacuum to completely remove the solvent.

[0102] The dried microspheres were placed in a chromatography column and washed alternately with 0.5 mol / L calcium nitrate solution and 0.3 mol / L diammonium hydrogen phosphate solution at a flow rate of 60 mL / h. This process was repeated 10 times. After washing the microspheres with purified water, they were calcined in a muffle furnace at 1000℃ for 15 h to remove the dextran template, resulting in a microsphere with an average particle size of 39.9 μm, a porosity of 83.7%, an average pore size of 28.4 nm, and a specific surface area of ​​74.4 m². 2 / g, biomimetic biomineralized raspberry-like biodegradable porous microspheres with a surface roughness of 65 nm and a high mesoporous ratio.

[0103] Example 9:

[0104] The organic continuous phase consisted of 2.45 L of n-octane containing 0.5% w / v sorbitan monolaurate. The organic continuous phase was injected into a 10 L three-necked glass reactor equipped with a heat-insulating jacket, a top stirrer, and an arc-bladed impeller, and connected to a syringe pump for reagent addition. The reaction temperature was controlled at 40°C using a thermostat.

[0105] The aqueous phase was prepared by adding 1 L of 4 mol / L NaOH solution to 2 L of 40% w / v dextran solution, and then homogenizing at 3000 rpm for 5 min using a dual centrifugal stirrer until completely mixed. After adding the aqueous phase to the reactor, it was pre-emulsified by stirring at 1300 rpm for 15 min to form a w / o emulsion and ensure that the surfactant was completely adsorbed on the droplet surface.

[0106] Set the stirring speed to 400 rpm and use a syringe pump to add 3.2 mol of glutaraldehyde dropwise to the reaction vessel at a rate of 4 mL / h. Stir the reaction for 18 h to ensure complete reaction.

[0107] Microspheres were separated from the reaction mixture by filtration through a 0.22 μm filter membrane and then washed twice, sequentially with n-octane and distilled water. The microspheres were then subjected to solvent exchange with acetone and / or ethanol and dried under vacuum to completely remove the solvent.

[0108] The dried microspheres were placed in a chromatography column and washed alternately with 0.5 mol / L calcium nitrate solution and 0.3 mol / L diammonium hydrogen phosphate solution at a flow rate of 60 mL / h. This process was repeated 10 times. After washing the microspheres with purified water, they were calcined in a muffle furnace at 1000℃ for 15 h to remove the dextran template, resulting in a microsphere with an average particle size of 75.5 μm, a porosity of 85.2%, an average pore size of 31.1 nm, and a specific surface area of ​​81.0 m². 2 / g, biomimetic biomineralized raspberry-like biodegradable porous microspheres with a surface roughness of 71 nm and a high mesoporous ratio. Example 10:

[0109] The organic continuous phase consisted of 2.45 L of n-octane containing 0.5% w / v sorbitan monolaurate. The organic continuous phase was injected into a 10 L three-necked glass reactor equipped with a heat-insulating jacket, a top stirrer, and an arc-bladed impeller, and connected to a syringe pump for reagent addition. The reaction temperature was controlled at 40°C using a thermostat.

[0110] The aqueous phase was prepared by adding 1 L of 2 mol / L NaOH solution to 2 L of 20% w / v dextran solution, and then homogenizing at 3000 rpm for 5 min using a dual centrifugal stirrer until completely mixed. After adding the aqueous phase to the reactor, it was pre-emulsified by stirring at 1300 rpm for 15 min to form a w / o emulsion and ensure that the surfactant was completely adsorbed on the droplet surface.

[0111] Set the stirring speed to 400 rpm and use a syringe pump to add 1.6 mol of glutaraldehyde dropwise to the reaction vessel at a rate of 4 mL / h. Stir the reaction for 18 h to ensure complete reaction.

[0112] Microspheres were separated from the reaction mixture by filtration through a 0.22 μm filter membrane and then washed twice, sequentially with n-octane and distilled water. The microspheres were then subjected to solvent exchange with acetone and / or ethanol and dried under vacuum to completely remove the solvent.

[0113] The dried microspheres were placed in a chromatography column and washed alternately with 0.5 mol / L calcium nitrate solution and 0.3 mol / L diammonium hydrogen phosphate solution at a flow rate of 60 mL / h. This process was repeated 10 times. After washing the microspheres with purified water, they were calcined in a muffle furnace at 1000℃ for 15 h to remove the dextran template, resulting in a microsphere with an average particle size of 26.0 μm, a porosity of 85.2%, an average pore size of 27.5 nm, and a specific surface area of ​​85.7 m². 2 / g, biomimetic biomineralized raspberry-like biodegradable porous microspheres with a surface roughness of 77 nm and a high mesoporous ratio. Example 11:

[0114] The organic continuous phase consisted of 2.45 L of n-octane containing 0.5% w / v sorbitan monolaurate. The organic continuous phase was injected into a 10 L three-necked glass reactor equipped with a heat-insulating jacket, a top stirrer, and an arc-bladed impeller, and connected to a syringe pump for reagent addition. The reaction temperature was controlled at 40°C using a thermostat.

[0115] The aqueous phase was prepared by adding 1 L of 2 mol / L NaOH solution to 2 L of 40% w / v dextran solution, and then homogenizing at 3000 rpm for 5 min using a dual centrifugal stirrer until completely mixed. After adding the aqueous phase to the reactor, it was pre-emulsified by stirring at 1300 rpm for 15 min to form a w / o emulsion and ensure that the surfactant was completely adsorbed on the droplet surface.

[0116] Set the stirring speed to 400 rpm and use a syringe pump to add 3.2 mol of glutaraldehyde dropwise to the reaction vessel at a rate of 4 mL / h. Stir the reaction for 18 h to ensure complete reaction.

[0117] Microspheres were separated from the reaction mixture by filtration through a 0.22 μm filter membrane and then washed twice, sequentially with n-octane and distilled water. The microspheres were then subjected to solvent exchange with acetone and / or ethanol and dried under vacuum to completely remove the solvent.

[0118] The dried microspheres were placed in a chromatography column and washed alternately with 0.5 mol / L calcium nitrate solution and 0.3 mol / L diammonium hydrogen phosphate solution at a flow rate of 60 mL / h. This process was repeated 10 times. After washing the microspheres with purified water, they were calcined in a muffle furnace at 1000℃ for 15 h to remove the dextran template, resulting in a microsphere with an average particle size of 26.0 μm, a porosity of 87.6%, an average pore size of 68.3 nm, and a specific surface area of ​​115.5 m². 2 / g, biomimetic biomineralized raspberry-like biodegradable porous microspheres with a surface roughness of 132 nm and high mesopority. Example 12:

[0119] The organic continuous phase consisted of 2.45 L of n-octane containing 1% w / v sorbitan monolaurate. The organic continuous phase was injected into a 10 L three-necked glass reactor equipped with a heat-insulating jacket, a top stirrer, and an arc-bladed impeller, and connected to a syringe pump for reagent addition. The reaction temperature was controlled at 40°C using a thermostat.

[0120] The aqueous phase was prepared by adding 1 L of 2 mol / L NaOH solution to 2 L of 40% w / v dextran solution, and then homogenizing at 3000 rpm for 5 min using a dual centrifugal stirrer until completely mixed. After adding the aqueous phase to the reactor, it was pre-emulsified by stirring at 1300 rpm for 15 min to form a w / o emulsion and ensure that the surfactant was completely adsorbed on the droplet surface.

[0121] The stirring speed was set to 400 rpm, and 2.4 mol of epichlorohydrin was added dropwise to the reaction vessel at a rate of 4 mL / h using a syringe pump. The reaction was stirred for 18 h to ensure complete reaction.

[0122] Microspheres were separated from the reaction mixture by filtration through a 0.22 μm filter membrane and then washed twice, sequentially with n-octane and distilled water. The microspheres were then subjected to solvent exchange with acetone and / or ethanol and dried under vacuum to completely remove the solvent.

[0123] The dried microspheres were placed in a chromatography column and washed alternately with 0.5 mol / L calcium nitrate solution and 0.3 mol / L diammonium hydrogen phosphate solution at a flow rate of 60 mL / h. This process was repeated 10 times. After washing the microspheres with purified water, they were calcined in a muffle furnace at 1000℃ for 15 h to remove the dextran template, resulting in a microsphere with an average particle size of 39.1 μm, a porosity of 81.0%, an average pore size of 24.4 nm, and a specific surface area of ​​62.4 m². 2 / g, biomimetic biomineralized raspberry-like biodegradable porous microspheres with a surface roughness of 65 nm and a high mesoporous ratio.

[0124] Example 13:

[0125] The organic continuous phase consisted of 2.45 L of n-octane containing 1% w / v sorbitan monolaurate. The organic continuous phase was injected into a 10 L three-necked glass reactor equipped with a heat-insulating jacket, a top stirrer, and an arc-bladed impeller, and connected to a syringe pump for reagent addition. The reaction temperature was controlled at 40°C using a thermostat.

[0126] The aqueous phase was prepared by adding 1 L of 2 mol / L NaOH solution to 2 L of 20% w / v dextran solution, and then homogenizing at 3000 rpm for 5 min using a dual centrifugal stirrer until completely mixed. After adding the aqueous phase to the reactor, it was pre-emulsified by stirring at 1300 rpm for 15 min to form a w / o emulsion and ensure that the surfactant was completely adsorbed on the droplet surface.

[0127] Set the stirring speed to 400 rpm and use a syringe pump to add 1.2 mol of epichlorohydrin dropwise to the reaction vessel at a rate of 4 mL / h. Stir the reaction for 18 h to ensure complete reaction.

[0128] Microspheres were separated from the reaction mixture by filtration through a 0.22 μm filter membrane and then washed twice, sequentially with n-octane and distilled water. The microspheres were then subjected to solvent exchange with acetone and / or ethanol and dried under vacuum to completely remove the solvent.

[0129] The dried microspheres were placed in a chromatography column and washed alternately with 0.5 mol / L calcium nitrate solution and 0.3 mol / L diammonium hydrogen phosphate solution at a flow rate of 60 mL / h. This process was repeated 10 times. After washing the microspheres with purified water, they were calcined in a muffle furnace at 1000℃ for 15 h to remove the dextran template, resulting in a microsphere with an average particle size of 25.6 μm, a porosity of 83.4%, an average pore size of 38.2 nm, and a specific surface area of ​​66.8 m². 2 / g, biomimetic biomineralized raspberry-like biodegradable porous microspheres with a surface roughness of 72 nm and a high mesoporous ratio.

[0130] Example 14:

[0131] The organic continuous phase consisted of 2.45 L of n-octane containing 0.5% w / v sorbitan monolaurate. The organic continuous phase was injected into a 10 L three-necked glass reactor equipped with a heat-insulating jacket, a top stirrer, and an arc-bladed impeller, and connected to a syringe pump for reagent addition. The reaction temperature was controlled at 40°C using a thermostat.

[0132] The aqueous phase was prepared by adding 1 L of 4 mol / L NaOH solution to 2 L of 20% w / v dextran solution, and then homogenizing at 3000 rpm for 5 min using a dual centrifugal stirrer until completely mixed. After adding the aqueous phase to the reactor, it was pre-emulsified by stirring at 1300 rpm for 15 min to form a w / o emulsion and ensure that the surfactant was completely adsorbed on the droplet surface.

[0133] Set the stirring speed to 400 rpm and use a syringe pump to add 1.2 mol of 1,4-butanediol diglycidyl ether dropwise to the reaction vessel at a rate of 4 mL / h. Stir the reaction for 18 h to ensure complete reaction.

[0134] Microspheres were separated from the reaction mixture by filtration through a 0.22 μm filter membrane and then washed twice, sequentially with n-octane and distilled water. The microspheres were then subjected to solvent exchange with acetone and / or ethanol and dried under vacuum to completely remove the solvent.

[0135] The dried microspheres were placed in a chromatography column and washed alternately with 0.5 mol / L calcium nitrate solution and 0.3 mol / L diammonium hydrogen phosphate solution at a flow rate of 60 mL / h. This process was repeated 10 times. After washing the microspheres with purified water, they were calcined in a muffle furnace at 1000℃ for 15 h to remove the dextran template, resulting in a microsphere with an average particle size of 43.2 μm, a porosity of 79.8%, an average pore size of 11.5 nm, and a specific surface area of ​​52.7 m². 2 / g, biomimetic biomineralized raspberry-like biodegradable porous microspheres with a surface roughness of 45 nm and a high mesoporous ratio.

[0136] Example 15:

[0137] The organic continuous phase consisted of 2.45 L of n-octane containing 1% w / v N,N-dimethylhexanoamide. The organic continuous phase was injected into a 10 L three-necked glass reactor equipped with a heat-insulating jacket, a top stirrer, and an arc-bladed impeller, and connected to a syringe pump for reagent addition. The reaction temperature was controlled at 40°C using a thermostat.

[0138] The aqueous phase was prepared by adding 1 L of 4 mol / L NaOH solution to 2 L of 40% w / v dextran solution, and then homogenizing at 3000 rpm for 5 min using a dual centrifugal stirrer until completely mixed. After adding the aqueous phase to the reactor, it was pre-emulsified by stirring at 1300 rpm for 15 min to form a w / o emulsion and ensure that the surfactant was completely adsorbed on the droplet surface.

[0139] Set the stirring speed to 400 rpm and use a syringe pump to add 3.2 mol of 1,4-butanediol diglycidyl ether dropwise to the reaction vessel at a rate of 4 mL / h. Stir the reaction for 18 h to ensure complete reaction.

[0140] Microspheres were separated from the reaction mixture by filtration through a 0.22 μm filter membrane and then washed twice, sequentially with n-octane and distilled water. The microspheres were then subjected to solvent exchange with acetone and / or ethanol and dried under vacuum to completely remove the solvent.

[0141] The dried microspheres were placed in a chromatography column and washed alternately with 0.5 mol / L calcium nitrate solution and 0.3 mol / L diammonium hydrogen phosphate solution at a flow rate of 60 mL / h. This process was repeated 10 times. After washing the microspheres with purified water, they were calcined in a muffle furnace at 1000℃ for 15 h to remove the dextran template, resulting in a microsphere with an average particle size of 74.5 μm, a porosity of 92.3%, an average pore size of 17.8 nm, and a specific surface area of ​​110.4 m². 2 / g, biomimetic biomineralized raspberry-like biodegradable porous microspheres with a surface roughness of 88 nm and a high mesoporous ratio.

[0142] Example 16:

[0143] The organic continuous phase consisted of 2.45 L of n-octane containing 0.05% w / v N,N-dimethylhexanoamide. The organic continuous phase was injected into a 10 L three-necked glass reactor equipped with a heat-insulating jacket, a top stirrer, and an arc-bladed impeller, and connected to a syringe pump for reagent addition. The reaction temperature was controlled at 40°C using a thermostat.

[0144] The aqueous phase was prepared by adding 1 L of 4 mol / L NaOH solution to 2 L of 40% w / v dextran solution, and then homogenizing at 3000 rpm for 5 min using a dual centrifugal stirrer until completely mixed. After adding the aqueous phase to the reactor, it was pre-emulsified by stirring at 1300 rpm for 15 min to form a w / o emulsion and ensure that the surfactant was completely adsorbed on the droplet surface.

[0145] Set the stirring speed to 400 rpm and use a syringe pump to add 2.4 mol of 1,4-butanediol diglycidyl ether dropwise to the reaction vessel at a rate of 4 mL / h. Stir the reaction for 18 h to ensure complete reaction.

[0146] Microspheres were separated from the reaction mixture by filtration through a 0.22 μm filter membrane and then washed twice, sequentially with n-octane and distilled water. The microspheres were then subjected to solvent exchange with acetone and / or ethanol and dried under vacuum to completely remove the solvent.

[0147] The dried microspheres were placed in a chromatography column and washed alternately with 0.5 mol / L calcium nitrate solution and 0.3 mol / L diammonium hydrogen phosphate solution at a flow rate of 60 mL / h. This process was repeated 10 times. After washing the microspheres with purified water, they were calcined in a muffle furnace at 1000℃ for 15 h to remove the dextran template, resulting in a microsphere with an average particle size of 57.10 μm, a porosity of 91.2%, an average pore size of 56.5 nm, and a specific surface area of ​​101.5 m². 2 / g, biomimetic biomineralized raspberry-like biodegradable porous microspheres with a surface roughness of 83 nm and a high mesoporous ratio.

[0148] Example 17:

[0149] The organic continuous phase consisted of 2.45 L of n-octane containing 2% w / v N,N-dimethylhexanoamide. The organic continuous phase was injected into a 10 L three-necked glass reactor equipped with a heat-insulating jacket, a top stirrer, and an arc-bladed impeller, and connected to a syringe pump for reagent addition. The reaction temperature was controlled at 40°C using a thermostat.

[0150] The aqueous phase was prepared by adding 1 L of 2 mol / L NaOH solution to 2 L of 10% w / v dextran solution, and then homogenizing at 3000 rpm for 5 min using a dual centrifugal stirrer until completely mixed. After adding the aqueous phase to the reactor, it was pre-emulsified by stirring at 1300 rpm for 15 min to form a w / o emulsion and ensure that the surfactant was completely adsorbed on the droplet surface.

[0151] Set the stirring speed to 400 rpm and use a syringe pump to add 6.4 mol of 1,4-butanediol diglycidyl ether dropwise to the reaction vessel at a rate of 2 mL / h. Stir the reaction for 18 h to ensure complete reaction.

[0152] Microspheres were separated from the reaction mixture by filtration through a 0.22 μm filter membrane and then washed twice, sequentially with n-octane and distilled water. The microspheres were then subjected to solvent exchange with acetone and / or ethanol and dried under vacuum to completely remove the solvent.

[0153] The dried microspheres were placed in a chromatography column and washed alternately with 0.5 mol / L calcium nitrate solution and 0.3 mol / L diammonium hydrogen phosphate solution at a flow rate of 60 mL / h. This process was repeated 10 times. After washing the microspheres with purified water, they were calcined in a muffle furnace at 1000℃ for 15 h to remove the dextran template, resulting in a microsphere with an average particle size of 57.10 μm, a porosity of 91.2%, an average pore size of 142.0 nm, and a specific surface area of ​​101.5 m². 2 / g, biomimetic biomineralized raspberry-like biodegradable porous microspheres with a surface roughness of 83 nm and a high mesoporous ratio.

[0154] Example 18:

[0155] The organic continuous phase consisted of 2.45 L of n-octane containing 2% w / v N,N-dimethylhexanoamide. The organic continuous phase was injected into a 10 L three-necked glass reactor equipped with a heat-insulating jacket, a top stirrer, and an arc-bladed impeller, and connected to a syringe pump for reagent addition. The reaction temperature was controlled at 40°C using a thermostat.

[0156] The aqueous phase was prepared by adding 1 L of 4 mol / L NaOH solution to 2 L of 80% w / v dextran solution, and then homogenizing at 3000 rpm for 5 min using a dual centrifugal stirrer until completely mixed. After adding the aqueous phase to the reactor, it was pre-emulsified by stirring at 1300 rpm for 15 min to form a w / o emulsion and ensure that the surfactant was completely adsorbed on the droplet surface.

[0157] Set the stirring speed to 400 rpm and use a syringe pump to add 1.2 mol of 1,4-butanediol diglycidyl ether dropwise to the reaction vessel at a rate of 4 mL / h. Stir the reaction for 18 h to ensure complete reaction.

[0158] Microspheres were separated from the reaction mixture by filtration through a 0.22 μm filter membrane and then washed twice, sequentially with n-octane and distilled water. The microspheres were then subjected to solvent exchange with acetone and / or ethanol and dried under vacuum to completely remove the solvent.

[0159] The dried microspheres were placed in a chromatography column and washed alternately with 0.5 mol / L calcium nitrate solution and 0.3 mol / L diammonium hydrogen phosphate solution at a flow rate of 60 mL / h. This process was repeated 10 times. After washing the microspheres with purified water, they were calcined in a muffle furnace at 1000℃ for 15 h to remove the dextran template, resulting in a microsphere with an average particle size of 57.10 μm, a porosity of 91.2%, an average pore size of 2.1 nm, and a specific surface area of ​​101.5 m². 2 / g, biomimetic biomineralized raspberry-like biodegradable porous microspheres with a surface roughness of 83 nm and a high mesoporous ratio.

[0160] Comparative Example 1:

[0161] Compared to Example 1, the organic continuous phase did not contain surfactants, while other conditions remained the same. During the synthesis reaction, the dextran aggregated and failed to form microspheres, thus it could not serve as a mineralization template for high-mesoporous raspberry-like biodegradable porous microspheres. Comparative Example 2:

[0162] Compared to Example 1, no cross-linking agent was added, and all other conditions were the same. After stirring was stopped and the emulsion was broken, the dextran was not completely cross-linked, and the solution was an uneven gel, which could not be used as a mineralization template for high mesoporous raspberry-like biodegradable porous microspheres. Comparative Example 3:

[0163] Compared to Example 1, the aqueous phase did not contain sodium hydroxide, while other conditions remained the same. After the synthesis reaction, the dextran was a two-phase solution and could not serve as a mineralization template for high-mesoporous raspberry-like biodegradable porous microspheres.

[0164] Comparative Example 4:

[0165] The organic continuous phase consisted of 2.45 L of n-octane containing 0.05% w / v polyethylene glycol (200) monolaurate. The organic continuous phase was injected into a 10 L three-necked glass reactor equipped with a heat-insulating jacket, a top stirrer, and an arc-bladed impeller, and connected to a syringe pump for reagent addition. The reaction temperature was controlled at 40°C using a thermostat.

[0166] The aqueous phase was prepared by adding 20 L of 0.1 mol / L NaOH solution to 8 L of 5% w / v dextran solution, and then homogenizing at 3000 rpm for 5 min using a dual centrifugal stirrer until completely mixed. After adding the aqueous phase to the reactor, it was pre-emulsified by stirring at 1300 rpm for 15 min to form a w / o emulsion and ensure that the surfactant was completely adsorbed on the droplet surface.

[0167] The stirring speed was set to 400 rpm, and 1.6 mol of epichlorohydrin was added dropwise to the reaction vessel at a rate of 4 mL / h using a syringe pump. The reaction was stirred for 18 h to ensure complete reaction. The microspheres were separated from the reaction mixture by filtration through a 0.22 μm filter membrane and then washed twice, successively with n-octane and distilled water. The microspheres were then solvent-exchanged with acetone and / or ethanol and dried under vacuum to completely remove the solvent.

[0168] The dried microspheres were placed in a chromatography column and washed alternately with 0.5 mol / L calcium nitrate solution and 0.3 mol / L diammonium hydrogen phosphate solution at a flow rate of 60 mL / h. This process was repeated 10 times. After washing the microspheres with purified water, they were calcined in a muffle furnace at 1000℃ for 15 h to remove the dextran template, resulting in a microsphere with an average particle size of 0.07 μm, a porosity of 98.9%, an average pore size of 182.0 nm, and a specific surface area of ​​392.3 m². 2 / g, biomimetic biomineralized raspberry-like biodegradable porous microspheres with a surface roughness of 2 nm and a high mesoporous ratio.

[0169] Comparative Example 5:

[0170] The organic continuous phase consisted of 2.45 L of n-octane containing 5% w / v polyethylene glycol (200) monolaurate. The organic continuous phase was injected into a 10 L three-necked glass reactor equipped with a heat-insulating jacket, a top stirrer, and an arc-bladed impeller, and connected to a syringe pump for reagent addition. The reaction temperature was controlled at 40°C using a thermostat.

[0171] The aqueous phase was prepared by adding 0.4 L of 10 mol / L NaOH solution to 0.667 L of 60% w / v dextran solution, followed by homogenization at 3000 rpm for 5 min using a dual centrifugal stirrer until completely mixed. After adding the aqueous phase to the reactor, pre-emulsification was performed by stirring at 1300 rpm for 15 min to form a w / o emulsion, ensuring complete adsorption of the surfactant onto the droplet surface.

[0172] Set the stirring speed to 400 rpm and use a syringe pump to add 2.4 mol of ethylene glycol diglycidyl ether dropwise to the reaction vessel at a rate of 4 mL / h. Stir the reaction for 18 h to ensure complete reaction.

[0173] Microspheres were separated from the reaction mixture by filtration through a 0.22 μm filter membrane and then washed twice, sequentially with n-octane and distilled water. The microspheres were then subjected to solvent exchange with acetone and / or ethanol and dried under vacuum to completely remove the solvent.

[0174] The dried microspheres were placed in a chromatography column and washed alternately with 0.5 mol / L calcium nitrate solution and 0.3 mol / L diammonium hydrogen phosphate solution at a flow rate of 60 mL / h. This process was repeated 8 times. After washing the microspheres with purified water, they were calcined in a muffle furnace at 900 °C for 18 h to remove the dextran template, resulting in a microsphere with an average particle size of 350 μm, a porosity of 23.3%, an average pore size of 161.0 nm, and a specific surface area of ​​101.2 m². 2 / g, biomimetic biomineralized raspberry-like biodegradable porous microspheres with a surface roughness of 189 nm and high mesopority.

[0175] As can be seen from Comparative Examples 1 to 3, when preparing the dextran microsphere template of the present invention, if a surfactant, crosslinking agent or sodium hydroxide is not added, it cannot be used as a mineralization template for raspberry-like biodegradable porous microspheres with high mesopority.

[0176] Table 1

[0177] .

[0178] Load testing experiment:

[0179] Take 0.1 g of the high mesoporous raspberry-shaped biodegradable porous microspheres prepared in Examples 1 and 2, and Comparative Examples 4 and 5, and immerse them in 100 mL of a 100 μg / L solution containing growth factors. After immersion for 3 h, remove them and measure the concentration of growth factors in the loaded solution using ultraviolet spectrophotometry to calculate the loading rate of growth factors. The growth factors include fibroblast growth factor and epidermal growth factor. Specific data are as follows:

[0180] Table 2

[0181]

[0182] Table 1 shows the range of parameters such as raw material dosage and concentration for Examples 1 to 18 and Comparative Examples 4 and 5. Combined with the loading test data in Table 2, it can be seen that the growth factor loading rates of Examples 1 and 2 are 48.7% and 45.2%, respectively, which are greater than the 17.2% and 5.8% of Comparative Examples 4 and 5.

[0183] Therefore, the raspberry-shaped biodegradable porous microspheres with high mesopority of the present invention have the beneficial effect of high drug loading.

[0184] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A raspberry-like biodegradable porous microsphere with high mesoporous content, characterized in that: The high mesoporous raspberry-like biodegradable porous microspheres are formed by the self-assembly of nano-sized calcium hydroxyphosphate particles under the thermodynamic behavior of reduced excess surface free energy, resulting in nano- to micro-sized microspheres with rough surfaces. The high mesoporous raspberry-like biodegradable porous microspheres are spherical or irregularly shaped and have a nano-sized pore structure, which has one or more combinations of cross-linked pores, through pores, and blind pores.

2. The high mesoporous raspberry-shaped biodegradable porous microsphere as described in claim 1, characterized in that: The surface roughness of the microspheres is 5–150 nm; the particle size of the microspheres ranges from 300 nm to 150 μm; the average pore size of the microspheres is 0.1 nm–150 nm; the porosity of the microspheres is 50%–95%; and the specific surface area of ​​the microspheres is 30–200 m². 2 / g.

3. A method for preparing raspberry-like biodegradable porous microspheres with high mesopority as described in claim 1, characterized in that, Includes the following steps: By mineralizing the dextran microsphere template, mineralized dextran microsphere templates are obtained; after calcining the dextran microsphere templates, raspberry-like biodegradable porous microspheres with high mesopority are prepared.

4. The method for preparing raspberry-like biodegradable porous microspheres with high mesopority as described in claim 3, characterized in that, The method for preparing the dextran microsphere template is as follows: S1. Add the dextran solution with a concentration of 10-50% W / V to the alkaline solution, mix well, and obtain the aqueous phase; S2. The aqueous phase is added to the organic phase, mixed and pre-emulsified to obtain an emulsion, wherein the organic phase is n-octane containing a surfactant; S3. Add a crosslinking agent to the emulsion, react, filter and separate the crosslinked product; S4. The crosslinked material is washed with n-octane and distilled water, then solvent exchanged with a volatile solvent, and then dried in a vacuum to obtain a dextran microsphere template.

5. The method for preparing raspberry-like biodegradable porous microspheres with high mesopority as described in claim 4, characterized in that: In step S1, the concentration of the dextran solution is 20–40% W / V; the weight-average molecular weight (Mw) of the dextran is 40,000–60,000, and its polymer dispersibility index (PDI) is <2.0; the alkaline solution is an aqueous NaOH solution with a concentration of 1–10 mol / L; the mixing in step S1 is performed by stirring at a speed of 2000–4000 rpm for 1–10 min.

6. The method for preparing raspberry-like biodegradable porous microspheres with high mesopority as described in claim 4, characterized in that: In step S2, the volume ratio of the aqueous phase to the organic phase is 1–5:1; the content of the surfactant in the organic phase is 0.1%–2% W / V; the surfactant is at least one selected from polyethylene glycol monolaurate, glycerol monolaurate, N,N-dimethylhexamethylene amide, polyglycerol fatty acid ester, polyethylene glycol dilaurate, diglycerol monolaurate, sorbitol monolaurate, nonylphenoxypolyethoxyethanol, and Span 80; the mixing in step S2 is performed by stirring at a speed of 1000–2000 rpm for 5–30 min; In step S3, the crosslinking agent is at least one selected from diisocyanate, glutaraldehyde, epichlorohydrin, epibromopropane, ethylene glycol diglycidyl ether, and 1,4-butanediol diglycidyl ether; the crosslinking agent is added dropwise using a syringe pump at a rate of 1–10 mL / h; the ratio of dextran to the crosslinking agent is 1:0.5–40 kg / mol; the reaction time is 12–48 h; the reaction temperature is 20°C–50°C; the reaction is stirred at a speed of 200–800 rpm; and the filtration in step S3 uses a microporous membrane with reduced pressure filtration, the membrane having a pore size of 0.22 μm–0.45 μm. The volatile solvent in step S4 is at least one of ethanol or acetone.

7. The method for preparing raspberry-like biodegradable porous microspheres with high mesopority as described in claim 3, characterized in that, The mineralization process is as follows: the dextran microsphere template is alternately rinsed with calcium salt aqueous solution and phosphate aqueous solution, and the number of alternating rinses is ≥2 times, followed by rinsing with purified water.

8. The method for preparing raspberry-like biodegradable porous microspheres with high mesopority as described in claim 7, characterized in that: The concentration of the phosphate aqueous solution is 0.1–2.0 mol / L; the flow rate of the phosphate aqueous solution during rinsing is 10–100 mL / h; the phosphate aqueous solution is one of diammonium hydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, ammonium dihydrogen phosphate, sodium dihydrogen phosphate, and potassium dihydrogen phosphate. The concentration of the calcium salt aqueous solution is 0.1–2.0 mol / L; the flow rate of the calcium salt aqueous solution during rinsing is 10–100 mL / h; the calcium salt aqueous solution is one of calcium chloride, calcium bromide, calcium nitrate, and dicalcium hydrogen phosphate.

9. The method for preparing raspberry-like biodegradable porous microspheres with high mesopority as described in claim 3, characterized in that: The calcination temperature is 600–1200 °C; the calcination time is 2–24 h.

10. The application of a high mesoporous raspberry-like biodegradable porous microsphere as described in any one of claims 1 to 9 in a drug sustained-release carrier or tissue engineering scaffold.

Images