CN107789332B - Calcium carbonate/calcium alginate composite microspheres capable of adjusting drug release rate and prepared based on double-aqueous-phase biomineralization technology - Google Patents
Calcium carbonate/calcium alginate composite microspheres capable of adjusting drug release rate and prepared based on double-aqueous-phase biomineralization technology Download PDFInfo
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/20—Pills, tablets, discs, rods
- A61K9/28—Dragees; Coated pills or tablets, e.g. with film or compression coating
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- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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Abstract
The invention discloses a calcium carbonate/calcium alginate composite microsphere with adjustable drug release rate prepared based on a double-aqueous-phase biomineralization technology and a preparation method thereof, belonging to the fields of high polymer materials and controlled release drug carriers. The preparation method disclosed by the invention is simple to operate, low in energy consumption and mild and nontoxic in system; by utilizing the selective distribution characteristic of the double water phase relative to the macromolecular protein, the macromolecular protein medicine can be well retained in the internal phase of the double water phase liquid drop template and is excellently encapsulated in the composite calcium alginate microspheres during solidification, so that the composite calcium alginate microsphere has excellent encapsulation efficiency on the protein medicine; moreover, calcium carbonate/calcium alginate microspheres with different compound degrees can be prepared by adjusting the concentration of urease of the system, so that the release rate of the drug embedded in the microspheres can be controlled; overcomes the defects that the traditional composite calcium alginate microsphere network has larger pores and the inclusion is easy to flow out. Can be widely applied to the fields of biological medical treatment, cosmetic industry, food industry, enzyme engineering and the like.
Description
Technical Field
The invention relates to the field of high polymer materials, in particular to a calcium carbonate/calcium alginate composite microsphere prepared by using a double-aqueous-phase biomineralization technology and a preparation method thereof.
The aqueous two-phase system is composed of two aqueous solutions of high-molecular polymer or one aqueous solution of high-molecular polymer and one salt solution. When the concentration of the two solutions exceeds a certain threshold value, the two mixed solutions can spontaneously phase separate,a two-phase system with the upper and lower phases being aqueous solution is formed, namely a two-water-phase system. The double water phase system has the advantages of extremely high biocompatibility to bioactive substances, and simultaneously has steric effect, and macromolecular substances are not easy to transfer across interfaces, so that the double water phase system has wide application in the aspect of biology. Cace, etc. uses two-phase polymer aqueous medium to simulate macromolecular crowding and intracellular environment division, and makes biomineralization in polyethylene glycol and dextran two-aqueous phase system, i.e. utilizes urease to catalyze CO produced by urea hydrolysis3 2-Production of calcium carbonate by reaction with calcium ions already present in the solution[1]David N.Cacace,ChrisstineD.keating,Biocatalyzed mineralization in an aqueous two-phase system:effectof background polymers and enzyme partitioning[J].Journal Material ChemistryB,2013,1,1794-1803)。
Biomineralization refers to the process of controlling the generation of inorganic minerals by organisms through self regulation, and is a simple method capable of preparing complex structures, and the comprehensive performance of the materials obtained by the method is much better than that of artificial synthesis. Xie et al utilize alginate to control the crystallization process of forming nano calcium carbonate and hydroxyapatite, and research results thereof are beneficial to the design of composite materials in the future ([2]M.Xie,M.
J.P.Andreassen,S.M.Selbach,B.L.Strand,P.SikorskiAlginate-controlled formation of nanoscale calcium carbonate andhydroxyapatite mineral phase within hydrogel networks[J].Acta Biomaterialia,2010,6(9):3665-3675)。
The calcium alginate microspheres are natural and non-toxic, have good biocompatibility and biodegradability, are simple, mild and safe in preparation process, and are widely applied to biological reaction engineering and medicine delivery systems at present. For example, Moebus et al, prepares calcium alginate microspheres loaded with Bovine Serum Albumin (BSA) by spray drying a polymer drug solution, but the spray drying equipment has high energy consumption and the preparation process is complicated ([3]Moebus K,Siepmann J,Bodmeier R.Novel preparation techniques foralginate-poloxamer microparticles controlling protein release on mucosalsurfaces.European Journal of Pharmaceutical Sciences,2012,45(3):358-366)。
However, the single calcium alginate hydrogel microsphere has the defects of large network pore size, poor mechanical strength, easy water absorption and swelling, and then breakage, so that the encapsulated substance flows out, and the reuse rate is not high. The preparation of chitosan-coated calcium alginate composite microspheres by using an emulsification method such as sony-Min improves the mechanical strength of calcium alginate, but the preparation process can not avoid using toxic organic reagents and can not maintain the activity of biomacromolecule drugs (the formula (I) (II))[4]Preparation of Songlimin, Li Ming, Niuxin, Ni sweet, high sprout, Wang Xiao bud, Zhang Bao Hua, beta-cypermethrin/sodium alginate/chitosan gel microsphere [ J]Application of chemical engineering, 2016, (11): 2118-2124).
Shi et al used aliphatic polyurethane-amines (PU) as the thermally responsive component at high pressure CO2Under the condition, adopting biomimetic mineralization technology to prepare alginate/calcium carbonate hybrid beads with pH and thermal response drug release characteristics, and using polyacrylic acid (PAA) as a crystal growth additive to control the structure of the hybrid beads (PAA)[5]Shi,J.；Zhang,Z.；Li,G.；Cao,S.Biomimetic Fabrication of Alginate/Caco3Hybrid Beads for Dual-ResponsiveDrug Delivery under Compressed CO2[J].Journal Material Chemistry B,2011,21(40)16028-16034.)
The method has violent reaction conditions and is also not beneficial to keeping the activity of the biomacromolecule medicine. Isiklan et al, which uses glutaraldehyde as cross-linking agent and hydrochloric acid as catalyst to prepare copolymer material of alginate grafted with Xintongding and itaconic acid for drug release ([6]Isiklan.N,Inal.M,Kursun.F,et al,pH responsive itaconic acidgrafted alginate microspheres for the controlled release of nifedipine[J].Carbohydrate Polymers,2011,84(3):933).
This method does not avoid the use of glutaraldehyde, which is toxic to humans, for crosslinking. Meanwhile, the calcium alginate microspheres have large gel grids, so that the encapsulation rate of encapsulated drugs is not high all the time, and waste of embedding substances such as drugs and enzymes is caused. Based on the above, the inventors designed a calcium carbonate/calcium alginate composite microsphere prepared by a biomineralization technique using a two-aqueous phase system in order to overcome the disadvantages of the conventional method for preparing composite microspheres and improve the disadvantages of a single calcium alginate microsphere. The composite microsphere utilizes the selective distribution characteristic of double water phases relative to macromolecular protein under the conditions of mild environment of a double water phase system and no use of other toxic and harmful reagents, can well retain macromolecular protein medicines in the internal phase of a double water phase liquid drop template, and is further encapsulated in the composite calcium alginate microsphere well during curing, so that the composite calcium alginate microsphere has excellent encapsulation efficiency, and the loss of raw materials is reduced. And the composite calcium alginate can change the compactness of calcium carbonate of the calcium carbonate/calcium alginate microspheres by adjusting the system reaction parameter-enzyme concentration, so that the internal drug release rate can be flexibly regulated and controlled. Compared with other methods, the method avoids coating the outer layer with a high polymer material by other methods, and is simpler and more convenient. The composite microspheres utilize a biomimetic mineralization method to enable calcium carbonate crystals to grow in the calcium alginate microsphere gel network, and overcome the defects that a single calcium alginate hydrogel microsphere network has large pores and the inclusion object is easy to flow out. Can be widely applied to the fields of biological medical treatment, cosmetic industry, food industry, enzyme engineering and the like.
Disclosure of Invention
The invention aims to prepare a calcium carbonate/calcium alginate composite microsphere by a double-aqueous-phase biomineralization technology, the microsphere has excellent encapsulation rate on protein drugs, different release rates of loaded drugs can be realized by changing an enzymatic reaction parameter, namely enzyme concentration, and the microsphere has better regulation performance compared with a single calcium alginate microsphere, so that the microsphere is applied to industrially constructing a step sustained-release drug system. The invention also aims to provide a preparation method for preparing the calcium carbonate/calcium alginate composite microspheres by the double-aqueous-phase biomineralization technology.
The technical scheme for realizing the purpose of the invention is as follows:
a calcium carbonate/calcium alginate composite microsphere with adjustable drug release rate is prepared based on PEG/Dex double-aqueous-phase biomineralization technology, the calcium carbonate/calcium alginate composite microsphere is a solid structure of a shell layer, the shell of the calcium carbonate/calcium alginate composite microsphere is a composite formed by blending calcium alginate and calcium carbonate, and the layer of the calcium carbonate/calcium alginate composite microsphere is an in-layer grid of the calcium alginate microsphere formed by mineralization reaction and can encapsulate macromolecular protein drugs; the particle size distribution of the composite microspheres is 200-600 mu m.
Further, by utilizing the selective distribution characteristic of the double-water phase relative to the macromolecular protein, the macromolecular protein medicine is encapsulated in the internal phase of the Dex double-water phase droplet template.
Further, forming microspheres by using the Dex liquid drops as templates by using a liquid drop template method; so that the macromolecular protein medicine is encapsulated in the composite calcium alginate microspheres during curing regulation.
Furthermore, microspheres with different calcium carbonate/calcium alginate composite degrees can be prepared by adjusting different enzyme reaction conditions, namely urease concentrations, and different drug release rates can be obtained.
Furthermore, the aqueous two-phase liquid drop template method is to disperse a certain amount of urea and a certain amount of calcium chloride in 8% of polyethylene glycol phase PEG (molecular weight of 8kDa), and disperse a certain amount of urease and a certain amount of sodium alginate in 8% of dextran phase Dex (molecular weight of 500 kDa); dropping Dex phase into PEG phase via capillary with 20-80 μm inner diameter to form calcium carbonate/calcium alginate composite microsphere.
A preparation method of calcium carbonate/calcium alginate composite microspheres with adjustable drug release rate based on PEG/Dex double aqueous phase biomineralization technology comprises the following specific steps:
the first step is as follows: preparation of PEG-Dex aqueous two phases:
(1) preparing PEG solution with the mass fraction of 10-20% and the molecular weight of 8 kDa;
(2) preparing Dex solution with the mass fraction of 10-20% and the molecular weight of 500 kDa;
(3) fully mixing the solutions in the step (1) and the step (2), standing and phase splitting to obtain an aqueous two-phase solution with an upper phase rich in PEG and a lower phase rich in Dex; respectively extracting the PEG solution and the Dex solution into two beakers for later use;
the second step is as follows: preparing calcium carbonate/calcium alginate composite microspheres by a liquid drop template method:
(1) solution A: weighing 0.01-0.10g of sodium alginate and 0.002-0.050g of urease, adding into 5-10mL of the Dex solution, uniformly mixing, and standing for later use;
(2) solution B: dissolving 0.1-0.5g of anhydrous calcium chloride and 0.1-0.5g of urea in 5-10mL of PEG solution, uniformly mixing, and standing for later use;
(3) sucking the solution A into a common commercially available injector, connecting a capillary tube with a sharp head of which the inner diameter is 20-80 mu m and a tube body of which the inner diameter is 500 mu m with the commercially available silicone tube, pushing the solution A into the sharp-head capillary tube through the injector, suspending the solution A through the sharp-head capillary tube, vertically dropping the solution A into the solution B at the speed of 2 seconds and a drop, wherein the distance between the sharp head of the capillary tube and the liquid level is 0.5-5cm, and forming calcium alginate microspheres with the diameter of 200-600 mu m after several seconds; standing at 20-60 deg.C for 1-10h, and allowing enzymatic reaction to proceed sufficiently to obtain calcium carbonate/calcium alginate composite microsphere with adjustable drug release rate.
Compared with the prior art, the invention has the following beneficial effects:
1. the calcium carbonate/calcium alginate composite microspheres introduce an aqueous two-phase system, and the aqueous two-phase macromolecular steric hindrance effect and the specific selectivity are utilized, so that the composite microspheres prepared by the liquid drop template method have extremely high encapsulation efficiency on macromolecular protein substances, for example, the encapsulation efficiency of BSA (bovine serum albumin) is 97.27%.
2. The composite microsphere has the advantages of biocompatibility, bioadhesion and the like of calcium alginate, is prepared by a biomineralization technology, has the advantage of high mechanical strength of bionic minerals, and is not easy to swell.
3. In the preparation process, the shell layer compactness of the calcium carbonate/calcium alginate microspheres can be changed by adjusting the concentration of system enzyme, so that the release rate of the calcium carbonate/calcium alginate microspheres can be adjusted, and the loaded drugs have different release rates.
4. The calcium carbonate/calcium alginate composite microspheres of the invention use reagents which are non-toxic and harmless, and the preparation process has mild conditions. Has wide application prospect in the fields of biological medical treatment, cosmetics industry, food industry, chemical materials and the like; especially can be applied to the construction of a step slow-release drug system.
5. Compared with other methods, the method avoids coating the outer layer with the high polymer material by other methods, and is simpler and more convenient. The bionic mineralization method is utilized to grow calcium carbonate crystals in the calcium alginate microsphere gel grid, and the defects that a single calcium alginate hydrogel microsphere grid is large in pore and easy to flow out of a wrapping object are overcome.
Drawings
FIG. 1 is a schematic diagram of the preparation of calcium carbonate/calcium alginate microspheres by the droplet template formation method of the present invention.
FIG. 2A1 shows urease concentration of 5 mg/mL-1The schematic diagram of the microphotograph of the prepared calcium carbonate/calcium alginate microspheres.
Fig. 2a2 is a partially enlarged microscopic view of fig. 2a 1.
FIG. 2B shows urease concentration of 5 mg/mL-1The schematic diagram of the fluorescence photograph of the FITC-BSA encapsulated in the prepared calcium carbonate/calcium alginate microsphere composite microsphere is shown.
FIG. 2C is a graph showing urease concentrations of 0-5 mg/mL-1The figure of the prepared calcium carbonate/calcium alginate microspheres is shown.
Fig. 3A is a graph showing the release effect of the small molecule neutral red embedded in the common calcium alginate sphere (i.e. no calcium carbonate mixed) in 0min, 10min and 20 min.
FIG. 3B is a graph showing the release effect of composite calcium alginate sphere-embedded small molecule neutral red (mixed with calcium carbonate) generated by urease reaction at 0min, 10min and 20 min.
FIG. 3C is a graph showing the release effect of the small molecule-embedded neutral red of the composite calcium alginate microspheres formed by the direct reaction of ammonium carbonate/calcium chloride (dense calcium carbonate mixed) at 0min, 10min and 20 min.
FIG. 4 is a bar chart showing the encapsulation efficiency of different concentrations of BSA encapsulated by the prepared calcium carbonate/calcium alginate microspheres according to the present invention.
FIG. 5 is a bar chart showing the release rate of BSA in calcium carbonate/calcium alginate microspheres prepared according to different enzyme concentrations.
Part of the apparatus: instrumental biomicroscope, model: XSP-24, manufacturer: phoenix optics stock limited, place of production: shangxi of China. Canon camera, model: SX260HS, manufacturer: canon (china) limited, origin: beijing, China.
Detailed Description
The invention is further specifically explained with reference to the drawings and the embodiments.
FIG. 1 depicts the mechanism and example of preparing calcium carbonate/calcium alginate microspheres by aqueous two-phase droplet template method. Forming microspheres by using the Dex liquid drops as templates by using a liquid drop template method; so that the macromolecular protein medicine is encapsulated in the composite calcium alginate microspheres when being solidified. Even though a certain amount of urea and a certain amount of calcium chloride are dispersed in 8% polyethylene glycol phase PEG (molecular weight 8kDa), a certain amount of urease and a certain amount of sodium alginate are dispersed in 8% dextran phase Dex (molecular weight 500 kDa); dropping Dex phase into PEG phase via capillary with 20-80 μm inner diameter to form calcium carbonate/calcium alginate composite microsphere.
From fig. 2a1, fig. 2a2, fig. 2B, and fig. 2C, it can be seen that the PEG/Dex aqueous two-phase biomineralization-based calcium carbonate/calcium alginate composite microspheres with adjustable drug release rate are prepared, and are of a solid shell structure, the shell of the calcium carbonate/calcium alginate composite microspheres is a composite formed by blending calcium alginate and calcium carbonate, and the layer of the calcium carbonate/calcium alginate composite microspheres is an in-layer mesh of calcium alginate microspheres formed by mineralization reaction, and can encapsulate macromolecular protein drugs. The particle size distribution of the composite microspheres is 200-600 mu m. By utilizing the selective distribution characteristic of the double-aqueous phase relative to the macromolecular protein, the macromolecular protein medicine is encapsulated in the internal phase of the Dex double-aqueous phase liquid drop template. FIG. 2A1 is a graph showing urease concentration of 5 mg/mL-1A photomicrograph of the prepared calcium carbonate/calcium alginate microspheres, wherein fig. 2a2 is a partially enlarged micrograph of fig. 2a1, in which the black particles are calcium carbonate particles and the transparent part is calcium alginate gel. FIG. 3B shows urease concentration of 5 mg/mL-1And the fluorescence photo of the FITC-BSA encapsulated in the prepared calcium carbonate/calcium alginate microsphere composite microsphere. The green part of the label represents the position of FITC-BSA. FIG. 3C shows urease concentrations of 0-5 mg. multidot.mL-1And then the obtained calcium carbonate/calcium alginate microspheres are taken as digital photos of appearance and appearance. The results illustrate the inventionThe prepared composite microspheres are formed by mixing calcium carbonate and calcium alginate, most of BSA in the composite microspheres is in an interface and an inner layer of the microspheres, so that the composite microspheres have good encapsulation property, and fig. 3A is a graph of release effects of embedded small-molecule neutral red in 0min, 10min and 20min when common calcium alginate spheres, namely calcium carbonate-free spheres, are mixed. FIG. 3B is a graph showing the release effect of embedded small molecule neutral red at 0min, 10min and 20min when the composite calcium alginate spheres produced by urease reaction, i.e. when calcium carbonate is mixed. Fig. 3C is a graph showing the release effect of small molecule neutral red in the composite calcium alginate microspheres formed by the direct reaction of ammonium carbonate/calcium chloride (i.e., when dense calcium carbonate is mixed). The results show that a composite microsphere whose release effect is modified by adjusting calcium carbonate can be prepared by the method of the present invention. In the experiment, the concentrations of the sodium alginate, the calcium chloride, the sodium carbonate and the urease are respectively 5 mg/mL-1，20mg·mL-1，20mg·mL-1，5mg·mL-1. The experimental data in fig. 4 shows that an aqueous two-phase system is introduced in the preparation of the calcium carbonate/calcium alginate composite microspheres, and the aqueous two-phase macromolecular steric hindrance effect and the specific selectivity are utilized to make the composite microspheres prepared by the droplet template method have a very high encapsulation rate for macromolecular protein substances, and the encapsulation rate of BSA measured in the examples is 97.27%. In the experiment, the concentrations of the sodium alginate and the calcium chloride are respectively 5 mg/mL-1，20mg·mL-1。
The data in fig. 5 illustrate that microspheres with different calcium carbonate/calcium alginate composite degree can be prepared by adjusting different enzyme reaction conditions-urease concentration, the calcium carbonate compaction degree on the calcium carbonate/calcium alginate microspheres can be changed, and different drug release rates can be obtained.
A preparation method of calcium carbonate/calcium alginate composite microspheres with adjustable drug release rate based on PEG/Dex double aqueous phase biomineralization technology comprises the following specific steps:
the first step is as follows: preparation of PEG-Dex aqueous two phases:
(1) preparing PEG solution with the mass fraction of 10-20% and the molecular weight of 8 kDa;
(2) preparing Dex solution with the mass fraction of 10-20% and the molecular weight of 500 kDa;
(3) fully mixing the solutions in the step (1) and the step (2), standing and phase splitting to obtain an aqueous two-phase solution with an upper phase rich in PEG and a lower phase rich in Dex; respectively extracting the PEG solution and the Dex solution into two beakers for later use;
the second step is as follows: preparing calcium carbonate/calcium alginate composite microspheres by a liquid drop template method:
(1) solution A: weighing 0.01-0.10g of sodium alginate and 0.002-0.050g of urease, adding into 5-10mL of the Dex solution, uniformly mixing, and standing for later use;
(2) solution B: dissolving 0.1-0.5g of anhydrous calcium chloride and 0.1-0.5g of urea in 5-10mL of PEG solution, uniformly mixing, and standing for later use;
(3) sucking the solution A into a common commercially available injector, connecting a capillary tube with a sharp head of which the inner diameter is 20-80 mu m and a tube body of which the inner diameter is 500 mu m with the commercially available silicone tube, pushing the solution A into the sharp-head capillary tube through the injector, suspending the solution A through the sharp-head capillary tube, vertically dropping the solution A into the solution B at the speed of 2 seconds and a drop, wherein the distance between the sharp head of the capillary tube and the liquid level is 0.5-5cm, and forming calcium alginate microspheres with the diameter of 200-600 mu m after several seconds; standing at 20-60 deg.C for 1-10h, and allowing enzymatic reaction to proceed sufficiently to obtain calcium carbonate/calcium alginate composite microsphere with adjustable drug release rate.
Example 1
Calcium carbonate/calcium alginate composite microspheres (enzyme concentration 5 mg. mL)-11 mg. mL in-1Encapsulation efficiency of BSA
1. Preparation of polyethylene glycol (PEG)/dextran (Dex) aqueous two-phase
(1) Precisely weighing 3.2g of PEG, putting the PEG into a 25mL beaker, adding 16.8mL of deionized water, stirring for 2 hours at the speed of 300r/min by adopting a magnetic stirrer, and preparing a PEG solution with the mass fraction of 16%;
(2) precisely weighing 3.2g of Dex, putting the Dex into a 25mL beaker, adding 16.8mL of deionized water, stirring for 2 hours at the speed of 300r/min by adopting a magnetic stirrer, and preparing a Dex solution with the mass fraction of 16%;
(3) and (3) mixing 10mL of the solutions obtained in the step (1) and the step (2) respectively to prepare 20mL of aqueous two-phase solution with the mass fraction of 8%, standing, and waiting for layering.
2. Method for preparing calcium carbonate/calcium alginate composite microspheres by using liquid drop template method
(1) Solution A: 0.025g of sodium alginate and 0.0050g of BSA are weighed, added into 5mL of 8% Dex solution, mixed uniformly, then 0.025g of urease is added, mixed uniformly and kept stand for later use.
(2) Solution B: 0.2g of anhydrous calcium chloride and 0.2g of urea are precisely weighed and dissolved in 10mL of 8% PEG solution for later use.
(3) Sucking the solution A obtained in the step (1) into a syringe with a needle with a sharp tip and an inner diameter of 50 mu m, slowly dropping 1mL of the solution A into the solution B obtained in the step (2) by using the syringe, and quickly forming a sphere. And standing at 40 ℃ for 4 hours, and carrying out enzymatic reaction to obtain the BSA embedded calcium carbonate/calcium alginate composite microspheres.
(4) 5mL of the solution B was collected, and the absorbance of BSA in the sample solution was measured by an ultraviolet spectrophotometer. Further, the encapsulation efficiency of BSA was determined to be 95.13% by comparison with the absorbance of the original BSA.
Example 2
Calcium carbonate/calcium alginate composite microspheres (enzyme concentration 5 mg. mL)-1Measured at 2 mg. mL-1Encapsulation efficiency of BSA
1. Preparation of polyethylene glycol (PEG)/dextran (Dex) aqueous two-phase
(1) Precisely weighing 3.2g of PEG, putting the PEG into a 25mL beaker, adding 16.8mL of deionized water, stirring for 2 hours at the speed of 300r/min by adopting a magnetic stirrer, and preparing a PEG solution with the mass fraction of 16%;
(2) precisely weighing 3.2g of Dex, putting the Dex into a 25mL beaker, adding 16.8mL of deionized water, stirring for 2 hours at the speed of 300r/min by adopting a magnetic stirrer, and preparing a Dex solution with the mass fraction of 16%;
(3) and (3) mixing 10mL of the solutions obtained in the step (1) and the step (2) respectively to prepare 20mL of aqueous two-phase solution with the mass fraction of 8%, standing, and waiting for layering.
2. Method for preparing calcium carbonate/calcium alginate composite microspheres by using liquid drop template method
(1) Solution A: weighing 0.025g of sodium alginate and 0.010g of BSA, adding the sodium alginate and the BSA into 5mL of 8% Dex solution, uniformly mixing, adding 0.025g of urease, uniformly mixing, and standing for later use.
(2) Solution B: 0.2g of anhydrous calcium chloride and 0.2g of urea are precisely weighed and dissolved in 10mL of 8% PEG solution for later use.
(3) Sucking the solution A obtained in the step (1) into a syringe with a needle with a sharp tip and an inner diameter of 50 mu m, slowly dropping 1mL of the solution A into the solution B obtained in the step (2) by using the syringe, and quickly forming a sphere. And standing at 40 ℃ for 4 hours, and carrying out enzymatic reaction to obtain the BSA embedded calcium carbonate/calcium alginate composite microspheres.
(4) 5mL of the solution B was collected, and the absorbance of BSA in the sample solution was measured by an ultraviolet spectrophotometer. Further, the encapsulation efficiency of BSA was found to be 97.31% by comparison with the absorbance of the original BSA.
Example 3
Calcium carbonate/calcium alginate composite microspheres (enzyme concentration 5 mg. mL)-1Measured at 3 mg. mL-1Encapsulation efficiency of BSA
1. Preparation of polyethylene glycol (PEG)/dextran (Dex) aqueous two-phase
(1) Precisely weighing 3.2g of PEG, putting the PEG into a 25mL beaker, adding 16.8mL of deionized water, stirring for 2 hours at the speed of 300r/min by adopting a magnetic stirrer, and preparing a PEG solution with the mass fraction of 16%;
(2) precisely weighing 3.2g of Dex, putting the Dex into a 25mL beaker, adding 16.8mL of deionized water, stirring for 2 hours at the speed of 300r/min by adopting a magnetic stirrer, and preparing a Dex solution with the mass fraction of 16%;
(3) and (3) mixing 10mL of the solutions obtained in the step (1) and the step (2) respectively to prepare 20mL of aqueous two-phase solution with the mass fraction of 8%, standing, and waiting for layering.
2. Method for preparing calcium carbonate/calcium alginate composite microspheres by using liquid drop template method
(1) Solution A: 0.025g of sodium alginate and 0.0150g of BSA are weighed, added into 5mL of 8% Dex solution, mixed uniformly, then 0.025g of urease is added, mixed uniformly and kept stand for later use.
(2) Solution B: 0.2g of anhydrous calcium chloride and 0.2g of urea are precisely weighed and dissolved in 10mL of 8% PEG solution for later use.
(3) Sucking the solution A obtained in the step (1) into a syringe with a needle with a sharp tip and an inner diameter of 50 mu m, slowly dropping 1mL of the solution A into the solution B obtained in the step (2) by using the syringe, and quickly forming a sphere. And standing at 40 ℃ for 4 hours, and carrying out enzymatic reaction to obtain the BSA embedded calcium carbonate/calcium alginate composite microspheres.
(4) 5mL of the solution B was collected, and the absorbance of BSA in the sample solution was measured by an ultraviolet spectrophotometer. Further, the encapsulation efficiency of BSA was found to be 97.27% by comparison with the absorbance of the original BSA.
Example 4
Calcium carbonate/calcium alginate composite microspheres (enzyme concentration 5 mg. mL)-1Measured at 4 mg. mL-1Encapsulation efficiency of BSA
1. Preparation of polyethylene glycol (PEG)/dextran (Dex) aqueous two-phase
(1) Precisely weighing 3.2g of PEG, putting the PEG into a 25mL beaker, adding 16.8mL of deionized water, stirring for 2 hours at the speed of 300r/min by adopting a magnetic stirrer, and preparing a PEG solution with the mass fraction of 16%;
(2) precisely weighing 3.2g of Dex, putting the Dex into a 25mL beaker, adding 16.8mL of deionized water, stirring for 2 hours at the speed of 300r/min by adopting a magnetic stirrer, and preparing a Dex solution with the mass fraction of 16%;
(3) and (3) mixing 10mL of the solutions obtained in the step (1) and the step (2) respectively to prepare 20mL of aqueous two-phase solution with the mass fraction of 8%, standing, and waiting for layering.
2. Preparation of calcium carbonate/calcium alginate composite microspheres by liquid drop method template
(1) Solution A: weighing 0.025g of sodium alginate and 0.020g of BSA, adding the sodium alginate and the BSA into 5mL of 8% Dex solution, uniformly mixing, adding 0.025g of urease, uniformly mixing, and standing for later use.
(2) Solution B: 0.2g of anhydrous calcium chloride and 0.2g of urea are precisely weighed and dissolved in 10mL of 8% PEG solution for later use.
(3) Sucking the solution A obtained in the step (1) into a syringe with a needle head with the inner diameter of 500 mu m, slowly dropping 1mL of the solution A into the solution B obtained in the step (2) by using the syringe, and quickly forming a sphere. And standing at 40 ℃ for 4 hours, and carrying out enzymatic reaction to obtain the BSA embedded calcium carbonate/calcium alginate composite microspheres.
(4) 5mL of the solution B was collected, and the absorbance of BSA in the sample solution was measured by an ultraviolet spectrophotometer. Further, when the absorbance of the BSA was compared with that of the original BSA, the encapsulation efficiency of the BSA was determined to be 96.85%.
Example 5
Calcium carbonate/calcium alginate composite microspheres (enzyme concentration 5 mg. mL)-1Measured at 5 mg. mL-1Encapsulation efficiency of BSA
1. Preparation of polyethylene glycol (PEG)/dextran (Dex) aqueous two-phase
(1) Precisely weighing 3.2g of PEG, putting the PEG into a 25mL beaker, adding 16.8mL of deionized water, stirring for 2 hours at the speed of 300r/min by adopting a magnetic stirrer, and preparing a PEG solution with the mass fraction of 16%;
(2) precisely weighing 3.2g of Dex, putting the Dex into a 25mL beaker, adding 16.8mL of deionized water, stirring for 2 hours at the speed of 300r/min by adopting a magnetic stirrer, and preparing a Dex solution with the mass fraction of 16%;
(3) and (3) mixing 10mL of the solutions obtained in the step (1) and the step (2) respectively to prepare 20mL of aqueous two-phase solution with the mass fraction of 8%, standing, and waiting for layering.
2. Method for preparing calcium carbonate/calcium alginate composite microspheres by using liquid drop template method
(1) Solution A: weighing 0.025g of sodium alginate and 0.0250g of BSA, adding the sodium alginate and the BSA into 5mL of 8% Dex solution, uniformly mixing, adding 0.025g of urease, uniformly mixing, and standing for later use.
(2) Solution B: 0.2g of anhydrous calcium chloride and 0.2g of urea are precisely weighed and dissolved in 10mL of 8% PEG solution for later use.
(3) Sucking the solution A obtained in the step (1) into a syringe with a needle with a sharp tip and an inner diameter of 50 mu m, slowly dropping 1mL of the solution A into the solution B obtained in the step (2) by using the syringe, and quickly forming a sphere. And standing at 40 ℃ for 4 hours, and carrying out enzymatic reaction to obtain the BSA embedded calcium carbonate/calcium alginate composite microspheres.
(4) 5mL of the solution B was collected, and the absorbance of BSA in the sample solution was measured by an ultraviolet spectrophotometer. Further, when the absorbance of BSA was compared with that of the original BSA, the encapsulation efficiency of BSA was determined to be 98.03%.
Example 6
Calcium carbonate/alginic acidCalcium composite microsphere (enzyme concentration 5 mg. mL)-1Envelope neutral red)
1. Preparation of polyethylene glycol (PEG)/dextran (Dex) aqueous two-phase
(1) Precisely weighing 3.2g of PEG, putting the PEG into a 25mL beaker, adding 16.8mL of deionized water, stirring for 2 hours at the speed of 300r/min by adopting a magnetic stirrer, and preparing a PEG solution with the mass fraction of 16%;
(2) precisely weighing 3.2g of Dex, putting the Dex into a 25mL beaker, adding 16.8mL of deionized water, stirring for 2 hours at the speed of 300r/min by adopting a magnetic stirrer, and preparing a Dex solution with the mass fraction of 16%;
(3) and (3) mixing 10mL of the solutions obtained in the step (1) and the step (2) respectively to prepare 20mL of aqueous two-phase solution with the mass fraction of 8%, standing, and waiting for layering.
2. Method for preparing template calcium carbonate/calcium alginate composite microspheres by liquid drop method
(1) Solution A: 0.025g of sodium alginate and 0.0050g of neutral red are weighed, added into 5mL of 8% Dex solution, mixed uniformly, then 0.025g of urease is added, mixed uniformly and kept stand for later use.
(2) Solution B: 0.2g of anhydrous calcium chloride and 0.2g of urea are precisely weighed and dissolved in 10mL of 8% PEG solution for later use.
(3) Sucking the solution A obtained in the step (1) into a syringe with a needle head with a sharp tip and an inner diameter of 50 mu m, and slowly dripping the solution A into the solution B obtained in the step (2) by using the syringe to quickly form balls. And standing at 40 ℃ for 4 hours, and carrying out enzymatic reaction to obtain the calcium carbonate/calcium alginate composite microspheres embedded with the micromolecular dye. The release of neutral red embedded in calcium carbonate/calcium alginate composite microspheres is shown in figure 1B.
Example 7
Calcium carbonate/calcium alginate composite microspheres (enzyme concentration is 0 mg. mL)-1Encapsulation BSA Release Rate measurement
Preparation of PEG-Dex aqueous two-phase
(1) Precisely weighing 3.2g of PEG, putting the PEG into a 25mL beaker, adding 16.8mL of deionized water, stirring for 2 hours at the speed of 300r/min by adopting a magnetic stirrer to prepare a PEG solution with the mass fraction of 16%;
(2) precisely weighing 3.2g of Dex, putting the Dex into a 25mL beaker, adding 16.8mL of deionized water, stirring for 2 hours at the speed of 300r/min by adopting a magnetic stirrer, and preparing a Dex solution with the mass fraction of 16%;
(3) and (3) mixing 10mL of the solutions obtained in the step (1) and the step (2) respectively to prepare 20mL of aqueous two-phase solution with the mass fraction of 8%, standing, and waiting for layering.
2. Preparation of BSA-calcium carbonate/calcium alginate composite microspheres by using droplet template method
(1) Solution A: weighing 0.025g of sodium alginate and 0.010g of BSA, adding the sodium alginate and the BSA into 5mL of 8% Dex solution by mass percent, uniformly mixing, adding 0g of urease, uniformly mixing, and standing for later use.
(2) Solution B: 0.2g of anhydrous calcium chloride and 0.2g of urea are precisely weighed and dissolved in 10mL of PEG solution with the mass percentage of 8% for later use.
(3) Sucking the solution A obtained in the step (1) into a syringe with a needle head with a sharp tip and an inner diameter of 50 mu m, and slowly dripping the solution A into the solution B obtained in the step (2) by using the syringe to quickly form balls. And standing at 40 ℃ for 4 hours, and carrying out enzymatic reaction to obtain the BSA-calcium carbonate/calcium alginate composite microspheres prepared at the enzyme concentration.
3. The calcium carbonate/calcium alginate composite microspheres release bovine serum albumin.
And (3) putting all the prepared BSA-calcium carbonate/calcium alginate composite microspheres into a glass bottle with a cover, adding 10mL of deionized water, putting the glass bottle on a rotary mixer at room temperature, keeping the rotation speed at 10r/min, taking out the clear liquid in the bottle after 2 hours, and measuring the absorbance of BSA in the sample liquid by using an ultraviolet spectrophotometer. The release of BSA was found to be 70.59%.
Example 8
Calcium carbonate/calcium alginate composite microspheres (enzyme concentration is 1 mg. mL)-1Encapsulation BSA Release Rate measurement
Preparation of PEG-Dex aqueous two-phase
(1) Precisely weighing 3.2g of PEG, putting the PEG into a 25mL beaker, adding 16.8mL of deionized water, stirring for 2 hours at the speed of 300r/min by adopting a magnetic stirrer to prepare a PEG solution with the mass fraction of 16%;
(2) precisely weighing 3.2g of Dex, putting the Dex into a 25mL beaker, adding 16.8mL of deionized water, stirring for 2 hours at the speed of 300r/min by adopting a magnetic stirrer, and preparing a Dex solution with the mass fraction of 16%;
(3) and (3) mixing 10mL of the solutions obtained in the step (1) and the step (2) respectively to prepare 20mL of aqueous two-phase solution with the mass fraction of 8%, standing, and waiting for layering.
2. Preparation of BSA-calcium carbonate/calcium alginate composite microspheres by using droplet template method
(1) Solution A: weighing 0.025g of sodium alginate and 0.010g of BSA, adding the sodium alginate and the BSA into 5mL of 8% Dex solution by mass percent, uniformly mixing, adding 0.005g of urease, uniformly mixing, and standing for later use.
(2) Solution B: 0.2g of anhydrous calcium chloride and 0.2g of urea are precisely weighed and dissolved in 10mL of PEG solution with the mass percentage of 8% for later use.
(3) Sucking the solution A obtained in the step (1) into a syringe with a needle head with a sharp tip and an inner diameter of 50 mu m, and slowly dripping the solution A into the solution B obtained in the step (2) by using the syringe to quickly form balls. And standing at 40 ℃ for 4 hours, and carrying out enzymatic reaction to obtain the BSA-calcium carbonate/calcium alginate composite microspheres prepared at the enzyme concentration.
3. The calcium carbonate/calcium alginate composite microspheres release bovine serum albumin.
And (3) putting all the prepared BSA-calcium carbonate/calcium alginate composite microspheres into a glass bottle with a cover, adding 10mL of deionized water, putting the glass bottle on a rotary mixer at room temperature, keeping the rotation speed at 10r/min, taking out the clear liquid in the bottle after 2 hours, and measuring the absorbance of BSA in the sample liquid by using an ultraviolet spectrophotometer. The release of BSA was found to be 67.37%.
Example 9
Calcium carbonate/calcium alginate composite microspheres (enzyme concentration is 2 mg. mL)-1Encapsulation BSA Release Rate measurement
Preparation of PEG-Dex aqueous two-phase
(1) Precisely weighing 3.2g of PEG, putting the PEG into a 25mL beaker, adding 16.8mL of deionized water, stirring for 2 hours at the speed of 300r/min by adopting a magnetic stirrer to prepare a PEG solution with the mass fraction of 16%;
(2) precisely weighing 3.2g of Dex, putting the Dex into a 25mL beaker, adding 16.8mL of deionized water, stirring for 2 hours at the speed of 300r/min by adopting a magnetic stirrer, and preparing a Dex solution with the mass fraction of 16%;
(3) and (3) mixing 10mL of the solutions obtained in the step (1) and the step (2) respectively to prepare 20mL of aqueous two-phase solution with the mass fraction of 8%, standing, and waiting for layering.
2. Preparation of BSA-calcium carbonate/calcium alginate composite microspheres by using droplet template method
(1) Solution A: weighing 0.025g of sodium alginate and 0.010g of BSA, adding the sodium alginate and the BSA into 5mL of 8% Dex solution by mass percent, uniformly mixing, adding 0.010g of urease, uniformly mixing, and standing for later use.
(2) Solution B: 0.2g of anhydrous calcium chloride and 0.2g of urea are precisely weighed and dissolved in 10mL of PEG solution with the mass percentage of 8% for later use.
(3) Sucking the solution A obtained in the step (1) into a syringe with a needle head with a sharp tip and an inner diameter of 50 mu m, and slowly dripping the solution A into the solution B obtained in the step (2) by using the syringe to quickly form balls. And standing at 40 ℃ for 4 hours, and carrying out enzymatic reaction to obtain the BSA-calcium carbonate/calcium alginate composite microspheres prepared at the enzyme concentration.
3. The calcium carbonate/calcium alginate composite microspheres release bovine serum albumin.
And (3) putting all the prepared BSA-calcium carbonate/calcium alginate composite microspheres into a glass bottle with a cover, adding 10mL of deionized water, putting the glass bottle on a rotary mixer at room temperature, keeping the rotation speed at 10r/min, taking out the clear liquid in the bottle after 2 hours, and measuring the absorbance of BSA in the sample liquid by using an ultraviolet spectrophotometer. The release of BSA was found to be 58.69%.
Example 10
Calcium carbonate/calcium alginate composite microspheres (enzyme concentration is 3 mg. mL)-1Encapsulation BSA Release Rate measurement
Preparation of PEG-Dex aqueous two-phase
(1) Precisely weighing 3.2g of PEG, putting the PEG into a 25mL beaker, adding 16.8mL of deionized water, stirring for 2 hours at the speed of 300r/min by adopting a magnetic stirrer to prepare a PEG solution with the mass fraction of 16%;
(2) precisely weighing 3.2g of Dex, putting the Dex into a 25mL beaker, adding 16.8mL of deionized water, stirring for 2 hours at the speed of 300r/min by adopting a magnetic stirrer, and preparing a Dex solution with the mass fraction of 16%;
(3) and (3) mixing 10mL of the solutions obtained in the step (1) and the step (2) respectively to prepare 20mL of aqueous two-phase solution with the mass fraction of 8%, standing, and waiting for layering.
2. Preparation of template BSA-calcium carbonate/calcium alginate composite microspheres by liquid drop method
(1) Solution A: weighing 0.025g of sodium alginate and 0.010g of BSA, adding the sodium alginate and the BSA into 5mL of 8% Dex solution by mass percent, uniformly mixing, adding 0.015g of urease, uniformly mixing, and standing for later use.
(2) Solution B: 0.2g of anhydrous calcium chloride and 0.2g of urea are precisely weighed and dissolved in 10mL of PEG solution with the mass percentage of 8% for later use.
(3) Sucking the solution A obtained in the step (1) into a syringe with a needle head with a sharp tip and an inner diameter of 50 mu m, and slowly dripping the solution A into the solution B obtained in the step (2) by using the syringe to quickly form balls. And standing at 40 ℃ for 4 hours, and carrying out enzymatic reaction to obtain the BSA-calcium carbonate/calcium alginate composite microspheres prepared at the enzyme concentration.
3. The calcium carbonate/calcium alginate composite microspheres release bovine serum albumin.
And (3) putting all the prepared BSA-calcium carbonate/calcium alginate composite microspheres into a glass bottle with a cover, adding 10mL of deionized water, putting the glass bottle on a rotary mixer at room temperature, keeping the rotation speed at 10r/min, taking out the clear liquid in the bottle after 2 hours, and measuring the absorbance of BSA in the sample liquid by using an ultraviolet spectrophotometer. The release rate of BSA was measured to be 52.81%.
Example 11
Calcium carbonate/calcium alginate composite microspheres (enzyme concentration is 4 mg. mL)-1Encapsulation BSA Release Rate measurement
Preparation of PEG-Dex aqueous two-phase
(1) Precisely weighing 3.2g of PEG, putting the PEG into a 25mL beaker, adding 16.8mL of deionized water, stirring for 2 hours at the speed of 300r/min by adopting a magnetic stirrer to prepare a PEG solution with the mass fraction of 16%;
(2) precisely weighing 3.2g of Dex, putting the Dex into a 25mL beaker, adding 16.8mL of deionized water, stirring for 2 hours at the speed of 300r/min by adopting a magnetic stirrer, and preparing a Dex solution with the mass fraction of 16%;
(3) and (3) mixing 10mL of the solutions obtained in the step (1) and the step (2) respectively to prepare 20mL of aqueous two-phase solution with the mass fraction of 8%, standing, and waiting for layering.
2. Preparation of BSA-calcium carbonate/calcium alginate composite microspheres by using droplet template method
(1) Solution A: weighing 0.025g of sodium alginate and 0.010g of BSA, adding the sodium alginate and the BSA into 5mL of 8% Dex solution by mass percent, uniformly mixing, adding 0.020g of urease, uniformly mixing, and standing for later use.
(2) Solution B: 0.2g of anhydrous calcium chloride and 0.2g of urea are precisely weighed and dissolved in 10mL of PEG solution with the mass percentage of 8% for later use.
(3) Sucking the solution A obtained in the step (1) into a syringe with a needle head with a sharp tip and an inner diameter of 50 mu m, and slowly dripping the solution A into the solution B obtained in the step (2) by using the syringe to quickly form balls. And standing at 40 ℃ for 4 hours, and carrying out enzymatic reaction to obtain the BSA-calcium carbonate/calcium alginate composite microspheres prepared at the enzyme concentration.
3. The calcium carbonate/calcium alginate composite microspheres release bovine serum albumin.
And (3) putting all the prepared BSA-calcium carbonate/calcium alginate composite microspheres into a glass bottle with a cover, adding 10mL of deionized water, putting the glass bottle on a rotary mixer at room temperature, keeping the rotation speed at 10r/min, taking out the clear liquid in the bottle after 2 hours, and measuring the absorbance of BSA in the sample liquid by using an ultraviolet spectrophotometer. The release rate of BSA was found to be 48.6%.
Example 12
Calcium carbonate/calcium alginate composite microspheres (enzyme concentration 5 mg. mL)-1BSA test Release Rate
Preparation of PEG-Dex aqueous two-phase
(1) Precisely weighing 3.2g of PEG, putting the PEG into a 25mL beaker, adding 16.8mL of deionized water, stirring for 2 hours at the speed of 300r/min by adopting a magnetic stirrer to prepare a PEG solution with the mass fraction of 16%;
(2) precisely weighing 3.2g of Dex, putting the Dex into a 25mL beaker, adding 16.8mL of deionized water, stirring for 2 hours at the speed of 300r/min by adopting a magnetic stirrer, and preparing a Dex solution with the mass fraction of 16%;
(3) and (3) mixing 10mL of the solutions obtained in the step (1) and the step (2) respectively to prepare 20mL of aqueous two-phase solution with the mass fraction of 8%, standing, and waiting for layering.
2. Preparation of BSA-calcium carbonate/calcium alginate composite microspheres by using droplet template method
(1) Solution A: weighing 0.025g of sodium alginate and 0.010g of BSA, adding the sodium alginate and the BSA into 5mL of 8% Dex solution by mass percent, uniformly mixing, adding 0.025g of urease, uniformly mixing, and standing for later use.
(2) Solution B: 0.2g of anhydrous calcium chloride and 0.2g of urea are precisely weighed and dissolved in 10mL of PEG solution with the mass percentage of 8% for later use.
(3) Sucking the solution A obtained in the step (1) into a syringe with a needle head with a sharp tip and an inner diameter of 50 mu m, and slowly dripping the solution A into the solution B obtained in the step (2) by using the syringe to quickly form balls. And standing at 40 ℃ for 4 hours, and carrying out enzymatic reaction to obtain the BSA-calcium carbonate/calcium alginate composite microspheres prepared at the enzyme concentration.
3. The calcium carbonate/calcium alginate composite microspheres release bovine serum albumin.
And (3) putting all the prepared BSA-calcium carbonate/calcium alginate composite microspheres into a glass bottle with a cover, adding 10mL of deionized water, putting the glass bottle on a rotary mixer at room temperature, keeping the rotation speed at 10r/min, taking out the clear liquid in the bottle after 2 hours, and measuring the absorbance of BSA in the sample liquid by using an ultraviolet spectrophotometer. The release rate of BSA was found to be 43.25%.
Claims (5)
1. A calcium carbonate/calcium alginate composite microsphere with adjustable drug release rate is prepared based on PEG/Dex double aqueous phase biomineralization technology, and is characterized in that the calcium carbonate/calcium alginate composite microsphere is of a solid structure of a shell layer, the shell of the calcium carbonate/calcium alginate composite microsphere is a composite formed by blending calcium alginate and calcium carbonate, and the layer of the calcium carbonate/calcium alginate composite microsphere is an in-layer grid of the calcium alginate microsphere formed by mineralization reaction and can encapsulate macromolecular protein drugs; the particle size distribution of the composite microspheres is 200-600 mu m; the preparation method comprises the following specific steps:
the first step is as follows: preparation of PEG-Dex aqueous two phases:
(1) preparing PEG solution with the mass fraction of 10-20% and the molecular weight of 8 kDa;
(2) preparing Dex solution with the mass fraction of 10-20% and the molecular weight of 500 kDa;
(3) fully mixing the solutions in the step (1) and the step (2), standing and phase splitting to obtain an aqueous two-phase solution with an upper phase rich in PEG and a lower phase rich in Dex; respectively extracting the PEG solution and the Dex solution into two beakers for later use;
the second step is as follows: preparing calcium carbonate/calcium alginate composite microspheres by a liquid drop template method:
(1) solution A: weighing 0.01-0.10g of sodium alginate and 0.002-0.050g of urease, adding into 5-10mL of the Dex solution, uniformly mixing, and standing for later use;
(2) solution B: dissolving 0.1-0.5g of anhydrous calcium chloride and 0.1-0.5g of urea in 5-10mL of PEG solution, uniformly mixing, and standing for later use;
(3) sucking the solution A into a common commercially available injector, connecting a capillary tube with a sharp head inner diameter of 20-80 microns and a tube body inner diameter of 500 microns with the commercially available injector by using a commercially available silicone tube, pushing the solution A into the capillary tube with the sharp head thereof being 0.5-5cm away from the liquid level by using the injector, suspending the solution A in the air by using the sharp head capillary tube, vertically dropping the solution A into the solution B at a speed of 2 seconds (one drop), thus forming a Dex double-aqueous-phase drop template, and forming calcium alginate microspheres with the diameter of 200-600 microns by using the Dex double-aqueous-phase drop template after several seconds; standing at 20-60 deg.C for 1-10h, and allowing enzymatic reaction to proceed sufficiently to obtain calcium carbonate/calcium alginate composite microsphere with adjustable drug release rate.
2. The calcium carbonate/calcium alginate composite microsphere with adjustable drug release rate according to claim 1, wherein the inner phase of the Dex aqueous two-phase droplet template is encapsulated with protein drugs with macromolecules by utilizing the selective distribution characteristic of the aqueous two-phase to macromolecular proteins.
3. The calcium carbonate/calcium alginate composite microsphere with adjustable drug release rate according to claim 1 or 2, wherein the microsphere is formed by the Dex aqueous two-phase droplet template; so that the macromolecular protein medicine is encapsulated in the composite calcium alginate microspheres during curing regulation.
4. The calcium carbonate/calcium alginate composite microspheres with adjustable drug release rate of claim 3, wherein microspheres with different calcium carbonate/calcium alginate composite degree can be prepared by adjusting different enzyme reaction conditions- (urease concentration), and different drug release rates can be obtained.
5. The calcium carbonate/calcium alginate composite microsphere capable of adjusting the drug release rate according to claim 1 or 2, wherein the Dex aqueous two-phase droplet template is prepared by dispersing a certain amount of urea and a certain amount of calcium chloride in polyethylene glycol (PEG) phase with a molecular weight of 8kDa and a mass percent of 8%, and dispersing a certain amount of urease and a certain amount of sodium alginate in dextran phase Dex with a molecular weight of 500kDa and a mass percent of 8%; dropping Dex phase into PEG phase via capillary with 20-80 μm inner diameter to form calcium carbonate/calcium alginate composite microsphere.
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| CN109701461B (en) * | 2018-10-25 | 2021-06-08 | 西南交通大学 | Preparation and application of PEG/Dex double aqueous phase-based calcium carbonate/calcium alginate composite microcapsule |
| CN109943558B (en) * | 2019-04-22 | 2023-02-17 | 西南交通大学 | Method for immobilizing enzyme based on biomimetic mineralization process of double-aqueous-phase system |
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| CN113929342B (en) * | 2020-06-29 | 2022-11-25 | 南京博特新材料有限公司 | Microcapsule type polycarboxylic acid superplasticizer and preparation method thereof |
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