CN110343288B - Porous calcium alginate microspheres using aqueous two-phase emulsion as template, preparation method and application thereof - Google Patents
Porous calcium alginate microspheres using aqueous two-phase emulsion as template, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a porous calcium alginate microsphere taking aqueous two-phase emulsion as a template, a preparation method and application thereof, and the preparation method comprises the following steps: preparation of mPEG-BSA particles, PEG-Dex double aqueous phase preparation, PEG-CaCl2Preparing a solution, and preparing porous calcium alginate microspheres; the porous calcium alginate microspheres prepared by the method avoid the residue of an oil phase in the process of forming a porous structure, and improve the biocompatibility of the porous microspheres; the stability of the emulsion is improved by utilizing the protein polymer particles, and the pore size of the porous microspheres is easier to control; has wide application prospect in the field of biological medicine.
Description
Technical Field
The invention relates to a preparation method of porous microspheres, in particular to porous calcium alginate microspheres taking aqueous two-phase emulsion as a template, a preparation method and application thereof.
Background
Porous microspheres are a new material with special internal structure, and are widely used in drug sustained release (Kim I, Byeon H J, Kim T H, et al. Donorubicin-loaded high density porous microspheres as a suspended-released system, 2012,33(22):5574 5583.), 3D cell culture (Kankala R K, Zhao J, Liu C, et al. high density microspheres for a Small interaction tissue scaffold, 78, 2019,1901397, etc.. because of their controllable pore size and internal structure, low density, high specific surface area, etc., 2013,3(19): 6871-6878), and the like.
The emulsion template method is a novel method for preparing porous microspheres developed in recent years. The emulsion is a system formed by two mutually insoluble liquids and dispersing one liquid in the other liquid in the form of liquid drops under the action of external force. Porous microspheres are generally prepared by forming an oil-in-water (O/W) emulsion by adding a forming Material (e.g., alginic acid, chitosan, and a polymer copolymer, etc.) to the external phase to form droplets with a plurality of emulsion enclosed therein and extruding the small oil droplets out of the interior by inducing volume-induced rapid contraction through gel crosslinking (Marco C, Jan G, Andrea B, et al. The pore diameter and density of the formed porous microsphere pores can be controlled by adjusting the size and density of the internal emulsion (Zhang Feng Ju. preparation research of porous calcium alginate gel microspheres by emulsion template method [ D ]. Tianjin university, 2003).
When the extrusion force generated by external stimulation and crosslinking of the gel is insufficient, oil droplets cannot be completely discharged, oil phase residues exist in the porous microspheres, the biocompatibility of the porous microspheres is reduced, and the application in the field of biomedicine is limited. Aqueous two-phase emulsions, also known as water-in-water emulsions, are formed by mixing two mutually immiscible aqueous solutions of high-molecular polymers or an aqueous solution of a high-molecular polymer and a salt solution in a certain ratio. Because the biological agent does not contain any organic reagent, the biological agent has the advantages of high biocompatibility, green, environmental protection and the like, and is widely applied to the field of biological medicine (Ma Q M, Song Y, Baier G, et al.Osmo-solubility of all-aqueous emulsion with enhanced preservation of protein activity [ J ]. Journal of Materials Chemistry B,2016,4(7): 1213-. The porous microspheres prepared by using the aqueous two-phase emulsion as a template have great potential. However, aqueous two-phase emulsions have very low surface tension and large interfacial thickness, which do not allow small molecule surfactants to span the entire phase interface (Buzza D M A, Fletcher P D I, Georgiou T K, et al, Water-in-water emulsions based on compatible polymers and stabilized by triblock copolymers-functionalized polymers [ J ]. Langmuir,2013,29(48): 14804-. Therefore, the double-aqueous-phase emulsion can not be stabilized by a surfactant unlike an oil-water emulsion, so that the size of the emulsion is not uniform, and the progress of various application researches of the double-aqueous-phase emulsion is greatly restricted. The preparation of porous microspheres using an aqueous two-phase emulsion as a template is a challenge.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a porous calcium alginate microsphere which utilizes protein polymer particles mPEG-BSA to improve the stability of emulsion and takes aqueous two-phase emulsion as a template, a preparation method and application thereof.
The technical scheme adopted by the invention is as follows:
a preparation method of porous calcium alginate microspheres with aqueous two-phase emulsion as a template comprises the following steps:
step 1: adding bovine serum albumin BSA and methoxypolyethylene glycol-acetaldehyde mPEG-CHO into an aqueous solution with the pH value of 5 according to the mass ratio of 1:1, fully dissolving and completely reacting, and drying to obtain mPEG-BSA particles;
step 2: fully mixing a polyethylene glycol (PEG) solution with the mass percentage concentration of 16% and a dextran Dex solution in equal volume, standing, splitting the phase, and taking an upper phase solution as a solution C;
and step 3: adding Alg alginate into the solution C obtained in the step 2, and preparing a PEG-Alg solution with the Alg mass percentage concentration of 2 wt% as a solution D;
and 4, step 4: adding calcium chloride CaCl into the solution C obtained in the step 22Preparing CaCl2PEG-CaCl with the mass percentage concentration of 10 wt%2The solution is E solution;
and 5: fully mixing PEG solution with mass percent concentration of 16% and Dex solution in equal volume, carrying out phase splitting, adding the mPEG-BSA particles obtained in the step 1 to form solution with the mass percent concentration of 0.5 wt%, and emulsifying to obtain emulsion A;
step 6: mixing the solution D obtained in the step 3 and the emulsion A obtained in the step 5 according to a set proportion, and fully and uniformly mixing to obtain an emulsion B;
and 7: and (3) dripping the emulsion B into the solution E at the speed of 3-5 seconds per drop to form the required porous calcium alginate microspheres.
Further, in the step 1, BSA and mPEG-CHO are dissolved and then sealed, reacted for 48 hours at room temperature, dried in an electrothermal constant-temperature air drying oven at 37 ℃, and dried to obtain the required mPEG-BSA particles.
Further, the molecular weight of PEG is 8kDa, and the molecular weight of Dex is 500 kDa.
Further, emulsifying for 15s at 2800-12000 rpm by using a vortex oscillator in the step 5 to obtain the emulsion A.
Further, in the step 7, the emulsion B is dripped at a position 3-5 cm away from the liquid level of the solution E by using an injector.
The porous calcium alginate microspheres have the advantages that calcium alginate hydrogel is arranged on the surfaces of the porous calcium alginate microspheres, the interior of the porous calcium alginate microspheres is of a porous structure with good connectivity, the pore size is 20-80 mu m, and the particle size of the microspheres is 2-3 mm.
The application of porous calcium alginate microspheres, which are used as drug carriers in the preparation of drugs.
The application of porous calcium alginate microspheres is used for 3D cell culture.
The invention has the beneficial effects that:
(1) the invention utilizes the double aqueous phase emulsion formed by two immiscible macromolecule dextran Dex and polyethylene glycol PEG solutions, avoids the residue of oil phase in the process of forming a porous structure, and improves the biocompatibility of the porous microsphere.
(2) The invention utilizes protein polymer particles mPEG-BSA which stabilize the double-aqueous phase emulsion to improve the stability of the emulsion and facilitate the control of the pore size of the porous microspheres.
(3) The invention utilizes the aqueous two-phase emulsion with stable protein polymer particles to prepare the porous calcium alginate microspheres with controllable porous structures in the full-water environment by a drip method, thereby improving the biocompatibility.
Drawings
FIG. 1 is a schematic view of the preparation process of the present invention.
Fig. 2 is a topography of the porous calcium alginate microspheres prepared in example 1 of the present invention, wherein a is a top view, b is a side view, c is a full-view micrograph, and d is a partial enlarged view.
Fig. 3 is an SEM image of the porous calcium alginate microspheres prepared in example 1 of the present invention, wherein b is a partially enlarged view of a.
FIG. 4 is a photograph in example 1 of the present invention, wherein a is a micrograph of the emulsion obtained in step 5, b is a micrograph of the emulsion obtained in step 6, c is an SEM photograph of the porous calcium alginate microspheres obtained, and d is a partial enlarged view of c;
FIG. 5 is a photograph of example 2 of the present invention, wherein a is a micrograph of the emulsion obtained in step 5, b is a micrograph of the emulsion obtained in step 6, c is an SEM photograph of the porous calcium alginate microspheres obtained, and d is a partial enlarged view of c.
FIG. 6 is a photograph of example 3 of the present invention, wherein a is a micrograph of the emulsion obtained in step 5, b is a micrograph of the emulsion obtained in step 6, c is an SEM photograph of the porous calcium alginate microspheres obtained, and d is a partial enlarged view of c.
FIG. 7 is a photograph of example 4 of the present invention, wherein a is a micrograph of the emulsion obtained in step 5, b is a micrograph of the emulsion obtained in step 6, c is an SEM photograph of the porous calcium alginate microspheres obtained, and d is a partial enlarged view of c.
FIG. 8 is a graph showing the frequency distribution of pore sizes of porous calcium alginate microspheres with different milk formation frequencies in the example of the present invention.
FIG. 9 is a frequency distribution diagram of pore sizes of porous calcium alginate microspheres with different volume ratios of aqueous two-phase emulsion and PEG-Alg solution in the embodiment of the present invention.
Detailed Description
The invention is further described with reference to the following figures and specific embodiments.
As shown in fig. 1, a method for preparing porous calcium alginate microspheres by using an aqueous two-phase emulsion as a template comprises the following steps:
step 1: adding Bovine Serum Albumin (BSA) and methoxypolyethylene glycol-acetaldehyde (mPEG-CHO) into an aqueous solution with the pH of 5 according to the mass ratio of 1:1, and fully dissolving the BSA and mPEG-CHO in the mixed solution to ensure that the mass concentration of the BSA and the mPEG-CHO is 10 wt%; and sealing the reaction solution, reacting for 48 hours at room temperature, drying the reaction solution in an electrothermal constant-temperature air blast drying oven at 37 ℃, and drying to obtain the mPEG-BSA particles.
Step 2: preparing a PEG solution with the molecular weight of 8kDa and the mass percent concentration of 16 percent as an A solution, and preparing a Dex solution with the molecular weight of 500kDa and the mass percent concentration of 16 percent as a B solution; weighing equal volumes of solution A and solution B, fully mixing the solutions on a rotary incubator, standing for 6 hours, then carrying out phase separation, wherein the upper phase is a PEG-rich phase, the lower phase is a Dex-rich phase, and extracting the upper phase and recording as solution C.
And step 3: adding Alg into the solution C obtained in the step 2, and preparing a PEG-Alg solution with the Alg mass percentage concentration of 2 wt% as a solution D;
and 4, step 4: adding CaCl into the solution C obtained in the step 22Preparing CaCl2PEG-CaCl with the mass percentage concentration of 10 wt%2The solution is E solution;
and 5: weighing equal volumes of PEG solution with mass percent concentration of 16% and Dex solution, fully mixing and standing for 6 hours on a rotary incubator, and then adding mPEG-BSA particles to form a solution with the mass percent concentration of 0.5 wt% of mPEG-BSA; then emulsifying for 15s at 12000rpm by using a vortex oscillator to obtain a Dex/PEG emulsion with Dex as a dispersed phase and PEG as a continuous phase, and recording as an A emulsion.
Step 6: and (3) mixing the solution D obtained in the step (3) with the emulsion A obtained in the step (5) in equal mass, and fully and uniformly mixing to obtain a Dex/PEG-Alg emulsion with a dispersed phase Dex and a continuous phase PEG-Alg, wherein the Dex/PEG-Alg emulsion is marked as emulsion B.
And 7: and (3) sucking the emulsion B by using a commercially available 10mL syringe, sleeving a syringe needle with the inner diameter of 0.8mm, keeping the syringe at a position 3-5 cm away from the receiving liquid surface of the solution E, and suspending and vertically. And (3) dropping the emulsion B into the solution E at the speed of 3-5 seconds per drop by pushing a push rod of the injector, and forming the porous calcium alginate microspheres with the pore size of 20-40 mu m and the diameter of 2-3 mm after 15-30 min.
Example 1
A preparation method of porous calcium alginate microspheres with aqueous two-phase emulsion as a template comprises the following steps:
step 1: BSA and mPEG-CHO were added to an aqueous solution having a pH of 5 at a mass ratio of 1:1, and the mixture was stirred to be sufficiently dissolved, and the BSA and mPEG-CHO concentration in the solution were 10 wt%. And sealing the reaction solution, reacting for 48 hours at room temperature, drying the reaction solution in an electrothermal constant-temperature air blast drying oven at 37 ℃, and drying to obtain the mPEG-BSA particles.
Step 2: preparing a PEG solution with the molecular weight of 8kDa and the mass percent concentration of 16 wt% as an A solution, and preparing a Dex solution with the molecular weight of 500kDa and the mass percent concentration of 16 wt% as a B solution; weighing equal volumes of solution A and solution B, fully mixing the solutions on a rotary incubator, standing for 6 hours, then carrying out phase separation, wherein the upper phase is a PEG-rich phase, the lower phase is a Dex-rich phase, and extracting the upper phase and recording as solution C.
And step 3: adding Alg into the solution C obtained in the step 2, and preparing a PEG-Alg solution with the Alg mass percentage concentration of 2 wt% as a solution D;
and 4, step 4: adding CaCl into the solution C obtained in the step 22Preparing CaCl2PEG-CaCl with the mass percentage concentration of 10 wt%2The solution is E solution;
and 5: weighing equal volumes of PEG solution with mass percent concentration of 16 wt% and Dex solution, fully mixing and standing for 6 hours on a rotary incubator, and then adding mPEG-BSA particles to form a solution with the mass percent concentration of 0.5 wt% of mPEG-BSA; then emulsifying for 15s at 12000rpm by using a vortex oscillator to obtain a Dex/PEG emulsion with Dex as a dispersed phase and PEG as a continuous phase, and recording as an A emulsion.
Step 6: and (3) slowly and uniformly mixing the solution D obtained in the step (3) with the emulsion A obtained in the step (5) in the same mass, and fully and uniformly mixing to obtain a Dex/PEG-Alg emulsion with a dispersed phase Dex and a continuous phase PEG-Alg, wherein the Dex/PEG-Alg emulsion is marked as emulsion B.
And 7: and (3) sucking the emulsion B by using a commercially available 10mL syringe, sleeving a syringe needle with the inner diameter of 0.8mm, keeping the syringe at a position 3-5 cm away from the receiving liquid surface of the solution E, and suspending and vertically. And (3) dropping the emulsion B into the solution E at the speed of 3-5 seconds per drop by pushing a push rod of the injector, and forming the porous calcium alginate microspheres with the pore size of 20-40 mu m and the diameter of 2-3 mm after 15-30 min.
The obtained porous calcium alginate microspheres are shown in figure 2; the microscopic observation results are shown in fig. 2a and 2b, and it can be seen from the figures that the microspheres have good monodispersity and uniform particle size distribution, and the particle size is 2-3 mm.
And (3) washing the porous calcium alginate microspheres prepared in the step (7) with deionized water for multiple times to remove an internal phase Dex, dewatering the washed microspheres, putting the microspheres into a 15mL test tube, freezing with liquid nitrogen, sealing with a porous preservative film, putting into a freeze dryer, and freeze-drying.
SEM images and partial enlarged images of the porous calcium alginate microspheres obtained after freeze drying are shown in figures 3a and 3 b. The surface structure of the porous calcium alginate microspheres can be seen.
The micrograph of emulsion A is shown in FIG. 4a and the micrograph of emulsion B is shown in FIG. 4B.
SEM is carried out on the porous calcium alginate microspheres obtained after freeze drying, and the internal structure can be seen as shown in figures 4c and 4 d.
Example 2
A preparation method of porous calcium alginate microspheres with aqueous two-phase emulsion as a template comprises the following steps:
step 1: BSA and mPEG-CHO were added to an aqueous solution having a pH of 5 at a mass ratio of 1:1, and the mixture was stirred to be sufficiently dissolved, and the BSA and mPEG-CHO concentration in the mixed solution were each 10 wt%. And sealing the reaction solution, reacting for 48 hours at room temperature, drying the reaction solution in an electrothermal constant-temperature air blast drying oven at 37 ℃, and drying to obtain the mPEG-BSA particles.
Step 2: preparing a PEG solution with the molecular weight of 8kDa and the mass percent concentration of 16 wt% as an A solution, and preparing a Dex solution with the molecular weight of 500kDa and the mass percent concentration of 16 wt% as a B solution; weighing equal volumes of solution A and solution B, fully mixing the solutions on a rotary incubator, standing for 6 hours, then carrying out phase separation, wherein the upper phase is a PEG-rich phase, the lower phase is a Dex-rich phase, and extracting the upper phase and recording as solution C.
And step 3: adding Alg into the solution C obtained in the step 2, and preparing a PEG-Alg solution with the Alg mass percentage concentration of 2 wt% as a solution D;
and 4, step 4: adding CaCl into the solution C obtained in the step 22Preparing CaCl2PEG-CaCl with the mass percentage concentration of 10 wt%2The solution is E solution;
and 5: weighing equal volumes of PEG solution with mass percent concentration of 16 wt% and Dex solution, fully mixing and standing for 6 hours on a rotary incubator, and then adding mPEG-BSA particles to form a solution with the mass percent concentration of 0.5 wt% of mPEG-BSA; then emulsifying for 15s at 2800rpm by using a vortex oscillator to obtain a Dex/PEG emulsion with Dex as a dispersed phase and PEG as a continuous phase, and recording as an A emulsion.
Step 6: and (3) slowly and uniformly mixing the solution D obtained in the step (3) with the emulsion A obtained in the step (5) in the same mass, and fully and uniformly mixing to obtain a Dex/PEG-Alg emulsion with a dispersed phase Dex and a continuous phase PEG-Alg, wherein the Dex/PEG-Alg emulsion is marked as emulsion B.
And 7: and (3) sucking the emulsion B by using a commercially available 10mL syringe, sleeving a syringe needle with the inner diameter of 0.8mm, keeping the syringe at a position 3-5 cm away from the receiving liquid surface of the solution E, and suspending and vertically. And (3) dropping the emulsion B into the solution E at the speed of 3-5 seconds per drop by pushing a push rod of the injector, and forming the porous calcium alginate microspheres with the pore size of 20-40 mu m and the diameter of 2-3 mm after 15-30 min.
And (3) washing the porous calcium alginate microspheres prepared in the step (7) with deionized water for multiple times to remove an internal phase Dex, dewatering the washed microspheres, putting the microspheres into a 15mL test tube, freezing with liquid nitrogen, sealing with a porous preservative film, putting into a freeze dryer, and freeze-drying.
The micrograph of emulsion A is shown in FIG. 5a and the micrograph of emulsion B is shown in FIG. 5B.
SEM is carried out on the porous calcium alginate microspheres obtained after freeze drying, and the internal structure can be seen as shown in figures 5c and 5 d.
Example 3
A preparation method of porous calcium alginate microspheres with aqueous two-phase emulsion as a template comprises the following steps:
step 1: BSA and mPEG-CHO were added to an aqueous solution having a pH of 5 at a mass ratio of 1:1, and the mixture was stirred to be sufficiently dissolved, and the BSA and mPEG-CHO concentration in the mixed solution were each 10 wt%. And sealing the reaction solution, reacting for 48 hours at room temperature, drying the reaction solution in an electrothermal constant-temperature air blast drying oven at 37 ℃, and drying to obtain the mPEG-BSA particles.
Step 2: preparing a PEG solution with the molecular weight of 8kDa and the mass percent concentration of 16 wt% as an A solution, and preparing a Dex solution with the molecular weight of 500kDa and the mass percent concentration of 16 wt% as a B solution; weighing equal volumes of solution A and solution B, fully mixing the solutions on a rotary incubator, standing for 6 hours, then carrying out phase separation, wherein the upper phase is a PEG-rich phase, the lower phase is a Dex-rich phase, and extracting the upper phase and recording as solution C.
And step 3: adding Alg into the solution C obtained in the step 2, and preparing a PEG-Alg solution with the Alg mass percentage concentration of 2 wt% as a solution D;
and 4, step 4: adding CaCl into the solution C obtained in the step 22Preparing CaCl2PEG-CaCl with the mass percentage concentration of 10 wt%2The solution is E solution;
and 5: weighing equal volumes of PEG solution with mass percent concentration of 16 wt% and Dex solution, fully mixing and standing for 6 hours on a rotary incubator, and then adding mPEG-BSA particles to form a solution with the mass percent concentration of 0.5 wt% of mPEG-BSA; then emulsifying for 15s at 2800rpm by using a vortex oscillator to obtain a Dex/PEG emulsion with Dex as a dispersed phase and PEG as a continuous phase, and recording as an A emulsion.
Step 6: and (3) slowly and uniformly mixing the solution D obtained in the step (3) with the emulsion A obtained in the step (5) in the same volume, and fully and uniformly mixing to obtain a Dex/PEG-Alg emulsion with a dispersed phase Dex and a continuous phase PEG-Alg, wherein the Dex/PEG-Alg emulsion is marked as emulsion B.
And 7: and (3) sucking the emulsion B by using a commercially available 10mL syringe, sleeving a syringe needle with the inner diameter of 0.8mm, keeping the syringe at a position 3-5 cm away from the receiving liquid surface of the solution E, and suspending and vertically. And (3) dropping the emulsion B into the solution E at the speed of 3-5 seconds per drop by pushing a push rod of the injector, and forming the porous calcium alginate microspheres with the pore size of 20-50 microns and the diameter of 2-3 mm after 15-30 min.
And (3) washing the porous calcium alginate microspheres prepared in the step (7) with deionized water for multiple times to remove an internal phase Dex, dewatering the washed microspheres, putting the microspheres into a 15mL test tube, freezing with liquid nitrogen, sealing with a porous preservative film, putting into a freeze dryer, and freeze-drying.
The micrograph of emulsion A is shown in FIG. 6a and the micrograph of emulsion B is shown in FIG. 6B.
SEM is carried out on the porous calcium alginate microspheres obtained after freeze drying, and the internal structure can be seen as shown in figures 6c and 6 d.
Example 4
A preparation method of porous calcium alginate microspheres with aqueous two-phase emulsion as a template comprises the following steps:
step 1: BSA and mPEG-CHO were added to an aqueous solution having a pH of 5 at a mass ratio of 1:1, and the mixture was stirred to be sufficiently dissolved, and the BSA and mPEG-CHO concentration in the mixed solution were each 10 wt%. And sealing the reaction solution, reacting for 48 hours at room temperature, drying the reaction solution in an electrothermal constant-temperature air blast drying oven at 37 ℃, and drying to obtain the mPEG-BSA particles.
Step 2: preparing a PEG solution with the molecular weight of 8kDa and the mass percent concentration of 16 wt% as an A solution, and preparing a Dex solution with the molecular weight of 500kDa and the mass percent concentration of 16 wt% as a B solution; weighing equal volumes of solution A and solution B, fully mixing the solutions on a rotary incubator, standing for 6 hours, then carrying out phase separation, wherein the upper phase is a PEG-rich phase, the lower phase is a Dex-rich phase, and extracting the upper phase and recording as solution C.
And step 3: adding Alg into the solution C obtained in the step 2, and preparing a PEG-Alg solution with the Alg mass percentage concentration of 2 wt% as a solution D;
and 4, step 4: adding CaCl into the solution C obtained in the step 22Preparing CaCl2PEG-CaCl with the mass percentage concentration of 10 wt%2The solution is E solution;
and 5: weighing equal volumes of PEG solution with mass percent concentration of 16 wt% and Dex solution, fully mixing and standing for 6 hours on a rotary incubator, and then adding mPEG-BSA particles to form a solution with the mass percent concentration of 0.5 wt% of mPEG-BSA; then emulsifying for 15s at 2800rpm by using a vortex oscillator to obtain a Dex/PEG emulsion with Dex as a dispersed phase and PEG as a continuous phase, and recording as an A emulsion.
Step 6: and (3) slowly and uniformly mixing the solution D obtained in the step (3) and the emulsion A obtained in the step (5) according to the volume ratio of 1:2, and fully and uniformly mixing to obtain a Dex/PEG-Alg emulsion with a dispersed phase of Dex and a continuous phase of PEG-Alg, which is marked as emulsion B.
And 7: and (3) sucking the emulsion B by using a commercially available 10mL syringe, sleeving a syringe needle with the inner diameter of 0.8mm, keeping the syringe at a position 3-5 cm away from the receiving liquid surface of the solution E, and suspending and vertically. And (3) dropping the emulsion B into the solution E at the speed of 3-5 seconds per drop by pushing a push rod of the injector, and forming the porous calcium alginate microspheres with the pore size of 20-70 mu m and the diameter of 2-3 mm after 15-30 min.
And (3) washing the porous calcium alginate microspheres prepared in the step (7) with deionized water for multiple times to remove an internal phase Dex, dewatering the washed microspheres, putting the microspheres into a 15mL test tube, freezing with liquid nitrogen, sealing with a porous preservative film, putting into a freeze dryer, and freeze-drying.
The micrograph of emulsion A is shown in FIG. 7a and the micrograph of emulsion B is shown in FIG. 7B.
SEM is carried out on the porous calcium alginate microspheres obtained after freeze drying, and the internal structure can be seen as shown in figures 7c and 7 d.
The porous calcium alginate microspheres prepared in example 1 were subjected to SEM, the internal structure thereof was observed, and 100 pores were randomly selected for measurement and recording. SEM is carried out on the porous calcium alginate microspheres prepared in the example 2, the internal structure of the microspheres is observed, and 100 holes are randomly selected for recording; and (4) counting the recorded data to obtain a pore size frequency distribution diagram of the porous calcium alginate microspheres with different milk forming frequencies, which is shown in fig. 8.
The porous calcium alginate microspheres prepared in example 3 were subjected to SEM, the internal structure thereof was observed, and 100 pores were randomly selected for measurement and recording. SEM is carried out on the porous calcium alginate microspheres prepared in the example 4, the internal structure of the microspheres is observed, and 100 holes are randomly selected for recording; the recorded data are counted to obtain the aqueous two-phase emulsion and PEG-Al shown in figure 9; g, the pore size frequency distribution diagram of the porous calcium alginate microspheres with different volume ratios of the solution.
The invention utilizes the stable aqueous two-phase emulsion of protein polymer particles to prepare the porous calcium alginate microspheres with controllable porous structures in the full-water environment. The porous microsphere has high biocompatibility and controllable pore size; the size of the aqueous two-phase emulsion is controlled by changing the emulsion forming frequency of the aqueous two-phase emulsion or the volume ratio of the aqueous two-phase emulsion to the polyethylene glycol-alginic acid (PEG-Alg) solution, so that the pore size of the porous microspheres is controlled; the biological compatibility of the porous microspheres is improved by using a double-aqueous-phase emulsion formed by two incompatible macromolecule Dex and PEG solutions; by utilizing the protein polymer particles, the emulsion is more stable, and the pore size of the porous microspheres is easier to control.
Claims (7)
1. A preparation method of porous calcium alginate microspheres with aqueous two-phase emulsion as a template is characterized by comprising the following steps:
step 1: adding bovine serum albumin BSA and methoxypolyethylene glycol-acetaldehyde mPEG-CHO into an aqueous solution with the pH value of 5 according to the mass ratio of 1:1, fully dissolving and completely reacting, and drying to obtain mPEG-BSA particles;
step 2: fully mixing a polyethylene glycol (PEG) solution with the mass percentage concentration of 16% and a dextran Dex solution in equal volume, standing, splitting the phase, and taking an upper phase solution as a solution C;
and step 3: adding Alg alginate into the solution C obtained in the step 2, and preparing a PEG-Alg solution with the Alg mass percentage concentration of 2 wt% as a solution D;
and 4, step 4: adding calcium chloride CaCl2 into the solution C obtained in the step 2, and preparing a PEG-CaCl2 solution with the CaCl2 mass percent concentration of 10 wt% as an E solution;
and 5: fully mixing PEG solution with mass percent concentration of 16% and Dex solution in equal volume, carrying out phase splitting, adding the mPEG-BSA particles obtained in the step 1 to form solution with the mass percent concentration of 0.5 wt%, and emulsifying to obtain emulsion A;
step 6: mixing the solution D obtained in the step 3 and the emulsion A obtained in the step 5 according to a set proportion, and fully and uniformly mixing to obtain an emulsion B;
and 7: and (3) dripping the emulsion B into the solution E at the speed of 3-5 seconds per drop to form the required porous calcium alginate microspheres.
2. The method for preparing porous calcium alginate microspheres using aqueous two-phase emulsion as a template according to claim 1, wherein bovine serum albumin and methoxypolyethylene glycol-acetaldehyde are dissolved in step 1, sealed, reacted at room temperature for 48 hours, dried in an electrothermal constant temperature air-blowing drying oven at 37 ℃, and dried to obtain the required mPEG-BSA particles.
3. The method for preparing the porous calcium alginate microspheres using the aqueous two-phase emulsion as the template according to claim 1, wherein the molecular weight of the PEG is 8kDa, and the molecular weight of the Dex is 500 kDa.
4. The preparation method of the porous calcium alginate microspheres using the aqueous two-phase emulsion as the template according to claim 1, wherein the emulsion A is obtained by emulsifying the mixture for 15s at 2800-12000 rpm by using a vortex oscillator in the step 5.
5. The method for preparing the porous calcium alginate microspheres using the aqueous two-phase emulsion as the template according to claim 1, wherein the emulsion B is dropwise added at a distance of 3-5 cm from the liquid level of the solution E by using an injector in the step 7.
6. The porous calcium alginate microspheres obtained by the preparation method of claim 1, wherein the surfaces of the porous calcium alginate microspheres are calcium alginate hydrogel, the interiors of the porous calcium alginate microspheres are porous structures with good connectivity, and the particle sizes of the microspheres are 2-3 mm.
7. The application of the porous calcium alginate microspheres prepared by the preparation method of any one of claims 1 to 5, wherein the porous calcium alginate microspheres are used as drug carriers in the preparation of drugs.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20110043344A (en) * | 2009-10-21 | 2011-04-27 | 한국과학기술연구원 | Micro/nanoparticles capable of generating and bearing gas, and preparation method and use thereof |
| CN105061772A (en) * | 2015-08-05 | 2015-11-18 | 西南交通大学 | Polyethylene glycol/glucan aqueous two-phase system emulsion stabilizer and preparation method thereof |
| CN107459664A (en) * | 2017-08-15 | 2017-12-12 | 华南理工大学 | A kind of method that spherex is prepared based on double-aqueous phase system |
| CN107789332A (en) * | 2017-08-31 | 2018-03-13 | 西南交通大学 | A kind of calcium carbonate/calcium alginate compounded microballoon that adjustable drug release rate is prepared based on aqueous two-phase biomineralization technology |
| CN108514896A (en) * | 2018-03-23 | 2018-09-11 | 西南交通大学 | A kind of preparation method and device of micro-fluidic aqueous two-phase monodisperse calcium alginate microsphere |
| CN109701461A (en) * | 2018-10-25 | 2019-05-03 | 西南交通大学 | A kind of preparation and its application of the calcium carbonate based on PEG/Dex aqueous two-phase/calcium alginate compounded micro-capsule |
-
2019
- 2019-07-19 CN CN201910654949.6A patent/CN110343288B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20110043344A (en) * | 2009-10-21 | 2011-04-27 | 한국과학기술연구원 | Micro/nanoparticles capable of generating and bearing gas, and preparation method and use thereof |
| CN105061772A (en) * | 2015-08-05 | 2015-11-18 | 西南交通大学 | Polyethylene glycol/glucan aqueous two-phase system emulsion stabilizer and preparation method thereof |
| CN107459664A (en) * | 2017-08-15 | 2017-12-12 | 华南理工大学 | A kind of method that spherex is prepared based on double-aqueous phase system |
| CN107789332A (en) * | 2017-08-31 | 2018-03-13 | 西南交通大学 | A kind of calcium carbonate/calcium alginate compounded microballoon that adjustable drug release rate is prepared based on aqueous two-phase biomineralization technology |
| CN108514896A (en) * | 2018-03-23 | 2018-09-11 | 西南交通大学 | A kind of preparation method and device of micro-fluidic aqueous two-phase monodisperse calcium alginate microsphere |
| CN109701461A (en) * | 2018-10-25 | 2019-05-03 | 西南交通大学 | A kind of preparation and its application of the calcium carbonate based on PEG/Dex aqueous two-phase/calcium alginate compounded micro-capsule |
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