CN113634208B - Method for preparing porous calcium alginate microspheres by taking micro-fluidic double-aqueous-phase emulsion as template - Google Patents
Method for preparing porous calcium alginate microspheres by taking micro-fluidic double-aqueous-phase emulsion as template Download PDFInfo
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- B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
- B01J13/02—Making microcapsules or microballoons
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- B01J13/14—Polymerisation; cross-linking
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- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0084—Guluromannuronans, e.g. alginic acid, i.e. D-mannuronic acid and D-guluronic acid units linked with alternating alpha- and beta-1,4-glycosidic bonds; Derivatives thereof, e.g. alginates
Abstract
The invention discloses a method for preparing porous calcium alginate microspheres by taking microfluidic double-aqueous-phase emulsion as a template, which comprises the following steps: taking PEG aqueous solution as an external aqueous phase and PEG/Dex-Alg emulsion as an internal aqueous phase, generating PEG/Dex-Alg emulsion droplets at the cone tip of the PEG/Dex-Alg emulsion droplets through a coaxial capillary device, wherein the external aqueous phase is continuously injected, the internal aqueous phase is periodically and intermittently injected, and introducing the generated PEG/Dex-Alg emulsion droplets into PEG-CaCl 2 The method comprises the steps of carrying out ionic crosslinking in a solution to generate porous calcium alginate microspheres, controlling the diameter size of the porous microspheres by changing the internal aqueous phase air pressure input into a microfluidic device, and controlling the pore size of the porous microspheres by changing the volume ratio of an aqueous emulsion forming solution and the oscillation frequency of emulsion forming.
Description
Technical Field
The invention relates to the technical field of preparation of porous calcium alginate microspheres, and particularly relates to a method for preparing porous calcium alginate microspheres by taking microfluidic double-aqueous-phase emulsion as a template.
Background
The porous microspheres are microspheres with certain interconnected or independent closed pores on the surface or in the sphere. The inner hole and the outer hole which are communicated with each other are interconnected, so that the porous microsphere has the advantages of very low mass density, huge specific surface area, adjustable pore diameter and good fluidity, and therefore, the porous microsphere has abundant application results in the fields of tissue regeneration supports, targeted drug delivery, drug screening, cell separation, immobilized enzymes, high-speed chromatography and the like.
The emulsion template method is a method for preparing porous microspheres which is developed vigorously in recent years. The emulsion template method has the advantages of convenience, high efficiency, high adjustability, low cost, mild conditions and the like, thereby being popular with the researchers. Emulsions are thermodynamically unstable systems formed by two mutually incompatible liquids when mixed under external force, one phase being dispersed in the other in the form of small droplets, the phase existing as small droplets being referred to as the internal or dispersed phase, the other phase being referred to as the external or continuous phase. Emulsions are generally classified into various types, such as oil-in-water type (O/W), water-in-oil type (W/O), water-in-oil-in-water type (W/O/W), and oil-in-water type (O/W/O), depending on the composition of the dispersed phase and the continuous phase. The preparation of the porous structure by the emulsion template method comprises two basic steps: the first step is the preparation of immiscible emulsions and the second step is the solidification of the continuous phase of the emulsion, during which the dispersed phases act like templates, which are removed after the continuous phase has solidified to obtain a porous structure.
In recent years, a microfluidic technology is often combined with an emulsion template method to prepare porous calcium alginate microspheres, however, most microfluidic methods mainly utilize oil-water phases to generate water-in-oil alginate drops to generate microspheres, but the introduction of oil phases and surfactants can reduce the biocompatibility of the microspheres, and in addition, the sphericity of the microspheres generated by a needle tube instillation method is not good, so that the preparation of the porous calcium alginate microspheres with good biocompatibility and flexibly controllable diameter and pore size of the microspheres is a challenge.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a method for preparing porous calcium alginate microspheres by using a microfluidic aqueous two-phase emulsion as a template, so as to solve the problems that the biocompatibility of the porous calcium alginate microspheres prepared by the existing preparation method is not high and the diameters and the pore diameters of the microspheres are not well controlled.
The technical scheme for solving the technical problems is as follows: the method for preparing the porous calcium alginate microspheres by using the microfluidic double-aqueous-phase emulsion as the template comprises the following steps:
taking polyethylene glycol (PEG) aqueous solution as an external water phase and polyethylene glycol/dextran-sodium alginate (PEG/Dex-Alg) emulsion as an internal water phase, generating PEG/Dex-Alg emulsion droplets at the cone tip of the PEG/dextran-sodium alginate (PEG/Dex-Alg) emulsion by a coaxial capillary device, wherein the external water phase is continuously injected, the internal water phase is periodically and intermittently injected, and introducing the PEG/Dex-Alg emulsion droplets into polyethylene glycol-calcium chloride (PEG-CaCl) 2 ) And in the solution, carrying out ionic crosslinking to obtain the porous calcium alginate microspheres.
The beneficial effects of the invention are as follows: the microfluidic technology is a technology for operating microfluid in a microscale space by using a microchannel, micron-sized microspheres with good monodispersity can be efficiently generated by independent operation of the microchannel through the microfluidic technology, the internal water phase is sheared into PEG/Dex-Alg emulsion droplets under the dual actions of shearing force generated by external water phase flow and intermittent sample injection of the internal water phase, and the PEG/Dex-Alg emulsion droplets are introduced into PEG-CaCl 2 And (3) carrying out ionic crosslinking in the solution, and discharging PEG liquid drops in the PEG/Dex-Alg emulsion liquid drops under the action of extrusion force generated by ionic crosslinking, namely generating the porous calcium alginate microspheres.
The invention adopts two hydrophilic nontoxic harmless, green environmental protection and mutually incompatible macromolecule solutions, namely Dex solution and PEG solution, takes Dex solution with higher viscosity as continuous phase and PEG solution with lower viscosity as disperse phase to form PEG/Dex double-water-phase emulsion with slow coalescence speed and longer stabilization time, simultaneously, sodium alginate with certain concentration is introduced into the continuous phase Dex solution because of the solidification needed for preparing the porous microspheres by emulsion template method, on one hand, the sodium alginate can be used as cross-linking agent to carry out cross-linking reaction to generate calcium alginate gel after solidification, on the other hand, the sodium alginate has certain viscosity after being dissolved, the viscosity of the emulsion can be increased so as to slow down the coalescence speed of the emulsion, the double-water-phase emulsion is more stable, the aperture size of the porous calcium alginate microspheres can be controlled more easily, the preparation process is completed by one-step method in full water solution by microfluidic technology, the preparation method is simple, and the prepared porous calcium alginate microspheres have high biocompatibility and wide application prospect in the fields of biology, medicine, tissue engineering and the like.
On the basis of the technical scheme, the invention can be further improved as follows:
furthermore, the external water phase is injected by an injection pump, and the injection speed is 60-70 mul/min.
The beneficial effect of adopting the further technical scheme is as follows: and the injection pump is adopted for sample injection, the sample injection is continuous, and the operation is simple and convenient.
Further, intermittently injecting the internal water phase by an air pump periodically with the injection pressure of 30-110KPa, and with the injection period of turning on the air pump for 0.1-0.2s and turning off for 5-6 s.
The beneficial effect of adopting the further technical scheme is as follows: the diameter size of the porous calcium alginate microspheres can be controlled by changing the sample injection pressure of the internal water phase.
Further, the PEG/Dex-Alg emulsion is dripped into PEG-CaCl 2 In the solution, the time for ion crosslinking is 4-8 h.
Further, PEG aqueous solution, PEG/Dex-Alg emulsion and PEG-CaCl 2 The solution was prepared by the following method:
(1) and (3) fully mixing the PEG solution and the Dex solution with the mass fraction of 15-18% in equal volume, standing, carrying out phase splitting, extracting the upper phase to obtain the PEG aqueous solution, and extracting the lower phase to obtain the Dex aqueous solution.
(2) Adding CaCl into the PEG aqueous solution obtained in the step (1) 2 Mixing to obtain PEG-CaCl 2 Solution of, in which, CaCl 2 The mass fraction is 9-12%;
(3) Adding Alg into the Dex aqueous solution obtained in the step (1), and uniformly mixing to obtain a Dex-Alg solution, wherein the mass fraction of Alg is 0.8-1.8%;
(4) taking the volume ratio of 1: 3-1: 5, emulsifying the aqueous solution of PEG and the Dex-Alg solution to obtain the PEG/Dex-Alg emulsion with PEG as a disperse phase and Dex-Alg as a continuous phase.
Further, the molecular weight of PEG in the step (1) is 7-9kDa, and the molecular weight of Dex is 400-600 kDa.
Further, PEG has a molecular weight of 8kDa and Dex has a molecular weight of 500 kDa.
Further, the emulsification frequency in the emulsification process in the step (4) is 2000-25000rpm/min, and the emulsification time is 28-32 s.
The beneficial effect of adopting the further technical scheme is as follows: the pore size of the porous calcium alginate microspheres can be controlled by changing the emulsification frequency of the PEG/Dex-Alg emulsion and the volume ratio of the two solutions required for emulsification.
The invention also provides the porous calcium alginate microspheres prepared by the method.
The invention also provides a device for preparing the porous calcium alginate microspheres by adopting the method, which is characterized by comprising an inner water phase feeding capillary tube and an outer water phase feeding capillary tube sleeved outside the inner water phase feeding capillary tube, wherein one end of the inner water phase feeding capillary tube is an inner water phase input port, the other end of the inner water phase feeding capillary tube is a conical output port, one end of the outer water phase feeding capillary tube is an outer water phase input port, the other end of the outer water phase feeding capillary tube is connected with an outer water phase output port, and the conical output port is arranged at one end, close to the outer water phase input port, in the outer water phase feeding capillary tube; the inner water phase input port is connected to a sealed glass bottle through a hose, the sealed glass bottle is connected to an air pump through a hard tube, the outer water phase input port is connected to a syringe needle through a hose, the syringe is arranged on the syringe pump, and the outer water phase output port is connected with a container filled with PEG-CaCl 2 A container for the solution.
Furthermore, the outer diameter of the inner water phase feeding capillary is 970-1050 μm, the inner diameter is 750-850 μm, the inner diameter of the tapered output port is 350-400 μm, and the inner diameter of the outer water phase feeding capillary is 1000-1200 μm.
The invention has the beneficial effects that: the method comprises the following steps that an injector is used for sucking an external water phase aqueous solution, an injection pump uniformly pushes the injector at a certain speed, the external water phase aqueous solution enters a gap between an external water phase feeding capillary and an internal water phase feeding capillary through an external water phase input port, compressed gas generated by an air pump is input into a glass bottle filled with internal water phase emulsion through a hard tube, then the compressed emulsion is input into an internal water phase input port through a hose and enters the internal water phase feeding capillary, the input internal water phase emulsion is also in an intermittent input state through air pressure intermittently input by the air pump, and the external water phase fluid is cutUnder the action of shearing force and air pressure for intermittently inputting internal water phase, the internal water phase emulsion is dispersed into emulsion in droplet form in a microfluidic device, and PEG-CaCl is dropped into the droplet 2 In the receiving liquid, porous calcium alginate microspheres are formed after ion crosslinking.
The device has the advantages of simple structure, flexible and convenient manufacture, easy adjustment of the flow rate of the injection pump and the air pressure of the air pump, simple operation and suitability for batch production.
Drawings
Fig. 1 is a 3D schematic of a microfluidic device of the present invention;
FIG. 2 is a schematic diagram of the structure of a microfluidic device according to the present invention;
FIG. 3 is a schematic diagram of a microfluidic device according to the present invention for generating a droplet-like emulsion;
FIG. 4 is a pictorial view of a microfluidic device according to the present invention;
FIG. 5 is a mechanism diagram of preparing porous calcium alginate microspheres by microfluidic technology;
FIG. 6 is a 4-fold microscopic image of the porous calcium alginate gel microspheres prepared in example 1;
FIG. 6a is a 10-fold microscope image of the overall appearance of the porous calcium alginate gel microspheres prepared in example 1;
FIG. 7 is a 4-fold microscopic image of the porous calcium alginate gel microspheres prepared in example 2;
FIG. 7a is a 10-fold microscope image of the overall appearance of the porous calcium alginate gel microspheres prepared in example 2;
FIG. 8 is a 4-fold microscopic image of the porous calcium alginate gel microspheres prepared in example 3;
FIG. 8a is a 10-fold microscope image of the overall appearance of the porous calcium alginate gel microspheres prepared in example 3;
FIG. 9 is a scanning electron microscope image of the surface of the porous calcium alginate microspheres prepared in this example 1;
FIG. 9a is a partially enlarged scanning electron micrograph of the porous calcium alginate microspheres prepared in this example 1;
FIG. 10 is a bar graph of the internal water phase input pressure and the diameter of porous calcium alginate microspheres;
FIG. 11 is a scanning electron microscope and a partial enlarged view of the cross-section of the porous calcium alginate microspheres prepared in example 4 after being cut open;
FIG. 12 is a scanning electron microscope and a partial enlarged view of the cross-section of the porous calcium alginate microspheres prepared in example 5 after being cut open;
FIG. 13 is a scanning electron microscope and a partial enlarged view of the cross-section of the porous calcium alginate microspheres prepared in example 6 after being cut open;
FIG. 14 is a scanning electron microscope and a partial enlarged view of the cross-section of the porous calcium alginate microspheres prepared in example 7 after being cut open;
FIG. 15 is a graph showing the correlation between the pore size of the porous calcium alginate microspheres prepared in examples 4 to 5 and the frequency of emulsion-forming oscillation;
FIG. 16 is a graph showing the correlation between the pore size of the porous calcium alginate microspheres prepared in examples 6 to 7 and the volume ratio of the emulsion to the emulsion solution.
Detailed Description
The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
As shown in fig. 1-5, the apparatus for preparing porous calcium alginate microspheres by the method of the present invention comprises an inner aqueous phase feeding capillary tube 100 and an outer aqueous phase feeding capillary tube 200 sleeved outside the inner aqueous phase feeding capillary tube 100, wherein one end of the inner aqueous phase feeding capillary tube 100 is an inner aqueous phase input port 101, the other end is a tapered output port 102, one end of the outer aqueous phase feeding capillary tube 200 is an outer aqueous phase input port 201, the other end is connected to an outer aqueous phase output port 202, and the tapered output port 102 is disposed at one end of the outer aqueous phase feeding capillary tube 200 close to the outer aqueous phase input port 201; the inner water phase input port 101 is connected into a closed glass bottle through a hose, the closed glass bottle is connected with an air pump through a hard tube, the outer water phase input port 201 is connected with a syringe needle through a hose, the syringe is arranged on the syringe pump, and the outer water phase output port 202 is connected with a container containing PEG-CaCl2 solution.
The device used by the invention adopts a structure of combining a glass capillary and a stainless steel dispensing machine needle, an external water phase feeding capillary 200 is a capillary glass tube with the length of 6cm, the outer diameter of 1500 mu m and the inner diameter of 1000-, the size of the device can be adjusted according to actual conditions.
Example 1:
a method for preparing porous calcium alginate microspheres by using microfluidic double-aqueous-phase emulsion as a template comprises the following steps:
(1) fully mixing PEG solution and Dex solution with equal volume and mass fractions of 16% on a rotary incubator, standing for 6 hours, splitting phases, extracting the upper phase to obtain PEG aqueous solution, and extracting the lower phase to obtain Dex aqueous solution, wherein the PEG has a molecular weight of 8kDa and the Dex has a molecular weight of 500 kDa;
(2) adding CaCl into the PEG aqueous solution obtained in the step (1) 2 After even mixing, CaCl is prepared 2 PEG-CaCl with mass fraction of 10% 2 A solution;
(3) adding Alg into the Dex aqueous solution obtained in the step (1), and uniformly mixing to obtain a Dex-Alg solution with the Alg mass fraction of 1%;
(4) measuring volume ratio of 1: 3, emulsifying the PEG aqueous solution and the Dex-Alg solution for 30s by using a portable high-speed disperser at the oscillation frequency of 2000rpm/min to obtain a PEG/Dex-Alg emulsion with a dispersed phase of PEG and a continuous phase of Dex-Alg;
(5) sucking PEG aqueous solution by a commercially available 10mL syringe, sleeving a syringe needle with the inner diameter of 0.8mm, placing the syringe needle on a syringe pump, connecting one end of a hose with the diameter of 1mm with the syringe needle, connecting the other end of the hose with the outer water phase input port, then filling PEG/Dex-Alg emulsion into a sealed glass bottle of an air pump, wherein the diameter of the hose for outputting the emulsion is 1mm, the diameter of a hard tube connected with the glass bottle and the air pump is 3mm, the outer diameter of an inner water phase feeding capillary is 970 mu m, the inner diameter of the inner water phase feeding capillary is 750 mu m, the inner diameter of an outlet of a conical tube is 350 mu m, and the inner diameter of an outer water phase feeding capillary is 1000 mu m;
(6) Setting the sample injection speed of an injection pump at 65 μ l/min, setting the sample injection pressure of an air pump at 30KPa, and the intermittent sample injection period of the air pump being 0.1s and 5.5s, forming PEG/Dex-Alg emulsion liquid drop in the external water phase feed capillary, introducing into PEG-CaCl 2 And (4) carrying out ion crosslinking for 6 hours in the receiving solution to obtain the porous calcium alginate microspheres.
Example 2:
a method for preparing porous calcium alginate microspheres by using microfluidic double-aqueous-phase emulsion as a template comprises the following steps:
(1) fully mixing PEG solution and Dex solution with equal volume and mass fractions of 16% on a rotary incubator, standing for 6 hours, carrying out phase splitting, extracting the upper phase to obtain PEG aqueous solution, and extracting the lower phase to obtain Dex aqueous solution, wherein the PEG molecular weight is 8kDa, and the Dex molecular weight is 500 kDa;
(2) adding CaCl into the PEG aqueous solution obtained in the step (1) 2 After even mixing, CaCl is prepared 2 PEG-CaCl with mass fraction of 10% 2 A solution;
(3) adding Alg into the Dex aqueous solution obtained in the step (1), and uniformly mixing to obtain a Dex-Alg solution with the Alg mass fraction of 1%;
(4) measuring volume ratio of 1: 3, emulsifying the PEG aqueous solution and the Dex-Alg solution for 30s by using a portable high-speed disperser at the oscillation frequency of 2000rpm/min to obtain a PEG/Dex-Alg emulsion with a dispersed phase of PEG and a continuous phase of Dex-Alg;
(5) Sucking PEG aqueous solution by a commercially available 10mL syringe, sleeving a syringe needle with the inner diameter of 0.8mm, placing on an injection pump, connecting one end of a hose with the diameter of 1mm with the syringe needle, connecting the other end of the hose with an outer water phase input port, filling PEG/Dex-Alg emulsion into a sealed glass bottle of an air pump, wherein the diameter of the hose for outputting the emulsion is 1mm, and the diameter of a hard tube connected with the air pump of the glass bottle is 3mm, wherein the outer diameter of an inner water phase feeding capillary is 970 mu m, the inner diameter is 750 mu m, the inner diameter of an outlet of a conical tube is 350 mu m, and the inner diameter of an outer water phase feeding capillary is 1000 mu m;
(6) setting the sample injection speed of an injection pump at 65 μ l/min, setting the sample injection pressure of an air pump at 70KPa, and the intermittent sample injection period of the air pump being 0.1s and 5.5s, forming PEG/Dex-Alg emulsion liquid drop in the external water phase feed capillary, introducing into PEG-CaCl 2 And (4) carrying out ion crosslinking for 6 hours in the receiving solution to obtain the porous calcium alginate microspheres.
Example 3:
a method for preparing porous calcium alginate microspheres by using microfluidic double-aqueous-phase emulsion as a template comprises the following steps:
(1) fully mixing PEG solution and Dex solution with equal volume and mass fractions of 16% on a rotary incubator, standing for 6 hours, carrying out phase splitting, extracting the upper phase to obtain PEG aqueous solution, and extracting the lower phase to obtain Dex aqueous solution, wherein the PEG molecular weight is 8kDa, and the Dex molecular weight is 500 kDa;
(2) Adding CaCl into the PEG aqueous solution obtained in the step (1) 2 Mixing to obtain CaCl 2 PEG-CaCl with mass fraction of 10% 2 A solution;
(3) adding Alg into the Dex aqueous solution obtained in the step (1), and uniformly mixing to obtain a Dex-Alg solution with the Alg mass fraction of 1%;
(4) measuring volume ratio of 1: 3, emulsifying the PEG aqueous solution and the Dex-Alg solution for 30s by using a portable high-speed disperser at the oscillation frequency of 2000rpm/min to obtain a PEG/Dex-Alg emulsion with a dispersed phase of PEG and a continuous phase of Dex-Alg;
(5) sucking PEG aqueous solution by a commercially available 10mL syringe, sleeving a syringe needle with the inner diameter of 0.8mm, placing on an injection pump, connecting one end of a hose with the diameter of 1mm with the syringe needle, connecting the other end of the hose with an outer water phase input port, filling PEG/Dex-Alg emulsion into a sealed glass bottle of an air pump, wherein the diameter of the hose for outputting the emulsion is 1mm, and the diameter of a hard tube connected with the air pump of the glass bottle is 3mm, wherein the outer diameter of an inner water phase feeding capillary is 970 mu m, the inner diameter is 750 mu m, the inner diameter of an outlet of a conical tube is 350 mu m, and the inner diameter of an outer water phase feeding capillary is 1000 mu m;
(6) setting the sample injection speed of an injection pump at 65 μ l/min, setting the sample injection pressure of an air pump at 110KPa, and the intermittent sample injection period at 0.1s and 5.5s, forming PEG/Dex-Alg emulsion liquid drop in the external water phase feed capillary, and introducing into PEG-CaCl 2 And (4) carrying out ion crosslinking for 6 hours in the receiving solution to obtain the porous calcium alginate microspheres.
Example 4:
a method for preparing porous calcium alginate microspheres by using microfluidic double-aqueous-phase emulsion as a template comprises the following steps:
(1) fully mixing PEG solution and Dex solution with equal volume and mass fraction of 15% on a rotary incubator, standing for 6 hours, then carrying out phase splitting, extracting the upper phase to obtain PEG aqueous solution, and extracting the lower phase to obtain Dex aqueous solution, wherein the PEG molecular weight is 7kDa, and the Dex molecular weight is 400 kDa;
(2) adding CaCl into the PEG aqueous solution obtained in the step (1) 2 After even mixing, CaCl is prepared 2 9% of PEG-CaCl by mass fraction 2 A solution;
(3) adding Alg into the Dex aqueous solution obtained in the step (1), and uniformly mixing to obtain a Dex-Alg solution with the Alg mass fraction of 0.8%;
(4) measuring volume ratio of 1: 4, emulsifying the PEG aqueous solution and the Dex-Alg solution for 28s by using a portable high-speed disperser at the oscillation frequency of 2000rpm/min to obtain a PEG/Dex-Alg emulsion with a dispersed phase of PEG and a continuous phase of Dex-Alg;
(5) sucking PEG aqueous solution by a commercially available 10mL syringe, sleeving a syringe needle with the inner diameter of 0.8mm, placing on an injection pump, connecting one end of a hose with the diameter of 1mm with the syringe needle, connecting the other end of the hose with an outer water phase input port, filling PEG/Dex-Alg emulsion into a sealed glass bottle of an air pump, wherein the diameter of the hose for outputting the emulsion is 1mm, and the diameter of a hard tube connected with the glass bottle and the air pump is 3mm, wherein the outer diameter of an inner water phase feeding capillary is 1000 mu m, the inner diameter is 800 mu m, the inner diameter of an outlet of a conical tube is 370 mu m, and the inner diameter of an outer water phase feeding capillary is 1100 mu m;
(6) Provided with an injection pumpThe sample injection speed is 65 mul/min, the sample injection pressure of an air pump is set to be 70KPa, the intermittent sample injection period is that the air pump is started for 0.15s and closed for 5s, and PEG/Dex-Alg emulsion liquid drops can be formed in the external water phase feed capillary and are introduced into PEG-CaCl 2 And (4) carrying out ion crosslinking for 4 hours in the receiving solution to obtain the porous calcium alginate microspheres.
Example 5:
a method for preparing porous calcium alginate microspheres by using microfluidic double-aqueous-phase emulsion as a template comprises the following steps:
(1) fully mixing PEG solution and Dex solution with equal volume and mass fraction of 15% on a rotary incubator, standing for 6 hours, then carrying out phase splitting, extracting the upper phase to obtain PEG aqueous solution, and extracting the lower phase to obtain Dex aqueous solution, wherein the PEG molecular weight is 7kDa, and the Dex molecular weight is 400 kDa;
(2) adding CaCl into the PEG aqueous solution obtained in the step (1) 2 After even mixing, CaCl is prepared 2 9% of PEG-CaCl by mass fraction 2 A solution;
(3) adding Alg into the Dex aqueous solution obtained in the step (1), and uniformly mixing to obtain a Dex-Alg solution with the Alg mass fraction of 0.8%;
(4) measuring volume ratio of 1: 4, emulsifying the PEG aqueous solution and the Dex-Alg solution for 28s by using a portable high-speed disperser at the oscillation frequency of 25000rpm/min to obtain a PEG/Dex-Alg emulsion with a dispersed phase of PEG and a continuous phase of Dex-Alg;
(5) Sucking PEG aqueous solution by a commercially available 10mL syringe, sleeving a syringe needle with the inner diameter of 0.8mm, placing on an injection pump, connecting one end of a hose with the diameter of 1mm with the syringe needle, connecting the other end of the hose with an outer water phase input port, filling PEG/Dex-Alg emulsion into a sealed glass bottle of an air pump, wherein the diameter of the hose for outputting the emulsion is 1mm, and the diameter of a hard tube connected with the glass bottle and the air pump is 3mm, wherein the outer diameter of an inner water phase feeding capillary is 1000 mu m, the inner diameter is 800 mu m, the inner diameter of an outlet of a conical tube is 370 mu m, and the inner diameter of an outer water phase feeding capillary is 1100 mu m;
(6) setting the sample injection speed of an injection pump to be 65 mul/min, setting the sample injection pressure of an air pump to be 70KPa, and setting the intermittent sample injection period to be that the air pump is turned on for 0.15s and turned off for 5ss, forming PEG/Dex-Alg emulsion droplets in the external water phase feeding capillary, and introducing the emulsion droplets into PEG-CaCl 2 And (4) carrying out ion crosslinking for 4 hours in the receiving solution to obtain the porous calcium alginate microspheres.
Example 6:
a method for preparing porous calcium alginate microspheres by using microfluidic double-aqueous-phase emulsion as a template comprises the following steps:
(1) fully mixing PEG solution and Dex solution with equal volume and mass fraction of 18% on a rotary incubator, standing for 6 hours, then carrying out phase splitting, extracting the upper phase to obtain PEG aqueous solution, and extracting the lower phase to obtain Dex aqueous solution, wherein the PEG molecular weight is 9kDa, and the Dex molecular weight is 600 kDa;
(2) Adding CaCl into the PEG aqueous solution obtained in the step (1) 2 After even mixing, CaCl is prepared 2 PEG-CaCl with mass fraction of 12% 2 A solution;
(3) adding Alg into the Dex aqueous solution obtained in the step (1), and uniformly mixing to obtain a Dex-Alg solution with the Alg mass fraction of 1.8%;
(4) measuring volume ratio of 1: 3, emulsifying the PEG aqueous solution and the Dex-Alg solution for 32s at the oscillation frequency of 2000rpm/min by using a portable high-speed disperser to obtain a PEG/Dex-Alg emulsion with a dispersed phase of PEG and a continuous phase of Dex-Alg;
(5) sucking PEG aqueous solution by a commercially available 10mL syringe, sleeving a syringe needle with the inner diameter of 0.8mm, placing on an injection pump, connecting one end of a hose with the diameter of 1mm with the syringe needle, connecting the other end of the hose with an outer water phase input port, filling PEG/Dex-Alg emulsion into a sealed glass bottle of an air pump, wherein the diameter of the hose for outputting the emulsion is 1mm, and the diameter of a hard tube connected with the air pump of the glass bottle is 3mm, wherein the outer diameter of an inner water phase feeding capillary is 1050 micrometers, the inner diameter of the inner water phase feeding capillary is 850 micrometers, the inner diameter of an outlet of a conical tube is 750 micrometers, and the inner diameter of an outer water phase feeding capillary is 1200 micrometers;
(6) setting the sample injection speed of an injection pump at 70 μ l/min, setting the sample injection pressure of an air pump at 70KPa, and the intermittent sample injection period of the air pump being 0.15s and 5s after the air pump is turned on to form PEG/Dex-Alg emulsion liquid drop in the external water phase feed capillary, introducing the PEG/Dex-Alg emulsion liquid drop into PEG-CaCl 2 In the receiving liquid, separateCrosslinking for 8h to obtain the porous calcium alginate microspheres.
Example 7:
a method for preparing porous calcium alginate microspheres by using microfluidic double-aqueous-phase emulsion as a template comprises the following steps:
(1) fully mixing PEG solution and Dex solution with equal volume and mass fraction of 18% on a rotary incubator, standing for 6 hours, then carrying out phase splitting, extracting the upper phase to obtain PEG aqueous solution, and extracting the lower phase to obtain Dex aqueous solution, wherein the PEG molecular weight is 9kDa, and the Dex molecular weight is 600 kDa;
(2) adding CaCl into the PEG aqueous solution obtained in the step (1) 2 After even mixing, CaCl is prepared 2 PEG-CaCl with mass fraction of 12% 2 A solution;
(3) adding Alg into the Dex aqueous solution obtained in the step (1), and uniformly mixing to obtain a Dex-Alg solution with the Alg mass fraction of 1.8%;
(4) measuring volume ratio of 1: 5, emulsifying the PEG aqueous solution and the Dex-Alg solution for 32s at the oscillation frequency of 2000rpm/min by using a portable high-speed disperser to obtain a PEG/Dex-Alg emulsion with a dispersed phase of PEG and a continuous phase of Dex-Alg;
(5) sucking PEG aqueous solution by a commercially available 10mL syringe, sleeving a syringe needle with the inner diameter of 0.8mm, placing on an injection pump, connecting one end of a hose with the diameter of 1mm with the syringe needle, connecting the other end of the hose with an outer water phase input port, filling PEG/Dex-Alg emulsion into a sealed glass bottle of an air pump, wherein the diameter of the hose for outputting the emulsion is 1mm, and the diameter of a hard tube connected with the air pump of the glass bottle is 3mm, wherein the outer diameter of an inner water phase feeding capillary is 1050 micrometers, the inner diameter of the inner water phase feeding capillary is 850 micrometers, the inner diameter of an outlet of a conical tube is 750 micrometers, and the inner diameter of an outer water phase feeding capillary is 1200 micrometers;
(6) Setting the sample injection speed of an injection pump at 70 μ l/min, setting the sample injection pressure of an air pump at 70KPa, and the intermittent sample injection period of the air pump being 0.15s and 5s after the air pump is turned on to form PEG/Dex-Alg emulsion liquid drop in the external water phase feed capillary, introducing the PEG/Dex-Alg emulsion liquid drop into PEG-CaCl 2 And (4) carrying out ion crosslinking for 8 hours in the receiving solution to obtain the porous calcium alginate microspheres.
And (4) detecting a result:
the specific detection method comprises the following steps: placing the prepared porous calcium alginate microspheres in PEG-CaCl 2 Observing the receiving solution by using a microscope and counting the diameter of the receiving solution; and (2) washing the prepared porous calcium alginate by using deionized water until internal phase PEG and redundant Dex are removed, then putting the porous calcium alginate into a culture dish, freezing the porous calcium alginate by using liquid nitrogen, sealing the culture dish by using a porous preservative film, finally putting the culture dish into a freeze dryer, carrying out freeze drying, carrying out scanning electron microscope analysis, and observing the surface appearance of the porous calcium alginate, or cutting the porous calcium alginate microspheres after freeze drying by using a blade, carrying out scanning electron microscope analysis, and observing the section appearance and the internal structure of the porous calcium alginate microspheres. The method comprises the following specific steps:
1. the porous calcium alginate microspheres prepared in the examples 1 to 3 are put in PEG-CaCl 2 Observing the received solution with 4 times microscope and 10 times microscope, and taking pictures with digital camera to obtain the results shown in figures 6-8 and 6a-8a, which show that PEG-CaCl 2 The solution is dispersed with porous calcium alginate microspheres with uniform size and good monodispersity.
2. After being washed and dried, the porous calcium alginate microspheres prepared in example 1 are analyzed by a scanning electron microscope, and the surface morphology of the microspheres is observed, and the results are shown in fig. 9 and 9a, which show that the surface of the microspheres is a relatively smooth surface with smaller pore diameters and has a typical calcium alginate hydrogel morphology.
3. The porous calcium alginate microspheres prepared in examples 1-3 were respectively placed in PEG-CaCl 2 Observing the received solution by using a microscope, counting the diameters of the received solution and averaging the diameters to obtain the average diameter of the porous calcium alginate microspheres, and drawing a graph according to the input pressure of the internal water phase to obtain a result shown in figure 10, wherein the graph shows that the average diameter of the microspheres is 496 microns when the input pressure of the internal water phase is 30KPa, the average diameter of the microspheres is 693 microns when the input pressure of the internal water phase is 70KPa, and the average diameter of the microspheres is 877 microns when the input pressure of the internal water phase is 110 KPa.
4. The porous calcium alginate microspheres prepared in examples 4 to 5 were washed, dried, cut, analyzed by scanning electron microscopy, and the cross-section was observed, as shown in fig. 11 to 12, which revealed that the porous calcium alginate microspheres had a dense porous structure inside.
5. The porous calcium alginate microspheres prepared in examples 6 to 7 were washed, dried, cut, analyzed by scanning electron microscopy, and the cross-section was observed, as shown in fig. 13 to 14, which revealed that the porous calcium alginate microspheres had a dense porous structure inside.
6. After being cleaned and dried, the porous calcium alginate microspheres prepared in the examples 4 to 5 are cut by a blade, then are analyzed by a scanning electron microscope, the internal structure of the microspheres is observed, then 100 holes are selected for measurement and recording, and the recorded data are analyzed and counted, so that a frequency distribution graph of the pore sizes of the porous calcium alginate gel microspheres prepared at different emulsification frequencies and a histogram of the average pore sizes of the porous calcium alginate gel microspheres prepared at different emulsification frequencies can be obtained, and the result is shown in fig. 15, which shows that when the oscillation frequency of the internal aqueous phase emulsion is 25000rpm/min, the average pore size of the prepared porous calcium alginate microspheres is 15 μm, and when the oscillation frequency of the internal aqueous phase emulsion is 2000rpm/min, the average pore size of the prepared porous calcium alginate microspheres is 25 μm.
7. After being cleaned and dried, the porous calcium alginate gel microspheres prepared in the embodiments 6 to 7 are cut by a blade, then the analysis is carried out by a scanning electron microscope, the internal structure is observed, then 100 holes are selected for measurement and recording, the recorded data is analyzed and counted, and a frequency distribution diagram of the pore size of the porous calcium alginate gel microspheres prepared by different volume ratios of emulsion forming solutions and a histogram of the average pore size of the porous calcium alginate gel microspheres prepared by different volume ratios of emulsion forming solutions can be obtained, the result is shown in figure 16, and the graph shows that the ratio of the emulsion forming volume ratios is 1: 3, the average pore diameter of the prepared porous calcium alginate microspheres is 24 microns, and the ratio of the formed milk volume to the formed milk volume is 1: and 5, the average pore diameter of the prepared porous calcium alginate microspheres is 17 mu m.
The porous calcium alginate microspheres prepared by the method have good monodispersity and a porous structure with good connectivity, the average pore size is 20-40 mu m, and the average diameter of the microspheres is 400-900 mu m.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (8)
1. A method for preparing porous calcium alginate microspheres by using microfluidic double-aqueous-phase emulsion as a template is characterized by comprising the following steps: taking PEG aqueous solution as an external water phase and PEG/Dex-Alg emulsion as an internal water phase, generating PEG/Dex-Alg emulsion droplets at the cone tip of the PEG/Dex-Alg emulsion droplets through a coaxial capillary device, wherein the external water phase is continuously injected, the internal water phase is periodically and intermittently injected, and introducing the PEG/Dex-Alg emulsion droplets into PEG-CaCl 2 Carrying out ion crosslinking in the solution to obtain porous calcium alginate microspheres;
the PEG aqueous solution, the PEG/Dex-Alg emulsion and the PEG-CaCl 2 The solution was prepared by the following method:
(1) fully mixing 15-18% of PEG solution and Dex solution in equal volume, standing, phase splitting, extracting the upper phase to obtain PEG aqueous solution, and extracting the lower phase to obtain Dex aqueous solution;
(2) Adding CaCl into the PEG aqueous solution obtained in the step (1) 2 Mixing to obtain PEG-CaCl 2 Solution of, wherein, CaCl 2 The mass fraction of (A) is 9-12%;
(3) adding Alg into the Dex aqueous solution obtained in the step (1), and uniformly mixing to obtain a Dex-Alg solution, wherein the mass fraction of Alg is 0.8-1.8%;
(4) measuring volume ratio of 1: 3-1: 5, emulsifying the aqueous solution of PEG and the Dex-Alg solution to obtain the PEG/Dex-Alg emulsion with PEG as a disperse phase and Dex-Alg as a continuous phase.
2. The method for preparing the porous calcium alginate microspheres by using the microfluidic aqueous two-phase emulsion as the template according to claim 1, wherein the external aqueous phase is injected by a syringe pump at a speed of 60-70 μ l/min.
3. The method for preparing the porous calcium alginate microspheres by using the microfluidic aqueous two-phase emulsion as the template according to claim 1, wherein the internal aqueous phase is intermittently injected by an air pump at a cycle of 30-110KPa, and the injection cycle is that the air pump is turned on for 0.1-0.2s and turned off for 5-6 s.
4. The method for preparing the porous calcium alginate microspheres by using the microfluidic aqueous two-phase emulsion as the template according to claim 1, wherein the ionic crosslinking time is 4-8 h.
5. The method for preparing porous calcium alginate microspheres by using the microfluidic aqueous two-phase emulsion as the template according to claim 1, wherein the molecular weight of the PEG in the step (1) is 7-9kDa, and the molecular weight of the Dex is 400-600 kDa.
6. The method for preparing the porous calcium alginate microspheres by using the microfluidic aqueous two-phase emulsion as the template according to claim 5, wherein the molecular weight of PEG is 8kDa, and the molecular weight of Dex is 500 kDa.
7. The method for preparing porous calcium alginate microspheres by using the microfluidic aqueous two-phase emulsion as the template according to claim 1, wherein the emulsification frequency in the emulsification process in the step (4) is 2000-25000rpm/min, and the emulsification time is 28-32 s.
8. The porous calcium alginate microspheres prepared by the method for preparing the porous calcium alginate microspheres by using the microfluidic aqueous two-phase emulsion as the template according to any one of claims 1 to 7.
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