CN115212813A - Full-aqueous phase double-layer porous gel microsphere and preparation method and application thereof - Google Patents
Full-aqueous phase double-layer porous gel microsphere and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a full-aqueous phase double-layer porous gel microsphere and a preparation method and application thereof, wherein the preparation method of the microsphere comprises the following steps: preparing polyethylene glycol and dextran into double-layer water phase solution; respectively taking an upper polyethylene glycol phase and a lower glucan phase, mixing the polyethylene glycol phase and the glucan phase, emulsifying, adding a cross-linking agent, and uniformly mixing to obtain an emulsion 1; respectively taking an upper polyethylene glycol phase and a lower glucan phase, mixing the polyethylene glycol phase and the glucan phase, emulsifying, adding a cross-linking agent, and mixing to obtain an emulsion 2; and (3) injecting the emulsion 2 into the liquid drop of the emulsion 1, then placing the liquid drop into calcium ion receiving liquid for crosslinking reaction, and taking out the liquid drop to obtain the calcium ion receiving liquid. The full-aqueous phase double-layer porous gel microsphere can effectively solve the problems that the interaction relation among a plurality of tissues cannot be researched and the multi-stage slow release of the medicine cannot be realized in the conventional single-layer porous gel microsphere.
Description
Technical Field
The invention belongs to the technical field of porous gel, and particularly relates to a full-aqueous-phase double-layer porous gel microsphere and a preparation method and application thereof.
Background
In recent years, the porous gel microspheres have been widely used in the fields of cell culture carriers, organoid construction, tissue injury repair, drug sustained release and the like due to the advantages of accelerating the material exchange rate, highly simulating the in vivo environment, being convenient to operate and the like. Therefore, many researchers have made porous gel microspheres with controllable size, good sphericity and controllable pore size by different methods.
Among the methods, the emulsion template method has been widely noticed by researchers because the pore size of the porous microspheres can be easily controlled. The emulsion template method generally operates by adding an emulsion stabilizer, a crosslinking agent and an initiator into a continuous phase of an oil-in-water emulsion, performing ultraviolet crosslinking to prepare gel microspheres, and discharging the emulsion template from the gel microspheres to prepare porous gel microspheres. However, the process of preparing the porous microspheres by the method adopts oil phase or other organic substances, which is difficult to directly encapsulate bioactive substances, and the operation steps are complicated. The recent emergence of the full aqueous phase emulsion template method has great advantages. The method has the advantages of high biocompatibility, direct cell encapsulation, simple preparation steps and the like, and the preparation material has wide sources and low price, reduces the preparation cost and is beneficial to large-scale production and use. However, the porous gel microspheres prepared by the full-aqueous phase emulsion template method only have a single-layer structure, so that the application prospect of the microspheres is limited.
In the aspects of organoid construction and tissue repair, the single-layer porous gel microspheres can only construct one tissue at most, but can not construct a plurality of tissues, and researchers can not study the interaction among the tissues by using the single-layer porous gel microspheres; in the aspect of drug slow release, the types of drugs which can be wrapped by the single-layer microspheres are limited, and the multi-stage release of the drugs cannot be realized.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides an all-water-phase double-layer porous gel microsphere and a preparation method and application thereof, and the all-water-phase double-layer porous gel microsphere can effectively solve the problems that the interaction relation among a plurality of tissues cannot be researched and the multi-stage sustained release of a medicament cannot be realized in the existing single-layer porous gel microsphere.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problem is as follows:
a preparation method of full-aqueous phase double-layer porous gel microspheres comprises the following steps:
(1) Mixing polyethylene glycol and dextran, and adding water to obtain double-layer water phase solution;
(2) Respectively taking an upper polyethylene glycol phase and a lower glucan phase, and mixing the polyethylene glycol phase and the glucan phase according to the ratio of 65-75%: mixing 35-25% of the mixture by volume ratio, emulsifying, adding a cross-linking agent, and uniformly mixing to obtain emulsion 1;
(3) Respectively taking an upper polyethylene glycol phase and a lower glucan phase, and mixing the polyethylene glycol phase and the glucan phase according to the ratio of 20-40%: mixing 80-60% of the raw materials according to a volume ratio, emulsifying, adding a cross-linking agent, and uniformly mixing to obtain an emulsion 2;
(4) And injecting the emulsion 2 into the liquid drop of the emulsion 1, then placing the liquid drop into the calcium ion receiving solution for cross-linking reaction, and taking out to obtain the calcium ion receiving solution.
In the scheme, the polyethylene glycol and the glucan are mixed and then added with water, and because the affinity of water molecules to the two substances is different, the water can be distributed into the two substances in different proportions to form two mutually incompatible phases so as to form a full water phase; the volume ratio of the polyethylene glycol phase in the emulsion 1 is greater than that of the glucan phase, so that the glucan phase in the formed emulsion is used as a dispersed phase, the polyethylene glycol phase is used as a continuous phase, and glucan particles are uniformly dispersed in the polyethylene glycol phase; the emulsion 2 is just opposite to the emulsion 1, wherein the volume ratio of the polyethylene glycol phase is smaller than that of the glucan phase, so that the glucan phase in the formed emulsion is used as a continuous phase, the polyethylene glycol phase is used as a dispersed phase, and the polyethylene glycol particles are uniformly dispersed in the glucan phase;
the cross-linking agents in the emulsion 1 and the emulsion 2 exist in continuous phases, when the double-layer liquid drops into the calcium ion receiving liquid, the cross-linking agents in the continuous phases and the calcium ions are subjected to cross-linking reaction to form gel, and in the reaction process, the gel absorbs water and expands to extrude the dispersed phase, so that a porous structure is formed in the continuous phases. The size of the formed pore structure is nearly equal to that of the dispersed liquid drops, the size of the dispersed phase can be adjusted through different emulsification frequencies, the emulsification frequency is high, the diameter of the dispersed phase liquid drops is small, the emulsification frequency is low, and the diameter of the dispersed phase liquid drops is large, so that the pore size of the double-layer porous gel microspheres can be adjusted and controlled according to needs.
Further, in the preparation process, respectively filling the emulsion 1 and the emulsion 2 into the injector 1 and the injector 2, wherein the injection needle of the injector 1 is vertically arranged, the injection needle of the injector 2 is horizontally arranged, the injector 1 is pushed, so that the emulsion 1 is in a liquid drop shape at the front end of the injection needle of the injector 1, the liquid drop covers the injection needle of the injector 2, then the injector 2 is pushed at the speed of 8-25 mul/min, so that the emulsion 2 enters the liquid drop formed by the emulsion 1, and the emulsion 1 and the emulsion 2 are continuously incompatible with each other, so that a liquid drop with a double-layer structure can be formed. Finally, the formed liquid drops with the double-layer structure drop into the calcium ion receiving liquid under the action of gravity to carry out crosslinking reaction.
Furthermore, the molecular weight of the polyethylene glycol is 8-10kDa, and the molecular weight of the glucan is 500-600kDa.
In the scheme, the molecular weight of the polyethylene glycol is obviously lower than that of the glucan, so that the polyethylene glycol and the glucan can form a double-layer structure full-water-phase solution after being mixed.
Further, the mass percentage of the polyethylene glycol and the glucan in the double-layer full-water phase solution in the step (1) is 3-15% to 3-15%.
Further, after emulsification in the step (2) and the step (3), the diameter of dispersed phase particles is 10-100 μm.
Further, the cross-linking agent in step (2) and step (3) is alginic acid, sodium carboxymethyl cellulose, matrigel or dextran.
Further, the addition amount of the cross-linking agent in the step (2) is 0.8-1.5 times of the total volume of the polyethylene glycol phase and the dextran phase.
Further, the addition amount of the cross-linking agent in the step (3) is 0.8-1.5 times of the total volume of the polyethylene glycol phase and the dextran phase.
Further, the densities of emulsion 1 and emulsion 2 were the same.
A full-water-phase double-layer porous gel microsphere is prepared by the method.
The full-aqueous phase double-layer porous gel microsphere is applied to 3D cell culture, organoid construction, drug delivery and tissue repair.
The beneficial effect that this application produced does:
1. the preparation method has the advantages that the double-layer porous gel microspheres which are stable in size, good in sphericity, controllable in pore size and adjustable in inner layer microsphere proportion are prepared by taking the full-water-phase emulsion as the template, the preparation process is simple, the operation is easy, and no oil phase or other organic substances are used in the preparation process.
2. The microsphere prepared in the application is of a double-layer structure, and different cells can be directly added into the continuous phase and the dispersed phase in the process of preparing the microsphere, so that one tissue can be constructed in the microsphere at the inner layer, one tissue can be constructed in the microsphere at the outer layer, and the effect of constructing various fine tissues by using the double-layer microsphere is achieved. The double-layer porous microspheres can study the interaction among various tissues, which is different from the porous microspheres with single-layer structures in that only one tissue can be constructed; meanwhile, by the same direct encapsulation method, the double-layer porous microsphere can also wrap various medicines, so that the slow release of the medicines is realized, and the application range of the microsphere is greatly improved.
3. The size of the inner layer structure of the microsphere is adjustable, and the inner layer structure can be adjusted according to the use requirement, so that the application range of the microsphere is further expanded.
4. The porous microsphere prepared by using the full water phase emulsion as the template has the advantages of high biocompatibility, direct cell encapsulation, low cost and the like. On the basis, a porous microsphere double-layer structure is endowed by using a simple double-needle-tube injection method, so that more complex tissues can be simulated, and the difficult problems in the field of current pathological model construction and tissue engineering are solved. And through data statistics, the size of the microsphere, the size of the pore diameter and the proportion of the inner layer microsphere are stable and controllable, so that the microsphere has great potential in the production or research field.
Drawings
FIG. 1 is a representation of the preparation process of the present application;
FIG. 2 is a schematic representation of the preparation process of the present application;
FIG. 3A is a micrograph of emulsion 1; FIG. 3B is a statistical chart of the particle diameters of the dispersed phase in emulsion 1;
FIG. 4A is a micrograph of emulsion 2; FIG. 4B is a statistical chart of the diameters of the dispersed phase particles in emulsion 2;
FIG. 5A is a statistical plot of the outer layer diameters of two-layer porous microspheres; FIG. 5B is a statistical plot of the inner layer diameter of a bilayer porous microsphere;
FIG. 6 is a statistical plot of the ratio of the inner microsphere structure throughout the microspheres;
FIG. 7 is a scanning electron micrograph of the final bilayer porous microspheres;
FIG. 8A is a statistical plot of the outer layer diameters of two-layer porous microspheres; FIG. 8B is a statistical plot of the inner layer diameter of a bilayer porous microsphere;
FIG. 9 is a statistical plot of the ratio of the inner microsphere structure throughout the microspheres;
FIG. 10 is a graph of injection time versus inner layer microsphere structure.
Detailed Description
The following detailed description of embodiments of the invention refers to the accompanying drawings.
Example 1
The preparation method of the full-aqueous phase double-layer porous gel microsphere comprises the following steps:
(1) Mixing polyethylene glycol and dextran, and adding water to obtain a double-layer full water phase solution with the mass ratio of polyethylene glycol of 8% and dextran of 8%, wherein the molecular weight of polyethylene glycol is 8kDa, and the molecular weight of dextran is 500kDa;
(2) Respectively taking an upper polyethylene glycol phase and a lower glucan phase, and mixing the polyethylene glycol phase and the glucan phase according to the ratio of 75%: mixing at a volume ratio of 25%, emulsifying at a vibration frequency of 500rpm to make the diameter of dispersed phase particles 40 μm, adding alginic acid which is 1 time of the total volume of the polyethylene glycol phase and the dextran phase, and mixing to obtain emulsion 1;
(3) Respectively taking an upper polyethylene glycol phase and a lower glucan phase, and mixing the polyethylene glycol phase and the glucan phase according to the ratio of 20%: mixing 80% of the mixture in volume ratio, emulsifying the mixture at the oscillation frequency of 600rpm to ensure that the diameter of dispersed phase particles is 25 mu m, adding alginic acid which accounts for 1 time of the total volume of the polyethylene glycol phase and the glucan phase, and uniformly mixing to obtain an emulsion 2;
(4) Respectively filling the emulsion 1 and the emulsion 2 into an injector 1 and an injector 2, wherein an injection needle of the injector 1 is vertically arranged, an injection needle of the injector 2 is horizontally arranged, the injector 1 is pushed to enable the emulsion 1 to be in a liquid drop shape at the front end of the injection needle of the injector 1, the liquid drop covers the injection needle of the injector 2, then the injector 2 is pushed at the speed of 13 mu l/min to enable the emulsion 2 to enter the liquid drop formed by the emulsion 1, the injection is carried out for 27s, finally, the formed liquid drop with the double-layer structure is dropped into a calcium ion receiving liquid containing 15wt% of calcium ions under the action of gravity to carry out crosslinking reaction for 80min, and the emulsion is taken out to obtain the calcium ion receptor.
Example 2
The preparation method of the full-aqueous phase double-layer porous gel microsphere comprises the following steps:
(1) Mixing polyethylene glycol and dextran, and adding water to obtain a double-layer full water phase solution with the mass ratio of the polyethylene glycol of 8% and the mass ratio of the dextran of 8%, wherein the molecular weight of the polyethylene glycol is 8kDa, and the molecular weight of the dextran is 500kDa;
(2) Respectively taking an upper polyethylene glycol phase and a lower glucan phase, and mixing the polyethylene glycol phase and the glucan phase according to the ratio of 75%: mixing at a volume ratio of 25%, emulsifying at an oscillation frequency of 500rpm to make the diameter of dispersed phase particles 30 μm, adding alginic acid 1 times of the total volume of polyethylene glycol phase and dextran phase, and mixing to obtain emulsion 1;
(3) Respectively taking an upper polyethylene glycol phase and a lower glucan phase, and mixing the polyethylene glycol phase and the glucan phase according to the ratio of 20%: mixing 80% of the mixture by volume ratio, emulsifying the mixture at the oscillation frequency of 600rpm to ensure that the diameter of dispersed phase particles is 25 mu m, adding alginic acid into the mixture, and uniformly mixing the alginic acid, wherein the alginic acid accounts for 1 time of the total volume of the polyethylene glycol phase and the glucan phase to obtain an emulsion 2;
(4) Respectively filling the emulsion 1 and the emulsion 2 into an injector 1 and an injector 2, wherein an injection needle of the injector 1 is vertically arranged, an injection needle of the injector 2 is horizontally arranged, the injector 1 is pushed to enable the emulsion 1 to be in a liquid drop shape at the front end of the injection needle of the injector 1, the liquid drop covers the injection needle of the injector 2, then the injector 2 is pushed at the speed of 13 mu l/min to enable the emulsion 2 to enter the liquid drop formed by the emulsion 1, the injection is carried out for 58s, finally, the formed liquid drop with the double-layer structure is dropped into a calcium ion receiving liquid containing 15wt% of calcium ions under the action of gravity to carry out crosslinking reaction for 80min, and the emulsion is taken out to obtain the calcium ion receptor emulsion.
Example 3
The preparation method of the full-aqueous phase double-layer porous gel microsphere comprises the following steps:
(1) Mixing polyethylene glycol and dextran, and adding water to obtain a double-layer full water phase solution with the mass ratio of polyethylene glycol of 15% and dextran of 7%, wherein the molecular weight of polyethylene glycol is 8kDa, and the molecular weight of dextran is 500kDa;
(2) Respectively taking an upper polyethylene glycol phase and a lower glucan phase, and mixing the polyethylene glycol phase and the glucan phase according to the ratio of 70%: mixing 30% of the mixture by volume ratio, emulsifying the mixture at the oscillation frequency of 400rpm to ensure that the diameter of dispersed phase particles is 30 mu m, adding alginic acid into the mixture, and uniformly mixing the alginic acid, wherein the alginic acid accounts for 1 time of the total volume of the polyethylene glycol phase and the glucan phase to obtain emulsion 1;
(3) Respectively taking an upper polyethylene glycol phase and a lower glucan phase, and mixing the polyethylene glycol phase and the glucan phase according to the ratio of 300%: mixing 70% of the mixture in volume ratio, emulsifying the mixture at the oscillation frequency of 700rpm to ensure that the diameter of dispersed phase particles is 25 mu m, adding alginic acid which accounts for 1 time of the total volume of the polyethylene glycol phase and the glucan phase, and uniformly mixing to obtain an emulsion 2;
(4) Respectively filling the emulsion 1 and the emulsion 2 into an injector 1 and an injector 2, wherein an injection needle of the injector 1 is vertically arranged, an injection needle of the injector 2 is horizontally arranged, the injector 1 is pushed to enable the emulsion 1 to be in a liquid drop shape at the front end of the injection needle of the injector 1, the liquid drop covers the injection needle of the injector 2, then the injector 2 is pushed at the speed of 13 mu l/min to enable the emulsion 2 to enter the liquid drop formed by the emulsion 1, the injection is carried out for 105s, finally, the formed liquid drop with the double-layer structure is dropped into a calcium ion receiving liquid containing 15wt% of calcium ions under the action of gravity to carry out crosslinking reaction for 80min, and the emulsion is taken out to obtain the calcium ion receptor.
Example 4
A full-aqueous phase double-layer porous gel microsphere is prepared by the following steps:
(1) Mixing polyethylene glycol and dextran, and adding water to obtain a double-layer full water phase solution with the mass ratio of polyethylene glycol of 3% and dextran of 10%, wherein the molecular weight of polyethylene glycol is 8kDa, and the molecular weight of dextran is 500kDa;
(2) Respectively taking an upper polyethylene glycol phase and a lower glucan phase, and mixing the polyethylene glycol phase and the glucan phase according to a ratio of 65%: mixing 35% of the mixture by volume ratio, emulsifying the mixture at the oscillation frequency of 400rpm to ensure that the diameter of dispersed phase particles is 30 mu m, adding alginic acid into the mixture, and uniformly mixing the alginic acid, wherein the alginic acid accounts for 1 time of the total volume of the polyethylene glycol phase and the glucan phase to obtain emulsion 1;
(3) Respectively taking an upper polyethylene glycol phase and a lower glucan phase, and mixing the polyethylene glycol phase and the glucan phase according to the ratio of 40%: mixing at a volume ratio of 60%, emulsifying at an oscillation frequency of 800rpm to obtain dispersed phase particles with a diameter of 25 μm, adding alginic acid 1 times of the total volume of polyethylene glycol phase and dextran phase, and mixing to obtain emulsion 2;
(4) Respectively filling the emulsion 1 and the emulsion 2 into an injector 1 and an injector 2, wherein an injection needle of the injector 1 is vertically arranged, an injection needle of the injector 2 is horizontally arranged, the injector 1 is pushed to enable the emulsion 1 to be in a liquid drop shape at the front end of the injection needle of the injector 1, the liquid drop covers the injection needle of the injector 2, then the injector 2 is pushed at the speed of 13 mu l/min to enable the emulsion 2 to enter the liquid drop formed by the emulsion 1, the injection is carried out for 60s, finally, the formed liquid drop with the double-layer structure is dropped into a calcium ion receiving liquid containing 15wt% of calcium ions under the action of gravity to carry out crosslinking reaction for 80min, and the emulsion is taken out to obtain the calcium ion receptor.
Test examples
Taking the preparation process and the prepared double-layer gel microspheres in the example 1 as an example, the dispersion particle size, the microsphere particle size and the like in the preparation process of the example 1 are detected, the specific results are shown in fig. 3-6, the finally prepared double-layer porous microspheres are washed, dried and then subjected to electron microscope scanning, and the specific results are shown in fig. 7.
It can be seen from FIG. 3 that the droplets of the dispersed phase in emulsion 1 are of uniform size and have diameters concentrated in the range of 20 to 40 μm.
It can be seen from FIG. 4 that the droplets in emulsion 2 are of uniform size and have diameters centered at 20-30 μm.
As can be seen from FIG. 5, the overall diameter of the microspheres in the double-layer porous microsphere structure is concentrated to 34-36mm, and the diameter of the microspheres in the inner layer is concentrated to 28-32mm.
As can be seen from FIG. 6, the volume ratio of the inner layer microspheres in the double-layer porous microsphere structure in the whole microsphere structure is concentrated in 60-75%.
As can be seen from fig. 7, the interior of the double-layer porous microsphere has a distinct double-layer structure, and a large amount of pore structures exist in both the inner layer and the outer layer.
Taking the preparation process in example 2 as an example, the particle size of the dispersed phase, the particle size of the microspheres, and the like in the preparation process in example 2 are detected, and specific results are shown in fig. 8-10.
As can be seen from FIG. 8, the overall diameter of the microspheres in the double-layer porous microsphere structure is concentrated to 33-34mm, and the diameter of the microspheres in the inner layer is concentrated to 18-21mm.
It can be known from fig. 9 that the volume ratio of the inner layer microspheres in the double-layer porous microsphere structure in the whole microsphere structure is concentrated in 60-70%.
It can be known from fig. 10 that the ratio of the inner layer microspheres in the microspheres is different along with the injection time, and the ratio of the inner layer microspheres is almost in direct proportion to the injection time, which proves that the preparation controllability of the microspheres is strong.
Claims (10)
1. A preparation method of full-aqueous phase double-layer porous gel microspheres is characterized by comprising the following steps:
(1) Mixing polyethylene glycol and dextran, and adding water to obtain double-layer water phase solution;
(2) Respectively taking an upper polyethylene glycol phase and a lower glucan phase, and mixing the polyethylene glycol phase and the glucan phase according to the weight ratio of 65-75%: mixing 35-25% of the mixture by volume ratio, emulsifying, adding a cross-linking agent, and uniformly mixing to obtain emulsion 1;
(3) Respectively taking an upper polyethylene glycol phase and a lower glucan phase, and mixing the polyethylene glycol phase and the glucan phase according to the ratio of 20-40%: mixing 80-60% of the mixture by volume ratio, emulsifying, adding a cross-linking agent, and uniformly mixing to obtain emulsion 2;
(4) And (3) injecting the emulsion 2 into the liquid drop of the emulsion 1, then placing the liquid drop into calcium ion receiving liquid for crosslinking reaction, and taking out the liquid drop to obtain the calcium ion receiving liquid.
2. The method for preparing the full-aqueous-phase bilayer porous gel microsphere of claim 1, wherein the molecular weight of the polyethylene glycol is 8-10kDa, and the molecular weight of the dextran is 500-600kDa.
3. The method for preparing the full-aqueous phase double-layer porous gel microsphere of claim 1, wherein the mass percentage ratio of the polyethylene glycol to the glucan in the double-layer full-aqueous phase solution in the step (1) is 3% -15% to 3% -15%.
4. The method for preparing full-aqueous-phase double-layer porous gel microspheres according to claim 1, wherein after emulsification in the step (2) and the step (3), the diameter of dispersed phase particles is 10-100 μm.
5. The method for preparing full aqueous phase bilayer porous gel microspheres according to claim 1, wherein the cross-linking agent in step (2) and step (3) is alginic acid, sodium carboxymethyl cellulose, matrigel or dextran.
6. The method for preparing full-aqueous phase double-layer porous gel microspheres according to claim 1, wherein the amount of the cross-linking agent added in the step (2) is 0.8 to 1.5 times of the total volume of the polyethylene glycol phase and the dextran phase.
7. The method for preparing full-aqueous phase double-layer porous gel microspheres according to claim 1, wherein the amount of the cross-linking agent added in the step (3) is 0.8 to 1.5 times of the total volume of the polyethylene glycol phase and the dextran phase.
8. The method for preparing full aqueous phase double-layer porous gel microspheres according to claim 1, wherein the densities of emulsion 1 and emulsion 2 are the same.
9. An all-aqueous phase double-layer porous gel microsphere, which is prepared by the method of any one of claims 1 to 8.
10. The use of the full aqueous bilayer porous gel microspheres of claim 9 in 3D cell culture, organoid construction, drug delivery and tissue repair.
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