CN115212813B - 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 an all-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 a double-layer full water phase solution; respectively taking an upper polyethylene glycol phase and a lower dextran phase, mixing the polyethylene glycol phase and the dextran phase, wherein the volume of the polyethylene glycol phase is larger than that of the dextran phase, emulsifying, adding a cross-linking agent into the mixture, and uniformly mixing to obtain emulsion 1; respectively taking an upper polyethylene glycol phase and a lower dextran phase, mixing the polyethylene glycol phase and the dextran phase, wherein the volume of the polyethylene glycol phase is smaller than that of the dextran phase, emulsifying, adding a cross-linking agent into the mixture, and uniformly mixing to obtain emulsion 2; injecting the emulsion 2 into the liquid drop of the emulsion 1, then placing the liquid drop into a calcium ion receiving liquid for crosslinking reaction, and taking out to obtain the emulsion. 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 studied, the multi-stage slow release of medicines cannot be realized and the like in the existing single-layer porous gel microsphere.
Description
Technical Field
The invention belongs to the technical field of porous gel, and particularly relates to an all-aqueous-phase double-layer porous gel microsphere, and a preparation method and application thereof.
Background
In recent years, the porous gel microsphere has been widely used in the fields of cell culture carriers, organoid construction, tissue injury repair, drug release and the like due to the advantages of being capable of accelerating the substance exchange rate, highly simulating the in vivo environment, being convenient to operate and the like. Therefore, a plurality of scholars utilize different methods to manufacture the porous gel microsphere with controllable size, good sphericity and adjustable pore size.
Among the methods, the emulsion template method is widely focused by researchers because the pore size of the porous microspheres can be easily controlled. The emulsion template method is generally operated by adding an emulsion stabilizer, a cross-linking agent and an initiator into a continuous phase of the oil-in-water-in-oil emulsion, preparing gel microspheres by ultraviolet cross-linking, and then discharging the emulsion template from the gel microspheres to prepare porous gel microspheres. However, in the process of preparing the porous microspheres by the method, oil phase or other organic substances are adopted, so that the bioactive substances are difficult to directly encapsulate, and the operation steps are complicated. The aqueous phase emulsion templating method has great advantages in recent years. The method has the advantages of high biocompatibility, direct cell encapsulation, simple manufacturing steps and the like, and the manufacturing materials are wide in source and low in price, so that the manufacturing cost is reduced, and the method is beneficial to mass production and use. However, the porous gel microsphere prepared by the full aqueous phase emulsion template method has only a single-layer structure, so that the application prospect of the microsphere is limited.
In the aspects of organoid construction and tissue repair, a single-layer porous gel microsphere can only construct one tissue at most, but can not construct a plurality of tissues, and researchers can not use the single-layer porous gel microsphere to study interaction among the tissues; in the aspect of drug slow release, the single-layer microsphere can wrap the limited drug types, and the multi-stage release of the drug cannot be realized.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides an all-aqueous-phase double-layer porous gel microsphere, and a preparation method and application thereof, and the all-aqueous-phase double-layer porous gel microsphere can effectively solve the problems that the interaction relationship among a plurality of tissues cannot be studied and multi-stage slow release of medicines cannot be realized in the existing single-layer porous gel microsphere.
In order to achieve the above purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
the preparation method of the full water phase double-layer porous gel microsphere comprises the following steps:
(1) Mixing polyethylene glycol and dextran, adding water, and making into double-layer total 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 65% -75%: mixing 35-25% by volume, 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 20% -40%: mixing 80% -60% by volume, emulsifying, adding a cross-linking agent, and uniformly mixing to obtain emulsion 2;
(4) Injecting the emulsion 2 into the liquid drop of the emulsion 1, then placing the liquid drop into a calcium ion receiving liquid for crosslinking reaction, and taking out to obtain the emulsion.
In the scheme, the polyethylene glycol and the glucan are mixed and then added with water, and as the affinities of water molecules for the two substances are different, the water can be distributed into the two substances according to different proportions to form mutually incompatible two phases, so that a full water phase is formed; the volume ratio of the polyethylene glycol phase in the emulsion 1 is larger than that of the dextran phase, so that the dextran phase in the formed emulsion is used as a disperse phase, the polyethylene glycol phase is used as a continuous phase, and the dextran particles are uniformly dispersed in the polyethylene glycol phase; in the emulsion 2, the volume ratio of the polyethylene glycol phase is just opposite to that of the emulsion 1, wherein the volume ratio of the polyethylene glycol phase is smaller than that of the dextran phase, so that the dextran phase in the formed emulsion is used as a continuous phase, the polyethylene glycol phase is used as a disperse phase, polyethylene glycol particles are uniformly dispersed in the dextran phase, and when the emulsion 2 is injected into the emulsion 1 due to the fact that the continuous phases of the two emulsions are different, the two liquid drops are mutually incompatible, and a double-layer liquid drop structure with distinct layers is formed;
the cross-linking agents in the emulsion 1 and the emulsion 2 are in continuous phases, and when the double-layer liquid drops drop into the calcium ion receiving liquid, the cross-linking agents in the continuous phases react with calcium ions in a cross-linking way to form gel, and in the reaction process, the gel absorbs water to expand and extrude the disperse phase, so that a porous structure is formed in the continuous phase. The size of the formed pore structure is approximately equal to the size of the dispersible liquid drops, the size of the disperse phase can be adjusted by different emulsification frequencies, the emulsification frequency is high, the diameter of the disperse phase liquid drops is small, the emulsification frequency is low, and the diameter of the disperse phase liquid drops is large, so that the pore size of the double-layer porous gel microsphere can be adjusted and controlled according to the requirement.
Further, in the preparation process, emulsion 1 and emulsion 2 are respectively filled into the injector 1 and the injector 2, 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 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 a speed of 8 mu l/min-25 mu l/min, so that the emulsion 2 enters into the liquid drop formed by the emulsion 1, and the emulsion 1 and the emulsion 2 are mutually incompatible, so that the liquid drop with a double-layer structure can be formed. And finally, the formed liquid drop with the double-layer structure is dripped into the calcium ion receiving liquid under the action of gravity to carry out a crosslinking reaction.
Further, the molecular weight of polyethylene glycol is 8-10kDa, and the molecular weight of dextran is 500-600kDa.
In the scheme, the molecular weight of polyethylene glycol is obviously lower than that of dextran, so that a full aqueous phase solution with a double-layer structure can be formed after the polyethylene glycol and the dextran are mixed.
Further, the mass percentage ratio of polyethylene glycol and dextran in the double-layer full-aqueous phase solution in the step (1) is 3% -15%, namely 3% -15%.
Further, after emulsification in step (2) and step (3), the diameter of the dispersed phase particles is 10-100. Mu.m.
Further, the crosslinking agent in the step (2) and the 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 are the same.
The full water phase double layer porous gel microsphere is prepared by the method.
The application of the full water phase double-layer porous gel microsphere in 3D cell culture, organoid construction, drug delivery and tissue repair.
The beneficial effects that this application produced are:
1. the preparation method has the advantages that the full aqueous phase emulsion is used as a template to prepare the double-layer porous gel microsphere with stable size, good sphericity, controllable pore size and adjustable inner layer microsphere ratio, and the preparation method has simple preparation process and easy operation, and oil phase or other organic substances are not 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 a continuous phase and a disperse phase in the process of preparing the microsphere, so that a tissue can be constructed in the inner-layer microsphere, and a tissue can be constructed in the outer-layer microsphere, so that the effect of constructing various fine tissues by using the double-layer microsphere is achieved. Unlike porous microspheres of single-layer structure, which can only build one tissue, the double-layer porous microspheres can study interactions among various tissues; meanwhile, by adopting the same direct encapsulation method, the double-layer porous microsphere can also wrap a plurality of medicines, thereby realizing slow release of the medicines and greatly improving the application range of the microsphere.
3. The inner layer structure of the microsphere in the application is adjustable in size and can be adjusted according to the use requirement, so that the application range of the microsphere is further improved.
4. The porous microsphere prepared by using the full water phase emulsion as a template has the advantages of high biocompatibility, capability of directly encapsulating cells, low cost and the like. On the basis, a simple double-needle tube injection method is utilized to endow the porous microsphere with a double-layer structure, so that the porous microsphere can simulate more complex tissues, and the problem of difficulty in the field of tissue engineering in the current pathological model construction is solved. And through data statistics, the sizes of the microspheres and the sizes of the apertures, the proportion of the inner microspheres is stable and controllable, so that the method has great potential in the production or research field.
Drawings
FIG. 1 is a diagram illustrating a preparation process of the present application;
FIG. 2 is a diagram of the preparation process of the present application;
FIG. 3A is a micrograph of emulsion 1; FIG. 3B is a graph showing particle diameter statistics of the dispersed phase in emulsion 1;
FIG. 4A is a micrograph of emulsion 2; FIG. 4B is a graph showing particle diameter statistics of the dispersed phase in emulsion 2;
FIG. 5A is a graph showing the outer diameter statistics of a bilayer porous microsphere; FIG. 5B is a graph showing statistics of inner layer diameters of bilayer porous microspheres;
FIG. 6 is a statistical plot of the occupancy of the inner microsphere structure throughout the microsphere;
FIG. 7 is a scanning electron microscope image of the final bilayer porous microsphere;
FIG. 8A is a graph showing the outer diameter statistics of a bilayer porous microsphere; FIG. 8B is a graph showing statistics of inner layer diameters of bilayer porous microspheres;
FIG. 9 is a graph showing statistics of the occupancy of the inner microsphere structure throughout the microsphere;
FIG. 10 is a graph showing the relationship between injection time and inner microsphere structure ratio.
Detailed Description
The following describes the embodiments of the present invention in detail with reference to the drawings.
Example 1
The preparation method of the full-aqueous-phase double-layer porous gel microsphere comprises the following steps:
(1) Adding water into polyethylene glycol and dextran according to the mixing ratio to prepare a double-layer full-aqueous phase solution with the mass ratio of the polyethylene glycol being 8% and the mass ratio of the dextran being 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 75%: mixing 25% by volume, emulsifying at 500rpm to obtain dispersed phase with particle diameter of 40 μm, adding alginic acid, mixing, wherein alginic acid is 1 times of total volume of polyethylene glycol phase and dextran 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 20%: mixing 80% by volume, emulsifying at 600rpm to obtain dispersed phase with particle diameter of 25 μm, adding alginic acid, mixing, wherein alginic acid is 1 times of total volume of polyethylene glycol phase and dextran phase to obtain emulsion 2;
(4) Respectively filling emulsion 1 and emulsion 2 into the injector 1 and the injector 2, arranging the injection needle of the injector 1 vertically, arranging the injection needle of the injector 2 horizontally, pushing the injector 1 to enable the emulsion 1 to be in a liquid drop shape at the front end of the injection needle of the injector 1, covering the injection needle of the injector 2 by the liquid drop, pushing the injector 2 at the speed of 13 mu l/min to enable the emulsion 2 to enter into the liquid drop formed by the emulsion 1, injecting for 27s, finally, dropping the formed liquid drop with a double-layer structure into a calcium ion receiving liquid containing 15wt% of calcium ions under the action of gravity to carry out a crosslinking reaction for 80min, and taking out to obtain the emulsion.
Example 2
The preparation method of the full-aqueous-phase double-layer porous gel microsphere comprises the following steps:
(1) Adding water into polyethylene glycol and dextran according to the mixing ratio to prepare a double-layer full-aqueous phase solution with the mass ratio of the polyethylene glycol being 8% and the mass ratio of the dextran being 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 75%: mixing 25% by volume, emulsifying at 500rpm to obtain dispersed phase with particle diameter of 30 μm, adding alginic acid, mixing, wherein alginic acid is 1 times of total volume of polyethylene glycol phase and dextran 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 20%: mixing 80% by volume, emulsifying at 600rpm to obtain dispersed phase with particle diameter of 25 μm, adding alginic acid, mixing, wherein alginic acid is 1 times of total volume of polyethylene glycol phase and dextran phase to obtain emulsion 2;
(4) Respectively filling emulsion 1 and emulsion 2 into the injector 1 and the injector 2, arranging the injection needle of the injector 1 vertically, arranging the injection needle of the injector 2 horizontally, pushing the injector 1 to enable the emulsion 1 to be in a liquid drop shape at the front end of the injection needle of the injector 1, covering the injection needle of the injector 2 by the liquid drop, pushing the injector 2 at the speed of 13 mu l/min to enable the emulsion 2 to enter into the liquid drop formed by the emulsion 1, injecting 58s, finally, dropping the formed liquid drop with a double-layer structure into a calcium ion receiving liquid containing 15wt% of calcium ions under the action of gravity to carry out crosslinking reaction for 80min, and taking out to obtain the emulsion.
Example 3
The preparation method of the full-aqueous-phase double-layer porous gel microsphere comprises the following steps:
(1) Adding water into polyethylene glycol and dextran according to the mixing ratio to prepare a double-layer full-aqueous phase solution with the mass ratio of polyethylene glycol being 15% and the mass ratio of dextran being 7%, 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 70%: mixing 30% by volume, emulsifying at 400rpm oscillation frequency to make the diameter of dispersed phase particle be 30 μm, adding alginic acid into the mixture, mixing uniformly, wherein alginic acid is 1 times of total volume of polyethylene glycol phase and dextran 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 300 percent: mixing 70% by volume, emulsifying at 700rpm to obtain dispersed phase with particle diameter of 25 μm, adding alginic acid, mixing, wherein alginic acid is 1 times of total volume of polyethylene glycol phase and dextran phase to obtain emulsion 2;
(4) Respectively filling emulsion 1 and emulsion 2 into the injector 1 and the injector 2, arranging the injection needle of the injector 1 vertically, arranging the injection needle of the injector 2 horizontally, pushing the injector 1 to enable the emulsion 1 to be in a liquid drop shape at the front end of the injection needle of the injector 1, covering the injection needle of the injector 2 by the liquid drop, pushing the injector 2 at the speed of 13 mu l/min to enable the emulsion 2 to enter into the liquid drop formed by the emulsion 1, injecting 105s, finally, dropping the formed liquid drop with a double-layer structure into a calcium ion receiving liquid containing 15wt% of calcium ions under the action of gravity to carry out crosslinking reaction for 80min, and taking out to obtain the emulsion.
Example 4
The preparation method of the full-aqueous-phase double-layer porous gel microsphere comprises the following steps:
(1) Adding water into polyethylene glycol and dextran according to the mixing ratio to prepare a double-layer full-aqueous phase solution with the mass ratio of polyethylene glycol being 3% and the mass ratio of dextran being 10%, 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 65%: mixing 35% by volume, emulsifying at 400rpm to obtain dispersed phase with particle diameter of 30 μm, adding alginic acid, mixing, wherein alginic acid is 1 times of total volume of polyethylene glycol phase and dextran 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 40%: mixing 60% by volume, emulsifying at 800rpm to obtain dispersed phase with particle diameter of 25 μm, adding alginic acid, mixing, wherein alginic acid is 1 times of total volume of polyethylene glycol phase and dextran phase to obtain emulsion 2;
(4) Respectively filling emulsion 1 and emulsion 2 into the injector 1 and the injector 2, arranging the injection needle of the injector 1 vertically, arranging the injection needle of the injector 2 horizontally, pushing the injector 1 to enable the emulsion 1 to be in a liquid drop shape at the front end of the injection needle of the injector 1, covering the injection needle of the injector 2 by the liquid drop, pushing the injector 2 at the speed of 13 mu l/min to enable the emulsion 2 to enter into the liquid drop formed by the emulsion 1, injecting for 60s, finally, dropping the formed liquid drop with a double-layer structure into a calcium ion receiving liquid containing 15wt% of calcium ions under the action of gravity to carry out a crosslinking reaction for 80min, and taking out to obtain the emulsion.
Test examples
Taking the preparation process in example 1 and the prepared double-layer gel microsphere as an example, the dispersion particle size, microsphere particle size and the like in the preparation process in example 1 are detected, the specific results are shown in fig. 3-6, the finally prepared double-layer porous microsphere is washed and dried, and then electron microscope scanning is carried out, and the specific results are shown in fig. 7.
As can be seen from FIG. 3, the dispersed phase in emulsion 1 has a uniform droplet size, with a droplet diameter centered between 20 and 40. Mu.m.
As can be seen from FIG. 4, the droplets in emulsion 2 were uniform in size and concentrated in 20-30 μm in diameter.
As can be seen from FIG. 5, the overall diameter of the microspheres in the double-layered porous microsphere structure is concentrated at 34-36mm, and the diameter of the microspheres in the inner layer is concentrated at 28-32mm.
As can be seen from fig. 6, the volume ratio of the inner layer microsphere in the whole microsphere structure in the double-layer porous microsphere structure is concentrated to 60-75%.
As can be seen from fig. 7, the inside of the double-layered porous microsphere has a distinct double-layered structure, and a large number of pore structures exist in both the inner and outer layers.
Taking the preparation process in example 2 as an example, the particle size of the dispersed phase, the particle size of the microspheres, etc. in the preparation process in example 2 were examined, and the specific results are shown in fig. 8 to 10.
As can be seen from FIG. 8, the overall diameter of the microspheres in the double-layered porous microsphere structure is concentrated at 33-34mm, and the diameter of the microspheres in the inner layer is concentrated at 18-21mm.
As can be seen from fig. 9, the volume ratio of the inner layer microsphere in the whole microsphere structure in the double-layer porous microsphere structure is concentrated to 60-70%.
As shown in fig. 10, the inner layer microspheres have different ratios in the whole microspheres along with the injection time, and the inner layer microspheres have almost proportional relation with the injection time, which proves that the manufacturing controllability of the microspheres is strong.
Claims (7)
1. The preparation method of the all-aqueous-phase double-layer porous gel microsphere is characterized by comprising the following steps of:
(1) Mixing polyethylene glycol and dextran, adding water into the mixture to prepare a double-layer full-aqueous-phase solution, wherein the mass percentage of the polyethylene glycol and the dextran in the double-layer full-aqueous-phase solution is 3% -15%, and the mass percentage of the polyethylene glycol and the dextran in the double-layer full-aqueous-phase solution is 3% -15%;
(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 65% -75%: mixing 35-25% by volume, 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 20% -40%: mixing 80% -60% by volume, emulsifying, adding a cross-linking agent, and uniformly mixing to obtain emulsion 2;
(4) Injecting the emulsion 2 into the liquid drops of the emulsion 1, then placing the liquid drops into a calcium ion receiving liquid for crosslinking reaction, and taking out to obtain the emulsion;
after emulsification in the step (2) and the step (3), the diameter of the dispersed phase particles is 10-100 mu m; the cross-linking agent in the step (2) and the step (3) is alginic acid, sodium carboxymethyl cellulose, matrigel or dextran.
2. The method for preparing the all-aqueous-phase double-layer porous gel microsphere according to claim 1, wherein the molecular weight of polyethylene glycol is 8-10kDa and the molecular weight of dextran is 500-600kDa.
3. The method for preparing the all-aqueous-phase double-layer porous gel microsphere according to claim 1, wherein the addition amount of the crosslinking agent in the step (2) is 0.8-1.5 times of the total volume of the polyethylene glycol phase and the dextran phase.
4. The method for preparing the all-aqueous-phase double-layer porous gel microsphere according to claim 1, wherein the adding amount of the crosslinking agent in the step (3) is 0.8-1.5 times of the total volume of the polyethylene glycol phase and the dextran phase.
5. The method for preparing the all-aqueous-phase double-layer porous gel microsphere according to claim 1, wherein the densities of the emulsion 1 and the emulsion 2 are the same.
6. An all aqueous phase bilayer porous gel microsphere prepared by the method of any one of claims 1-5.
7. Use of the all aqueous phase bilayer porous gel microsphere according to claim 6 in 3D cell culture, organoid construction, drug delivery and tissue repair.
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