CN113975250A - Preparation and application of double-water-phase porous islet microcapsules with core-shell structure - Google Patents

Preparation and application of double-water-phase porous islet microcapsules with core-shell structure Download PDF

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CN113975250A
CN113975250A CN202111230390.8A CN202111230390A CN113975250A CN 113975250 A CN113975250 A CN 113975250A CN 202111230390 A CN202111230390 A CN 202111230390A CN 113975250 A CN113975250 A CN 113975250A
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core
phase
porous
microcapsule
shell structure
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李玲
赵远锦
魏琼
刘小钰
余筠如
王月桐
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Zhongda Hospital of Southeast University
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Abstract

The invention discloses a preparation method and application of a double-aqueous-phase porous islet microcapsule with a core-shell structure. The internal phase is a carboxymethyl cellulose (CMC) solution. The microcapsule has good biocompatibility, target cells are added into an internal phase solution to obtain the cell-loaded microcapsule, and due to the internal liquid environment and the porous gel outside, the three-dimensional culture of the cells can be realized, the cell survival rate is improved, and the cell-loaded microcapsule can be transplanted to realize the cell treatment effect. The preparation method of the core-shell structure porous islet microcapsules based on microfluidic electrospray has the advantages of reusability, low cost, simplicity in operation, convenience, reliability and the like.

Description

Preparation and application of double-water-phase porous islet microcapsules with core-shell structure
Technical Field
The invention belongs to the field of biological materials, and particularly relates to a porous islet microcapsule with a core-shell structure based on microfluidic electrospray and a preparation method thereof.
Background
The common cell transplantation modes include hepatic portal vein, kidney capsule, abdominal cavity and the like, and the microcapsule is one of the cell transplantation methods capable of being transplanted into the abdominal cavity. The microcapsule is of a double-water-phase core-shell structure, the shell layer is porous hydrogel, small molecular substances can be allowed to pass through the microcapsule, and macromolecular substances such as immune cells, immune globulin and the like can be prevented, so that a certain immune isolation effect is achieved; the cellulose solution with the inner phase in liquid form provides a 3D liquid environment to enhance cell viability. The method can improve survival rate of encapsulated cells, and improve activity and effect of transplanted cells. Compared with other cell encapsulating technologies, the method has the unique advantages of being a better cell encapsulating technology and improving the effect of cell transplantation.
The traditional hydrogel packaging technology comprises a mechanical stirring method, an emulsion solvent volatilization method or a method of forming hydrogel microspheres or microcapsules by cutting an organic oil phase of a microfluidic system, most of the hydrogel microspheres or microcapsules are hydrogel solid microspheres, and the preparation method of the main structure of the hydrogel solid microspheres is complex, high in technical requirement, large in size distribution, nonuniform in particle size and poor in monodispersity. In addition, the traditional prepared microspheres and microcapsules are not beneficial to the material exchange of cells due to the solid structure, influence the growth of the cells and limit the application of the microspheres and microcapsules.
PEO is used as a pore-making agent and is mixed with alginate of an external phase, when the alginate is crosslinked with calcium ions in a collected liquid to form hydrogel, PEO does not gel and is dispersed in the hydrogel to be dissolved in water to form a porous structure in the hydrogel, and the porous structure is uniformly dispersed in the hydrogel in the order of nanometers. The size of the porous hydrogel can be adjusted by changing the concentration and the proportion of PEO, and the method is flexible and convenient.
Disclosure of Invention
In order to solve the defects of reduced cell activity, low transplantation survival rate and complex preparation of hydrogel microspheres of the traditional hydrogel-coated cells, the invention provides a double-aqueous-phase porous islet microcapsule with a core-shell structure and a preparation method thereof. In the invention, the porous islet microcapsules with the core-shell structure are designed and invented by taking ALG and PEO mixed liquor as an external phase and CMC as an internal phase, can wrap cells, realize 3D culture and can be transplanted to treat immune-related diseases.
In order to solve the technical problems, the invention adopts the technical scheme that:
a preparation method of a double-aqueous-phase porous islet microcapsule with a core-shell structure comprises the following steps:
(1) designing a microfluidic electrojet double-emulsion device:
the microfluidic electronic injection chip is prepared by adjusting the pipe diameter and the alignment mode so as to ensure that the two-phase solution can smoothly pass through the pipeline and has proper proportion;
(2) preparing an inner phase solution and an outer phase solution, and preparing the microcapsule:
the inner and outer phase solution need to have different viscosity, and the non-gel component is added into the outer phase solution; under the action of an external high-voltage electric field, the internal and external phase solutions form an immiscible core-shell structure, and the outer layer of the core-shell structure is formed into glue through ion exchange or photocuring; the above steps are repeated to prepare the microcapsule, and because the substance which does not form the gel acts as the pore-forming agent, the shell layer of the obtained microcapsule is of a porous structure, and finally the double-aqueous-phase porous islet microcapsule with the core-shell structure is prepared.
In the step (1), the microfluidic electronic injection chip is a coaxial device and can be filled with double emulsions.
The hydrogel of the outer shell is made into a porous structure by adding a pore-forming agent polyethylene oxide (PEO) solution into an alginic Acid (ALG) solution of the outer shell, and the inner phase is a solution with biocompatibility and certain viscosity, such as carboxymethyl cellulose or methyl cellulose.
The shell layer of the microcapsule is porous hydrogel, and the concentration range of the porous hydrogel is 0.5-10% w/v.
The shell layer of the microcapsule is one material or a composite of more than two materials of polyethylene oxide (PEO), polyethylene glycol (PEG), Alginate (ALG), polyethylene glycol diacrylate (PEGDA), methacrylate gelatin (GelMA), silk fibroin, hyaluronic acid, Polydimethylsiloxane (PDMS), chitosan, gelatin, lactose, galactose, dextrin, maltose, polylactic acid (PLA), polyvinylpyrrolidone (PVP) and polyvinyl alcohol (PVA).
Microcapsules with different sizes and core-shell proportions are obtained by adjusting the voltage electric field, the flow velocity, the collection distance and the concentration of the internal and external phase solutions; wherein an increase in voltage causes a corresponding decrease in the diameter of the microcapsules and a decrease in voltage causes a corresponding increase in the diameter of the microcapsules.
The invention also protects the double-water-phase porous islet microcapsules with the core-shell structure, which are prepared by the method, and the microcapsules have two obvious core-shell parts, wherein the shell layer of the outer phase is of a porous structure, and the core of the inner phase is of a liquid state.
Further, it contains a liquid polymer solution having a viscosity different from that of the shell layer, forming a liquid core capable of dispersing the objective cells in the internal phase.
Furthermore, the diameter of the double-water-phase porous islet microcapsule with the core-shell structure is 100-1200 mu m.
Preferably, the PEO concentration is in the range of 0.5% to 2% (w/v).
Preferably, the sodium alginate in the shell layer has a concentration of 0.5-4% (w/v), and the sodium carboxymethyl cellulose is arranged inside and is loaded with cells.
Preferably, the diameter of the double-water-phase porous islet microcapsules with the core-shell structure is 200-950 μm.
The invention also protects the application of the double-aqueous-phase porous islet microcapsules with the core-shell structure in the preparation of the transplantable cell-loaded microcapsules for realizing the cell therapy effect.
Compared with the prior art, the invention has the beneficial effects that:
(1) the invention prepares the microcapsule with a core-shell structure by a microfluidic electrospray technology, the shell layer is hydrogel, the core layer is liquid, the method is simple, the operation is convenient, the price is low, the repeatability is high, the high technical requirement is not needed, and the size of the microcapsule is easy to control.
(2) The invention designs a porous islet microcapsule with a core-shell structure, wherein a non-gelling component is added into a shell layer to obtain a microcapsule shell with a porous structure, so that macromolecular substances such as immune cells, immune globulin and the like are isolated while exchange of small molecular substances is allowed.
(3) The aqueous two-phase porous islet microcapsules with the core-shell structure can be used for cell 3D culture, cell wrapping transplantation, tissue engineering and the like.
Drawings
FIG. 1 is a schematic diagram of a preparation process of porous islet microcapsules with a core-shell structure; the outer phase is a mixed solution of alginate and PEO, the inner phase is a carboxymethyl cellulose solution, a high-voltage electric field is applied to the outside, and the collection solution is a calcium chloride solution.
In the process of forming the porous islet microcapsules with the core-shell structure shown in fig. 2, the two-phase solution can be clearly seen to form an inner phase and an outer phase, so that the microcapsules with the core-shell structure are obtained.
FIG. 3 is a light and electron microscope image of the porous islet microcapsules with core-shell structure obtained in one example; wherein, the drawing a is a forming process drawing, the drawing b is a light mirror drawing, and the drawings c-d are electron microscope drawings.
Figure 4 effect of several key factors on microcapsule diameter.
Detailed Description
The above-mentioned contents of the present invention are further described in detail by way of examples below, but it should not be understood that the scope of the above-mentioned subject matter of the present invention is limited to the following examples, and any technique realized based on the above-mentioned contents of the present invention falls within the scope of the present invention.
The experimental procedures used in the examples below are conventional procedures unless otherwise specified, and the reagents, methods and equipment used therein are conventional in the art unless otherwise specified.
Example 1
Preparation of porous islet microcapsules with core-shell structure
(1) Designing a microfluidic chip:
firstly, designing a microfluidic electronic injection chip, selecting a capillary glass tube with the diameter of 800 micrometers and 300 micrometers, adjusting the outlet of an external phase capillary tube to be 500 micrometers by drawing the tube, nesting the capillary tubes with two diameters into a capillary tube with a coaxial outlet by adopting a coaxial method, sticking and fixing the capillary tube with the coaxial outlet at a corresponding position by using a needle head, and reserving an inlet of a two-phase solution. As shown in fig. 1. And repeatedly washing with ultrapure water to obtain the microfluidic chip.
(2) Preparing a core-shell structure porous islet microcapsule:
in order to enable the shell layer hydrogel to have a porous structure, preparing 1% (w/v) sodium alginate solution, adding a small amount of 1% (w/v) PEO solution, and stirring overnight to fully and uniformly mix the two solutions; preparing 1% (w/v) sodium carboxymethylcellulose solution, building a microfluidic electronic injection platform comprising a high-voltage direct-current power supply and two flow pumps, and finally setting and adjusting flow and voltage to obtain the porous islet microcapsules with the core-shell structure, as shown in fig. 2.
Example 2
Preparation of porous islet microcapsules with core-shell structure
(1) Designing a microfluidic chip:
firstly, designing a microfluidic electronic injection chip, selecting a capillary glass tube with the diameter of 800 micrometers and 300 micrometers, adjusting the outlet of an external phase capillary tube to be 500 micrometers by drawing the tube, nesting the capillary tubes with two diameters into a capillary tube with a coaxial outlet by adopting a coaxial method, sticking and fixing the capillary tube with the coaxial outlet at a corresponding position by using a needle head, and reserving an inlet of a two-phase solution. As shown in fig. 1. And repeatedly washing with ultrapure water to obtain the microfluidic chip.
(2) Preparing a core-shell structure porous islet microcapsule:
in order to enable the shell layer hydrogel to have a porous structure, polyethylene glycol diacrylate (PEGDA) solutions with different concentration ranges are prepared, a small amount of 1% (w/v) PEO solution is added, and the mixture is stirred overnight to fully and uniformly mix the two solutions; preparing 1% (w/v) sodium carboxymethylcellulose solution, building a microfluidic electronic injection platform comprising a high-voltage direct-current power supply and two flow pumps, and finally setting and adjusting flow and voltage to obtain the porous islet microcapsules with the core-shell structure, as shown in fig. 2.
Example 3
Preparation of porous islet microcapsules with core-shell structure
(1) Designing a microfluidic chip:
firstly, designing a microfluidic electronic injection chip, selecting a capillary glass tube with the diameter of 800 micrometers and 300 micrometers, adjusting the outlet of an external phase capillary tube to be 500 micrometers by drawing the tube, nesting the capillary tubes with two diameters into a capillary tube with a coaxial outlet by adopting a coaxial method, sticking and fixing the capillary tube with the coaxial outlet at a corresponding position by using a needle head, and reserving an inlet of a two-phase solution. As shown in fig. 1. And repeatedly washing with ultrapure water to obtain the microfluidic chip.
(2) Preparation of core-shell structure porous islet microcapsules
In order to enable the shell layer hydrogel to have a porous structure, methacrylate gelatin (GelMA) solutions with different concentrations are prepared, a small amount of 1% (w/v) PEO solution is added, and the mixture is stirred overnight to fully and uniformly mix the two solutions; preparing 1% (w/v) sodium carboxymethylcellulose solution, building a microfluidic electronic injection platform comprising a high-voltage direct-current power supply and two flow pumps, and finally setting and adjusting flow and voltage to obtain the porous islet microcapsules with the core-shell structure, as shown in fig. 2.
Adding a small amount of 1% (w/v) PEO solution into 1% (w/v) sodium alginate solution to serve as an external phase; 1% (w/v) sodium carboxymethylcellulose solution and islet cells are used as internal phases, a microfluidic electrospray platform is built to prepare the porous islet microcapsules with good monodispersity and a core-shell structure, and the experimental results show that the islet cells can grow well in the microcapsules, the survival rate is more than 90%, and the islet cells have the effect of reducing the blood sugar of a mouse after being transplanted into a diabetic mouse.
The above description is only a preferred embodiment of the present invention, and should not be taken as limiting the invention in any way, and any person skilled in the art can make any simple modification, equivalent replacement, and improvement on the above embodiment without departing from the technical spirit of the present invention, and still fall within the protection scope of the technical solution of the present invention.

Claims (10)

1. A preparation method of a double-aqueous-phase porous islet microcapsule with a core-shell structure is characterized by comprising the following steps:
(1) designing a microfluidic electrojet double-emulsion device:
the microfluidic electronic injection chip is prepared by adjusting the pipe diameter and the alignment mode so as to ensure that the two-phase solution can smoothly pass through the pipeline and has proper proportion;
(2) preparing an inner phase solution and an outer phase solution, and preparing the microcapsule:
the inner and outer phase solution need to have different viscosity, and the non-gel component is added into the outer phase solution; under the action of an external high-voltage electric field, the internal and external phase solutions form an immiscible core-shell structure, and the outer layer of the core-shell structure is formed into glue through ion exchange or photocuring; the above steps are repeated to prepare the microcapsule, and because the substance which does not form the gel acts as the pore-forming agent, the shell layer of the obtained microcapsule is of a porous structure, and finally the double-aqueous-phase porous islet microcapsule with the core-shell structure is prepared.
2. The preparation method of the aqueous two-phase porous pancreatic islet microcapsule with the core-shell structure according to claim 1, is characterized in that: in the step (1), the microfluidic electronic injection chip is a coaxial device and can be filled with double emulsions.
3. The preparation method of the aqueous two-phase porous pancreatic islet microcapsule with the core-shell structure according to claim 1, is characterized in that: the hydrogel of the outer shell is made into a porous structure by adding a pore-forming agent polyethylene oxide (PEO) solution into an alginic Acid (ALG) solution of the outer shell, and the inner phase is a solution with biocompatibility and certain viscosity, such as carboxymethyl cellulose or methyl cellulose.
4. The preparation method of the aqueous two-phase porous pancreatic islet microcapsule with the core-shell structure according to claim 3, is characterized in that: the shell layer of the microcapsule is porous hydrogel, and the concentration range of the porous hydrogel is 0.5-10% w/v.
5. The preparation method of the aqueous two-phase porous pancreatic islet microcapsule with the core-shell structure according to claim 3, is characterized in that: the shell layer of the microcapsule is one material or a composite of more than two materials of polyethylene oxide (PEO), polyethylene glycol (PEG), Alginate (ALG), polyethylene glycol diacrylate (PEGDA), methacrylate gelatin (GelMA), silk fibroin, hyaluronic acid, Polydimethylsiloxane (PDMS), chitosan, gelatin, lactose, galactose, dextrin, maltose, polylactic acid (PLA), polyvinylpyrrolidone (PVP) and polyvinyl alcohol (PVA).
6. The preparation method of the aqueous two-phase porous pancreatic islet microcapsule with the core-shell structure according to claim 1, is characterized in that: microcapsules with different sizes and core-shell proportions are obtained by adjusting the voltage electric field, the flow velocity, the collection distance and the concentration of the internal and external phase solutions; wherein an increase in voltage causes a corresponding decrease in the diameter of the microcapsules and a decrease in voltage causes a corresponding increase in the diameter of the microcapsules.
7. The aqueous two-phase porous islet microcapsules with a core-shell structure, prepared by the method of any one of claims 1 to 6, is characterized in that: the composite material has two obvious core-shell parts, the shell layer of the outer phase is in a porous structure, and the core of the inner phase is in a liquid state.
8. The aqueous two-phase porous islet microcapsules with core-shell structure according to claim 7, characterized in that: the polymer solution in liquid state has viscosity different from that of the shell layer, forms a liquid core, and can disperse the target cells in the inner phase.
9. The aqueous two-phase porous islet microcapsules with core-shell structure according to claim 7, characterized in that: the diameter of the double-water-phase porous islet microcapsule with the core-shell structure is 100-1200 mu m.
10. The use of the aqueous two-phase porous islet microcapsules with a core-shell structure according to claim 7 for the preparation of implantable cell-loaded microcapsules for the realization of a cell therapeutic effect.
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