CN114832742B - Emulsion template and polymer porous microsphere derived from same - Google Patents
Emulsion template and polymer porous microsphere derived from same Download PDFInfo
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Classifications
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- 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
Abstract
The invention particularly relates to an emulsion template and a polymer porous microsphere derived from the emulsion template, which belong to the technical field of porous microspheres, wherein the template comprises an oil phase system and a water phase system, and the density relationship between the water phase system and the oil phase system accords with a set relationship so that the water phase system can stably suspend in the oil phase system; by making the densities of the water phase system and the oil phase system very close to each other, the external force is reduced as much as possible, the surface tension of the water-oil interface after emulsification can maintain the water-in-oil structure of the emulsion, phase separation can not occur in a proper environment theoretically, the product can be kept stable for a long time in actual use, the uniformity of the product is extremely good, only specific porous microspheres can be obtained, and the problem of non-uniformity of the appearance and the size of the existing porous microspheres is solved.
Description
Technical Field
The invention belongs to the technical field of porous microspheres, and particularly relates to an emulsion template and a polymer porous microsphere derived from the emulsion template.
Background
Porous microspheres refer to micron-sized spherical particles with interconnected or non-interconnected pores within and/or on the surface, generally having a very high specific surface area, and are commonly used as carriers. Polymeric porous microspheres have a wide range of applications, for example in the pharmaceutical field for drug loading and controlled release, in the chemical field for catalyst loading or immobilized enzymes, and in the biomedical field for cell and tissue culture. However, the existing porous microsphere preparation method has some defects, which limit the application of the method: the porous microspheres prepared by the traditional emulsification-solvent volatilization method have wide particle size distribution and uneven morphology, and often need to be screened, thus increasing the production cost; the porous microspheres prepared by the nano precipitation method have the same problem of non-uniform particle size and morphology; the microfluidic method is a newer microsphere preparation method, and by enabling the continuous phase and the discontinuous phase to be intersected in a microfluidic device or a microfluidic chip to form uniform liquid drops and further form microspheres, the method greatly improves the uniformity of the solid microspheres, and is further convenient for the design and mass preparation of the functionalized solid microspheres. However, for porous microspheres, the micro-fluidic method cannot solve the problem of poor uniformity, because the traditional emulsion as a discontinuous phase is rapidly layered, so that the size and composition of droplets formed in a micro-fluidic device are also changed, and finally, the porous microspheres with different particle sizes and morphologies are difficult to accurately design the morphology of the microspheres and prepare in large quantities. Among the above methods, the microfluidic method is a more complex but most stable microsphere preparation method, but the porous microspheres prepared by the current method are not uniform in size and morphology.
Disclosure of Invention
The application aims to provide an emulsion template and a polymer porous microsphere derived from the emulsion template, so as to solve the problem of non-uniform size and morphology of the existing porous microsphere.
The embodiment of the invention provides an emulsion template, which comprises an oil phase system and a water phase system, wherein the density relationship between the water phase system and the oil phase system accords with a set relationship, so that the water phase system can stably suspend in the oil phase system.
Optionally, the absolute value of the density difference between the oil phase system and the water phase system is less than or equal to 15g/L.
Optionally, the absolute value of the density difference between the oil phase system and the water phase system is less than or equal to 10g/L.
Optionally, the oil phase system at least comprises two organic solvents, at least one of the two organic solvents has a density greater than that of the aqueous phase system, and at least one of the two organic solvents has a density less than that of the aqueous phase system.
Optionally, the oil phase system comprises two organic solvents.
Alternatively, the two organic solvents are ethyl acetate and dichloromethane, respectively.
Optionally, the aqueous phase system further comprises a soluble salt to adjust the density of the aqueous phase system.
Optionally, the solute of the oil phase system is a biodegradable polymer material.
Optionally, the biodegradable polymer material comprises at least one of polycaprolactone, a polycaprolactone-polyethylene glycol block copolymer and a polylactic acid-glycolic acid copolymer.
Based on the same inventive concept, the embodiment of the invention also provides a polymer porous microsphere, wherein the microsphere is prepared by adopting the emulsion template.
One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:
according to the emulsion template provided by the embodiment of the invention, the densities of the water phase system and the oil phase system are very close, so that the external force is reduced as much as possible, the surface tension of the water-oil interface after emulsification can maintain the water-in-oil structure of the emulsion, phase separation can not occur in a proper environment theoretically, the emulsion template can be kept stable for a long time in actual use, the uniformity of a product is excellent, only specific porous microspheres can be obtained, and the problem of non-uniform size of the existing porous microspheres is solved.
The foregoing description is only an overview of the present invention, and is intended to be implemented in accordance with the teachings of the present invention in order that the same may be more clearly understood and to make the same and other objects, features and advantages of the present invention more readily apparent.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic illustration of an emulsion template provided in accordance with an embodiment of the present invention;
FIG. 2 is a photo-microscopic view of porous microspheres according to example 1 of the present invention;
FIG. 3 is a photomicrograph of porous microspheres provided in example 2 of the present invention;
FIG. 4 is a photomicrograph of the porous microspheres provided in example 3 of the present invention;
FIG. 5 is a photomicrograph of the porous microspheres provided in example 4 of the present invention;
FIG. 6 is a photomicrograph of the porous microspheres provided in example 5 of the present invention;
FIG. 7 is a photomicrograph of the porous microspheres provided in example 6 of the present invention;
FIG. 8 is a photomicrograph of the porous microspheres provided in comparative example 1 of the present invention;
FIG. 9 is a graph showing the particle size statistics of porous microspheres provided in examples 1-6 and comparative example 1 of the present invention;
FIG. 10 is a scanning electron microscope image of the porous microspheres provided in example 1 of the present invention;
FIG. 11 is a scanning electron microscope image of porous microspheres provided in example 2 of the present invention;
FIG. 12 is a scanning electron microscope image of the porous microspheres provided in example 3 of the present invention;
FIG. 13 is a scanning electron microscope image of the porous microspheres provided in example 4 of the present invention;
FIG. 14 is a scanning electron microscope image of the porous microspheres provided in example 5 of the present invention;
FIG. 15 is a scanning electron microscope image of the porous microspheres provided in example 6 of the present invention;
FIG. 16 is a scanning electron microscope image of the porous microspheres provided in comparative example 1 of the present invention;
FIG. 17 is a graph showing the cytotoxicity test results of the porous microspheres according to example 1 of the present invention;
FIG. 18 is a graph showing the result of culturing BMSCs on porous microspheres provided in example 1 of the present invention;
FIG. 19 is a graph showing the results of culturing BMSCs on dopamine modified porous microspheres provided in example 1 of the present invention;
fig. 20 is a schematic diagram of the layering of a water-in-oil emulsion as provided in the background.
Detailed Description
The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.
Abbreviations and key term definitions:
polycaprolactone (polycaprolactone): PCL (PCL)
Polylactic acid-glycolic acid copolymer (poly (lactic-co-glycolic acid): PLGA
Polycaprolactone-polyethylene glycol block polymer: PECL
Double emulsion template: double-breast template
Isopycnic emulsion: equal density emulsion
Polyvinyl alcohol (polyvinyl alcohol): PVA (polyvinyl alcohol)
The technical scheme of the embodiment of the application aims to solve the technical problems, and the overall thought is as follows:
applicants found during the course of the invention that: the porous microspheres are often prepared using a common double emulsion as a template, with the simplest and most common method being to disperse the emulsion directly into the mobile phase and volatilize the solvent to prepare the porous microspheres. The method comprises the following specific steps: preparing an inner water phase and a single oil phase, mixing and emulsifying according to a certain proportion to obtain a common water-in-oil emulsion; adding the common water-in-oil emulsion into the outer water phase according to a certain proportion and emulsifying to obtain the common water-in-oil-in-water emulsion, namely a common double-emulsion template; adding a plurality of common double-emulsion templates into a plurality of external water phases, continuously stirring, and volatilizing an organic solvent in an oil phase to obtain cured microspheres; and finally removing the inner water phase, washing and freeze-drying to obtain the porous microspheres.
In addition to obtaining an emulsified double emulsion template by direct dispersion, a water-in-oil emulsion can be introduced into a microfluidic device to obtain a double emulsion template and produce porous microspheres. The method can disperse the discontinuous phase into uniform liquid drops, the process is controllable and mild, and the quality of the porous microsphere is better, but the operation difficulty is also higher. The method comprises the following specific steps: preparing an inner water phase and a single oil phase, mixing and emulsifying according to a certain proportion to obtain a common water-in-oil emulsion; introducing the water-in-oil emulsion into a microfluidic device, and dispersing the water-in-oil emulsion into uniform liquid drops in an external water phase by a microfluidic method, so as to obtain a more uniform water-in-oil-in-water emulsion, namely a common double-emulsion template; continuously stirring the outer water phase to volatilize the organic solvent in the oil phase, thereby obtaining solidified microspheres; and finally removing the inner water phase, washing and freeze-drying to obtain the porous microspheres.
Because the common emulsion is very unstable, the emulsion can be layered in a short period of time due to phase separation, so that the obtained double-emulsion template is very non-uniform, and finally the microsphere is in a different form and has poor quality. For the direct emulsification method, although the time required for preparing the common double emulsion is short, the double emulsion template can be obtained fastest and the organic solvent is volatilized for solidification, the size of the microspheres obtained by the direct emulsification method is different, and the process is rough, which may be unfavorable for the maintenance of the double emulsion template structure. As for the microfluidic method, although it is theoretically possible to produce double emulsion template droplets with uniform size, because the method takes a long time, the water-in-oil emulsion can be rapidly layered (as shown in fig. 18), resulting in a large difference in composition of the obtained double emulsion template droplets, and some of the double emulsion template droplets are broken into many small droplets due to excessive internal water phase, and some of the double emulsion template droplets cannot form a porous structure due to insufficient internal water phase, and finally, porous microspheres have different sizes and different morphologies.
If the problem of instability of the traditional emulsion can be overcome, the method can be used for producing uniform porous microspheres in a large scale, and the particle size and morphology of the porous microspheres can be accurately designed by regulating and controlling various preparation parameters.
Aiming at the problem of non-uniformity of a product caused by poor stability of an emulsion template in a microfluidic-double-emulsion template method, the application provides an isopycnic emulsion template, and the stability of the emulsion template in the preparation process is improved, so that the uniformity of the product is excellent, and only specific porous microspheres can be obtained; the method is also suitable for direct emulsification-double-emulsion template method, membrane emulsification-double-emulsion template method and other methods based on emulsion template, and can be used for large-scale production; in addition, the method can improve the pore-forming of the polymer through the amphoteric polymer, so that the method can be applicable to the polymer difficult to pore-form and has better universality.
According to an exemplary embodiment of the present invention, there is provided an emulsion template including an oil phase system and an aqueous phase system, the density relationship of the aqueous phase system and the oil phase system conforming to a set relationship so that the aqueous phase system can be stably suspended in the oil phase system.
By making the densities of the water phase system and the oil phase system very close to each other, the external force is reduced as much as possible, the surface tension of the water-oil interface after emulsification can maintain the water-in-oil structure of the emulsion, phase separation can not occur in a proper environment theoretically, the product can be kept stable for a long time in actual use, the uniformity of the product is extremely good, only specific porous microspheres can be obtained, and the problem of nonuniform size of the existing porous microspheres is solved.
In some embodiments, the absolute value of the difference in density between the oil phase system and the aqueous phase system is less than or equal to 15g/L, and more preferably, the absolute value of the difference in density between the oil phase system and the aqueous phase system is less than or equal to 10g/L.
In some embodiments, the oil phase system comprises at least two organic solvents, at least one of the two organic solvents having a density greater than the aqueous phase system and at least one of the two organic solvents having a density less than the aqueous phase system.
The multi-oil phase system composed of multiple organic solvents is adopted to replace a single oil phase, the density of the oil phase is regulated by changing the proportion of different organic solvents, so that the oil phase is very similar to the density of the water phase serving as a pore-forming agent, the external force is reduced as much as possible, the surface tension of the water-oil interface after emulsification can maintain the water-in-oil structure of the emulsion, phase separation can not occur in a proper environment theoretically, and the emulsion can be kept stable for a long time in actual use
In this example, the oil phase system comprises two organic solvents. Specifically, the two organic solvents may be ethyl acetate and methylene chloride, respectively.
In some embodiments, the aqueous phase system further comprises a soluble salt to adjust the density of the aqueous phase system.
The density of the aqueous phase can also be adjusted theoretically with a soluble salt to obtain an isopycnic emulsion template.
In some embodiments, the solute of the oil phase system is a biodegradable polymeric material. Specifically, the biodegradable polymer material may be at least one selected from the group consisting of polycaprolactone, polycaprolactone-polyethylene glycol block copolymers, and polylactic acid-glycolic acid copolymers.
Polylactic-co-glycolic acid (PLGA) is one of the most commonly used materials for preparing polymeric porous microspheres, mainly because PLGA microspheres are relatively prone to form pores. PLGA is prepared by randomly polymerizing lactic acid and glycolic acid, is a biodegradable polymer with good biocompatibility, can adjust the degradation rate by changing the ratio of lactic acid to glycolic acid, and is commonly used for preparing drug-loaded microspheres with controllable release. However, most of the PLGA microspheres currently used are solid microspheres, and porous microspheres have not been widely used because the existing preparation methods of porous microspheres are difficult to meet the production requirements.
The preparation of porous microspheres from Polycaprolactone (PCL) is currently one research focus, because polycaprolactone not only has good biocompatibility and biodegradability, but also is safer in degradation products, but the polycaprolactone is difficult to form into porous microspheres due to the material characteristics.
According to another exemplary embodiment of the present invention, there is provided a polymeric porous microsphere prepared using the emulsion template as described above.
It will be apparent to those skilled in the art that various emulsification devices (agitators, microfluidic devices, membrane emulsifiers, etc.) and modifications and variations of formulation or preparation parameters can be made without departing from the technical principles of the present invention, but the scope of the present invention should be considered as long as an isopycnic emulsion template is used in the process of preparing the porous polymer microspheres.
The preparation of the isopipe template is not limited to a specific emulsifying device, and a microfluidic device is selected to prepare the isopipe template for the purpose of exhibiting the best effect. The invention will be described in further detail with reference to specific embodiments and accompanying drawings. The following examples are provided to illustrate the invention but do not limit the scope of the invention.
Example 1
This example is directed to the preparation of polycaprolactone porous microspheres having a diameter of about 300 μm and a plurality of interconnected pores, and is prepared by the following method:
(1) Assembled microfluidic device
The microfluidic device was assembled according to the current method, with a 27G right angle needle and a 0.5mm x 2mm glass capillary tube selected.
Two 50mL syringes were mounted on the two thrust pumps, respectively; a syringe connected with the silicone tube, and a glass capillary tube connected with the tail end of the syringe and used as a continuous phase channel; the other syringe is connected with a right-angle needle, and the needle penetrates the silicone tube and is just inserted into the mouth of the glass capillary to be used as a discontinuous phase channel.
(2) Preparation of 1% aqueous polyvinyl alcohol solution
Slowly adding 10g of polyvinyl alcohol into a beaker filled with 1L of distilled water while stirring, stirring in a water bath at 60 ℃ for 0.5h and in a water bath at 85 ℃ for 2h to thoroughly dissolve the polyvinyl alcohol, supplementing 1L of volume with distilled water, filtering impurities with a 50 mu m screen, and cooling to room temperature; the syringe connected with the silicone tube is taken down, 1% polyvinyl alcohol solution is filled and put back, the rest polyvinyl alcohol solution is poured into a 1L beaker and placed under the glass capillary tube, and the position is adjusted so that the liquid level is beyond the outlet of the capillary tube.
(3) Preparation of an isopycnic emulsifying System
0.135g of polycaprolactone and 0.027g of polycaprolactone-polyethylene glycol block copolymer were weighed and dissolved in 5.238g of a mixed oil phase consisting of ethyl acetate and methylene chloride, wherein the mass ratio of ethyl acetate to methylene chloride was 8/5, yielding 5.4g of a mixed oil phase containing 2.5wt% of polycaprolactone and 0.5wt% of polycaprolactone-polyethylene glycol block copolymer.
3g of 10wt% gelatin water solution is dripped into the mixed oil phase, and the mass ratio of the water phase to the oil phase is 1/1.8; carefully dropwise adding ethyl acetate or dichloromethane to adjust the density of the oil phase until the water phase is stably suspended in the mixed oil phase; preliminary emulsification is carried out for 3min by a magnetic stirrer at 2200rpm, then homogenization is carried out for 24s by a high-speed homogenizer, an isopycnic emulsifying system is obtained, and the emulsion is filled into a syringe.
(4) Preparation of polycaprolactone porous microspheres
The collected phase was added with 10mL of ethyl acetate and stirred in an ice bath at 200rpm, the propeller pump was adjusted to moderate the flow rates of the continuous phase and the discontinuous phase, and uniform droplets with moderate spacing were formed in the glass capillary; in this case the continuous and discontinuous phase flow rates were 25mL/h and 2mL/h, respectively.
The droplets are stirred overnight in the collection phase, and the organic solvent is volatilized to form microspheres; the microspheres were collected and stirred in a 45 ℃ water bath for 2 hours to dissolve gelatin, washed and freeze-dried to obtain porous microspheres.
Example 2
This example was conducted to prepare polycaprolactone porous microspheres with interconnected pores of about 150 μm in diameter, and the procedure of this example was the same as that of example 1, except that: a 32G needle and a 0.25mm by 2mm glass capillary are selected when the microfluidic device is assembled; the continuous phase and the collection phase use 2% polyvinyl alcohol solution; when the isodensity emulsifying system is prepared, the mass ratio of the water phase to the oil phase is 1/2.4; when the microspheres are prepared, the flow rates of the continuous phase and the discontinuous phase are respectively 1mL/h and 6mL/h.
Example 3
This example was conducted to prepare polycaprolactone porous microspheres having closed cells of about 180 μm in diameter, and the procedure of this example was the same as in example 1, except that: selecting a 30G needle and a glass capillary with an inner diameter of 0.3mm when assembling the microfluidic device; distilled water is used for the collecting phase to replace the polyvinyl alcohol solution; when the isodensity emulsifying system is prepared, the concentrations of the polycaprolactone and the polycaprolactone-polyethylene glycol block copolymer in the oil phase are respectively 5 weight percent and 1 weight percent, and the mass ratio of the water phase to the oil phase is 1/4; when the microspheres are prepared, the flow rates of the continuous phase and the discontinuous phase are respectively 1mL/h and 6mL/h.
Example 4
This example, which is identical to the procedure in example 1 except that polycaprolactone porous microspheres having small pores with a diameter of about 280 μm were prepared: selecting a 28G needle and a glass capillary with an inner diameter of 0.4mm when assembling the microfluidic device; when the isopycnic emulsifying system is prepared, the concentrations of the polycaprolactone and the polycaprolactone-polyethylene glycol segmented copolymer in the oil phase are 5wt% and 1wt%, respectively, the mass ratio of the water phase to the oil phase is 1/4, and the oil phase is homogenized for 2min at high speed by using a homogenizer; when preparing the microsphere, the flow rates of the continuous phase and the discontinuous phase are respectively 1.5mL/h and 10mL/h.
Example 5
This example, which is identical to the procedure in example 1 except that polycaprolactone porous microspheres having small surface pores were prepared with a diameter of about 100 μm: a 32G needle and a 0.25mm by 2mm glass capillary are selected when the microfluidic device is assembled; when the discontinuous phase does not use an isopycnic emulsifying system, the oil phase component is methylene dichloride solution containing 4wt% of polycaprolactone; when preparing the microsphere, the flow rates of the continuous phase and the discontinuous phase are respectively 4mL/h and 6mL/h.
Example 6
In this example, porous microspheres of polylactic acid-glycolic acid copolymer having interconnected pores with a diameter of about 300 μm were prepared, which was the same as in example 1 except that: when the isodensity emulsifying system is prepared, 2.5 weight percent of polycaprolactone and 0.5 weight percent of polycaprolactone-polyethylene glycol block copolymer in the oil phase are replaced by 3 weight percent of polylactic acid-glycolic acid copolymer, and the mass ratio of the water phase to the oil phase is 1/2.4.
Comparative example 1
The comparative example was prepared using a microfluidic-generic emulsion template method to prepare porous microspheres of polylactic acid-glycolic acid copolymer, the comparative example being identical to the procedure in example 6, except that: instead of using the isopycnic emulsification method, a methylene chloride solution containing 3wt% of the polylactic acid-glycolic acid copolymer was used as the oil phase.
Experimental example
The porous microspheres prepared in examples 1 to 6 and comparative example 1 were observed for shape by an inverted fluorescence microscope and photographed. Fig. 2 to 8 are obtained.
As can be seen from fig. 2 to 8, the porous microspheres prepared by the method provided in the examples of the present application are round and regular in shape and substantially uniform in size, while the morphology of the porous microspheres prepared by the common emulsion template provided in comparative example 1 is not as uniform as that of example 6.
The particle size distribution of the polycaprolactone porous microspheres in fig. 2-8 was counted using mathematical software mathmatics to give fig. 9.
As can be seen from fig. 9, the particle size distribution of the porous microspheres prepared using the method provided in the examples of the present application was quite concentrated, whereas the particle size distribution of the porous microspheres prepared using the conventional emulsion template provided in comparative example 1 was significantly wider than that of example 6.
The morphology of the porous microspheres prepared in examples 1 to 6 and comparative example 1 was observed by a field emission scanning electron microscope, and FIGS. 10 to 16 were obtained.
As can be seen from fig. 10 to 16, the porous microspheres prepared by the method provided in the example of the present application have a large number of pores having a diameter of 1 to 20 μm on the surface and inside, and the pores are interconnected, whereas the pores of the porous microspheres prepared by the conventional emulsion template provided in comparative example 1 are not uniform.
The porous microspheres prepared in example 1 were co-cultured in direct contact with bone marrow mesenchymal stem cells (BMSCs) to determine if they were cytotoxic, and cell activity was determined using CCK-8 kit after a series of time points. The results are shown in FIG. 17, where pure polycaprolactone solid microspheres were used as a control.
As can be seen from fig. 17, the polycaprolactone porous microsphere provided by the embodiment of the invention has good cell compatibility, and the cell activity and proliferation rate of the BMSCs co-cultured with the polycaprolactone porous microsphere are close to those of the pure polycaprolactone group and the negative control group.
The porous microspheres prepared in example 1 were co-cultured with bone marrow mesenchymal stem cells (BMSCs) to verify their cell adhesion properties, stained with cytoskeletal staining kit and DAPI after a series of time points. The confocal microscopy photograph was obtained as shown in fig. 18.
As can be seen from fig. 18, the polycaprolactone porous microsphere provided by the embodiment of the invention has the characteristic of cell adhesion resistance, because the polycaprolactone-polyethylene glycol block copolymer can form an anti-fouling layer, and has potential for antibacterial application and the like.
The porous microsphere prepared in the embodiment 1 is used as a platform for surface modification, so that various functions can be obtained, and the requirements of more applications are met. Alternatively, the porous microspheres prepared in example 1 were modified with dopamine and co-cultured with bone marrow mesenchymal stem cells (BMSCs) to verify their cell adhesion properties, stained with cytoskeletal staining kit and DAPI after a series of time points. The confocal microscopy photograph was obtained as shown in fig. 19.
As can be seen from fig. 19, the polycaprolactone porous microsphere provided by the embodiment of the invention shows good cell compatibility and cell adhesion performance after simple dopamine modification, and has potential for application such as cell co-culture.
One or more technical solutions in the embodiments of the present invention at least have the following technical effects or advantages:
(1) The emulsion template provided by the embodiment of the invention enables the densities of the water phase and the oil phase to be very close by adjusting the densities of the multi-oil phase or multi-water phase system, so that the emulsion template is kept stable for a long time, and uniform porous microspheres are obtained;
(2) The method provided by the embodiment of the invention has the advantages that the particle size and the morphology of the produced porous microspheres are more uniform through an improved isodensity emulsification method and the existing microfluidic technology, so that the specific porous microspheres can be produced by adjusting the preparation parameters; the emulsifier is not needed, and the product has no residue of the emulsifier; the method can be used for large-scale production of single products of various microfluidic devices without screening, improves production quality, reduces production cost and is suitable for large-scale production; for polymers that are not prone to pore formation (polycaprolactone as used in the example) this can be improved by the addition of an amphiphilic polymer;
(3) The emulsion template provided by the embodiment of the invention adopts a multi-oil-phase system consisting of a plurality of organic solvents to replace a single oil phase, the density of the oil phase is regulated by changing the proportion of different organic solvents, so that the density of the oil phase is very close to that of a water phase serving as a pore-forming agent, the external force is reduced as much as possible, the surface tension of an emulsified water-oil interface can maintain the water-in-oil structure of the emulsion, phase separation can not occur in a proper environment theoretically, and the emulsion template can be kept stable for a long time in practical use;
(4) The emulsion template provided by the embodiment of the invention adopts soluble salt to adjust the density of the water phase so as to obtain the isopycnic emulsion template.
Finally, it is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (1)
1. A method for preparing polylactic acid-glycolic acid copolymer porous microspheres with a diameter of 300 mu m and communicated pores is characterized in that,
(1) Assembled microfluidic device
A 27G right angle needle and a 0.5mm x 2mm glass capillary were selected;
two 50mL syringes were mounted on the two thrust pumps, respectively; a syringe connected with the silicone tube, and a glass capillary tube connected with the tail end of the syringe and used as a continuous phase channel; the other syringe is connected with a right-angle needle, and the needle penetrates through the silicone tube and is just inserted into the mouth of the glass capillary to be used as a discontinuous phase channel;
(2) Preparation of 1% aqueous polyvinyl alcohol solution
Slowly adding 10g of polyvinyl alcohol into a beaker filled with 1L of distilled water while stirring, stirring in a water bath at 60 ℃ for 0.5h and in a water bath at 85 ℃ for 2h to thoroughly dissolve the polyvinyl alcohol, supplementing 1L of volume with distilled water, filtering impurities with a 50 mu m screen, and cooling to room temperature; taking down a syringe connected with a silicone tube, filling 1% polyvinyl alcohol solution, filling back, pouring the rest polyvinyl alcohol solution into a 1L beaker, placing under a glass capillary tube, and adjusting the position to enable the liquid level to be over the outlet of the capillary tube;
(3) Preparation of an isopycnic emulsifying System
Weighing polylactic acid-glycolic acid copolymer, dissolving in 5.238g of mixed oil phase composed of ethyl acetate and methylene dichloride, wherein the mass ratio of the ethyl acetate to the methylene dichloride is 8/5, and obtaining 5.4g of mixed oil phase containing 3wt% of polylactic acid-glycolic acid copolymer;
3g of 10wt% gelatin water solution is dripped into the mixed oil phase, and the mass ratio of the water phase to the oil phase is 1/2.4; carefully dropwise adding ethyl acetate or dichloromethane to adjust the density of the oil phase until the water phase is stably suspended in the mixed oil phase; preliminary emulsifying for 3min by a magnetic stirrer at 2200rpm, homogenizing for 24s by a high-speed homogenizer to obtain an isopycnic emulsifying system, and loading into a syringe;
(4) Preparation of polylactic acid-glycolic acid copolymer porous microspheres
Adding 10mL of ethyl acetate into the collected phase, stirring at 200rpm in an ice bath, regulating a propelling pump to ensure that the flow rates of the continuous phase and the discontinuous phase are 25mL/h and 2mL/h respectively, and forming uniform droplets with moderate intervals in a glass capillary;
the droplets are stirred overnight in the collection phase, and the organic solvent is volatilized to form microspheres; the microspheres are collected and stirred in a water bath at 45 ℃ for 2 hours to dissolve gelatin, and the polylactic acid-glycolic acid copolymer porous microspheres are obtained after washing and freeze-drying.
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