KR102482010B1 - Two-Phase System (ATPS) Based Polymersomes for Single Particle Level Isolation and Intensification - Google Patents

Abstract

The present invention relates to a particle separation and aggregation technology at a single particle level using an aqueous biphasic-based polymersome.

Description

Single particle level separation and integration technology using aqueous phase system based polymersome {Two-Phase System (ATPS) Based Polymersomes for Single Particle Level Isolation and Intensification}

The present invention relates to a particle separation and aggregation technology at a single particle level using an aqueous biphasic-based polymersome.

Biomolecules such as proteins, nucleic acids, and proteins are widely used in pharmaceutical applications, diagnostic medicine, and cosmetics. In order to effectively apply biomolecules, a technique for isolating and purifying biomolecules is required. Indeed, the technology to isolate and assemble biomolecules down to the single-particle level is the most important and costly step.

Among various biomolecules, active research is being conducted on methods for isolating extracellular vesicles (EVs), collectively referred to as exosomes and vesicles composed of nano-sized lipid films. Since these EVs play a role in communication between cells and between antigen-antibody reactions, EVs can exert their potential in diagnostic medicine if they are isolated, purified and analyzed with high efficiency. However, EVs vary greatly in size distribution, from 30 nm to 1000 nm, and their biological diversity can vary depending on conditions. In addition, since EVs coexist with other molecules such as proteins and floating biomolecules in blood and body fluids, the need for an effective method for removing these substances and integrating only EVs is increasing.

Existing methods for isolating EVs have used approaches such as ultracentrifugation, immunoseparation, and polymer precipitation, but they require relatively complex processes without going through a single step, and the process time is long.

Korean Patent Publication No. 10-2018-0081354 discloses a device for continuous separation of biomolecules including exosomes and a separation method using the same, in which a buffer solution is additionally injected in addition to a sample, and a configuration using the flow of the buffer solution is disclosed, Korean Patent Publication No. 10-2017-0048188 discloses a configuration for separating nanovesicles irrelevant to antibody specificity in a short time without an ultracentrifuge using a centrifugal force-based nanoparticle separation device based on low centrifugal force and size, and Korea Publication No. 10-2016-0017374 discloses a method for isolating microvesicles using a microfluidic device using two types of buffers with different densities and a density gradient, but conventional methods do not provide a sufficiently satisfactory EVs recovery yield. There were many limitations to commercialization due to the disadvantage that it could not be seen and that EVs could not be selectively extracted.

An object to be solved by the present invention is to provide a microfluidic device capable of preparing aqueous phase-based polymersomes.

Another problem to be solved by the present invention is to provide a method for preparing aqueous biphasic polymersomes using the microfluidic device and separating biomolecules using the same.

In order to solve the above problems, the present invention is a square columnar capillary;

a cylindrical injection capillary tube disposed in the inner space of the square columnar capillary tube and having a tapered structure at one end;

a cylindrical collection capillary disposed in the inner space of the square columnar capillary and having a tapered structure at one end; and

Including; the innermost injection capillary disposed in the inner space of the injection capillary,

Provided is a microfluidic device in which a distal end having a tapered structure of an inlet capillary and a distal end having a tapered structure of a collection capillary are disposed facing each other at a spaced distance from each other.

In the present invention, the inner and outer walls of the collection capillary and the outer wall of the injection capillary may be surface-modified to be hydrophilic.

In the present invention, the inner wall of the injection capillary may be surface-modified to be hydrophobic.

In the present invention, the hydrophilic surface modification may be 2-[methoxy(polyethyleneoxy)propyl]trimethoxysilane treatment.

In the present invention, the hydrophobic surface modification may be octadecyltrimethoxysilane treatment.

In the present invention, the separation distance between the distal end having a tapered structure of the injection capillary and the distal end having a tapered structure of the collection capillary may be 30 μm to 500 μm.

In the present invention, the inner diameter (e) of the tapered end of the collection capillary may be wider than the diameter (d) of the tapered end of the inlet capillary.

In the present invention, the end of the innermost capillary may be located within the tapered region of the injection capillary.

In the present invention, the inner diameter (a) of the innermost injection capillary may be 10 μm to 60 μm, and the outer diameter (a′) may be 15 μm to 100 μm.

In the present invention, the inner diameter (b) of the injection capillary may be 50 μm to 250 μm, and the outer diameter (b′) may be 60 μm to 280 μm.

In the present invention, the inner diameter (g) of the collection capillary may be 100 μm to 380 μm, and the outer diameter (g′) may be 110 μm to 500 μm.

In the present invention, the inner diameter (c) of the square columnar capillary may be 200 μm to 1,500 μm.

In the present invention, the first fluid is injected into the innermost injection capillary,

The second fluid is injected into the inside of the injection capillary and into the outer space of the innermost injection capillary,

A third fluid is injected into the interior of the square columnar capillary and into the external space of the injection capillary,

A fourth fluid is injected into the interior of the square columnar capillary and into the external space of the collection capillary,

The injected first fluid, second fluid, third fluid, and fourth fluid are introduced into the collection capillary and discharged to the outside, and the first fluid discharged from the innermost capillary in the tapered region of the collection capillary is a droplet A core is formed, the second fluid forms a first shell with the outer shell of the droplet core, and the third fluid forms a second shell with the outer shell of the first shell, so that triple droplets having a core/shell/shell structure are formed. The triple droplet may be transferred to the outside through the inner passage of the collecting capillary along with the fourth fluid.

In addition, the present invention is a method for preparing an aqueous biphasic-based polymersome using the microfluidic device,

preparing a first fluid, a second fluid, and a fourth fluid, which are aqueous two-phase polymer solutions, and a third fluid, which is a hydrophobic polymer solution containing a polymer-soluble organic solvent;

A first fluid is injected into the innermost injection capillary, and a second fluid is injected into a space inside the injection capillary and external to the innermost injection capillary, and into a space inside the square columnar capillary and external to the injection capillary. injecting a third fluid, and injecting a fourth fluid into a space inside the square columnar capillary and external to the collecting capillary;

forming, within the tapered region of the collecting capillary, a droplet core from a first fluid discharged from the innermost capillary and forming a first shell surrounding the droplet with a second fluid;

Forming a triple droplet of a core-shell/shell structure to form a second shell with the outer shell of the double droplet formed with a third fluid;

transferring the formed triple droplet to the outside through a collection capillary together with a fourth fluid; and

Forming a stabilized polymer assembly film by self-assembly of polyethylene glycol and polybutadiene-polyethylene glycol block copolymer at the boundary between the first shell and the second shell of the triple droplet while transferring the triple droplet through the collecting capillary; It provides a method for preparing an aqueous biphasic-based polymersome comprising

In the present invention, the step of peeling off the second shell while the polymersome is transported through the collection capillary may be further included.

In the present invention, it may further include the step of rupturing the polymer assembled membrane using osmotic pressure and obtaining droplets composed of a single component of the first fluid.

In the present invention, the first fluid may be a dextran aqueous solution, the second fluid may be a polyethylene glycol aqueous solution, the third fluid may be a polybutadiene-polyethylene glycol block copolymer solution, and the fourth fluid may be a polyvinyl alcohol aqueous solution.

In the present invention, biomolecules may be injected together with the first fluid.

In the present invention, biomolecules may be separated at the level of single particles.

In the present invention, the biomolecule may be selected from the group consisting of proteins, nucleic acids, peptides, amino acids, carbohydrates, lipids, enzymes, antibodies, and antibody fragments.

In addition, the present invention is a core composed of a dextran aqueous solution; an aqueous first shell containing an aqueous polyethylene glycol solution surrounding the core; and a hydrophobic second shell comprising a polybutadiene-polyethylene glycol block copolymer surrounding the first core.

According to the present invention, the interface between the first shell and the second shell may include a polymer assembly membrane formed by self-assembly of polyethylene glycol and polybutadiene-polyethylene glycol block copolymer, and the polymer assembly membrane formed may be destroyed by a change in osmotic pressure. there is.

According to the present invention, the core may further include biomolecules.

The present invention provides a new method for efficiently separating biomolecules by effectively using a microfluidic device and a high surface-to-volume ratio provided by a droplet-based microfluidic technology in an aqueous biphasic-based polymersome prepared using the same. In the present invention, a polymersome that is monodisperse and stably supports biomolecules was easily prepared using a triple droplet system using the designed microfluidic sheath. The polymersome prepared in this way is an aqueous biphasic system (ATPS) that has high biocompatibility and low interfacial tension and can stably store molecules without damaging biomolecules. can be easily separated. In addition, a method of extracting a single particle including a biomolecule by rupturing the membrane of the biomolecule supported on the polymersome using a difference in osmotic pressure is further provided. The present invention is advantageous for industrial application because it provides a technology for separating and integrating biomolecules at a single particle level with a simple method and high yield.

1 is a cross-sectional view of a microfluidic device according to the present invention.
2 is a cross-sectional view of the microfluidic device of the present invention, where 20 is an injection capillary, 30 is a collection capillary, 501 is a hydrophobic surface modification film, and 502 is a hydrophilic surface modification film.
3 is a cross-sectional view of the microfluidic device of the present invention, where a is the inner diameter of the innermost injection glass tube, b is the inner diameter of the injection capillary, c is the inner diameter of the square column glass tube, and d is the inside of the tapered end of the injection capillary The diameter, e is the inner diameter of the tapered end of the collecting capillary, f is the separation distance between the tapered end of the inlet capillary and the tapered end of the collecting capillary, and g is the inner diameter of the collecting capillary.
4 is a cross-sectional view of the microfluidic device of the present invention, where a' indicates the outer diameter of the innermost injection glass tube, b' indicates the outer diameter of the injection capillary, and g' indicates the outer diameter of the collection capillary.
5 schematically shows the flow of first to fourth fluids and the formation of triple droplets in the microfluidic device of the present invention. The first fluid is sky blue, the second fluid is light green, the third solution is yellow, and the fourth solution is light blue.
6 is an optical microscope image of the flow of the first to fourth fluids and the formation of triple droplets in the microfluidic device of the present invention (scale bar: 100 μm). In the spaced apart space between the injection capillary and the collection capillary, the interface between the third fluid and the fourth fluid is observed, and the core droplet (first fluid and biomolecule), the first shell (second fluid) and the second fluid enter the collection capillary. A triple droplet flow in which a shell (third fluid) was formed was observed.
Figure 7 shows that each phase is spread by the difference in interfacial tension while the triple droplet is transported through the collecting capillary, and at the boundary between the first shell and the second shell, the polymer of the first shell and the block cavity of the second shell This is an image taken with an optical microscope of the self-assembly of the coalescence to form a stabilized polymer assembly film and the removal of the second shell (scale bar: 100 μm).
8 is an image taken with an optical microscope of a plurality of polymersomes separated at the level of a single particle using the microfluidic device and the aqueous biphasic-based polymersome of the present invention (scale bar: 100 μm).
9 is a schematic diagram showing the chemical structure of the aqueous biphasic-based polymersome. The core droplet (first fluid) is shown in blue, the first shell (second fluid) is shown in green, and the polymer membrane having a hydrophilic-hydrophobic-hydrophilic structure is It is marked as sky blue-red-sky blue.
10 is a fluorescence image of an aqueous biphasic-based polymersome according to the present invention, taken with an optical microscope (scale bar: 1 μm).
FIG. 11 is a schematic diagram of the rupture of the polymer assembly membrane of aqueous biphasic polymersomes using osmotic pressure and an image taken with an optical microscope (scale bar: 100 μm).
12 shows polystyrene beads surface-modified with amino fluorescent crystal (FITC) and aminodextrin (DEX) and dissolution properties.
FIG. 13 shows the results of fluorescence imaging of the behavior of aqueous biphasic polymersomes according to changes in osmotic pressure (scale bar: 100 μm).

The present invention relates to a microfluidic device for preparing aqueous biphasic-based polymersomes, a method for preparing polymersomes using the microfluidic device, and a technology for separating and integrating biomolecules at a single particle level using the microfluidic device.

Hereinafter, the present invention will be described in detail.

The present invention provides a microfluidic device for preparing aqueous biphasic-based polymersomes. A microfluidic device according to the present invention is shown in FIGS. 1 to 4 .

The microfluidic device 100 according to the present invention includes a square columnar capillary 10; a cylindrical injection capillary 20 disposed in the inner space of the square columnar capillary and having a tapered structure at one end; a cylindrical collection capillary 30 disposed in the inner space of the square columnar capillary and having one end tapered; and an innermost injection capillary 40 disposed in the inner space of the injection capillary, wherein the distal end having a tapered structure of the injection capillary and the distal end having a tapered structure of the collection capillary are spaced apart from each other at a distance f. placed face to face

According to the present invention, each capillary constituting the microfluidic device may be made of glass.

According to the present invention, the microfluidic device is designed to be driven as follows. First, a first fluid can be injected into the innermost injection capillary, and a second fluid can be injected into the interior of the injection capillary and the outer space of the innermost injection capillary. , The third fluid may be injected into the outer space of the injection capillary, and the fourth fluid may be injected into the outer space of the collection capillary and inside the square columnar capillary. The injected first fluid, second fluid, third fluid, and fourth fluid are merged in the spaced apart space between the tapered distal end of the inlet capillary and the tapered distal end of the collection capillary, and into the collection capillary. A triple droplet structure as it is transported, for example, a droplet core composed of an innermost first fluid, a core of a second fluid (first shell) surrounding the first fluid, and a third fluid (second shell) surrounding the second fluid/ To complete the shell/shell shape, the distal end having a tapered structure of the injection capillary and the distal end having a tapered structure of the collecting capillary are disposed facing each other at a distance f.

The separation distance (f) may be 30 μm to 500 μm, preferably 35 μm to 200 μm. If the separation distance is less than the above range, it is not easy to form triple droplets, and if it exceeds the above range, it is not easy to form and transport triple droplets.

In one embodiment of the present invention, the diameter (e) of the tapered end of the inlet capillary or the area of the inlet is larger than the diameter (d) of the tapered end of the inlet capillary or the area of the outlet. If the diameter of the tapered end of the injection capillary or the area of the discharge port is larger than the diameter of the tapered end of the collection capillary or the area of the inlet, it is not easy to form a triple droplet structure, and it is difficult for the generated triple droplets to flow into the collection capillary. .

In one embodiment of the present invention, the outer wall of the injection capillary 20 is provided with a hydrophilic surface modification layer 501 to facilitate fluid movement and droplet formation, and the inner wall of the injection capillary 20 is provided with a hydrophobic surface modification layer 502 ) and; The outer and inner walls of the collection capillary 30 each have a hydrophilic surface modification layer 501. Hydrophilic surface modification may be performed using a surface modifier known to be capable of imparting hydrophilicity to the glass surface, preferably hydrophilic surface modification with 2- [methoxy (polyethyleneoxy) propyl] trimethoxysilane, , Hydrophobic surface modification may be performed using a surface modifier known to be able to impart hydrophobicity to the glass surface, and preferably may be hydrophobic surface modification with octadecyltrimethoxysilane.

In one embodiment of the present invention, the end of the innermost infusion capillary is preferably located within the tapered region of the infusion capillary. This structure gives a fluid flow in the form of a double layer in which the first fluid is surrounded by the second fluid before the first fluid and the second fluid are discharged from the injection capillary, and when discharged from the injection capillary, immediately by the third fluid It enables the formation of triple droplets with a core/shell/shell structure.

In one embodiment of the present invention, the cross-sectional area of the innermost injection capillary, the injection capillary, and the square columnar capillary may be appropriately sized according to the size of the particle to be separated, for example, the size of the biomolecule.

The microfluidic device according to the present invention can be used to separate any one or more selected from the group consisting of biomolecules, such as proteins, nucleic acids, peptides, amino acids, carbohydrates, lipids, enzymes, antibodies, and antibody fragments. It is also possible to fabricate a microfluidic device in a suitable scale according to the size and quantity of biomolecules. For example, the inner diameter (a), outer diameter (a') and length of the innermost inlet capillary, the inner diameter (b), outer diameter (b') and length of the inlet capillary, the inner diameter (c) and It is possible to adjust the length, and the inner diameter (g), outer diameter (g') and length of the collection capillary.

In one embodiment of the present invention, a' < b, b' < c, and g' < c. Illustratively, the inner diameter (a) of the innermost injection capillary of the microfluidic device according to the present invention may be 10 μm to 60 μm, the outer diameter (a′) may be 15 μm to 100 μm, and the inside of the injection capillary The diameter (b) is between 50 μm and 250 μm, the outer diameter (b′) can be between 60 μm and 280 μm, the inner diameter (g) of the collection capillary is between 100 μm and 380 μm, and the outer diameter (g′) may be 110 μm to 500 μm, and the inner diameter (c) of the square columnar capillary may be 200 μm to 1,500 μm.

In the microfluidic device according to the present invention, for example, the inner diameter (a) of the innermost injection capillary may be 20 μm to 40 μm, the outer diameter (a′) may be 45 μm to 70 μm, and the innermost injection capillary may have an inner diameter (a) of 45 μm to 70 μm. The diameter (b) is between 80 μm and 150 μm, the outer diameter (b′) can be between 100 μm and 175 μm, the inner diameter (g) of the collection capillary is between 150 μm and 280 μm, and the outer diameter (g′) may be 170 μm to 300 μm, and the inner diameter (c) of the square columnar capillary may be 1,300 μm to 1,050 μm.

The present invention also provides a method for preparing aqueous biphasic-based polymersomes using the microfluidic device.

The method includes preparing a first fluid, a second fluid, and a fourth fluid that are aqueous two-phase polymer solutions, and a third fluid that is a hydrophobic polymer solution containing a polymer-soluble organic solvent;

A first fluid is injected into the innermost injection capillary, and a second fluid is injected into a space inside the injection capillary and external to the innermost injection capillary, and into a space inside the square columnar capillary and external to the injection capillary. injecting a third fluid, and injecting a fourth fluid into a space inside the square columnar capillary and external to the collection capillary;

forming a droplet from a first fluid discharged from the innermost capillary in the tapered region of the collection capillary, and forming a first shell with the outer shell of the formed droplet core with a second fluid; a triple droplet formation step of forming a second shell with an outer shell of the double droplet formed using a third fluid; transferring the formed triple droplet to the outside through a collection capillary together with a fourth fluid; And while the triple droplet formed through the collecting capillary is transported, polymer-block copolymer self-assembly is formed at the boundary between the first shell and the second shell using the self-assembly phenomenon between polymers to obtain polymersomes having a stabilized polymer assembly film. manufacturing step; includes.

The aqueous two-phase system consists of two aqueous solutions in which equal polymer materials, polymers or salts exist at a certain concentration or higher, and at this time, it means that two layers are formed that do not mix by the electrostatic repulsive force between each material.

5 schematically shows the formation of triple droplets, red is a hydrophilic surface modification layer, dark blue is a hydrophobic surface modification layer, the first fluid is light blue, the second fluid is light green, the third solution is yellow, and the fourth The solution appeared in light blue color.

For example, the formed triple droplet spreads due to the difference in interfacial tension between the respective phases, and forms a polymersome having a stable polymer assembly film through a single membrane assembly process of block copolymers through dewetting.

In one embodiment of the present invention, while the polymersome is transported through the collection capillary, the step of peeling the second shell is further included, through which aqueous biphasic polymersomes protected by a stabilized polymer assembly film can be prepared. .

7 shows an image taken with an optical microscope of the above process.

In one embodiment of the present invention, biomolecules may be injected together with the first fluid. In this case, biomolecules are included in droplets formed by the first fluid.

The biomolecule may be selected from the group consisting of proteins, nucleic acids, peptides, amino acids, carbohydrates, lipids, enzymes, antibodies, and antibody fragments.

In addition, in another embodiment of the present invention, the aqueous biphasic polymersome protected by the stabilized polymer assembly membrane may rupture the stabilized polymer assembly membrane using osmotic pressure. The use of osmotic pressure is well illustrated in FIG. 11 . (i) In an isotonic environment, the form of aqueous biphasic polymersomes protected by a polymer assembly membrane is maintained. (ii) In a hypotonic environment, moisture flows into the polymer membrane, causing the polymersome to swell and the aqueous biphasic system to disappear. On the other hand, (iii) in a hypertonic environment, moisture escapes into the polymer membrane, the polymersome shrinks, and finally only the droplet formed by the first fluid (core droplet) remains inside, collecting the core droplet. By doing so, it is possible to separate and integrate components, such as biomolecules, supported in the droplet core at the level of a single particle. Membrane rupture of the polymer assembly membrane of the aqueous biphasic polymersome using the above-described osmotic pressure is shown in FIG. 11 .

In one embodiment of the present invention, the first fluid is an aqueous solution of dextran, preferably a 10 to 30% by weight aqueous solution of dextran having a molecular weight of 350,000 to 650,000, more preferably a dextran 15 to 15 having a molecular weight of 450,000 to 550,000 It may be a weight % aqueous solution, most preferably, a 20% aqueous solution of dextran having a molecular weight of 450,000 to 550,000.

In one embodiment of the present invention, the second fluid is a polyethylene glycol aqueous solution, preferably a 5 to 30% by weight aqueous solution of polyethylene glycol having a molecular weight of 3,000 to 15,000, more preferably a polyethylene glycol 7 to 25 having a molecular weight of 5,000 to 12,000 It may be a weight % aqueous solution, more preferably a 7 to 25 weight % aqueous solution of polyethylene glycol having a molecular weight of 7,000 to 9,000, and most preferably a 10 to 20% aqueous solution of polyethylene glycol having a molecular weight of 8,000.

In one embodiment of the present invention, the third fluid is a hydrophobic polymer solution containing a polymer-soluble organic solvent. The hydrophobic polymer may be a polybutadiene-polyethylene glycol block copolymer, preferably a block copolymer of polybutadiene having a molecular weight of 4,200 to 6,200 and polyethylene glycol having a molecular weight of 3,500 to 5,500, more preferably having a molecular weight of It may be a block copolymer (PBD _5.2k -PEO _4.5k ) of polybutadiene having a molecular weight of 5,000 and polyethylene glycol having a molecular weight of 4,500; The polymer-soluble organic solvent may be at least one selected from the group consisting of chloroform, cyclohexane, pentane, toluene, dichloromethane, carbon tetrachloride, benzene, and xylene, preferably a mixed solution of chloroform and cyclohexane, More preferably, chloroform:cyclohexane may be 26-46:54-74 vol%, and most preferably chloroform:cyclohexane 36:64 vol%; The content of the hydrophobic polymer in the organic solvent may be 0.01 to 0.5% by weight, preferably 0.05 to 0.3% by weight, and most preferably 0.1% by weight. When the content of the hydrophobic polymer in the third fluid is less than the above range, the content of the hydrophobic polymer is too low and the polymer-block copolymer polymer assembly formed by self-assembly of the second fluid and the third fluid at the boundary between the first shell and the second shell It is difficult to form a membrane, and if the above range is exceeded, the membrane rupture using osmotic pressure may not be smoothly performed because the polymer density of the third shell is high.

According to the present invention, the fourth fluid may be a polyvinyl alcohol aqueous solution, preferably a 5 to 15% by weight aqueous solution of polyvinyl alcohol having a molecular weight of 9,000 to 27,000, more preferably a polyvinyl alcohol 7 to 11,000 to 25,000 molecular weight It may be a 13% by weight aqueous solution, more preferably a 9 to 10% by weight aqueous solution of polyvinyl alcohol having a molecular weight of 13,000 to 23,000, and most preferably a 9.5% by weight aqueous solution of polyvinyl alcohol having a molecular weight of 13,000 to 23,000.

The concentration of the above-mentioned fluid is not limited, but it was confirmed through research that it is a concentration capable of stably supporting biomolecules.

The aqueous biphasic polymersome according to the present invention prepared by the above method includes a core composed of an aqueous solution of dextran; an aqueous first shell containing an aqueous polyethylene glycol solution surrounding the core; and a polybutadiene-polyethylene glycol block copolymer surrounding the first core, and a hydrophobic second shell including a polyethylene glycol block copolymer.

The aqueous biphasic polymersome according to the present invention may include a polymer assembly membrane formed by self-assembly of polyethylene glycol and polybutadiene-polyethylene glycol block copolymer at the interface between the first shell and the second shell, and the aqueous biphasic polymersome by using osmotic pressure change. The polymersome may be contracted, thereby forming a first fluid-rich state, for example, a condensed first fluid state, and furthermore, a single-component first fluid may be provided by membrane rupture of the polymer assembled membrane.

Specifically, (i) in an isotonic environment, the form of aqueous biphasic polymersomes protected by a polymer assembly membrane is maintained. (ii) In a hypotonic environment, moisture flows into the polymer membrane, causing the polymersome to swell and the aqueous biphasic system to disappear. On the other hand, (iii) in a hypertonic environment, moisture escapes into the polymer membrane, the polymersome shrinks, and finally only the droplet formed by the first fluid (core droplet) remains inside, collecting the core droplet. By doing so, it is possible to separate and integrate components, such as biomolecules, supported in the core liquid droplet at the level of a single particle.

Hereinafter, preferred embodiments are presented to aid understanding of the present invention, but the following examples are merely illustrative of the present invention, and various changes and modifications are possible within the scope and spirit of the present invention. It is obvious to those skilled in the art, It goes without saying that these variations and modifications fall within the scope of the appended claims.

reagent

Dextran (DEX, molecular weight 450,000-550,000) and polyethylene glycol (PEG, molecular weight 8,000) were purchased from SigmaAldrich, USA. A block copolymer of polybutadiene (molecular weight 5,200)-polyethylene glycol (molecular weight 4,500) (PBD _{5.2k -PEO 4.5k} ) was purchased from _{PolymerSource} , Canada. Chloroform (>99.5%) and cyclohexane (>99.5%) were purchased from SigmaAldrich, USA. Polyvinyl alcohol (PVA, molecular weight 13,000-23,000) was purchased from SigmaAldrich, USA. Polystyrene (PS) beads were purchased from Thermo Fisher Scientific, USA, amino dextran (molecular weight 70,000) was purchased from Thermo Fisher Scientific, USA, and amino fluorescent nanocrystals were purchased from Cytodiagnostics, Canada. purchased from

Preparation Example 1.

As the first fluid, a 20% by weight aqueous solution of dextran was prepared.

Preparation Example 2.

As the second fluid, an aqueous solution of 10% by weight of polyethylene glycol was prepared.

Preparation Example 3.

As a third fluid, it was prepared by dissolving 0.1% by weight of PBD _{5.2k -PEO 4.5k} block copolymer in a solution _obtained by mixing chloroform:cyclohexane at a ratio of 36:64 volume%.

Preparation Example 4.

As a third fluid, an aqueous polymer solution in which 9.5% by weight of polyvinyl alcohol was dissolved was prepared.

Preparation Example 5. Synthesis of Model Particles for Separation of Single Particles

Preparation of Buffer Solution A: Buffer Solution A was prepared by dissolving 0.3 g of boric acid in 50 mL of distilled water and adjusting the pH to 9.5 with 5M NaOH.

Preparation of Buffer Solution C: Buffer Solution C was prepared by adding 2 g of ammonium sulfate to 50 mL of Buffer Solution A.

Synthesis of model particles: After centrifuging the suspension containing polystyrene beads at 1,000 rpm for 10 minutes, only the bottom particles were extracted, washed three times with 1 mL buffer solution A, and transferred to a 1 mL centrifugal tube. Next, 450 μl of buffer solution A and 300 μl of buffer solution C were stirred, 1.5 mg of aminodextran and 1.5 mg of aminofluorescent crystal were dissolved, and the prepared solution was put into a centrifugal separation tube prepared above, and maintained at 37 ° C. and stirred for 18 hours. Amino fluorescent crystals were used to obtain fluorescence images of the prepared particles. After completion of the reaction, the particles whose bottom surface was modified were extracted by centrifugation at 1,000 ppm for 10 minutes, transferred to distilled water, and stored at 4°C.

Fabrication Example 1. Fabrication of glass capillary microfluidic device

The cylindrical capillary having one end tapered structure was produced by cutting the cylindrical capillary to an appropriate length and then stretching it to have a tapered end using a pipette puller or burner. (Outside diameter of tapered end 137 μm) The other was used as a collection capillary (outside diameter of tapered end 300 μm). The outer wall of the injection capillary and the inner and outer walls of the collection capillary were treated with 2-[methoxy(polyethyleneoxy)propyl]trimethoxysilane to form a hydrophilic surface treatment layer, and the inner wall of the injection capillary was treated with octadecyltrimethoxysilane. to form a hydrophobic surface treatment layer. The prepared injection capillary and collection capillary were placed in a square prism capillary (inner diameter of 1.05 mm), and the innermost injection capillary was embedded in the injection capillary and fixed with epoxy. Fluid flow rate was controlled using a syringe pump (KDS Legato™ 100), and droplet generation was recorded using a microscope (Eclipse Ts2, Nikon) equipped with a high-speed camera (FASTCAM Mini UX50, Photron).

Example 1.

The first fluid of Preparation Example 1 was injected into the innermost injection capillary, the second fluid of Preparation Example 2 was injected into the gap between the innermost injection capillary and the injection capillary, and Preparation Example 3 was injected into the gap between the injection capillary and the square columnar capillary. The third fluid of was injected, and the fourth fluid of Preparation Example 4 was injected into the gap between the collection capillary and the square columnar capillary.

Since the third fluid and the fourth fluid have high interfacial tension, the double droplet composed of the first fluid/second fluid (referred to as ATPS) is encapsulated by the third fluid to form triple droplets with a core/shell/shell structure. makes it possible The phenomenon that occurs as the generated triplex droplet moves to the collecting capillary was photographed with an optical microscope equipped with a high-speed camera.

6 is an image taken with an optical microscope of the formation of triple droplets using the microfluidic device according to the present invention. In the spaced apart space between the injection capillary and the collection capillary, the interface between the third fluid and the fourth fluid is observed, and the core droplet (first fluid and biomolecule), the first shell (second fluid) and the second fluid enter the collection capillary. A triple droplet flow in which a shell (third fluid) was formed was observed.

7 shows that each phase is spread by the difference in interfacial tension while the triple droplet is transported through the collecting capillary, and at the boundary between the first shell and the second shell, self-assembly between the second fluid and the third fluid This is an image taken with an optical microscope of a process in which a stabilized polymer assembly film is formed and then the second shell is removed (scale bar: 100 μm).

Figure 8 is an image taken with an optical microscope of a plurality of polymersomes separated at the level of a single particle using a microfluidic device and aqueous biphasic polymersomes according to the present invention, and Figure 9 is a schematic diagram of the chemical structure of aqueous biphasic polymersomes. 10 is a fluorescence image of an aqueous biphasic-based polymersome according to the present invention, taken with an optical confocal microscope. It can be confirmed that the aqueous biphasic polymersome was successfully synthesized at the single particle level.

Example 2. Observation of aqueous biphasic polymersome behavior according to osmotic pressure change 1

Using osmotic pressure, the polymer assembly membrane of aqueous biphasic polymersomes was ruptured, and through this, it was confirmed that separation and integration at the single particle level were possible.

FIG. 11 is a schematic diagram of the rupture of the polymer assembly membrane of aqueous biphasic polymersomes using osmotic pressure and an image taken with an optical microscope (scale bar: 100 μm). (i) In an isotonic environment, the morphology of the aqueous biphasic polymersomes protected by the polymer assembly membrane was maintained, and the polymer assembly membrane of the polymersome was maintained, as can be seen in the optical microscope image on the right. (ii) In a hypotonic environment, moisture flows into the polymer membrane, causing the polymersome to swell and the aqueous biphasic system to disappear. As can be seen in the right optical microscope image, it was observed that the aqueous biphasic polymersome was destroyed. (iii) In a hypertonic environment, moisture escapes into the polymer membrane, the polymersome shrinks, and finally only the droplet formed by the first fluid (core droplet) remains inside. As can be seen in the right optical microscope image, the polymer assembly membrane of the polymersome disappeared, but it was observed that the dextrin core droplet was maintained.

Example 3. Observation of aqueous biphasic polymersome behavior according to osmotic pressure change 2

In order to observe the behavior of the aqueous biphasic polymersome according to the change in osmotic pressure according to the present invention by fluorescence analysis, the model particles prepared in Preparation Example 5 were used. As shown in Figure 12, the surface of the polystyrene beads of the particles of Preparation Example 5 was surface-modified with amino fluorescent crystals (FITC) and aminodextrin (DEX), and whether or not fluorescent labeling was confirmed through an optical microscope, confirmed in the right vial tube Possibly, it was present in the dextrin layer (DEX-rich phase).

The model particles of Preparation Example 5 were injected together with one fluid, droplets were formed by the method of Example 1, and a hypotonic osmotic pressure environment was provided.

As shown in FIG. 13, (a) the shape of the aqueous phase-based polymersome was maintained under the isotonic condition, but (b) under the hypotonic condition, the polymersome contracted and the volume of the dextrin decreased.

100 Microfluidic Devices
10 Square Column Capillary
20 injection capillary
30 collection capillary
501 hydrophilic surface modification layer
502 hydrophobic surface modification layer
40 Innermost injection capillary
a Inside diameter of the innermost injection capillary
a' innermost injection capillary outer diameter
b injection capillary inner diameter
b' injection capillary outer diameter
c Square prism capillary inner diameter
d injection capillary tapered end inner diameter
e collection capillary tapered end inner diameter
f Separation distance between inlet and collection capillaries
g collection capillary inner diameter
g' collection capillary outer diameter

Claims (18)

delete delete delete delete delete delete As a method for producing an aqueous biphasic-based polymersome using a microfluidic device,
preparing a first fluid, a second fluid, and a fourth fluid, which are aqueous two-phase polymer solutions, and a third fluid, which is a hydrophobic polymer solution containing a polymer-soluble organic solvent;
A first fluid is injected into the innermost injection capillary, and a second fluid is injected into a space inside the injection capillary and external to the innermost injection capillary, and into a space inside the square columnar capillary and external to the injection capillary. injecting a third fluid, and injecting a fourth fluid into a space inside the square columnar capillary and external to the collection capillary;
forming, within the tapered region of the collecting capillary, a droplet core from a first fluid discharged from the innermost capillary and forming a first shell surrounding the droplet with a second fluid;
Forming a core/shell/shell triple droplet structure of forming a second shell with the outer shell of the double droplet formed with a third fluid;
transferring the formed triple droplet to the outside through a collection capillary together with a fourth fluid; and
Forming a stabilized polymer assembly film by self-assembly of polyethylene glycol and polybutadiene-polyethylene glycol block copolymer at the boundary between the first shell and the second shell of the triple droplet while transferring the triple droplet through the collecting capillary; A method for producing an aqueous biphasic-based polymersome comprising: According to claim 7,
The method of producing an aqueous biphasic-based polymersome, further comprising the step of peeling off the second shell while the polymersome is transferred through the collection capillary. According to claim 8,
A method for preparing aqueous biphasic-based polymersomes, further comprising the step of rupturing the polymer assembly membrane using osmotic pressure and obtaining droplets composed of a single component of the first fluid. According to claim 7,
The first fluid is an aqueous solution of dextran,
The second fluid is a polyethylene glycol aqueous solution,
The third fluid is a polybutadiene-polyethylene glycol block copolymer solution,
The fourth fluid is a polyvinyl alcohol aqueous solution, a method for producing an aqueous phase-based polymersome. According to claim 7,
A method for preparing aqueous biphasic-based polymersomes, wherein biomolecules are injected together with a first fluid. According to claim 11,
A method for preparing aqueous biphasic-based polymersomes in which biomolecules are separated at the level of single particles. According to claim 11,
wherein the biomolecule is selected from the group consisting of proteins, nucleic acids, peptides, amino acids, carbohydrates, lipids, enzymes, antibodies and antibody fragments. a core composed of an aqueous solution of dextran; an aqueous first shell containing an aqueous polyethylene glycol solution surrounding the core; and a hydrophobic second shell comprising a polybutadiene-polyethylene glycol block copolymer surrounding the first core. According to claim 14,
An aqueous biphasic polymersome having a core/shell/shell triple droplet structure, wherein the interface between the first shell and the second shell includes a polymer assembly film formed by self-assembly of polyethylene glycol and polybutadiene-polyethylene glycol block copolymer. According to claim 14,
An aqueous biphasic polymersome having a core/shell/shell triple droplet structure, wherein the core further comprises a biomolecule. According to claim 16,
The biomolecule is selected from the group consisting of proteins, nucleic acids, peptides, amino acids, carbohydrates, lipids, enzymes, antibodies and antibody fragments, core / shell / shell triple droplet structure aqueous biphasic polymersome. According to claim 15,
The polymer assembly membrane is destroyed by a change in osmotic pressure, an aqueous biphasic polymersome of a core / shell / shell triple droplet structure.

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