AU2020234406A1 - Microfluidic system for intracellular delivery of materials and method therefor - Google Patents

Abstract

Provided is a microfluidic system for delivering external substances into cells by cell perforation performed using inertia, the system comprising a fluid channel structure through which a solution containing cells and external substances continuously flows, wherein the fluid channel structure includes a junction between one or more channels, a local vortex occurs near the interface of the junction, the cells are deformed by the vortex, the vortex causes a temporary discontinuity in the cell membrane, and the external substances are introduced into the cells by solution exchange between the cells and the fluid around the cells.

Description

WO 2020/184992 PCT/KR2020/003411

DESCRIPTION MICROFLUIDIC SYSTEM FOR INTRACELLULAR DELIVERY OF SUBSTANCES AND METHOD THEREFOR TECHNICAL FIELD

[0001] The present disclosure relates to a microfluidic system for intracellular delivery of materials and a method therefor.

BACKGROUND ART

[0002] Intracellular delivery of materials is one of the most key steps in cell engineering, in which materials are traditionally delivered by using a carrier or by physically

making nanopores in a cell/nuclear membrane. For virus or lipid carrier techniques, it is possible to deliver materials with high efficiency when optimized, but there are drawbacks such as low safety, slow delivery speed, labor/cost-intensive

carrier preparation process, and low reproducibility.

[0003] On the other hand, for methods of physically making a nanopore by applying energy to a cell membrane (e.g., electroporation or microneedle), there is an advantage that

relatively various materials are able to be delivered to various cell lines. However, low cell viability, denaturation of delivery material, and low throughput, which are caused by the invasiveness of the methods, are pointed

out as major limitations.

[0004] To address the above-mentioned drawbacks, microfluidic devices capable of processing a large number of

cells are prominently used. Typically, there is a platform that creates nanopores in cell membranes through physical

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deformation of cells when the cells pass through the

bottleneck section. However, the approach has major

drawbacks such as clogging of the bottleneck section itself

during the experiment and inconsistent material delivery

efficiency.

[0005] For example, US Patent No. 2014-0287509 discloses a

technology for inducing cell deformation by applying pressure

to cells through a channel having a bottleneck structure.

However, in this case, there is a drawback that the cells

block the bottleneck structure, and there is a drawback that

it is possible to deliver materials only to cells smaller

than the size of a constriction. Furthermore, there is also

a drawback that the cost rises because a channel having a

fine diameter has to be used.

[0006] Therefore, it is urgent to develop an innovative

next-generation intracellular material delivery platform

capable of delivering various materials into cells uniformly

and with high efficiency while making use of the high

processing function of the microfluidic device. DISCLOSURE OF THE INVENTION TECHNICAL PROBLEM

[0007] Accordingly, in order to solve the above-mentioned

problems, an object of the present disclosure is to provide a

system and method capable of delivering materials to a large

number of cells with high efficiency without using a new

active delivery means. TECHNICAL SOLUTION

[0008] In order to solve the problems described above,

according to an aspect of the present disclosure, there is

provided a microfluidic system delivering external materials

into a cell by cell mechanoporation using inertia and

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inertial effects, the microfluidic system including a fluidic

channel structure through which a solution containing a cell

and external materials flows continuously, in which the

fluidic channel structure includes a junction between one or

more channels, a localized vortex is generated near an

interface of the junction, the cell is deformed by the vortex,

and transient discontinuities are generated in a cell

membrane by fluidic cell deformation and the external

materials are introduced into the cell via solution exchange

between the cell and fluid around the cell.

[0009] In an embodiment of the present disclosure, the

fluidic channel structure including the junction between one

or more channels may include a junction including a T, Y,

cross shape, or a combination thereof.

[0010] In an embodiment of the present disclosure, the

fluidic channel structure may include a cavity near a fluid

stagnation point when the fluidic channel structure is a

channel of the T or Y shape.

[0011] In an embodiment of the present disclosure, the

cavity may have a shape of a circle, an ellipse, an elongate

slit, a square, a rectangle, a trapezoid, a polygon, and a

combination thereof, and a modification thereof.

[0012] In an embodiment of the present disclosure, a

diameter of the cavity may be determined according to a

diameter of the cell.

[0013] In an embodiment of the present disclosure, the

microfluidic system may further include fluid control unit

for allowing a solution to flow in the fluidic channel

structure, and the fluid control unit may allow the solution

to flow in the fluidic channel at a velocity that is at a

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level capable of generating a localized vortex near the

interface of the junction.

[0014] In an embodiment of the present disclosure, the fluid

control unit may be a syringe pump or pneumatic system.

[0015] In an embodiment of the present disclosure, a

Reynolds number (Re) of the solution may be 1 to 1000.

[0016] In an embodiment of the present disclosure, the

vortex feature may be determined by the Reynolds number.

[0017] In an embodiment of the present disclosure, the

vortex may be in a form of a closed or open recirculating

flow.

[0018] In an embodiment of the present disclosure, the

microfluidic system may be formed by combining the

microfluidic system according to any one of claims 1 to 12 in

series, parallel, or a combination thereof.

[0019] According to another aspect of the present disclosure,

there is provided a method of delivering external materials

into a cell by cell mechanoporation using inertia and

inertial effects, the method including: allowing a solution

containing the cell and external materials to continuously

flow a fluidic channel; forming a vortex by vortex generating

means near the junction; deforming the cell by the vortex;

and allowing the external materials to be introduced into the

cell through a pore created in a cell membrane by the

deforming of the cell.

[0020] in an embodiment of the present disclosure, the

vortex generating means may be a junction structure of the

fluidic channels.

[0021] In an embodiment of the present disclosure, the

fluidic channel may include a junction including a T, Y,

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cross shape, or a combination thereof. ADVANTAGEOUS EFFECTS

[0022] According to the present disclosure, a vortex is

generated by allowing a cell and external materials to flow

into a fluidic channel structure including at least one

junction, and the resulting inertia and inertial effects

deform the cell to induce transient discontinuity in a cell

membrane, thereby perforating the cell membrane. Then, a

solution exchanges between the cell and fluid around the cell

occurs through the perforated cell membrane, and as a result,

the external materials are introduced into the cell. Thus,

the present disclosure does not require vectors or active

cell delivery means (e.g., electric fields). Therefore, the

present disclosure may directly deliver external materials

(e.g., genes, plasmids, nanoparticles, or the like) into

cells with high efficiency and low cost only by solution and

channel structure features.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 is a diagram for illustrating a formation of a

spiral vortex in a + junction channel.

[0024] FIG. 2 is a schematic diagram of cell deformation

caused by a spiral vortex (1) and shows high-speed microscopy

images showing rotational cell motion (scale bar: 10 pm)(2).

[0025] FIG. 3 is a diagram for describing a mechanism of

material delivery with cell deformation according to an

embodiment of the present disclosure.

[0026] FIG. 4 is a schematic diagram of two types of channel

structures according to an embodiment of the present

disclosure and a diagram for describing cell deformation in

each channel.

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[0027] FIG. 5 is a schematic diagram of a microfluidic

system in which two types of fluidic channels of FIG. 4 are

connected in series in a fluid flow direction.

[0028] FIG. 6 shows a result of an experiment on behavior

according to a Reynolds number of fluid flowing in opposite

directions toward a junction (intersection) in a + junction

channel.

[0029] FIG. 7 shows high-speed microscopy images showing a

cell (MDA-MB-231) trapping behavior in the + junction

channel.

[0030] FIG. 8 shows a graph of normalizing cell trapping and

cell deformation time by vortex breakdown of FIG. 7 as a

function of the Reynolds number.

[0031] FIG. 9 is a diagram for illustrating an intracellular

material delivery platform according to an embodiment of the

present disclosure.

[0032] FIGS. 10 to 12 are diagrams for illustrating a method

for intracellular delivery of materials using the

intracellular material delivery platform according to an

embodiment of the present disclosure.

[0033] FIG. 13 is a schematic diagram of a T-junction

channel according to an embodiment of the present disclosure.

[0034] FIG. 14 shows high-speed microscopy images

illustrating a cell deformation and vortex deformation

mechanism of a T-junction channel of a cavity structure.

Here, each arrow indicates a fluid flow direction.

[0035] FIG. 15 shows a vortex deformability index (VDI)

analysis result in a fluid having different Reynolds numbers.

[0036] FIG. 16 is a diagram for illustrating an

intracellular material delivery platform according to an

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embodiment of the present disclosure.

[0037] FIGS. 17 to 19 are diagrams for illustrating a method

for intracellular delivery of materials using the

intracellular material delivery platform according to an

embodiment of the present disclosure.

[0038] FIGS. 20 and 21 show analysis results of the amount

of materials delivered into cells.

[0039] FIG. 22 shows a result of measuring cell viability by

varying the Reynolds number under the same conditions as in

FIG. 19.

[0040] FIG. 23 shows an analysis result of delivery

efficiency for various dextran sizes.

[0041] FIG. 24 shows a result of measuring dextran delivery

efficiency in the complex microfluidic system of FIG. 5 (a

combination of the + junction channel and the T-junction

channel).

[0042] FIG. 25 shows a comparison result of the delivery

efficiency of electroporation in the related art (a neon

transfection system (Thermo Fisher Scientific, Waltham, MA,

USA)) and the method for delivery of materials based on

inertia (hydroporation) according to the present disclosure.

[0043] FIGS. 26 and 27 are photographs for analyzing the

intracellular delivery effect of gold nanoparticles (GNPs),

and a result of counting scattering spots, respectively.

[0044] FIG. 28 shows a result of measuring cell viability

depending on GNP delivery.

[0045] FIG. 29 shows a result of calculating a yield

normalized by multiplying the number of scattering spots by

the cell viability.

[0046] FIG. 30 shows an analysis result of intracellular

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delivery efficiency of mesooporous silica nanoparticles (MSN)

used as a drug, protein, and nucleic acid nanocarrier instead

of gold nanoparticles.

[0047] FIG. 31 shows an analysis result of a time-dependent

cell viability for the analyte of FIG. 30.

[0048] FIG. 32 is a histogram of fluorescence intensity

obtained by measuring a delivery effect to K562 cells using

mRNA (996-nucleotide mRNA fragment, green fluorescent

protein) as an external material.

[0049] FIG. 33 shows fluorescence images after delivery of

mRNA into K562 by (C) endocytosis and (D) the microfluidic

system according to the present disclosure.

[0050] FIG. 34 shows a result of measuring delivery

efficiency (E) and viability (F) when mRNA is delivered

according to the present disclosure as shown in FIGS. 32 and

33.

[0051] FIG. 35 shows fluorescence images after mRNA (996

nucleotide mRNA fragment, green fluorescent protein) is

delivered into K562 cells by endocytosis and a method of

Embodiment 2.

[0052] FIG. 36 shows an analysis result of (b) mRNA

transfection efficiency and (c) mean fluorescence intensity

depending on mRNA concentration.

[0053] FIG. 37 shows fluorescence images after delivery of

plasmid DNA into HEK293t by the endocytosis (control) and the

method of Embodiment 2.

[0054] FIG. 38 shows a graph of (e) analyzing plasmid DNA

(pDNA) transfection efficiency of HEK293t cells and (f) mean

intensity of HEK293t cells depending on plasmid DNA

concentration.

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[0055] FIGS. 39 and 40 are a result of western blot analysis

of the ITGal gene knockdown effect of HeLa cells (al subunit

of an integrin transmembrane receptor) into which siRNA is

delivered, and a result of comparing relative expression

levels, respectively.

[0056] FIG. 41 shows an analysis result of transfection

yields by Lipofectamine 3000, electroporation, and the

present disclosure.

[0057] FIGS. 42 to 44 are analysis results of transfection

efficiencies, cell viability, and transfection yields for

human MSC, human ADSC, and mouse BMDC.

[0058] FIG. 45 shows confocal microscopy images showing

delivery of quantum dots (qdot625) into MDA-MB-231 cells by

the present disclosure (p-Hydroporator), electroporator, and

Lipofectamine 3000.

[0059] FIG. 46 shows an analysis result of spot number

counts of quantum dots (Qdot 625) per cell.

[0060] FIG. 47 is a fluorescence intensity histogram of

Embodiment (p-Hydroporator, Ncelu = 5,000 per sample) in which

nanospheres (green fluorescent silica nanospheres, Micromod,

Germany) are delivered into K562 cells, by a negative control

(nontreated K562 cells), a positive control (K562 cells co

incubated with nanospheres, the same time and concentration

as those in Embodiment), and the present disclosure (p

Hydroporator).

[0061] FIG. 48 shows a result of measuring relative mean

fluorescence intensity.

[0062] FIGS. 49 and 50 show confocal images of two of the

groups of FIGS. 47 and 48.

BEST MODE FOR CARRYING OUT THE INVENTION

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[0063] Hereinafter, preferred embodiments of a system for intracellular delivery of materials based on inertia according to the present disclosure will be described in detail with reference to the accompanying drawings. For reference, it should be understood that the terms used in the

specification and the appended claims should not be construed as limited to general and dictionary meanings, but should be interpreted based on the meanings and concepts corresponding to technical aspects of the present disclosure on the basis

of the principle that the inventor is allowed to define terms appropriately for the best explanation. In addition, the embodiment disclosed in the present disclosure and configurations shown in the accompanying drawings are just

one preferred embodiment of the present disclosure and do not represent all technical ideas of the present disclosure. Therefore, it should be understood that the present disclosure covers various modifications and variations

provided they come within the scope of the appended claims and their equivalents at the time of filing of this application.

[0064] In order to solve the problems described above, the present disclosure provides a microfluidic system delivering external materials into a cell by cell mechanoporation using inertia and inertial effects, the microfluidic system including a fluidic channel structure through which a

solution containing a cell and external materials flows continuously, in which the fluidic channel structure includes a junction between one or more channels, a localized vortex

is generated near an interface of the junction, the cell is deformed by the vortex, and a transient discontinuous shape

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of a cell membrane is generated by the vortex and the external materials are introduced into the cell by solution exchange between the cell and fluid around the cell.

[0065] In addition, the present disclosure provides a method of delivering external materials into a cell by cell

mechanoporation using inertia and inertial effects, the method including: allowing a solution containing the cell and external materials to continuously flow a fluidic channel; forming a vortex by a vortex generating means near the

junction; deforming the cell by the vortex; and allowing the external materials to be introduced into the cell through a pore created in a cell membrane by the deforming of the cell. MODE FOR CARRYING OUT THE INVENTION

[0066] A method and system for intracellular delivery of materials according to an embodiment of the present disclosure are based on a technical feature of generating a vortex in a fluidic channel and deforming cells by the vortex

to create a pore in cell membranes. In particular, the present disclosure improves the intracellular material delivery efficiency by cell deformation caused by the vortex and cell deformation by subsequent vortex breakdown, when

cells flow into the vortex. In an embodiment of the present disclosure, the vortex is generated through a physical structure of a fluidic channel, such as a junction, but the scope of the present disclosure is not limited thereto.

[0067] According to an embodiment in which a vortex generating means is the junction, the present disclosure provides a microfluidic system having a fluidic channel

structure including one or more junctions at which at least two channels are connected, as a system for delivering an

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external material outside a cell into the cell. In the

present disclosure, "junction" means a point at which each

channel meets another channel when two or more channels are

connected in the form of T, Y, + or a combination thereof,

and in the present disclosure, at least one junction may be

provided in a single channel defined by an inlet and an

outlet of a fluid.

[0068] In the microfluidic system according to an embodiment

of the present disclosure, a vortex is generated at an

interface of the junction as the solution containing cells

and external materials flows into the junction, and at this

time, cells that continuously experience the vortex and

vortex breakdown are continuously trapped and deformed by

inertia and inertial effects, and then pores in cell

membranes, which are pathways for intracellular delivery of

materials, are formed one after another as the deformation

progresses. That is, the microfluidic system according to

the present disclosure may greatly improve the intracellular

material delivery efficiency by utilizing a fluid property

such as a Reynolds number (1 to 1000) and the channel

structure in the form of T, Y, + or a combination thereof.

[0069] In the microfluidic system of the following

embodiments of the present disclosure, a mold with channels

according to the present disclosure formed is first made by

etching a SU-8 mold or a silicon wafer through a normal

photolithography process. Then, a polydimethylsiloxane

(PDMS)-based chip is made through PDMS, and at this time, an

inlet and an outlet are made in the chip made as mentioned

above, and general slide glass is combined using plasma

treatment (Cute, Femto Science, South Korea), thereby making

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a platform device. Then, cells in the suspended state and

materials to be delivered are mixed, and then the mixture is

injected into the made chip by using a syringe pump. In this

case, the intracellular delivery of materials may be

controlled by adjusting the flow rate of the syringe pump,

and after the delivery, only the cells are separated by using

a centrifuge, and then cultured or analyzed or used according

to the purpose. However, the scope of the present disclosure

is not limited to the embodiments themselves.

[0070]

[0071] Embodiment 1: + Junction Structure Microfluidic

System

[0072] FIG. 1 is a diagram for illustrating a formation of a

spiral vortex in a + junction channel.

[0073] Referring to FIG. 1, as fluid flows in opposite

directions at both ends of the + channel, the fluid exits

into two other channels intersecting the channel, and at this

time, fluid instability near a stagnation point induces a

strong spiral localized vortex near a proximal point. The

local vortex explains the reasons for the phenomena of the

cell deformation and intracellular delivery of materials

described below. In the present disclosure, "near" refers to

a region in which a vortex of a fluid flowing into a junction

may be generated due to a junction structure.

[0074] FIG. 2 is (1) a schematic diagram of cell deformation

caused by a spiral vortex and shows high-speed microscopy

images (scale bar: 10 pm) showing rotational cell motion.

[0075] Referring to FIG. 2, in the present disclosure, when

allowing fluid to flow to a junction channel with a medium

Reynolds number (1 to 1000), cells move spirally instead of

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symmetrical cell elongation, and at this time, the cells show

a large deformation as the cells approach a stagnation point

of the vortex flow.

[0076] FIG. 3 is a diagram for describing a mechanism of

material delivery with cell deformation according to an

embodiment of the present disclosure.

[0077] Referring to FIG. 3, a nanopore is created in a cell

membrane by the cell deformation on the left, and the

external materials are introduced into the cell by solution

exchange between the cell and fluid around the cell. Then,

within 1 to 10 minutes, the cell membrane self-recovers and

closes, and the recovery time may be controlled by adjusting

the concentration of an electrolyte such as calcium in the

solution.

[0078] FIG. 4 is a schematic diagram of two types of channel

structures according to an embodiment of the present

disclosure and a diagram for describing cell deformation in

each channel.

[0079] The two types of channel structures in FIG. 4 are a

+ structure in which oppositely flowing fluid collides near the junction to form a vortex near the junction, and a T

shaped structure in which the introduced cells collide with a

channel wall.

[0080] FIG. 5 is a schematic diagram of a microfluidic

system in which two types of fluidic channels of FIG. 4 are

connected in series in a fluid flow direction.

[0081] Referring to FIG. 5, the microfluidic system of the

present disclosure has (1) a + junction channel (cross

junction) and (2) a T-junction channel (T-junction) in which

a fluid guided while passing through the vortex from the +

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junction channel collides with the channel wall.

[0082] For the above-mentioned system, cells out of the

stagnation point in the + junction channel (cross-junction)

are again guided to a channel center by inertia and collide

with the channel wall again. That is, the T, Y, and + shaped microfluidic structures according to the present

disclosure may be designed in series, parallel, or a

combination thereof with respect to fluid flow, all of which

fall within the scope of the present disclosure. In this

case, a plurality of junctions may be formed that connects a

plurality of unit channels on a single channel defined by an

inlet and an outlet in terms of cells.

[0083] FIG. 6 shows a result of an experiment on behavior

according to the Reynolds number of fluid flowing in opposite

directions toward a junction (intersection) in the

+ junction channel.

[0084] Referring to Fig. 6, at the lowest Reynolds number,

the interface between the two fluid streams remains clearly

symmetrical, and the three-dimensional vortex motion clearly

starts at the Reynolds number 37.9 (critical Reynolds number).

Then, with the increase in the Reynolds number, the

intersection between the two fluid streams becomes stronger

and more complex (see (1)).

[0085] (2) and (3) in FIG. 6 are confocal microscopy images,

where, with the increase in the Reynolds number, the

counterclockwise spiral vortex develops beyond the critical

Reynolds number, and the spiral expands vertically and

horizontally. The above result means that the vortex is

generated near the junction and its shape, pattern, velocity,

and the like vary depending on the Reynolds number of the

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fluid. This also suggests that delivery efficiency may be

improved by varying the Reynolds number depending on the

desired cell type and the type of materials to be delivered.

[0086] The system according to the present disclosure has a

specific additional effect of trapping cells and inducing

deformation thereof by cell deformation due to vortex

formation and vortex breakdown after the delivery of

materials.

[0087] FIG. 7 shows high-speed microscopy images showing a

cell (MDA-MB-231) trapping behavior in the + junction

channel.

[0088] Referring to FIG. 7, a specific trapping phenomenon

of cells is observed for a certain period of time as the

cells leave a cell deformation region by the first vortex.

That is, slightly above the stagnation point, the cells

repeatedly move up and down for approximately 30 ps and then

go out again toward the outlet. This means that immediately

after leaving a vortex region, cells are deformed for a

certain period of time and then stay in a region near the

junction (a region near the stagnation point) by the inertia

caused by the vortex breakdown, and thus the intracellular

material delivery efficiency may be greatly improved.

[0089] FIG. 8 shows a graph of normalizing cell trapping and

cell deformation time by vortex breakdown of FIG. 7 as a

function of the Reynolds number.

[0090] Referring to FIG. 8, with the increase in the

Reynolds number, the cell trapping and deformation times

increase, and cell deformation for a certain period of time

after the formation of the vortex can greatly improve the

intracellular material delivery efficiency.

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[0091] More specifically, the + junction structure microfluidic system according to the present disclosure will

be described.

[0092] FIG. 9 is a diagram for illustrating an intracellular

material delivery platform according to an embodiment of the

present disclosure.

[0093] Referring to FIG. 9, the intracellular material

delivery platform according to an embodiment of the present

disclosure includes: a first channel 100 through which a

fluid including cells and delivery material flows; a second

channel 200 that vertically intersects with the first channel

100; and a first fluid control unit 300 provided on one side

of the first channel 100 to control a fluid velocity in the

first channel 100 in a first direction.

[0094] According to an embodiment of the present disclosure,

the fluid in the first channel 100 flows in opposite

directions to the point where the first channel 100

vertically intersects with the second channel 200, and the

first fluid control unit 300 applies, to cells in the first

channel 100, kinetic energy for causing cell membrane

deformation at a level at which nanopores are formed in the

cells by the vortex formed at the point where the first

channel 100 and the second channel 200 vertically intersect

each other.

[0095] In addition to that, a second fluid control unit 300'

for controlling the fluid velocity in the first channel 100

in a second direction may be further included on the other

side of the first channel 100.

[0096] In particular, in the first channel 100, the vortex

may be formed at the point where the first channel 100 and

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the second channel 200 vertically intersect each other by the

first and second fluid control units 300 and 300' performing

controls in opposite directions, and due to the inertial

force and inertial flow that are generated in this way,

physical deformation may occur in the cell and the cell

membrane may be deformed accordingly.

[0097] Meanwhile, the intracellular material delivery

platform according to the present disclosure may deliver

nucleic acids, proteins, transcription factors, vectors,

plasmids, genetic-scissors materials, nanoparticles, and the

like. However, the present disclosure is not limited thereto.

[0098] Furthermore, the intracellular material delivery

platform is not limited in application to regenerative

medicine, cancer immunotherapy, genomic editing, or other

fields.

[0099]

[00100] FIGS. 10 to 12 are diagrams for illustrating a method

for intracellular delivery of materials using the

intracellular material delivery platform according to an

embodiment of the present disclosure.

[00101] Referring to FIG. 10, the fluid containing cells and

delivery material flows in the first channel 100 by the first

fluid control unit 300. At this time, the second fluid

control unit 300' formed on the other side of the first

channel 100 also operates such that the delivery material

flows in a direction opposite to the fluid controlled by the

first fluid control unit 300.

[00102] In an embodiment of the present disclosure, the

delivery material includes all materials that may be

delivered into cells, and specifically, genetic-scissors

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materials, plasmids, nucleic acids, proteins, nanoparticles,

and the like may all the delivery materials.

[00103]

[00104] Referring to FIG. 11, the fluid accelerated by the

first fluid control unit 300 forms a vortex along the

interface with the opposing fluid near the junction, and the

cells trapped by the vortex are deformed. Then, a nanopore

is formed in the cell by the cell deformation. The delivery

material is delivered into the cell through the nanopore.

Accordingly, it is desirable that the first and second fluid

control units 300 and 300' apply kinetic energy having a

Reynolds number of a level at which the vortex of the fluid

is formed near the junction.

[00105] According to another embodiment of the present

disclosure, vortex breakdown formed after passing through the

vortex formed in the fluid makes another cell deformation.

[00106] Referring to FIG. 12, cells passing through the

vortex experience the vortex breakdown, and cell deformation

occurs again due to inertia. Through the nanopore formed in

the cell membrane by another cell deformation occurring at

this time, the delivery material is again delivered into the

cell, which greatly improves intracellular material delivery

efficiency.

[00107]

[00108] Embodiment 2: T or Y Junction Structure Microfluidic

System

[00109] Another embodiment of the present disclosure provides

an intracellular material delivery system using a T or Y

junction channel having a cavity. In the present disclosure,

a cavity refers to an empty space formed in a channel at a

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stagnation point, which is a structure in which when the

cells of the solution and the channel wall collide at the

junction, a collision area between the cells and a channel

wall is eliminated or reduced.

[00110] In this case, similar to the + junction channel

described above, a localized vortex is formed near the T

junction, and sequentially, cell deformation, formation of

the nanopore in the cell membrane, intracellular delivery of

external materials, and closure of the nanopore in the cell

membrane are performed.

[00111] FIG. 13 is a schematic diagram of a T-junction

channel according to an embodiment of the present disclosure.

[00112] Referring to FIG. 13, it can be seen that a channel

shaped cavity 1 is formed near a junction of a T-channel. In

an embodiment of the present disclosure, cells mixed with

external materials delivered into the cells are injected into

the microfluidic channel of FIG. 13 by using a syringe pump

in the related art (PHD 2000, Harvard Apparatus, USA), as a

fluid control unit, at an appropriate Reynolds number.

[00113] Upon injection, the cells are concentrated in the

channel center by inertia, and the cells collided with the

channel wall. Each cell penetrates a portion of the above

mentioned cavity ((1) of FIG. 13) and is deformed (refer to

the photographs on the right of FIG. 13). Then, after the

first cell deformation by the collision, the cell is trapped

in a localized vortex near the stagnation point (point (2) in

FIG. 13) and is hydrodynamically deformed to form nanopores

in the cell membrane, as described in FIG. 3.

[00114] Then, the cell that has passed through the vortex

region are trapped and then deformed by vortex breakdown

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again ((3) of Fig. 13). An advantage of such additional cell

trapping and deformation is the improvement of intracellular

material delivery efficiency as described above in FIG. 7.

[00115] In an embodiment of the present disclosure, the

cavity is used in the T-junction channel structure, the

advantage of which is to reduce cell damage due to collision

with a rigid solid channel wall by allowing cells to collide

with the channel wall of a fluid form, instead of the channel

wall of a rigid solid form. Another advantage is to

virtually prevent cell clogging by creating a stagnation

point upstream in the fluid flow direction to support complex

fluid behavior patterns. Accordingly, the form, size, shape,

and the like of the cavity structure may vary depending on

the cell. For example, the cavity may include not only an

elongated slit structure as shown in FIG. 13 but also a

circular, oval, elongated slit, square, rectangular,

trapezoidal, polygonal, a combination thereof, and a modified

form thereof, and at least as long as the introduced cells do

not collide with the channel wall as they are, they all

belong to the cavity of the present disclosure.

[00116] In addition, the cavity diameter is determined

depending on the cell diameter, and in particular, the cavity

diameter is preferably on the order of 10% to 5 times the

cell size. The cavity diameter according to the present

disclosure is within the scope of the present disclosure, at

least as long as the cavity diameter can reduce the collision

force caused by the collision between the cells, which are

introduced to the junction through the solution, and the

channel wall.

[00117] FIG. 14 shows high-speed microscopy images

WO 2020/184992 PCT/KR2020/003411

illustrating a cell deformation and vortex deformation

mechanism of a T-junction channel of a cavity structure.

Here, each arrow indicates a fluid flow direction.

[00118] Referring to FIG. 14, trapping by the localized

vortex near the stagnation point ((1) in FIG. 14) and

trapping of the cell passing through the stagnation point by

vortex breakdown due to fluid instability near the stagnation

point ((2) in FIG. 14) can be confirmed, which shows that the

vortex, the trapping by the vortex breakdown, and the

deformation occur continuously, similar to the channel of the

+ channel described above. In particular, in (2) of FIG. 14, it can be seen that the cell maintains a static state for

about 20 ps and then exits downstream.

[00119] FIG. 15 shows a vortex deformability index (VDI)

analysis result in a fluid having different Reynolds numbers.

Here, VDI is defined as the following formula, which can be

interpreted as the degree of cell deformability index and the

duration of cell membrane permeation, which in turn indicates

the intracellular material delivery efficiency.

[00120] VDI=t(1-c)U/D

[00121] where t is the cell trapping time in the vortex, c is

the circularity (c , where A and P are the area and

radius in the state with maximum deformation, respectively),

U is the average velocity of the fluid, and D is the cell

diameter.

[00122] Referring to FIG. 15, with the increase in the

Reynolds number, the VDI increases during (1) the vortex and

(2) the vortex breakdown, indicating that a high Reynolds

WO 2020/184992 PCT/KR2020/003411

number has a higher intracellular material delivery

efficiency.

[00123] Hereinafter, the T-junction structure microfluidic

system according to the present disclosure will be described

in more detail.

[00124] FIG. 16 is a diagram for illustrating an

intracellular material delivery platform according to an

embodiment of the present disclosure.

[00125] The intracellular material delivery platform

according to the present disclosure includes: a third channel

101 forming a pathway through which a fluid including cells

and delivery material moves; a fourth channel 201 vertically

extending to both sides of the third channel 101 at an end of

the third channel 101; and a fluid control unit 301 provided

at the third channel 101 to control a fluid velocity in the

third channel 101.

[00126] FIGS. 17 to 19 are diagrams for illustrating a method

for intracellular delivery of materials using the

intracellular material delivery platform according to an

embodiment of the present disclosure.

[00127] Referring to FIG. 17, the fluid containing cells and

delivery material flows in the third channel 101 by the fluid

control unit 301. In an embodiment of the present disclosure,

the delivery material includes all materials that may be

delivered into cells, and genetic-scissors materials,

plasmids, nucleic acids, proteins, nanoparticles, and the

like are all the delivery materials.

[00128] Referring to FIG. 18, the cells in the third channel

101 accelerated by the fluid control unit 301 are trapped by

the vortex formed near the junction and then deformed. This

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is the same as that described with reference to FIGS. 13 and

14. Then, the cells collide with a partition wall of the

fourth channel 201 connected to the end of the third channel

101.

[00129] At this time, the fourth channel 201 is provided with

a slit-shaped cavity 401 formed in the same direction as the

fluid flow direction in the third channel 101, and the cavity

produces effects of 1) preventing cell damage by physical

collision, 2) preventing clogging, and 3) forming the

stagnation point upstream.

[00130] In the present disclosure, as described above, a

plurality of unit microfluidic systems may be connected in

series or parallel or a combination thereof to construct an

entire system. In this case, the vortex may occur in the

recirculated stream in a circulation mode, where the vortex

may be a closed or open stream.

[00131]

[00132] Experimental examples

[00133] Experimental Example 1

[00134] In the experimental example, delivery characteristics

of the microfluidic system based on Embodiment 1 were

analyzed.

[00135] FIG. 19 shows a result of comparing the intracellular

material delivery efficiencies after 18 hours by the

endocytosis (control group) and the intracellular mass

transfer method according to the present disclosure (spiral

hydroporation). Here, 3-5 kDa fluorescein isothiocyanate

(FITC)-conjugated dextran was delivered into MDA-MB-231 cells,

and the result was shown as a fluorescence image (scale bar:

pm). In particular, it should be noted here that the

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delivery of dextran into MDA-MB-231 cells is known to be very

difficult. In the experiments described below, unless

otherwise noted, cells are MDA-MB-231.

[00136] Referring to FIG. 19, the intracellular material

delivery effect according to the present disclosure can be

confirmed through multiple FITC signals on the right.

[00137] FIGS. 20 and 21 show analysis results of the amount

of materials delivered into cells. In the analysis, the

delivery efficiency was defined as a fraction of fluorescence

signals equal to or greater than 5%, which corresponds to the

red dotted line.

[00138] Referring to FIGS. 20 and 21, it can be seen that at

Reynolds number 366, a delivery efficiency of approximately

96.5% was achieved, and the fluorescence intensity increases

with the increase in Re and the histogram profile shifted to

the right. This means that the amount of materials delivered

into cells may be adjusted by adjusting the Reynolds number.

[00139] FIG. 22 shows a result of measuring cell viability by

varying the Reynolds number under the same conditions as in

FIG. 19.

[00140] Referring to FIG. 22, with regard to cell viability,

a decrease in viability was observed at a higher Reynolds

number, presumably because the higher the Reynolds number,

the greater the cell perturbation.

[00141] Furthermore, in order to further investigate cell

viability, a standard MTT assay was performed via metabolic

function and the result is shown in FIG. 22. The overall

trends of the trypan blue exclusion method and the MTT assay

were in good agreement with each other, whereas the MTT assay

showed slightly lower viability values at high Reynolds

WO 2020/184992 PCT/KR2020/003411

numbers.

[00142] FIG. 23 shows an analysis result of delivery

efficiency for various dextran sizes. In the analysis,

dextrans of molecular weights ranging from 3 to 2000 kDa,

corresponding to a hydraulic diameter of 2 to 55 nm, were

used, where FITC-dextrans (3 to 5, 70, 150, 500, and 2000

kDa) of 5 different sizes and the same concentration were

delivered into MDA-MB-231 cells in the same manner as in

Embodiment 1 under the same flow conditions (Re = 366) and

the efficiency was calculated.

[00143] Referring to FIG. 23, it was shown that relatively

small dextran (<70 kDa) had higher delivery efficiency than

relatively large dextran. This is because, for small dextran,

convective and diffuse transport of dextran across the cell

membrane occurs, whereas for large dextran, convective

transport is the dominant transport.

[00144] FIG. 24 shows a result of measuring dextran delivery

efficiency in the complex microfluidic system of FIG. 5 (a

combination of the + junction channel and the T-junction

channel).

[00145] Referring to FIG. 24, it can be seen that the complex

microfluidic system of FIG. 5, in which continuous cell

deformation was performed, exhibited relatively higher

delivery efficiency than the stand-alone one.

[00146] FIG. 25 shows a comparison result of the delivery

efficiency of electroporation in the related art (a neon

transfection system (Thermo Fisher Scientific, Waltham, MA,

USA)) and the method for delivery of materials based on

inertia (hydroporation) according to the present disclosure.

In the experimental example, 500 and 2000 kDa FITC-dextrans

WO 2020/184992 PCT/KR2020/003411

were used as external materials.

[00147] Referring to FIG. 25, for the 2000 kDa FITC-dextran,

the efficiency of the present disclosure was approximately 4

times higher than that of electroporation. In particular,

the method according to the present disclosure greatly

improves the delivery efficiency of macromolecules that are

difficult to be delivered into cells by electroporation,

which suggests the possibility of intracellular delivery of

molecular weight materials that is not possible to be

achieved by electroporation techniques in the related art.

[00148] FIGS. 26 and 27 are photographs for analyzing the

intracellular delivery effect of gold nanoparticles (GNPs),

and a result of counting scattering spots, respectively.

Here, (A) shows a result of hydroporation (SH) according to

the present disclosure, (B) shows a result of electroporation

(EP), and (C) shows a result of endocytosis (EC).

[00149] Referring to FIGS. 26 and 27, it can be seen that

very large GNPs (200 nm in diameter) were successfully

delivered into MDAMB-231 cells through the fluidic system

according to the present disclosure (A). On the other hand,

it can be seen that only a few scattering points were

detected although electroporation was able to deliver GNPs

(B). Furthermore, the endocytosis mechanism exhibited lower

particle delivery efficiency than that of electroporation (C).

[00150] FIG. 28 shows a result of measuring cell viability

depending on GNP delivery.

[00151] Referring to FIG. 28, it can be seen that the method

and system for intracellular delivery of materials according

to the present disclosure exhibited higher viability than

that of electroporation.

WO 2020/184992 PCT/KR2020/003411

[00152] FIG. 29 shows a result of calculating a yield

normalized by multiplying the number of scattering spots by

the cell viability (FIG. 5F).

[00153] Referring to FIG. 29, it can be seen that the

material delivery efficiency according to the present

disclosure was three times higher than that of

electroporation or endocytosis.

[00154] FIG. 30 shows an analysis result of intracellular

delivery efficiency of mesoporous silica nanoparticles (MSN)

used as a drug, protein, and nucleic acid nanocarrier instead

of gold nanoparticles. In the experiment, doxorubicin (DOX),

an anticancer drug, was loaded into MSNs and the MSNs were

delivered into MDA-MB-231 cells.

[00155] Referring to FIG. 30, it can be seen that the method

according to the present disclosure (spiral control)

exhibited a number of bright fluorescence points compared to

endocytosis. This suggests that a carrier carrying a

functional material can be effectively delivered into the

cell by the method according to the present disclosure.

[00156] FIG. 31 shows an analysis result of a time-dependent

cell viability for the analyte of FIG. 30.

[00157] Referring to FIG. 31, DOX cytotoxicity to MDA-MB-231

cells was measured by the trypan blue exclusion method, and

it can be seen that approximately 85% of cancer cells were

killed after 6 hours. The result indicates that the

microfluidic system according to the present disclosure may

investigate DOX-induced cell death without using a surface

ligand-based DOX delivery method in the related art.

[00158] FIG. 32 is a histogram of fluorescence intensity

obtained by measuring a delivery effect to K562 cells using

WO 2020/184992 PCT/KR2020/003411

mRNA (996-nucleotide mRNA fragment, green fluorescent

protein) as an external material. In FIG. 32, (A) shows a

result of endocytosis and (B) shows a result of the

microfluidic system according to the present disclosure,

where EGFP protein expression was assessed based on mRNA

delivery (2 pg/mL) using a flow cytometer.

[00159] Referring to FIG. 32, it can be seen that the method

according to the present disclosure exhibited a very high

protein expression level. This means that the method and

system for intracellular delivery of materials according to

the present disclosure may be used for chimeric antigen

receptor-expressing T-cells (CAR-T) without the use of

vectors or vaccines.

[00160] FIG. 33 shows fluorescence images after delivery of

mRNA into K562 by (C) endocytosis and (D) the microfluidic

system according to the present disclosure.

[00161] Referring to FIG. 33, in the method according to the

present disclosure, strong EGFP signals were detected

compared to endocytosis, which results in higher mRNA

delivery efficiency.

[00162] FIG. 34 shows a result of measuring delivery

efficiency (E) and viability (F) when mRNA is delivered

according to the present disclosure as shown in FIGS. 32 and

33.

[00163] Referring to FIG. 34, the delivery efficiency of up

to approximately 92% was achieved without sacrificing cell

viability, which represents the high potential of the

platform for use of the platform for immunotherapy research

although further investigations are needed to test human

immune cells in the future.

WO 2020/184992 PCT/KR2020/003411

[00164]

[00165] Experimental Example 2

[00166] In the experimental example, delivery characteristics

of the microfluidic system (p-Hydroporator) based on

Embodiment 2 were analyzed.

[00167] FIG. 35 shows fluorescence images after mRNA (996

nucleotide mRNA fragment, green fluorescent protein) is

delivered into K562 cells by endocytosis and the method of

Embodiment 2.

[00168] Referring to FIG. 35, it can be seen that, unlike

endocytosis (control), green fluorescence was detected when

mRNA was delivered into cells by the method of Embodiment 2.

This proves that the T-junction channel system according to

the present disclosure (Embodiment 2) enables intracellular

delivery of mRNA with high efficiency as in Embodiment 1

described above.

[00169] FIG. 36 shows an analysis result of (b) mRNA

transfection efficiency and (c) mean fluorescence intensity

depending on mRNA concentration.

[00170] Referring to FIG. 36, different concentrations of

mRNAs were accessed based on flow cytometry analysis, and in

2 pg/ml, transfection efficiency was achieved up to

approximately 93%. This is an efficiency that is not

possible in compression-based microfluidic systems such as

bottleneck structures in the related art.

[00171] Unlike mRNA, which binds to the cell substrate first,

DNA has to first pass through the cell membrane and enter a

nuclear membrane through a nuclear pore. Furthermore, in

terms of material delivery, naked plasmid DNA has a drawback

in that it is easily degraded by nucleases and has a high

WO 2020/184992 PCT/KR2020/003411

viscosity. In addition, high-density cytoplasm does not

provide favorable conditions for long, twisted DNA to reach

the nucleus purely by diffusion. In order to overcome the

above-mentioned drawback, the present inventors provide a

fluid-based microfluidic system according to the present

disclosure as a method for delivering plasmid DNA to a

nucleus. To this end, in the experimental example, an

experiment was performed to encode copepod GFP and deliver

7.9 kbp plasmid DNA to HEK293t cells.

[00172] FIG. 37 shows fluorescence images after delivery of

plasmid DNA into HEK293t by the endocytosis (control) and the

method of Embodiment 2.

[00173] Referring to FIG. 37, it can be seen that the method

according to the present disclosure exhibited a very strong

fluorescence image.

[00174] FIG. 38 shows a graph of analyzing plasmid DNA (pDNA)

transfection efficiency (E) of HEK293t cells and mean

intensity (F) of HEK293t cells depending on plasmid DNA

concentration.

[00175] Referring to FIG. 38, it can be seen that with an

increase in the pDNA concentration, the transfection

efficiency increases, and the mean fluorescence intensity

also increases.

[00176] FIGS. 39 and 40 are a result of western blot analysis

of the ITGal gene knockdown effect of HeLa cells (al subunit

of an integrin transmembrane receptor) into which siRNA is

delivered, and a result of comparing relative expression

levels, respectively. Here, the knockdown effect of the

ITGal gene was compared using cationic Lipofectamine 3000 in

the related art, which is known as the siRNA delivery method,

WO 2020/184992 PCT/KR2020/003411

as a comparative example.

[00177] Referring to FIGS. 39 and 40, when the present

disclosure was used, ITGA1 expression (197 kDa) was almost

eliminated; whereas, in Lipofectamine 3000, the comparative

example, only partial knockdown was observed. According to

the result, the present disclosure has at least three times

greater knockdown efficiency (97% vs. 30%), suggesting that

the present disclosure has a high potential for use in gene

editing.

[00178] In an experiment below, non-functional materials or

extremely small molecules (e.g., calcein, propidium iodide,

and 3 kDa dextran) were delivered to cell lines. In the

experiment below, mRNA was chosen as a delivery target, since

protein expression after mRNA delivery occurs in the

cytoplasm and is guided to fat, is well controllable, and is

easily comparable by dose-dependent transfection.

[00179] In the experiment, EGFP mRNA was delivered into

Harton's jelly human umbilical cord mesenchymal stem cells

(MSCs), human adipose derived stem cells (ADSCs), and mouse

bone marrow derived dendritic cells (BMDC) by using the

method of Embodiment 2 (electroporator), Lipofectamine 3000,

and the electroporation neon transfection system

(electroporator; Thermo Fisher Scientific).

[00180] FIG. 41 shows an analysis result of transfection

yields by Lipofectamin 3000, electroporation, and the present

disclosure.

[00181] Referring to FIG. 41, it can be seen that the present

disclosure exhibited a very high transfection yield compared

to Lipofectamine 3000 and electroporation. In the analysis

result, the transfection yield was defined as the product of

WO 2020/184992 PCT/KR2020/003411

transfection efficiency and cell viability, which can be

understood as the ratio of viable cells to cells transfected

by material delivery.

[00182] FIGS. 42 to 44 are analysis results of transfection

efficiencies, cell viability, and transfection yields for

human MSC, human ADSC, and mouse BMDC.

[00183] Referring to FIGS. 42 to 44, it can be seen that

lipofection exhibited improved cell viability in all cell

types compared to the present disclosure (p-Hydroporator) and

electroporation, but exhibited substantially low transfection

efficiency in all cell types.

[00184] In addition, for MSC and ADSC, the transfection

efficiency was slightly higher in the electroporator than in

the present disclosure (p-Hydroporation). However, for all

cell types, the present disclosure (p-Hydroporator) exhibited

higher cell viability without the use of special stabilized

buffer required for the electroporator. Furthermore, the

cell viability of the present disclosure may further increase

cell viability simply by adding trehalose or polymer to the

cell media.

[00185] For BMDCs, the present disclosure exhibited higher

transfection efficiency and cell viability than

electroporation, indicating that the present disclosure has a

high potential to be used for cancer immunotherapy.

[00186] The present disclosure has several advantages over

electroporation in the related art with respect to immune

cell therapy. First, electroporation is known to have a side

effect of altering important properties of primary T cells

(e.g., non-specific cytokine burst and blunted IFN-y

response), lowering the therapeutic performance. However,

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the low scalability of electroporation (treating 104 to 105

cells per run) is a drawback regarding its potential clinical

usage in cancer immunotherapy, which generally requires the

treatment of 108 cells.

[00187] However, in the present disclosure, 1 x 106 cells/min

may be processed while the same level of delivery efficiency

is maintained and this throughput is based on a single

microchannel, and thus the present disclosure may achieve the

cell throughput required for cancer immunotherapy through

multiplexing and parallelization of microchannels.

[00188] In an experiment below, quantum dots (Dibenzo

cyclooctyne (DOBI)) and silica nanospheres, which are widely

used as target molecules, were determined as intracellular

delivery materials, and delivery properties to cells (MDA-MB

231) were analyzed.

[00189] FIG. 45 shows confocal microscopy images showing

delivery of quantum dots (qdot625) into MDA-MB-231 cells by

the present disclosure (p-Hydroporator), electroporator, and

Lipofectamine 3000.

[00190] Referring to FIG. 45, all the methods showed

excellent delivery efficiency, but the result in which

quantum dots were well dispersed in the cytoplasm was shown

when intracellular delivery was performed by the method

according to the present disclosure. In cells treated with

electroporation and Lipofectamine 3000, multiple red spots

were observed, indicating the possibility of aggregation or

endosome entrapment of the quantum dots. Since such quantum

dot aggregation eventually causes a decrease in the

efficiency of intracellular delivery of materials, the result

means that the method for intracellular delivery of materials

WO 2020/184992 PCT/KR2020/003411

according to the present disclosure is also useful for

intracellular delivery of micro-materials.

[00191] FIG. 46 shows an analysis result of spot number

counts of quantum dots (Qdot 625) per cell.

[00192] Referring to FIG. 46, the analysis result represents

the degree of aggregation of quantum dots, and it can be seen

that in the present disclosure, the number of quantum dots

per cell was the lowest. That is, electroporation or

lipofection exhibited three- and four-fold levels of quantum

dot count compared to the present disclosure. This is also

consistent with the analysis result of FIG. 45, indicating

that when intracellular delivery of particles such as quantum

dots is performed according to the present disclosure,

quantum dots are well dispersed in the cytoplasm by rapid

solution exchange through the cell membrane after cell

deformation.

[00193] FIG. 47 is a fluorescence intensity histogram of

Embodiment (p-Hydroporator, Nceui = 5,000 per sample) in which

nanospheres (green fluorescent silica nanospheres, Micromod,

Germany) are delivered into K562 cells, by a negative control

(nontreated K562 cells), a positive control (K562 cells co

incubated with nanospheres, the same time and concentration

as those in Embodiment), and the present disclosure (p

Hydroporator).

[00194] Further, FIG. 48 shows a result of measuring relative

mean fluorescence intensity.

[00195] Referring to FIGS. 47 and 48, on average, higher

fluorescence intensity was observed in cells treated

according to the present disclosure, but high fluorescence

signals were detected in the co-incubation group in flow

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cytometry analysis. Considering the short incubation time

and the excessively large nanosphere size for endocytosis,

the fluorescence in the positive control presumably seems to

be obtained from nanospheres adhering to the cell surfaces.

[00196] FIGS. 49 and 50 show confocal images of two of the

groups of FIGS. 47 and 48. Here, the cell membrane was

visualized by using DiD lipophilic carbocyanine dye.

[00197] As shown in FIGS. 49 and 50, the green fluorescence

signals from the positive control (co-incubation) were

present only in the cell membrane, whereas bright green

fluorescence from the cytoplasm was only observed in cells

treated according to the present disclosure. This indicates

that only the system for intracellular delivery of materials

according to the present disclosure is possible as a

technology capable of delivering nanospheres into cells,

considering the positive control in which only the action of

nanospheres adhering to the cell surfaces by electrostatics

is confirmed.

[00198]

[00199] INDUSTRIAL APPLICABILITY

[00200] The microfluidic system for delivering external

materials into cells by cell mechanoporation using inertia

according to the present disclosure has industrial

applicability in the bio and medicine fields requiring

delivery of materials into cells.

[00201]

[00202]

Claims (16)

WO 2020/184992 PCT/KR2020/003411 CLAIMS

1. A microfluidic system delivering external

materials into a cell by cell mechanoporation using inertia,

the microfluidic system comprising:

a fluidic channel structure through which a solution

containing a cell and external materials flows continuously,

wherein the fluidic channel structure includes a

junction between one or more channels,

a localized vortex is generated near an interface of

the junction,

the cell is deformed by the vortex, and

transient discontinuities are generated in a cell

membrane by the vortex and the external materials are

introduced into the cell by solution exchange between the

cell and fluid around the cell.

2. The microfluidic system of claim 1, wherein the

fluidic channel structure including the junction between one

or more channels includes a junction including a T, Y, cross

shape, or a combination thereof.

3. The microfluidic system of claim 2, wherein the

fluidic channel structure includes a cavity near a fluid

stagnation point when the fluidic channel structure is a

channel of the T or Y shape.

4. The microfluidic system of claim 3, wherein the

cavity has a shape of a circle, an ellipse, an elongate slit,

a square, a rectangle, a trapezoid, a polygon, and a

WO 2020/184992 PCT/KR2020/003411

combination thereof, and a modification thereof.

5. The microfluidic system of claim 3, wherein a

diameter of the cavity is determined according to a diameter

of the cell.

6. The microfluidic system of claim 3, wherein the

cavity has a structure for eliminating or reducing a

collision area between the cell and a channel wall when the

cell of the solution collides with the channel wall at the

junction.

7. The microfluidic system of any one of claims 1 to

6, further comprising a fluid control unit for allowing a

solution to flow in the fluidic channel structure,

wherein the fluid control unit allows the solution to

flow in the fluidic channel at a velocity that is at a level

capable of generating a localized vortex near the interface

of the junction.

8. The microfluidic system of claim 7, wherein the

fluid control unit is a syringe pump or pneumatic system.

9. The microfluidic system of any one of claims 1 to

8, wherein a Reynolds number (Re) of the solution is 1 to

1000.

10. The microfluidic system of claim 9, wherein the

vortex is determined by the Reynolds number.

WO 2020/184992 PCT/KR2020/003411

11. The microfluidic system of any one of claims 1 to

8, wherein the vortex is in a form of a closed or open

recirculating flow.

12. The microfluidic system of any one of claims 1 to

8, wherein the fluidic channel has a plurality of the

junctions at least in a channel between an inlet and an

outlet of the solution.

13. The microfluidic system of claim 10, wherein the

microfluidic system is formed by combining the microfluidic

system according to any one of claims 1 to 12 in series,

parallel, or a combination thereof.

14. A method of delivering external materials into a

cell by cell mechanoporation using inertia, the method

comprising:

allowing a solution containing the cell and external

materials to continuously flow a fluidic channel;

forming a vortex by a vortex generating means near the

junction;

deforming the cell by the vortex; and

allowing the external materials to be introduced into

the cell through a pore created in a cell membrane by the

deforming of the cell.

15. The method of claim 14, wherein the vortex

generating means is a junction structure of the fluidic

channels.

WO 2020/184992 PCT/KR2020/003411

16. The method of claim 15, wherein the fluidic

channel includes a junction including a T, Y, cross shape, or

a combination thereof.

[DRAWINGS]

[FIG. 1]

[FIG. 2]

[FIG. 3]

[FIG. 4]

[FIG. 5]

[FIG. 6]

[FIG. 7]

[FIG. 8]

[FIG. 9]

[FIG. 10]

[FIG. 11]

[FIG. 12]

[FIG. 13]

[FIG. 14]

[FIG. 15]

[FIG. 16]

[FIG. 17]

[FIG. 18]

[FIG. 19]

[FIG. 20]

[FIG. 21]

[FIG. 22]

[FIG. 23]

[FIG. 24]

[FIG. 25]

[FIG. 26]

[FIG. 27]

[FIG. 28]

[FIG. 29]

[FIG. 30]

[FIG. 31]

[FIG. 32]

[FIG. 33]

[FIG. 34]

[FIG. 35]

[FIG. 36]

[FIG. 37]

[FIG. 38]

[FIG. 39]

[FIG. 40]

[FIG. 41]

[FIG. 42]

[FIG. 43]

[FIG. 44]

[FIG. 45]

[FIG. 46]

[FIG. 47]

[FIG. 48]

[FIG. 49]

[FIG. 50]