U.S. patent application number 11/134078 was filed with the patent office on 2006-04-06 for highly controllable electroporation and applications thereof.
Invention is credited to Sadeg M. Faris.
Application Number | 20060073597 11/134078 |
Document ID | / |
Family ID | 32713474 |
Filed Date | 2006-04-06 |
United States Patent
Application |
20060073597 |
Kind Code |
A1 |
Faris; Sadeg M. |
April 6, 2006 |
Highly controllable electroporation and applications thereof
Abstract
The controllable electroporation system and method described
herein allows control over the size, the number, the location, and
the distribution of aqueous pores, thus increasing flexibility of
use. The herein described system and method for controllable
electroporation generally employs at least two actuating
sub-systems and sub-processes. One sub-system and sub-process
employs a relatively broad effect in order to weaken the membrane,
a broad effect sub-system. Another sub-system and sub-process
employs a relatively narrow effect in order to localize the
position of the pore in the membrane, a narrow effect
sub-system.
Inventors: |
Faris; Sadeg M.;
(Pleasantville, NY) |
Correspondence
Address: |
Reveo, Inc.
85 Executive Blvd.
Elmsford
NY
10523
US
|
Family ID: |
32713474 |
Appl. No.: |
11/134078 |
Filed: |
May 20, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10755709 |
Jan 12, 2004 |
6962816 |
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11134078 |
May 20, 2005 |
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60439387 |
Jan 10, 2003 |
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Current U.S.
Class: |
435/461 ;
210/295 |
Current CPC
Class: |
C12M 35/02 20130101 |
Class at
Publication: |
435/461 ;
210/295 |
International
Class: |
C12N 15/87 20060101
C12N015/87 |
Claims
1. A system for controllable electroporation of a membrane
comprising: a broad energy sub-system operably coupled to the
membrane and a narrow energy sub-system operably coupled to the
membrane, wherein a pore is opened or created at a position
corresponding to the position of the narrow energy.
2. The system as in claim 1, wherein the broad energy sub-system is
selected from the group of weakening systems consisting of electric
fields, microwave energy, other electromagnetic radiation, low
energy laser beams, or any combination comprising at least one of
the foregoing weakening systems.
3. The system as in claim 1, wherein the energy magnitude of the
broad energy sub-system is lower than the energy magnitude of
electroporation systems without the narrow effect sub-system
whereby random pore opening occur.
4. The system as in claim 1, wherein the area of the broad energy
sub-system encompasses an area larger than the desired pore
size.
5. The system as in claim 1, wherein the area of the broad energy
sub-system encompasses the membrane of a cell.
6. The system as in claim 1, wherein the area of the broad energy
sub-system encompasses membranes of an array of cells.
7. The system as in claim 1, wherein the area of the broad energy
sub-system encompasses a region of a membrane.
8. The system as in claim 1, wherein the narrow energy sub-system
is selected from the group of position localization systems
consisting of laser beams, electrode tips, or any combination
comprising at least one of the foregoing position localization
systems.
9. The system as in claim 1, wherein the area of the narrow energy
sub-system corresponds to the dimensions of the pore opening.
10. The system as in claim 1, wherein the pore has sub-micron
dimensions.
11. The system as in claim 1, wherein the pore has dimensions of
about 100 nanometers or less.
12. A controllable electroporation system comprising: a broad
energy sub-system operably coupled for providing broad energy to
the membrane and a narrow energy sub-system operably coupled to the
membrane, wherein a pore is opened when both the broad energy
sub-system and the narrow energy sub-system are activated.
13-23. (canceled)
24. A cell pore opening system comprising: a microrobotic device
for holding a cell and a system as in claim 1 for controllably
opening a pore in the cell.
25. The cell pore opening system as in claim 24, wherein the broad
energy sub-system comprises an electrode plate and a switchable
voltage source.
26. The cell pore opening system as in claim 24, wherein the narrow
energy sub-system comprises a laser.
27. A cell pore macromolecule system comprising: a microrobotic
device for holding a cell; a system as in claim 1 for controllably
opening a pore in the cell; and a macromolecule injection device
for injecting a macromolecule into the cell via the pore.
28-29. (canceled)
30. A system for filtering molecules or macromolecules comprising:
a plurality of membrane layers, each membrane layer including a
system as in claim 1 for controllably opening a pore in the cell,
each membrane layer opened to a different size to create a pore
size gradient.
31. A system for filtering molecules or macromolecules comprising:
a plurality of membrane layers, a broad energy sub-system
associated with each membrane layer, wherein each layer includes a
position having a defect whereby said position is closed without
activation of the broad energy sub-system and said position is
opened upon activation of the broad energy sub-system.
32. The system as in claim 31, wherein said defect at each layer
controls the size of the pore.
33. The system as in claim 31, wherein the magnitude of the energy
of the broad energy sub-system controls the size of the pore.
34. A system for filtering molecules or macromolecules comprising:
a membrane layer including a system as in claim 1 for controllably
opening a pore in the cell, wherein the narrow energy sub-system
comprises an array of narrow energy sub-sub-systems.
35. A system for filtering molecules or macromolecules comprising:
a membrane layer including a system as in claim 1 for controllably
opening a pore in the cell, wherein the narrow energy sub-system
comprises an array of lasers.
36. A system for filtering molecules or macromolecules comprising:
a membrane layer including a system as in claim 1 for controllably
opening a pore in the cell, wherein the narrow energy sub-system
comprises a beam steering device associated with a source of
electromagnetic energy.
37-47. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. .sctn.
119(e) of U.S. Provisional Application No. 60/439,387 filed on Jan.
10, 2003, which is herein incorporated by reference
BACKGROUND
[0002] The membrane of a cell serves the vital function of
partitioning the molecular contents from its external environment.
The membranes are largely composed of amphiphilic lipids, which
self-assemble into highly insulating structures and thus present a
large energy barrier to trans-membrane ionic transport.
[0003] However, the lipid matrix can be disrupted by a strong
external electric field leading to an increase in trans-membrane
conductivity and diffusive permeability, a well-known phenomenon
known as electroporation. These effects are the result of formation
of aqueous pores in the membrane. More particularly,
electroporation process involve permeation of cell membranes upon
application of short duration electric field pulses, traditionally
between relatively large plate electrodes (Neumann, et al.,
Bioelectrochem Bioenerg 48, 3-16 (1999); Ho, et al., Crit Rev
Biotechnol 16, 349-62 (1996)).
[0004] For example, FIGS. 2A and 2B show a conventional
electroporation system 20, whereby impression of an electric field
(e.g., shown by closing a circuit 22) creates random pores 26 in a
cell membrane 24. The distribution of such pores, in terms of size
and number only, is determined by the strength and duration of the
applied electric field. The stronger and the longer the electric
field is applied, the more numerous and larger the pores are.
However, the exact location of such pores cannot be controlled, and
thus the final distribution of pores is somewhat random.
Researchers lose control over where compounds are introduced into
the intracellular matrix, an oft-important ingredient in
biochemical pathways. Thus researchers often have to rely on the
cell's own natural mechanisms, a far slower and difficult process
to utilize.
[0005] Electroporation is used for introducing macromolecules,
including DNA, RNA, dyes, proteins and various chemical agents,
into cells. Large external electric fields induce high
trans-membrane potentials leading to the formation of pores (e.g.,
having diameters in the range of 20-120 nm). During the application
of the electric pulse, charged macromolecules, including DNA, are
actively transported by electrophoresis across the cell membrane
through these pores (Neumann, et al., Biophys J 712 868-77 (1996)).
Uncharged molecules may also enter through the pores by passive
diffusion. Upon pulse termination, pores reseal over hundreds of
milliseconds as measured by recovery of normal membrane conductance
values (Ho, 1996, supra).
[0006] This procedure is often used in laboratory settings to
inject chemical and biological compounds into a cell, avoiding the
reliance on the cell's own protein receptors and trans-membrane
channels for transport across the cell membrane. This allows
researchers to easily study the biological affect of compounds, be
it a potentially life-saving cancer drug or a deadly biological
toxin. However, current electroporation techniques are limited.
[0007] Therefore, it would be desirable to provide a method and
system to overcome these and other limitations of conventional
electroporation.
SUMMARY
[0008] The above-discussed and other problems and deficiencies of
the prior art are overcome or alleviated by the several methods and
apparatus of the present invention for controllable
electroporation. The controllable electroporation system and method
allows control over the size, the number, the location, and the
distribution of aqueous pores, thus increasing flexibility of use.
The herein described system and method for controllable
electroporation generally employs at least two actuating
sub-systems and sub-processes. One sub-system and sub-process
employs a relatively broad effect in order to weaken the membrane,
a broad effect sub-system. Another sub-system and sub-process
employs a relatively narrow effect in order to localize the
position of the pore in the membrane, a narrow effect
sub-system.
[0009] The above-discussed and other features and advantages of the
present invention will be appreciated and understood by those
skilled in the art from the following detailed description and
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIGS. 1A and 1B show operation of the controllable
electroporation system described herein;
[0011] FIGS. 2A and 2B show operation of conventional
electroporation systems;
[0012] FIGS. 3A-3D show a general embodiment of a cell injection
system;
[0013] FIG. 4A-4B shows an example of a cell injection system;
[0014] FIG. 5A-5B shows an example of a cell injection system for
an array of cells;
[0015] FIG. 6 shows a separation device using multi-phospholipid
layers and electroporation;
[0016] FIG. 7 shows a separation device using multiple layers,
electroporation and a microfluid array;
[0017] FIG. 8 shows a separation device using a single layer
capapble of having different pores at different locations; and
[0018] FIG. 9 shows an exemplary electrode grid that may be used in
various embodiments described herein.
DETAILED DESCRIPTION
[0019] Herein described is an electroporation system and method
providing control over the size, the number, the location, and the
distribution of aqueous pores, thus increasing flexibility of use.
Referring generally to FIGS. 1A and 1B, the herein described system
10 and method for controllable electroporation of a membrane 12
generally employs at least two actuating sub-systems and
sub-processes. FIG. 1A shows a portion of a membrane 12, and FIG.
1B shows the controllable electroporation system 10 described
herein.
[0020] Note that in general, one of the actuating sub-systems alone
will not suffice to open or create a pore 14 in the membrane--both
actuating sub-systems are employed, thereby functioning in a
similar manner as a logical "and" gate. A broad effect sub-system
16 employs a relatively broad effect in order to weaken the
membrane, and the narrow effect sub-system 18 employs a relatively
narrow effect in order to localize the position of the pore 14 in
the membrane 12. Employing both the broad effect sub-system 16 and
the narrow effect sub-system 18 enables highly localized and
controlled electroporation and hence opening of pore 14.
[0021] Therefore, for example as compared to conventional
electroporation processes of cellular membranes, described in the
Background and with respect to FIGS. 2A and 2B, in addition to the
applied electric field of conventional electroporation technology,
the herein disclosed electroporation system and method employs a
narrow effect sub-system 18 directed at a specific location on the
cellular membrane, enabling positional control over the pore 14.
The narrow effect sub-system 18 will excite the phospholipid
molecules, thus reducing the amount of energy required from the
broad effect sub-system 16 to create the aqueous pores. Thus, for
example, a very weak electric field can be applied to a system, in
which typically, electroporation would not occur. However, this
weak electric field can open aqueous pores in places of the
cellular membrane already excited by the laser beam.
[0022] The broad effect sub-system or sub-process 16 may be
selected from any suitable membrane weakening systems and/or
processes. Such weakening systems and/or processes may be selected
from the group consisting of electric fields (in certain preferred
embodiments uniform electric fields), microwave energy, other
electromagnetic radiation, relatively low energy laser beams (i.e.,
lower energy than that required to commence random
electroporation), or any combination comprising at least one of the
foregoing weakening systems and/or processes. The energy magnitude
of the broad effect sub-system or process 16 is generally lower
than the energy magnitude of conventional electroporation systems
whereby random pore opening occur. Further, the area (e.g.,
cross-sectional area) of the weakening systems and/or processes 16
generally encompasses an area larger than the desired pore size. In
certain embodiments, this area encompasses the entire cell membrane
or an array of cell membranes. In other embodiments, this area is a
region of a membrane.
[0023] The narrow effect sub-system or sub-process 18 may be
selected from any suitable membrane pore position localization
systems and/or processes. Such position localization systems and/or
processes may be selected from the group consisting of laser beams,
electrode tips, or any combination comprising at least one of the
foregoing position localization systems and/or processes. The area
(e.g., cross-sectional area) of the position localization systems
and/or processes 18 is generally narrow, e.g., corresponding to the
desired dimensions of the pore opening. Thus, for example,
controlled pore openings having of sub-micron or nanometer (e.g.,
1-100 nm) magnitude are enabled, since existing and developing
laser and electrode tip technologies are capable of such
sub-micron-scale and nano-scale dimensions.
Applications
Cell Injection
[0024] The herein described controllable electroporation system and
process may be used to inject macromolecules, including DNA, RNA,
dyes, proteins and various chemical agents, in a controlled manner.
Without intending to limit the applications of the present
controllable electroporation system, FIGS. 3A-5B show various
embodiments of cell pore opening systems employing the controllable
electroporation system.
[0025] Referring now to FIGS. 3A-3D, a system 30 is shown for
controllable injecting macromolecules into a cell. FIG. 3A depicts
the system 30 including a mechanism 32 for holding a cell 24.
Mechanism 32 is a suitable microrobotic device including associated
Microsystems as are generally known in the biotechnology arts. Such
as device 32 preferably is capable of holding individual cells or
controlled groups of cells. Further, mechanism 32 may also be used
to obtain biological, electrical, optical, or other data from the
cell 24.
[0026] Referring now to FIG. 3B, the system 30 is shown including
the mechanism 32 holding the cell 24, and a controllable
electroporation system including the broad effect sub-system 16 and
the narrow effect sub-system 18, whereby the narrow effect
sub-system 18 is focused at a location on the cell 24 to induce
opening of a pore 34.
[0027] Referring now to FIG. 3C, a macromolecule 38 is introduced,
for example, via a nano-nozzle or other suitable injection device
36. When the narrow effect sub-system 18 and/or the broad effect
sub-system 16 is removed, the pore will close, resulting in cell
24' having macromolecule 38 therein.
[0028] FIGS. 4A and 4B show one embodiment of a controllable
electroporation system 40 for introducing controllably opening a
pore 34 in a cell 24, e.g., for introduction of macromolecules as
described above. The system 40 includes a broad effect sub-system
in the form of an electric field producing apparatus 42, 44, 46,
and a laser beam 48 from a suitable source (not shown). The
electric field producing apparatus is in the form of an electrode
plate 42 coupled to a switchable (via a switch 44) voltage source
46. As shown, the laser beam may be focused, and the electric field
applied to active the pore opening mechanism, akin to a logical
"and" circuit as described above.
[0029] With the system and method described with respect to FIGS.
3A-3D and 4A-4B, researchers could only expose a few cells in a
tissue construct to a biological compound and observe how the
signal is propagated to its neighboring cells. Alternatively,
researchers could study if asymmetric cells such as neurons and
gastrointestinal mucosa cells react differently to compounds
injected at different places.
[0030] Referring now to FIGS. 5A and 5B, a system 50 is shown that
operates similar to that of FIGS. 3A-3D or 4A-4B in conjunction
with an array 52 of cells 24. When the broad effect sub-system 16
and the narrow effect sub-system 18 are operated, pores 34 are
formed in the cells 24. Such pores may be used for selective
introduction of macromolecules into the cells 24.
Separation Device Referring now to FIGS. 6-10, various embodiments
of filtration/separation devices are provided using the
controllable electroporation system herein.
[0031] FIG. 6 depicted one embodiment of a system 60, e.g., a
molecular sieve. System 60 includes plural membrane layers 62,
e.g., phospholipid bilayers. Each membrane layer 62 may be subject
to a narrow effect sub-system during application of the broad
effect sub-system, alternatively the location of the pores 64 may
be predetermined upon assembly and or manufacture, e.g., with
suitable micro- or nano-defects, or may be identical whereby
different voltage levels at each layer determined the pore size.
Application of different voltages (e.g. V1, V2 and V3) at each
layer creates a filter gradient from large pores 64 to small pores
64, allowing passage of molecules 66 through suitable layers.
[0032] Using a phospholipid bilayer, which is extremely cheap to
manufacture, the same filter 60 can be used repeatedly and adapted
for any size requirements using electroporation and carefully
controlling the electric field that is applied. Instead of
depending on multiple filters, a single filter could be used and
configured for any situation.
[0033] Referring to FIG. 7, a system 70 is shown with a molecular
sieve functioning similar to that of FIG. 6 associated with a
biochip array 72 having channels 74 therein. Channels 74 may serve
to collect macromolecules and molecules at each level based on
size. Further, channels 74 may incorporate or be associated with a
gradient system, e.g., pressure, thermal, electrical, or other
gradient to induce macromolecules and molecules toward the array
72. Array 72 may be any suitable microfluidic or nanofluidic
device. For example, methods of manufacturing such devices are
described in Reveo Inc. PCT Application No. PCT/US03/37304 filed
Nov. 30, 2003 entitled "Three Dimensional Device Assembly and
Production Methods Thereof", which is incorporated by reference
herein.
[0034] FIG. 8 shows another example dynamic filtration device,
wherein an array of lasers provides positional control over the
pore openings. FIG. 80 shows a filtration system 80 including a
membrane layer 82 associated with a broad effect energy sub-system
16 and a narrow effect sub-system 18. For example, the narrow
effect sub-system 18 may be generated with a laser array 88.
Alternatively, instead of an array of lasers 88, a beam steering
device may be incorporated, allowing use of only one laser source.
When a laser is activated from the array associated with a certain
position on the membrane 82, a pore 84 will open. The size of the
pores may be controlled by predetermined membrane characteristics,
area or magnitude of the narrow effect energy sub-system, or
magnitude of the broad effect energy sub-system.
[0035] Cells, proteins, enzymes, DNA molecules, RNA molecules, and
other macromolecules or molecules may be collected via an array of
containers 86, e.g., on a suitable microfluidic device. Thus,
separation device 80 may be made extremely compact and highly
flexible for any purpose.
[0036] FIG. 9 shows an example of an electrode suitable for
providing the broad effect energy sub-system in various embodiments
shown herein. By providing electrodes in a grid pattern, a suitable
field generating system may be provided to allow access for various
purposes including the narrow effect sub-system, macromolecule
introduction, filtration, or any other purpose.
[0037] In addition to filtering based on size, the aforementioned
separation devices may also separate on the basis of ionic charge,
since the applied voltage will drive only one type of ions across
the membrane.
[0038] While preferred embodiments have been shown and described,
various modifications and substitutions may be made thereto without
departing from the spirit and scope of the invention. Accordingly,
it is to be understood that the present invention has been
described by way of illustrations and not limitation.
* * * * *