Highly controllable electroporation and applications thereof
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.
This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 60/439,387 filed on Jan. 10, 2003, which is herein incorporated by reference
BACKGROUNDThe 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.
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)).
For example,
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).
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.
Therefore, it would be desirable to provide a method and system to overcome these and other limitations of conventional electroporation.
SUMMARYThe 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.
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
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
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.
Therefore, for example as compared to conventional electroporation processes of cellular membranes, described in the Background and with respect to
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.
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
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,
Referring now to
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With the system and method described with respect to
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Separation Device Referring now to
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.
Referring to
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.
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.
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.
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)
Type: Application
Filed: May 20, 2005
Publication Date: Apr 6, 2006
Inventor: Sadeg Faris (Pleasantville, NY)
Application Number: 11/134,078
International Classification: C12N 15/87 (20060101);