Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Cell membrane or cell surface is target
Reexamination Certificate
2000-04-25
2003-02-18
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Cell membrane or cell surface is target
C435S461000, C600S373000, C600S377000, C600S378000, C600S381000, C600S395000
Reexamination Certificate
active
06521430
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a highly spatially resolved method for permeabilisation of cell structures, organelle structures and cell-like structures in order to transfer compounds into or out of the structures. It also relates to use of this method.
BACKGROUND OF THE INVENTION
During the last two decades there has been a tremendous growth in experimental methods that allow for biochemical and biophysical investigations of single cells. Such methods include patch clamp recordings which can be used for measurement of transmembrane currents through a single ion channel (O. P. Hamill, A. Marty, E. Neher, B. Sakman, F. J. Sigworth,
Pfleugers Arch
. 391, 85-100 (1981)); laser confocal microscopy imaging techniques that can be used to localise bioactive components in single cells and single organelles (S. Maiti, J. B. Shear, R. M. Williams, W. R Zipfel, W. W. Webb,
Science
, 275, 530-532 (1997)); use of near field optical probes for pH measurements in the cell interior; and use of ultramicroelectrodes for measurement of release of single catechol- and indol-amine-containing vesicles (R. H. Chow, L. von Ruden, E. Neher,
Nature
, 356, 60-63 (1992) and R. M. Wightman, J. A. Jankowski, R. T. Kennedy, K. T. Kawagoe, T. J. Scroeder, D. J. Leszczyszyn, J. A. Near, E. J. Diliberto Jr., O. H. Viveros,
Proc. Natl. Acad. Sci. U.S.A
., 88, 10754-10758 (1991)). Although numerous high-resolution techniques exist to detect, image and analyse the contents of single cells and subcellular organelles, few methods exist to control and manipulate the biochemical nature of these compartments. Most compounds for biological and medical use that are of interest to include in cells are polar. Polar solutes are cell-impermeant and unable to pass biological membranes unless specific transporters exist. Often in experimental biology as well as in biochemical and clinical work, polar solutes need to be administered to the cytoplasm of cells or to the interior of organelles. Examples of such polar solutes are nanoparticles, dyes, drugs, DNAs, RNAs, proteins, peptides, and amino acids. At present, it is extremely difficult, for example, to label a cell in a cell culture with a dye, or transfect it with a gene without labelling or transfecting its adjacent neighbour. It is even more difficult to introduce polar molecules into organelles because of their size which many times is smaller than the resolution limit of a light microscope, or at least less than a few micrometers in diameter.
Microinjection techniques for single cells and single nuclei have also been described (see e.g. M. R. Capecchi,
Cell
, 22, 479-488 (1980), but these get increasingly difficult to implement as the size of the cell or organelle decreases. For cells and organelles measuring only a few micrometers in diameter or less, microinjection techniques become virtually impossible to use.
It has for a long time been recognised that cell membranes can be permeabilised by pulsed electric fields (see e.g. Zimmermann, U.
Biochim. Biophys Acta
, 694, 227-277 (1982); Tsong, T. Y.
Biophys. J
., 60, 297-306 (1991); Weaver, J. C.
J. Cell. Biochem
., 51, 426-435 (1993)). This technique is called electroporation. The membrane voltage, V
m
, at different loci on phospholipid bilayer spheres during exposure in a homogenous electric field of duration t, can be calculated from:
V
m
=1.5r
c
Ecos&agr;[1-exp (−&tgr;/
t
)] (1)
where E is the electric field strength, r
c
is the cell radius, &agr;, the angle in relation to the direction of the electric field, and &tgr; the capacitive-resistive time constant. Pore-formation will result at spherical coordinates exposed to a maximal potential shift, which is at the poles facing the electrodes (cos&agr;=1 for &agr;=0; cos&agr;=−1 for &agr;=&pgr;). Generally, electric field strengths on the order of from 1 to 1.5 kV/cm for durations of a few &mgr;s to a few ms are sufficient to cause transient permeabilisation in 10-&mgr;m-outer diameter spherical cells. A recent study shows that isolated mitochondria, because of their correspondingly smaller size, require 7-10-fold higher electric field strengths to incorporate a 7.2-kilobase plasmid DNA (J-M. Collombet, V. C. Wheeler, F. Vogel, & C. Coutelle
J. Biol. Chem
., 272, 5342-5347 (1997)). Mitochondrial outer-membrane fusion at lower electric field strengths of about 2.5 kV/cm has also been observed.
Traditionally, electroporation is made in a batch mode allowing for administration of polar solutes into several millions of cells simultaneously. The electrodes producing such fields can be several square centimetres and the distance between the electrodes several centimetres, thus requiring high-voltage power sources to obtain the needed electrical field strength to cause electrically induced permeabilisation of biological membranes.
One advantage of electroporation compared to microinjection techniques is that electroporation can be extremely fast, and precisely timed (see e.g. K. Kinosita, K., Jr., I. Ashikawa, N. Saita, H. Yoshimura, H. Itoh, K. Nagayama, & A. Ikegami
J. Biophys
., 53, 1015-1019 (1988); M. Hibino, M. Shigemori, H. Itoh, K. Nagayama, & K. Kinosita, K., Jr.,
Biophys. J
., 59, 209-220 (1991)) which is of importance in studying fast reaction phenomena.
Instrumentation that can be used for electroporation of a small number of cells in suspension (K. Kinosita, Jr., & T. Y. Tsong, T.
Biochim. Biophys. Acta
, 554, 479-497 (1979); D. C Chang,
J. Biophys
., 56, 641-652 (1989; P. E. Marszalek, B. Farrel, P. Verdugo, & J. M. Fernandez,
Biophys. J
., 73, 1160-1168 (1997)) and for a small number of adherent cells grown on a substratum (Q. A. Zheng, & D. C. Chang,
Biochim. Biophys. Acta
, 1088, 104-110 (1991); M. N. Teruel, & T. Meyer
Biophys. J
., 73, 1785-1796 (1997)) have also been described. The design of the electroporation device constructed by Marszalek et al. is based on 6 mm long 80 &mgr;m diameter platinum wires that are glued in a parallel arrangement at a fixed distance of 100 &mgr;m to a single glass micropipette. The design by Kinosita and Tsong uses fixed brass electrodes spaced with a gap distance of 2 mm, the microporator design of Teruel and Meyer relies on two platinum electrodes that are spaced with a gap distance of about 5 mm, and the electroporation chamber design by Chang uses approximately 1 mm-long platinum wires spaced at a distance of 0.4 mm. It is obvious, that these electroporation devices, which are optimized for usage in vitro, create electric fields that are several orders of magnitude larger than the size of a single cell which typically is 10 &mgr;m in diameter, and thus can not be used for exclusive electroporation of a single cell or a single organelle or for electroporation inside a single cell. The techniques do not offer a sufficient positional and individual control of the electrodes to select a single cell, or a small population of cells. Furthermore, these techniques are not optimized for electroporation in vivo or for electroporation of remote cells and tissue. Electroporation devices for clinical and in vivo applications have also been designed. Examples include devices for electroporation-mediated delivery of drugs and genes to tumours (WO 96/39226) and to blood cells (U.S. Pat. No. 5,501,662) and to remote cells and tissue (U.S. Pat. No. 5,389,069). Likewise, these can not be used to create a highly localised electric field for electroporation of a single cell, a single organelle, or a population of organelles within a cell.
SUMMARY OF THE INVENTION
One of the major disadvantages with the known techniques is that they are not applicable for permeabilisation of single cells or single intracellular organelles.
The present invention provides a highly spatially resolved technique to alter the biochemical content of single cells and organelles, based on permeabilisation of phospholipid bilayer membranes by pulsed electric fields, i.e. so called electroporation.
An advantage the method according to the present invention co
Chiu Daniel
Eriksson Peter
Lundqvist Anders
Orwar Owe
Cellectricon AB
Conlin David C.
Edward & Angell LLP
Katcheves Konstantina
Ketter James
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