Chemistry: molecular biology and microbiology – Treatment of micro-organisms or enzymes with electrical or... – Cell membrane or cell surface is target
Reexamination Certificate
2000-04-11
2001-12-04
Wang, Andrew (Department: 1636)
Chemistry: molecular biology and microbiology
Treatment of micro-organisms or enzymes with electrical or...
Cell membrane or cell surface is target
C600S012000
Reexamination Certificate
active
06326177
ABSTRACT:
BACKGROUND OF THE INVENTION
Biological cells consist of cytoplasm surrounded by a membrane. The cytoplasm is conducting, the membrane, which is made up of a lipid bilayer, can be considered a dielectric. The application of electric fields to biological cells causes buildup of electrical charge at the cell membrane, and consequently a change in voltage across the membrane. For eukaryotic cells the transmembrane voltage under equilibrium condition is approximately 70 mV. In order to affect membrane processes by means of external electric fields, the amplitude of these fields (“E”) must be such that it generates a potential difference (“V
m
”) at least on the same order as the resting potential. The amplitude of the electric field is:
E=V
m
/fa
(1)
where a is the radius of the cell and f is a form factor which depends on the shape of the cell. For spherical cells, f is 1.5; for cylindrical cells of length l, with hemispheres of diameter d at each end, the form factor is
f=l
/(
l−d/
3) (2)
For a biological cell with an assumed radius of about 5 &mgr;m and a spherical shape, the external electric field required to generate a voltage of the same amplitude as the resting potential across the membrane is on the order of 100 V/cm. Due to their smaller size, the electric field required to affect the membrane permeability of bacteria is much higher, on the order of kV/cm.
For external electric fields of a magnitude such that the change in membrane potential is on the order of the resting potential, voltage induced opening of channels in the membrane causes flux of ions through the membrane. This leads to changes in the ion concentration close to the cell membrane, and consequently causes cell stress. The stress lasts on the order of milliseconds, and generally does not cause permanent cell damage. If the strength of the electric field is increased such that the voltage across the cell membrane reaches levels on the order of one volt, the membrane permeability increases to such a level that either the cell needs from seconds to hours to recover (reversible breakdown), or cell death may occur. The mechanism of the membrane breakdown is not well understood. A common hypothesis is that pores are generated in the membrane. The pores can be of sizes which allow the exchange of macromolecules. If the transmembrane voltages are sufficiently high the pores will not close anymore. The use of the reversible breakdown effect has been reported in electroporation and in biofouling prevention. The irreversible effect has been employed in the debacterialization of water and food.
The effect of electric fields on biological cells is not simply dependent on the magnitude of the applied electric field, but also on its duration. This can be understood by considering a model for the electrical equivalent circuit of the cell, shown schematically in FIG.
1
. The model shown in
FIG. 1
does not take the effect of structures inside the cell into account. The cell (in suspension) is modeled by a resistance and capacitance. For a pulse duration which is long compared to the dielectric relaxation time of the suspension, the capacitive component of the suspension impedance can be neglected. For many cell suspensions and seawater (i.e., aqueous solutions with relatively high ionic strengths) the dielectric relaxation time is on the order of nanoseconds. The cell membrane can be modeled as capacitor, the cytoplasm as a resistor. The outer membrane contains channels which are affected by the applied voltage and allow flow of ions through the membrane, representing a leakage current. The voltage-gated channels can be modeled as variable, voltage-dependent resistors.
When a voltage pulse is applied to the cell, charges accumulate at the membrane and the membrane voltage is increased. The charging time constant of the cell membrane may be represented by equation (3):
&tgr;=(&rgr;
1
/2+&rgr;
2
)Cr (3)
with &rgr;
1
being the resistivity of the suspending medium, e.g. water, &rgr;
2
being the resistivity of the cytoplasm, C the capacitance per unit area, and r the cell radius (spherical cell). Using typical data for cells, the duration of the electric field pulses required to generate a potential difference of 1 V across the membrane can be calculated. The energy, W, dissipated in the suspension is given by:
W=E
2
&tgr;/&rgr;
1
(4)
Electric field and energy density are plotted in
FIG. 2
versus pulse duration for spherical cells of radius 5 &mgr;m in a suspension with a resistivity of 50 &OHgr;cm. The resistivity of the cytoplasm is assumed to be 100 &OHgr;cm. The curves show a minimum at 100 nsec. This is the pulse duration where the stunning or killing of these kind of biological cells is predicted to be most effective. Experimental studies have reported which confirm the presence of such a minimum.
Modifications of cells which lead to rupture of the cell membrane can lead to cell death via necrosis, a nonphysiological type of cell destruction. It would be advantageous to be able to initiate cell death via apoptosis in a selective manner. This would allow the destruction of cells without engendering the non-specific damage to surrounding tissues due to inflammation and scarring that is normally observed with necrosis. The ability to selectively modify cells in ways that lead to apoptosis could provide a new method for the selective destruction of undesired cells/tissue (e.g., cancer cells, fat cells or cartilage cells) while minimizing side effects on surrounding tissue.
SUMMARY OF THE INVENTION
The present invention relates to a method for modifying cells by intracellular electro-manipulation. The method includes applying one or more ultrashort electric field pulses to target cells. The ultrashort electric field pulse generally has at least a sufficient amplitude and duration when applied as a sequence of pulses to modify subcellular structures in the target cells. The amplitude of individual pulses do not exceed the irreversible breakdown field of the target cells. The amplitude and duration of the ultrashort electric field pulse(s) are typically chosen so as to be insufficient to permanently alter permeability of surface membranes of the target cells, e.g., by rupturing the surface membranes.
The targeting of substructures of cells rather than cell membranes can have a utility in treatments involving the selective destruction of cells (e.g., tumor cells) without substantially damaging surrounding tissue(s). Most therapeutic applications of the pulsed electric field method require that the fields be applied to tissues rather than single cells. With the comparatively long pulses currently employed in such treatments (e.g., several hundred microseconds in length), however, the electric field seems to be less effective in treating tissue compared to single cells. It is known that although generalized tissue can be regarded as an aggregate of cells, with different types of cell-cell interconnections, tissue electroporation consists of electroporation of individual cells. There are two major differences between the electroporation of individual cells in a suspension and the electroporation of tissue. In tissue, the local extracellular electric field depends in a complicated way on the many neighboring cells. In addition, for tissues the ratio of the extra- to intracellular volume is usually small, just the opposite of most in vitro electroporation conditions. This means that if chemical exchange between the intra- and extracellular volumes is the main cause of cell stress, and therefore cell death, tissue electroporation with microsecond pulses may be intrinsically less damaging than most in vitro electroporation conditions. Since ultrashort pulses affect the interior of the cell, such pulses are expected to have roughly the same effect on tissues as on individual cells.
Another advantage of using ultrashort pulses of the type employed in the present method is the low energy of these pulses. Although the electrical power of the pulses m
Beebe Stephen J.
Buescher E. Stephen
Schoenbach Karl H.
Eastern Virginia Medical School of the Medical College of Hampto
Foley & Lardner
Ketcheves Konstantina
Wang Andrew
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