Surgery: light – thermal – and electrical application – Light – thermal – and electrical application – Electrical therapeutic systems
Reexamination Certificate
2000-11-22
2003-04-01
Jastrzab, Jeffrey R. (Department: 3762)
Surgery: light, thermal, and electrical application
Light, thermal, and electrical application
Electrical therapeutic systems
C607S074000
Reexamination Certificate
active
06542778
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a process for the manipulation or processing of biological objects by means of electrical pulses, in particular, for the permeation and/or fusion of cells or of synthetic, membrane-encased structures such as liposoma or vesicles, or for the permeation of membrane or layered materials in electrode structures, and devices for the implementation of the process.
BACKGROUND
With many biotechnological, medical, or genetic tasks, the short-term and reversible increase in the permeability of the covering of living cells suspended in a fluid is of interest (overview in “Electromanipulation of Cells”, U. Zimmermann, G. A. Neil, CRC, 1996). In addition to chemical and virus-based methods, the simplicity and definability of the application have caused permeabilization by means of short electrical pulses to come to prominence. The process is designated as “electroporation,” “electropermeation,” or also “electropermeabilization,” and is implemented with commercially available equipment from a number of manufacturers.
In conventional electroporation devices, the cells which are to be manipulated are suspended in a solution in a cuvette between electrodes for the application of electrical pulses. What all the devices have in common is the use of one or more electrical pulses of an amplitude of between 10 V up to several 1000 V. The pulse form depends on the device. The pulse duration is selected dependent on the object (dependent on the size in particular), and lies in the range from a few &mgr;s up to several 100 ms. The electrodes (in most cases two) are in each case permanently connected in pairs, as cathode and anode, respectively.
One serious problem of electropermeabilization which has not hitherto been solved lies in the fact that the pH in the vicinity of the electrodes changes locally due to electrolysis manifestations which are not temporally stationary, with the result that sustained interference in the poration conditions occurs. Depending on the composition of the solution, subjecting the electrodes to electrical pulses leads to creation of H
+
(protons) or OH
−
(hydroxyl ions) enrichment (“clouds”) and, therefore, to a local acidic or alkaline area. As the pulse duration increases, a front of low or high pH value migrates from one electrode to the other. Even if the pH changes in the areas of the two electrodes are virtually neutralised over the entire fluid filling of the cuvette, the cells of the individual spatial areas are subjected to a pH gradient and change of up to 5 pH units, which can lead to a substantial influence on the life processes, up to the stage of devitalization and initiation of apoptosis.
In addition to this, the local pH changes may cause undesirable electrode reactions. If, for example, aluminium is used as the electrode material, there is the possibility of aluminium being resolved when the pulses are applied, and poisoning the cell suspension. This problem can indeed be solved by use of noble metals as the electrode materials. However, the result of this is that an electroporation device becomes very expensive.
With the known pulse techniques (capacitor discharge, square-wave pulse, delta pulse, etc.) it is not possible for the pH stress of the cells to be avoided, since this involves fundamental phenomenon at the anode and cathode. There have indeed been attempts before now to minimize the electrolysis effect by application of very short pulses. As a result, however, usability of the electroporation process is limited.
Particularly with objects with widely deposited layers, such as bacteria, longer pulse times are, however, more effective, so that pH changes have a particularly negative effect, and there is a need for alternative techniques without losing the advantage of the electrical field application.
A method for cell poration and fusion using electrical high frequency pulses is known from EP 0 710 718 A1. For pulse treatment, an electrode pair is subjected to high frequency voltages, whereby one electrode of the electrode pair has ground potential while the other electrode is subjected to a high frequency alternating voltage.
Thus, it would be advantageous to provide a process which is an improvement on conventional permeabilization systems, based on the electrical pulse technology. This would be characterised in particular by a reduced or almost compensated pH change stress on the cells, and the suppression of electrode reactions. It would be advantageous to provide a device for implementing the process, with which the possibilities of use of the electropermeation techniques referred to can be extended and the efficiency of the permeation or cell fusion can be increased.
SUMMARY OF THE INVENTION
The invention relates to a process for treating a biological or synthetic object subjected to an electrical field in a surrounding medium for a predetermined pulse time (t
1
), the electrical field being formed by at least two electrodes, whereby during the pulse time (t
1
) each of the electrodes is actuated at least once as the anode and at least once as the cathode, to cause at each electrode a polarity reversal and alternating electrolytic increases and reductions in pH of at least a portion of the object, wherein the object is subjected during the pulse time (t
1
) to a number of electrical partial pulses which possess partial pulse durations, with sequentially reversing polarity or field direction, whereby the partial pulses possess sequentially varying partial pulse durations (t
11
, t
12
), pulse forms, and/or pulse amplitudes, and the partial pulse durations (t
11
, t
12
), pulse forms, and/or pulse amplitudes are selected such that, due to the electrolytic increases and reductions in pH value, substantially equal H
+
and OH
−
ion concentrations are created.
The invention also relates to a device for treating a biological or synthetic object in a surrounding medium between at least two electrodes, which are connected to a pulse generator, wherein the pulse generator is connected to the electrodes via a control circuit arranged to impose on the electrodes, during a predetermined pulse time, at least two partial pulses of alternating reversed polarity or field direction, whereby the pulse generator provides the partial pulses in digitalized form for creation of asymmetries in pulse height, pulse path, or pulse length.
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patent: 4822470 (1989-04-01), Change
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patent: 6029090 (2000-02-01), Herbst
patent: 6264815 (2001-06-01), Pethig et al.
patent: 44 11 333 (1995-08-01), None
patent: 0 126 389 (1984-11-01), None
patent: 0 710 718 (1996-05-01), None
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patent: 60-251877 (1985-12-01), None
patent: 61-111680 (1986-05-01), None
patent: 63-049071 (1988-01-01), None
patent: WO 98/56893 (1998-12-01), None
Fuhr Günter
Hagedorn Rolf
Zimmermann Ulrich
Evotec OAI AG.
Jastrzab Jeffrey R.
Oropeza Frances P.
Schnader Harrison Segal & Lewis LLP
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