WO2001080946A1

WO2001080946A1 - Electroporation device with measurement of electrical properties

Info

Publication number: WO2001080946A1
Application number: PCT/IT2001/000197
Authority: WO
Inventors: Damijan Miklavcic; Lluis Mir
Original assignee: Igea S.R.L.
Priority date: 2000-04-21
Filing date: 2001-04-20
Publication date: 2001-11-01
Also published as: ITTO20000387A0; IT1320520B1; AU5873001A; ITTO20000387A1

Abstract

A method for electroporation of a substrate (35) containing cells, the method including the steps of generating (110) and applying an adjustable number (N) of pulses to the substrate, and regulating the number on the basis of the impedance measured in the subsrate (35); the impedance being a function of the permeabilization of the cell membranes.

Description

ELECTROPORATION DEVICE WITH MEASUREMENT OP ELECTRICAL PROPERTIES

ELECTROPORATION DEVICE AND METHOD, WHERE THE POST-PULSE MEASUREMENT OF ELECTRIC PROPERTIES OF THE SAMPLE ALLOWS TO STOP THE TRAIN OF PULSES WHEN CELL ELECTROPERMEABILIZATION IS ACHIEVED

TECHNICAL FIELD

The present invention relates to an electroporation device and method, where the post-pulse measurement of electric properties of the sample allows to stop the train of pulses when cell electropermeabilization is achieved.

BACKGROUND ART

As is known, recent biological, microbiological and pharmacological applications involve introducing molecules into cells, which is done by inserting the molecules through the cell membranes .

The molecules may be inorganic substances (e.g. drugs) or organic molecules (cells are known to be inserted, for example, with DNA molecules) .

Molecules are introduced using various methods, including: viral vectoring : associating the molecule with a virus, which is then introduced into the cell; chemical vectoring : associating the molecule with a chemical substance for reducing the resistance of the cell membrane and so permitting introduction of the molecule into the cell; and ballistic methods : accelerating the molecule so that it strikes and penetrates the cell membrane.

Known methods involve several drawbacks, including: risk of immune reaction to the vector; production difficulties and poor stability of the vector itself

(viral vectoring) ; ineffectiveness, toxicity and poor selectivity (chemical vectoring) . As for ballistic methods, these only apply to surface ceils.

New so-called electroporation methods have recently been devised, which provide for applying an electric field to the cells to permeabilize, and so enable substances to penetrate the cell membrane.

Electroporation methods normally; involve emitting a number of voltage pulses, which are applied to electrodes close to the cells to direct a pulsating electric field onto the cells. > One problem posed by known electroporation methods is establishing the number of pulses to be applied, which is normally determined by trial and error. As a result, the number of pulses applied may be too low, thus resulting in incomplete permeabilization of the cell membranes, or too high, thus possibly resulting in damage to the cells.

DISCLOSURE OF INVENTION

It is an object of the present invention to provide an electroporation device and method designed to eliminate the drawbacks of known electroporation devices and methods .

According to the present invention, there is provided an electroporation device as described in Claim 1.

The present invention also relates to an electroporation method as described in Claim 6.

BRIEF DESCRIPTION OF THE DRAWINGS A preferred, non-limiting embodiment of the invention will be described by way of example with reference to the accompanying drawings, in which:

Figure 1 shows, schematically, an electroporation device in accordance with the teachings of the present invention;

Figure 2 shows a logic operating diagram of the Figure 1 device;

Figure 3 shows a graph of a quantity controlled by the device according to the present invention. BEST MODE FOR CARRYING OUT THE INVENTION

Number 1 in Figure 1 indicates as a whole an electroporation device .

Device 1 comprises a signal generator, in particular a voltage pulse generator 3 having at least two output electrodes 5; a measuring system 7 connected to output electrodes 5; and an electronic control unit 10 for controlling voltage pulse generator 3 and measuring system 7. Electronic control unit 10 comprises at least one microprocessor 12 cooperating with memory devices, e.g. a RAM memory 14 and EPROM memory 16; and interface devices 18. Pulse generator 3 comprises a digital-analog D/A converter 20, which receives a control signal CNTRL from unit 10 and cooperates at the output with a preamplifying circuit 21; preamplifying circuit 21 has an output connected to the input of a power amplifier 22 in turn having an output communicating with electrodes 5; and electrodes 5, in the example embodiment shown, are each defined by a flat, rectangular metal blade to which the output signal from power amplifier 22 is applied.

The electrodes may, of course, differ in shape, structure and size from those shown, e.g. may be designed for use in a laparoscopy process .

Measuring system 7 comprises an oscillating circuit 24 for supplying electrodes 5 with an excitation signal; and a converting circuit 26 supplied by electrodes 5 with a signal in response to the excitation signal . Converting circuit 26 cooperates with a memory 28 (e.g. a RAM memory) which is also connected to a known measuring circuit 30, which also cooperates with converting circuit 26 and with oscillating circuit 24. Figure 2 shows a block diagram of the operations performed by electroporation device 1 under the control of electronic unit 10.

When device 1 is activated, a first block 100 measures the impedance value between electrodes 5. More specifically, impedance Zt(ω) is measured in known manner by measuring system 7, which may determine one of several of the following parameters for instance the absolute impedance value |Zt(ω)|, the real impedance part Zr, the imaginary part jZo, or angle α = arctg( Zo/Zr) .

Alternatively or in addition of the measure of the impedance the device may also measure different electric characteristics such as: admittance, resistivity or conductivity including dynamic resistance or dynamic conductivity. The device may also measure current at constant voltage and vice versa.

Block 100 is followed by a block 110, which generates a control signal CNTRL for pulse generator 3, which, in response, produces one voltage pulse II which is applied to electrodes 5.

Pulse II is preferably rectangular, and has a predetermined time width and predetermined constant amplitude. Alternatively block 110 may also generate a rectangular current pulse instead of a voltage pulse.

Block 110 is followed by a block 120, which measures the instantaneous impedance value Zt+1 (ω) between electrodes 5. Value Zt+1 (ω) is measured at instant t+1 after instant t at which the measuring and generating operations in respective blocks 100 and 110 are performed, so that impedance Zt+l(ω) is measured after pulse II is emitted.

It is also clear that also other characteristics may be measured after the pulse has been emitted by block 110; for instance block 120 may measure resistivity, admitance, conductivity, dynamic conductivity or resistivity, also through measuring current at constant voltage or vice versa.

Block 120 is followed by a block 130, which calculates the variation in impedance ΔZ(ω) between electrodes 5 between instants t and t+1, i.e. the difference between the impedance (Zt (ω) ) measured before pulse II is emitted, and the impedance (Zt+1 (ω) ) measured after the pulse is emitted, i.e.:

Block 130 is followed by a block 140, which determines whether impedance variation ΔZ(co) is strictly above zero, i.e.: Δz(ω) > 0 If impedance variation ΔZ(ω) is greater than zero:

then impedance decreases following emission of pulse II . Conversely, if impedance variation Δz (ω) is substantially equal to zero:

Zt(ω) = zt+l(ω) then there is no variation in impedance following emission of pulse II. In the first case (Δz(ω) > 0), block 140 goes back to block 110 to emit a further pulse II. Otherwise (Δz(ω) = 0), pulse emission is terminated.

In actual use, electrodes 5 are applied to, and form an electric contact with, a tissue portion 35 (shown schematically in Figure 1) containing live cells. The tissue portion may be one forming part of a live being (human, animal or vegetable) or one containing cells removed from a live being (human, animal or vegetable) . Tissue portions are also understood to include cultures of uni- or multicellular organisms. In other words, a tissue portion is intended, to mean, in general, a substrate of any nature on which live cells or cellular organisms are present .

Tissue portion 35 is also applied with a substance

(organic or inorganic) 37 to be introduced into the cells. The substance may be applied in a number of different ways, some of which are listed below by way of non-limiting examples: direct application of the substance to the tissue portion, e.g. by applying the tissue portion with a fluid containing the substance; indirect application of the substance, e.g. by introducing the substance into the circulatory system of the tissue portion; injecting the substance, e.g. using needlelike electrodes 5 , each having an inner conduit containing the substance to be injected into the tissue portion. Needles separate from electrodes 5 may, of course, also be used.

The substance introduced may be inorganic or organic, e.g. a DNA molecule containing one or more regulatory sequences and/or sequences coding for therapeutic genes or genes of interest for biomedical or biotechnological purposes ,- . an oligonucleotide, whether natural (phosphodiesters) or modified (inside the backbone of the oligonucleotide, such as phosphosulfates, or at the extremities, by addition of groups to protect the oligonucleotides from digestion by nucleases) - the description of oligonucleotide modification being non-limiting; . a protein or peptide, whether natural or genetically or chemically modified, obtained by natural means or by synthesis, or a molecule mimicking the structure of a protein or peptide, whatever its chemical backbone; . a cytotoxic agent; in particular, of cytotoxic agents, the antibiotic bleomycin or cisplatinum; . a penicilli ,- . a nucleic acid; . a pharmacological agent other than a nucleic acid.

Device 1 is activated to immediately determine (block 100) the initial impedance of tissue 35, the value of which depends essentially on the electrode geometry, the tissue type and permeability of the cell membranes.

A first voltage pulse is then generated (block 110) and applied to electrodes 5 to produce an electric field directed into tissue 35, and which initiates permeabilization of the tissue 35 cell membranes. Following permeabilization, the impedance of tissue 35 decreases. Tissue impedance is then measured (block 120) at an instant following that at which pulse II is emitted; which impedance, for the reasons given above, is typically lower than the initial impedance, so that impedance variation Δz(ω) is positive and block 110 is reselected (block 140) to emit a further pulse II. The device remains in the loop defined by blocks 110, 120, 130, 140 until impedance variation Δz(ω) equals zero. A variable N number of pulses II is thus generated until no significant variation in impedance is detected, thus protecting the tissue by preventing the application of pulses to an already highly permeabilized tissue.

The N number of pulses generated is regulated automatically by the device on the basis of the impedance measured in substrate 35, and therefore corresponds with that required to achieve complete permeabilization of the tissue cells. Substance 37 is then introduced into the cells of substrate 35.

Figure 3 , plotted on the basis of tests conducted by the Applicant, shows how permeabilization of the tissue varies as a function of the number of pulses generated.

More specifically, the x axis shows the number of pulses applied to the tissue, and the y axis the percentage of cells permeabilized. As can be seen, the percentage of permeabilized cells increases rapidly with emission of the first few pulses, and then levels off following emission of a given number of further pulses (in this case six pulses) . At which point (roughly 100% of the cells permeabilized and no significant variation in impedance) , the device according to the present invention arrests pulse emission, so that only the number of pulses strictly required to permeabilize all the cells is generated.

Clearly, changes may be made to the device as described herein without, however, departing from the scope of the present invention.

As opposed to being measured using the same pair of electrodes 5 used to apply the electric field to substrate 35, as in the embodiment described above, the impedance of substrate 35 may also, obviously, be measured by a separate pair of auxiliary electrodes (not shown) close to electrodes 5 and placed in contact with tissue portion 35 to be permeabilized.

Claims

1) An electroporation device comprising signal generating means (3) connectable at the output to electrodes (5) fittable to a substrate (35) comprising cells; said electrodes (5) producing, in said substrate (35) , an electric field which induces permeabilization of the membranes of said cells to facilitate introduction of substances (37) into the cells,- characterized in that said signal generating means (3) comprise pulse generating means (110) for generating an adjustable number (N) of pulses; said number (N) being regulated automatically on the basis of at least one electric characteristic (Z(ω)) measured in said substrate (35); and said electric characteristic (Z(ω)) changing as function of the permeabilization of the membranes of said cells .

2) A device as claimed in Claim 1, characterized by comprising: - first measuring means (100) for determining the electric characteristic (Z(ω)) in said substrate prior to emission of a pulse;

- second measuring means (120) for determining the electric characteristic (Z(co)) in said substrate following emission of said pulse; calculating means (130) for calculating the variation in the electric characteristic determined by the first and second measuring means (100, 120) ; and - repeating means (140) for reselecting said pulse generating means (110) to emit at least one further pulse in the event said variation is significant, in particular, in the event said variation is greater than zero; said repeating means (140) arresting pulse generation in the event said variation is insignificant, in particular, in the event said variation equals zero.

3) A device as claimed in Claim 1 or 2, characterized in that said electric characteristic is the impedance of said substrate (35) .

4) A device as claimed in any one of the foregoing Claims, characterized in that said signal generating means (3) generate voltage pulses.

5) A device as claimed in Claim 4, characterized in that said voltage pulses are rectangular in shape.

6) A method for electroporation of a substrate (35) containing cells, for introducing at least one substance (37) into the cells; characterized by comprising the steps of: - generating (110) and applying an adjustable number (N) of pulses to the substrate (35) ; and

- automatically regulating said number (N) on the basis of at least one electric characteristic (Z(ω)) measured in said substrate (35) ,- said electric characteristic (Z(ω)) being a function of the permeabilization of the membranes of said cells.

7) A method as claimed in Claim 6, characterized by comprising the steps of -. determining (100) the electric characteristic (Z(ω)) in said substrate prior to emission of a pulse;

- emitting (110) said pulse (II) ; determining (120) the electric characteristic (Z(ω)) in said substrate following emission of said pulse;

- calculating (130) the variation in the electric characteristic determined before and after emission of said pulse,- and - generating a further pulse (140, 110) in the event said variation is significant, in particular, in the event said variation is greater than zero; and arresting

(140) pulse generation in the event said variation is insignificant, in particular, in the event said variation equals zero .

8) A method as claimed in Claim 6 or 7, characterized in that said electric characteristic comprises the impedance of said substrate.

9) A method as claimed in one of Claims 6 to 8, characterized in that said step of generating a pulse comprises the step of generating a voltage pulse.

10) A method as claimed in Claim 5, characterized in that said substance is selected from a list comprising:

. a nucleic acid; . a DNA molecule;

. an oligonucleotide;

. a protein;

. a peptide; a cytotoxic agent, in particular the antibiotic bleomycin or cisplatinum; . a penicillin; . a pharmacological agent other than a nucleic acid.