ELECTROPORATION DEVICE AND METHOD, DELIVERING A MODULATED SIGNAL UNDER CONTINUOUS CONTROL OF CELL ELECTROPERMEABILIZATION

Abstract: An electroporation device having a signal generating circuit (3) connectable at the output to electrodes (5) fittable to a substrate (35) having cells. The electrodes (5) produce an electric field which induces permeabilization of the cell membranes to facilitate introduction of substances into the cells. The amplitude of the signal (I2) applied to the electrodes is closed-loop controlled on the basis of the impedance (Z( omega )) of the substrate, so as to regulate the amplitude of the signal following a reduction in impedance caused by permeabilization of the cell membranes.

Description: ELECTROPORATION DEVICE AND METHOD, DELIVERING A MODULATED SIGNAL UNDER CONTINUOUS CONTROL OF CELL ELECTROPERMEABILIZATION TECHNICAL FIELD The present invention relates to an electroporation device and method, delivering a modulated signal under continuous control of cell electropermeabilization. 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 immunity 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 cells. New so-called electroporation methods have recently been devised, which provide for briefly applying a strong electric field to the cells to permeabilize, and so enable substances to penetrate, the cell membrane. One problem posed by known electroporation methods is establishing the value of the electric field applied, which must be high enough to permeabilize the cell membrane, but not so high as to cause irreversible damage to the cell. More specifically, known electroporation devices and methods employ a fixed output voltage value (determined, for example, experimentally) , so that, in certain operating conditions, the electric field may be too low, thus preventing introduction of the substances, and/or too high, thus resulting in irreversible damage to the cell. 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 13. 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; Figures 3 and 3a show signals produced by the Figure 1 device; Figure 4 shows a variation of the Figure 3, 3a signals . 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 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 pulse generator 3 and measuring system 7. Electronic control unit 10 comprises at least one microprocessor 12 co-operating 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 co-operates 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. Electrodes 5 may also comprise a number of electric terminals (Figure 1) arranged, for example, along a circular (triangular or hexagonal) closed-loop path C and supplied sequentially in pairs with the output signal from power amplifier 22. In which case, a multiplexer circuit (not shown) is provided to select and supply pairs of electrodes, e.g. pairs of electrodes on opposite sides of circular path C. The multiplexer circuit may also be associated with a circuit (not shown) for determining the impedance between pairs of electrodes in the number; and a circuit (not shown) for directing the signal to an automatically selected pair of electrodes . The pair may be selected automatically by comparing the measured impedance and selecting the pair (or the pairs) of electrodes whose impedance has a predetermined relationship with, e.g. is greater than, the impedance of the other pairs. 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 co-operates with a memory 28 (e.g. a RAM memory) which is also connected to a known measuring circuit 30, which also co-operates with converting circuit 26 and with oscillating circuit 24. It is noted that the measurement of the impedance may be done both in the frequency domain or in the time domain. 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 generates a first control signal CNTRLl for pulse generator 3, which, in response, produces a first number of first voltage pulses II which are applied to electrodes 5 (Figure 3) . First pulses II, which are preferably rectangular, are of constant amplitude and width and equally spaced in the time. The voltage value of each first pulse depends on the geometry of electrodes 5, while the total application time TI of the first pulses depends on the instant at which a change in impedance is detected. More specifically, block 100 is followed by a block 105, - which measures the impedance Z(ω) between electrodes 5. More specifically, block 105 calculates the impedance variation Δz(ω) between two successive instants t-1 and t, i.e. ΔZ(ω) = Δz(ω) t - ΔZ(ω) t-1 . When the impedance variation Δz (ω) remains substantially equal to zero (i.e. no noticeable change in impedance), block 105 goes back to block 100. When the impedance variation is less than zero (i.e. impedance decreasing and therefore changing with time) , block 105 is followed by a block 110. Block 110 generates a second control signal CNTRL2 for pulse generator 3, which, in response, produces a second number of second pulses 12 which are applied to electrodes 5 (Figure 3) . The amplitude of second pulses 12 depends on the impedance Z(ω) measured between electrodes 5. Impedance Z(ω) is measured in known manner by measuring system 7, and the measured impedance value is supplied to electronic control unit 10, which, in response, controls the amplitude of second pulses 12. More specifically, the amplitude of second pulses 12 decreases alongside a reduction in impedance, increases alongside an increase in impedance, and may also increase when impedance remains constant for a given length of time . The impedance measurement may comprise measuring the absolute impedance value |Z(ω)| or calculating the real impedance part Zr, the imaginary part jZo and angle α = arctg(Zo/Zr) . -Instead or in addition of impedance calculating the device could also measure other electric characteristics for instance, admittance, resistivity or conductivity including dynamic resistance or dynamic conductivity. The device could also measure current at constant voltage and vice versa. On average, second pulses 12 have a lower amplitude than the first pulses . Second pulses 12 are generated until the measured impedance reaches a predetermined lower threshold value zp indicating permeability of the cell membranes has been attained and no further changes in impedance between successive pulses are detected (block 115 following block 110) . Once the lower threshold value is reached, block 115 goes on to a block 120, thus providing for closed- loop control of the amplitude of pulses 12. Alternatively, block 110 may produce a single pulse Iu2 (Figure 3a) of an amplitude depending on the impedance Z(ω) measured between electrodes 5, thus providing for closed-loop control of the amplitude of pulse Iu2. More specifically, like pulses 12, the amplitude of single pulse Iu2 decreases alongside a reduction in impedance, increases alongside an increase in impedance, and may also increase when impedance remains constant for a given length of time. More specifically, pulse Iu2 normally decreases in amplitude as impedance decreases with time, and the closed-loop control regulates, by decreasing, the amplitude of pulse Iu2. The amplitude of pulse Iu2 may also increase when impedance remains constant for a given length of time. Block 120 generates a third control signal CNTRL3 for pulse generator 3, which, in response, produces a third number of third pulses 13 which are applied to electrodes 5 (Figure 3) . Third pulses 13 have a much lower amplitude than first and second pulses II and 12; are, for example, rectangular in shape; are applied for a predetermined time T3 ; are each of a time width greater than that of pulses II and 12; and may also comprise a single pulse 13. Block 120 is followed by a block 130, which enquires whether another electroporation cycle is to be performed. If it is, block 130 goes back to block 100; if it is not, electroporation is interrupted. In actual use, electrodes 5 are applied to 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 (prokaryotes or eukaryotes) . 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 or biopolymeric) 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. The substance may also be injected using needles separate from the electrodes . The substance introduced may be inorganic or organic or biopolymeric, e.g. . a nucleic acid; a DNA molecule containing regulatory sequences and sequence 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 of nucleoasis,- the description of oligonucleotide modifications being non-limiting) ; . a protein or peptide, whether natural or genetically or chemically modified, extracted from natural sources or obtained by synthesis, or a molecule simulating the structure of a protein or peptide, whatever its structure,- a cytotoxic agent, in particular, the antibiotic bleomycin or cisplatinum; . a penicillin; . a pharmacological agent other than a nucleic acid. Device 1 is activated to generate first pulses II, which are applied to electrodes 5 to produce an electric field, which is directed into the tissue portion to commence permeabilization of the tissue cell membranes. Permeabilization is more intense close to electrodes 5 (where the electric field is stronger) and becomes gradually less as the distance from the electrodes increases . Following permeabilization of the cell membranes, the electric characteristics of the tissue change. More specifically, conductivity of the tissue increases, so that tissue impedance decreases. Second pulses 12 (or pulse Iu2) are then applied, the value of which depends on the tissue impedance measured. Permeabilization of the cell membranes is therefore followed by a fall in impedance, which is detected by measuring system 7 to enable an automatic closed-loop reaction of device 1 to reduce the amplitude of pulses 12 (or pulse Iu2) and hence the intensity of the electric field applied. Similarly, poor permeabilization of the cell membranes is marked by little variation in impedance, which is detected by measuring system 7 to enable an automatic closed-loop reaction of device l to increase the amplitude of pulses 12 (or pulse Iu2) and hence the intensity of the electric field applied. The amplitude of the pulses (or single pulse) is therefore regulated according to the instantaneous electric characteristics of the tissue, which in turn depend on the extent to which the cell membranes are permeabilized. This therefore prevents damage to the cells caused by too high an electric field being applied to an already permeabilized tissue. At the same time, in the event the tissue fails to "respond" to the pulses applied, i.e. permeabilization of the cell membranes is not initiated, the amplitude of the pulses (or single pulse) and of the electric field is increased. Generating the second pulses (or pulse) ensures the whole of the tissue portion area between the electrodes is permeabilized. Pulses 13 are then applied to facilitate introduction of charged high-molecular-mass substances, e.g. DNA. Substance 37 is then introduced into the cells. The knowledge gathered by the Applicant indicates that applying at least one pulse of an amplitude adjustable according to the measured impedance provides for achieving a high degree of permeabilization of the cell membranes, while at the same time preventing damage to the cell tissue. 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 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 5a (shown schematically with dotted lines in figure 1) close to electrodes 5, electrically separated from electrodes 5 and placed in contact with tissue portion 35 to be permeabilized. Continuous signals, as opposed to pulses, may be generated. In which case, as opposed to producing a number of separate pulses (II, 12, 13), signal generator 3 may generate a single continuous analog signal (Figure 4) comprising three adjacent portions respectively corresponding to pulses II, 12, 13. The continuous analog signal may comprise a first high- amplitude portion corresponding to first pulses II; a second portion (corresponding to pulses 12) having an amplitude varying as a function of measured impedance Z(ω); and a third lower-amplitude portion corresponding to pulses 13. pulse sequence. The sequences may also be of the exponential type and may also comprise bipolar pulses to avoid the polarixation of the electrodes.

Claims: 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 by comprising regulating means (7, 10, 3) for controlling characteristic quantities of the signal (12) applied to the electrodes on the basis of instantaneous electric characteristics (Z(ω)) measured in the substrate (35); said electric characteristics (Z(ω)) being functions of the degree of permeabilization attained by the membranes of said cells. 2) A device as claimed in Claim 1, wherein said regulating means (7, 10, 3) control the amplitude of the signal applied to the electrodes (5) on the basis of said electric characteristics (Z(ω)). 3) A device as claimed in Claim 1, wherein said regulating means (7, 10, 3) control said characteristic quantities of the signal applied to the electrodes on the basis of an impedance (Z(ω)) measured in the substrate (35) . 4) A device as claimed in Claim 3, wherein said regulating means (7, 10, 3) reduce the amplitude of said signal as the impedance (Z(ω)) decreases. 5) A device as claimed in Claim 3 or 4, wherein said regulating means (7, 10, 3) increase the amplitude of said signal when said impedance (Z(ω)) increases or remains noticeably constant . 6) A device as claimed in any one of the foregoing Claims, wherein said signal generating means comprise pulse generating means (3) . 7) A device as claimed in Claim 6, wherein said pulse generating means produce a number of pulses (12) , each having an amplitude adjustable on the basis of said electric characteristics (Z(ω)) measured in the substrate (35) . 8) A device as claimed in Claim 6, wherein said pulse generating means produce a single pulse (Iu2) having an amplitude adjustable on the basis of said electric characteristics (Z (ω) ) measured in the substrate (35) . 9) A device as claimed in Claim 6, wherein said signal generating means (3) generate: - a number of first pulses (II) applied to the electrodes (5) ; each first pulse having a constant amplitude and preferably being rectangular; - at least one second pulse (12; Iu2) applied to the electrodes (5); said second pulse (12; Iu2) having an amplitude which is a function of the impedance (Z(ω)) measured in the substrate (35) ,- and - at least one third pulse (13) applied to the electrodes (5) ; said third pulse (13) having a much lower amplitude than the first pulses (II) and the second pulse (12; Iu2) . 10) A device as claimed in Claim 9, wherein test means (115) are provided permitting generation of said second pulse until the measured impedance reaches a predetermined lower threshold value indicating permeability of the cell membranes has been achieved; said test means (115) initiating generation of said third pulse upon the lower threshold value being reached. 11) A device as claimed in any one of the foregoing Claims, wherein said electrodes (5) comprise a number of electrodes arranged along a closed path, in particular a circular path, and supplied sequentially in pairs with the output signal of said signal generator (3) . 12) A device as claimed in Claim 11, wherein are provided means for determining the impedance between pairs of electrodes in said number,- and means for directing said signal to an automatically selected pair of electrodes; said pair being selected automatically by comparing the impedances measured, and by selecting the pair of electrodes whose impedance has a predetermined relationship with respect to, e.g. is greater than, the impedances of the other pairs . 13) An electroporation method comprising the steps of : - generating (3) an electric signal; and - applying (5) said electric signal to a substrate (35) comprising cells, to produce, in said substrate (35) , an electric field which induces permeabilization of the membranes of said cells to facilitate introduction of a substance (37) into the cells; characterized by comprising the step of regulating (110) characteristic quantities of the signal (12) on the basis of electric characteristics (Z(ω)) measured in the substrate (35); said electric characteristics (Z(ω)) being functions of the degree of permeabilization attained by the membranes of said cells . 14) A method as claimed in Claim 13, wherein said regulating step (110) comprises the step of controlling the amplitude of the signal on the basis of said electric characteristics (Z (ω) ) . 15) A method as claimed in Claim 13 or 14, wherein said regulating step (110) comprises the step of: - measuring (7) the impedance of said substrate (35) ; and - controlling the characteristic quantities of the signal on the basis of said impedance (Z(ω)) . 16) A method as claimed in Claim 15, wherein said regulating step (110) comprises the step of reducing the amplitude of said signal as said impedance decreases . 17) A method as claimed in Claim 15 or 16, wherein said regulating step (110) comprises the step of increasing the amplitude of said signal when said impedance increases or remains noticeably constant . 18) A method as claimed in any one of Claims 13 to 17, wherein said step of generating a signal comprises the step of generating a number of pulses (12) ; each pulse-having an amplitude adjustable on the basis of said electric characteristics (Z(ω)) measured in the substrate (35) . 19) A method as claimed in any one of Claims 13 to 17, wherein said step of generating a signal comprises the step of generating a single pulse (Iu2) ; said single pulse having an amplitude adjustable on the basis of said electric characteristics (Z(ω)) measured in the substrate (35) . 20) A method as claimed in Claim 13, wherein said step of generating an electric signal comprises the steps Of: - generating a number of first pulses (II) ; each first pulse having a constant amplitude and preferably being rectangular; - generating at least one second pulse (12; Iu2) having an amplitude which is a function of the impedance (Z(ω)) measured in said substrate (35); and - generating at least one third pulse (13) having a much lower amplitude than the first pulses (II) and said second pulse (12; Iu2) . 21) A method as claimed in Claim 20, characterized in that said step of generating at least one second pulse (Iu2) is performed until the measured impedance reaches a predetermined lower threshold value indicating permeability of the cell membranes has been achieved; said third pulse being generated upon the lower threshold value (Zp) being reached. 22) A method as claimed in any one of Claims 13 to 21, characterized in that said substance comprises an organic compound selected from the series including: . a nucleic acid; . a DNA molecule; . an oligonucleotide; . a cytotoxic agent; . a penicillin.

Inventors: MIKLAVCIC DAMIJAN, MIR LLUIS, NEUMANN EBERHARD, PERSSON BERTIL, MIKLAVCIC, DAMIJAN, MIR, LLUIS, NEUMANN, EBERHARD, PERSSON, BERTIL
Applicants: IGEA SRL, MIKLAVCIC DAMIJAN, MIR LLUIS, NEUMANN EBERHARD, PERSSON BERTIL, IGEA S.R.L., MIKLAVCIC, DAMIJAN, MIR, LLUIS, NEUMANN, EBERHARD, PERSSON, BERTIL
Application year: 2001
Main classification: C12M3
IPC codes: C12M3