WO2006018528A1 - Monitoring and control of an electroporation - Google Patents

Monitoring and control of an electroporation Download PDF

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Publication number
WO2006018528A1
WO2006018528A1 PCT/FR2005/001914 FR2005001914W WO2006018528A1 WO 2006018528 A1 WO2006018528 A1 WO 2006018528A1 FR 2005001914 W FR2005001914 W FR 2005001914W WO 2006018528 A1 WO2006018528 A1 WO 2006018528A1
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Prior art keywords
cell
electroporation
data
characterized
assembly
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Application number
PCT/FR2005/001914
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French (fr)
Inventor
Lionel Cima
Lluis Mir
Original Assignee
Centre National De La Recherche Scientifique (Cnrs)
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Priority to FR0408176 priority Critical
Priority to FR0408176A priority patent/FR2873385B1/en
Application filed by Centre National De La Recherche Scientifique (Cnrs) filed Critical Centre National De La Recherche Scientifique (Cnrs)
Publication of WO2006018528A1 publication Critical patent/WO2006018528A1/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02Electrical or electromagnetic means, e.g. for electroporation or for cell fusion

Abstract

The invention relates to an assembly which is intended for the electroporation of a cell structure containing at least one cell. The invention comprises means for monitoring the power supplied to the electroporation electrodes. The invention is characterised in that the monitoring means comprise an estimator which can estimate the effective part of the power supply delivered to one or more determined cell parts of the cell structure, using data which is received by the estimator and which comprises data that are representative of the power supply and data relating to the assessed bioimpedance components of each determined cell part. The invention also relates to methods and uses for the assembly that is intended for electroporation.

Description

MONITORING AND CONTROL OF ELECTROPORATION

The invention relates to an assembly for electroporation of a cell group consisting of at least one cell, comprising means for monitoring and optionally control the power supply of electroporation electrodes.

Electroporation is generating electropores, in cell membranes, resulting from the application (conventionally between two electrodes) of an electric field greater than a predetermined threshold value. These electropores are not selective and any chemical species can then enter the cell. This technique, used to transfer various sized chemicals (small molecules or macromolecules, typically RNA molecules or DNA), opens the way to many applications in biomedical fields, such as non-viral transfer genes, for example. Electroporation is attributed to a reversible destabilization of the membrane causing the penetration of water molecules and a local reduction in the electrical impedance. If the electric field continues to grow, the destabilization becomes irreversible and causes destruction of the cell.

It is therefore important to control the degree of electroporation to free themselves of cell destruction, while ensuring internalisation of chemicals of various sizes (transfer DNA macromolecules, RNA or therapeutic small molecules).

In addition, a control of the size of electropores would ensure an opening large enough to internalize the substance, while protecting the one or more cell (s).

There are known techniques for monitoring and control of electroporation.

monitoring methods are based on measuring the bioimpedance during electroporation, as for example described in WO 99/52589. It may also attempt to control in real time by monitoring electroporation one hand the impedance variations of the electroporated tissue, and secondly by controlling in turn the power supply electrodes in these impedance variations, as described in US 2003/0194808 and WO 01/81533.

These control techniques seem to thereby detect in real time the exceeding of different thresholds electroporation (onset thresholds electropores and cell destruction limit) measuring a characteristic impedance drop, and then be able to retro-act in a manner fitted on the applied electric field.

However, these control techniques through a simple impedance measurement are unsatisfactory, electroporation is a dynamic phenomenon, nonlinear, and therefore difficult to model.

In particular, the threshold voltages themselves (occurrence of electropores threshold and cell destruction limit) are closely related to the geometry of the electrodes, the quality of the touch electrode - tissue, for the influence of the extracellular medium in bioimpedance depends on many electrical parameters (as many parameters that need to control the shape and amplitude of the applied electrical quantities), etc. Moreover, these techniques do not actually propose methods to monitor in real time the status of the electroporated biological cells.

An object of the invention to improve the real-time control over the state of the art, the degree of cell electroporation, to maximize the number of electroporated cells and to minimize the number of destroyed cells, playing particular size electropores within cell membranes.

A second objective of the invention is to overcome all external parameters Electroporation (electrode geometry, quality of electrode-tissue contact, influence of the extracellular medium, etc.). A third object of the invention is to estimate in real time of eigenstates variables electropores tissue (ie the mean transmembrane electric field and the mean density of the current through the electropores).

A fourth object of the invention is to control in real time the electric field delivered by the electrodes to the tissue, taking into account the estimated electrical values ​​during the monitoring, in order to self-adapt electroporation to electro-chemical phenomena appearing in the treated cells.

A fifth object of the invention is the implementation of a simple method to implement to achieve the foregoing objectives.

A sixth object of the invention is to implement a device and an electroporation method adapted regardless of the biological nature of the sample, whatever the configuration in which the sample is located (in vivo, ex vitro or in vitro), and regardless of the medium in which is located the biological sample.

A seventh object of the invention is to adapt the shape of the pulse at the considered bioburden (ie biological sample considered), this pulse is not necessarily square, such as conventionally encountered in the prior art.

To this end, the invention provides, in a first aspect an assembly for electroporation of a cell group consisting of at least one cell, comprising means for monitoring the power supply electrodes electroporation, characterized in that the monitoring means comprises an estimator adapted to estimate the effective portion of the power supply that is supplied to one or more cell specific parts of the cell set, from data received by the estimator comprising data representative of the power supply and data relating to the assessed components of bio-impedance of each cell fixed part.

Other features of the invention are:

- said data relating to said measured components of bio-impedance are independent of the data representative of the power supply, and are stored in memory, - the bio-impedance components have been previously evaluated in the absence of electroporation,

- each determined cell portion is selected from intracellularly, extracellularly, cell membranes, the pores formed cell membrane of said cell assembly,

- the estimator is adapted to estimate, from said data comprising data that it receives, and relationships to one another, at least one of the following electrical parameters: the mean intensity of the average current I c issued solely cell the cell assembly, the average voltage Uj ntra and the average intensity of the average current I applied to the GTW only intracellularly, the mean voltage Um applied to the single cell membrane, the average intensity of the electric current I supplied to only p pores formed in the cell membrane,

- the estimator is adapted to estimate, from data including said data it receives data relating to the average cell thickness / of data relating to the average thickness h of cell membrane, data relating to the distance e between the electrodes electroporation, and relation between these data, the electric field applied to only cell membranes of cell assembly,

- the estimator is adapted to estimate, from said data comprising data that it receives, of the surface S to the data of the electrodes used for electroporation, and relation between these data, the mean density of the electric current delivered through the pores formed;

- the assembly further comprises the electrodes used for electroporation, a generator electrically powering the electrodes and adapted to generate a pulse voltage U and / or current / pulse suitable for electroporation, means for measuring the voltage U and / or the current intensity / providing the input of the estimator said data representative of the power supply, and means for controlling the values ​​of U and / or / provided by the generator to the data output of the estimator, so as to thus embody a control electroporation; - the assembly further comprises a generator adapted to generate a voltage and a current below the threshold voltage, respectively, and the threshold current from which the electroporation begins, means for measuring the bio-impedance of the cell assembly subjected to electric field of electrodes supplied by the generator with a voltage and / or current below the threshold voltage, respectively, and means for evaluating the bio-impedance components of each cell determined part of the cell set, based on data provided output means for measuring the bio-impedance;

- the assembly further comprises switching means for switching the power supply electrodes of a first of the two generators in the second of the two generators, so as to thus achieve bioimpedance measurements between two electric pulses with the same electrodes;

- use this assembly for electroporation of the cell together, for the administration of drugs.

In a second aspect, the invention provides a method for monitoring a cell electroporation comprising the steps of:

(A) receiving data representative of the electric field applied or the power supply provided during electroporation; (B) receiving data relating to the assessed components bio¬ impedance of one or more cell specific portions of cell assembly;

(C) calculating an estimate of the effective part of the power supply that is supplied to each cell determined part, from the data received in step (a) and in step (b).

Other features of the monitoring method are:

- said data relating to said measured components of bio-impedance are independent of the data representative of the electric field, and are stored, - the method further comprises at least an evaluation step, prior to step (a), components of bio-impedance, in the absence of electroporation, carried out prior to electroporation and / or between two electrical pulses,

- each determined cell portion is selected from intracellularly, extracellularly, cell membranes, the pores formed cell membrane of said cell assembly,

- step (c) gives an estimate, based on data comprising said received data in (a) and (b), and relationships between them, at least one of the following electrical parameters: the mean voltage U ex t r has applied to only the extracellular medium, the average voltage applied to Uintra only intracellularly, the mean voltage Um applied to the single cell membrane, the average electric current I p issued only to the pores formed in the cell membrane,

- step (c) gives an estimate, based on data comprising said received data in (a) and (b) data relating to the average cell thickness / of data relating to the average thickness h cell membrane, data relating to the distance e between the electrodes used for electroporation, and relation between these data, the average electric field applied to only cell membranes of cell assembly,

- step (c) gives an estimate, based on data comprising said received data in (a) and (b), data relating to the surface S of the electrodes used for electroporation, and relation between these data, the electric current average density delivered through the pores formed,

- the method further comprises, after step (c), a control step of the electric field to the estimation made in step (c), - the method further comprises, prior to step (a ), a step of measuring characteristics of the applied electric field for electroporation, or characteristics representative of the power supply electrodes (correlated with the electric field), for providing said data representative of the applied electric field or power supply power supplied received in step (a), - the method further comprises, prior to step (a), a measure of the bio¬ impedance of a cell assembly in the absence of electroporation; evaluation of bio-impedance components of each cell determined portion of the cell assembly is then carried out, starting from this measurement of bio-impedance.

According to a third aspect, the invention provides a method of electroporation of a cell group consisting of at least one cell, comprising the steps of:

(A) applying one or more electric field pulses to the cell assembly, adapted to cause electroporation in the cell assembly;

(B) measuring data representative of the applied electric field or the electric power supplied to the electrodes for electroporation;

(C) carrying out said method for monitoring.

Other features of the electroporation method are:

- the method further comprises, prior to step (a) and / or during step (a) between two electrical pulses, the steps of: applying an electric field to the cell together, the amplitude of which is less than the threshold electric field above which the membrane pores are formed by electroporation; measuring the bio-impedance of the cell assembly; evaluate bioimpedance components of one or more cell specific parts of the cell assembly, from the measurement results of step (b), so as to provide said data relating to the measured components of the bio-impedance part of each cell determined from the cell together,

- use of the electroporation method for the administration of drugs.

Other features, objects and advantages of the invention will become apparent from the following description, which is purely illustrative and not restrictive, and that will be read in conjunction with the accompanying drawings wherein:

1 shows schematically an assembly for electroporation according to the invention.

2 shows an estimator according to the invention. 3 shows an electrical circuit modeling the electrical characteristics of a cell or a set of multiple cells in the absence of électrop oration.

4 shows an electrical circuit modeling the electrical characteristics of a cell or a set of several electroporated cells (s).

Figure 5 is a graph showing variation of an average membrane voltage U 1n a function of time of application of a biological tissue to an electric field electroporation, calculated according to the invention.

Figure 6 is a graph showing variation of an average intensity I p through the electropores a electroporated membrane depending on the application time of a biological tissue to an electric field electroporation, calculated according to invention.

Referring to Figure 1, an assembly 1 for cell electroporation according to the invention includes electrodes 51 and 52 placed around the cell assembly 100 (in one or more cells) supplied by a generator 30 adapted to generate a electric power suitable for the electroporation of the cell assembly 100, to a conventionally know impulse voltage U and a current / pulse suitable for electroporation. The assembly 1 also comprises means for measuring electrical quantities, such as an ammeter 61 and a voltmeter 62, representative of the power supply electrodes 51-52 by the generator 30, these means then outputting 65 66 data representative of the power supply, such as data relating to U and /.

Upstream of the generator 30, a command and control unit 20 via the connection 25, the generator 30 so that it delivers to the electrodes a voltage of 51-52 U profile and / or electric / desired current. Upstream of the control unit 20, instructions (here transmembrane field E 1n and medium density J p of the electric current passing through the pores) are sent automatically or after a manual input (by a user via an interface sample type keyboard or computer mouse) to the control unit 20 which takes them into account when correcting the control unit to send to the generator 30. According to the invention, these instructions are changed to 15 and

16 according to the data (Ê m and J ^) output 11 and 12 of an estimator 10.

This estimator 10 is adapted to estimate the effective portion of the power supply that is supplied to one or more cell specific parts of the cell assembly 100, a cellular portion typically being intracellularly, extracellularly, the cell membrane, or the pores formed in the cell membrane, cell assembly 100. This estimator 10 primarily base its estimation on the data (U, I) it receives (via connections 65 and 66) measuring means 61-62, and data relating to the assessed components of bio-impedance of each cell fixed portion (initially stored in memory and received via the link 95), as well as the relationships between them.

The assembly 1 may further include an apparatus for evaluating the latter data relating to bio-impedance components in each fixed cellular portion. This apparatus comprises a second generator 40 adapted to generate a voltage and a current below the threshold voltage, respectively, and the threshold current above which electroporation begins. The assembly 1 can in this case be provided with switching means 81-82 and 71-72 adapted to switch the power supply of the electrodes 51-52 of the second generator 40 of the first generator 30, so that the electrodes 51- 52 first provide an electric field adapted not to electroporate the cell assembly 100 and an electric field capable to electroporate.

A measuring unit 90 is connected upstream of the second generator 40, and comprises: - means for measuring the bio-impedance of the entire cell 100 in the absence of electroporation, when the second generator 40 delivers its low voltage,

- means for evaluating the components of bio-impedance (s) cell (s) determined (s) (ie extracellular, intracellular, membrane, and / or other) of the cell set, based on data provided by means for measuring the bio-impedance, - means for storing data relating to measured components of bio-impedance portion (s) cell (s) determined (s), so that these data can be used (via link 95) by the estimator 10 in F electroporation. Optionally, the measuring unit 90 further allows to evaluate other parameters as electrical components, such as parameters related to the configuration of the electrodes (surface S, an inter-electrode distance), and / or parameters relating to cell assembly 100 electroporated (average thickness h of cell membranes, average thickness / biological cells). Referring to Figure 2, the present invention may also relate to, not a complete assembly 1 for electroporation as shown in Figure 1, but only a monitoring device comprising an estimator 10 adapted to estimate the effective portion of the power supply that is supplied to one or more cell specific parts of the cell 100 electroporated together. The monitoring device may typically include a microprocessor for performing calculations in particular according to the invention, a memory for storing data such as electrical data and mathematical formulas, and ports or input links (65, 66, 95 ) which allow input of user data and ports or output links (11, 12) that allow an output estimated data. The estimator 10 receives for this purpose (via connections 65 and 66) data representative of the power delivered to the electrodes 51-52 for electroporation of a cell assembly 100 or data representative of the applied electric field (such as for example the amplitudes of the pulse electric fields, pulse voltages, pulse current, pulse frequencies, pulse widths, and / or others).

The estimator 10 further receives (via link 95) data relating to measured components of bioimpedance (s) cell (s) determined (s), and optionally information relating to the configuration of the electrodes and the biology of the cell 100 electroporated together. To understand the electrical characteristics of a biological cell, it may be necessary, first, to know that the biological tissue (or cell assembly 100 any) are composed of live cells immersed in an extracellular medium. The intracellular environment is protected by a cell membrane which selects the passage of certain chemical species, the cell membrane is a lipid bilayer having a high electrical insulation power. Referring to Figure 3, it may then model the electrical characteristics of a cell 100 (or a set multi-cell 100) by an electrical circuit whose extracellular portion 130 would be represented by a χ R e tra and a resistance tra capacitor C ex in parallel, the intracellular portion 120 by a Rintra resistance, and the cell membrane 110 by a resistance R m and a capacitor C n, in parallel. The intracellular portion 110 is in series with the intracellular portion 120, and the set of these two cell parts is in parallel with the extracellular portion 130.

However it would be wrong to consider the membrane 110 as a simple RC circuit. Indeed, a coefficient called "coefficient of Cole-Cole" (see in particular J. Chem. Phys. 9341, (1941) and KS COIE COIE HR) involved in the expression of the electrical parameters of the membrane 110, this coefficient taking into account the influence of the mode of the distribution of cells and their size dispersion (in the case of a cellular multi-cell assembly 100) to the bio-impedance. Therefore the electrical characteristics further comprises the coefficient of Cole-Cole. These electrical characteristics form so many bio¬ impedance components that may have been previously identified, for example, from a measurement of bioimpedance, a method of least squares.

For a sinusoidal electric field delivered at a frequency / = ω / 27r in the cell assembly 100 to an amplitude less than that beyond which the membrane is likely to present electropores, bio-impedance Z t (ω ) of the cell assembly 100 may then be written from the following formula:

Figure imgf000013_0001

This bio-impedance Z (ω) measured for an applied electric field low (ie not liable to cause electroporation of the cell assembly 100) and has a linear behavior. From the measurement of t Z (oi), and ω, we can evaluate Rextr components a, C ex tra Rfatra, R m, C m and the cell assembly 100.

These components of bio-impedance are then advantageously stored in memory. When then applies an electric field (or voltage U and / or a current / to electrodes used for electroporation) may create electroporated into the cell assembly 100 of electropores are formed in the membranes 110 which thus become permeable to molecules, showing then this time, a non-linear behavior of electroporated cells.

The Applicant proposes, with reference to Figure 4, an equivalent electrical circuit modeled with electrical characteristics of a cell assembly 100 and electroporated. The Applicant proposes to model the electrical part linked to electropores by a shunt 112 of the part non-altered membrane 111, whose charge is a non-linear dipole D p. Part of the electric current I c that feeds directly into the cells will therefore go to the non-altered membrane portion 111, and another part of / c, denoted by I p, is only the current through the membrane part altered 112 through electropores.

In this context, the estimator 10 is then adapted to estimate the effective portion of the power supply (UJ) which is fourme to the electrodes or the electric field applied to the cell specific parts of the cell assembly 100.

It should be noted that if the data received via the links 65-66 are from U measuring voltage and / or current / made upstream of the electrodes 51-52, it is preferable that the frequency of the sinusoidal signal then chosen for this voltage and / or the current is not too low, so that the effects of tissue-electrode contact does not significantly disturb the results of estimation calculations to be performed (listed below). Typically, the frequency is selected higher than 50 Hz.

Thus, the average voltage applied to the O ext ra single extracellular medium 130 may be calculated as follows:

Uexra U (2) Thus, the average electric current / c cells alone delivered to the cell assembly 100, can be calculated as follows:

1 C - 1 p "extra Taj

The formula (3) eliminates the effects related to the extracellular medium. Thus, the average voltage Ui ntra and the mean current I ntra issued only intracellular environment of the cell assembly 100, can be calculated as follows:

Mntra - * c> Uintra K-intra ± intra vv

Thus, the mean voltage Um applied to the single cell membrane of the cell assembly 100, can be calculated as follows:

U n = U n-Ru I 0 (5)

The formula (5) can eliminate the effects associated with intracellular medium 120.

The average current I p issued only to the pores formed in the cell membrane of the cell assembly 100, can be calculated by modeling in the field firéquentiel non-altered membrane portion 111 by an admittance constant phase element (also called admittance " CPE ", the acronym

"Constant Phase Element"). This model takes into account a distribution of the electric time constants of the membrane and can be used at a cellular tissue (in vivo, ex vivo) or cell (s) in an artificial environment (in vitro). In the time domain, the non-altered membrane can then be characterized by its impulse response ycpε (t) calculated by the inverse Laplace transform of this admittance, which may be expressed as:

there CPE (t) ≈ Tr \ ~] (6)

where: TL '1 represents the inverse Laplace Transform, τ = R m C m = p / ω, where "y" indexes the imaginary part of a complex number. We then obtain, for i p, the following expression:

I p (t) = I c (t) -y CPE (t) * U m (t) (7) where the product symbolized by "*" is a convolution product between the average transmembrane voltage U m (t) and its impulse response ycpεit).

Formula (7) allows to eliminate the influence of the non-altered membrane 111, to maintain while the average intensity of the current related to electropores (altered membrane 112).

It should be noted that all previous electrical quantities (t JJex m, Ic, ntra Ui, Ii ntm, U m, I p) correspond to macroscopic quantities. Thus, for example, U 1n corresponds to the estimated average voltage applied across the membranes of cells comprising the cell assembly 100. In the absence of electroporation., I c (t) is naturally equal to including PE ( ή * U m (t), and

I p (t) becomes zero, as if the non-linear dipole D p then had an infinite impedance. The model established by the Applicant (in reference to Figure 4) therefore also works well for a cell assembly 100 undergoing electroporation for cell assembly 100 undergoing no electroporation.

Optionally, the estimator 10 also receives, via the connection 95, information on the configuration of the electrodes and the biological characteristics of the cell 100 electroporated together to estimate other electrical quantities representative of the distribution of the feed into the cell together.

Thus, if the estimator 10 receives data relating to the average cell thickness / of data relating to the average thickness h of cell membrane (which is typically between about 5 and about 6 nanometers), and data relating to the inter-electrode distance e from the estimator 10 may compute the average electric field estimated Ê m applied to only cell membranes 110 of the cell assembly 100 by means of the following formula:

Figure imgf000016_0001

The formula (8) standardizes the average transmembrane pressure compared with the sizes of the cells and the inter-electrode distance to define the mean transmembrane electric field. This estimated average electric field E m represents the average electric field applied to each cell membrane of the cell assembly 100.

Thus, if the estimator 10 receives data relating to the electrode surface S used for electroporation, the estimator 10 can calculate the estimated average density J p of the electrical current delivered through the pores formed in the cell assembly 100 by means of the following formula:

Λ p = ~ S (9)

J p is a macroscopic size.

The estimator 10 can thus outputting one or more of these electrical quantities (estimated using the above formulas), and / or other electrical parameters within the same concept.

For example, the estimator 10 may be provided on outputs 11 and 12 one or more of the following pairs of values: (Ui ntra, Iintra), (U m Jp), (e m, J p).

The output (here 11 and 12) of the estimator 10 can then be connected to a display terminal type, or other calculation means, or a memory in which the data it provides is stored, or another system such as an electric power control device of the electroporation electrodes.

This estimator 10 therefore allows real-time monitoring of the degree of electroporation of the entire cell 100, since it provides data for adapting (manually or automatically by a control system for a self-adaptation of the system) the electric field electroporation according to the electro-chemical phenomena observed. This will attempt to maximize the number of electroporated cells and minimize the number of destroyed cells, by varying the size of electropores within cell membranes.

This estimator 10 can also allow to overcome all external parameters Electroporation (electrode geometry, quality of electrode-tissue contact, influence of the extracellular medium, etc.), if it provides for example at the output an estimate average voltage U m membrane and / or an estimate of the average intensity I p of the pore current.

This estimator 10 provides further real-time variables eigenstates electropores to the cell assembly 100 (ie the mean transmembrane electric field estimated Ê m, and the estimated average density J p of the current through the electropores).

This estimator 10 can operate regardless of the biological nature of the cell 100 to electroporate together, regardless of the configuration in which the cell 100 is set (in vivo, ex vivo, or in vitro), and whatever the extracellular medium in which it is immersed.

According to a first variant of the invention, the monitoring device comprising the estimator 10 (and possibly a display and / or other) part of a control device for electroporation of a cell assembly 100. This device control then comprises, with reference to Figure 1, the estimator 10 and said control unit 20. It further comprises means for generating instructions upstream of the control unit (at 17 and 18), these means being a user interface by means of which the latter enters the desired instructions, or means for automatically generating these instructions according to specific settings, or means able to pick up in memory at a certain frequency, the pre-recorded instructions . Furthermore, this control device comprises means 15 and 16 to change the desired instructions (from 17 and 18) based on estimation data output (11 and 12) of the estimator 10.

This control device then allows tracking electroporation F estimate of 10 F estimator.

According to a second variant of the invention, the control device forms part of an assembly for electroporation F further comprising said generator 30 of voltage U and / or current pulse I F adapted to electroporation, the electrodes 51-52 electroporation, as well as means 61-62 for measuring electrical quantities of the power provided by the generator 30 or the electrodes 51-52, these measuring means providing information representative of the applied electric field or of the power supply supplied to the electrodes for electroporation, to the input of the estimator 10 (via connections 65 and 66).

The generator 30 may be a controlled power generator, for outputting the voltage levels and / or current (possibly higher) in order to trigger an electroporation "bioburden" (ie cell assembly 100). This generator 30 may deliver pulses of any shape, since the estimator 10 may be adjusted (by adjusting the setpoint sent to the control unit 20) the shape of the pulse at the considered bioburden (ie cell assembly 100 ). This impulse is not necessarily square.

51-52 electrodes may be a pair of electrodes for imposing an electric field electroporation on a "bioburden" (ie cell assembly 100) which can be a tissue in vivo or ex vivo, or cells in vitro (or a single cell) or cell tissue placed in a bio-chip. The means 61-62 may comprise measurement means for measuring electrical quantities representative of the power supplied to the electrodes for electroporation, thus including for example a current meter 61 measuring the intensity of the current absorbed by the cell assembly 100 and a voltmeter 62 measuring the inter-electrode voltage. The means 61-62 may also comprise measurement of the measurement electrodes disposed on the cell assembly 100, for directly measuring the electric field applied to the cell together, and s' freeing so the influence on the outcome of measures of the electrode contacting tissue.

This set allows such enslave own variables to electropores on a particular target, to control the degree of electroporation of cells without destroying them. Control of an average transmembrane electric field E n, can trigger the electroporation phenomenon without destroying the membranes. Control of the average current density J p crossing electropores helps control Ia average surface electropores. Prior to the implementation of electroporation, it can be implemented in an assessment of bio-impedance components (Rextra, C e χtra 3 Rintra Cintra "Rm, C m, a, ...) of each part determined cell (extracellular medium 130, intracellular medium 120, membrane 110) of the cell assembly 100, from a measurement of bio-impedance of the cell assembly 100 in the absence of electroporation. To this end, a device according to the invention to realize such functions is provided, incorporated or not to said monitoring device (with reference to figure 2), or said control device according to the first variant of the invention, or said assembly for electroporation according to the second variant of the invention.

This device requires a second generator 40, low-voltage circuitry to determine the linear behavior of the "biological load" (Le. The cell assembly 100) and electrodes (51-52) for measuring the bioimpedance Z (ω ) in the absence of electroporation.

In addition, this device comprises the measurement unit 90 previously described (with reference to Figure 1). The treated biological tissue and the electrodes are therefore characterized by a first impedance measuring small signals. This measure implements a low frequency electronic voltage, which does not cause electroporation.

In the event that this impedance components evaluation device is incorporated into the rest of the assembly for electroporation F, we get the assembly 1 of Figure 1.

Optionally, switching means 71-72 and 81-82 are provided between the generators 30 and 40 and the electrodes 51-52, to switch the electrodes supplying a generator 30 or 40 to the generator 40 or other 30.

This assembly can then allow to supply the low voltage electrodes 51-52 using the generator 40 between two electric high-voltage pulses supplied by the generator 30. It is thus possible bio-impedance measurements between two electric pulses electroporation, with the same electrodes 51-

52, without affecting the latter.

This assembly therefore makes it possible to observe any changes that may undergo electrical components of bio-impedance (in the absence of electroporation) progressively as the pulse number data to the cellular tissue 100 increases, thereby updating the respective values of these components. This arrangement can be particularly useful in in vivo electroporation, cells can then possibly swell under the effect of electroporation and thus see their electrical components slightly change over time).

The cell assembly 100 to be electroporated according to the invention can be a living tissue in vivo were placed around which the electrodes 51-52, or a tissue removed (ex vivo) or tissue placed in an artificial environment (in vitro) or a cellular tissue placed in a bio¬ chip. There can be vegetable, animal or human. Electroporation can return to play active products such as inorganic drugs, proteins or peptides (nucleic acid, DNA or RNA molecule with sequences encoding therapeutic genes or genes related to health or for biotechnological purposes, natural oligonucleotide or modified, ...) to be administered to the cell assembly 100. experimental study: the applicant present below the results obtained after the implementation of an electroporation method according to the invention made on the fabric 100 of a potato.

The fabric 100 is placed between two electrodes 51 and 52 of stainless steel, having a surface area S 2 of 30 mm and separated by a distance e of about 6 mm. The bio-tissue impedance is measured using an impedance analyzer

HP4192A and a low sinusoidal voltage generator (i.e. a generator capable of delivering sinusoidal voltages below the threshold voltage beyond which electropores appear) to 1 V amplitude and frequencies between 50Hz and 10MHz. The latter frequency range is preferably chosen because it allows to detect the components of bio¬ impedance without these are significantly disturbed by the effects of cell-tissue contacting electrode.

Linear components of bio-impedance are then evaluated by means of a calculation using a least squares identification module of the bio-impedance measured, and are as follows:

R EXTM = l8 kQ; i ^ ra = 151 Ω; R m = l, l kW; C m ≈7,2 nF; a ≈ 0.1677 Good agreement was observed between the measured impedance and the impedance reconstituted according to formula (1) from the identified components.

Once these known components of the voltages and currents of unipolar pulse length of approximately 150μs each, by varying the amplitudes between 0 V and 300 V are applied to electrodes 51-52 by a suitable generator 30.

The waveforms of the intensities I of the current supplied to electrodes 51-52, when measured, then revealed linear behavior for voltages below 100V 5 while a large nonlinearity appeared beyond 125 V.

The threshold voltage at which the electropores begin to appear would be between 100 V and 125 V in the conditions of the experiment.

5 shows the macroscopic average transmembrane voltage U m as a function of application time of the fabric 100 to the pulsed electric field, calculated using the formula (5).

Figure 6 shows macroscopic average intensity I p flowing through the pores as a function of the application time of the fabric 100 to the pulsed electric field, calculated using the formula (7).

M It can be seen that U (t) increases with U (t), and I p (t) appears from the threshold voltage (between 100 V and 125 V) and it becomes important as soon as applied a voltage U (t) much greater than this threshold voltage (here U (t) = 200V during the pulse).

One can observe that the average macroscopic membrane voltage U m (t) does not vary linearly with the voltage U (t). These measures are in line with the results of theoretical analytical models providing dynamic developments of electropores from transmembrane voltage values, including donated by JC Neu and W. Krassowska (Phys. Rev. E 59, 3471 (1999)) and KA DeBruin and W. Krassowska (Ann. Biomed. Eng. 26, 584 (1998)).

Claims

1. An assembly for electroporation of a cell group consisting of at least one cell comprising means for electrical power monitoring electroporation electrodes, characterized in that the monitoring means comprises an estimator adapted to estimating the effective part of the power supply that is supplied to one or more cell specific parts of the cell set, from data received by the estimator comprising data representative of the power supply and data relating to components evaluated bio-impedance of each cell fixed part.
2. Assembly according to the preceding claim, characterized in that it further comprises a memory storing said data relating to said components evaluated bio-impedance, these being independent of the data representative of the power supply.
3. Assembly according to the preceding claim, characterized in that it further comprises means for evaluating the components of bio-impedance in the absence of electroporation.
4. Assembly according to one of the preceding claims, characterized in that each fixed cellular moiety is selected from intracellularly, extracellularly, cell membranes, the pores formed cell membrane of said cell assembly.
5. Assembly according to the preceding claim, characterized in that the estimator is arranged to estimate, from said data comprising data that it receives and relationships to one another, at least one of the following electrical parameters: the mean intensity the current Ic delivered to cells alone of cellular overall average voltage Uj n t r and the average intensity of the current applied to Iintr has only intracellularly, the mean voltage Um applied to the. single cell membrane, the average intensity of the electric current I p issued only to the pores formed in the cell membrane.
6. Assembly according to one of the preceding claims, characterized in that the estimator is arranged to estimate the average electric field applied to each cell membrane of the cell set, from data including said data that it receives, of data relating to the average cell thickness / of data relating to the average thickness h of cell membrane, data relating to the distance e between the electroporation electrodes, and relationship between these data.
7. Assembly according to one of the preceding claims, characterized in that the estimator is arranged to estimate the average electric current density delivered through the pores formed, from data including said data it receives, the data to the surface S of the electrodes used for electroporation, and relationship between these data.
8. Assembly according to one of the preceding claims, characterized in that it further comprises:
- the electrodes used for electroporation,
- a generator electrically powering the electrodes and adapted to generate a pulse voltage U and / or current / pulse suitable for electroporation, - means for measuring the voltage U and / or the current intensity / providing the input estimator said data representative of the power supply; and
- means for controlling the values ​​of U and / or I supplied by the generator to the data output of the estimator, so as to perform. and control over electroporation. 9. An assembly according to one of Claims 3 to 8, characterized in that said means for evaluating the bio-impedance of components in the absence of électroporatipn include:
- adapted to generate a voltage generator and / or a current below the threshold voltage, respectively, and the threshold current from which the electroporation begins;
- means for measuring the bio-impedance of the cell assembly subjected to the electric field of electrodes supplied by the generator with a voltage and / or current below the threshold voltage, respectively, and the threshold current; and
- means for estimating bioimpedance components of each cell determined portion of the cell assembly, from the data output means for measuring the bio-impedance.
lO.Ensemble according to the two preceding claims, characterized in that it further comprises switching means for switching the power supply electrodes of a first of the two generators in the second of the two generators, so as to be able to realize of bioimpedance measurements between two electrical pulses with the same electrodes.
ll.Ensemble according to one of the preceding claims, characterized in that it is for administration of drugs in the cell together.
H.A method for monitoring a cell electroporation comprising the steps of:
(A) receiving data representative of the electric field applied or the power supplied to the electrodes during electroporation;
(B) receiving data relating to the assessed components of bio- impedance of one or more cell specific portions of cell assembly; (C) calculating an estimate of the effective part of the power supply that is supplied to each cell determined part, from the data received in step (a) and in step (b).
13. A monitoring method according to the preceding claim, characterized in that said data relating to said measured components of bio-impedance are independent of the data representative of the electric field, and are stored.
14.A method of monitoring according to the preceding claim, characterized in that it further comprises at least an evaluation step, prior to step (a), bio-impedance components, in the absence of electroporation .
15.A method of monitoring according to the preceding claim, characterized in that a step of evaluating bioimpedance components is carried out prior to electroporation.
lβ.Procédé monitoring device according to one of the preceding claims, characterized in that the data received in step (a) are representative of the application of a pulsed electric field applied or a pulse electric power supplied to carry out the electroporation, and in that an evaluation stage of bioimpedance components is performed between two electrical pulses.
17.A method of monitoring according to one of claims 12 to 16, characterized in that each fixed cellular moiety is selected from intracellularly, extracellularly, cell membranes, the pores formed cell membrane of said cell assembly.
18.A method of monitoring according to the preceding claim, characterized in that step (c) gives an estimate, based on data comprising said received data in (a) and (b) and the relationships between them, of at least one of the following electrical parameters: the mean intensity of the current I 0 supplied to the (x) one (s) cell (s) of the entire cell electroporated, the mean voltage U applied to the GTW single extracellular medium, the average voltage U m applied to the single cell membrane, the average intensity of the electric current I p issued only to the pores formed in the cell membrane.
19.A method of monitoring according one of the two preceding claims, characterized in that step (c) gives an estimate of the average electric field applied to each cell membrane of the cell set, based on data comprising said received data in (a) and (b) data relating to the average cell thickness / of data relating to the average thickness h of cell membrane, data relating to the distance e between the electrodes used for electroporation, and relationship between these data.
20.A method of monitoring according one of the three preceding claims, characterized in that step (c) gives an estimate of the average density of electrical current delivered through the pores formed, from data including said data received (a) and (b), data relating to the surface S of the electrodes used for electroporation, and relationship between these data.
21.A method of monitoring according to one of claims 12 to 20, characterized in that it further comprises, after step (c), an electrical field of the servo step applied to the estimate made during step (c).
22.A method of monitoring according to one of claims 12 to 21, characterized in that it further comprises, before step (a), a step of measuring electric field characteristics applied to electroporation, or characteristics representative of the power supply electrodes (correlated with the electric field), for providing said data representative of the electric field applied or the power supplied to the electrodes received in step (a).
23.A method of monitoring according to one of claims 14 to 22, characterized in that it further comprises a measure of the bio-impedance of a cell assembly in the absence of electroporation; said evaluating step of the bio-impedance components of each cell determined portion of the cell assembly is then made from this measurement of bio-impedance.
24.A method of electroporation of a cell group consisting of at least one cell, comprising the steps of:
(A) applying one or more electric field pulses to the cell assembly, adapted to cause electroporation in the cell assembly; (B) measuring data representative of the applied electric field or the electric power supplied to the electrodes for electroporation; (C) implementing the monitoring method according to one of claims 12 to 21.
25.The electroporation according to the preceding claim, characterized in that it further comprises, before step (a) and / or during step (a) between two electrical pulses, the steps of:
- applying an electric field to the cell together, whose amplitude is smaller than the threshold electric field above which the membrane pores are formed by electroporation;
- measuring the bio-impedance of the cell assembly;
- evaluating bioimpedance components of one or more cell specific parts of the cell assembly, from the measurement results of step (b), so as to provide said data relating to the evaluated components of bio- impedance of each cell determined portion of the cell assembly.
26.Use the electroporation method according to one of the two preceding claims for the administration of drugs.
PCT/FR2005/001914 2004-07-23 2005-07-25 Monitoring and control of an electroporation WO2006018528A1 (en)

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