Electroporation apparatus and method

Abstract

To provide an electroporation probe drive, apparatus, and method for supplying with sharp turn-on and turn-off times and stable amplitude.SOLUTION: An electroporation apparatus (1) has an electroporation probe (26) for linking with electrodes. A foam is injected at the treatment site to displace blood rather than mixing with it, increasing the contact time of a higher concentration of active agent with the tissue and thus resulting in greater efficacy. With foam solutions, a lower concentration of agent can be used to obtain the same therapeutic effect as in their liquid counterpart, reducing the prevalence of side effects associated with higher concentrations. A foam solution compared to an equivalent liquid solution enables more efficient cell electroporation particularly where bipolar pulses have been employed by mitigating an increase in tissue conductivity as would normally be observed with a comparable liquid solution.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to electroporation.

Background Art

[0002] U.S. Patent Application No. 2006 / 089674 (Walters et al.) and WO2018 / 200800 (Bowers et al.) describe electroporation devices.

[0003] Electroporation is a medical and molecular biology technique that applies an electric field to cells to increase the permeability of cell membranes, enabling the introduction of molecules that were previously impervious to the cells. Electroporation has many potential applications and can be used when the effects on the cell membrane are applied reversibly and irreversibly. In a typical electroporation treatment, short, intense electrical pulses are generated to temporarily permeabilize the cell membrane. For example, in reversible electroporation, which may be used in connection with enabling intracellular passive diffusion of chemotherapy, the electric field is below a certain voltage threshold, allowing the cell membrane to repair after treatment. Reversible electroporation may involve introducing molecules such as drugs or genes into cells or molecules that are normally not permeable to this substance without inducing cell death by the electric field alone. The electric field threshold of cells varies from cell to cell. In irreversible electroporation, the electric field is higher than this voltage threshold, creating permanent nanopores in the cell membrane, disrupting the cell's homeostasis, and

[0004] its consequences Ultimately, cells die through a combination of apoptosis and necrosis, which result from the disruption of normal cellular functions.

[0005] The present invention relates to providing electroporation probe drivers, apparatus, and methods for supplying high-voltage, ultrashort electroporation pulses (preferably in the microsecond range, and possibly down to the nanosecond range) with a stable amplitude, sharp turn-on and turn-off times, when applied to a biological load, which are close to the theoretical ideal.

[0006] A further objective is to avoid the need for physically large drive units. A further objective is to achieve a more desirable therapeutic environment surrounding the cells being treated.

[0007] References References: BLIbey et al., Bipolar nanosecond electric pulses are less efficient at electropermeabilization and killing cells than Monopolar pulses (bipolar nanosecond electrical pulses are less efficient than unipolar pulses in electrical permeation and cell death), Biochem. Biophys. Res. Commun., Vol. 443, No. 2, p. 568, pp. 73, January 2014, 10.1016 / j.bbrc.2013.12.004.

[0008] References: Polajzer T, Dermol-Cerne J, Rebersek Written by M, O'Connor R, Miklavcic D, Cancellation The counteracting effect is present in high-frequency reversible and irreversible electroporation, Bioelectrochemistry, April 2020;132:107442, doi:10.1016 / j.bioelechem.2019.107442.Epub December 24, 2019, PubMed PMID:31923714. [Overview of the project] [Means for solving the problem]

[0009] We describe the electroporation apparatus described in claims 1 to 29, the method of operating the apparatus described in claims 30 to 42, the electroporation method described in claims 43 to 70, the foam for use in electroporation described in claims 71 to 94, the electroporation apparatus described in claims 95 to 100, the method of operating the apparatus described in claims 101 to 103, and the calcium ion-containing solution for electroporation described in claims 104 and 105.

[0010] Typically, electroporation therapy incorporates pulse lengths ranging from 50 to 100 μs, using pulses alone (irreversible electroporation) or in combination with chemotherapy (electrochemotherapy) to induce cell death. More recently, it has been discovered that shorter microsecond (<10 μs) high-frequency irreversible electroporation and nanosecond (<1 μs) pulses, when using bipolar pulses, have a counteracting effect on the degree of cell permeabilization compared to unipolar pulses.

[0011] This invention provides apparatus and methods for achieving an improved environment around cells to be treated. These improvements arise from the understanding that, in the short microsecond range (<10 μs), there is a counteracting effect (reduced cell permeability) when using bipolar pulses, which is partly determined by the conductivity of the solution surrounding the cells. The effectiveness of cell permeability (creation of pores on the cell membrane) produced by short bipolar electrical pulses (<10 μs) is affected by conductivity, as lower conductivity allows for more efficient electrical pulse-induced cell membrane permeability. The conductivity around cells is partly affected by the amount of liquid in the area and by the local injection of solutions, such as local anesthetics or other ion-containing solutions. Increased conductivity surrounding cells to be electroporated results in higher currents, which are detrimental to treatment and reduce the patient's cell permeability and pain sensation. In highly conductive environments, electroporation treatment may generate gases during treatment and produce pressure waves (referred to as discharges, sparks, or arc discharges) accompanied by severe acoustic symptoms and mechanical tissue damage. The cell lysis and ion release that occur during the electroporation process itself cause an increase in tissue conductivity, leading to high currents and potential mechanical failure and improper pulse supply.

[0012] Below, we describe an improved electroporation apparatus for providing pulses, a substance for use in improving the environment surrounding cells being treated, and an improved probe head for both the injection of the substance and the application of electroporation pulses in any desired order, inserted into the tissue with the same needle.

[0013] We describe an electroporation apparatus comprising a plurality of electroporation probe terminals, a transformer for providing a boosted voltage, and a switching circuit for supplying pulses to the probe terminals for electroporation. The switching circuit preferably has switches for connecting high voltage and low voltage or ground level to the probe terminals, and a controller configured to control the switches according to a control scheme for supplying pulses to the probe terminals, where the pulses are intervals It is supplied to a group consisting of at least two probe electrode terminals for the probe electrode which is positioned and placed.

[0014] Preferably, the pulse is bipolar with respect to the pair of electrodes. Preferably, each terminal of the pair is sequentially driven by the voltage amplitude and grounded in phase with the corresponding probe terminal. Preferably, the controller applies a ramp-up (t) of less than 0.5 μs to each probe electrode terminal, up to a plateau (U) amplitude in the range of 100 V to 3000 V with a duration in the range of 1 μs to 5 μs. r ) Duration, and ramp-down from the voltage less than 0.5 μs (t f The duration is configured to apply.

[0015] Optionally, the amplitude is in the range of 700V to 1600V, the pulse plateau duration is in the range of 1μs to 3μs, and preferably the energization on time for supplying pulses is in the range of 100μs to 300μs. Preferably, pulses are supplied immediately and continuously. Preferably, the bipolar frequency is in the range of 100kHz to 500kHz.

[0016] Preferably, the controller is configured to apply a plateau with a near-zero duration after the electroporation operation, allowing ramp-down decay in the pulse, and preferably the decay rate is set by a permanent or adjustable value of the overall resistance of the charged capacitor.

[0017] Optionally, the controller comprises mapping data defining the relative physical positions of the probe terminals connected to the terminals, and is configured to direct a pulse for applying a voltage across the space defined by the probe electrodes.

[0018] Preferably, the controller is configured to simultaneously drive a first group of terminals at the same potential and a second opposing group at a different potential to apply charge across and between the planes defined by the first and second mapped probe locations.

[0019] Optionally, the first group is driven at a high potential and the second group is grounded. Preferably, the controller is configured to immediately reverse the direction across the plane, the first group is applied the potential previously applied to the second group, and vice versa, and the first and second groups are redefined such that charge is applied in one direction across the space and then immediately reversed.

[0020] Preferably, after reversing the direction across the plane, the controller is configured to drive third and fourth groups defining the plane, wherein the direction between the third and fourth groups is different from the direction between the first and second groups.

[0021] Preferably, the directions are substantially orthogonal. Preferably, the mapping data relates to a plane defined by at least one quadrilateral bounded by four probes. Preferably, the mapping data relates to a plurality of quadrilaterals.

[0022] Optionally, the mapping data defines a quadrilateral and some probes serve to define the sides of different adjacent quadrilaterals. Preferably, the switching circuit includes switches dedicated to each voltage level of each terminal. Preferably, the switching circuit includes a switch dedicated to the high voltage level applied as a pulse to the terminal and a switch dedicated to grounding the terminal.

[0023] Preferably, the switching circuit includes a driver circuit dedicated to each switch. Preferably , each driver circuit can be individually addressed by the controller. Optionally, each driver circuit includes an independent floating power supply. Preferably, the switch includes a FET and / or an IGBT.

[0024] Preferably, the controller is configured to first execute a probe query to determine a desired drive profile from the probe memory. Preferably, the probe query is executed by the controller to set the optimal parameters of the probe.

[0025] Preferably, the controller is configured to measure the impedance in the biological load by applying an AC signal to the probe electrode across the frequency spectrum. Preferably, the frequency spectrum ranges from 1 kHz to 100 kHz. Preferably, the impedance measurement is performed before electroporation driving, and the controller is configured to automatically adjust the drive parameters according to the measured impedance to achieve a current across a pair of electrodes preferably less than 500 mA.

[0026] We also describe an electroporation device of any example described herein and a plurality of probe electrodes connected to the probe terminals. In one example, the probe electrode is hollow and located in a probe head comprising at least one electrode needle having at least one opening for the inflow of material into the tissue before and / or during and / or after electroporation. Preferably, the controller is configured to control the flow of material to one or more needles, including driving the needles with pulses.

[0027] We also describe a method of operating such an apparatus, which includes the steps of inserting the probe electrode into a biological load and the apparatus applying a bipolar voltage pulse to the electrode and the load for electroporation.

[0028] Preferably, the controller applies pulses bipolarly to the opposing probe electrodes. Preferably, the pulse plateau (U) duration (Δt) is in the range of 1 μs to 5 μs, preferably 1 μs to 3 μs. Preferably, the pulse plateau voltage amplitude (U) is in the range of 100 V to 3000 V, preferably 700 V to 1600 V.

[0029] Preferably, the duration of the active treatment in which the probe electrode is pulsed is in the range of 100 μs to 300 μs, and the bipolar pulse frequency based on a cycle of two consecutive pulses is in the range of 100 kHz to 500 kHz.

[0030] Preferably, the electrodes are driven such that the current flowing through the probe electrode and biological load is less than 500 mA for the transfer of DNA to the permeabilized cells. Preferably, irreversible electroporation is performed in which a hyperosmolar solution is injected into the site to assist in the cell excision process, and the solution may contain calcium.

[0031] Preferably, calcium ions (Ca++) are injected into the tumor at a concentration ranging from 2 mMol / L to 250 mMol / L, more preferably from 2 mMol / L to 150 mMol / L.

[0032] Preferably, a solution containing calcium ions (Ca++) at a concentration of 2 mMol / L to 150 mMol / L is injected for temporary permeabilization by electroporation with an applied pulse voltage of 500 V / cm to 1500 V / cm, a pulse plateau duration of 1 μs to 3 μs, and a pulse count in the range of 1,000 to 10,000.

[0033] In other recommended cases, calcium ions (Ca++) are injected into the tumor at concentrations ranging from 2 mMol / L to 250 mMol / L, preferably from 2 mMol / L to 150 mMol / L.

[0034] In other recommended examples, a solution containing calcium ions (Ca++) at concentrations ranging from 2 mMol / L to 150 mMol / L is injected for transient permeabilization by electroporation with an applied pulse voltage of 500 V / cm to 1500 V / cm, a pulse plateau duration of 1 μs to 3 μs, and a pulse count ranging from 1,000 to 10,000.

[0035] In one example, (a) Injection of DNA into the site, (b) Insertion of probe electrode, (c) Driving a pulse to an electrode for permeabilizing cells, the drive comprising a bipolar pulse with a frequency in the range of 100 kHz to 500 kHz, a pulse plateau duration in the range of 1 μs to 5 μs, preferably 1 μs to 3 μs, and a voltage amplitude in the range of 100 V to 3000 V, (d) Reversible electroporation to supply DNA to cells, consisting of a step in which low-frequency electrode drive draws DNA or drug into cells with a low-voltage pulse of less than 5V during an electrophoretic phase with a pulse duration of 10ms to 100ms.

[0036] In one example, a drug such as a chemotherapy agent is injected into the site, electroporation is performed, and the drug moves into the cells, causing cell excision. In one example, the controller discharges the accumulated charge with a pulse of nearly zero duration after the electrode is pulsed, followed by exponential decay.

[0037] In one example, the controller measures the impedance of the biological load between at least one pair of electrodes and automatically sets the pulse voltage amplitude to avoid excessive current during electroporation.

[0038] We also describe a method for performing electroporation in a subject, which comprises the steps of injecting a fluid into a target environment surrounding the cells to be treated and supplying pulses to that site with an electroporation probe.

[0039] The liquid is preferably in the form of a bubble having liquid and gas bubbles. Preferably, the bubble is supplied to achieve a higher impedance than the liquid, and therefore a lower conductivity than the liquid. Preferably, the bubble contains a blowing agent to aid in the mixing of the gas. The gas in the bubble may include air and / or CO2. Preferably, the ratio of gas to liquid is in the range of approximately 1:2 to 1:10 by volume. Preferably, the blowing agent contains one or more of albumin and human serum albumin. Preferably, the blowing agent concentration is in the range of 5% to 80% w / w. Preferably, the blowing agent contains polidocanol or sodium tetradecyl sulfate (STS).

[0040] Preferably, the liquid contains an activating therapeutic composition. Preferably, the composition contains one or more selected from calcium, potassium, bleomycin, cisplatin, DNA, and RNA. Preferably, the therapeutic composition contains one or more of calcium ions, potassium ions, bleomycin, cisplatin, DNA, or RNA.

[0041] The liquid preferably has a certain concentration of ions, such as calcium or potassium ions at a concentration of 2 mMol to 250 mMol, or more preferably 2 mMol to 150 mMol, for several applications.

[0042] Preferably, the electrical pulse has a pulse length in the range of 0.05 μs to 5 μs. Preferably, in many treatments, the electrical pulse is bipolar. Preferably, the electrical pulses are supplied in the train with an "on" energization time of 1 μs to 1000 μs per train, repeated up to 1000 times at a frequency in the range of 1 kHz to 1000 kHz.

[0043] In some cases, the treatment is electrochemotherapy, with pulse voltages ranging from 500 V / cm to 1500 V / cm, and / or pulse durations ranging from 50 μs to 100 μs, and / or pulse frequencies ranging from 1 Hz to 5000 Hz, and / or pulse counts ranging from 4 to 8.

[0044] In some cases, the treatment is irreversible electroporation, with a pulse voltage ranging from 1500 V / cm to 3000 V / cm, and / or a pulse duration ranging from 70 to 100 μs, and / or a pulse frequency ranging from 0.5 Hz to 10 Hz, and / or a pulse count ranging from 90 to 200.

[0045] In some cases, the treatment is high-frequency irreversible electroporation, with pulse voltages ranging from 2500 V / cm to 5000 V / cm, and / or pulse durations ranging from 1 μs to 5 μs, and / or pulse frequencies ranging from 100 kHz to 500 kHz, and / or pulse counts exceeding 100.

[0046] In some cases, the treatment is electroporation and electrolysis (E2), the pulse voltage is in the range of 100 V / cm to 3000 V / cm, and / or the supplied charge is greater than 100 μF, and / or the pulse is an exponentially decaying wave.

[0047] We also describe how the treatment is a DNA or RNA supply, the pulse voltage is in the range of 1 V / cm to 1000 V / cm, and / or the pulse is a unipolar square wave.

[0048] In one embodiment, the liquid provides dispersion of a local anesthetic to the tissue to be treated, preferably in the form of foam. In one embodiment, the local anesthetic comprises lignocaine (also known as "lidocaine"). In one embodiment, the local anesthetic comprises lignocaine in a proportion of 5 to 20 mg / ml, with or without adrenaline. In one embodiment, the local anesthetic comprises mepivacaine. In one embodiment, the local anesthetic comprises mepivacaine in a proportion of 10 to 30 mg / ml.

[0049] In one embodiment, preferably, a liquid having an anesthetic in the form of foam is administered in combination with selected molecules such as one or more of calcium ions, potassium ions, bleomycin, and DNA.

[0050] We also describe foam for use in assisting cell permeabilization or excision during electroporation. Preferably, the foam contains a therapeutic composition. Preferably, the composition contains one or more selected from calcium, potassium, bleomycin, cisplatin, DNA, and RNA.

[0051] Preferably, the foam has an ion concentration. Preferably, the foam contains calcium ions at a concentration of 2 mMol to 150 mMol. One recommended example is foam containing calcium ions, potassium ions, bleomycin, and DN. Includes an anesthetic in combination with one or more selected molecules, such as A.

[0052] We also describe an electroporation probe head comprising at least one electrode needle that is hollow and has at least one opening for the inflow of material into the tissue before, and / or during, and / or after electroporation.

[0053] Preferably, there are multiple needles. The needles may have a maximum inner width dimension in the range of 0.1 mm to 1.8 mm and a maximum outer width dimension in the range of 0.25 mm to 2.5 mm, and the opening may have a maximum width dimension in the range of 0.05 mm to 1.5 mm.

[0054] In one example, multiple needles are present, with spacing between them ranging from 2 mm to 3 cm. The openings may be arranged spirally or staggered along the length of the needle. The openings may have a greater distribution on the side where at least one needle faces another.

[0055] We also describe an electroporation apparatus comprising a pulse generator connected to a probe head, which has a hollow electrode needle having at least one opening for the inflow of material into the tissue before, and / or during, and / or after electroporation.

[0056] Preferably, the device includes a controller adapted to control the supply of pulses to the needle and the supply of material to the needle in a desired manner. Preferably, the controller is configured to sequentially cause the supply of the first substance to at least some needles, the pulsing of the needles, and the supply of the second substance to the needles. Preferably, the first substance has a lower conductivity than the second substance. In one example, the first substance contains bubbles. In one example, the first substance contains bubbles, the second substance contains bubbles, and the bubbles of the first substance have a higher gas concentration than the bubbles of the second substance.

[0057] We also describe a method of operating an electroporation apparatus, which involves pumping a substance through a needle so that it flows out from an opening, and applying pulses to the needle. This method may include supplying the substance through the needle before and / or after the application of pulses.

[0058] The present invention will be better understood from the following description of some embodiments, which are given only as examples with reference to the following appended drawings. [Brief explanation of the drawing]

[0059] [Figure 1] This is a perspective view of an electroporation device. [Figure 2] This is a block diagram of the device. [Figure 3] This diagram shows a high-voltage generator equipped with a transformer. [Figure 4] This diagram shows the pulse switching and control block in detail. [Figure 5] This diagram shows the flow of electricity to the biological load during use. [Figure 6] This diagram shows the electrical characteristics of the probe. [Figure 7] This is a flowchart of the pulse generator algorithm implemented by the controller. [Figure 8] This is a diagram illustrating the spatial application of voltage by a probe under biological stress. [Figure 9] This is a diagram of the electroporation pulses supplied by the generator, and their parameter values ​​are shown in Table 1 below. [Figure 10] This is an image of a sample pulse captured by a CRO. [Figure 11] This is a series of plots showing tissue impedance before and after electroporation. [Figure 12] This is a series of plots showing tumor volume changes in four scenarios for comparison purposes. [Figure 13]This is a plot of muscle contraction induced by electroporation, demonstrating the advantages of shorter duration and higher frequency. [Figure 14] This is a plot of a pulse waveform for one example. [Figure 15] This is a plot of propidium iodide uptake by electroporation. [Figure 16] This plot shows the impedance and current in tissue containing an injected liquid solution compared to the injected foam solution. [Figure 17] This is a plot of cell permeation versus electric field intensity for low-conductivity buffers and high-conductivity buffers. [Figure 18] This is a plot of cell survival against low and high conductivity buffers. [Figure 19] This is a diagram showing the foam injection head of an electroporation device. [Figure 20] This plot shows the decrease in electroporation current resulting from the use of bubbles at various voltage drive levels. [Modes for carrying out the invention]

[0060] As shown with reference to Figures 1 and 2, the electroporation apparatus 1 includes a main controller 2 connected to a user interface 3 and a probe drive circuit 4, all of which are mounted in a housing 9. The controller 2 and interface 3 provide user-side functions, while the drive circuit 4 provides pulses to the probe 26.

[0061] The drive circuit 4 is, To prevent high voltage from being inadvertently transmitted to low-voltage control electronic equipment, the pulse control unit 5 is isolated from the rest of the drive unit 4 by an optical isolator 18. A high-voltage generator 6, equipped with a transformer and providing a maximum of 1.5kV across capacitors, as will be described in more detail below with reference to Figure 3, As will be explained in more detail with reference to Figure 4, a pulse switching controller 7 is used to supply high-frequency bipolar pulses to an array of seven probe electrodes 26, It includes a voltage setting and maintenance circuit 8.

[0062] The touchscreen interface 3 is operably coupled to controller 2, which generates high voltage and controls pulses, as well as managing voltage, pulse duration, polarity, and orientation. This level of control is achieved through a series of control circuits in blocks 5-8.

[0063] A high-voltage generator 6 is shown in Figure 3. A switch controller 5 receives a low-voltage DC input, for example, 48V. This input level sets the resulting pulse intensity level supplied to the probe 26, as described below. The switch controller 5 controls the output voltage of the capacitor 14 by adjusting the time the primary side 1 of the transformer is energized. Feedback from the output to the switch controller 5 (with proper isolation via a medical-grade optocoupler device 18) allows the voltage to be precisely set and maintained. Thus, the output voltage of the capacitor 14 can vary over a wide range (typically from 100V to about 1.5KV).

[0064] In this example, transformers 11 and 12 boost the input voltage to 1.5kV, which is then rectified by a conventional rectifier 13. This high voltage level (currently DC) appears across a balanced resistor 15 and a 1000μF capacitor 14 as an output to the probe electrode (26). In this example, each of the four capacitors 14 handles a maximum of 450V. The charging time is less than 10 seconds.

[0065] Diode 13 withstands the total output voltage as well as the peak reverse voltage generated by switching in the transformer. This is typically several times the forward voltage. A high-voltage capacitor 14 is placed in series to enable operation at the required voltage and, together with a resistor 15 in parallel with the capacitor, helps to level the voltage between them. For clarity, only two windings 12 are shown at the transformer output, but in reality, this number may be greater. The recommended parameter range for these components is 2 to 10.

[0066] The high-voltage generator 6 provides this level of DC voltage to the pulse switching controller 7, which then applies this voltage to the probe electrode 26 via terminal 27 in a controlled pattern for optimal electroporation treatment. This will be explained in more detail below.

[0067] As shown with reference to Figures 4 and 5, the pulse switching controller 7 and the voltage setting and maintenance circuit 8 are shown in more detail. The driver circuit 22, with an independent floating power supply 20, is individually addressable by the controller 5, so that the IGBTs 24 and 25 supply high-voltage pulses to the probe terminals 26 and 27, and the polarity and duration of the voltage supplied to the probe are optimized.

[0068] To achieve the required switching of the probes, the driver circuit 22 connects each probe electrode 26 to either a high-voltage rail HV or ground GND, and the voltage duration and polarity of the pin pairs can be set by software. To achieve the required high voltage, insulated-gate bipolar transistors (IGBTs) (or FETs in other examples) are used. For even higher voltages, the number of transistors can be increased. Only seven circuits are shown, but the number of outputs can be increased or decreased as needed.

[0069] As described above, the pulse switching controller 7 comprises seven top driver circuits and seven low driver circuits 22, each with a dedicated link to a power supply 20 (mainly the capacitor 14 of the high-voltage generator 6). There are a series of pairs of IGBTs 24 / 25 (seven in this example), each pair for one probe electrode terminal 27. For example, the first IGBT pair 24 / 25 (leftmost in Figure 4) connects the first probe electrode P1 to the high voltage from the rail HV or to ground GND. At the same time, the probe electrode P2 of the second IGBT pair is connected to ground or the HV rail, conversely. Thus, current passes through the biological load in either direction.

[0070] At any given time, one probe electrode will have either a high or low voltage (+1.5kV or -1.5kV), and the opposite electrode will be grounded. The fact that the voltage on one electrode of the pair is 1.5kV lower than that on the other electrode gives a relatively negative pulse. Therefore, looking at the pair of opposing electrodes, the voltage is bipolar, and the relative voltage switches from +1.5kV to -1.5kV.

[0071] The voltage setting and maintenance circuit 8 comprises a comparison device and an isolation control circuit that ensures the output voltage is maintained at a set voltage for pulse train supply. As shown with reference to Figure 6, the probe 26 has a memory device 40 that stores probe type, configuration, and output setting data. A single-line memory I / O line exists to the memory device 50.

[0072] As shown with reference to Figure 7, the pulse switching control algorithm 100 is performed by the controller 5 for pulse generation and application. There is an operation start sequence 50 in which the probe 26 memory device 40 is read for probe type, configuration, and output setting data. In the probe drive sequence 100, the low-side driver 22 The high-side driver 22 is then turned on. The controller waits for the required pulse width and compensation time, and then turns off the associated high-side driver 22. The controller waits for the off time and compensation time, and then turns off the low-side driver 22. Negative polarity pulses can then be generated as needed.

[0073] The controller manages control pulses to rotate the voltage source by 180° using a dual-pin probe contact technique, as shown in Figure 8, where the pin probe electrodes are individually labeled P1 through P7.

[0074] The switching controller 5 queries the probe 26 to first verify that it is authorized and to discover an authorized pulse profile for a specific application. The probe identity is evaluated and the appropriate pulse profile and sequence are defined. When the probe 26 is plugged in, the sense wire activates the microcontroller by pulling the sense line low. The microcontroller can then communicate with the memory device 40 to read the probe parameters. Several copies of the parameters are stored in the probe memory 40 along with error detection to verify that the reading is correct. The sequence for reading the probe identity is as follows:

[0075] A sense probe is attached. Communicate with the memory device and read the first set of parameters. Verify the error detection code.

[0076] If verified, complete the setup. If not validated, read the next block of parameters. Verify the error detection code.

[0077] If a suitable parameter set cannot be found, an error message will be generated. The probe type is displayed on the screen as a visual indicator for the user. As illustrated by referring again to Figure 8, rotating the voltage pulse as shown in the example results in comprehensive electroporation of the affected tissue. In this scheme, the electrodes are energized in pairs, and the direction of the pulse can be adjusted in a controlled manner by manipulation. Electrodes P1 and P2 are positive (connected to HV via upper IGBT 24), and electrodes P7 and P3 are negative (connected to GND via lower IGBT 25), giving the marked field marked 1 in Figure 8. Reversing the polarity of the pair (i.e., P1 and P2 negative, P7 and P3 positive) gives the opposite field. If electrodes P1 and P7 are positive and electrodes P2 and P3 are negative, the field marked 5 in Figure 8 crosses the field marked 1. Again, reversing the polarity of the probes gives the opposite field. The same applies to other possibilities.

[0078] Cancer cells exhibit characteristic fractal patterns, and solid tumors are composed of millions of these cells. The geometric shape of solid tumors can be modeled as ellipsoids ranging from flattened to oblong. Small tumors can be treated in a single run at a single location, but for tumors whose size is greater than the distance between electrodes, it is necessary to rotate the electrodes around the tumor surface to maximize cell permeability and subsequent treatment. The device described here offers advantages in ease of use and efficiency by reducing the number of probe repositioning steps, as the device rotates the electric field around the tumor surface.

[0079] In addition, the uniform electric field supply from multiple directions across the entire tissue from a single location offers clinically significant advantages, increasing the potential for cell membrane poration and allowing for the removal of chemotherapeutic drugs such as bleomycin and cisplatin, or calcium, potassium, and gene therapy. The therapeutic effect is maximized when combined with other treatments such as ointment.

[0080] Furthermore, it is important to note that to obtain the optimal therapeutic effect, pulses must be delivered during the pharmacokinetic peak. This device reduces the number of times electrodes need to be repositioned, thus shortening the time it takes to deliver pulses across the entire tumor surface. In addition, as will be explained in more detail below with reference to Figures 19 and 20, the direct delivery of substances such as therapeutic agents through electrodes used through pores along their length helps to optimally ensure their accurate and efficient delivery.

[0081] In addition, these fields can rotate around the tumor surface, allowing for the use of milder field conditions. The scheme shown in Figure 8 is based on at least four probe electrodes arranged in a square, thereby defining the space in the cross-section between them. Two electrodes defining one side of the square (e.g., P1 and P7) are positive, and two electrodes defining opposite sides (e.g., P2 and P3) are negative, and this state is immediately reversed. This is then carried out in the orthogonal direction (e.g., P7 and P3 are positive, P1 and P2 are negative). The same pattern is repeated for other squares defined by electrodes P1-P6-P5-P7 and probes P5-P7-P3-P4. Of course, many of the electrodes play the role of different squares.

[0082] The effect of this scheme is that the tissue between electrodes 26 is grouped into three zones bounded by rectangles, each of which is comprehensively treated in four nearly orthogonal charge directions through the tissue, as indicated by the arrows within the rectangles in Figure 8.

[0083] Generally, the controller is configured to simultaneously drive a first group of probe terminals 27 having the same potential (e.g., 1.5kV) and an opposing second group (e.g., P2, P3) having a different potential (e.g., ground) to apply a charge across and between (e.g., orthogonal) planes defined by first and second probe locations. Since the device 1 is for driving an external set of probes, it is programmed to drive the terminals 27 using a probe location preset. This preset may be called mapping data, which can take the form of a series of instructions for the driving scheme or may indicate physical locations. This preset may arise from probe queries and / or user input.

[0084] The controller advantageously reverses the direction across the plane instantly, so that the first group is applied with the potential previously applied to the second group, and vice versa, and the first and second groups are redefined so that the charge is applied in one direction across space and then instantly reversed. The controller also drives third and fourth groups (e.g., P1, P2 and P3, P7) that define the plane after reversing the direction across the plane, but the directions between the third and fourth groups are different from the directions between the first and second groups, and are nearly orthogonal in one example, for example, as shown by the arrows in the rectangle P1-P2-P3-P7.

[0085] Another advantageous aspect of the electroporation scheme is that there is a ramp-up, plateau, and ramp-down sequence to drive pulses to a pair of probes. Each ramp duration is less than 0.5 μs, and typically less than 0.25 μs, due to the short turn-on time of the switches used and the fact that each is driven by an individual driver circuit 22 dedicated to the individual switch, each individually addressable by the controller. Thus, the minimum required for each pulse for therapeutic purposes If the duration is, for example, 2 μs, the total duration required is only 2.5 μs, at which point the direction is instantly reversed. Therefore, the total duration for one axis through the tissue is 5 μs in both directions at 180°. By immediately switching the direction by 180°, optimal treatment is possible, minimizing patient discomfort caused by muscle contraction. This often avoids the need for general anesthesia, for example, in the treatment of skin cancer. To improve the results, pulses can also be generated orthogonally across the tissue in both directions, also with a duration of 5 μs.

[0086] As shown in Figure 9, the main parameters of the drive pulse are shown. In these examples, the values ​​of these parameters are as follows: Table 1

[0087] [Table 1]

[0088] In the table above, Example 1 is recommended. Examples 2 and 3 are examples of different electrode configurations with different depths and separation distances, as outlined in the table. These differences in electrode configurations result in longer ramp durations. In extreme cases, as described above, the ramp-down value can approach 1 μs, which is generally not recommended, although a ramp duration of less than 0.5 μs is recommended.

[0089] As shown in Figure 10, Table 1 contains image captures of typical pulses from a pair of opposing electrodes obtained during the experiment, specifically 1000V, 2μs bipolar pulses. ΔU sIt is not visible in this image due to the electrode position and the impedance of the potato tissue. There is a slight delay of less than 1 μs due to the dead time for one transistor pair to turn off before the other transistor pairs turn on (preventing a direct short circuit through the system). The circuit design is engineered to minimize this dead time. The oscilloscope shows a bipolar pulse (+1000V, -1000V) with sharp rise and fall times, where the amplitude of the electroporation pulse remains at the expected level for the duration of the pulse. The features (U, tr, tf, U / 2) in Figure 9 can be identified in this image.

[0090] The multi-stage transformer approach and output switching are understood to provide high voltage / high current and flexible polarity control within a compact housing. Furthermore, the integration of probe detection and automatic parameter setting ensures the safe delivery of application-specific doses.

[0091] Switch-mode transformers 11 / 12 operate efficiently at low voltages, but tend to have design trade-offs when high voltages are applied. This problem is avoided in the present invention, particularly for the following reasons.

[0092] - Use of transformer drive circuits designed to minimize dead time losses across a wide range of operating conditions and component variations. - A predefined voltage Vout is supplied by a modular circuit Vmod using n secondary windings in series, where Vout = Vmod * n.

[0093] - Selection of core and winding materials for transformers. - Use of a feedback circuit (8) to monitor and maintain this voltage. The device will be understood to generate safe and effective high-voltage pulses with precise and reliable control of the pulse profile, i.e., time, duration, polarity, and direction. This is particularly advantageous for ensuring effective treatment without the need for burning sensations or undesirable levels of anesthetic. The level of control is also provided by sensing the connected probe and automatically verifying the configuration of the permitted operating parameters and generator parameters. It is particularly advantageous that the device performs a machine start query on the probe to verify compatibility and selects the appropriate pulse generation profile from an internal lookup table.

[0094] In another example, controller 2 may generate a research interface screen that provides access to more pulse configuration setting options available under controlled conditions. This allows researchers to modify pulse parameters up to the maximum value allowed for the probe in order to investigate the optimal parameters.

[0095] Examples of device usage The above apparatus can be used for various therapeutic methods of either irreversible or reversible electroporation. Some of these methods can be performed using other electroporation apparatus known in the art, provided that the indicated electrical parameters can be provided.

[0096] As stated at the beginning of this specification, electroporation is a medical and molecular biological technique that applies an electric field to a cell or molecule to increase the permeability of the cell membrane, allowing chemicals, drugs, or DNA to be introduced into the cell. Electroporation has many applicable medical fields and can be used both reversibly and irreversibly. In a typical electroporation procedure, a short, strong electrical pulse is generated to temporarily permeate the cell membrane.

[0097] For example, in reversible electroporation, which may be used in conjunction with chemotherapy, the electric field is lowered below the electric field threshold, allowing the cell membrane to be repaired after treatment. Reversible electroporation may involve allowing molecules such as drugs or genes to enter cells or molecules that are normally impermeable to this substance without inducing cell death. The electric field threshold of a cell varies from cell to cell.

[0098] In irreversible electroporation (for cell excision), the electric field is higher than a certain electric field threshold, which creates permanent nanopores in the cell membrane, disrupting cellular homeostasis and consequently forcing the cell into irreversible cell death pathways such as apoptosis or necrosis mechanisms. Hit the target.

[0099] We describe an electroporation method which includes the steps of inserting probe electrodes into a biological load, applying voltage pulses to the electrodes and the load for electroporation, preferably by a controller applying pulses bipolarly to opposing probe electrodes.

[0100] Preferably, the pulse plateau (U) duration (Δt) is in the range of 1 μs to 5 μs, preferably 1 μs to 3 μs. Preferably, the pulse plateau voltage amplitude (U) is in the range of 100 V / cm to 3000 V / cm, preferably 500 V / cm to 2000 V / cm. Alternatively, the individual pulse length may be less than 1 μs, in the range of 0.03 to 0.99 μs, and the pulse plateau voltage amplitude (U) may be in the range of 5 KV / cm to 50 KV / cm, preferably 10 KV / cm to 20 KV / cm.

[0101] Preferably, the duration of the active treatment in which the probe electrode is pulsed is in the range of 100 μs to 300 μs, and the bipolar pulse frequency based on a cycle of two consecutive pulses is in the range of 100 kHz to 500 kHz.

[0102] The electrodes can be driven so that a current flows within the probe electrode, and the biological load is less than 500 mA for the transfer of DNA to the permeabilized cells. In some cases, irreversible electroporation is performed in which a substance, such as a liquid solution including a hyperosmolar solution, is injected into the site to assist in the process of cell excision, and the solution may contain calcium or potassium. Preferably, calcium ions (Ca++) are injected directly into the tissue at a concentration ranging from 2 mMol / L to 250 mMol / L, preferably from 2 mMol / L to 150 mMol / L.

[0103] The composition of the material can be selected, at least partially, to induce a desired level of tissue conductivity adjacent to and between the electrodes. For example, the material may contain deionized water to reduce conductivity. Alternatively or additionally, as will be described in more detail below with reference to Figures 15 to 18, the material may contain a foam solution that preferentially increases resistance (ohms) in the tissue environment, as opposed to a more conductive liquid solution. As a result, the current (amperes) generated during the supply of electroporation pulses is reduced in the tissue to which the foam solution is injected, allowing for safer and more effective treatment.

[0104] Injection of material For transient permeabilization by electroporation, a solution containing calcium ions (Ca++) at concentrations of 2 mMol / L to 150 mMol / L may be injected with applied pulse voltages of 800 V / cm and 2000 V / cm, pulse plateau durations of 1 μs to 3 μs, and pulse counts ranging from 1,000 to 10,000. The calcium ion-containing solution is just one example; other examples are given below.

[0105] In one example, (a) Injection of DNA into the site, (b) Insertion of probe electrode, (c) Driving a pulse to an electrode for permeabilizing cells, the drive comprising, for example, a bipolar pulse with a frequency in the range of 100 kHz and 500 kHz, a pulse plateau duration in the range of 1 μs to 5 μs, preferably 1 μs to 3 μs, and a voltage amplitude in the range of 100 V / cm to 3000 V / cm. (d) Each step consists of a low-frequency electrode drive that draws DNA or drug into cells with a low-voltage pulse of less than 5V during an electrophoretic phase with a pulse duration of 10ms to 100ms, and DNA is supplied to the cells by reversible electroporation.

[0106] One example of DNA supply is, DNA injection into the site, and / or, Supply of one or more unipolar pulses ranging from 800 V / cm to 1600 V / cm, then, For example, treatment using a bipolar HF with a frequency in the range of 100 kHz to 500 kHz, a pulse plateau duration in the range of 1 μs to 5 μs, preferably 1 μs to 3 μs, and a voltage amplitude in the range of 100 V / cm to 3000 V / cm, and / or There is a supply of one or more unipolar pulses ranging from 1 V / cm to 200 V / cm.

[0107] A single-pole pulse with a higher voltage of 800V to 1600V may have a pulse duration in the range of 50μs to 250μs, preferably in the range of 100μs to 200μs. The voltage is more preferably in the range of 1000V / cm to 1200V / cm.

[0108] A single-pole pulse at lower voltages from 1 V / cm to 200 V / cm may have a pulse duration of 10 ms to 10 s, preferably 20 ms to 100 ms. This voltage is preferably in the range of 100 V / cm to 150 V / cm.

[0109] The second unipolar pulse is preferably supplied within 1 second of the first unipolar pulse. In one example, a drug is injected into the site, electroporation is performed, and the drug, such as a chemotherapeutic agent, moves into the cells, causing cell excision.

[0110] In one example, the controller discharges the accumulated charge with a pulse of nearly zero duration after the electrode is pulsed, followed by exponential decay. Opposing electrodes are inserted into the biological load at intervals of 1 mm to 30 mm, more preferably 1 mm to 10 mm, and more preferably 2 mm to 8 mm. The probe electrode may be a needle electrode, but is not necessarily required.

[0111] The controller of the electroporation apparatus used is programmed to provide high-frequency operating parameters within the following range: The pulses from opposing probes or electrodes are preferably bipolar.

[0112] The frequency (bipolar frequency, the cycle which consists of both positive and negative pulses) is preferably in the range of 100 kHz to 500 kHz. In this specification, this is referred to as the “high frequency.” This corresponds to a pulse length (plateau) of about 1 μs to 5 μs, although in many applications, this range is more preferably in the range of 1 μs to 3 μs, and more preferably about 2 μs.

[0113] The voltage amplitude is in the range of 100 V / cm to 3000 V / cm, preferably about 800 V / cm to 2000 V / cm. Electroporation pulses may be effective in the presence of high-osmotic pressure solutions or foaming agents, as detailed below.

[0114] The duration of the active treatment in which the probe electrode is pulsed is preferably in the range of 0.1 ms to 1 s, and more preferably in the range of 0.2 ms to 10 ms. The pulses are supplied in bursts (single energization) with durations ranging from 100 μs to 300 μs within this duration. For example, if the pulse length is 2 μs, there will be approximately 25 to 75 pulse cycles for the energization on time.

[0115] The probe current should be less than 1A, preferably less than 500mA. However, the current is a value derived depending on the biological load. For DNA transfer into cells, a current of less than approximately 500mA is more preferable, but it is less critical in other methods. In this example, the current when supplying high-frequency pulses can range from 5A to 40A. For DNA supply, the applied voltage is preferably less than 5V and the current is less than 1A.

[0116] When the medication is administered to the site, it is done by any well-known injection technique. Fluid injection: Hyperosmolar therapy for cell excision A liquid solution injected locally into the target tissue facilitates irreversible electroporation and cell death, enabling lower voltage pulses in the high-frequency drive outlined above. Such a solution may, for example, contain calcium ions to induce cell death.

[0117] In one example, calcium ions (Ca++) are injected into the tumor at concentrations ranging from 2 mMol / L to 150 mMol / L, preferably at the lower end of this range for the high-frequency drive outlined above.

[0118] Liquid injection: DNA supply (reversible electroporation) Reversible electroporation includes the following: (a) Injection of DNA into the site. (b) Insertion of the probe. (c) High-frequency treatment according to the high-frequency parameters described above to permeate the cells. This is called the electroporation phase. (d) Low-frequency therapy to draw DNA into cells. This is called the electrophoresis phase. In this phase, there is a low-frequency, low-voltage pulse of less than 5V during a pulse duration of 10ms to 100ms. This enables effective uptake of DNA into cells, mainly due to the negative charge of the DNA.

[0119] Liquid injection: Drug supply The drug is injected into the site, electroporation is performed, and the drug's intracellular passive diffusion is enabled. In one example, the drug is selected to induce cell death (resection), such as a chemotherapeutic agent. The passive diffusion of the chemotherapeutic agent is enabled by the effect of high-frequency electroporation, leading to cell death. For example, the drug could be bleomycin or cisplatin.

[0120] Generally, in the case of drug delivery, unlike in the case of DNA delivery, there is no low-frequency stage (as described in (d) above). As shown with reference to Figures 11 to 13, these figures are Effects of high-frequency versus standard low-frequency electroporation pulses on potato cells. The effects of high-frequency electroporation versus standard low-frequency electroporation (pulse length approximately 100 μs) in mouse tumor models when combined with drugs, and This shows a decrease in muscle contraction due to high-frequency electroporation. The data in Figures 11 to 13 clearly demonstrate effective results at clinically manageable voltages (<1500V) that allow for the absorption of drugs, ions, and DNA.

[0121] Figure 11 Healthy biological tissue exhibits different electrical impedances than abnormal precancerous tissue; that is, current flows more easily across abnormal precancerous tissue, and therefore the measured impedance is lower than that of healthy biological tissue.

[0122] Tissue impedance and changes in tissue impedance can, in some cases, be measured by the instrument across the frequency spectrum. This allows the controller to control its operation. It automatically modifies the system to generate user information, for example, to distinguish between abnormal precancerous tissue and healthy tissue. This is done before and during treatment, providing diagnostic capabilities and enabling the treating healthcare professional to receive actionable feedback on the treated and planned areas. The controller can also automatically avoid adverse effects from significantly reduced impedance by automatically adjusting the voltage level to ensure that the current does not become excessive.

[0123] The controller measures impedance by applying an AC signal to the tissue in a suitable frequency spectrum, typically between 1 kHz and 100 kHz. This is done immediately before and / or after high-frequency electroporation.

[0124] Figure 11 shows the effects of high-frequency (2 μs bipolar, approximately 217 kHz) versus low-frequency (100 μs monopolar, 1 Hz) and 70 μs monopolar, 1 Hz) electroporation pulses on potato tissue. This shows the tissue impedance measured using a potato tissue model. A pair of probe needle electrodes were inserted 0.4 cm apart, and the tissue impedance was measured before and after electroporation (the experiment was performed three times).

[0125] Impedance changes were monitored using the following parameters: - A 2μs bipolar (approximately 217KHz) with a current duration of 200μs (50 pairs) supplied 50 times at 1000V / cm, - A 100μs unipolar (1Hz) supplied 8 times at 1000V / cm, - A 70μs unipolar (1Hz) pulse supplied 90 times at 1500V / cm.

[0126] Changes in tissue impedance are caused by the opening of the ion gate after the electroporation pulse and the release of electrolytes into the surrounding tissue. This indicates the degree of electroporation. The largest differential change occurred with a change of 130 in the irreversible electroporation parameters of group C (right plot), followed by a change of 118 in group A (left plot). Standard electroporation parameters (group B) (center plot) as a control for drug delivery provided a net change of 72. The data show the degree of tissue electroporation achieved with the bipolar high-frequency parameters that occur, despite the observation of reduced muscle contraction with high-frequency delivery. This data indicates cell permeabilization, and the left plot shows that high-frequency operation of the device achieves good permeabilization while avoiding the risk of patient discomfort due to high-frequency operation.

[0127] Figure 12 Figure 12 shows the effects of high-frequency (217 kHz, 2 μs pulse plateau) electroporation versus low-frequency (1 Hz) electroporation in combination with drugs in a mouse tumor model.

[0128] Preclinical results performed with device 1 using a mouse tumor colorectal model are shown. The tumor was 0.1 cm in mice. 3 They grew to this stage and were subsequently treated. Four groups were studied, with six mice in each group. A) Control group, no treatment. B) Controlled by ECT (electrotherapy). C) ePORE (device 1) containing bleomycin, and D) ePORE containing calcium (device 1).

[0129] ECT control (B): Standard electroporation parameters using direct injection of bleomycin (250 IU) with an applied electric field of 1000 V / cm and polar pulses totaling 8,100 μs at 1 Hz.

[0130] ePORE Bleomycin (C): A pulse delivered as a bipolar pulse at a frequency of 217 kHz. Bleomycin (250 IU) was directly injected. ePORE Calcium (D): The same applies to ePORE Bleomycin as above, but it uses calcium injected into the tumor at a concentration of 9 mg / ml and pulses supplied as bipolar pulses at a frequency of 217 kHz.

[0131] Short ramp times (<0.25 μs up to 1000 V) allow for more uniform electric field penetration through the cell membrane, and at higher frequencies, it becomes possible to more efficiently take up large macromolecules and ions (including Ca++) that directly pass through the cell membrane.

[0132] In one example, DNA uptake across the cell membrane was achieved using high-frequency (>200 kHz) bursts combined with low-current (<1 Amp) pulses. A bipolar × 50 bipolar square wave high-voltage high-frequency burst with a length of 1–2 μs (on-time of 100–200 μs) was followed within 1 minute by a unipolar square wave low-voltage low-current pulse of less than 5V <250 μAmp with a duration of 1–10 m.

[0133] This device enhances DNA endocytosis and uptake, improving cell transfection and DNA expression. In another example, cell death is achieved by supplying high-frequency electrical pulses (>200 kHz) to a hyperosmolar solution. Such solutions can be prepared from sucrose, glycerol, xylose, mannitol, or fructose at concentrations of 125 to 300 mMol / L. Such solutions may also contain up to 99 mMol / L of Ca2+ or Mg2+ or K+.

[0134] In another example, transient permeabilization is performed by electroporation using a solution containing calcium ions (Ca++) at a concentration of at least 2 mMol / L, with a voltage of 500–1500 V / cm, pulse length of 0.1 μs–3 μs, pulse count of 1000–10,000, and pulse frequency of 200 kHz–500 kHz.

[0135] Figure 13 This figure shows the reduction in muscle contraction due to high-frequency electroporation by device 1. Accelerometer data was calculated from porcine tissue when electroporation was delivered to colorectal tissue. Muscle contraction was 0.5 μm between each pulse. The results were observed when using a 1000V 2μs bipolar pulse (approximately 217kHz) with a gap of less than s, a 1000V 2μs bipolar pulse (125kHz) with a 2μs gap between each pulse, a 1000V 100μs monopolar pulse (1Hz), and a 1500V 100μs monopolar pulse (1Hz). The observed peak accelerations were 0.04g at 217kHz, 0.08g at 125Hz, 0.75g at 1000V 100μs, and 0.83g at 1500V 100μs.

[0136] Figure 14 As described above, the device drives pulses to a pair of probe electrodes in ramp-up, plateau, and ramp-down sequences. Each ramp duration is less than 0.5 μs, or preferably less than 0.25 μs, due to the short turn-on times of the switches used and the fact that they are driven by individual driver circuits 22.

[0137] In the modified version, the controller drives at least some pulses with the ramp up as described above, but the switch is turned off and the plateau is set to 0 before the pulse is achieved. This achieves an exponentially decaying pulse. The capacitor is charged normally, and then the pulse is activated and it discharges freely. The decay rate shown in Figure 14 is controlled by the values ​​of the balance resistor 15 and the capacitor 14. Thus, the controller controls the electrodes U max The amplitude is driven, and the pulse is turned off. That is, the pulse duration is set to 0. Therefore, for part of the treatment time, the electroporation device drives the pulse in a shape that decays exponentially. This operation can be useful after high-frequency operation. It effectively discharges the capacitor.

[0138] Figure 15 Flow cytometry using propidium iodide markers The relationship between the effectiveness / cell permeation of electroporation and the medium used as the cell buffer was determined using (ry).

[0139] We use standard isotonic buffer (~270 mOsm). Using a high-temperature (V / cm) electric field (V / cm) and a hyperosmotic buffer (~600 mOsm / l) containing mannitol or sucrose, the range of electric field strength (V / cm) is measured. We evaluated the use of electroporation with an ultrashort bipolar pulse length (2 μs) that induces cell permeabilization across the entire area.

[0140] The electric field intensity (V / cm, the distance between electrodes) was adjusted from 500 V / cm to 1400 V / cm at 100 V intervals, and the degree of PI uptake and the resulting cell permeabilization was determined.

[0141] To evaluate the effectiveness of electroporation delivered to cells in vitro using ultrashort pulses, we utilized a flow cytometer (FacsCalibur®, Becton Dickinson, USA) and propidium iodide (PI), a fluorescent dye that fluoresces only when it binds to DNA. Due to its size, living cell membranes are impermeable to propidium iodide, and therefore its entry into the electroporated cells and subsequent fluorescence upon binding to DNA in the nucleus can be detected. The FacsCalibur® instrument has the ability to quantify the fluorescence of individual cells, which changes depending on the effectiveness of the electroporation (i.e., the number of pores opened in the cell membrane by electroporation; these pores allow PI to enter the cell nucleus, making fluorescence from the cell detectable).

[0142] A FACSCalibur® flow cytometer with CELLQUEST® software was used for cell size and fluorescence analysis. Cell samples were acquired during the completion of the electroporation experiment. Briefly, the measured ratio of electroporated cells to unelectroporated cells was calculated as follows: The "Acquisition Criteria" was set to the "Number of Events or Time" position in the Acquisition and Save dialog box, and the acquisition time was selected. The flow rate was set and the "Voltage," "Amplitude Gain," and threshold were adjusted to ensure reliable cell detection. Forward scattering (FSC) and side scattering (SSC) were acquired in linear mode, and FL2 (PI fluorescence) was set in logarithmic mode. Data acquisition could not be started until the sample voltage stabilized. A total of 10,000 sample points (cells) were collected per data point. FSC vs. FL2-H contour plots of negative control (unelectroporated cells) helped to define the following three regions representing intact cells, cell membrane-permeabilized cells, and nuclear membrane-permeabilized cells. The remaining events with a low number of channels were recorded as fragments.

[0143] foam injection This substance may be in the form of bubbles, which are liquids containing very small amounts of gas. Bubbles can be used to enhance the permeability effect of electroporation, yielding particularly beneficial results for high-frequency electroporation (above 100 kHz). The use of bubbles is described in detail below. In this specification, the relative concentrations of liquid and gas within a bubble are expressed in volume at atmospheric pressure, as in an injector filled with air and liquid.

[0144] The foam can be formed by any suitable means, and in fact, it can be done manually by a clinician in an injector. The main advantage of using foam for direct injection into target tissues to be electroporated is that foam can act as a carrier for selected molecules when needed, while having a less impact on tissue conductivity compared to liquids, minimizing the increase in conductivity. The air or gaseous component of foam bubbles has minimal conductivity compared to liquids, enabling a more preferred environment, especially for high-frequency (>100kHz) pulses, minimizing the supplied current and increasing cell permeability.

[0145] The use of high-frequency (>100 kHz) bipolar electrical pulses is advantageous for direct cell excision or cell permeabilization due to passive diffusion of molecules. In some cases, combination with foam provides an advantage in the effectiveness of the procedure (compared to using an equivalent liquid solution).

[0146] Figure 16 shows a comparison of impedance and current using liquid versus foam. Liquid has low impedance (high conductivity), resulting in a high supplied current (A). On the other hand, foam has high impedance / resistance and low conductivity, resulting in a low supplied current (A). When foam is used, the pulse width may be wider than described above, sometimes in the range of 0.05 μs, and the voltage may exceed 10 KV / cm.

[0147] As an example, Figure 17 shows cell permeability from 0% to 100% as the applied electric field (V / cm) is increased. When using a low-conductivity buffer, cells are permeated at lower electric field intensities compared to a high-conductivity buffer. Our understanding is that bubbles generate lower conductivity around cells than when the corresponding liquid solution is used.

[0148] Figure 18 shows the expected cell viability as the electric field (V / cm) increases. Low conductivity leads to cell death at lower electric field intensities compared to buffers with high conductivity. Non-foaming liquids, upon injection, are rapidly diluted by the circulating blood volume. Interaction with blood reduces the effectiveness of the liquid solution because it ultimately decreases the number of active molecules through binding with plasma proteins. Foam, on the other hand, can replace blood rather than mix with it, allowing for higher concentrations of the active agent and longer contact time with tissue, resulting in a greater effect. With foam, lower concentrations of the drug can be used to achieve the same therapeutic effect as the liquid counterpart, while reducing the prevalence of side effects associated with high concentrations.

[0149] Because of the presence of gas bubbles such as air, the bubbles have lower conductivity than the corresponding liquid solution. As a result, the patient's electrical current decreases, cell permeability increases, and the sensation of pain is reduced. In some cases, foam can be produced by mixing albumin, gas, and a liquid solution, for example, in a ratio of 1:4:1 by volume. Preferably, the ratio of gas (e.g., room air or CO2 gas) to liquid is in the range of 1:2 to 1:10 by volume.

[0150] Preferably, the foam used is Albumin, human serum albumin; concentration 10-50% by volume, preferably 15-30%, plus one of polidocanol (0.5-5% by volume) or sodium tetradecyl sulfate (STS) (0.5-5% by volume). This includes multiple substances. STS and polidocanol are sclerosing agents individually, but albumin is not, although polidocanol is also a local anesthetic.

[0151] Albumin alone is a foaming agent, but polidocanol and STS are both foaming and sclerosing agents (these are irritants and directly induce cell death). The activator (the molecule introduced) in the solution is, It may contain one or more of the following: calcium ions, Ca++ (2 mMol to 150 mMol), potassium (2 mMol to 100 mMol), bleomycin, cisplatin, DNA, and / or RNA.

[0152] Preferably, the electroporation pulse is advantageous, The parameters include a bipolar pulse with a pulse length of 0.05 μs to 5 μs supplied by a train that has an "on" energization time of 0.1 μs to 1000 μs for each train, which is repeated up to 1000 times at a frequency of 1 kHz to 1000 kHz.

[0153] Injecting bubbles directly into the environment surrounding cells, rather than just using a liquid substance containing the same activator, creates a low-conductivity environment, enabling more efficient cell permeability that supports the effectiveness of electroporation-based therapies.

[0154] The effectiveness of cell permeabilization (the creation of pores on the cell membrane) generated by short bipolar electrical pulses (<50 μs) is affected by the conductivity of the cells. If the conductivity of the liquid solution surrounding the cells is high, the current will be high, negatively impacting the treatment, reducing cell permeabilization, and causing the patient to experience pain.

[0155] The increase in conductivity around cells is partially induced by the amount of fluid in that region and by the local injection of an electroporation solution that may contain selected therapeutic molecules (such as calcium, potassium, bleomycin, and cisplatin) and high concentrations of ions.

[0156] When a foaming agent is used to deliver the therapeutic agent, the effect of high conductivity on the effectiveness of electroporation pulses that permeate cells is reduced. Bubbles, which are mainly composed of gas or air, have lower conductivity than the corresponding liquid solution. As a result, the current is lower, cell permeability is increased, and patient pain is reduced.

[0157] The use of foam injected into an electroporated environment will beneficially enhance the degree of treatment and cell permeation by not increasing conductivity to the same degree as that of an equivalent liquid solution. The table below shows some recommended parameter ranges for foam injection, although these ranges are more favorably applied to liquid injection.

[0158] [Table 2]

[0159] Foam can also be used to facilitate the dispersion of the local anesthetic into the tissue being treated. The local anesthetic can be lignocaine 5-20 mg / ml, with or without adrenaline. Mepivacaine 10-30 mg / ml is another example of a local anesthetic that can be used. Foam and local anesthetics can be administered in combination with selected molecules such as calcium or potassium ions, bleomycin, or DNA, or they can be provided alone.

[0160] A device for injecting substances (liquids only or foam) As shown with reference to Figure 19, the injection device is used to deliver a substance to target tissue via a needle electrode, which can also facilitate the uniform distribution of the injected substance through "pores" or openings along its conductive length. In one example, the device 200 comprises a reservoir 201 and an array of needles 202 having walls 203 and pores 204. The reservoir 201 is connected to any desired terminal such as a peristaltic pump or injector. It is equipped with a pump of type 90. The needle gauge size is preferably in the range of 14GA to 30GA (outer diameter 0.3 mm to 2.1 mm, inner diameter 0.15 mm to 1.8 mm). More generally, it is recommended that the needles have a maximum inner width dimension in the range of 0.1 mm to 1.8 mm and a maximum outer width dimension in the range of 0.25 mm to 2.5 mm. Preferably, the opening has a maximum width dimension in the range of 0.05 mm to 1.5 mm, and preferably the distance between needles to the nearest needle is in the range of 2 mm to 3 cm.

[0161] In other examples, the openings may be in the form of slots or holes of any desired shape to achieve the desired flow and distribution of the injected material. The needle may have an insulating sleeve to cover the openings and / or to electrically insulate where voltage is not applied to the patient's tissue. Whether insulating or not, the sleeve may be removed after material delivery and before pulsing. The pattern of the openings is preferably helical (staggered), as shown in Figure 19. The number of openings is unevenly distributed, weighted more toward the needle side facing other needles / electrodes, so that the majority of the volume is distributed in the space between electrodes. A depth indicator may be used to convey the depth of the openings relative to the depth of the tissue.

[0162] The probe head 200 can be used with a pulse generator as described above, or with a known third-party electroporation generator. The needles may all be connected to the same pump or reservoir, or they may have independent pump or valve configurations to ensure that different substances are supplied through different needles. One or more needles may have their own solid trocar tips.

[0163] The substance injected may be foam, but it could also be a liquid-only substance, such as a non-conductive substance like deionized water. The test data show that we evaluated the current generated during the supply of electroporation pulses while increasing the voltage in viable test cells (animal liver). The total pulse energization "on time" was 6 ms. When bubbles were injected into the tissue immediately before pulsing, the reduction in current generated per supplied pulse was approximately 40%. Figure 20 shows that in the test, the electroporation current decreased significantly with respect to the given pulse voltage amplitude. In this plot, the unit of current is amperes.

[0164] By using the probe shown in Figure 19, the electrode supplies electrical pulses and also delivers a substance (liquid only or bubbles) to the tissue through the pores 204. This allows for a more uniform distribution of the substance to the environment surrounding the electrode. It also enables the application of a coordinated substance delivery and pulsing method, which can be optimally controlled by an electronic controller that directs the pulse generator and pump and / or valve for substance delivery to the needle. For example, one programmed method involves injection to provide a desired level of conductivity, followed by pulsing, and then injection for access to the porated cells. The first substance may be bubbles with relatively high levels of gas bubbles, and the solution after pulsing may contain therapeutic agents such as calcium, potassium, bleomycin or cisplatin or DNA or RNA.

[0165] Pore ​​204 directly delivers bubbles or other substances, such as a non-conductive solution (e.g., deionized water), to the tissue immediately before the electroporation pulse is supplied, thereby reducing the current that would otherwise be generated. High currents generated during the procedure negatively affect the effectiveness of the electroporation pulses that porate the tissue. The generation of high currents also presents technical and safety challenges, such as current "arc discharge," tissue burning, and premature termination of the procedure, to protect the patient and the generator.

[0166] Using a needle electrode 202 having pores 204, a non-conductive solution can be delivered immediately before the supply of an electrical pulse, and in addition, once the electrical pulse is supplied, a therapeutic solution can be immediately administered using the same electrode. In practice, for example, the needle electrode is placed in the target tissue and, partially based on impedance feedback, bubbles (or other solutions) are injected directly into the target tissue through the pores in the needle electrode. This is followed by the supply of an electrical pulse through the same needle electrode, and post-electroporation injection of a therapeutic substance, such as calcium or a chemotherapy solution, can be directly administered through the needle electrode.

[0167] This optimizes the effects of electroporation pulses while simultaneously enabling the direct delivery of therapeutic substances to the target tissue. In the example in Figure 19, there are two electrodes, positive and negative. In other examples, there may be a bipolar single electrode where the negative and positive polarities are supplied to the same electrode. Alternatively, there may be more than two electrodes in the array, as shown above, referring to Figure 8.

[0168] advantage Generally speaking, the present invention minimizes the risk of patient discomfort and provides highly effective electrotherapy. It will be understood that electroporation can be achieved. Below is a summary of some of the benefits.

[0169] In particular, DNA uptake is improved when low-frequency and low-voltage pulses are used as described above, following an initial high-frequency, high-voltage pulse (100kHz~500kHz; 800V / cm~1600V / cm bipolar). The high-frequency characteristics of the high-voltage pulse are particularly advantageous as they are painless to the patient. This device provides high-frequency pulses for the purpose of permeabilization of cell membranes and viscera, causing ablation alone or in combination with locally injected drugs / molecules. We have described using low concentrations of calcium injected as a liquid or foam in the range of 2~150 mMol / L, preferably aiming for the lower end of this range, to uptake calcium. Furthermore, the high-frequency pulse generates a reduced current by utilizing foam in the environment being treated.

[0170] This device will be understood to achieve the safe delivery of high-voltage, ultrashort pulses (ranging from 0.1 μs to 5.0 μs) to human or animal subjects while counteracting the inefficiencies inherent in the design of switch-mode transformers.

[0171] This device also delivers ultrashort pulses (microseconds) to a highly osmotic solution, enabling more efficient cell electroporation. Furthermore, this device enables a combination of ultrashort pulses (microseconds) and sharp turn-on and turn-off times, maximizing the synergistic effect with the resulting capacitive discharge in the range of 10–500 μF to induce localized cell death. The plateau, while pronounced, remains stable for a short duration of 1 to 3 μs, which is particularly beneficial, thereby achieving highly effective treatment with short current-on times.

[0172] Furthermore, the apparatus and method achieve precise coverage of the tissue with a sufficiently large electric field, avoiding the problem of uneven electric field distribution in tumor cells caused by their heterogeneity. Lower conductivity can be achieved by using appropriate substances injected into the tissue, such as deionized liquids and / or bubbles. Since the distribution of the electric field within the tissue is determined by the flow of current through the tissue, the lower the conductivity, the stronger the electric field; therefore, the above methods and apparatus help to generate a more uniform electric field, resulting in more consistent results.

[0173] The present invention is not limited to the embodiments described, and the structure and details may be modified.

Claims

1. Electroporation device Multiple probe terminals (27) and Multiple probe electrodes connected to the probe terminal, and the probe electrodes (P1 to P7) are adapted for insertion into a biological load. A transformer (6) for supplying a boosted voltage, A switching circuit (7) having switches (24, 25) for connecting high voltage and low voltage or ground level to the probe terminal (27), and A controller (2, 4, 5) configured to control the switch according to a control scheme for supplying pulses to the probe terminal (27), wherein the pulses are supplied to a group consisting of a pair of spaced probe electrodes, The aforementioned device is (a) A bipolar pulse is supplied with a frequency in the range of 100 kHz to 500 kHz, a pulse duration in the range of 1 μs to 5 μs, and a voltage in the range of 100 V to 3000 V. (b) Supply a unipolar pulse with a voltage in the range of 1V to 200V and a pulse duration in the range of 10ms to 10s. An electroporation apparatus configured in such a way.

2. The electroporation apparatus according to claim 1, configured to supply the unipolar pulse of step (b) with a voltage in the range of 100V to 150V and a pulse duration of 20ms to 100ms.

3. The electroporation apparatus according to claim 1, wherein the pair of probe electrodes are sequentially driven by the voltage and are grounded in phase with their corresponding probe terminals.

4. The electroporation apparatus according to claim 1, wherein the controller includes mapping data that defines the relative physical positions of probe electrodes (P1 to P7) connected to the probe terminal (27), and is configured to direct pulses for applying a voltage across a space whose boundaries are defined by the probe electrodes.

5. The electroporation apparatus according to claim 4, wherein the controller is configured to simultaneously drive a first group of probe electrodes (P1, P2) having the same potential and a second group of opposing probe electrodes (P3, P7) having different potentials, in order to apply a charge across and between the plane defined by the four mapped probe electrodes, the first group being driven at a high potential and the second group being grounded.

6. The electroporation apparatus according to claim 5, wherein the mapping data relates to a plane defined by at least one quadrilateral whose boundaries are defined by four probes.

7. The electroporation apparatus according to claim 5, wherein the switching circuit (7) comprises switches (24, 25) dedicated to each voltage level of each terminal, and selectively, the switching circuit (7) comprises a switch dedicated to a high voltage level applied to the terminal as a pulse and a switch dedicated to grounding the terminal.

8. The electroporation apparatus according to claim 7, wherein the switching circuit comprises a dedicated driver circuit (20, 22) for each switch, each driver circuit (22) is individually addressable by the controller (6), and an independent floating power supply (20).

9. The electroporation apparatus according to any one of claims 1 to 5, wherein the controller is configured to first query the probe (26) and determine a desired drive profile from the probe's memory (40), and the query to the probe is selectively performed by the controller to set the optimal parameters of the probe.

10. The electroporation apparatus according to any one of claims 1 to 5, wherein the controller is configured to measure the impedance in a biological load by applying an AC signal to probe electrodes across a frequency spectrum, the impedance measurement is performed before electroporation is driven, and the controller is configured to automatically adjust the drive parameters according to the measured impedance to achieve a current of less than 500 mA across a pair of probe electrodes.

11. The electroporation apparatus according to any one of claims 1 to 5, wherein the controller is adapted to measure the impedance of the biological load between at least one pair of probe electrodes and to automatically set a pulse voltage to avoid excessive current during electroporation.

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