Systems and methods for improving tissue-sensing-based electroporation

Abstract

This application relates to systems and methods for improving tissue-sensing-based electroporation. This disclosure relates to an adaptive control method for controlling EP pulse parameters during EP in cells or tissue using an electroporation (EP) system, comprising: providing a system for adaptive control to optimize EP pulse parameters including EP pulse parameters; applying voltage and current excitation signals to the cell; obtaining data from current and voltage measurements; processing the data to separate desirable data from undesirable data; extracting relevant features from the desirable data; applying at least a portion of the relevant features to a trained diagnostic model; estimating EP pulse parameters based on the results of the applied relevant features, wherein the initialized EP pulse parameters are based on the trained model and the relevant features, thereby optimizing the EP pulse parameters; and applying a first EP pulse by a generator based on the first pulse parameters.

Description

[0001] This application is a divisional application of patent application No. 201680026625.2, filed on March 31, 2016, entitled "System and Method for Improved Tissue Sensing-Based Electroporation".

[0002] Reference to relevant applications

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 214,807, filed September 4, 2015, entitled "System and Method for Optimized Electroporation," and U.S. Provisional Patent Application No. 62 / 214,872, filed September 4, 2015, entitled "System and Method for Optimized Catheter-Based Electroporation," each of which relates to U.S. Provisional Patent Application No. 62 / 141,142, filed March 31, 2015, entitled "Focused Pulse Addition Electroporation," and U.S. Provisional Patent Application No. 62 / 141,142, filed March 31, 2015, entitled "Electrochemical Tissue Sensing." The disclosures of U.S. Provisional Patent Application No. 62 / 141,182 entitled “All-in-one Device for Improved Therapeutic Agent Delivery”, filed March 31, 2015, and U.S. Provisional Patent Application No. 62 / 141,256 entitled “Device for Improved Therapeutic Agent Delivery”, filed March 31, 2015, are expressly incorporated herein by reference in their entirety. Technical Field

[0004] The present invention generally relates to the use of a control system to improve the electroporation process and increase cell permeability, and more specifically to a method and apparatus for the optimized application of a controlled electric field for delivering a therapeutic portion into cells via electroporation therapy (EPT), also known as cell perforation therapy (CPT) and electrochemical therapy (ECT). Background Technology

[0005] In the 1970s, it was discovered that pores could be created in cells using an electric field without causing permanent damage. This discovery made it possible to insert macromolecules into the cytoplasm of cells. It is well known that therapeutic components, such as pharmacological compounds, can be incorporated into living cells through a process called electroporation. Genes or other molecules are injected into living cells and short pulses of a high electric field are applied. The cell membrane briefly becomes porous, and the gene or molecule enters the cell, where it can modify the cell's genome.

[0006] In chemotherapy for certain types of cancer, it is necessary to use sufficiently high doses of the drug to kill cancer cells without killing an unacceptably high number of normal cells. This can be achieved if the chemotherapy drug can be inserted directly into the cancer cells. Some anticancer drugs, such as bleomycin, are often unable to effectively penetrate the membranes of some cancer cells. However, electroporation makes it possible to insert bleomycin into the cells.

[0007] Treatment is typically performed by injecting anticancer drugs directly into the tumor and applying an electric field to the tumor between a pair of electrodes. The field strength must be precisely and appropriately adjusted so that electroporation of the tumor cells occurs without damage to any normal or healthy cells, or at least with minimal damage. This is often easily achieved for external tumors by applying electrodes to opposite sides of the tumor so that the electric field is between the electrodes. Once the field is uniform, the distance between the electrodes can then be measured, and a suitable voltage can then be applied to the electrodes according to the formula E=V / d (E = field strength in V / cm; V = voltage in volts; and d = distance in cm). Properly positioning the electrodes and measuring the distance between them is not easy when treating large or internal tumors.

[0008] Treatment of the host using cell perforation therapy offers a means of avoiding the harmful effects typically associated with the administration of anticancer or cytotoxic agents. This treatment allows the introduction of these agents to selectively damage or kill unwanted cells while avoiding surrounding healthy cells or tissue. However, a problem with using electroporation techniques is that diseased tissue, especially cancerous tissue, can be quite heterogeneous, requiring adjustments to the electroporation conditions. Therefore, this invention provides the use of electrochemical impedance spectroscopy in conjunction with adaptive control methods for EP to maximize electroporation of the desired tissue while minimizing tissue damage. Summary of the Invention

[0009] Therefore, a control system is needed that uses tissue-sensing feedback to optimize the EP process using tumor-specific measurements acquired before and between each EP pulse.

[0010] According to some embodiments, a system for providing adaptive control to optimize EP pulse parameters during EP in cells and tissues using an electroporation (EP) device includes a measuring device, an initialization module, a generator, a controller, and a memory module. The measuring device is configured to measure the dielectric and conductive properties of cells and tissues and includes: a voltage sensor for measuring a voltage across the tissue generated by each of an excitation signal and an EP pulse applied to the tissue; and a current sensor for measuring a current across the tissue generated by the excitation signal and at least one applied EP pulse. The initialization module is configured to initialize EP pulse parameters for performing electroporation in cells or tissues, wherein the initialized EP pulse parameters are at least partially based on at least one trained model. The generator is configured to apply at least one of an excitation signal and an EP pulse to the tissue. The voltage and current sensors of the measuring device measure the voltage and current across the tissue of the cells in response to the application of the excitation signal. The controller is configured to receive signals corresponding to at least one of the excitation signal and EP pulses, associated with the measured sensor data, from the measuring device to fit the data to at least one trained model and process the data into diagnostic and updated control parameters. The controller includes: a preprocessing module for receiving signals related to data from current and voltage measurements, and processing the data to separate desirable data from undesirable data; a feature extraction module for extracting relevant features from the desirable data; a diagnostic module for applying at least a portion of the relevant features of the desirable data to at least one trained diagnostic model; and a pulse parameter estimation module for estimating at least one of an initialized pulse parameter and subsequent pulse parameters based on the measured data, the result of at least one of the diagnostic module and the feature extraction module. A memory module stores the desirable and undesirable data, sensor data, and the trained model for the controller to perform feature extraction.

[0011] In some embodiments, the EP device includes a central probe, an applicator, and at least two electroporation electrodes (EPEs) with opposite charges. The central probe defines at least a central lumen and extends proximally to a distal end, at least a portion of the central probe having a helical geometry to create a channel for delivery of a treatment portion to tissue. The portion of the central probe has at least one injection port positioned along the helical geometry. The proximal end of the central probe is configured to receive a treatment portion delivered to the central probe, and the distal end of the central probe is open to define an opening for delivery of the treatment portion to tissue and has a shape configured to puncture tissue. The applicator at least partially houses the central probe and has a distal end, the portion of the central probe being configured to extend through the distal end to the exterior of the applicator to contact tissue and retract into the applicator. The at least two oppositely charged EPEs are configured to surround tissue and are positioned and adapted to extend proximally to the distal end. The distal end has a needle shape configured to puncture tissue. A measuring device is coupled to the EPEs, and the EPEs are adapted to be coupled to a generator to receive at least one of an excitation signal and an electrical waveform for an EP pulse.

[0012] In some embodiments, the EP device includes a central probe, at least one take-up line, a ramp, an electrical connector, a small-aperture connector, a handle, and at least two oppositely charged electrodes. The central probe defines at least a central lumen and has a proximal end and a closed distal end. The distal end has a needle-shaped tip configured to pierce tissue and at least one exit port positioned at a predetermined location from the distal end. The exit port fluidly connects the central lumen to the outside of the central probe. The at least one take-up line is positioned within the central lumen and is slidable within the central probe, having a proximal end positioned within the central probe and a distal end configured to extend to the outside of the central probe and retract into the central lumen via the exit port. The distal end of the take-up line has a shape configured to pierce through tissue and define an opening through which at least a portion of the take-up line enters the tissue to create a fluid channel through which a treatment portion is delivered to the tissue. The treatment portion is delivered from the central lumen into the channel via the exit port. A ramp is integrally formed with or coupled to the inner surface of a central probe, the inner surface defining a central lumen, and the ramp is configured to contact and guide the take-up line to allow the central probe to exit outside the central probe. An electrical connector electrically connects the central probe and the take-up line to a generator. A small-aperture connector is connected to the central probe for delivery of the treatment portion. A handle at least partially accommodates the electrical connector and is coupled to the proximal ends of the central probe and the take-up line to facilitate penetration depth at the distal ends of the central probe and the take-up line. The at least two oppositely charged electrodes are configured to surround tissue and are positioned extending from the proximal end to the distal end. The distal tip has a needle shape configured to pierce the tissue. The electrodes are adapted to couple to the generator, receive at least one electrical waveform from the generator, and supply at least one excitation signal and at least one EP pulse to the tissue. A measuring device is coupled to the electrodes.

[0013] In some embodiments, the EP device includes a cannula comprising a cannula and an obturator, at least two oppositely charged electrodes, and a central probe. The cannula extends proximally to an open distal end and defines a first lumen configured to receive the obturator. The obturator extends proximally to a distal end. The distal end has a sharp shape configured to pierce through the skin, penetrate into a body cavity, and form a path through which the cannula can be at least partially inserted into the lumen. The obturator is configured to slide within the first lumen, and the distal end of the obturator is configured to extend beyond the first lumen through the open distal end of the cannula. The at least two oppositely charged electrodes are retractably positioned at the distal end of an anchor and configured to surround tissue for positioning. A measuring device is coupled to the electrodes, and the electrodes are adapted to be coupled to a generator, receiving at least one electrical waveform from the generator and supplying at least one excitation signal and an EP pulse to the region. The central probe is retractably positioned at the distal end of the anchor and has an inner surface that defines a central lumen and extends from the distal end of the anchor. At least a portion of the central probe has a helical geometry configured to create a channel for delivery of the treatment portion to the tissue. The distal end of the central probe has a shape configured to pierce the tissue and is open to define an opening for delivery of the treatment portion to the tissue.

[0014] In some embodiments, the EP device includes an electroporation rod housing comprising an array of electroporation electrodes (EPEs), an array of electrical measurement electrodes (EMEs), wherein the EPEs and EMEs are offset; and a rod delivery system comprising at least one injection probe defining a first lumen. The injection probe extends from its proximal end to its distal end and has an elongated cylindrical shape. The distal end of the injection probe has a needle shape and is open for delivering a therapeutic portion to cells. A generator is configured to supply EP pulses of multiple waveforms to the array of EPEs and is configured to supply excitation signals of multiple waveforms to the array of EMEs. The EP device further includes an electrical connector electrically connecting the arrays of EPEs and EMEs to the generator, and a switching mechanism between the electrical connector and the generator.

[0015] In some embodiments, both the EPE and EME are configured as EPEs, i.e., all electrodes are EPEs capable of switching between EP and electrochemical impedance spectroscopy (EIS) modes. The generator is configured to supply the EPE with EP pulses of the plurality of waveforms in EP mode and with excitation signals of the plurality of waveforms in EIS mode. The measurement device is coupled to the EPE, and a switching mechanism is adapted to switch the generator between EIS and EP modes.

[0016] According to some embodiments, an adaptive control method for controlling EP pulse parameters during EP in cells or tissues using an electroporation (EP) system includes: a) providing any of the EP devices described herein; b) initializing EP pulse parameters for performing EP in cells or tissues via an initialization module, the initialized EP pulse parameters being at least partially based on at least one trained model; c) applying voltage and current excitation signals to cells and tissues via a generator, and measuring voltage and current across cells and tissues corresponding to the applied excitation signals via a measuring device; d) obtaining data from the current and voltage measurements via a controller, and processing the data to separate desirable data from undesirable data; e) extracting relevant features from the desirable data via the controller; f) applying at least a portion of the relevant features of the desirable data to at least one trained diagnostic model via the controller; g) estimating EP pulse parameters via the controller based on the results of the applied relevant features to the trained model, wherein the initialized EP pulse parameters are based on at least one trained model and relevant features, thereby optimizing the EP pulse parameters; and h) applying a first EP pulse via a generator based on first pulse parameters.

[0017] In some embodiments, the adaptive control method further includes predicting parameters for subsequent EP pulses after a first EP pulse has been applied by using a controller to predict parameters for subsequent EP pulses after the first EP pulse has been applied, by using a trained model based on previous EP pulses and changes to at least one of the relevant features between the applied EP pulses.

[0018] In some embodiments, the adaptive control method further includes generating a diagnostic response by a controller at least in part based on the application. The diagnostic response includes a) tissue detection, b) tumor type detection, c) needle placement detection, d) co-localization detection, and e) cell penetration detection.

[0019] In some embodiments, the adaptive control method further includes: f) applying a subsequent EP pulse based on subsequent EP pulse parameters via a generator; and g) repeating the application of voltage and current excitation signals, repeating the measurement of cells or tissues, repeating the acquisition of data and separating desirable data from undesirable data; repeating the extraction of relevant features; and repeating the application until i) a predetermined limit on the number of cycles of the EP pulse sequence or EP pulses is reached, or ii) a diagnostic response prompts a diagnostic decision to terminate the adaptive control method.

[0020] In some embodiments, the adaptive control method further includes storing desired data in a memory module.

[0021] In some embodiments, at least one trained model is trained using empirical data observed during the initial operation of the EP system using fixed EP pulse parameters.

[0022] In some embodiments, the adaptive control method further includes determining the dielectric and conductive properties of cells and tissues generated by the applied excitation signal.

[0023] In some embodiments, dielectric and conductivity properties are determined by applying a band-limited signal that is repeated over a fixed frequency range.

[0024] In some embodiments, the adaptive control method further includes verifying the current and voltage sensors of the measuring device, thereby obtaining measured data to evaluate the quality of the data, and the verification includes statistically analyzing the quality of the measured data.

[0025] In some embodiments, separating desirable data from undesirable data includes at least one of the following: a) denoising the sensor signal, b) removing the DC bias from the sensor signal, c) scaling the data based on a normalized value, wherein the normalized value includes the standard deviation, d) filtering the mean, and e) removing outliers from the data.

[0026] In some embodiments, the features are derived from a parametric model fitting of the magnitude and phase measurements of the voltage and current signals, and are selected from the group consisting of intracellular resistance, extracellular resistance, solution resistance, thin-film capacitance, admittance, constant phase element exponent, and charging time constant.

[0027] In some embodiments, the parametric model fitting of the magnitude and phase measurements of the excitation voltage and current signals applied to cells and tissues is determined by cross-correlating the excitation voltage and current signals with known reference signals stored in a memory module.

[0028] In some embodiments, the dielectric and conductive properties of a cell or tissue are determined by the ratio of the magnitudes of the excitation voltage and current applied to the cell or tissue and the phase difference between them.

[0029] In some embodiments, the features are derived from the magnitude ratio or phase difference of the excitation voltage and current signals. The features include: a) the values ​​of the magnitude ratio and phase difference of the excitation voltage and current signals at a fixed frequency; b) at least one of the following: i) the magnitude ratio or phase difference of the excitation voltage and current signal magnitudes over a narrow frequency band; ii) the magnitude ratio or phase difference of the excitation voltage and current signal phases over a wide frequency band; and c) the curvature, slope, and noise of the magnitude ratio or phase difference of the excitation voltage and current signals relative to frequency.

[0030] According to some embodiments, a system for electroporation (EP) of cells in a host tissue includes: a) an electroporation rod housing comprising i) an array of electroporation electrodes (EPEs); and ii) an array of electrochemical impedance spectroscopy (EIS) electrodes (EISEs), wherein the EPEs and EISEs are offset; b) an EP power supply configured to supply electrical signals of multiple waveforms to the array of EPEs; c) an EIS power supply configured to supply electrical signals of multiple waveforms to the array of EISEs; d) an electrical connector electrically connecting the array of EPEs to the EP power supply; e) an electrical connector electrically connecting the array of EISEs to the EIS power supply; and f) an EIS sensor.

[0031] According to some embodiments, the system further includes a rod delivery system configured to deliver a therapeutic portion to cells, the delivery system including at least one injection probe defining a first lumen, the injection probe extending from its proximal end to its distal end and having an elongated cylindrical shape, wherein the distal end of the injection probe has a needle shape and is open for delivering the therapeutic portion to cells.

[0032] According to some embodiments, a system for electroporation (EP) of cells in a body tissue includes: a) an electroporation rod housing including an array of electrodes, b) an EP power supply configured to supply electrical signals of multiple waveforms to the array of electrodes, c) an EIS power supply configured to supply electrical signals of multiple waveforms to the array of electrodes, d) an electrical connector electrically connecting the array of electrodes to the EP power supply, e) an electrical connector electrically connecting the array of electrodes to the EIS power supply, f) a switching mechanism between the electrical connector and the power supply, and g) an EIS sensor.

[0033] In some embodiments, the system therefore further includes a rod delivery system configured to deliver a therapeutic portion to cells, the delivery system including at least one injection probe defining a first lumen, the injection probe extending from its proximal end to its distal end and having an elongated cylindrical shape, wherein the distal end of the injection probe has a needle shape and is open for delivering the therapeutic portion to cells.

[0034] In some embodiments, the electrode is a needle configured to penetrate the skin and contact cells in an electric field region.

[0035] In some embodiments, the electrodes are non-penetrating contacts.

[0036] According to some embodiments, a method for electroporating cells in a patient’s tissue includes: a) providing any of the EP systems described herein, b) inserting electrodes into the tissue, c) applying at least one voltage pulse from an EIS power supply to an EIS electrode to determine tissue parameters, d) calculating the voltage pulse to be used for electroporation using an electronic signal processing device, and e) applying at least one voltage pulse between multiple pairs of electrodes in an EP electrode array inserted into the tissue to establish an electric field in the cells of the tissue sufficient to cause electroporation of the cells in the tissue.

[0037] In some embodiments, the method further includes: a) providing a rod delivery system configured to deliver a therapeutic portion (TM) to cells, the delivery system including at least one injection defining a first lumen, an injection probe extending from its proximal end to its distal end and having an elongated cylindrical shape, wherein the distal end of the injection probe has a needle shape and is open for delivering the therapeutic portion to cells; and b) delivering the TM to cells.

[0038] In some embodiments, the TM is delivered before, simultaneously with, or after electroporation.

[0039] In some embodiments, TM is locally injected into the tissue.

[0040] In some embodiments, the method is performed in vivo.

[0041] In some embodiments, TM is a nucleic acid.

[0042] In some embodiments, the cells are tumor cells.

[0043] In some embodiments, the cells are melanoma or basal cell carcinoma cells.

[0044] In some embodiments, the electric field ranges from approximately 10 V / cm to about 2000 V / cm.

[0045] In some embodiments, the number of applied electrical pulses ranges from 1 to 100.

[0046] In some embodiments, the duration of each electrical pulse ranges from about 10 µs to about 100 ms.

[0047] In some embodiments, at least one electrical pulse is selected from the group consisting of: square wave pulse, exponential wave pulse, monopolar vibration wave form, and bipolar vibration wave form.

[0048] In some embodiments, each electrical pulse includes a square wave pulse.

[0049] According to some embodiments, a method of electroporating a reagent into the cells of a tissue includes: a) introducing a therapeutic agent into the tissue of a patient requiring treatment; b) performing tissue impedance sensing to determine a suitable EP protocol; and c) using an electrode device positioned in contact with the tissue to deliver voltage pulses that establish an electric field sufficient to introduce the therapeutic agent into the cells of the tissue by means of electroporation. The electrode device includes i) a support member on which two or more opposing pairs of needle electrodes are disposed, the needle electrodes being arranged relative to each other to form an electrode array; and ii) a power supply electrically connected to the needle electrode pairs disposed in the support member, wherein the power supply provides voltage pulses to at least two of the opposing pairs of needle electrodes to achieve electroporation.

[0050] In some embodiments, an apparatus for delivering a therapeutic portion to cells in a treatment area of ​​a tissue includes: a) a central probe defining at least a central lumen and extending from a proximal end to a distal end, at least a portion of the central probe having a helical geometry to create a channel for delivery of the therapeutic portion to the tissue, the portion of the central probe having at least one injection port positioned along the helical geometry. The proximal end of the central probe is open and fluidly connects the first central lumen to the lumen of an injector through which a therapeutic agent is delivered to the central probe. The distal end of the central probe is open to define an opening for delivery of the therapeutic portion into the tissue and has a shape configured to pierce the tissue. The apparatus for delivery further includes: b) an applicator at least partially receiving the central probe, the applicator having a distal end, the portion of the central probe being configured to extend through the distal end to the exterior of the applicator to contact the tissue and retract into the applicator.

[0051] In some embodiments, the device further includes at least one electrode pair positioned on the portion of the central probe.

[0052] In some embodiments, the distal end of the central probe is closed.

[0053] In some embodiments, at least one of the diameter of the first lumen of the central probe, the outer diameter of the central probe, the helical diameter, and the spacing is adjustable to change the distribution and volume of the delivered treatment portion.

[0054] In some embodiments, the central probe is actuated to move forward toward and through the distal end of the central probe and through the tissue.

[0055] In some embodiments, the device further includes: a) an electrical connector that electrically connects the center probe to a power source, and b) a handle that houses the electrical connector and is coupled to the applicator.

[0056] In some embodiments, the proximal end of the central probe is formed of or coated with a non-conductive material to prevent or reduce the generation of an electric field at the portion.

[0057] In some embodiments, the apparatus further includes an electroporation system comprising at least two opposing charged electroporation electrodes positioned to surround the region, the electrodes being adapted to extend from a proximal end to a distal end, the distal end having a needle-shaped tip configured to puncture tissue. The electrodes are adapted to be coupled to an electrode power supply, receiving at least one electrical waveform from the power supply and supplying a pulsed electric field sufficient for electroporation to the region.

[0058] In some embodiments, the electrode is at least partially housed in the applicator, positioned around the central probe, and configured to expand out of the applicator to surround the area.

[0059] In some embodiments, the handle includes a power supply interface for supplying power from a power source to actuate the extension and retraction of the center probe, and to actuate the extension and retraction of the electroporation electrode.

[0060] In some embodiments, the device further includes a sensor system configured to sense the capacitance of the cell membrane. The sensor system includes: a) a pair of capacitive or EIS sensing electrodes powered by a low-voltage power supply; b) a voltage sensor configured to sense voltage or voltage drop across the cell membrane; c) a current sensor configured to sense current across the cell membrane; and d) an electronic signal processing device configured to process the voltage drop and current across the cell membrane and determine the capacitance of the cell membrane.

[0061] In some embodiments, the center probe is an electrode probe connected to an electrode power supply configured to generate an electric field between the center probe and the electroporation electrode to facilitate electroporation.

[0062] In some embodiments, the device further includes at least a second probe having a second lumen defining at least a second lumen and extending from a proximal end to a distal end of the other probe, at least a portion of the other probe having a helical geometry configured to create at least a second channel for delivery of a treatment portion into tissue. The proximal end of the other probe is open and fluidly connects the second lumen to the lumen of an injector through which a therapeutic agent is delivered to the other probe. The distal end of the other probe is open to define an opening for delivery of the treatment portion into tissue and has a shape configured to puncture tissue. The other probe is received within an applicator, and the portion of the other probe is configured to extend outside the applicator to contact tissue and retract into the applicator.

[0063] According to some embodiments, an apparatus for delivering a therapeutic portion to cells in a treatment area of ​​a tissue includes: a) a central probe defining at least a first lumen and extending from a proximal end to a distal end, at least a portion of the central probe having a helical geometry configured to enhance anchoring of the central probe in the tissue and create a channel for delivery of the therapeutic portion to the tissue. The portion of the central probe is formed of or coated with a conductive material. The proximal end of the central probe is open and fluidly connects the first lumen to the lumen of an injector through which a therapeutic agent is delivered to the central probe. The distal end of the central probe is open to define an opening for delivery of the therapeutic portion into the tissue and has a shape configured to pierce the tissue. The apparatus further includes: b) an applicator housing the central probe, the applicator having a distal end, the portion of the central probe being configured to extend through the distal end to the exterior of the applicator to contact the tissue and retract into the applicator; and c) at least one distal electrode located at the distal end of the applicator and configured to generate an electric field with the portion of the central probe.

[0064] In some embodiments, the at least one distal electrode is configured based on a ring configuration, a straight line configuration, a spiral configuration, or a retractable ring configuration.

[0065] In some embodiments, the device further includes at least one injection port located on the portion of the central probe.

[0066] In some embodiments, the distal electrode is configured to be positioned outside the tissue.

[0067] In some embodiments, the distal electrode is configured to be positioned below the surface of the tissue.

[0068] In some embodiments, the distal electrode is formed by a helical configuration and positioned below the surface of the tissue, with the helices of the central probe and the distal electrode wound in opposite directions.

[0069] In some embodiments, the apparatus further includes an electroporation system comprising at least two opposing charged electroporation electrodes positioned to surround the region, the electrodes being adapted to extend from a proximal end to a distal end, the distal end having a needle-shaped tip configured to puncture tissue. The electrodes are adapted to be coupled to an electrode power supply, receiving at least one electrical waveform from the power supply and supplying a pulsed electric field sufficient for electroporation to the region.

[0070] In some embodiments, the electrode is housed in an applicator, positioned around a central probe, and configured to expand out of the applicator to surround the area.

[0071] In some embodiments, the device further includes a sensor system configured to sense the capacitance of the cell membrane. The sensor system includes: a) a pair of capacitance sensing or EIS electrodes powered by a low-voltage power supply; b) a voltage sensor configured to sense voltage or voltage drop across the cell membrane; c) a current sensor configured to sense current across the cell membrane; and d) an electronic signal processing device configured to process the voltage drop and current across the cell membrane and determine the capacitance of the cell membrane.

[0072] In some embodiments, the handle includes a power supply interface for supplying power from a power source to actuate the extension and retraction of the center probe, and to actuate the extension and retraction of the electroporation electrode.

[0073] In some embodiments, the device further includes a sensor system configured to sense the capacitance of the cell membrane. The sensor system includes: a) a pair of capacitive or EIS sensing electrodes powered by a low-voltage power supply; b) a voltage sensor configured to sense voltage or voltage drop across the cell membrane; c) a current sensor configured to sense current across the cell membrane; and d) an electronic signal processing device configured to process the voltage drop and current across the cell membrane and determine the capacitance of the cell membrane.

[0074] According to some embodiments, an apparatus for delivering a therapeutic portion to cells in a treatment area of ​​a tissue includes: a) a central probe having an inner surface defining at least a first central lumen and extending from a proximal end of the central probe to a distal end; at least a portion of the central probe having a helical geometry configured to enhance anchoring of the central probe in the tissue and create a channel for delivery of the therapeutic portion to the tissue, wherein said portion of the central probe is formed of or coated with a conductive material. The proximal end of the central probe is open and fluidly connects the central lumen to the lumen of an injector through which a therapeutic agent is delivered to the central probe. The distal end of the central probe is open to define an opening for delivery of the therapeutic portion into the tissue and has a shape configured to pierce the tissue. The device further includes: b) an applicator housing a central probe, the applicator having a distal end, the portion of the central probe being configured to extend through the distal end to the outside of the applicator to contact tissue and retract into the applicator; c) at least one straight probe having an open proximal end and a distal end for delivery of a treatment portion to tissue, and a vertical axis coaxially aligned with the central axis of the diameter of the central probe, and configured to generate an electric field with the portion of the central probe.

[0075] In some embodiments, the device further includes at least one injection port located on the portion of the central probe.

[0076] In some embodiments, the spiral probe is configured to transmit acoustic energy received from an acoustic horn mounted at the distal end of the applicator.

[0077] In some embodiments, the device further includes a sensor system configured to sense the capacitance of a cell membrane, the sensor system comprising:

[0078] According to some embodiments, a method for delivering a therapeutic portion to a treatment area of ​​tissue includes a) providing means for delivering a therapeutic portion to a treatment area of ​​tissue. The means includes i) a central probe and ii) an applicator. The central probe has at least a first central lumen extending from a proximal end to a distal end, and at least a portion of the central probe has a helical geometry configured to enhance anchoring of the central probe in tissue and create a channel for delivery of the therapeutic portion to the tissue. The portion of the central probe has a plurality of injection ports positioned along the helical geometry. The proximal end of the central probe is open and fluidly connects the central lumen to the lumen of an applicator through which a therapeutic agent is delivered to the central probe. The distal end of the central probe is open to define an opening for delivery of the therapeutic portion into the tissue and has a shape configured to pierce the tissue. The applicator receives the central probe and has a distal end, the portion of the central probe being configured to extend through the distal end to the exterior of the applicator to contact the tissue and retract into the applicator. The method further includes: b) contacting a central probe with diseased cells in the treatment area of ​​the tissue; c) actuating and extending the central probe in the axial direction from the applicator; d) piercing the tissue with at least a portion of the central probe to create an opening, the at least portion of the central probe entering the tissue through the opening to create a fluid channel for delivery of the treatment portion to the tissue; and e) injecting the treatment portion into a first central lumen and delivering the treatment portion to the tissue through the at least one injection port and the open distal end of the central probe.

[0079] In some embodiments, the method further includes f) providing an electroporation system comprising at least two opposing charged electroporation electrodes positioned to surround the region. The electroporation electrodes are adapted to extend from a proximal end to a distal end, the distal end having a needle-shaped tip configured to puncture tissue, and the electroporation electrodes are adapted to be coupled to a power source. The method further includes g) contacting the region of tissue with the electroporation electrodes, h) delivering an electrical pulse from the power source to the electrodes, and i) applying a pulsed electric field sufficient for electroporation from the electroporation electrodes to the region.

[0080] In some embodiments, the method further includes providing a sensor system for sensing the capacitance of a cell membrane. Capacitance sensing includes: a) contacting tissue with at least one pair of capacitance sensing electrodes powered by a low-voltage power supply; b) transmitting a low-power interrogation signal via the low-voltage power supply to the at least one pair of capacitance sensing electrodes to generate a low-intensity electric field excitation in the region; c) sensing a voltage or voltage drop across the cell membrane by a voltage sensor; d) sensing a current across the cell membrane by a current sensor; and e) determining the capacitance of the cell membrane by an electronic signal processing device based on the voltage drop and current across the cell membrane.

[0081] According to some embodiments, a method for delivering a therapeutic portion to a treatment area of ​​tissue includes a) providing means for delivering a therapeutic portion to a treatment area of ​​tissue. The means includes i) a central probe connected to a power source and having an inner surface defining at least a first central lumen and extending from a proximal end to a distal end of the central probe. At least a portion of the central probe has a helical geometry configured to enhance anchoring of the central probe in tissue and create a channel for delivery of the therapeutic portion to the tissue. The portion of the central probe is formed of or coated with a conductive material. The proximal end of the central probe is open and fluidly connects the central lumen to the lumen of an injector through which a therapeutic agent is delivered to the central probe. The distal end of the central probe is open to define an opening for delivery of the therapeutic portion into the tissue and has a shape configured to pierce the tissue. The device further includes: ii) an applicator housing a central probe having a distal end, the portion of the central probe being configured to extend through the distal end to the outside of the applicator to contact tissue and retract into the applicator; and iii) at least one distal electrode located at the distal end of the applicator, connected to a power source and configured to generate an electric field with the portion of the central probe. The method further includes b) contacting the central probe and distal electrode with diseased cells in the treatment area of ​​the tissue, c) actuating and extending the central probe and distal electrode in the axial direction from the applicator, d) piercing the tissue with the distal electrode and at least a portion of the central probe to create an opening, the at least portion of the central probe entering the tissue through the opening to create a fluid channel for delivery of the treatment portion to the tissue, e) injecting the treatment portion into a first central lumen and delivering the treatment portion to the tissue through at least one injection port and an open distal end of the central probe, f) delivering electrical pulses from a power source to the distal electrode and central probe, g) applying a pulsed electric field sufficient for electroporation to the area from the distal electrode and central probe, and h) retracting the distal electrode and central probe from the tissue.

[0082] According to some embodiments, an apparatus for delivering a therapeutic portion to a region of target cells in a tissue includes: a) a central probe defining at least a first lumen and having a proximal end and a closed distal end, the distal end having a needle-shaped portion configured to pierce tissue and having at least one exit port positioned at a predetermined location from the distal end, the exit port fluidly connecting the first lumen to the outside of the central probe; and b) at least one take-up line positioned within the first lumen and slidable within the central probe, the take-up line having a proximal end positioned within the central probe and a distal end configured to extend to the outside of the central probe and retract into the first lumen via the exit port, the distal end of the take-up line having a shape configured to pierce through tissue and define an opening, at least a portion of the take-up line entering the tissue through the opening to create a fluid channel through which the therapeutic portion is delivered to the tissue. The therapeutic portion is delivered from the first lumen into the channel via the exit port. The device further includes: c) a ramp integrally formed or coupled with the first lumen, the ramp being configured to contact and guide the take-up line to allow the central probe to exit outside the central probe; d) an electrical connector electrically connecting the central probe and the take-up line to a power source; e) a small aperture connector connecting the central probe to a syringe for delivery of a treatment portion; and f) a handle at least partially housing the electrical connector and coupled to the proximal ends of the central probe and the take-up line to facilitate penetration depth at the distal ends of the central probe and the take-up line.

[0083] In some embodiments, the device further includes an electroporation system comprising at least two oppositely charged electrodes positioned to surround the region of a target cell, the electrodes being adapted to extend from a proximal end to a distal end, the distal end having a needle shape configured to puncture the tissue, wherein the electrodes are adapted to couple to the power source, receive electrical waveforms from the power supply, and supply a pulsed electric field sufficient for electroporation to the region of the target cell.

[0084] In some embodiments, the electrodes surround the central probe.

[0085] In some embodiments, the device includes a plurality of exit ports and a plurality of take-off lines configured to simultaneously extend to the exterior of the central probe and to retract into the central lumen of the central probe via the exit ports.

[0086] In some embodiments, the handle includes a power supply interface for supplying power from a power source to actuate the extension and retraction of the take-off line, and to actuate the extension and retraction of the electrodes.

[0087] In some embodiments, the device further includes a catheter shaft that surrounds the outer surface of the central probe to support and protect the central probe during insertion into a body containing tissue.

[0088] In some embodiments, the take-off line includes a cutting blade located at the tip of the distal end of the take-off line.

[0089] In some embodiments, the cutting blade at the distal end is configured to enter the tissue and to rotate about the central axis of the cutting blade to form a fluid channel.

[0090] In some embodiments, the angle of the ramp contact with the take-up line is adjustable to change the trajectory angle of the take-up line exiting the central lumen.

[0091] According to some embodiments, an apparatus for delivering a therapeutic portion to a region of target cells in a tissue includes: a) a central probe defining at least a first lumen and having a proximal end and an open distal end, the distal end having a needle-shaped tip configured to pierce tissue, and the open distal end fluidly connecting the first lumen to the outside of the central probe; b) at least one take-up line positioned within the first lumen and slidable within the central probe, having a proximal end positioned within the central probe and a distal end configured to extend to the outside of the central probe and retract through the distal end of the central probe back into the central lumen, the take-up line comprising a hyperelastic material configured for curved heat setting, wherein the take-up line is adapted to elastically straighten when positioned within the central lumen and adapted to curvedly bend when extended to the outside of the central probe to form a channel extending into the cells, the take-up line having an elongated cylindrical shape and its distal end further configured to pierce through tissue and define an opening, at least a portion of the take-up line entering the tissue through the opening to create a fluid channel through which the therapeutic portion is delivered to the tissue. The therapeutic portion is delivered from the first lumen into the channel via an exit port. The device further includes: b) a ramp integrally formed or coupled to the inner surface of the central probe, the ramp being configured to contact and guide the take-up line to allow the central probe to exit to the outside of the central probe; c) an electrical connector electrically connecting the central probe and the take-up line to a power source; d) a small aperture connector connecting the central probe to a syringe for delivery of a treatment portion; and e) a handle at least partially housing the electrical connector and coupled to the proximal ends of the central probe and the take-up line to facilitate penetration depth at the distal ends of the central probe and the take-up line.

[0092] In some embodiments, the superelastic material is any one or a combination of materials selected from the group consisting of NiTi, Cu-Al-Ni, Fe-Mn-Si, NiTi-Zr, Cu-Zr, Ni-Al and Cu-based alloys.

[0093] In some embodiments, the device includes a plurality of exit ports and a plurality of take-off lines configured to simultaneously extend to the exterior of the central probe and to retract into the central lumen of the central probe via the exit ports.

[0094] In some embodiments, the device further includes at least two oppositely charged electrodes positioned to surround a region of target cells for cell therapy, the electrodes being adapted to extend from a proximal end to a distal end, the distal end having a needle shape configured to pierce the tissue, wherein the electrodes are adapted to be coupled to a power source, to receive electrical waveforms from the power supply, and to supply a pulsed electric field sufficient for electroporation to the target tissue region.

[0095] In some embodiments, the handle includes a power supply interface for supplying power from a power source to actuate the extension and retraction of the take-off line, and to actuate the extension and retraction of the electrodes.

[0096] In some embodiments, the device further includes a catheter shaft that surrounds the outer surface of the central probe to support and protect the central probe during insertion into a body containing tissue.

[0097] According to some embodiments, an apparatus for delivering a therapeutic portion to a region of target cells in a tissue includes: a) an injection probe defining at least a first lumen, the injection probe extending from its proximal end to its distal end and having an elongated cylindrical shape, the distal end having a needle shape and being open for delivering the therapeutic portion to said region; b) a central probe coupled to the injection probe and having an inner surface defining at least a second lumen, the central probe having a proximal end and a closed distal end, the tip of the distal end having a needle shape configured to pierce tissue and having at least one exit port located at a predetermined distance between the distal and proximal ends of the central probe, the exit port fluidly connecting the second lumen to the outside of the central probe; c) at least one take-up line located in the second lumen and slidable within the central probe, the take-up line having a proximal end located in the central probe for... The distal end of the take-up line is configured to extend outside the central probe and retract into the second lumen via an exit port. The tip of the distal end of the take-up line has a shape configured to pierce through tissue and define an opening. At least a portion of the take-up line enters the tissue through the opening to create a fluid channel. A treatment portion is injected into the area by an injection probe through the fluid channel. d) A ramp integrally formed or coupled to the inner surface of the central probe, defining the inner surface of the second lumen, and the ramp is configured to contact and guide the take-up line to allow the central probe to exit outside the central probe. e) An electrical connector that electrically connects the central probe and the take-up line to a power source. f) A handle that at least partially accommodates the electrical connector and is coupled to the proximal end of the central probe and the proximal end of the injection probe and the take-up line to facilitate penetration depth of the injection probe and the distal end of the central probe.

[0098] In some embodiments, the device includes a plurality of exit ports and a plurality of take-off lines configured to simultaneously extend to the exterior of the central probe and to retract into the central lumen of the central probe via the exit ports.

[0099] In some embodiments, the device further includes at least two oppositely charged electrodes positioned to surround a target tissue region for cell therapy, the electrodes being adapted to extend from a proximal end to a distal end, the distal end having a needle shape configured to pierce the tissue, wherein the electrodes are adapted to couple to a power source, receive electrical waveforms from the power supply, and supply a pulsed electric field sufficient for electroporation to the target tissue region.

[0100] In some embodiments, the angle of the ramp contact with the take-up line is adjustable to change the corresponding trajectory angle of the take-up line exiting the second lumen.

[0101] According to some embodiments, a method for delivering a treatment portion to a region of target cells in tissue includes a) providing means for delivering the treatment portion to a region of target cells in tissue. The means includes: i) a central probe having an inner surface defining at least a first central lumen and having a proximal end and a closed distal end, the distal end having a needle-shaped portion configured to pierce tissue and having at least one exit port positioned at a predetermined location from the distal end, the exit port fluidly connecting the central lumen to the outside of the central probe; ii) at least one take-up line positioned within the central lumen and slidable within the central probe, the take-up line having a proximal end positioned within the central probe and a distal end configured to extend to the outside of the central probe and retract into the central lumen via the exit port. The distal end of the take-up line has a shape configured to pierce through tissue and define an opening, at least a portion of the take-up line entering the tissue through the opening to create a fluid channel through which the treatment portion is delivered to the tissue. The treatment portion is delivered from the first central lumen into the channel via the exit port. The device further includes: iii) a ramp integrally formed or coupled to the inner surface of the central probe, the inner surface defining a central lumen, and the ramp being configured to contact and guide the take-up line to allow the central probe to exit outside the central probe; iv) an electrical connector that electrically connects the central probe and the take-up line to a power source; v) a small aperture connector that connects the central probe to a syringe for delivery of a treatment portion; and vi) a handle that houses the electrical connector and is coupled to the proximal ends of the central probe and the take-up line to facilitate penetration depth at the distal ends of the central probe and the take-up line. The method further includes: b) inserting a central probe into a diseased cell in a region of target cells; c) actuating and extending a take-up line from a central lumen in the axial direction of the central probe, the distal tip of the take-up line having a needle shape that pierces through tissue and forms an opening, at least a portion of the take-up line entering the tissue through the opening and creating a fluid channel through which a treatment portion is delivered; d) actuating a ramp integrally formed or coupled to the inner surface of the central probe, the ramp contacting the take-up line and guiding the trajectory of the take-up line through an exit port toward the distal end of the central probe, the exit port fluidly connecting the central lumen to the outside of the central probe; e) piercing the tissue with the take-up line and creating an opening, at least a portion of the take-up line entering the tissue through the opening to create a fluid channel for delivery of the treatment portion to the tissue; f) retracting the take-up line back into the central lumen; and g) injecting the treatment portion into the central lumen and delivering the treatment portion to the tissue through the fluid channel.

[0102] In some embodiments, the method further includes: a) rotating the device for delivery at least once and puncturing the tissue with a guideline to create an additional fluid channel for delivery of the treatment portion to the tissue, then injecting the treatment portion through the fluid channel and delivering the treatment portion to the tissue.

[0103] In some embodiments, the method further includes: a) providing an electroporation system comprising at least two oppositely charged electroporation electrodes positioned to surround a region of a target cell, wherein the electroporation electrodes are adapted to extend from a proximal end to a distal end, the distal end having a needle-shaped tip configured to puncture tissue, and the electroporation electrodes being adapted to be coupled to a power source; b) contacting the region of the target cell with the electroporation electrodes; c) delivering an electrical pulse from the power source to the electrodes; and d) applying a pulsed electric field sufficient for electroporation from the electroporation electrodes to the region of the target cell.

[0104] According to some embodiments, a method for delivering a therapeutic portion to a region of target cells in a tissue includes a) providing means for delivering the therapeutic portion to the region of target cells in the tissue. The means includes: a) an injection probe defining at least a first lumen, the injection probe extending from its proximal end to its distal end and having an elongated cylindrical shape, the distal end having a needle shape and being open for delivering the therapeutic portion to the region; b) a central probe coupled to the injection probe and having an inner surface defining at least a second lumen, the central probe having a proximal end and a closed distal end, the distal end having a needle shape configured to pierce tissue and having at least one exit port located at a predetermined distance between the distal and proximal ends of the central probe, the exit port fluidly connecting the second lumen to the outside of the central probe; c) at least one take-up line located in the second lumen and slidable within the central probe, the take-up line having a proximal end located in the central probe and configured to extend to the central probe. The external portion of the central probe and the distal end retracting into the second lumen via the exit port, the distal tip of the take-up line having a shape configured to pierce through tissue and define an opening, at least a portion of the take-up line entering the tissue through said opening to create a fluid channel, the treatment portion being injected into the area by the injection probe through said fluid channel, d) a ramp integrally formed or coupled to the inner surface of the central probe, defining the inner surface of the second lumen, and said ramp being configured to contact and guide the take-up line to allow the central probe to exit to the outside of the central probe, e) an electrical connector electrically connecting the central probe and the take-up line to a power source, and f) a handle at least partially housing the electrical connector and coupled to the proximal end of the central probe and the proximal end of the injection probe and the take-up line to facilitate penetration depth of the injection probe and the distal end of the central probe. The method further includes: b) inserting a central probe into a diseased cell in a region of target cells; c) actuating and extending a take-up line from a central lumen in the axial direction of the central probe, the distal tip of the take-up line having a needle shape that pierces through tissue and forms an opening, at least a portion of the take-up line entering the tissue through the opening and creating a fluid channel through which a treatment portion is delivered; d) actuating a ramp integrally formed or coupled to the inner surface of the central probe, the ramp contacting the take-up line and guiding the trajectory of the take-up line through an exit port toward the distal end of the central probe, the exit port fluidly connecting the central lumen to the outside of the central probe; e) piercing the tissue with the take-up line and creating an opening, at least a portion of the take-up line entering the tissue through the opening to create a fluid channel for delivery of the treatment portion to the tissue; f) retracting the take-up line back into the central lumen; and g) injecting the treatment portion into the central lumen and delivering the treatment portion to the tissue through the fluid channel.

[0105] The method further includes: a) rotating the device for delivering at least once and piercing the tissue along a guideline to create an additional fluid channel for delivery of the treatment portion to the tissue, then injecting the treatment portion through the fluid channel and delivering the treatment portion to the tissue.

[0106] The method further includes: a) providing an electroporation system comprising at least two opposing charged electroporation electrodes positioned to surround a region of a target cell, wherein the electroporation electrodes are adapted to extend from a proximal end to a distal end. The distal tip has a needle shape configured to puncture tissue. The electroporation electrodes are adapted to be coupled to a power source. The method further includes b) contacting the region of the target cell with the electroporation electrodes, c) delivering an electrical pulse from the power source to the electrodes, and d) applying a pulsed electric field sufficient for electroporation from the electroporation electrodes to the region of the target cell.

[0107] According to some embodiments, a system for electroporating cells at electroporation sites in an electric field region within a tissue in a body includes a) an electroporation rod housing. The housing includes i) a first pair of electroporation electrodes, and ii) at least a second pair of electroporation electrodes housed within the rod housing, the first and second pairs of electroporation electrodes being configured to be charged oppositely, offset from each other at a predetermined angle, and configured to define the outer periphery of the electric field region. The system further includes: b) a power supply configured to supply electrical signals of multiple waveforms to the first and second pairs of electroporation electrodes, and c) an electrical connector electrically connecting each of the first and second pairs of electroporation electrodes to the power supply.

[0108] In some embodiments, the system further includes a rod delivery system configured to deliver a treatment portion to an electroporation site, the delivery system including at least one injection probe defining a first lumen, the injection probe extending from its proximal end to its distal end and having an elongated cylindrical shape, wherein the distal end of the injection probe has a needle shape and is open for delivering the treatment portion to the electroporation site.

[0109] In some embodiments, the system includes two pairs of electroporation electrodes at an angle of approximately 90 degrees.

[0110] In some embodiments, a first pair of electroporation electrodes is configured to receive a first electrical signal represented by a first waveform from a power supply, and a second pair of electroporation electrodes is configured to receive a second electrical signal represented by a second waveform from a power supply.

[0111] In some embodiments, the first and second pairs of electroporation electrodes are needles configured to penetrate the skin and contact cells in an electric field region.

[0112] In some embodiments, the first and second pairs of electroporation electrodes are non-penetrating contacts.

[0113] According to some embodiments, a method for electroporating cells at electroporation sites in an electric field region in a tissue of a body includes: a) providing an electroporation system comprising i) an electroporation rod housing including 1) a first pair of electroporation electrodes, and 2) at least a second pair of electroporation electrodes housed within the rod housing, the first and second pairs of electroporation electrodes being charged oppositely, offset from each other at a predetermined angle, and configured to define an outer periphery of an electric field region; ii) a power supply configured to supply electrical signals of multiple waveforms to the first and at least the second pair of electroporation electrodes; and iii) an electrical connector electrically connecting the pair of electroporation electrodes to the power supply. The method further includes: b) contacting the electroporation rod housing with the tissue such that an electric field region is formed between the pairs of electroporation electrodes; c) applying a first signal of a first waveform to the first pair of electroporation electrodes from a power supply and applying a second signal of a second waveform to the second pair of electroporation electrodes from a power supply, wherein the first waveform has a predetermined phase difference with the second waveform; d) applying a pulsed electric field from the first pair of electroporation electrodes to the electric field region, the pulsed electric field being based on the first signal, wherein the pulsed electric field and each subsequent pulsed electric field of the first pair of electroporation electrodes have a voltage and duration lower than a minimum threshold for electroporation; e) applying another pulsed electric field from the second pair of electroporation electrodes to the electric field region, the other pulsed electric field being based on the second signal, wherein the other pulsed electric field and each subsequent pulsed electric field of the second pair of electroporation electrodes have a voltage and duration lower than a minimum threshold for electroporation. The paths of the pulsed electric fields of the first and second pairs of electroporation electrodes intersect at the electroporation site, and the application of each pulsed electric field from the first pair of electroporation electrodes to the electroporation site alternates with the application of each pulsed electric field from the second pair of electroporation electrodes to the electroporation site, to total a continuous pulsed electric field with a voltage and duration sufficient for electroporation to be applied to the cells at the electroporation site. The application of each pulsed electric field from the first pair of electroporation electrodes to tissue adjacent to the first pair of electroporation electrodes but outside the electroporation site alternates with a resting period, such that tissue adjacent to the first pair of electroporation electrodes but outside the electroporation site receives the alternating on and off pulsed electric fields from the first pair of electroporation electrodes, having a voltage and duration below the minimum threshold for electroporation. The application and resting period of each pulse electric field of the second pair of electroporation electrodes to tissue adjacent to the second pair of electroporation electrodes but outside the electroporation location is alternated, such that tissue adjacent to the second pair of electroporation electrodes but outside the electroporation location receives the alternating on and off pulse electric fields of the second pair of electroporation electrodes, having a voltage and duration below the minimum threshold for electroporation.

[0114] In some embodiments, the method further includes delivering a treatment portion to an electroporation site using a rod delivery system, the delivery system including at least one injection probe defining a first lumen, the injection probe extending from its proximal end to its distal end and having an elongated cylindrical shape. The distal end of the injection probe has a needle shape and is open for delivering the treatment portion to the electroporation site.

[0115] In some embodiments, the method further includes providing a sensor system for sensing the capacitance of a cell membrane. Capacitance sensing includes: a) contacting tissue with at least one pair of capacitance sensing electrodes powered by a low-voltage power supply; b) transmitting a low-power interrogation signal via the low-voltage power supply to the at least one pair of capacitance sensing electrodes to generate a low-intensity electric field excitation at the electroporation site; c) sensing a voltage or voltage drop across the cell membrane by a voltage sensor; d) sensing a current across the cell membrane by a current sensor; and e) determining the capacitance of the cell membrane by an electronic signal processing device based on the voltage drop and current across the cell membrane.

[0116] In some embodiments, the capacitance of the cell membrane is determined before and between the application of the pulsed electric field.

[0117] In some embodiments, the method further includes adjusting the pulse width of the pulsed electric field based on a time constant associated with the thin film capacitance after determining the capacitance of the cell membrane between the pulsed electric fields.

[0118] In some embodiments, the first and second waveforms have the same wavelength.

[0119] In some embodiments, the voltage of the power supply is variable from about 50 V to 1000 V.

[0120] In some embodiments, each pulse electric field of the first and second electrode pairs has a pulse width that varies from 1 µs to 1 ms.

[0121] In some embodiments, each pair of electroporation electrodes emits a pulsed electric field during a time period of 1 / (the number of electrode pairs) of the period of the wavelength of each corresponding waveform.

[0122] In some embodiments, the cells are selected from the group consisting of: pancreas, larynx, pharynx, lips, throat, lungs, kidneys, muscles, breasts, colon, uterus, prostate, thymus, testes, skin, and ovarian cells.

[0123] In some embodiments, the cells are prostate tumor cells.

[0124] In some embodiments, the cell is a mammalian cell.

[0125] In some embodiments, the cell is a human cell.

[0126] In some embodiments, the pulsed electric field range of the first and second pairs of electroporation electrodes is from about 200 to 500 mV.

[0127] In some embodiments, the pulsed electric field of the first and second pairs of electroporation electrodes is applied as an electric pulse of about 1 to about 5.

[0128] In some embodiments, the first and second pulsed electric fields are selected from the group consisting of: square wave pulses, exponential wave pulses, finite-duration monopolar vibration wave forms, and finite-duration bipolar vibration wave forms.

[0129] In some embodiments, the first and second pulsed electric fields comprise square wave pulses.

[0130] In some embodiments, the treatment component is selected from the group consisting of nucleic acids, peptides, and chemotherapeutic agents.

[0131] In some embodiments, the chemotherapeutic agents are selected from the group consisting of bleomycin, cisplatin, and mitomycin C.

[0132] In some embodiments, the housing of the electroporation rod is composed of a non-conductive device.

[0133] In some embodiments, the non-conductive appliance is made of plastic.

[0134] In some embodiments, each pair of electroporation electrodes determines the field vector and current path of the corresponding electric field.

[0135] In some embodiments, the first and second waveforms have a predetermined phase difference.

[0136] According to some embodiments, a system for electroporation (EP) of cells in a body tissue includes: a) a cannula and b) an EP device. The cannula includes: i) an insertion cannula extending proximally to an open distal end and defining a first lumen configured to receive an obturator; and ii) an obturator extending proximally to a distal end, the proximally end including a handle mounted thereon, and the distal end including a blade configured to pierce through the skin, penetrate into a body cavity, and form a path through which the insertion cannula can be at least partially inserted into the lumen. The obturator is configured to slide within the first lumen, the distal end of the obturator being configured to extend to the outside of the first lumen through the open distal end of the insertion cannula. The EP device is slidably mounted and retractable within the insertion cannula to reach cancer cells and includes: i) an anchor extending proximally to a distal end; and ii) at least two oppositely charged electrodes retractably positioned at the distal end of the anchor and configured to surround an area of ​​the target cell. The electrodes are adapted to be coupled to a generator, from which at least one electrical waveform is received, and at least one of an excitation signal and an EP pulse is supplied. The EP device further includes: iii) a central probe retractably disposed at the distal end of the anchor and having an inner surface defining at least a central lumen and extending from the distal end of the anchor; at least a portion of the central probe has a helical geometry configured to enhance anchoring of the central probe in tissue and create a channel for delivery of a treatment portion into the tissue. The distal end of the central probe is open to define an opening for delivery of a treatment portion into the tissue and has a shape configured to puncture the tissue.

[0137] In some embodiments, the blade of the obturator is configured to extend to the outside of the cannula through an opening at the distal end of the cannula.

[0138] In some embodiments, the EP device electrode is adapted to extend from a proximal end to a distal end, the distal end having a needle-shaped tip configured to pierce tissue. The electrode is adapted to be coupled to a power supply, from which it receives electrical waveforms and supplies at least one of an excitation signal and an EP pulse to a region of the target cell.

[0139] In some embodiments, an adaptive control method for controlling EP pulse parameters during EP in cells or tissues using an electroporation (EP) system includes: a) providing any of a system for providing adaptive control to optimize EP pulse parameters during EP in cells and tissues using any of the electroporation (EP) devices described herein; b) initializing EP pulse parameters for performing EP in cells or tissues via an initialization module, the initialized EP pulse parameters being at least partially based on at least one trained model; c) applying voltage and current excitation signals to cells and tissues via a generator, and measuring the values ​​corresponding to the applied excitation signals via a measuring device. d) Obtain data from current and voltage measurements across cells and tissues via a controller, and process the data to separate desirable data from undesirable data, e) Extract relevant features from the desirable data via a controller, f) Apply at least a portion of the relevant features of the desirable data to at least one trained diagnostic model via a controller, g) Estimate EP pulse parameters via a controller based on the results of the applied relevant features to the trained model, wherein the initialized EP pulse parameters are based on the at least one trained model and the relevant features, thereby optimizing the EP pulse parameters, h) Apply a first EP pulse via a generator based on the first pulse parameters.

[0140] In some embodiments, the method further includes predicting parameters for subsequent EP pulses after a first EP pulse has been applied by a controller using a trained model based on previous EP pulses and changes to at least one of the relevant features between applied EP pulses.

[0141] In some embodiments, the method further includes generating a diagnostic response by a controller at least in part based on the application, wherein the diagnostic response includes a) tissue detection, b) tumor type detection, c) needle placement detection, d) co-localization detection, and d) cell penetration detection.

[0142] In some embodiments, the method further includes: a) applying a subsequent EP pulse based on subsequent EP pulse parameters via a generator; and b) repeating the application of voltage and current excitation signals, repeating the measurement of cells or tissues, repeating the acquisition of data and separating desirable data from undesirable data, repeating the extraction of relevant features; and repeating the application until i) a predetermined limit on the number of cycles of the EP pulse sequence or EP pulses is reached, or ii) a diagnostic response prompts a diagnostic decision to terminate the adaptive control method. Attached Figure Description

[0143] Figure 1 This is a simplified schematic diagram depicting some components of an EP device according to the present invention for applying electrical pulses for EIS.

[0144] Figure 2 An EP electrode array with four electrodes (two pairs or groups) and an EM electrode array with four electrodes (two pairs or groups) are depicted, each attached to a suitable power source via connectors and circuitry. Again, each array is a four-electrode array, but more electrodes may be used. Furthermore, in these embodiments using two different electrode arrays, the number of electrodes need not be equal in every case; an EP array with four electrodes and an EM array with six electrodes may be used, etc.

[0145] Figure 3 A schematic diagram is depicted for EIS determination using 4 EP and 4 EME electrodes. Figure 3 A top view depicting the electrodes of a device inserted into hypothetical tissue containing blood vessels and irregularly shaped tumors (note that EMs can also be applied to non-penetrating electrode devices). For non-limiting illustrative purposes, two sets of electroporation electrodes (EPEs) and two sets of electrochemical impedance spectroscopy electrodes (EMEs) are shown, but other numbers and geometries are contemplated. These electrode sets in Figure 3 The arrangement is shown as substantially equidistant, but as those skilled in the art will understand, any number of groups conforming to the present invention can be used.

[0146] Figure 4A and 4B Two embodiments are shown that utilize inserted EP electrodes with insulating material to generate different electric field regions (for simplicity, only a single pair of electrodes is shown). Figure 4A A single pair of electrodes is shown, having alternating regions of insulating material and bare electrodes; in other words, the electrodes have conductors that are alternately and uniformly spaced along the length of the electrodes, wherein each conductor is separated by an insulating material. Figure 4B A similar group is shown, but in this case each conductor is not uniformly spaced, making it possible to generate an asymmetric electric field.

[0147] Figure 5 This is a diagram illustrating the hardware architecture of an EP generator used to generate pulsed electric fields for EPE pairs A and B. The EP device can be based on a digital signal processor (DSP), microprocessor, field-programmable gate array (FPGA), application-specific integrated circuit (ASIC), central processing unit (CPU), or any multi-purpose programmable device that accepts analog / digital data as input, processes the input according to instructions stored in memory, and provides an output as a result. Switching sequence routines for electrode pairs A and B are programmed and stored in memory. A data bus can be used to display and modify pulse parameters. High-voltage isolation allows the hardware to be used with a high-voltage power supply when plugged into a PC. A low-voltage power supply can be used to power all auxiliary circuitry, such as EMEs for capacitance and impedance measurements, analog-to-digital converters, digital-to-analog converters, repeaters, DSPs, optical switches, etc.

[0148] Figure 6A , 6B The 6C describes three different configurations of EPE and EME. Figure 6C In this system, a single set of electrodes is used, connected to the corresponding EPE and EME power sources via a switching mechanism. The switching mechanism switches on and off when a small excitation voltage is applied across its control terminals. These switches utilize coupling mechanisms incorporating electromagnetic, electromechanical, piezoelectric, and photoelectric elements. Figure 6A In this process, two sets of electrodes, EPE and EME, are used, and each is configured and connected to a suitable power source. Figure 6B They are similar, but the difference is that the EPE and EME are offset from each other at a predetermined angle, depending on the number of each electrode type used. In this embodiment, tissue in the region can be interrogated in different ways. For example, tissue directly against the EPE will experience damage (e.g., tissue in the "kill zone"). Figure 6B In this process, measuring the impedance (including capacitance) between EME #1 and EME #2 can help determine tissue damage, for example, at EPE #1, or alternatively, it can be used for a kind of "electron tumor computed tomography," as described more fully below relative to EM.

[0149] Figure 7A , 7B 7C and 7D depict different EPE configurations, but for simplicity, only a single pair of electrodes is depicted. Figure 7A A set of non-penetrating solid EPEs is depicted, applied to the surface of the skin. An additional set of EPEs is not shown, but is included. Figure 7B A set of solid EPEs penetrating into the tissue is depicted; in this embodiment, the tips of the EPEs are generally pointed to facilitate insertion into the tissue, such as a solid needle tip. In this embodiment, the electric field region is "deep" within the tissue, for example, below the surface. This results in a three-dimensional electric field along the length and radial dimensions between the electrodes. Generally, these penetrating EPEs can range from about 1 to about 20 mm, depending on the geometry and physiological condition of the tissue to be treated. Figure 7C In this process, the penetrating solid EPE is coated with an insulating (non-conductive) material, so that only the distal portion of the electrode is exposed. Figure 7A , 7B In embodiments like 7C, the TM delivery system typically uses a needle (not shown) that is shallowly inserted into the EP position between the EPEs. Figure 7D In the figure, the penetrating EPE is hollow, having a lumen for TM delivery and an open tip connected to the lumen. On the left, the penetrating electrode has a portion coated with an insulating material along its axis. As those skilled in the art will appreciate, the EPE can be used as an electrical measuring electrode (EME) when capacitance measurement is completed, or it can exist as a separate set of EMEs, as generally depicted in Figure 6.

[0150] Figure 8A, 8B 8C and 8D depict the components of the EP device of the present invention (which all rely on cylindrical needles, but other geometries may also be used; and, only a single pair of EP electrodes are depicted). Figure 8A and 8B A group of EPEs (the second group is not shown) with TM delivery (TMD) systems are depicted. Figure 8A An EPE and TM delivery system inserted into tissue is shown, wherein the hollow TMD is required to have an open end, the lumen is used for TM delivery, and the TM is being delivered in an abbreviated manner. Figure 8B The lower side of the device is shown, which may be located in the distal end of the rod. Alternatively, as... Figure 8C As shown, the TMD system may include a standard syringe, which is manually inserted by the administering physician during the procedure. In this embodiment, the syringe may have an optional needle stop to physically prevent deeper penetration at depths related to the depth of the electric field region. Figure 8D The diagram depicts a TM delivery needle with multiple openings for TM delivery. This can be useful when delivering larger biomolecules such as plasmids and antibodies, as larger molecules (and often charged) generally diffuse more slowly in tissues than other molecules. Therefore, having multiple delivery gene sites within the EP location enables a higher percentage of cells in the region to absorb the TM. Figure 8D Describe three openings or ports, but any number can be used. Additionally, Figure 8D An opening is depicted on one "side" of the needle, but the opening can be located on any part of the outer surface of the needle, thus forming a spiral or other shape.

[0151] Figure 9 This is a schematic diagram of an EP device according to the present invention, comprising a rod housing having a first pair and a second pair of electrodes.

[0152] Figure 10 This is a schematic illustration of the first and second pairs of EPEs defining the electric field region and the electroporation location according to the present invention.

[0153] Figure 11 This is an illustration of a pair of non-penetrating EPEs according to the present invention.

[0154] Figure 12 This is a schematic diagram of the offset angle produced by multiple EPE pairs according to the present invention.

[0155] Figure 13 The diagrams are illustrations of a first waveform corresponding to a first pair of EPEs and a second waveform corresponding to a second pair of EPEs according to the present invention.

[0156] Figure 14A and Figure 14BThis is a diagram illustrating a continuous pulsed electric field at the electroporation location and an alternating switching on and off pulsed electric field in the electric field region but outside the electroporation location, according to the present invention.

[0157] Figure 15 This is a simplified schematic diagram illustrating an adaptive control system according to the invention for optimizing electroporation (EP) pulse parameters during electroporation (EP) of cells in a body's tissue.

[0158] Figure 16 This invention describes an EP system used in an adaptive control system during electroporation (EIS) of cells in the body's tissue, according to the present invention.

[0159] Figure 17A This is a schematic illustration of an exemplary EP device having electrodes integrated around a central injection element and a central portion delivery probe for use in an adaptive control system for optimizing electroporation (EP) pulse parameters. Figure 17B This is a bottom view of the EP device.

[0160] Figure 18A and Figure 18B These are schematic illustrations of perspective and bottom views of an exemplary EP device having electrodes integrated around an injection element and a partial delivery probe for use in an adaptive control system for optimizing electroporation (EP) pulse parameters.

[0161] Figure 19 This is a schematic diagram of multiple electrodes positioned on the delivery probe of the spiral portion of the EP device according to the present invention.

[0162] Figure 20A This is an illustration of multiple center injection probes of the EP device according to the present invention, each comprising a helical blade for creating a channel. Figure 20B It is surrounded by multiple electrodes. Figure 20A A schematic diagram of the EP device with a central injection probe, and Figure 20C yes Figure 20B The diagram is a bottom view.

[0163] Figure 21 This invention describes an EP device with a spiral center probe and a spiral electrode according to the present invention.

[0164] Figure 22A , Figure 22B and Figure 22C This is a schematic diagram of an EP device with a distal electrode and a central probe according to the present invention.

[0165] Figure 23A , Figure 23B and Figure 23C , Figure 23D , Figure 23E and Figure 23F Various EP devices according to the present invention are described.

[0166] Figure 24A , Figure 24B and Figure 24C This invention describes the EP system of a cannula-based direct rod applicator.

[0167] Figure 25 and Figure 26 This invention describes the catheter / endoscope-based EP device according to the present invention.

[0168] Figure 27 This is a schematic illustration of an EP device according to the present invention for delivering a therapeutic portion to a region of target cells in a tissue.

[0169] Figure 28 It is based on the invention having Figure 27 A schematic diagram of the exit port and take-off line at the tip of the blade of the EP device.

[0170] Figure 29 It is based on the invention Figure 27 A schematic diagram of the ramp and take-off line for the EP device's ramp guidance.

[0171] Figure 30 This is a schematic diagram of the injection probe and the center probe coupled to each other according to the present invention.

[0172] Figure 31 It is based on the present invention Figure 27 A diagram of the curved route of the EP device.

[0173] Figure 32 This is a schematic illustration of a capacitive sensing (CS) / EIS sensing system according to the present invention.

[0174] Figure 33 This is an illustration of a method according to the present invention for delivering a therapeutic portion to a region of target cells in a tissue using an EP device.

[0175] Figure 34A , 34B And 34C describes the invention according to ( Figure 27 (The) EP device has multiple configurations. Figure 34A In this process, multiple channel lines are used, all unfolded at once, to create a "star" pattern. Figure 34B In this process, a single channel line is used, which is expanded, retracted, rotated, and re-expanded to produce the same "star" pattern, but sequentially. Figure 34C In this process, a single channel line is used, but after the channel line is unfolded and retracted, the outer shell of the rod is slightly removed and the channel line is unfolded again, thus forming a "comb" structure.

[0176] Figure 35 A housing containing curved channel lines is shown, which remains rigid when in place, but returns to its curved shape and forms curved channels for a fluid reservoir when the lines are unfolded.

[0177] Figure 36 This is a flowchart illustrating the control routine of the adaptive control method for controlling EP pulse parameters during the use of an EP system according to the present invention.

[0178] Figure 37 This describes the use of the present invention. Figure 36 The flowchart shows the leading-forward control routine for optimizing EP pulse parameters.

[0179] Figure 38 This is a diagram illustrating the initial training phase of a model for estimating impulse parameters according to the present invention.

[0180] Figure 39 This is an illustration of a trained model according to the present invention for estimating the parameters of the first impulse (initialization).

[0181] Figure 40 This is a flowchart illustrating the EP diagnostic routine in the method for adaptive control of EP pulse parameters according to the present invention.

[0182] Figure 41A and Figure 41B This is a flowchart illustrating the method for adaptive control of EP pulse parameters according to the present invention.

[0183] Figure 42A This illustrates the distribution of the time constant as a percentage of the applied electric field crosses the lipid bilayer. Figure 42B Explain the distribution of the time constant measured before EP. Figure 42C This illustrates the effect of modulating the pulse width based on pre-pulse EIS data, where the pulse duration is set as a multiple of the time constant used for each tumor. Figure 42D The present invention illustrates the data showing the relative change of the time constant calculated after EP with respect to the resulting luminescence.

[0184] Figure 43A This demonstrates the solution resistance data derived from the model fitting of normal C57BL / 6J mice and transgenic PDGF-C mice. Figure 43B This describes the admittance data derived from model fitting in normal C57BL / 6J mice and transgenic PDGF-C mice. Figure 43C This describes the data derived from the constant phase element (CPE) model fitting of normal C57BL / 6J mice and transgenic PDGF-C mice. Figure 43DThis indicates the calculated time constant data derived from the model fitting of normal C57BL / 6J mice and transgenic PDGF-C mice.

[0185] Figure 44 Histogram showing the percentage decrease in solution resistance after plasmonic DNA injection.

[0186] Figure 45 This describes the luminescence data observed 48 hours after intratumoral EP with 50 μg of plasso DNA expressing luciferase. EP conditions were set at 500 V / cm, with 8 pulses applied, and the duration set as a multiple of the calculated mean time constant.

[0187] Figure 46 : Luminescence data observed 48 hours after intratumoral EP with 50 μg of plasso DNA expressing luciferase. EP conditions were set at 350 V / cm, with 8 pulses applied, and the duration was set as a multiple of the calculated time constant for each individual tumor.

[0188] Figure 47 The plotted luminescence data varies with the calculated time constant after electroporation. Longer pulses lead to a decrease in the calculated time constant, with more than 20% of the groups showing a significant difference from the control group. Shorter pulses lead to an increase in the calculated time constant.

[0189] Figure 48 Equivalent circuit model based on the CPE organizational model. Detailed Implementation

[0190] I. Overview

[0191] This invention is generally directed to improved and ideally optimized apparatus, systems, and methods for controlling electroporation (EP) pulse parameters for use in patient cells and tissues. As further described herein, the invention has a variety of uses, including but not limited to, the ability to insert therapeutic portions (including small molecule drugs, plasmid-encoded therapeutic proteins, etc.) into cells. The invention is particularly suitable for oncology applications. The invention allows for the real-time determination of appropriate EP conditions and / or EP protocols using electrophysiological measurements (EM), including but not limited to electrochemical impedance spectroscopy (EIS). The invention aims to improve the EP process through integrated feedback control mechanisms. Therefore, the systems and methods of the present invention can be used with any EP device / applier and any method, such as those outlined in U.S. Provisional Patent Applications Nos. 62 / 214,807, 62 / 214,872, 62 / 141,142, 62 / 141,182, 62 / 141,256, and 62 / 141,164, all of which are expressly incorporated herein by reference in their entirety, including, and particularly, the accompanying drawings, illustrations, and descriptions of the drawings and components therein.

[0192] The EP parameters currently used in clinical trials are empirically established in preclinical mouse studies using homogeneous allogeneic tumor models. Typically, the electrical parameter that yields the highest increase in average expression of the injected electroporated nucleic acid alone is selected. Previous studies in this field have analyzed the effects of pDNA concentration, electric field (e-field) intensity, pulse, tissue type, electrical conditions, injection volume, molecule of interest, concentration, and applicator geometry on expression. Each of these parameters has been determined to significantly influence the resulting expression.

[0193] To maximize the efficacy of EP, a quantitative measure of membrane integrity that can be measured in real time is desirable. Electrochemical impedance spectroscopy (EIS) is a method for characterizing physiological and chemical systems and can be performed using standard EP electrodes. This technique measures the electrical response of a system over a frequency range to reveal energy storage and dissipation properties. In biological systems, the extracellular and intracellular matrix resists current and can therefore be represented electrically as resistors. Lipids in intact cell membranes and organelles store energy and can be represented as capacitors. Impedance is the sum of these resistive and capacitive elements over a frequency range. To quantify each of these parameters, tissue impedance data can be fitted to an equivalent circuit model. Real-time monitoring of the electrical properties of tissues will enable feedback control of EP parameters and lead to optimal transfection in heterogeneous tumors. Using EIS feedback will allow (1) real-time adjustment of delivery parameters, (2) delivery of only the pulses necessary to generate a therapeutic response, and (3) thus reduction of overall EP-mediated tissue damage.

[0194] Various embodiments of the present invention are directed to providing a closed-loop EP control system that uses tissue-sensing-based feedback to optimize the EP process using tumor-specific measurements acquired before and between each EP pulse. Tissue sensing is used to measure the film charge time of a specific tumor to adjust each EP pulse for optimal treatment.

[0195] As those skilled in the art will understand, a successful EP (epidemic response) occurs when the cell membrane ruptures, resulting in a change in capacitance. Therefore, by monitoring and measuring electrical properties, such as impedance (including capacitance) before, during, and / or after the EP pulse, relevant empirical data can be collected during the initial training phase and used to generate a model.

[0196] Various embodiments of the present invention are adaptive control methods and systems for improving or optimizing controlled EP pulse parameters using the aforementioned closed-loop EP control system and apparatus during EP in cells and tissues.

[0197] In some embodiments, the control system may include a measuring device, an initialization module, a signal generator, a controller, and a memory module. The control method described herein is implemented in the control system.

[0198] In one aspect, the measuring device measures tissue / cell conditions, such as the dielectric and conductive properties of cells and tissues. The measuring device may include one or more different measuring devices to facilitate the measurement of tissue / cell conditions. For example, the measuring device may include a voltage sensor / device and / or a current sensor / device. The voltage sensor may be configured to measure the voltage across a cell or tissue when an excitation signal and / or EP pulse is applied to the cell or tissue. The current sensor measures the current across a cell or tissue when an excitation signal and / or EP pulse is applied to the cell or tissue. The results of the measurements (e.g., measured data) may be sent to a controller for further processing.

[0199] The initialization module can be configured to initialize EP pulse parameters for performing electroporation (EP) on cells and tissues. The EP pulse parameters can be predetermined EP pulse parameters established empirically based on previous experimental / clinical trials. Alternatively, the predetermined EP pulse parameters can be based at least in part on one or more trained models. A signal generator can generate excitation signals and / or electroporation pulses applied to the cells and tissues. A measuring device measures tissue / cell conditions, such as the dielectric and conductive properties of the cells and tissues, in response to the application of the excitation signals and / or electroporation pulses.

[0200] As mentioned, the controller receives measured data corresponding to measurements of tissue / cell conditions. The controller then processes the measured data to facilitate the diagnosis / identification of tissue and cell characteristics and / or determine updated control parameters for the system. For example, it can assess the heterogeneity or homogeneity of tissues. The controller may include (in any combination) a preprocessing module, a feature extraction module, a diagnostic module, and a pulse parameter estimation module.

[0201] The preprocessing module obtains measured data from the measuring device and preprocesses the measured data to separate desirable data from undesirable data. For example, undesirable data may include noise or DC bias. Preprocessing may include scaling the measured data based on a standardized value such as standard deviation, performing digital filtering on the measured data, and verifying the measured data.

[0202] The feature extraction module extracts information such as relevant features from the desired data. Relevant features can be quantitative information. For example, quantitative information can be extracted using the computational routines described herein. The relevant features of the desired data are sent to the diagnostic module for further processing. For example, the diagnostic module applies at least a portion of the desired relevant features to one or more trained diagnostic models to determine whether the next step is to select the next applied EP pulse parameter or to stop the control process if a diagnostic problem is detected, such as an electrode not being placed in the tissue. The pulse parameter estimation module is configured to select or generate the next applied EP pulse parameter based on the results from the diagnostic module and the feature extraction module.

[0203] In some embodiments, the present invention relates to "lead-ahead feedforward control." "Lead-ahead feedforward control" means that prior to applying a first EP pulse, a parameter estimation routine initializes initial control parameters for the first pulse based on a model trained in an initial training phase, using empirical data from previously conducted experiments. These previously conducted experiments may be based on tissue samples, for example, of tumors with similar characteristics to those of the current tissue to which the control method of the present invention will be subjected. For example, the type, size, or location of melanoma tumors can be used to build a dataset to serve as the basis for the initial model. An initial excitation signal, comprising voltage and current signals, is applied via a signal generator (e.g., a proprietary signal generator described herein). A measuring device measures the tissue's response to the excitation signal. The controller derives "features" based on the measurements and compares the extracted features with older features derived from empirical data obtained from previously conducted experiments using a trained model. The older and derived "features" are obtained from tissue sensing measurements, such as EIS. The model can be trained based on the tissue or tumor type identified by a diagnostic module in the diagnostic phase and then used to select the optimal parameters / conditions for the first EP pulse. These first pulse parameters are thus “feeded forward” to be applied as the first pulse for the control routine, in contrast to conventional EP systems and methods where the parameters / conditions of the first pulse are based on fixed or static conditions. In this sense, the method of the present invention utilizes feedforward control to provide optimal EP parameters based on the sensed tissue type, combined with feedback control to sense cellular conditions, such as permeability, and adjust the pulse parameters accordingly.

[0204] Variations in tumor characteristics, such as tumor location, size, and the degree of angiogenesis, fibrosis, and necrosis that typically influence treatment outcomes, lead to poor predictability of effective electrostatic field (EP) conditions for gene delivery, and thus variable treatment outcomes. Conventional EP systems employ open-loop control systems with static parameters that rely on prior knowledge determined through preclinical studies in homogeneous, syngeneic tumor models. However, preliminary data have shown that even in homogeneous tumors, the time required to apply an electrostatic field across the cell membrane follows a log-normal distribution. Even in homogeneous models, applying static parameters to different tumors results in a wide range of electrostatic field application across the cell membrane, leading to treatment variability. This invention overcomes the aforementioned shortcomings of the prior art by implementing a control method employing a closed-loop control system that uses tissue-sensing feedback to optimize the EP process using tumor-specific measurements acquired before and between each EP pulse. Therefore, by combining EIS feedback control with "lead-ahead feedforward control," this invention can more effectively predict the effective parameters for EP, taking into account variations in tumor characteristics that typically affect treatment.

[0205] II. Electrochemical Impedance Spectroscopy (EIS)

[0206] The systems and methods of the present invention may include electrochemical impedance spectroscopy (EIS) (or tissue sensing) measurements, which can be performed using an EP device. In some embodiments, the EP device may include an electroporation electrode (EPE) for applying an EP pulse and an electrometry electrode (EME) for applying a low-voltage interrogation signal to the cell. In some embodiments, the electrodes of the EP device act as both the EME and the EPE, and a solid-state repeater can be used to switch between a high-voltage EP pulse circuit and a low-voltage EIS interrogation circuit, such as... Figure 1 As explained in the text. Figure 1 This is a simplified schematic diagram depicting some components of an EP device according to the present invention for applying electrical pulses for EIS. Although an electrode array of four electrodes is shown, this is not limiting, and arrays of electrode pairs comprising 2, 4, 6, 8, 10, and 12 or more electrodes are all applicable to the present invention. Furthermore, although the electrodes are shown as having a straight shape, this is not limiting, as the electrodes can have curved or spiral shapes, as will be described below with respect to various EP devices that can be used in the systems and methods of the present invention. Figure 1 The diagram depicts the scenario where the array of electrodes acts as EP electrodes when connected to an EP circuit or as electrical measurement (EM) electrodes when connected to a tissue sensing / EIS circuit. As discussed herein, when the electrodes of the EP device act as both EME and EPE, the electrodes switch between EPE and EME modes via a relay switch. That is, a solid-state repeater is used to switch between a high-voltage EP pulse circuit and a low-voltage EIS interrogation circuit, such as... Figure 1As described herein, the proprietary generator of this invention can supply both high-voltage pulses and low-voltage interrogation signals to the EP device when necessary. In other embodiments where the EP device has separate EPE and EME, the EP device can be connected to two power supplies via a switching mechanism that is switched on and off when a small excitation voltage is applied across its control terminals. These switches utilize coupling mechanisms that incorporate electromagnetic, electromechanical, piezoelectric, and photoelectric mechanisms.

[0207] Due to general knowledge of electroporation conditions, capacitance and resistance measurements obtained before applying the EP pulses provide prior knowledge of conditions that would cause instability in capacitive elements such as cell membranes. Measuring capacitance between pulses allows adjustment of electrical conditions based on time constants associated with the film's capacitance and resistance, including the pulse width (which can be calculated from the associated time constant). Furthermore, this information allows the process to be stopped when an ideal decrease in the time constant is reached, for example, when film integrity has been compromised, thus allowing for the introduction of therapeutic portions.

[0208] In some embodiments, the EP device uses different sets of EPE and EME, such as Figure 3 The general description is as follows. In some cases, as summarized more fully below, when using different sets of EPE and EME sets, the EME and EPE will be offset, thus allowing impedance measurements in different regions of the electric field region, as described below. Figure 3 The illustrations are generally discussed. In these embodiments, in addition to the higher voltage EPE power supply, an additional low voltage EME power supply, along with appropriate circuitry and connectors, is used.

[0209] In other preferred embodiments, as described above and as will be used to illustrate the system and method of the invention, the EP device uses a single set of electrodes for both EP and EIS measurements. EIS measurements can be performed using an EPE without adversely affecting tissue properties. It is ideal to use the same electrodes to perform low-power EIS measurements and high-power EP pulses because this reduces the number of electrodes required and allows for direct measurement of the tissue response. EIS is a low-power technique capable of real-time monitoring of tissue. This technique is performed by applying a series of low-voltage excitation signals across a pair of electrodes and measuring the response current over a frequency range. The magnitude and phase of each applied excitation are then calculated and fitted to an equivalent circuit model of the tissue as described below (e.g., Figure 48 As shown in the figure, it will be referred to below as the "CPE-based organizational model".

[0210] The impedance measurement value can be obtained using the following equation:

[0211]

[0212] In the above equation, Z(f) is Tissue impedance in ohms The frequency is measured in Hertz; It means The constant; The admittance of Siemens (in) =1Hz); The resistance is measured in ohms; and It is a unitless constant phase element (CPE).

[0213] As illustrated in the model, the resistive element (R) I and R E These are due to the intracellular and extracellular matrix, and the lipid structure is composed of constant phase elements (CPEs) of tissues and cells. M (This indicates that) CPE M It represents the charge or capacitance of the lipid bilayer (by Q). M This is represented by (α) and a scalar (denoted by α) representing the non-ideal properties of the capacitor, ranging from 0 to 1. As will be discussed further below, the time constant used for charging the lipid bilayer can then be calculated as... The calculation of the time constant in this manner is integral to the method of the present invention, because the time constant is subsequently used to identify the optimal EP pulse duration before, during, and / or after each treatment.

[0214] By using an array of EPE and / or additional electrical measurement electrodes (EMEs), it is possible to interrogate the tissue within the area surrounded by the electrode array. This information can be used to guide, for example, EP conditions. That is, by using different EISs to interrogate the input signal, such as a linear frequency modulated pulse (or many other signals summarized below), the output signal allows the device to fit a tissue model to determine the nature of the tissue and the EP signal to be used. For example, refer to... Figure 3 After electrode insertion, various inquiries can be performed. For example, comparing the impedance between electrodes 1 and 2 and between 1 and 8 can help determine that the tissue between electrodes 1 and 8 is "normal" tissue, rather than "abnormal" or "disease-prone" tissue between 1 and 2. Similarly, inquiries between electrodes 7 and 8 or 6 and 7 can help determine that electrode 7 is near or within a blood vessel and therefore should not be used for electroporation. Thus, for example, these measurements can be used to address the following four questions, as well as any other relevant inquiries depending on the data necessary based on the experimental scope.

[0215] 1) Is there good contact between each electrode and the tissue? As those skilled in the art will understand, using electrodes in hard-to-reach areas or on skin with specific compliance can lead to uncertainty when both electrodes are fully inserted into the tissue to be treated. This results in a heterogeneous electric field and poor delivery. 2) Is the electrode inserted into feasible tissue? Inserting electrodes into abnormal tissue, such as specific tumor tissues which may be heterogeneous in terms of texture and / or cellular integrity, with many tumors having necrotic and / or apoptotic cellular regions, can lead to electrodes being inserted in locations that may not result in a good and / or even non-existent electric field, and therefore such electrodes cannot be used in the procedure of this invention. 3) Is the treatment portion (TM) or drug in the correct position? In this embodiment, this measurement can be performed before and after the injection of the TM solution, and the difference can indicate whether more TM solution should be injected. 4) Are there electrodes that should not be used due to their position and / or contact? Again, refer to Figure 3 These EIS measurements allow for the determination of irregular electrode placement (e.g., in or near blood vessels) or poor electrical contact due to tissue heterogeneity and / or integrity. 5) Does the tissue (e.g., tumor) adequately achieve electroporation? This is similar to tissue sensing, as it measures cell membrane integrity. Therefore, these measurements can be performed before EP (to establish a baseline), during EP, and after EP to ensure that EP actually occurs.

[0216] Furthermore, these EIS measurements can be used to determine ideal EP conditions, as described below, relative to the adaptive control method of the present invention, for providing improved or optimized EP pulse parameters. In some embodiments, the method of the present invention may include contacting the electric field region 100 or the electroporation location 110 with a pair of EPE / EME 120 contacts. Figure 10 The organization is shown in the diagram. A low-voltage power supply electrically connected to the EPE / EME is used to apply a low-voltage interrogation signal to the EPE / EME. Methods for sensing impedance and / or capacitance may include (but are not limited to) waveforms such as phase-locked loops, square wave pulses, high-frequency pulses, and linear frequency modulated pulses. Voltage and current sensors are used to sense the voltage drop and current flowing through the circuit, and these parameters can then be obtained from, for example,... Figure 1 The controller processing described herein is used to determine the average impedance of all cells in the measurement area.

[0217] As described above, capacitance and resistance measurements are indicators of cell health and can be used to determine the length of the electrical pulse to be applied to disrupt the cell membrane and provide sufficient conditions for electroporation. Once the average impedance of the cell has been determined, it becomes possible to determine several characteristics of the measured cell, including, but not limited to, the initial conditions of the cell or tissue, such as whether the cell is diseased (indicated by below-average capacitance), whether the cell is healthy, the positioning of the electrode—whether the electrode surrounds the area of ​​interest / cells properly positioned / and whether the electrode is in the correct location for effective electroporation, and the time constant, as briefly discussed above for cells (described further below).

[0218] In some embodiments, impedance measurements can be performed across several EI sensing electrode pairs to determine whether the average of all cells in the electric field region 100 is consistent and for more accurate readings at a specific location. If the EI measurements are inconsistent across several electrode pairs, this can indicate inconsistencies in cell homogeneity, thus requiring different time constants to be applied to electrodes in different sets. The time constant provides an indication of the pulse width to be applied to the cells for electroporation to occur. Charging the capacitor to its maximum value (i.e., the case where the capacitor / cell cannot store energy due to electroporation, where good transfection occurs) takes approximately 5 times the time constant. Therefore, it is possible to determine the pulse width used to charge the capacitor to the point just before electroporation occurs, thus determining that the capacitor should be charged to at least 5 times the time constant (τ). C The necessary pulse width is determined. After determining the time constant, the pulse width is set accordingly for each set of EPEs based on the time constant determined for the cells in the region surrounded by the EPE.

[0219] The time constant can be based on the circuit model described above and derived from the series of equations below. For the purposes of this invention, the time constant is described as the potential applied across the terminals (…). Drive the CPE to half of the applied potential. The amount of time (τ) required.

[0220] (1)

[0221] (2)

[0222] (3)

[0223] (4)

[0224] Here, It is the voltage across the CPE, and It is the voltage applied across the terminal or film. At (2), The substitution results in step (3) of calculating f, and f is replaced by f. Replace with the equation to derive the final time constant used in the calculation of the ideal pulse width for the EP pulse.

[0225] Therefore, the method and system of the present invention utilize electrical-based measurements and feedback to significantly improve the EP process, as will be further described below. Since the EPE is used for both EPE and EME, the feedback is provided by the EPE, thus requiring no additional hardware. Fitting electrical data to a modified Randles model circuit allows for parameter monitoring of the thin film's conditions. Tumor tissue can therefore be fitted to the modified Randles equation in real time. The modification involves replacing the constant phase element (CPE) with a capacitive / resistive element. The CPE provides a realistic representation of the thin film, where Q = admittance; and 0 ≤ α ≤ 1.

[0226] Figure 10 This is a schematic illustration of the first and second pairs of EPEs defining the electric field region and the electroporation location according to the present invention. Figure 14A and Figure 14B This is an illustration of a continuous pulsed electric field at the electroporation location according to the invention, and alternating switching on and off of the pulsed electric field in the electric field region but outside the electroporation location. As those skilled in the art will understand, successful electroporation occurs when the cell membrane breaks, resulting in changes in capacitance and resistance. When subjected to an electric field, the cell essentially acts as a capacitor. When the electric field is applied for a sufficiently long period (depending on cell nature, health, size, etc.), charge accumulates at the cell membrane until it reaches a certain threshold and causes a breach in membrane integrity. In embodiments where the EPE and EME are different electrodes, the EME can be powered by a low-voltage interrogation circuit. The invention also includes voltage sensors and current sensors, such as... Figure 15 As described herein, current and voltage across cell membranes and tissues are measured, and the controller processes the voltage and current to determine the average capacitance of the cells in electric field region 100.

[0227] Impedance can be measured based on charge redistribution within cells in response to low-frequency electric field excitation from a low-voltage interrogation circuit. Impedance can be measured before, during, and after the application of the electroporation electric field to determine cell conditions, including but not limited to cell health, the placement of the electrodes relative to the cells for optimal electroporation, and, most importantly, the time constant, which can be used to determine the pulse width of the electric field to be applied to the cells in the electric field region. As previously described, generally, charging the capacitor to its maximum value, i.e., exactly before electroporation occurs, takes five time constant periods; therefore, the pulse width of the initial electroporation electric field pulse can be set to five times the time constant. This pulse width is insufficient to cause electroporation in cells outside electroporation location 110, as described above, but sufficient to cause electroporation in cells of tissue at electroporation location 110 that have undergone the additive effect of electric fields from all sets of EPEs being applied as a continuous electric field. Impedance measurements can be applied again after the first EP electric field has been applied, and the percentage decrease in impedance or time constant can be calculated and compared with a predetermined value to determine whether the cells at the electroporation location have been adequately electroporated. If not, the system and method of the present invention adjust the pulse width of the next set of electroporations through the pulsed electric field based on the calculated percentage decrease in capacitance until sufficient EP has occurred at the determined EP location. Therefore, impedance measurements between pulses allow adjustment of the electrical conditions, i.e., the pulse width, based on the time constant associated with cell membrane capacitance and resistance, and the electroporation process can be stopped when ideal support for capacitance, time constant, or film integrity is achieved.

[0228] Various embodiments of the present invention are directed to control systems and methods for electroporating cells in the EP location of tissue using the various electroporation devices of the present invention described herein.

[0229] III. The Adaptive Control System of the Invention

[0230] Various embodiments of the present invention are directed to systems implemented in electroporation (EP) devices for providing adaptive control to optimize controlled parameters during EP in cells and tissues. In some embodiments, such as Figure 15 and Figure 16 As described in the document, the adaptive control system includes measuring devices configured to measure the dielectric and conductive properties of cells and tissues.

[0231] Examples of dielectric and conductive properties may include capacitance, resistance, and impedance. In some embodiments, the measuring device includes a voltage sensor configured to measure the voltage across the cell or tissue generated by each of the excitation signal and each of the at least one applied EP pulse, and a current sensor configured to measure the current in the cell or tissue generated by each of the excitation signal and each of the at least one applied EP pulse. Voltage and current measurements indicate and are used to calculate various dielectric and conductive properties of the cell and tissue.

[0232] Figure 15 This is a simplified schematic diagram illustrating an adaptive control system for optimizing electroporation (EP) pulse parameters during electroporation (EP) of cells in a body tissue according to the present invention, and Figure 16 This describes the EP system used in an adaptive control system during electroporation (EIS) of cells in a body's tissue, according to the present invention. In some embodiments, such as... Figure 16 As described herein, the EP system of the present invention includes (A) an EP generator equipped with EIS that has data recording capabilities (e.g., Figure 15 The generator 1530, (B) a graphical user interface for programming pulse conditions, setting feedback criteria, and downloading EIS and pulse performance characteristics, (C) a proprietary applicator (EP device) consisting of two electrodes surrounding the central injection lumen, and (D) a foot pedal switch for remotely activating the EP process. Although Figure 16 The EP system described herein is one type of EP device, but it should be understood that the control system of the present invention can be incorporated into any EP device described herein to perform the adaptive control method described herein.

[0233] In some embodiments, the adaptive control system includes an initialization module 1520 configured to initialize EP pulse parameters for performing electroporation in cells or tissues. The EP pulse parameters may include (but are not limited to) voltage magnitude, repetition rate, and pulse width. The initialized EP pulse parameters are based at least in part on at least one trained model. The trained model may be, but is not limited to, a physics-based model, an empirical model, or a data-driven model. The EP pulse parameters may include (but are not limited to) pulse width, number of pulses, amplitude / field strength, and frequency.

[0234] In some embodiments, the adaptive control system further includes a signal generator 1530 configured to generate an excitation signal and deliver EP pulses to cells and tissues via (EPE / EME). The signal generator 1530 may be a pulse generator equipped with an EIS that provides an initial excitation signal with a predetermined pulse width based on experimental data observed offline, i.e., data from previous electroporation experiments with tissues / cells having similar properties to those to be subjected to the control methods of the present invention. In some embodiments, the signal generator 1530 is capable of supplying both a low-voltage excitation (interrogation) signal and a high-voltage signal for the EP pulse. An example of this generator is... Figure 16 The proprietary generator (A) described herein is capable of performing real-time feedback control based on EIS data before and between each EP pulse. The generator can output a minimum of 10 V and a maximum of 300 V for pulse durations ranging from 100 µs to 10 ms. The EIS data captured before and between pulses is obtained by the generator at 10 data points acquired every ten times the range from 100 Hz to 10 kHz. The acquisition of EIS data on this spectrum is achieved in 250 ms, which is fast enough to: (1) execute routines to determine the time constant for the next pulse; (2) store the EIS data for post-analysis; and (3) not interrupt clinically used EP conditions. The generator may be capable of having a minimum output load impedance of 20 ohms and a maximum open-circuit load impedance. The custom generator interfaces with a variety of standard EP device applicators and can support up to six electrodes. A solid-state repeater can be used to switch between a high-voltage EP pulse circuit and a low-voltage EIS interrogation circuit. To allow for hands-free operation of the generator, a foot pedal can be added to trigger, pause, or abort the EP process.

[0235] The measuring device 1510, which includes voltage and current sensors, measures voltage and current across cells and tissues in response to the application of an excitation signal and / or an EP pulse. In some embodiments, the measuring device is incorporated into the electrodes of the EP device 1540 of the present invention, but is not limited thereto. In other embodiments, the measuring device may be separate from the electrodes and implemented elsewhere in a control system.

[0236] In some embodiments, the control system of the present invention includes a controller 1505 configured to receive sensor data from a measuring device corresponding to measured cellular or tissue properties (i.e., dielectric and conductivity, such as capacitance, resistance, and impedance) and process the data into diagnostic and updated control parameters. In some embodiments, the controller comprises four modules, including a preprocessing module 1550, a feature extraction module 1570, a diagnostic module 1580, and a pulse parameter estimation module 1560. The preprocessing module obtains data from current and voltage measurements and preprocesses the data to separate desirable data from undesirable data. Undesirable data may include, but is not limited to, outliers, out-of-range values, and missing values. The controller is used to fit the data collected from EIS measurements to a tissue impedance model, i.e., the CPE-based tissue model described above, in real time. The controller, for example, has a microprocessor with a reduced instruction set computing architecture.

[0237] In some embodiments, the feature extraction module uses computational routines to extract quantitative information from desired data. These computational routines may include (but are not limited to) linear curve fitting parameters, nonlinear curve fitting parameters, cross-correlation, curvature, mean, average, median, range, standard deviation, variance, and kurtosis. When operating in feedback mode, the features of the measured EIS data can be used to control parameters associated with the EP process.

[0238] In some embodiments, the diagnostic module applies at least a portion of the relevant features of the desired data to at least one trained diagnostic model. The diagnostic model, along with the relevant features, is used to make a decision regarding the applied pulse. The diagnostic module may combine several features to identify whether the EP device is correctly positioned, whether the drug or gene is located between the EPE pairs, whether the EP pulse is effectively applied to transfection, and whether another pulse can be applied to the same electrode pair.

[0239] In some embodiments, the pulse parameter estimation module is used to generate the next applied EP pulse parameters based on the results of the diagnostic module and the feature extraction module. In some embodiments, the control system of the present invention further includes a memory module for storing processed device / sensor data and the trained model for feature extraction by the controller.

[0240] IV. Electroporation Apparatus and Methods

[0241] A. EP electrode configuration

[0242] The EP device of the present invention is applied to two main treatment areas: delivery of the treatment portion and tissue electroporation / ablation. Generally, and for many of the embodiments summarized herein, patients with diseases localized in specific tissues, such as cancer, will benefit from intracellular delivery of the treatment portion (TM). Alternatively, in some embodiments, it is desirable to kill small cellular sites within the tissue (sometimes referred to in the context of electroporation as "irreversible electroporation" or "electroporation ablation"). As is known in this art, one advantage of irreversible electroporation is that it induces apoptosis rather than necrosis as in other common ablation techniques. While most of the discussion herein relates to the former, FPA systems and methods without TM delivery are always anticipated.

[0243] The EP device and method of the present invention are used to electroporate cells in the tissues of a patient or subject and to deliver TM to the electroporation site for treatment. Generally, the EP device of the present invention is used to treat diseased or abnormal tissues, such as cancerous tissue. The term "cancer" encompasses a wide range of diseases generally characterized by inappropriate cell proliferation, abnormal or excessive cell proliferation. Examples of cancer include, but are not limited to, breast cancer, colon cancer, prostate cancer, pancreatic cancer, skin cancer (including melanoma, basal cell carcinoma, and squamous cell carcinoma), lung cancer, ovarian cancer, kidney cancer, brain cancer, or sarcoma. Therefore, cancerous tissues comprising skin tissue, connective tissue, adipose tissue, etc., can be treated using the system of the present invention. These cancers may be caused by chromosomal abnormalities, degenerative growth and developmental disorders, cell division-promoting agents, ultraviolet radiation (UV), viral infection, inappropriate tissue expression of genes, alteration of gene expression, or carcinogenic agents. The term "treatment" includes (but is not limited to) the inhibition or reduction of cancer cell proliferation, the destruction of cancer cells, the prevention of cancer cell proliferation or the prevention of malignant cell initiation, or the inhibition or reversal of the progression of precancerous cells to malignant disease, or the improvement of disease. The terms "subject" or "patient" refer to any animal, preferably a mammal such as a human. Use in livestock is also desirable to be included in this invention.

[0244] The systems and methods of this invention are applicable to electroporation of cells in tissues. As used herein, the terms “electroporation,” “electroosmosis,” or “electro-enhanced” (“EP”) interchangeably refer to the use of transmembrane electric field pulses to induce microscopic pathways (pores) in biological membranes; their presence allows therapeutic components (including, but not limited to, biomolecules such as plasmids, oligonucleotides, siRNAs, drugs, ions, and water) to be transferred from one side of the cell membrane to the other. This is achieved by applying an electric field over a time period, according to the formula Vm = 1.5 × radius × E. ext The cell membrane accumulates charge and generates a transmembrane voltage, where the radius is the radius of the cell, and E extThis is the external electric field of the cell. Generally, the cell membrane ruptures at approximately one volt (e.g., forming pores), but in addition to the cell's placement in the electric field, cell size and shape can also contribute to the difference. For example, long muscle cells have a higher capacitance across the cell width compared to along their length. Similarly, larger cells generally electroporate at lower voltages. As discussed herein, the use of the EM or capacitance sensing technology of the present invention can help optimize EP pulses and durations by determining the bulk properties of the cell in the electric field.

[0245] "Electroporated cells" include those cells that have momentarily open pores in their cell membranes, which close as the charge on the cell membrane dissipates ("open-pore cells"), and those cells that have undergone electroporation so that the cells now contain exogenously added therapeutic portions and have closed pores (e.g., intact again).

[0246] refer to Figure 9 The following describes an electroporation apparatus according to various embodiments of the present invention, generally designated by the numeral 10. The electroporation apparatus 10 may generally comprise an electroporation rod housing 12, optionally in the form of a cylindrical tube (but other geometries may also be used), a first pair of electroporation electrodes A housed within the rod housing, and at least a second pair of electroporation electrodes B. The rod housing may optionally include other components, including systems for TM delivery, switching circuitry, etc.

[0247] In some embodiments, the rod housing is shaped to facilitate physician use, for example, having a molded handle portion or grip, an optional illumination element at the distal end, a camera for observing and recording treatment sites, biopsy forceps, tissue scissors, a joining device, a suturing system, etc.

[0248] The term "electroporation electrode pair" or "EPE" as used herein refers to a pair of two electrodes configured to be energized in opposite directions when connected to a power supply. The first and second pairs of electroporation electrodes may be stationary or retractable within an electroporation rod housing 12. The electroporation rod housing 12 may further include a circuit board 16 having multiple sliding sockets through which electroporation electrodes A and B are slidably retracted and extended. Electrode pairs A and B are mounted within the electroporation housing 12, which is slidably engaged with an indicator or gauge 11. As the electroporation rod housing 12 moves along the gauge 11, it alternately extends and retracts electrode pairs A and B. The device indicator or gauge 11 can provide indication of the extension length of electrode pairs A and B. The electroporation device may further include an electrical connector 14 to electrically connect each of the first A and second B electrode pairs to a power supply 18, such as a pulse generator. The electrical connector, depending on the number of electroporation electrodes, includes four or more wires for transmitting electrical signals from the power supply to each of the electroporation electrodes. These signals may include pin voltage setpoint, pulse width, pulse shape, number of pulses, and switching sequence. As described above, and as those skilled in the art will understand and more fully described below, the EPE can also act as an EME, in which case the generator may be able to supply both a high-voltage EP pulse and a low-voltage EIS interrogation signal, or a second low-voltage power supply used with a suitable switching mechanism to allow delivery of a higher-voltage EP signal, and then delivery of a lower-voltage EIS signal.

[0249] In some embodiments of the invention, one or more of the EPEs may be non-penetrating electrodes, which may have an open distal end for applying a therapeutic portion to tissue, such as Figure 7A and Figure 11 As described herein, the non-penetrating electrode can be any suitable shaped conductor, such as a button or plate for contacting surface tissue. The injectors can be spaced apart from each other and in close contact with the surface area of ​​the body tissue. The portion of the non-penetrating electrode in contact with the tissue surface is conductive and electrically connected to a power supply, such as power supply 18, via an electrical connector, for example, electrical connector 14, so that EP is achieved by delivering current through the tissue area through a circuit between the conductive distal ends of the non-penetrating EPE.

[0250] EPE can be formed from conductive materials, but optional insulating coatings can be used as discussed herein. EPE can be made from any conductive material capable of carrying the large instantaneous current density associated with the applied high-voltage pulse, including, but not limited to: certain metals and their oxides, including gold, platinum, palladium, silicon, and aluminum; metal oxide electrodes, including platinum oxide, titanium oxide, tin oxide, indium tin oxide, palladium oxide, silicon oxide, aluminum oxide, molybdenum oxide (Mo2O6), tungsten oxide (WO3), and ruthenium oxide; and carbon (including glassy carbon electrodes, graphite, and carbon paste). Preferred electrodes comprise AgCl, cobalt-chromium, titanium, stainless steel, platinum, gold, or highly conductive metals plated with gold or platinum.

[0251] In some embodiments, such as when using non-penetrating electrode pairs, the distal end of the electrode is exposed for generating an electric field, but its proximal end may be coated with a non-conductive material to limit the application of the electric field to only the distal end of the electrode adjacent to the tissue.

[0252] In some embodiments, the EP electrode configured for insertion may be similarly coated with an insulating material so that the electric field is generated at the distal end of the electrode and not along the length of the electrode, for example to allow the EP to be "deeper" in the tissue rather than in a "shallow" area.

[0253] In some embodiments, the inserted EP electrode may have areas of alternating insulating material and bare electrode, such as... Figure 4A and 4B The electric field is generally depicted in this document. In this embodiment, the electrodes can be coated with the same pattern, resulting in a more uniform electric field, or coated with different patterns, resulting in an asymmetric electric field. Similarly, for all electrode configurations herein, the electrodes can have the same length or different lengths.

[0254] The pulsed electric field generated by these partially insulated electroporation electrodes is primarily concentrated in the area between and near the exposed tips of the electrodes at their distal ends during treatment, and is small in the area between and near the insulated portions. Partially insulated needle arrays can be used to limit electroporation in the target area with a tumor and significantly shield skin and tissue beyond the target area from the electroporation process. This provides protection for healthy skin and tissue that may be at risk due to unwanted or even adverse effects when some treatment portions are injected into healthy surface tissue above the target area.

[0255] Figure 7A , 7B 7C and 7D depict different EPE configurations, but for simplicity, only a single pair of electrodes is depicted. Figure 7A A set of non-penetrating solid EPEs is depicted, applied to the surface of the skin. An additional set of EPEs is not shown, but is included. Figure 7BA set of solid EPEs penetrating into the tissue is depicted; in this embodiment, the tip of each EPE is generally pointed to facilitate insertion into the tissue, such as a solid needle tip. In this embodiment, the electric field region is "deep" within the tissue, for example, below the surface. This results in a three-dimensional electric field along the length and radial dimensions between the electrodes. Generally, these penetrating EPEs can range from about 1 to about 20 mm, depending on the geometry and physiological condition of the tissue to be treated. It should be noted that this measurement is the depth of insertion and not the total length of the electrodes; generally, a portion of the electrode is present extending upwards from the point of contact with the tissue and into the rod housing for attachment to appropriate circuitry, holding the electrode in the correct spatial configuration, etc. Figure 7C In this process, the penetrating solid EPE is coated with an insulating (non-conductive) material, so that only the distal portion of the electrode is exposed. Figure 7A , 7B In embodiments like 7C, the TM delivery system typically uses a needle (not shown) that is shallowly inserted into the EP position between the EPEs. Figure 7D In the diagram, the penetrating EPE is hollow, having a lumen for TM delivery and an open tip connected to the lumen. On the left, the penetrating electrode has a portion coated with an insulating material along its axis. As those skilled in the art will appreciate, when capacitance measurements are completed, the EPE can be used additionally as an electrical measuring electrode (EME) or may exist as a separate assembly of EMEs, as generally depicted in Figure 6.

[0256] Figure 8A , 8B 8C and 8D depict the components of the EP device of the present invention (which all rely on cylindrical needles, but other geometries may also be used; and, only a single pair of EP electrodes are depicted). Figure 8A and 8B A group of EPEs (the second group is not shown) with TM delivery (TMD) systems are depicted. Figure 8A An EPE and TM delivery system inserted into tissue is shown, wherein the hollow TMD is required to have an open end, the lumen is used for TM delivery, and the TM is being delivered in an abbreviated manner. Figure 8B The lower side of the device is shown, which may be located in the distal end of the rod. Alternatively, as... Figure 8C As shown, the TMD system may include a standard syringe, which is manually inserted by the administering physician during the procedure. In this embodiment, the syringe may have an optional needle stop to physically prevent deeper penetration at depths related to the depth of the electric field region. Figure 8D The diagram depicts a TM delivery needle with multiple openings for TM delivery. This can be useful when delivering larger biomolecules such as plasmids and antibodies, as larger molecules (and often charged) generally diffuse more slowly in tissues than other molecules. Therefore, having multiple delivery gene sites within the EP location enables a higher percentage of cells in the region to absorb the TM. Figure 8D Describe three openings or ports, but any number can be used. Additionally, Figure 8D An opening is depicted on one "side" of the needle, but the opening can be located on any part of the outer surface of the needle, thus forming a spiral or other shape.

[0257] In some embodiments, the electroporation electrodes have a generally long length to completely surround the tissue to be treated. In a preferred embodiment, all the assembled electrodes (an “array” of electrodes) have the same length within the array, but in some cases the use of electrodes of different lengths results in altered and asymmetrical electric fields.

[0258] In many embodiments, the width and cross-sectional shape of the electrodes used for insertion are configured to minimize pain. Therefore, the width of the electrodes can range from 0.05 to 1 to 2 mm, and can depend on when the electrodes are also used for TM delivery. Generally, when the electrodes are hollow and used for TM delivery, they are generally large enough to accommodate the lumen for TM delivery.

[0259] Furthermore, the electrode and rod housings are preferably made of sterilizable materials and configured to similarly minimize microbial retention should the electrode array and rod housings be reused. In some embodiments, at least the electrode array is disposable, and in some embodiments, the entire rod housing is also disposable.

[0260] In some embodiments of the invention, multiple sets of electrode pairs are used. That is, as depicted in the figures, two sets (two pairs, four electrodes) can be used, for example, utilizing first and second pairs of electroporation electrodes. The first and second pairs of electroporation electrodes are offset from each other by a predetermined angle. For a set of two pairs of electrodes, the two electrode pairs are offset from each other by an angle of approximately 90 degrees, such as... Figure 1 As described herein, 90 degrees is preferred in some embodiments. The electrodes may also be positioned at a distance of 1 to 10 mm and define the outer perimeter of the electric field region.

[0261] In some embodiments, one or more of the electrodes may be hollow needles used for introducing the treatment portion, as discussed below.

[0262] The tissue surrounding the EP electrodes is sometimes referred to as the "burn-off zone." A "burn-off" zone means that the area is occupied by tissue that is adjacent to and / or in contact with each of the individual electrodes. It is called a "burn-off" zone because the cells are in direct contact with the electrodes, which are heated due to the high voltage signal from the power supply, and thus the cells are damaged by overheating. However, by using the alternating pulse device of the present invention, damage to cells in the burn-off zone can be minimized by reducing heat and field by 50% (in the case of two sets of electrodes; even more if more sets are used). Furthermore, because the electric field strength is focused / increased at the electrodes, the higher voltage causes EP-mediated cell death in a manner independent of heat.

[0263] Figure 5 This is a diagram illustrating the hardware architecture used to generate pulsed electric fields for electroporation electrode pairs A and B. The electroporation device can be based on a digital signal processor (DSP), microprocessor, field-programmable gate array (FPGA), application-specific integrated circuit (ASIC), central processing unit (CPU), or any multi-purpose programmable device that accepts analog / digital data as input, processes the input according to instructions stored in memory, and provides an output as a result. Switching sequence routines for electrode pairs A and B are programmed and stored in memory. A data bus can be used to display and modify pulse parameters. High-voltage isolation allows the hardware to be used with a high-voltage power supply when plugged into a PC. A low-voltage power supply can be used to power all auxiliary circuitry, such as capacitance or impedance sensing electrodes, analog-to-digital converters (ADCs), digital-to-analog converters (DACs), repeaters, DSPs, optical switches, etc.

[0264] In some embodiments, such as Figure 5 As explained, the first and second pairs of electrodes can be further connected to a generator capable of supplying electrical signals of various waveforms to each corresponding EPE pair. The first pair of EPEs A can be supplied with a waveform having a predetermined phase difference from the waveform supplied to the second pair of electroporation electrodes B by a power supply. For example, the first and second pairs of EPEs can receive waveforms with a 180-degree phase difference, such as... Figure 13 The rectangular electrode pair A waveform and electrode pair B waveform are illustrated in the diagram. As those skilled in the art will appreciate, and described more fully below, during EIS, the generator or power supply is capable of delivering both a high-voltage EP pulse and a low-voltage interrogation signal, and if not, provides an additional low-voltage EIS power supply.

[0265] In some embodiments, a highly specialized medical-grade fast-switching high-voltage / high-current solid-state repeater is used to switch a generator from a low-voltage EIS mode supplying an interrogation signal for EIS to a high-voltage EP mode supplying EP pulses for EP using an optically coupled repeater driver. Each repeater driver may be connected between a high-voltage power supply and a corresponding pair of electroporation electrodes. Each repeater channel may be implemented in a push-pull configuration to ensure the removal of stray charge from each of the electrode pair during a disconnection event.

[0266] A power supply with first and second waveform generators can be electrically connected to solid-state high-voltage relay channels A and B to control and output first and second electrical signals, wherein the first and second waveforms reach the corresponding electroporation electrodes A and B.

[0267] B. Treatment delivery system

[0268] In some embodiments, the EP device of the present invention may include a therapeutic portion (TM) delivery system. The TM delivery system may be integrated into the EP device in the form of a central probe or channel for TM delivery. In some embodiments, as described above, the EPE may be formed as a hollow electrode with an open distal end for delivery of the TM therethrough. The term "therapeutic portion" ("TM") herein means a portion suitable for EP capable of treating diseased tissue, comprising a cytotoxic agent, chemotherapeutic agent, toxin, radioisotope, intercytokine, or other therapeutically active agent. The TM may be a small molecule drug, nucleic acid (including those encoding a therapeutic target protein), or a biologically active protein (including polypeptides and peptides).

[0269] In some embodiments, TM is a drug; drugs intended for use in the methods of the present invention are typically chemotherapeutic agents with antitumor or cytotoxic effects. These drugs or agents include bleomycin, neomycin, suramin, doxorubicin, carboplatin, paclitaxel, mitomycin C, and cisplatin. Other chemotherapeutic agents will be known to those skilled in the art (see, for example, the Merck index). EP facilitates the entry of bleomycin or other similar drugs into tumor cells by creating pores in the cell membrane.

[0270] In some embodiments, the TM is a nucleic acid. Generally, a TM that is a nucleic acid has two distinct functional types. In one embodiment, the nucleic acid encodes a protein used to treat a disease; in other embodiments, the nucleic acid is the TM, for example, when the nucleic acid is siRNA or snRNA. The terms "nucleic acid" or "oligonucleotide" or their grammatical equivalents herein mean at least two nucleosides covalently linked together. The nucleic acids of the present invention will generally contain phosphodiester bonds, but in some cases, as summarized below, include nucleic acid analogs that may have an alternating backbone, including, for example, phosphoramides (Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein; Letsinger, Journal of Organic Chemistry 35:3800 (1970); Sprinzl et al., European Journal of Biochemistry 81:579 (1977); Letsinger et al., Nucleic Acids Res. 14:3487 (1986); Sawai et al., Chem. Lett. 805 (1984); Letsinger et al., Journal of the American Chemical Society 110:4470). (1988); and Pauwels et al., *Chemica Scripta* 26:141 91986, thiophosphates (Mag et al., *Nucleic Acid Research* 19:1437 (1991); and US Patent No. 5,644,048), dithiophosphates (Briu et al., *Journal of the American Chemical Society* 111:2321 (1989), O-methylaminophosphate bonds (see Eckstein, *Oligonucleotides and Analogues: A Practical Approach*, Oxford University Press), and peptide nucleic acid backbones and bonds (see Egholm, *Journal of the American Chemical Society* 114:1895 (1992); Meier et al., *Chem. Int. Ed. Engl.* 31:1008 (1992); Nielsen, *Nature* 365:566). (1993); Carlsson et al., Nature 380:207 (1996), all of which are incorporated herein by reference.Other similar nucleic acids include those with positive backbones (Denpcy et al., Proceedings of the National Academy of Sciences of the United States of America 92:6097 (1995); nonionic backbones (US Patent Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., Journal of the American Chemical Society 110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994); ASC Conference Series 580, Carbohydrate Modifications in Antisense Studies). Research), Chapters 2 and 3, edited by YS Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., Journal of Biomolecular NMR 34:17 (1994); Tetrahedron Letters 37:743 (1996), and nonribose backbones, including those described in U.S. Patents 5,235,033 and 5,034,506 and ASC Conference Series 580, Carbohydrate Modifications in Antisense Studies, Chapters 6 and 7 (edited by YS Sanghui and P. Dan Cook). Nucleic acids containing one or more carbocyclic sugars are also included in the definition of nucleic acids (see Jenkins et al., Chemical Science Review). Rev. (1995), pp. 169-176. Several nucleic acid analogs are described in Rawls' *Chemical & Engineering News*, June 2, 1997, p. 35. All these references are hereby explicitly incorporated herein by reference. These modifications to the ribophosphate backbone can be made to increase the stability and half-life of these molecules in physiological environments, for example, when the nucleic acid is siRNA, etc.

[0271] In some embodiments, nucleic acids are DNA or RNA encoding therapeutic biomolecules, including proteins and antibodies.

[0272] In some embodiments, the nucleic acid encodes an interleukin, which can be used to stimulate the patient's immune system and / or induce apoptosis or necrosis of cells by nucleic acid conversion. Suitable interleukins include (but are not limited to) IL-12.

[0273] In some embodiments, the nucleic acid encodes a chemotherapeutic antibody. Generally, in this embodiment, two nucleic acids are present, one encoding a heavy chain and the other encoding a light chain, which are electroporated into the tissue. In some cases, these may be in a single expression vector or two expression vectors may be used.

[0274] The term "antibody" is used generally. Antibodies suitable for use in this invention can take many forms as described herein, including conventional antibodies as described below, as well as antibody derivatives, fragments, and mimics. Conventional antibody structural units typically comprise tetramers. Each tetramer typically consists of two identical pairs of polypeptide chains, each pair having a "light chain" (typically having a molecular weight of about 25 kDa) and a "heavy chain" (typically having a molecular weight of about 50-70 kDa). Human light chains are classified as κ and λ light chains. This invention relates to IgG classes, which have several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4, the former being particularly suitable for many applications, especially oncology. Therefore, as used herein, "isotype" means any of the immunoglobulin subclasses defined by the chemical and antigenic characteristics of its constant region. It should be understood that therapeutic antibodies may also comprise hybrids of isotypes and / or subclasses.

[0275] The amino-terminal portion of each chain includes a variable region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition, commonly referred to in the art and herein as the “Fv domain” or “Fv region”. Within the variable region, each V domain of both the heavy and light chains aggregates three loops to form an antigen-binding site. Each loop is called a complementarity-determining region (hereinafter referred to as “CDR”), where the amino acid sequence variation is most significant. “Variable” refers to the fact that the sequence of certain segments of the variable region differs widely from that of the antibody. The variability within the variable region is not uniformly distributed. In contrast, the V region consists of a relatively invariant segment of 15–30 amino acids called a framework region (FR), which is separated by shorter, highly variable regions (referred to as “hypervariable regions”) each having a length of 9–15 amino acids or longer.

[0276] In some embodiments, the antibody is a full-length antibody. Hereinafter, “full-length antibody” means a structure constituting the native biological form of the antibody, containing variable and constant regions, and optionally including one or more amino acid modifications as known in the art. Alternatively, antibodies can be a variety of structures, including, but not limited to, antibody fragments, monoclonal antibodies, bispecific antibodies, microantibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimics”), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as “antibody conjugates”), and fragments of each. Specific antibody fragments include (but are not limited to) (i) Fab fragments consisting of VL, VH, CL, and CH1 domains; (ii) Fd fragments consisting of VH and CH1 domains; (iii) Fv fragments consisting of the VL and VH domains of a single antibody; (iv) dAb fragments consisting of a single variable composition (Ward et al., 1989 Nature 341:544-546, incorporated in full); (v) isolated CDR regions; (vi) F(ab')2 fragments, including bivalent fragments comprising two linked Fab fragments; (vii) single-chain Fv molecules (scFv) in which the VH and VL domains are linked by peptide linkers that allow the two domains to associate to form an antigen-binding site (Bird et al., 1988 Nature 242:423-426; Huston et al., 1988 Proceedings of the National Academy of Sciences 85:5879-5883, incorporated in full); (viii) bispecific single-chain Fv (WO 03 / 11161, hereafter incorporated by reference), and (ix) “bifunctional antibodies” or “trifunctional antibodies”, multivalent or multispecific fragments constructed through gene fusion (Tomlinson et al., 2000 Methods Enzymol. 326:461-479; WO94 / 13804; Holliger et al., 1993 Proceedings of the National Academy of Sciences 90:6444-6448, all hereafter incorporated by reference). Antibody fragments can be modified. For example, molecules can be stabilized by incorporating disulfide bridges linking the VH and VL domains (Reiter et al., 1996 Nature Biotech. 14:1239-1245, hereafter incorporated by reference).

[0277] As will be understood by those skilled in the art, nucleic acid™ can be incorporated into plasmids and / or expression vectors, containing additional components, including but not limited to expression promoters.

[0278] In some embodiments, the delivery system may include a rod delivery system configured to deliver TM to an electroporation site. The delivery system may include at least one injection probe defining a first lumen, and the injection probe may be cylindrical in shape and have a tip at its end. The tip may be hollow and have an open end for delivering TM to the electroporation site. In some embodiments, TM is injected into the middle of the outer periphery defined by the EPE, and electroporation is performed into cells at the electroporation site using any of the EP devices described herein.

[0279] It should be understood that EP of a tissue can be performed in vitro, in vivo, or in vitro. EP can also be performed using a single cell, such as a single-cell suspension, in vitro, or in a cell culture.

[0280] The EP rod housing, such as rod housing 12, is grasped and the EPE is inserted into the tissue to the desired depth. Subsequently, a suitable generator or power supply, as described herein, is connected to the EPE and an appropriate voltage is applied to each of the EPE pair. A suitable amount of a therapeutic component, such as a gene or molecule of a suitable chemical or pharmaceutical agent for tissue treatment, is then injected into the tissue using the aforementioned rod delivery system, followed by the application of EP pulses.

[0281] In some embodiments, the delivery system may include at least one injection probe defining a first lumen, and the injection probe may be cylindrical in shape and have a needle tip at its end. The needle tip may be hollow and have an open end for delivering a treatment portion to the electroporation site. In some embodiments, the treatment portion is injected into the middle of an outer periphery defined by electroporation electrode pairs A and B, and electroporated into cells in electroporation site 110 using the EP device of the present invention.

[0282] C. Electroporation method

[0283] Various embodiments of the present invention are for use with electroporation systems, such as system 10 of the present invention (in... Figure 9 (See description) A method for electroporating cells at the electroporation site in a tissue. Various embodiments of the invention are directed to the use of focused pulse addition (FPA) electroporation. "Focused pulse addition (FPA) electroporation" herein means applying short electric field pulses to an electric field region via at least first and second pairs of electroporation electrodes to create transient pores in the cell membrane without causing permanent damage to the cells. "Electroporated cells" include those cells having transiently open pores in the cell membrane that close upon dissipation of charge on the cell membrane ("open-pore cells"), and those cells that have undergone electroporation such that the cell now contains an exogenously added therapeutic portion and has closed pores (e.g., re-intact).

[0284] The term "electroporation electrode pair" as used herein refers to a pair of two electrodes configured to be charged in opposite directions when connected to a power supply. The method may involve contacting the electroporation rod housing 12 with tissue such that an electric field region 100 is defined by the area encompassed by the electroporation electrode pairs A and B, as illustrated in the figures. The first and second pairs of electroporation electrodes may be stationary or retractable within the electroporation rod housing 12. The electroporation rod housing 12 may further include a circuit board 16 having a plurality of sliding sockets through which the electroporation electrodes A and B are slidably retracted and extended. Electrode pairs A and B are mounted within the electroporation housing 12, which is slidably engaged with an indicator or gauge 11. As the electroporation rod housing 12 moves along the gauge 11, it alternately extends and retracts the electrode pairs A and B, as shown in the figures. Figure 2 As described herein, the device indicator or gauge 11 can provide an indication of the extension length of electrode pairs A and B. The electroporation system may further include an electrical connector 14 to electrically connect each of the first A and second B electrode pairs to a power supply 18, such as a pulse generator. The electrical connector, depending on the number of electroporation electrodes, includes four or more wires for transmitting electrical signals from the power supply to each of the electroporation electrodes. These signals may include needle voltage setpoints, pulse widths, pulse shapes, the number of pulses, and switching sequences. As those skilled in the art will appreciate, and described more fully below, the EP electrode can also act as a CS or EIS electrode, in which case a second low-voltage power supply is used with a suitable switching mechanism to allow delivery of a higher voltage EP signal, and then delivery of a lower voltage CS or EIS signal.

[0285] In some embodiments of the invention, one or more of the electroporation electrode pairs A and B may be non-penetrating electrodes, which may or may not have an open distal end for applying a therapeutic portion to the tissue, such as Figure 11 As described herein, the non-penetrating electrodes can be conductors of any suitable shape, such as buttons or plates for contacting surface tissue. The injectors can be spaced apart from each other and in close contact with the surface area of ​​the tissue. The portions of the non-penetrating electrodes that contact the tissue surface are conductive and electrically connected to a power supply 18 via an electrical connector 14, such that electroporation is achieved by delivering current through the area of ​​the tissue through a circuit completed between the conductive distal ends of the non-penetrating electroporating electrodes.

[0286] In some embodiments, such as Figure 12 As explained, more than two pairs of electrodes can be used and the offset angle can be adjusted accordingly. The larger the number of electrodes, the more electroporation sites the electric fields of all electrode pairs pass through become focused. In addition, a large number of electrode pairs allows for shorter pulses from each electrode pair, thereby substantially reducing or even eliminating cell death and ablation around the electrodes in the "burn-out zone".

[0287] The tissue surrounding the EP electrodes is sometimes referred to as the "burn-off zone." A "burn-off zone" means that the area is occupied by tissue that is adjacent to and / or in contact with each of the individual electrodes. It is called a "burn-off zone" because the cells are in direct contact with the electrodes, which are heated due to the high voltage signal from the power supply, and thus the cells are damaged by overheating. However, by using the alternating pulse system of the present invention, damage to cells in the burn-off zone can be minimized by reducing heat and field by 50% (in the case of two sets of electrodes; even more if more sets are used). Furthermore, because the electric field strength is focused / increased at the electrodes, the higher voltage causes EP-mediated cell death in a manner independent of heat.

[0288] In some embodiments, such as Figure 13 As explained, the first and second pairs of electrodes can be further connected to an EP power supply (EPP) capable of supplying electrical signals of various waveforms to each corresponding electroporation electrode pair. That is, the power supply can be a high-voltage power supply suitable for waveform generation. The first pair of electroporation electrodes A can be supplied with a waveform having a predetermined phase difference from the waveform supplied to the second pair of electroporation electrodes B. For example, the first and second pairs of electroporation electrodes can receive waveforms from a first waveform generator and a second waveform generator of the power supply, respectively, the waveforms having a 180-degree phase difference, such as... Figure 13 and Figure 14A and Figure 14B The rectangular electrode pair A waveform and electrode pair B waveform are illustrated in the diagram. As those skilled in the art will appreciate, and as described more fully below, a low-voltage power supply may optionally be used when capacitance sensing is complete.

[0289] The method may include contacting the electroporation rod housing 12 with the tissue such that the electric field region 100 is defined by the regions contained in A and B by the electroporation electrodes, such as... Figure 10 and Figure 14A and 14B As described in the document. In some embodiments, the electroporation method may further include applying a first signal generated from a power supply to a first pair of electroporation electrodes A with a first waveform and applying a second signal from a power supply to a second pair of electroporation electrodes B with a second waveform, wherein the first waveform has a predetermined phase difference with the second waveform.

[0290] During operation, the electroporation system 10 sends multiple independent electrical signals to selected electrode pairs A and B, which, upon contact with tissue, cause electroporation in the cell membrane. When the first and second electrode pairs A and B are in electrical contact with the tissue, a first electrical signal and a second electrical signal, having a first frequency, combine to produce a constant waveform with a frequency and amplitude sufficient to temporarily open the pores of the cells for therapeutic purposes, optionally introduced into the cells of the tissue without permanently damaging the cells and minimizing pain.

[0291] The nature of the tissue, the size of the selected tissue, and its location can determine the nature of the generated electrical signal. Ideally, the electric field should be as homogeneous as possible and have the correct amplitude. Excessive electric field strength can lead to cell death, while insufficient field strength can result in inefficient electroporation of cells, thus reducing the efficiency of reagent delivery into the cell.

[0292] The method may further include applying a pulsed electric field from a first pair of electroporation electrodes A to an electric field region 100, the pulsed electric field being based on a first signal, wherein the pulsed electric field of the first pair of electroporation electrodes A and each subsequent pulsed electric field have a voltage and duration lower than a minimum threshold for electroporation. Then, another pulsed electric field is applied to the electric field region 100 from a second pair of electroporation electrodes B, the other pulsed electric field being based on a second signal, wherein the other pulsed electric field of the second pair of electroporation electrodes B and each subsequent pulsed electric field have a voltage and duration lower than a minimum threshold for electroporation.

[0293] In some embodiments, the first and second pulsed electric fields are selected from the group consisting of: square wave pulses, exponential wave pulses, finite-duration monopolar vibration wave forms, and finite-duration bipolar vibration wave forms.

[0294] According to the method of the present invention, the paths of the pulsed electric fields of the first and second pairs of electroporation electrodes A and B intersect at the electroporation location 110, and the application of each pulsed electric field of the first pair of electroporation electrodes to the electroporation location alternates with the application of each pulsed electric field of the second pair of electroporation electrodes to the electroporation location, and together form a continuous pulsed electric field having a voltage and duration sufficient for applying electroporation to the cells at the electroporation location, such as... Figure 14A and 14B As explained in the text.

[0295] On the other hand, the application and resting period of each pulse electric field of the first pair of electroporation electrodes to tissue adjacent to the first pair of electroporation electrodes but outside the electroporation location alternates, such that the tissue adjacent to the first pair of electroporation electrodes but outside the electroporation location receives alternating on and off pulse electric fields from the first pair of electroporation electrodes, having a voltage and duration lower than the minimum threshold for electroporation. Similarly, the application and resting period of each pulse electric field of the second pair of electroporation electrodes to tissue adjacent to the second pair of electroporation electrodes but outside the electroporation location alternates, such that the tissue adjacent to the second pair of electroporation electrodes but outside the electroporation location receives alternating on and off pulse electric fields from the second pair of electroporation electrodes, having a voltage and duration lower than the minimum threshold for electroporation. Figure 14A and 14B As explained in the text.

[0296] Therefore, the advantage of the electroporation method of the present invention is that the duration of the electrical pulse experienced by healthy cells outside the electroporation location but within the electric field region 100 is only half that of those cells at the electroporation location and insufficient for electroporation, thus maintaining minimal damage to these cells to the point of no permanent damage. Furthermore, since cells outside the electroporation location 110 but within the electric field region 100 are only subjected to short pulses, this minimizes the degree of damage to cells in the "burn-out" region immediately adjacent to the electroporation electrode.

[0297] The nature of the tissue, the size of the selected tissue, and its location can determine the nature of the generated electrical signal. Ideally, the electric field should be as homogeneous as possible and have the correct amplitude. Excessive electric field strength can lead to cell death, while insufficient field strength can result in inefficient electroporation of cells, thus reducing the efficiency of reagent delivery into the cell.

[0298] In some embodiments, the pulsed electric field is selected from the group consisting of: square wave pulses, exponential wave pulses, finite-duration monopolar vibration wave forms, and finite-duration bipolar vibration wave forms.

[0299] According to various methods of the present invention, such as Figure 10 As described, the paths of the pulsed electric fields of the first and second pairs of EPEs A and B intersect at electroporation site 110, and the application of each pulsed electric field of the first pair of EPEs to electroporation site 110 alternates with the application of each pulsed electric field of the second pair of EPEs to electroporation site 110, resulting in a continuous pulsed electric field with sufficient voltage and duration for applying electroporation to the cells in electroporation site 110, as shown in the diagram. Figure 14A and 14B As explained in the text.

[0300] V. Preferred Device Embodiment

[0301] (i) Integrated EP device

[0302] Therefore, the present invention provides an apparatus and method for improved delivery of cells from a treatment portion to a patient's tissue. An integrated apparatus for improved delivery of cells from a treatment portion to a treatment area of ​​tissue is described. The apparatus includes at least a helical probe 1702 having an inner surface; in some embodiments, the helical probe may be a central probe, such as… Figure 17A As described herein, and in some embodiments, at least one additional probe 1702 may be included, such as Figure 18A and 18B As described in the document. Each of the central probe and the additional probes can define one or more central lumens 1704 (e.g., a first central lumen).

[0303] A first central lumen 1704 extends from the proximal end 1706 of the central probe 1702 to the distal end 1708. In some embodiments, the proximal end of the central probe may be formed of or coated with a non-conductive material to prevent or reduce the generation of an electric field at the proximal end. The proximal end 1706 of the central probe 1702 may define an opening to fluidly connect the central lumen to the lumen of an injector through which therapeutic agents can be delivered to the central probe 1702. In some embodiments, the distal end 1708 of the central probe also defines an opening for delivering a therapeutic portion into tissue. Alternatively, the distal end 1708 may be closed, as... Figures 22A to 22C As described in the description, one or more portions of the distal end of the central probe 1702 may have a shape configured to puncture tissue.

[0304] The central lumen 1704, or a portion thereof, includes a helical geometry configured to enhance anchorage of the central probe in the tissue and create a channel 1734 for delivering TM to the tissue via a jet port positioned on the central probe 1702. For example, a portion of the central probe 1702 may include one or more jet ports 1710 positioned along the geometry, such as… Figure 17B As explained in the text.

[0305] In some embodiments, the central probe 1702 may be at least partially housed within the applicator 1712. The applicator may include a distal end through which a portion of the central probe extends beyond the applicator 1712 to contact tissue and retract into the applicator 1712. For example, the EP device may include an actuator to advance the central probe 1702 toward and through the distal end of the applicator and through tissue 1714.

[0306] One or more diameters defined along the inner and / or outer surface of the central probe can be adjustable to change the distribution and volume of the delivered treatment portion. Similarly, the helical diameter and spacing of the central probe are adjustable to change the distribution and volume of the delivered treatment portion.

[0307] In some embodiments, the EP device may also include an electrical connector 1716 for electrically coupling or connecting the center probe 1702 to a power source. The electrical connector may be included or housed in a handle 1718.

[0308] In some embodiments, the EP device may also include an electroporation system comprising two or more opposing charged electroporation electrodes (EPEs) 1720. The two or more electrodes are configured and positioned such that they substantially surround the treatment area 1722 during treatment. The electrodes 1720 are adapted to extend from a proximal end to a distal end. One or more of the distal tips 1724 of the electrodes comprise a needle shape for puncturing tissue. The electrodes may be coupled to an electrode power supply (e.g., in…). Figure 16 The generator A described herein is configured to cause the electrodes to receive one or more electrical waveforms from the power supply for supplying electrical pulses 1730 to generate sufficient pulses for treating the treatment area 1722 as described above. Figure 14A The pulsed electric field of electroporation described in the paper.

[0309] Similar to a central probe, the electrode can be housed within the applicator. Electrode 1720 can be positioned around the central probe 1702 and configured to extend from the applicator 1712 to the treatment area 1722. For example, the electrode can advance from the applicator toward the treatment site and retract back into the applicator.

[0310] In some embodiments, the extension and retraction of the electrodes can be powered by a power supply interface included in the handle 1718. For example, the power supply interface can supply power to actuate the extension and retraction of the center probe 1702 and EPE 1720.

[0311] In other embodiments, such as Figure 19 As described, the central probe 1702 may include electrodes positioned on a helical geometry of the central probe 1702. In these embodiments, the electrodes may be integrally formed with the central probe 1702 or may be detachably mounted thereon. The electrodes on the central probe 1702 may be used in combination with electrode 1720 to generate the desired electric field configuration.

[0312] In other embodiments, such as Figure 20A and 20B As described, the center probe can be electrode probe 1750, which is connected to an electrode power supply, for example... Figure 16 Generator A is configured to generate an electric field between the center probe 1750 and the EPE 1720 to facilitate electroporation. In some embodiments, such as Figure 20A As explained, the central probe 1750 may include a helical blade for creating channels and for better anchoring of the central probe 1750 in the tissue. Figure 20B This is a schematic diagram of a center 1750 surrounded by multiple electrodes 1720, and Figure 20C yes Figure 20B The diagram is a bottom view.

[0313] The one or more central probes may include a second helical probe, similarly defined to the central probe 1720 described herein. In this case, the second probe supplies a second channel for delivery of the treatment portion to the tissue.

[0314] In some embodiments, such as Figure 22AAs described, one or more distal electrodes 1752 may be positioned at the distal end of the applicator 1712. These distal electrodes 1752 may be configured to generate an electric field with one or more portions of the central probe 1702. The one or more distal electrodes may be configured based on a loop configuration, a pen-line configuration, a spiral configuration, or a retractable loop configuration. As mentioned, the device may include a jet port located on one or more portions of the central probe. The distal electrodes may be configured to be positioned outside the tissue to generate an electric field experienced by the tissue externally. Alternatively, the distal electrodes 1752 may be configured to be positioned below the surface of the tissue. In some embodiments, such as Figure 21 As described, the distal electrode 1754 can be formed based on a helical configuration so that the helical electrode is positioned below the surface of the tissue. The helix of the central probe 1702 and the helix of the distal electrode 1754 can be wound in opposite directions, such as... Figure 21 As explained in the text.

[0315] In some embodiments, the electrodes described herein (including distal electrodes) are housed in the applicator 1712 around the central probe 1702 so that they can be deployed accordingly from the applicator 1712 to substantially surround the treatment area.

[0316] In other embodiments, one of the probes does not include a helical geometry. For example, one probe is a straight probe with open proximal and distal ends for delivery of the treatment portion to tissue. The vertical axis of the straight probe is coaxially aligned with the central axis of the diameter of the central probe. The straight probe can be configured to generate an electric field with a portion of the central probe. A central probe with a helical geometry can be configured to transmit acoustic energy received from an acoustic horn mounted to the distal end of the applicator.

[0317] a. Sensor system

[0318] In some embodiments, the present invention may include a sensor system. As those skilled in the art will understand, successful electroporation occurs when a cell membrane ruptures, resulting in a change in capacitance. When subjected to an electric field, a cell generally acts as a capacitor. When an electric field is applied for a sufficiently long period (depending on cell nature, health, size, etc.), charge accumulates at the cell membrane until it reaches a certain threshold and causes a breach in membrane integrity. The capacitance sensor system may be a low-voltage interrogation or excitation circuit, and may include a pair of capacitance sensing electrodes powered by a low-voltage power source, a voltage sensor, a current sensor, and electronic signal processing means to process the voltage and current to determine the average capacitance of the cell in the region.

[0319] In these embodiments, the sensor system is used to perform impedance measurements of the cell membrane of a tissue and includes a low-voltage power supply (e.g., in...). Figure 16The generator A described herein is powered by a pair of capacitive sensing electrodes (e.g., electrode 1720). The sensor system may further include a voltage sensor (integrated into electrodes 1720, 1752, and / or 1754) configured to sense a voltage or voltage drop across the cell membrane. Additionally, the sensor system may include a current sensor (integrated into electrode 1720) configured to sense a current across the cell membrane, and electronic signal processing devices, such as in… Figure 15 The controller 1505 described herein. An electronic signal processing device (e.g., controller 1505) processes voltage drops and currents across the cell membrane to determine the cell membrane impedance.

[0320] In some embodiments, the above-described method for sensing impedance (EIS) may include applying waveforms such as phase-locked loops, square wave pulses, high-frequency pulses, linear frequency modulated pulses, etc., as described in more detail below. When exposed to an electric field, the cell membrane acts as a capacitor. The capacitance can be measured based on charge redistribution within the cell in response to a low-frequency electric field excitation from a low-voltage interrogation circuit, and an impedance measurement can be derived from the capacitance. The capacitance can be measured before, during, and after the application of the electroporation electric field to determine cell conditions, including, but not limited to, cell health, the placement of the electrodes relative to the cell for optimal electroporation, and, most importantly, the time constant, which can be used to determine the pulse width of the electric field to be applied to the cell in the electric field region. Generally, charging the capacitor to its maximum value, i.e., exactly before electroporation occurs, takes five time constant periods; therefore, the pulse width of the initial electroporation electric field pulse can be set to five times the time constant. The pulse width is insufficient to cause electroporation in cells outside the electroporation site, as described above, but sufficient to cause electroporation in cells of tissue at the electroporation site that have undergone the additive effect of the electric fields from all the combined electroporation electrodes being applied as a continuous electric field. Capacitance measurements can be repeated after the first electroporation electric field has been applied, and the percentage decrease in capacitance can be calculated and compared to a predetermined value to determine whether the cells at the electroporation site have been sufficiently electroporated. If not, the pulse width can be adjusted for the next combined electroporation pulse electric field based on the calculated percentage decrease in capacitance until sufficient electroporation is determined to have occurred at the electroporation site.

[0321] An electronic signal processing device (e.g., controller 1505) can fit tissue impedance data to an equivalent circuit model, namely the CPE-based tissue model described above, in order to predict the optimal pulse parameters for the next step. As described above, impedance is the sum of resistive and capacitive elements over a frequency range, and therefore, in order to quantify each of these parameters, tissue impedance data can be fitted to a CPE-based tissue model. Thus, capacitance measurements performed between pulses by electrodes 1720, 1752, and 1754 with integrated sensors allow for adjustment of electrical conditions, such as pulse width, based on time constants associated with cell membrane capacitance, and the electroporation process can be stopped when an ideal decrease in capacitance or membrane integrity is achieved. It is assumed that real-time monitoring of the electrical properties of the tissue will enable feedback control of EP parameters and lead to optimal transfection in heterogeneous tumors. Using EIS feedback will allow (1) real-time adjustment of delivery parameters, (2) delivery of only pulses necessary to generate a therapeutic response, and (3) reduction of overall EP-mediated tissue damage.

[0322] b. Treatment component delivery method

[0323] Various embodiments of the present invention are directed to methods for delivering a therapeutic portion to cells in a treatment area of ​​a tissue using a delivery device integrated into an electrophysiological device (EP) with electrodes, such as a central probe 1702 having a jet port 1710, or a central probe 1750 having a jet port 1751. In some embodiments, the method for delivering a therapeutic portion to a treatment area of ​​a tissue may include providing an EP device with a central probe as a delivery device. In some embodiments, the delivery device includes a central probe 1702, 1750 having an inner surface that defines at least a first central lumen and extends from a proximal end to a distal end of the central probe 1702, 1750. In some embodiments, at least a portion of the delivery device 1702 has a helical geometry configured to enhance anchoring of the delivery device 1702 in the tissue and create a channel for delivery of the therapeutic portion to the tissue. A portion of the central probe delivery device 1702 may have a plurality of jet ports positioned along the helical geometry, wherein the proximal end of the central probe delivery device 1702 is open and fluidly connects the central lumen to which the therapeutic agent is delivered to the cells or tissue. The distal end of the central probe delivery device 1702 is open to define an opening / jet port 1710 for delivering a treatment portion into the tissue, and has a shape configured to puncture the tissue.

[0324] In other embodiments, Figures 20A to 20CThe central probe delivery device 1750 described herein has a straight tube shape and includes a blade 1753 with a helical geometry configured to enhance the anchoring of the delivery device 1750 in tissue and create a channel for delivery of the therapeutic portion to the tissue. At least a portion of the central probe delivery device 1750 may have at least one injection port 1751 positioned thereon to connect the central lumen to which the therapeutic agent is delivered to fluidly connect to cells or tissue.

[0325] The method further includes contacting a central probe with diseased cells in a treatment area of ​​the tissue, actuating and extending a central probe delivery device 1702, 1750 in an axial direction from an applicator, such that at least a portion of the central probe delivery device 1702, 1750 punctures the tissue and creates an opening, at least a portion of the central probe entering the tissue through the opening to create a fluid channel for delivery of the treatment portion to the tissue, injecting the treatment portion into a central lumen and delivering the treatment portion to the tissue through the at least one injection port 1751 and the open distal end of the central probe.

[0326] In some embodiments, the method further includes providing an electroporation system or apparatus comprising at least two oppositely charged electroporation electrodes, such as an electrode 1720 positioned to surround a region of tissue, wherein the electroporation electrode is adapted to extend from a proximal end to a distal end, the distal end having a tip with a needle shape configured to puncture the tissue, and the electroporation electrode is adapted to be coupled to a power source. The method further includes contacting a region of tissue with the electroporation electrodes, delivering an electrical pulse from the power source to the electrodes, and applying a pulsed electric field sufficient for electroporation from the electroporation electrodes to the region.

[0327] In some embodiments, a method for delivering a treatment portion to a treatment area of ​​tissue includes providing means for delivering the treatment portion to the treatment area of ​​tissue. The method further includes contacting a central probe (e.g., 1702) and a distal electrode 1752 to diseased cells in the treatment area of ​​the tissue; actuating and extending the central probe 1702 and the distal electrode 1752 in an axial direction from an applicator; piercing the tissue with at least a portion of the distal electrode 1752 and the central probe 1702 to create an opening; at least a portion of the central probe entering the tissue through the opening to create a fluid channel 1734 for delivery of the treatment portion to the tissue; injecting the treatment portion into a central lumen 1704 and delivering the treatment portion to the tissue through at least one of a jet port of the central probe and an open distal end; delivering electrical pulses from a power source to the distal electrode and the central probe; applying a pulsed electric field sufficient for electroporation to the area from the distal electrode and the central probe; and retracting the distal electrode 1752 and the central probe 1702 from the tissue.

[0328] In some embodiments, as described above, the method for delivering a treatment portion to a region may further include coupling an EPE to a power source, contacting a region of the tissue with the EPE, delivering electrical pulses from the power source to an electrode, and applying a pulsed electric field from the EPE sufficient for electroporation to the region of the tissue. Embodiments of the present invention add the advantage of opening the pores of cells to the delivery method of the present invention, thereby allowing cells to absorb a larger volume of treatment portion and producing better therapeutic results.

[0329] In some embodiments, the pulsed electric field is selected from the group consisting of: square wave pulses, exponential wave pulses, finite-duration monopolar vibration wave forms, and finite-duration bipolar vibration wave forms.

[0330] In some embodiments, as described above, the method for delivering a therapeutic portion to the region may further include providing a capacitance sensing system and method in conjunction with an electroporation system and method optimized for electroporation parameters, as described in more detail below. When exposed to a low-frequency, low-intensity electric field, cells generally exhibit an insulating structure surrounded by an ion cloud that compensates for the fixed charge present in the membrane. The electric field polarizes the ion cloud and generates an electric dipole, causing the cell to act as a capacitor. Healthy cells act as stronger capacitors than dead or diseased cells with damaged membrane structures, resulting in stronger capacitive coupling between the cell and the capacitance sensing electrode. Therefore, these properties can be used as an indicator of the cell's membrane integrity, which in turn leads to the determination of the degree of electroporation of the cell.

[0331] The method for delivering a therapeutic portion to a region of tissue may further include sensing the cell membrane capacitance of the tissue in order to optimize the electroporation process.

[0332] In some embodiments, the method of the present invention may include contacting tissue in a region of the tissue with the capacitance sensing electrode (e.g., 1720). A low-voltage power supply (e.g., generator A) electrically connected to the capacitance sensing electrode is used to apply a low-voltage interrogation signal to the capacitance sensing electrode. The method for sensing capacitance may include (but is not limited to) waveforms such as phase-locked loops, square wave pulses, high-frequency pulses, and linear frequency modulated pulses. Voltage and current sensors are used to sense voltage drops and current flowing through the circuit, and these parameters may then be processed by an electronic signal processing device to determine the average capacitance of all cells in the measurement region. As described above, the measured capacitance is an indicator of the health of the cells and is used to determine how long an electrical pulse should be applied to disrupt the cell membrane and provide conditions sufficient for electroporation.

[0333] Figure 23A , Figure 23B and Figure 23C , Figure 23D , Figure 23E and Figure 23FThis invention describes various EP devices with a central probe having the spiral geometry described above.

[0334] (ii) EP device based on cannula needle device

[0335] Therefore, the present invention provides an improved EP system for accessing cavities within the body that are not easily accessible. Figure 24A , Figure 24B and Figure 24C This invention describes a cannula-based direct rod applicator (EP) system. The cannula-based direct rod applicator (EP) system is designed to allow for the delivery of immunotherapeutic agents and genes to tumors inaccessible to electroporation devices. Examples of such cases include lung, liver, breast, or any tumor located more than 10 cm below the skin. The EP system offers the advantage of improved co-localization of DNA and electric fields for efficient gene delivery and reduced user-induced variations.

[0336] In some embodiments, a system for electroporation (EP) of cells in a body's tissue may include a cannula 2402 and an obturator 2404, and an EP device 2406 slidably mounted and retracted within the cannula 2402 to reach cells or tissue. In some embodiments, the cannula 2402 extends from a proximal end to an open distal end 2408 and defines a first lumen configured to receive the obturator 2404, the obturator extending from a proximal end 2410 to a distal end 2412. The proximal end of the obturator may include a handle mounted thereon, and the distal end of the obturator may include a blade or sharp tip 2414 configured to pierce through the skin, penetrate into a body cavity, and form a path through which the cannula 2402 can be inserted at least partially into the body cavity. In some embodiments, the obturator 2404 is configured to slide within the first lumen, and the distal end 2412 of the obturator is configured to extend beyond the first lumen through the open distal end of the cannula 2402.

[0337] In some embodiments, the EP device 2406 includes an anchor 2418 extending from a proximal end to a distal end 2420, at least two oppositely charged electrodes 2422, and a central probe 2424 retractably disposed at the distal end 2420 of the anchor (which may be configured in the same manner as a helical probe 1702 having an open distal end 1708). In some embodiments, the at least two oppositely charged electrodes 2422 are retractably disposed at the distal end 2420 of the anchor 2418 and configured to surround a region of a target cell for positioning, for example... Figure 17A Section 1722. In some embodiments, the measuring device is coupled to an electrode. The electrode is adapted to be coupled to a generator, for example... Figure 16A generator A receives at least one electrical waveform from the generator and supplies at least one of an excitation signal and an EP pulse to the tissue in the region. A central probe may have an inner surface defining at least a central lumen and extending from the distal end of an anchor. At least a portion of the central probe 2424 may have a helical geometry configured in a manner similar to that described in 17A to 22C of the invention to enhance the anchoring of the central probe in the tissue and create a channel for delivery of the treatment portion to the tissue. The distal end 2428 of the central probe 2424 may be open to define an opening for delivery of the treatment portion to the tissue and may have a shape configured to puncture the tissue. When the central probe is deployed, an anchor 2418 may be coupled to the proximal end of the central probe 2424.

[0338] In some embodiments, each spiral of the center probe 2424 can vary in diameter from 1 mm to 6 mm, typically from about 1 mm to 3 mm, more typically from 1.2 mm to 2.3 mm, and in some cases approximately 1.5 mm. In some embodiments, the electrodes can be spaced from 2 mm to 10 mm, more typically from 2 mm to 5 mm, and in some cases approximately 2 mm. In some embodiments, the length of the center probe and the length of the electrodes can vary from 5 mm to 15 mm, more typically from 7 mm to 10 mm, and in some cases approximately 8 mm. Although stated in terms of certain ranges, it will be understood that all ranges from the lowest lower limit to the highest upper limit are included within these complete ranges or any specifically stated ranges, including all intermediate ranges or particular measurements.

[0339] In some embodiments, the anchor is configured to engage with a 12ga biopsy needle to achieve a depth of 10 cm via the biopsy needle. In this manner, the EP device can be anchored to soft tumors for increased DNA dispersion. An advantage of the EP device of the present invention is that only 87 V needs to be applied across a 2.5 mm gap between the electrodes to achieve a field strength of 350 V / cm. This magnitude of electric field strength has been associated with a significant enhancement in TM delivery.

[0340] In some embodiments, the blade or sharp end 2414 of the obturator 2404 is configured to extend beyond the cannula 2402 through an opening at the distal end 2408 of the cannula 2402. The EP device electrode 2422 may be adapted to extend from the proximal end to the distal end, the distal tip of which may have a needle shape configured to pierce tissue, and the electrode 2422 may be adapted to couple to a generator, receive at least one electrical waveform from the generator, and supply at least one of an excitation signal and an EP pulse to the region of the target cell.

[0341] Various embodiments of the present invention are directed to providing a method for delivering a therapeutic portion to cells in a tissue and to EP of cells using the EP device of the foregoing embodiments. In some embodiments, the method includes (i) inserting a central probe into an anchoring device, (ii) deploying an electrode, (iii) partially withdrawing the central probe, and (iv) injecting a therapeutic portion into the lumen of the central probe for delivery of the therapeutic portion to the tissue via its distal end. In some embodiments, the method may further include (v) withdrawing the central probe; and (vi) applying an electrical pulse from a generator 1530 to the electrode for electroporation; and (vii) removing the device.

[0342] (iii) A device for improved therapeutic agent delivery

[0343] Therefore, the present invention provides an apparatus and method for improved delivery of therapeutic components to cells in a patient's tissue. Figures 25 to 33 This invention describes an EP device for improved therapeutic agent delivery according to the present invention.

[0344] like Figure 27 As depicted, the EP device includes a central probe 2710 having an inner surface 2712 defining a first central lumen 2715, with at least one access line 2720 extending through the first central lumen to the outside of the central probe 2710 and retractable into the first central lumen 2715. The central probe 2710 further includes an exit port 2730 that fluidly connects the first central lumen 2715 to the outside of the central probe 2710, and through which injected therapeutic portions flow from the first central lumen 2715 into channels within cells. The EP device also includes a ramp 2760 integrally formed with or coupled to the inner surface of the central probe to guide the access line 2720 outside the central probe 2710 to reach diseased tissue or cells.

[0345] In some embodiments, the central probe 2710 has a closed distal and proximal lumen. The distal tip of the probe 2710 is shaped to any form designed for puncturing tissue. Proximal to the distal tip, an exit port 2730 exposes the first central lumen 2715 to the outside of the central probe / needle 2710. A puncture line 2720, also having puncture features shaped in its distal tip, is sized such that it is slidable within the first central lumen 2715 and exits through the exit port 2730. The puncture line 2720 is adapted to advance into the tumor tissue and create a channel through the tissue that serves as a fluid path for a later-period injection portion of the treatment. The puncture line 2720 is guided outward by a ramp 2760 within the central probe 2710, as... Figures 25 to 33As described herein, the take-up line 2720 is adapted to retract into the central probe 2710 and the EP device can be rotated to a new orientation. The take-up line 2720 can be repeatedly advanced into the cell to create additional channels for delivery of the treatment portion. The channels created by the take-up line 2720 enhance the retention of the injected treatment portion in the tissue and allow for injection of a larger volume than is possible from a typical needle / syringe of similar size.

[0346] In some embodiments, the EP device may further include a handle that automates the extension, retraction, and rotation of the center probe / needle 2710 and the take-off line 2720 to facilitate sufficient depth of penetration.

[0347] In other embodiments, for example Figure 25 and 26 The catheter- or endoscope-based EP device will include a similar central probe / needle 2710 as described in the main embodiment.

[0348] In other embodiments, for example Figure 30 The EP device will include a similar center probe / needle 2710 as described in the main embodiment. This embodiment will have multiple exit ports 2730 through which multiple take-off lines can exit the device simultaneously.

[0349] In such Figure 29 In some other embodiments described herein, the access line 2720 includes a line having a cutting blade 2773 shaped in its distal end. The blade may extend into the tumor and then be rotated to create a disc-shaped incision in the tumor to form a channel through which a treatment portion is delivered to the cells.

[0350] In yet another embodiment, such as Figure 31 As described, the center probe has an open distal end similar to that of a typical syringe / needle. The take-up line 2720 can be formed of a shape memory alloy, such as a superelastic material (e.g., nitinol), to allow the curve to be thermally shaped into the line (sometimes referred to as a "shape memory"). When the take-up line is in the center probe 2710, the line is elastically straightened. Upon withdrawal from the center probe / needle, the take-up line 2720 is immediately allowed to return to its curved shape, as... Figure 31 As explained in the document, this creates a channel that extends outward from the device.

[0351] In yet another embodiment, such as Figure 30 As described, the EP device includes an injection probe 2745 with an injection needle at its distal end for injecting a therapeutic agent, and a central probe 2755 with a separate lumen for guiding the take-off line 2720. The inner surface 2712 of the central probe 2755 may contain a ramp 2760, for example... Figure 25 , 29As described in section 30, the guide line 2720 is used to guide the patient from the central probe 2755 outwards into the tumor. The two components that create the two lumens are joined side-by-side by a method suitable to their construction materials. For example, if the two lumens are made of metal, they can be spot-welded together, and if the two lumens are made of a material such as hard plastic, they can be ultrasonically welded together. Alternatively, a single component with multiple lumens formed integrally can achieve the same purpose. This configuration has advantages in treatments where the treatment portion requires a larger injection lumen for delivery.

[0352] refer to Figure 27 According to some embodiments of the present invention, an apparatus for improved delivery of a therapeutic portion to cells in tissue further comprises: an electrical connector 2770 that electrically connects a central probe 2710 and a take-up line 2720 to a power source 2780; a small aperture connector 2795 configured to connect the central probe 2710 to a syringe for delivery of the therapeutic portion; and a handle 2790 that receives the electrical connector 2770 and is coupled to the proximal end of the central probe 2710 and the take-up line 2720 to facilitate penetration depth of the distal end of the take-up line 2710 of the central probe.

[0353] like Figure 26 , Figure 27 , Figure 28 and Figure 29 As described, the central probe 2710 extends vertically outward from its proximal end to a closed distal end, and is configured in a needle shape at the distal end to provide initial penetration into the tumor / tissue. The inner surface 2712 of the central probe 2710 defines a first central lumen 2715 and is configured with a ramp 2760 that guides the take-off line 2720 outward from the EP device. The first central lumen 2715 provides the path along which the injected therapeutic portion flows before being delivered to diseased cells or tissue.

[0354] In some embodiments, an exit port 2730 is positioned at a predetermined distance distal to the side surface of the central probe 2710, through which the treatment portion is delivered to diseased cells or tissue. The exit port 2730 fluidly connects the central lumen to the outside of the central probe. The central probe 2710 may be formed of a low-conductivity material coated distally with an insulating (non-conductive) material to prevent interference with an electric field optionally applied using the EPE to facilitate cellular uptake of the treatment portion. The central probe 2710 can measure from about 1 mm to about 10 mm, depending on the geometry and physiology of the tissue to be treated and the depth to which the access line 2720 and EPE 2750 need to be inserted into the tissue.

[0355] In some embodiments, the access line is positioned within a central lumen and is slidable within a central probe. The access line may have a proximal end positioned within the central probe and a distal end with a needle-like puncture, configured to extend beyond the central probe via an exit port 2730 to reach and penetrate disease cells and create a fluid channel through which a treatment portion can be delivered to tissue. The distal end of the access line may be formed of a low-conductivity material coated with a conductive material or an insulating (non-conductive) material to avoid interference with an electric field optionally applied using the EPE. The access line 2720 can be measured from about 1 mm to about 20 mm, depending on the geometry and physiology of the tissue to be treated and how deep the access line 2720 and EPE 2750 need to be inserted into the tissue.

[0356] like Figure 29 As described, the ramp can be integrally formed or coupled to the inner surface 2712 of the central probe, and can be adapted to contact and guide the take-up line out of the central probe to the outside of the central probe. The ramp 2760 can be formed or coupled to the inner surface of the central probe at a predetermined angle, which may or may not be adjusted based on the extension angle necessary for the take-up line 2720 to reach the disease cells.

[0357] refer to Figure 27 To supply power to the EP delivery device, electrical connector 2770 electrically connects the center probe 2710 and the take-up line 2720 to a power source 2780. In some embodiments, the power source may be, for example, the present invention. Figure 16 The generator described herein is an example of such a generator. The power source may be a high-voltage power source to facilitate the application of high-voltage electrical pulses to an optional EPE for generating an electric field on the open pores of disease cells.

[0358] Handle 2790 at least partially accommodates electrical connector 2770 and is coupled to the proximal end of the center probe and take-up line to facilitate penetration depth at the distal end of the center probe and take-up line. Handle 190 can provide proximal termination points for various components (e.g., take-up line, first central lumen), connection points for center probe 2710 and small aperture connector fitting 2795. Handle also serves as the primary user interface to the device and may include one or more user input buttons electrically connected to the take-up line and / or optional electrodes for actuation or deployment of the take-up line and / or optional electrodes. Handle also accommodates electrical connector 2770 connected to power source 2780. Handle allows control of device orientation and direction, deployment and retraction of the take-up line, deployment and retraction of the center probe / needle, deployment and retraction of optional electrodes, and remote triggering of electroporation pulse delivery (optional). Additionally, as described above, handle is configured to facilitate penetration depth of the needle, take-up line, and electrodes.

[0359] In some embodiments, the handle 2790 is shaped for use by a physician, for example having a molded handle portion or grip, an optional illumination element at the distal end, a camera for observing and recording treatment sites, biopsy forceps, tissue scissors, a joining device, a suturing system, etc.

[0360] Additionally, the electrodes 2750 and handle 2790 are preferably made of sterilizable materials and configured to minimize microbial retention, similarly in cases where the electrode array and rod housing will be reused. In some embodiments, at least the electrode array is disposable, and in some embodiments, the entire handle is also disposable.

[0361] In some embodiments, such as Figure 25 and 26 As illustrated in Figure 8, the EP delivery device may further include a catheter shaft that surrounds the outer surface of the central probe to support and protect the central probe during insertion into a body containing tissue.

[0362] a. Electroporation electrode

[0363] As described above, the EPE 2750 electrically connects to the EP power supply 2780. The electrical connector 2770 may contain four or more conductive wires (depending on the number of EPEs) for transmitting electrical signals from the power supply to each of the EPEs. These signals may include pin voltage setpoints, pulse widths, pulse shapes, the number of pulses, and switching sequences. As those skilled in the art will appreciate, and described more fully below, the EP electrodes may also function as capacitance sensing (CS) or impedance sensing (EIS) electrodes, in which case a second low-voltage power supply is used with a suitable switching mechanism to allow delivery of a higher voltage EP signal, and then delivery of a lower voltage CS or EIS signal, such as... Figure 1 As explained in the text.

[0364] The EPE electrode 2750 is formed of a conductive material, but optional insulating coatings may be used as discussed herein. The electrode can be made of any conductive material capable of carrying the large instantaneous current density associated with the applied high-voltage pulse, including, but not limited to: certain metals and their oxides, including gold, platinum, palladium, silicon, and aluminum; metal oxide electrodes, including platinum oxide, titanium oxide, tin oxide, indium tin oxide, palladium oxide, silicon oxide, aluminum oxide, molybdenum oxide (Mo2O6), tungsten oxide (WO3), and ruthenium oxide; and carbon (including glassy carbon electrodes, graphite, and carbon paste). Preferred electrodes comprise AgCl, cobalt-chromium, titanium, stainless steel, platinum, gold, or highly conductive metals plated with gold or platinum.

[0365] Additionally, the electrodes, TM delivery device, and rod housing are preferably made of sterilizable materials and configured to minimize microbial retention, even if the electrode array and rod housing are to be reused. In some embodiments, at least and / or the TM delivery and electrode array are disposable, and in some embodiments, the entire rod housing is also disposable.

[0366] In some embodiments, such as when the distal end of the EPE is exposed to generate an electric field, its proximal end may be coated with a non-conductive material to limit the electric field to the distal end of the EPE only adjacent to the tissue and not along the length of the electrode, for example, allowing the EPE to be applied in a "deeper" but not "shallow" region of the tissue. In some embodiments, the EPE may have alternating areas of insulating material and bare electrodes, such as... Figure 4A and 4B The electric field is generally depicted in this document. In this embodiment, the electrodes can be coated with the same pattern, resulting in a more uniform electric field, or coated with different patterns, resulting in an asymmetric electric field. Similarly, for all electrode configurations herein, the electrodes can have the same length or different lengths.

[0367] The pulsed electric field generated by these partially insulated EPEs is mainly concentrated in the area between and near the exposed tip portions at the distal end of the electrode during treatment, and is small in the area between and near the insulated portions.

[0368] In some embodiments, the EPE has a generally long length to completely surround the tissue to be treated. In a preferred embodiment, all the assembled electrodes (an “array” of electrodes) have the same length within the array, but in some cases the use of electrodes of different lengths results in altered and asymmetrical electric fields.

[0369] In many embodiments, the length of the electrode varies from 1 mm to 20 mm. It should be noted that this measurement is the insertion depth and not the total length of the electrode 2750; generally, a portion of the electrode is present that extends upward from the point of contact with the tissue and into the handle 2790 for attachment to appropriate circuitry, holding the electrode in the correct spatial configuration, etc.

[0370] In many embodiments, the width and cross-sectional shape of the electrode for insertion are configured to minimize pain. Therefore, the electrode width can range from about 0.5 mm to 1 mm to 20 mm, with 1 mm to 15 mm being preferred.

[0371] b. Treatment component delivery method

[0372] Various embodiments of the present invention are methods for delivering therapeutic portions to cells in a region of target cells in a tissue using the delivery device 100 of the present invention.

[0373] In some embodiments, a method for delivering a treatment portion to a region of target cells includes providing means for delivering a treatment portion of any embodiment of the invention described herein to a region of target cells in a tissue. Figure 33 This is an illustration of a method according to the present invention for delivering a therapeutic portion to a region of target cells in a tissue using an EP device.

[0374] In some embodiments, a method for delivering a treatment portion into cells may include inserting a central probe / needle (e.g., 2710) into the cells. In some embodiments, for example... Figure 25 and 25 As explained, the EP device can be used for endoscopic purposes to reach hard-to-access cavities in the body. The guide wire 2720 extends from inside the central probe 2710 to the outside via an exit port 2730 on the side wall of the central probe 2710, thereby creating a fluid channel within cells or tissue. The guide wire 120 is then removed, the central probe 2710 is rotated, and the guide wire is extended again. Multiple probe extensions create a fluid channel within the tumor. With the guide wire removed, a therapeutic agent is injected, allowing it to flow into the fluid channel.

[0375] An embodiment of the invention provides a method for delivering a therapeutic portion to a region of target cells, further comprising inserting a central probe 2710 into a diseased cell within the region of the target cells, actuating a central lumen and extending a take-up line 2720 in an axial direction of the central probe 2710, and piercing the cell or tissue with a needle-shaped distal end of the take-up line. The method may further comprise forming an opening due to the piercing, through which at least a portion of the take-up line 2720 enters the tissue and creates a fluid channel for delivery of the therapeutic portion. The method may further comprise actuating a ramp 2760 integrally formed with or coupled to the inner surface of the central probe 2710, and contacting the take-up line with the ramp to guide the trajectory of the take-up line through an exit port toward the distal end of the central probe 2710. Upon exiting the central probe, the take-up line 2710 immediately extends to pierce the tissue and create an opening through which at least a portion of the take-up line enters the tissue to create a fluid channel for delivery of the therapeutic portion to the tissue. The take-up line may retract into the central lumen, and the therapeutic portion may subsequently be injected into the central probe using a syringe. Once injected into the central probe, the treatment portion travels out through the exit port and enters the channels created in the cells by the insertion of the take-up line.

[0376] In other embodiments, the take-up line may have a blade shape, and the method of creating the channel may further include rotating the take-up line while it is in the cell to create a hollow cylindrical channel with a large area for receiving a larger amount of treatment portion.

[0377] VI. The adaptive control method of the present invention

[0378] Various embodiments of the present invention are adaptive control methods for controlling EP pulse parameters during EP of cells in tissue using an EP device. Figure 36 This is a flowchart illustrating the control routine of the adaptive control method according to the present invention for controlling EP pulse parameters during the use of an EP system, and Figure 37 This describes the use of the present invention. Figure 36 The flowchart describes a leading-forward control routine that optimizes the EP pulse parameters. In some embodiments, the adaptive control method can be implemented using any EP device described herein. However, the method of the invention is not limited thereto, but can also be practiced on any of the EP systems and devices / appliers and any methods outlined in, for example, U.S. Provisional Patent Applications Nos. 62 / 214,807, 62 / 214,872, 62 / 141,142, 62 / 141,182, 62 / 141,256, and 62 / 141,164, all of which are expressly incorporated herein by reference in their entirety, including, and particularly, the accompanying drawings, illustrations, and descriptions of the drawings and components therein.

[0379] The apparatus, system, and method of the present invention will improve the process of EP-based gene therapy. Current EP systems employ open-loop control systems using static parameters that rely on prior knowledge determined through preclinical studies in homogeneous, allogeneic tumor models. However, preliminary data have shown that even in homogeneous tumors, the time required to apply an electrostatic field across the cell membrane follows a log-normal distribution. Even in homogeneous models, applying static parameters to different tumors results in a wide range of electrostatic field application across the cell membrane, leading to treatment variability. One potential remedy is to define static parameters for applying sufficiently long EP pulses covering 95% of the known film charging time. However, due to variations in charging time, the average tumor will be overtreated by 4-fold, increasing the likelihood of adverse effects such as necrosis and apoptosis. The present invention provides a solution to the aforementioned problems by implementing a closed-loop control system that uses tissue-sensing-based feedback control to optimize the EP process with tumor-specific measurements acquired before and between each EP pulse. In some embodiments, tissue sensing is used to measure the film charging time of a specific tumor to adjust each EP pulse for optimal treatment. Constraint boundaries are applied to the EP pulse parameters to ensure feedback convergence. The necessary conditions for implementing a closed-loop control system for enhancing EP are (1) the ability to apply electrical force to the tissue to drive it toward a desired state, and (2) the ability to measure the state of the tissue. This can be achieved by measuring the bioelectrical changes resulting from the applied electrical excitation signal.

[0380] The feedback adaptive control method of this invention employs a closed-loop feedback control mechanism to adjust the EP (Electronic Precipitation) by monitoring the physiological properties of the tumor before and between EP pulses. The physiological properties are determined in real-time by fitting EIS tissue data to the equivalent circuit model described herein using a nonlinear least-squares curve fitting routine. Fitting the data to the tissue model allows for the quantification of cell membrane integrity, represented by CPE, for the tissue to be treated. The duration of the EP pulse is modulated based on the CPE model fitting parameters, thereby allowing EP to be stopped when a relative change in the CPE parameters reaches a level associated with therapeutically beneficial pDNA expression. The control device, system, and method of this invention will allow the user to inject therapeutic molecules, characterize the baseline state of the tissue, deliver an optimized EP pulse for said tissue, and stop the pulse when a relative decrease in membrane integrity is achieved. This removes any ambiguity associated with EP and ensures successful delivery of immunotherapeutic agent genes regardless of changes in tumor properties. Therefore, EIS represents a significant advancement in the hardware currently used for clinical intratumoral immunotherapy.

[0381] Various aspects of the present invention address the need for advancements in the practice of EP by implementing a dynamic feedback control system as described above. In vivo EP for gene therapy has been clinically used for inoculation and oncology indication of many different tissue types and tumors. As described above, EIS is a low-power technique capable of real-time monitoring of tissues. This technique is performed by applying a series of low-voltage excitation signals across a pair of electrodes and measuring the response current over a frequency range. The magnitude and phase of each applied excitation are then calculated and fitted to an equivalent circuit model of the tissue. A common equivalent circuit for tissues is described above. In this model, resistive elements (R... I and R E These are due to the intracellular and extracellular matrix, respectively, and the lipid structure is composed of constant phase elements (CPE). M (This indicates that) CPE M It represents the charge or capacitance of the lipid bilayer (by Q). M The time constant for charging the lipid bilayer is calculated as a function of a scalar (denoted by α) ranging from 0 to 1, representing the non-ideal properties of the capacitor. This can be used to identify the optimal EP pulse duration before each treatment.

[0382] In some embodiments, the adaptive control method for controlling EP pulse parameters during electroporation (EP) includes providing any of the EP systems described herein. Various embodiments of the EP systems and apparatus of the present invention utilize the same electrodes to perform low-power EIS measurements and high-power EP pulses. The aforementioned configuration is ideal because it reduces the number of electrodes required and directly measures the tissue response. The adaptive control method further includes initializing the EP pulse parameters for performing EP in the tissue, and the initialized EP pulse parameters are at least partially based on, for example... Figure 38 At least one trained model as described herein. Figure 38 This is an illustration of the initial training phase of a model for estimating pulse parameters according to the present invention. As previously described, the model can be a physics-based model, an empirical model, or a data-driven model. In some embodiments, the trained model is trained using empirical data observed during the initial operation of the EP device using fixed EP pulse parameters. The model can be trained using supervised learning routines employing machine learning methods. In some embodiments, a specific implementation for the model prediction phase can be a decision support tree, which generates a set of logical rules for parameter estimation and diagnosis using the adaptive control method according to the present invention.

[0383] In some embodiments, the present invention relates to "single-step feedforward control." "One-step feedforward control" means that before applying the first EP pulse, a parameter estimation routine initializes initial control parameters for the first pulse based on a model trained in an initial training phase, using empirical data from previously conducted experiments. These previously conducted experiments may be based on tissue samples, for example, of tumors with similar characteristics to those of the current tissue to be subjected to the control methods of the present invention. The initialization may be an offline initial training phase. The parameter estimation routine (described more fully below) is first generated during the initial model training phase using, for example, empirical data collected from several experiments / trials. This can be done offline via the operating system without any feedforward or feedback control (fixed pulse parameters). The empirical data may include various fixed pulse settings, resulting features, and corresponding biological outcomes generated from these experiments / trials. Based on the previously trained model and measured features derived from tissue sensing measurements in the initial training phase and the tissue or tumor type identified in the diagnostic phase, the controller uses the parameter estimation routine to select the optimal parameters / conditions for the first EP pulse. These first pulse parameters are therefore “feeded forward” to be applied as the first pulse for the control routine, in contrast to conventional EP systems and methods where the parameters / conditions of the first pulse are based on fixed conditions. In this sense, the method of the present invention utilizes feedforward control to provide optimal EP parameters based on the sensed tissue type, combined with feedback control to sense cellular conditions, such as permeability, and adjust the pulse parameters accordingly.

[0384] Figure 41A and Figure 41B This is a flowchart illustrating the adaptive control method for EP pulse parameters according to the present invention. Figure 41A As described herein, the adaptive control method further includes applying voltage and current excitation signals from signal generator 1530 to the cell using the i-th electrode pair of EP device 1540, and measuring the voltage and current across the cell and tissue corresponding to the applied excitation signals to obtain the dielectric and conductive properties of the cell and tissue, including but not limited to capacitance, resistance, and impedance. Here, i=1 for measurements performed across the first set of electrodes. In some embodiments, current and voltage measurements can be performed using voltage sensors and current sensors, such as... Figure 15 The measurement device 1510 of the control system is described. Current and voltage sensors (which may be integrated into the electrodes of the EP device or contained separately elsewhere in the control system of the present invention) act as transducers, sensing the current and voltage across the cell membrane and detecting any changes in quantity, and providing an output signal to controller 1505 for the controller to perform a function corresponding to the signal received from the sensor, i.e., predicting the first pulse parameters. An excitation voltage signal is initially applied, and then applied between each set of EP pulses, for example between the first and second EP pulses, and the measurement is performed across the tissue. This signal may be a bandwidth-limited signal. The corresponding current signal is measured. This sensor data is time-dependent and internally stored for use during data preprocessing as described below.

[0385] The adaptive control method further includes obtaining sensor data from the measuring device 1510 corresponding to the measured cell or tissue properties, and processing the data into diagnostic and updated control parameters. In these embodiments, a preprocessing module 1550 of the controller 1505 preprocesses the data to separate desirable data from undesirable data. In some embodiments, the voltage and current sensors 1510 transmit signals to the controller 1505 for voltage and current measurements, and the controller derives impedance data from these measurements.

[0386] like Figure 41A As described, the preprocessing module 1550 of controller 1505 preprocesses and separates the measured data into desirable and undesirable data. Controller 1505 can run algorithms to process data obtained from various measurements and internally stored, allowing for curve plotting and various other statistical analyses to find the set of EP parameters that produce the best EP results. In some embodiments, undesirable data is stored in a memory module as an additional safeguard to mark subsequently collected data with similar properties as "undesirable" data.

[0387] In some embodiments, data preprocessing may include data mining. Loose control over data collection methods often leads to out-of-range values, impossible data combinations, missing values, etc., and thus analytical data that has not been carefully screened for these undesirable data can produce misleading results. Therefore, the data preprocessing implemented by the controller of this invention provides necessary assurance of the quality of data representation. To this end, sensor data is cleaned by removing outliers, out-of-range values, missing values, removing biases, scaling, cross-correlation, and applying denoising routines. In some embodiments, sensor verification routines are used to determine or evaluate data quality before the controller extracts features to estimate improved and more ideally optimized EP pulse parameters.

[0388] In some embodiments, the controller's preprocessing module may use any of the following to preprocess the data:

[0389] Noise Reduction Filter – A digital filter that removes noise from sensor signals. The filter can be implemented as an Infinite Impulse Response (IIR) or Finite Impulse Response (FIR) filter. It can also be implemented as an analog filter or part of an EP circuit. Debiasing – AC signals measured by sensors can be preprocessed by removing a DC bias from each of the signals. Scaling – Data can be standardized based on, for example, standard deviation. Median Filtering – Data can be filtered using nonlinear digital filtering techniques. Outliers – Data can be processed to remove extreme values ​​by identifying one or more values ​​outside the range of a specified number of standard deviations from the rest of the dataset. Sensor Verification – The controller can execute routines by running algorithms that analyze the quality of the measured data using statistical measures such as standard deviation, the number of outliers, skewness, and kurtosis, or any other known statistical measures used to analyze data quality. For example, if the standard deviation of the data exceeds a threshold, the data is labeled as “undesirable data” or an unusable dataset.

[0390] The adaptive control method of the present invention further includes extracting relevant features from desirable data by a feature extraction module 1570. "Feature" means a value derived from preprocessed data, which is intended to be informative and non-redundant based on various characteristics of the preprocessed data. The features of the system can be estimated and controlled, thus demonstrating that the system can be stabilized by the controller 1505.

[0391] In some embodiments, the feature extraction module is configured to execute certain software instructions to derive relevant features from preprocessed desirable data using computational routines. The computational routines can be used to obtain characteristics of the preprocessed desirable data, including, but not limited to, data descriptive statistics, data descriptive models, time-independent transformations, time series transformations, and domain-dependent feature extraction.

[0392] In some embodiments, descriptive statistics for sensor data may include, but are not limited to, mean, standard deviation, peak-to-peak, root mean square (RMS), variance, kurtosis, crest factor, correlation coefficient, autocorrelation, and cross-correlation. For events, descriptive statistics may include counts, occurrence rates, durations, and time delays. Descriptive models may include distribution models, such as parameter distributions, histograms, regression models (using model parameters or modeling errors): curve fitting, autoregressive (AR) models, classification / clustering models (using class labels as features), sequence matching probabilities, and pattern recognition classifiers (Fischer discriminant, Bayes' theorem). Time-independent transformations may include explicit mathematical operations, such as difference, summation, ratio, logarithm, exponentiation, principal component analysis, and independent component analysis. Time series transformations may include frequency domain, time-frequency domain, and wavelet domain. Domain-dependent feature extraction may include physics-based features, such as expected input-output or output-output relationships, derived hidden states, and special procedures for data processing such as operational mechanism segmentation, as well as envelope analysis.

[0393] In some embodiments, the features are derived from a parametric model fitting of the magnitude ratio or phase difference of the excitation voltage and current signals. These data include, but are not limited to, intracellular resistance, extracellular resistance, solution resistance, film capacitance, admittance, constant phase element exponent, and charging time constant. Feature extraction by the feature extraction module may include determining the capacitance or impedance of the cell membrane of the cell generated by the applied excitation signal. In these embodiments, impedance can be determined from measurements of the dielectric and conductive properties of the cell and tissue generated by the applied excitation signal by applying a band-limited signal repeated over a fixed frequency range. The dielectric and conductive properties of the cell or tissue are determined by the magnitude ratio and phase difference of the excitation voltage and current applied to the cell or tissue. Controller 1505 can calculate the magnitude and phase of each applied excitation and fit these to an equivalent circuit model of the tissue. In the model, resistive elements (R... I and R E These are due to the intracellular and extracellular matrix, respectively, and the lipid structure is composed of constant phase elements (CPE). M (This indicates that) CPE M It represents the charge or capacitance of the lipid bilayer (by Q). M The time constant for charging the lipid bilayer is calculated as a function of a scalar (denoted by α) ranging from 0 to 1, representing the non-ideal properties of the capacitor. It can be used by the pulse parameter estimation module to estimate the optimal EP pulse duration before each treatment.

[0394] The magnitude ratio and phase difference between the excitation voltage and current signals applied to cells and tissues are determined by cross-correlating the excitation voltage and current signals with a known reference signal stored in the memory module. Examples of features include (but are not limited to) the following: a) the values ​​of the magnitude ratio and phase difference of the excitation voltage and current signals at a fixed frequency; b) at least one of the following average, median, maximum, and minimum values: i) the magnitude ratio or phase difference of the excitation voltage and current signal magnitudes over a narrow frequency band; ii) the magnitude ratio or phase difference of the excitation voltage and current signal phases over a wide frequency band; c) the curvature, slope, and noise of the magnitude ratio or phase difference of the excitation voltage and current signals relative to frequency; d) a constant phase fundamental parameter; e) a high-frequency resistor (e.g., at 100 Hz), a low-frequency resistor (e.g., at 1 kHz); and f) a capacitor.

[0395] In some embodiments, the control method of the present invention includes applying at least a portion of relevant features of desirable data to at least one trained diagnostic model by a diagnostic module, such as... Figure 40 As explained in the text, prior histological diagnostics are important in predicting successful EP. For example... Figure 40 As described herein, the features are used as input to a series of diagnostic models for (i) tissue detection, (ii) tumor type detection, (iii) injection detection, and (iv) penetration detection. Based on the results of these models, the system will (i) terminate treatment due to penetration, (ii) proceed to estimate the parameters of the next pulse, or (iii) stop and warn the operator of a diagnostic event (e.g., no tissue detected, no tumor detected, no injection detected). One or more statistical inference routines (e.g., Bayesian inferencers) will be used to combine or fuse multiple features used for each diagnostic module. The system will include several diagnostic modules for making decisions on control inputs (applying pulses). In some embodiments, the control method includes fitting or applying the derived features to a trained diagnostic model by a controller, and fitting observed data where poor fitting or correlation is an indicator of a diagnostic problem, such as improper electrode placement, or electrode corrosion, for example, in necrotic or fibrotic tissue. In some embodiments, the criteria for tissue fitting to models such as CPE-based tissue models are R 2 >0.98. The diagnostic module 1580 of the controller 1505 generates a diagnostic response at least in part based on the results of the fitting or application, as described above, wherein the diagnostic response includes tissue detection, tumor type detection, needle placement detection, cell penetration detection, co-localization diagnosis, pulse test, and repetitive pulse diagnosis.

[0396] The diagnostic routines described in detail above play a crucial role in the control method of this invention. One particularly important area is co-localization detection. Alignment of the electric field with the injection is essential for the success of the EP procedure. Electrical measurements ensure that abnormalities do not interfere with treatment. Examples of problems causing poor co-localization include, but are not limited to, injection deeper than the effective electric field, deflection of the injection element, and biological abnormalities in the tissue or cells. Experiments and studies have shown that good co-localization is characterized by a decrease of at least 10% in solution resistance. This invention aims to achieve good or ideal co-localization, at least in part, by integrating the treatment delivery device with the EP electrode in a single EP device or applicator. The diagnostic routines of this invention, executed by the diagnostic module, ensure that EP is performed only after good co-localization is observed, tissue is detected, tumor is detected, and injection is detected. When the foregoing conditions are met, the controller then applies the relevant features to a CPE-based tissue model to estimate the initial pulse parameters.

[0397] In some embodiments, as described above, the adaptive control method further includes estimating a first pulse parameter by a pulse parameter estimation module based on the result of applying relevant features to a CPE-based tissue model after the execution of the aforementioned diagnostic routine and feature extraction. That is, if tissue is detected, if a tumor is detected, and if injection is detected, then the EP pulse parameter is initialized based on the at least one trained model and the measured features to estimate improved or ideally optimized first EP pulse parameters. In some embodiments, such as Figure 36 and 37 As explained, features combined with past features will be used to determine future impulse parameters. The estimator can include state-space estimators, artificial neural networks, autoregressive (AR) estimators, and autoregressive moving average (ARMA) estimators.

[0398] In some embodiments, the control method further includes applying a first EP pulse based on estimated improved / optimized first pulse parameters. Various embodiments of the pulse sequence are described below. Figure 41A and 41B The description states that i = pulse sequence (i = 0 for the excitation signal, i = 1 for the first applied EP pulse), and N = number of electrode pairs. The adaptive control method may further include predicting subsequent EP pulse parameters after the first EP pulse has been applied, based on changes in at least one of the characteristics described above, such as previous EP pulse parameters, voltage and current measurements of the cell's response to the first EP pulse, and the characteristics between applied EP pulses. Figure 41B As explained in the document, the tissue sensing routine described above is repeated between applied EP pulses until the optimized EP pulse parameters are achieved or until the pulse limit is reached.

[0399] In some embodiments, the control method may further include a) applying subsequent EP pulses based on predicted subsequent EP pulse parameters, and b) repeatedly applying voltage and current excitation signals to cells and tissues, repeatedly measuring cells or tissues, repeatedly acquiring data and separating desirable data from undesirable data; repeatedly extracting relevant features, and repeating the application until i) a predetermined limit on the number of EP pulse sequences or the cycle of EP pulses is reached, or ii) a diagnostic response prompts a diagnostic decision to terminate the adaptive control method, such as Figure 41A and 41B As described above. In some embodiments, the control method can terminate without applying further EP pulses when the time constant decreases by 50%. At this point, expression in all groups is statistically determined to be significantly different from the control. As described above, the duration of the EP pulse is modulated according to CPE-based model fitting parameters, so that EP is stopped when the relative change in the CPE parameters reaches a level associated with therapeutically beneficial pDNA expression. This technique will allow clinicians to infuse therapeutic molecules, characterize the baseline state of the tissue, deliver optimized EP pulses for said tissue, and stop the pulses when a relative decrease in membrane integrity is achieved. This removes any ambiguity associated with EP and ensures successful delivery of immunotherapeutic agent genes regardless of changes in tumor nature.

[0400] VII. Treatment portion for delivery

[0401] This invention provides an improved device and method for the therapeutic delivery of cells to a patient's tissue. Generally, the system of this invention is intended for treating diseased or abnormal tissue, such as cancerous tissue. The term "cancer" encompasses a wide range of diseases generally characterized by inappropriate, abnormal, or excessive cell proliferation. The device is intended for use in patients suffering from cancer or other non-cancerous (benign) growths. These growths can manifest themselves as any of the following: lesions, polyps, vesicles (e.g., papillary urethral vesicles), papillomas, malignancies, tumors (e.g., Kraskin tumors, hilar tumors, noninvasive papillary urethral lesions, germ cell tumors, Ewing's tumors, Askin's tumors, primitive neuroectodermal tumors, Ledich cell tumors, Wilms' tumors, Sertoli cell tumors), sarcomas, carcinomas (e.g., squamous cell carcinoma, cloacal carcinoma, adenocarcinoma, adenosquamous carcinoma, cholangiocarcinoma, hepatocellular carcinoma, traumatic papillary urethral lesion, squamous urethral lesion), masses, or any other type of cancerous or non-cancerous growth. Tumors treated by the devices and methods of embodiments of the present invention can be any of the following: noninvasive, traumatic, superficial, papillary, squamous, metastatic, localized, unicentric, multicentric, low-grade, and high-grade.Examples of cancers include, but are not limited to, breast cancer, colon cancer, prostate cancer, pancreatic cancer, skin cancer (including melanoma, basal cell carcinoma, and squamous cell carcinoma), lung cancer, ovarian cancer, kidney cancer, brain cancer, or sarcoma, adrenal cortex cancer, anal cancer, bile duct cancer (e.g., peritoneal cancer, distal bile duct cancer, intrahepatic bile duct cancer), bladder cancer, benign and cancerous bone cancers (e.g., osteoma, osteoid osteoma, osteoblastoma, osteochondroma, hemangioma, chondromycinoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of bone, chordoma, lymphoma, multiple lesions). Multiple myeloma), brain and central nervous system cancers (e.g., meningioma, astrocytoma, oligodendroglioma, ependymoma, glioma, neuroblastoma, ganglioglioma, schwannoma, germ cell tumor, craniopharyngioma), breast cancer (e.g., ductal carcinoma in situ, invasive ductal carcinoma, invasive lobular carcinoma, lobular carcinoma in situ, gynecomastia), Kassmann's disease (e.g., giant lymph node hyperplasia, angiofollicular lymph node hyperplasia), cervical cancer, colorectal cancer, endometrial cancer (e.g., endometrial adenocarcinoma, adenosine, papillary serous carcinoma). Adenocarcinoma, clear cell carcinoma), esophageal cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoids (e.g., choriocarcinoma, choriocarcinoma destruction), Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, renal cancer (e.g., renal cell carcinoma), laryngeal and hypopharyngeal cancers, liver cancer (e.g., hemangioma, hepatic adenoma, focal nodular hyperplasia, hepatocellular carcinoma), lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancers (e.g., nasal glioma, midline granuloma), nasopharyngeal carcinoma, neuroblastoma, oral cavity and oral cavity cancers. Pharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g., embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer (melanoma and non-melanoma skin cancer), gastric cancer, testicular cancer (e.g., seminoma, non-seminomatous germ cell cancer), thymic cancer, thyroid cancer (e.g., follicular carcinoma, degenerative carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma, thyroid lymphoma), vaginal cancer, vulvar cancer, and uterine cancer (e.g., uterine leiomyosarcoma). Therefore, cancerous tissues including skin tissue, connective tissue, adipose tissue, etc., can be treated using the system of the present invention. These cancers may be caused by chromosomal abnormalities, degenerative growth and developmental disorders, cell division promoters, ultraviolet radiation (UV), viral infection, inappropriate tissue expression of genes, alteration of gene expression, or carcinogenic agents.

[0402] The term "treatment" includes (but is not limited to) the inhibition or reduction of cancer cell proliferation, the destruction of cancer cells, the prevention of cancer cell proliferation or the prevention of malignant cell initiation, or the inhibition or reversal of the progression of precancerous cells to malignant disease, or the improvement of disease. The terms "subject" or "patient" refer to any animal, preferably a mammal such as a human. Use in livestock is also desirable to be included in this invention.

[0403] The systems and methods of this invention deliver a therapeutic portion to cells in tissue within an electroporation zone. As used herein, “therapeutic portion” or TM means a portion of diseased tissue suitable for electroporation, comprising a cytotoxic agent, chemotherapeutic agent, toxin, radioisotope, intercytokine, or other therapeutically active agent. The therapeutic portion may be a small molecule drug, nucleic acid (including those encoding a therapeutic target protein), or a biologically active protein (including polypeptides and peptides), as more fully summarized herein.

[0404] In some embodiments, TM is a drug; drugs contemplated for use in the methods of the present invention are typically chemotherapeutic agents with antitumor or cytotoxic effects. These drugs or agents include bleomycin, neomycin, suramin, doxorubicin, carboplatin, paclitaxel, mitomycin C, and cisplatin. Other chemotherapeutic agents will be known to those skilled in the art (see, for example, the Merck index). Electroporation facilitates the entry of bleomycin or other similar drugs into tumor cells by creating pores in the cell membrane. This local delivery provides significant benefits because the normal systemic toxicity typically associated with these drugs is minimized through local administration of the EP method described herein.

[0405] In some embodiments, TM is a biomolecule that includes nucleic acids and proteins.

[0406] In some embodiments, the TM is a nucleic acid. Generally, a TM that is a nucleic acid has two distinct functional types. In one embodiment, the nucleic acid encodes a protein used to treat a disease; in other embodiments, the nucleic acid is the TM, for example, when the nucleic acid is siRNA or snRNA. The terms "nucleic acid" or "oligonucleotide" or their grammatical equivalents herein mean at least two nucleosides covalently linked together. The nucleic acids of the present invention will generally contain phosphodiester bonds, but in some cases, as summarized below, include nucleic acid analogs that may have alternating backbones, including, for example, phosphoramides (Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein; Letsinger, Journal of Organic Chemistry 35:3800 (1970); Sprinzl et al., European Journal of Biochemistry 81:579 (1977); Letsinger et al., Nucleic Acids Res. 14:3487 (1986); Sawai et al., Chem. Lett. 805 (1984); Letsinger et al., Journal of the American Chemical Society 110:4470 (1988); and Pauwels et al., Chemical Technology Scripta) 26:141 91986), thiophosphates (Mag et al., Nucleic Acid Research 19:1437 (1991); and US Patent No. 5,644,048), dithiophosphates (Briu et al., JAMC 111:2321 (1989), O-methylaminophosphate bonds (see Eckstein, Oligonucleotides and Analogues: APractical Approach, Oxford University Press), and peptide nucleic acid backbones and bonds (see Egholm, JAMC 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature 365:566 (1993); Carlsson et al., Nature 380:207 (1996), all of the above are incorporated by reference.Other similar nucleic acids include those with a positive backbone (Denpcy et al., Proceedings of the National Academy of Sciences of the United States of America 92:6097 (1995); nonionic backbones (US Patent Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., Journal of the American Chemical Society 110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994); ASC Conference Series 580, Carbohydrate Modifications in Antisense Studies). Research), Chapters 2 and 3, edited by YS Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., Journal of Biomolecular NMR 34:17 (1994); Tetrahedron Letters 37:743 (1996), and non-ribose backbones, including those described in U.S. Patents 5,235,033 and 5,034,506 and Chapters 6 and 7 of ASC Conference Series 580, Carbohydrate Modifications in Antisense Studies (edited by YS Sanghui and P. Dan Cook). Nucleic acids containing one or more carbocyclic sugars are also included in the definition of nucleic acids (see Jenkins et al., Chemical Science Review). Rev. (1995), pp. 169-176. Several nucleic acid analogs are described in Rawls' *Chemical & Engineering News*, June 2, 1997, p. 35. All these references are hereby explicitly incorporated herein by reference. These modifications to the ribophosphate backbone can be made to increase the stability and half-life of these molecules in physiological environments, for example, when the nucleic acid is siRNA, etc.

[0407] In many embodiments, the nucleic acid of the present invention is contained within one or more expression vectors, the expression vectors containing additional nucleic acid sequences that bring functionality to the expression vectors, including, but not limited to, promoters, regulatory sequences, etc.

[0408] In some embodiments, nucleic acids are DNA or RNA encoding therapeutic protein portions, such as antibodies and interferons.

[0409] In some embodiments, nucleic acids encode immunostimulatory cytokines, as summarized herein. The phrase “immunostimulatory cytokines” includes cytokines that mediate or enhance immune responses to foreign antigens comprising viruses, bacteria, or tumor antigens. Innate immunostimulatory cytokines may include, for example, TNF-α, IL-1, IL-10, IL-12, IL-15, type I interferons (IFN-α and IFN-β), IFN-γ, and chemokines. Adaptive immunostimulatory cytokines include, for example, IL-2, IL-4, IL-5, TGF-β, IL-10, and IFN-γ. Examples of immunostimulatory cytokines are provided in Table 1 below.

[0410] Table 1: Accession numbers of immune-stimulating cytokines

[0411]

[0412] The specific immunostimulatory cytokine applicable to this invention is IL-12.

[0413] In some embodiments, the nucleic acid encodes a therapeutic antibody. Generally, in this embodiment, two nucleic acids are present, one encoding a heavy chain and the other encoding a light chain, which are electroporated into the tissue. In some cases, these may be in a single expression vector or two expression vectors may be used, as described more fully below.

[0414] The term "antibody" is used generally. Antibodies suitable for use in this invention can take many forms as described herein, including conventional antibodies as described below, as well as antibody derivatives, fragments, and mimics. Conventional antibody structural units typically comprise tetramers. Each tetramer typically consists of two identical pairs of polypeptide chains, each pair having a "light chain" (typically having a molecular weight of about 25 kDa) and a "heavy chain" (typically having a molecular weight of about 50-70 kDa). Human light chains are classified as κ and λ light chains. This invention relates to IgG classes, which have several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4, the former being particularly suitable for many applications, especially oncology. Therefore, as used herein, "isotype" means any of the immunoglobulin subclasses defined by the chemical and antigenic characteristics of its constant region. It should be understood that therapeutic antibodies may also comprise hybrids of isotypes and / or subclasses.

[0415] The amino-terminal portion of each chain includes a variable region of approximately 100 to 110 or more amino acids primarily responsible for antigen recognition, commonly referred to in the art and herein as the “Fv domain” or “Fv region”. Within the variable region, each V domain of both the heavy and light chains aggregates three loops to form an antigen-binding site. Each loop is called a complementarity-determining region (hereinafter referred to as “CDR”), where the amino acid sequence variation is most significant. “Variable” refers to the fact that the sequence of certain segments of the variable region differs widely from that of the antibody. The variability within the variable region is not uniformly distributed. In contrast, the V region consists of a relatively invariant segment of 15–30 amino acids called a framework region (FR), which is separated by shorter, highly variable regions (referred to as “hypervariable regions”) each having a length of 9–15 amino acids or longer.

[0416] In some embodiments, the antibody is a full-length antibody. Hereinafter, “full-length antibody” means a structure constituting the native biological form of the antibody, containing variable and constant regions, and optionally including one or more amino acid modifications as known in the art. Alternatively, antibodies can be a variety of structures, including, but not limited to, antibody fragments, monoclonal antibodies, bispecific antibodies, microantibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimics”), chimeric antibodies, humanized antibodies, antibody fusions (sometimes referred to as “antibody conjugates”), and fragments of each. Specific antibody fragments include (but are not limited to) (i) Fab fragments consisting of VL, VH, CL, and CH1 domains; (ii) Fd fragments consisting of VH and CH1 domains; (iii) Fv fragments consisting of the VL and VH domains of a single antibody; (iv) dAb fragments consisting of a single variable composition (Ward et al., 1989 Nature 341:544-546, incorporated in full); (v) isolated CDR regions; (vi) F(ab')2 fragments, including bivalent fragments comprising two linked Fab fragments; (vii) single-chain Fv molecules (scFv) in which the VH and VL domains are linked by peptide linkers that allow the two domains to associate to form an antigen-binding site (Bird et al., 1988 Nature 242:423-426; Huston et al., 1988 Proceedings of the National Academy of Sciences 85:5879-5883, incorporated in full); (viii) bispecific single-chain Fv (WO 03 / 11161, hereafter incorporated by reference), and (ix) “bifunctional antibodies” or “trifunctional antibodies”, multivalent or multispecific fragments constructed through gene fusion (Tomlinson et al., 2000 Methods Enzymol. 326:461-479; WO94 / 13804; Holliger et al., 1993 Proceedings of the National Academy of Sciences 90:6444-6448, all hereafter incorporated by reference). Antibody fragments can be modified. For example, molecules can be stabilized by incorporating disulfide bridges linking the VH and VL domains (Reiter et al., 1996 Nature Biotech. 14:1239-1245, hereafter incorporated by reference).

[0417] As those skilled in the art will understand, a wide variety of suitable therapeutic antibodies are available for use in this invention, depending on the type and location of the cancer. Suitable therapeutic antibodies include (but are not limited to) human, humanized, or chimeric antibodies for therapeutic use in humans, including currently approved antibodies that are identical to or similar to moromonas, abciximab, rituximab, dalizumab, baliximab, palizumab, infliximab, trastuzumab, gemtuzumab, alemtuzumab, isbemumab, adalimumab, omalizumab, tosimomab, efalizumab, cetuximab, bevacizumab, natezumab, nivorumab, pelizumab, and plus rituximab MPDL328OA (ROCHE), as well as antibodies in clinical development, specifically those for oncology applications.

[0418] Additionally, this invention provides an EP method and apparatus for delivering therapeutic antibodies to immune checkpoint inhibitors. As used herein, "immune checkpoint" molecules refer to a group of immune cell surface receptors / ligands that induce T cell dysfunction or apoptosis. These immunosuppressive targets attenuate excessive immune responses and ensure self-restraint. Tumor cells utilize the inhibitory effects of these checkpoint molecules. Immune checkpoint target molecules include (but are not limited to) the checkpoint targets described in Table 2.

[0419] Table 2: Checkpoint Target Login Number

[0420]

[0421] The phrase "immune checkpoint inhibitor" includes molecules that prevent immunosuppression by blocking the effects of immune checkpoint molecules. Checkpoint inhibitors may include antibodies and antibody fragments, nanobodies, bifunctional antibodies, scFvs, soluble binding partners of checkpoint molecules, small molecule therapies, peptide antagonists, etc. Inhibitors include (but are not limited to) the checkpoint inhibitors described in Table 2.

[0422] In some embodiments, the EP method and apparatus are used in combination therapies, such as for the delivery of two different TMs of higher efficacy. As those skilled in the art will appreciate, the combination can be any of the TMs summarized herein, including, but not limited to, a) nucleic acids encoding a therapeutic biomolecule (including an expression vector described more fully below) and a small molecule drug, such as plastids encoding IL-12 and a drug as summarized above; b) a first nucleic acid encoding a first therapeutic biomolecule and a second nucleic acid encoding a second therapeutic biomolecule (e.g., an expression vector encoding IL-12 and two nucleic acids encoding an anti-immune checkpoint inhibitor antibody as described herein); and c) a first nucleic acid encoding a first biomolecule and a second protein molecule, such as an anti-immune checkpoint inhibitor antibody; and d) two small molecule oncology drugs.

[0423] In some embodiments, the EP method and apparatus of the present invention are used in immuno-oncology combination therapy. In this embodiment, a combination therapy of immunostimulatory cytokine therapy (as described above) and checkpoint inhibitors is administered to the patient.

[0424] In one embodiment, immunostimulatory cytokines are administered in the form of plastids containing nucleic acids encoding immunostimulatory cytokines, and checkpoint inhibitors are administered to cells and tissues as proteins (e.g., antibodies against checkpoint inhibitors).

[0425] In another embodiment, the immunostimulatory cytokines are administered in the form of an expression vector plasmid containing nucleic acids encoding the immunostimulatory cytokines, and the checkpoint inhibitors are administered similarly to one or more expression vectors, the expression vectors comprising a first nucleic acid encoding a heavy chain of an anti-checkpoint inhibitor antibody and a second nucleic acid encoding a light chain of an anti-checkpoint inhibitor antibody.

[0426] In this embodiment, one, two, or three vectors may be used: if one is used, it contains coding sequences (and appropriate regulatory sequences) to express the heavy and light chains of immunostimulatory cytokines and anti-checkpoint inhibitor antibodies. Alternatively, three expression vectors may be used, each encoding one of the above. Two expression vectors may also be used, one containing a single component (e.g., immunostimulatory cytokines) and the other containing two components (e.g., the heavy and light chains of anti-immune checkpoint inhibitor antibodies).

[0427] Alternatively, small molecule drugs can be delivered using any of the above combinations.

[0428] In addition, administration of anti-checkpoint inhibitors (and / or small molecule drugs as summarized above) can be done systemically rather than as EP therapy to achieve the same efficacy.

[0429] Combined therapy can be administered either alone through electroporation or in combination with systemic delivery.

[0430] Other anticipated combination therapies include checkpoint inhibitors in combination with the following: TLR promoters (e.g., flagellin, CpG); IL-10 antagonists (e.g., anti-IL-10 or anti-IL-10R antibodies); TGF-β antagonists, CD3 promoters; telomerase antagonists, etc.

[0431] VIII. Examples

[0432] Example 1:

[0433] OncoSec builds on Figure 16The EP generator A described herein is capable of performing real-time feedback control based on EIS data before and between each EP pulse. This system is capable of outputting a minimum of 10V and a maximum of 300V with pulse durations ranging from 100 µs to 10 ms. The EIS data captured before and between pulses is obtained at 10 data points acquired every tenth of the pulse in the range of 100 Hz to 10 kHz. Acquisition of EIS data on this spectrum is achieved in 250 ms, which is fast enough to: (1) execute routines to determine the time constant for the next pulse; (2) store the EIS data for post-analysis; and (3) not interrupt clinically used EP conditions. The data collected from the EIS system is fitted to the aforementioned tissue impedance model in real time using an embedded advanced RISC machine (ARM) microprocessor (STM32F407, STMicroelectronics). When operating in feedback mode, the characteristics of this data can be used to control parameters associated with the EP process. The custom generator interfaces with a variety of standard EP applicators and will support up to six electrodes. Solid-state repeaters are used to switch between high-voltage EP pulse circuits and low-voltage EIS interrogation circuits. To allow hands-free operation of the generator, a foot pedal is added to trigger, pause, or abort the EP process. Figure 16 The image shown is of this generator and its accessories.

[0434] Unpublished in vivo studies investigated the effects of pulse width alteration based on time constant data obtained from EIS spectra. These studies concerned the execution of MC38 tumors implanted in the flank skin of 8-week-old albino B6 mice. The average tumor volume at treatment was 75 mm. 3 50 μg of pDNA encoding luciferase protein was injected into the tumor under the control of CMV promoter. Figure 16 The prototype applicator C shown, incorporating an EP device, is used to perform both injection and EP. This applicator contains two EP electrodes surrounding a central injection lumen; the injection lumen retracts during EP. Electroporation is performed at an electric field strength of 350 V / cm, and the pulse width is modulated in real-time from 0.1 to 20.0 times the time constant calculated from EIS data collected before pulse each MC38 tumor. A total of eight pulses are applied to each tumor, as this was previously associated with high transfection rates. Luminescence data are acquired at 48 hours by injecting 200 µl of a 15 mg / ml D-fluorescein solution prepared in DPBS and performing in vivo optical imaging. Figures 42A to 42D Summary data from this experiment is shown in the figure.

[0435] Figure 42A This illustrates the distribution of the time constant with respect to the applied electric field across the lipid bilayer. Figure 42B This describes the distribution of the time constants measured before EP. Figure 42CThis demonstrates the effect of modulating the pulse width based on pre-pulse EIS data, where the pulse duration is set as a multiple of the time constant used for each tumor. Figure 42D The illustration shows the data on the relative change of the calculated time constant with respect to the resulting luminescence after EP. Data that are statistically significant at α = 0.05 are indicated by asterisks.

[0436] These data show that pDNA expression depends on the applied pulse width. The original hypothesis was that as the applied pulse width increases relative to the measured time constant, the percentage of the electric field applied across the lipid bilayer will vary. Figure 42A This is especially important because the time constant calculated from the EIS data prior to each EP treatment follows... Figure 42B The log-normal distribution shown is illustrated. Figure 42C The experimental results showed that as the applied pulse width increased relative to the measured time constant, the luminescence also increased. This phenomenon supports the hypothesis that the capacitor approaches charge saturation at 5 times the time constant, and the measured expression reaches its upper limit. Data sets obtained above two time constants experienced significantly (p<0.05) higher luminescence compared to injection alone. As the pulse width becomes longer and more energy is dissipated through the tissue, expression begins to attenuate due to irreversible tissue damage.

[0437] Furthermore, this experiment demonstrates a potential criterion for stopping the EP process before reaching a pre-determined number of pulses. As cell membranes begin to permeate, their ability to retain charge decreases, which in turn leads to a decrease in the time constant associated with charging CPE. A high correlation was observed between the change in time constant and the measured luminescence, supporting this theory. Tumors with a time constant decrease greater than 20% were associated with significantly (p<0.05) higher expression of pDNA. This measurement can be used to stop the pulsed process when conditions for successful gene therapy are present. Of concern, the group with shorter pulse durations resulted in an increase in the time constant due to compression of the lipid bilayer, which causes increased capacitance. For this study, we propose using a technology prototype generator to explore variables for controlling EP and to validate this technique in both homogeneous and heterogeneous tumors. We hypothesize that EP-based gene delivery can be optimized for each tumor by measuring tissue properties and adjusting the width of each applied pulse. Inquiry into real-time changes in membrane capacitance will result in (1) regenerative transfection efficiency; (2) increased duration of gene expression; (3) enhanced efficacy; and (4) reduced tissue damage.

[0438] Example 2:

[0439] Experiments were performed to determine whether electrochemical impedance spectroscopy (EIS) could distinguish data obtained from fibrotic or necrotic tissue from data obtained from healthy tissue. In these experiments, livers from 9-month-old transgenic mice expressing human platelet-derived growth factor C (PDGF-C) were used to represent fibrotic tissue. PDGF-C transgenic mice at this age are known to have enlarged livers with significant fibrosis, fatty infiltration, and cellular dysplasia. Data obtained from the livers of transgenic animals were compared to livers from healthy or wild-type 3-month-old C57BL / 6J mice. To obtain this data, mice were anesthetized to allow surgical access to the liver. Parallel electrodes spaced 3 mm apart were inserted 5 mm into the left lateral lobe of the liver. Data collected from these experiments were fitted to a constant-phase element model used to electrically represent biological tissue. Parameters derived from the model fitting revealed that liver fibrosis leads to an increase in admittance, an increase in the calculated time constant, and a decrease in the constant-phase element. These data are shown in Figure 43.

[0440] Example 3:

[0441] A second experiment was performed to determine whether EIS could detect the presence of the injected solution in tumor tissue. In this experiment, tumors were implanted subcutaneously into the flank skin of 8-week-old albino B6 mice by injecting 50 μl of phosphate-buffered saline with 106 MC38 cells. After approximately 10 days, the tumors reached an average volume of 100 mm³. At this point, a two-electrode applicator with a central injection lumen was inserted 7 mm into the tumor. Baseline EIS measurements of the initial tumor conditions were then performed. Following this measurement, a 50 μl volume of a 1 mg / ml solution of plasmid DNA prepared in physiological saline was injected into the tumor. A second EIS measurement was performed after injection. These data were again fitted to a constant phase element model used to represent biological tissue. A decrease in solution resistance of at least 10% was observed after injection of the plasmid DNA solution into the tumor. Figure 44 Provides a histogram overview of the percentage reduction in solution resistance observed from model fitting parameters after plasmonic DNA injection.

[0442] Example 4:

[0443] In addition to detecting tissue viability and the presence of injection, EIS can also inform the user of the optimal pulse width for performing electroporation. To demonstrate this, a study was conducted in which the pulse width was varied based on time constant data obtained from model fitting of the EIS spectrum. This study was performed using MC38 tumors implanted in the flank skin of 8-week-old albino B6 mice. The average tumor volume at treatment was 75 mm. 350 μg of pDNA encoding luciferase protein was injected into the tumor under the control of a CMV promoter. A two-electrode applicator with a central injection lumen was used to perform injection and electroporation (EP). During EP, the injection lumen was retracted from the tumor. Electroporation was performed at a field strength of 500 V / cm, with the pulse width modulated near the average time constant obtained a priori from 10 tumors. This average calculated time constant was 0.50 ms, and the pulse widths selected for this experiment were 0.1, 0.5, 2.0, and 10.0 times the average time constant. A total of 8 pulses were applied to each tumor. Luminescence data were acquired 48 hours after injection of 200 µl of a 15 mg / ml D-luciferin solution prepared in D-PBS. This data was collected using in vivo optical imaging. Data from this experiment show the maximum increase in luminescence in tumors treated with pulse widths of 10 times the average time constant or a total of 5 ms. Additionally, these data show a significant increase in luminescence in the group treated with two or more times the time constant compared to injection alone. Figure 45 Summary data from this experiment is shown in the figure.

[0444] Example 5:

[0445] Following the experiments conducted in Example 4, a study was performed to determine whether EIS could be used in real-time to enhance the optimal pulse width for each individual tumor. This would allow for adjustment of each electroporation sequence based on the initial conditions of each individual tumor. MC38 tumors were again implanted into the flank skin of 8-week-old albino B6 mice. When the tumor reached 75 mm³, 50 μg of pDNA encoding the luciferase protein was injected into the tumor. The same two-electrode applicator with a central injection lumen was used to perform the injection and EP. For this experiment, the field strength was reduced to 350 V / cm, and the pulse width was modulated in real-time using a time constant calculated for each tumor being treated. The pulse width was modulated from 0.1 to 20.0 times the calculated time constant. A total of 8 pulses were applied to each tumor. Luminescence data were acquired via in vivo optical imaging at 48 hours by injecting 200 µl of a 15 mg / ml D-luciferin solution. Data from this experiment showed a significant increase in luminescence for all tumors treated with time constants of 2.0 times and above. No statistical differences were observed between groups at 5.0, 10.0, and 20.0 times the calculated time constant. Figure 46 The data from this experiment is shown in the figure.

[0446] Post-processing of data acquired during this experiment suggests a potential criterion for stopping the EP process before reaching a pre-determined number of pulses. As cell membrane permeation begins, its ability to retain charge decreases, which in turn causes a decrease in the time constant associated with charging CPE. A high correlation was observed between the change in time constant and measured luminescence, supporting this theory. Tumors exhibiting a decrease in time constant greater than 20% were associated with significantly higher pDNA expression. This measurement can be used to stop the pulse process when conditions for successful gene therapy are present. Of concern, the group with shorter pulse durations caused an increase in the time constant due to compression of the lipid bilayer, which leads to increased capacitance. Figure 47 These data are shown in the figure.

[0447] Example 6: (Expected Experiment)

[0448] The objective (Objective 1) is to evaluate feedback parameters leading to the desired outcomes of intratumoral immunotherapy. Based on preliminary studies, EP with integrated EIS feedback control has the potential to reduce the rate of change between treatments. To evaluate the histological effects of EP on pDNA expression and changes in calculated time constants, in vivo tumor studies will be performed in a homogeneous contralateral mouse melanoma model. Briefly, B16 / OVA cells (1 x 10⁶ / site) will be subcutaneously implanted in the flank of B6 mice (n = 10 / group). When the tumor reaches a volume of 75 mm³, they will be injected with dual reporter plasmids (1 mg / ml, 50 μl per tumor) expressing both luciferase and mCherry. This will allow for non-invasive, longitudinal bioluminescence imaging and spatially specific cell-specific gene expression. Figure 16 The dual-electrode applicator C in the experiment pulses the tumor with FCEP. The electrode will operate at 350 V / cm, with the pulse width set for each individual pulse to five calculated time constants. Cells will continue to receive EP pulses until a relative decrease of 20%, 40%, 60%, or 80% of the time constant is reached. Operational limits for the generator will be set to ensure safety, with the maximum permissible pulse width fixed at 10 ms and the maximum pulse size set to 10. Control animals for this experiment will include no treatment, pDNA injection only, and pDNA injection followed by uncontrolled EP with 10 pulses over a 10 ms duration using the same electrode at an electric field strength of 350 V / cm.

[0449] Bioluminescence will be quantified at 24 hours via injection of D-fluorescein (intraperitoneal, 200 μl at 15 mg / ml). The luminescence of these tumors will be captured at 24, 48, and 72 hours using an in vivo imaging system (Lago, a spectroscopic instrument). Tumor tissue will be collected, longitudinally bisected, with half frozen in an optimal cutting temperature compound (OTC) and the other half fixed in formalin for routine histological analysis. Three independent experiments will be performed, with each experimental group consisting of twelve biological replicas. Data will be analyzed using one-way ANOVA (i.e., Kruskal-Wallis, GraphPad Prizm).

[0450] Routine histological and immunohistochemical (IHC) analyses will be performed on tumor segments to assess necrosis and specific forms of cell death, such as apoptosis spatially associated with mCherry expression. TdT-mediated dUTP cleavage terminator (TUNEL) and active cassinoprotein 3 IHC will be performed, and segments will be evaluated to score the degree of apoptosis. Semi-quantitative analyses will be performed using the ImageJ script as described. H&E-stained slides will be used to assess the extent of inflammatory infiltration and necrosis.

[0451] Expected Outcomes – FCEP is expected to reduce the rate of change in expression among treated tumors. Larger amounts of pDNA transfection are anticipated to be associated with a higher relative decrease in the calculated time constant. Furthermore, increased apoptosis and inflammation are expected as the relative decrease in the time constant increases.

[0452] Example 7: (Expected Experiment)

[0453] The objective (Objective 2) is to test the feedback control system in vivo by conducting intratumoral immunotherapy experiments aimed at tumor regression.

[0454] Following in vivo characterization, a series of experiments will be performed using the FCEP system to examine tumor regression and the durability of expression. To compare with published studies and control for rate of change, a homogeneous melanoma model with contralateral tumors will be used. B16 / OVA melanoma cells (1 x 10⁶ / injection site) will be subcutaneously implanted in the flank of albino B6 mice (n=10 / group). When the tumor is 75 mm³, one tumor per mouse will be injected with a polycistronic plasmid encoding interleukin-12 (IL-12), luciferase, and mCherry (in 50 μl at 1 mg / ml). Expression of this plasmid allows for immunotherapy and long-term bioluminescent quantification. The tumor will be pulsed using the FCEP system at 350 V / cm, with the pulse width set to five time constants for each individual pulse. The EP stopping criterion for the first group will be selected based on the feedback control group in Target 1, where maximum pDNA expression is independent of observed histological features. The second feedback group will be selected from Target 1 by choosing the group exhibiting significant expression levels with minimal tissue damage. The control animals for this experiment will include no treatment, pDNA injection only, and pDNA injection followed by conditions optimized for this tumor model. To apply these conditions, a MedPulser with a 6-electrode applicator will be used to apply 6 rotating pulses, each with a duration of 100 μs and an electric field strength of 1,500 V / cm.

[0455] Data from these experiments will be collected in two distinct ways. Tumor growth rates of both the treated and contralateral tumors will be collected every 48 hours post-treatment using a two-dimensional caliper. This will be achieved by administering D-fluorescein (intraperitoneally, in 200 μl at 15 mg / mL) starting 48 hours post-treatment and subsequently every 4 days. Tumor volume and luminescence data will continue to be monitored for up to 30 days or until the tumor burden exceeds 1,000 mm³, at which point the animals will be euthanized according to the established IACUC protocol. Three independent experiments will be performed, each containing 12 animals. Data will be analyzed using one-way ANOVA (Kruskal-Wallis).

[0456] In addition to monitoring tumor growth rate, the response to the tumor-specific neoantigen CD8 will be determined by harvesting spleens at the end of the study. The spleens will be mechanically dissected and erythrocytes will be lysed by suspension in ACK buffer. The isolated spleen cells will be purified using a cell separation medium (lympholysin-M, Cedarline) prior to staining. The purified cells will then be mixed with a tetramer solution (e.g., SIINFEKL, TS-5001-2C, MBL). CD8 positive T cells will then be identified by flow cytometry analysis (LSR II, BD).

[0457] Expected Outcomes – The FCEP device is expected to produce greater levels of IL-12 and IFN-γ compared to the published EP method. The longer duration of plasmid expression and the longer survival of tumor-bearing animals treated with FCEP are attributed to the assurance of treatment success. Enhanced survival will serve as an additional metric for evaluating this system, and should be higher than the conventional EP treatment group with an expected long-term survival of approximately 47% based on similar studies, while the control group may not respond to treatment.

[0458] Potential issues and alternatives – A potential issue is that CD8 positive T cells may be difficult to assess 30 days post-treatment. In this case, a separate group of tumor-bearing animals will be treated with the conditions described in this objective. Tumors will be excised from euthanized animals 14 days post-treatment.

[0459] Example 8: (Expected Experiment)

[0460] Objective (Objective 3) is to demonstrate the feedback control system by performing intratumoral EP in a heterogeneous spontaneous breast cancer model.

[0461] Following optimization and validation in homogeneous tumor models, a set of experiments will be performed to confirm FCEP using heterogeneous models. These experiments will use transgenic mouse models expressing the intermediate T antigen of a polyomavirus in the direction of mouse mammary tumor virus promoter (MMTV-PyVT), which develop spontaneously palpable mammary tumors at ages 8 to 10 weeks. Plastids expressing IL-12, luciferase, and mCherry (in 50 μl at 1 mg / mL) will be delivered to the mammary tumors of MMTV-PyVT mice at 10 weeks of age. Tumors will be treated with pulses of 350 V / cm using the stopping criteria from Target 2, resulting in the longest mean survival. The control group will consist of no treatment, pDNA injection only, and pDNA injection followed by six pulses of current clinical parameters applied at 1,500 V / cm for 100 μs. A total of 10 tumors will be treated with each treatment condition, with two of these tumors treated in each mouse. The experiments will be run a total of three times.

[0462] Utilizing each of the proteins encoded by the plasmids will allow for the generation of multiple data streams. Efficacy will be quantified by in vivo imaging every 72 hours for up to 21 days following D-luciferin injection (intraperitoneal, 200 μl at 15 mg / ml). A population of five animals will be euthanized and tumors will be collected at days 7, 14, and 21. The collected tumors will be split equally to directly assess IL-12 expression and determine the percentage of transfected cells. A portion of these excised tumors will be aggregated and homogenized. IL-12 expression will be directly quantified from these tumors sampled by ELISA (Andy Biotech). The other half of the tumors will be isolated (tumor isolation kit, Miltenyl Biotec) and run using a flow cytometer (LSR II, BD) using optical elements specifically designed for mCherry proteins. This will allow for the determination of the percentage of transfected cells. One-way ANOVA will be used to analyze the data.

[0463] Expected Outcomes – FCEP is expected to produce more regenerative transfections of these heterogeneous tumors than current clinical EP protocols. This will be directly measured by chemiluminescence data and IL-12 expression. Additionally, this novel approach is expected to correlate with the highest percentage of transfections.

[0464] Potential problems and alternatives – A potential problem that may arise during this study is that IL-12 expression may be difficult to assess in tumors. In this case, an ELISA will be performed to directly measure luciferase levels. Alternatively, the downstream cytokine interferon-γ will be directly assessed as an alternative to IL-12 expression.

[0465] Timeline. The completion of Phase I efforts will be executed within a 12-month period. Goal 1 is expected to last a total of 3 months. Goal 2 will be completed within 5 months. Finally, Goal 3 will be completed within 4 months. This timeline is summarized in Table 1.

[0466] Table 1. Timeline of Targets (in months)

[0467]

Claims

1. An electroporation (EP) system, comprising: At least two electroporation electrodes (EPEs) are configured to be located in or adjacent to the tissue; A signal generator electrically connected to the at least two EPEs and generating at least one electrical signal based on at least one control parameter, wherein the at least one electrical signal is applied to the tissue via the at least two EPEs, and wherein the at least one electrical signal includes an excitation signal and an electroporation pulse; A measuring device, electrically connected to the at least two EPEs and generating measured sensor data, wherein the measuring device is configured to measure the dielectric and conductive properties of the tissue, wherein the measuring device includes a voltage sensor and a current sensor, wherein the voltage sensor is configured to measure the voltage across the tissue when the excitation signal and / or the electroporation pulse is applied to the tissue, and the current sensor is configured to measure the current across the tissue when the excitation signal and / or the electroporation pulse is applied to the tissue, and the measured sensor data includes the voltage measured by the voltage sensor and the current measured by the current sensor; as well as The controller is configured as follows: Receive the measured sensor data from the measuring device, and The at least one control parameter is updated at least in part based on the measured sensor data, wherein the at least one control parameter includes a time constant of the pulse width of the electroporation pulse to be applied to the tissue.

2. The EP system as described in claim 1, further comprising: At least one probe is configured to receive one or more treatment portions and deliver the one or more treatment portions to the tissue.

3. The EP system as described in claim 2, wherein, The controller determines whether one or more treatment portions are present at or adjacent to the at least two EPEs based on multiple resistance or impedance measurements from the sensor data measured.

4. The EP system as described in claim 3, wherein, The controller is configured to detect the presence of one or more treatment portions if a decrease in resistance or impedance is detected based on the plurality of resistance or impedance measurements.

5. The EP system as described in claim 2, wherein, The one or more therapeutic components contain plasmid DNA.

6. The EP system as described in claim 1, wherein, The controller is configured to apply the measured sensor data to at least one trained diagnostic model.

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