WO2016193403A1

WO2016193403A1 - Electroporation device

Info

Publication number: WO2016193403A1
Application number: PCT/EP2016/062575
Authority: WO
Inventors: Owe Orwar; Carolina TRKULJA; Gavin Jeffries
Original assignee: Oblique Therapeutics Ab
Priority date: 2015-06-02
Filing date: 2016-06-02
Publication date: 2016-12-08

Abstract

A device for electroporating cells is provided that comprises a distal portion configured to be inserted into a tissue sample and comprising a plurality of fluid openings, one or more networks of microfluidic channels located within said device at said distal portion and in fluid communication with said fluid openings, and one or more electrodes that extend to the distal portion of the device, for generating an electric field across the tissue sample at said distal portion.

Description

ELECTROPORATION DEVICE CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority from and the benefit of United States patent application No. 62/169,604 filed on 2 June 2015. The entire content of this application is incorporated herein by reference. FIELD OF THE INVENTION

The present invention relates generally to devices and methods for electroporating cells within a tissue sample. BACKGROUND

The production of monoclonal antibodies by the hybridoma technique was first developed in 1975 (Kohler, G.; Milstein, C. Nature 1975, 256, 495). Briefly, mammals are injected with an antigen that triggers their immune response. Splenocytes from the animals spleen are then removed and later fused with immortalised myeloma cells. The cells are diluted down to single cells and separated into multi-well plates. Since one cell give rise to each separate colony, the produced antibodies in a single well will be monoclonal. The final step is to screen all of the different wells for the best candidate for binding to the antigen.

Today, strategies towards developing and finding successful immunotherapies are not limited to full size monoclonal antibodies. Due to advances in protein engineering, a wide variety of engineered antibody fragments have been derived during the two last decades such as Fab fragments, ScFv fragments, diabodies, tetrabodies, antibody fragments functionalized with protein conjugates as well as bispecific fragments binding to two antigens (Holliger, P.; Hudson, P. J. Nat. Biotechnol. 2005, 23, 1 126; Nelson, A. L; Reichert, J. M. Nat. Biotechnol. 2009, 27, 331 ). These new constructs provide us with a much larger toolbox when trying to develop antibodies and antibody-derived biologies with high specificity and affinity, deep tissue penetration, high stability and low toxicity.

Intrabodies can be constructed to target different cellular compartments by fusing the genetic sequence of the intrabody with intracellular trafficking signals. The need for efficient delivery methods is nonetheless a crucial step in intrabody therapy since the genetic material encoding the intrabody still needs to be delivered to the target cell (Lo, A. S.-Y.; Zhu, Q.; Marasco, W. A. In Therapeutic antibodies. Handbook of Experimental

Pharmacology 181 ; Chernajovsky, Y.; Nissim, A., Eds.; Springer-Verlag: Berlin, 2008; pp. 343-373).

A huge advantage with smaller antibody fragments compared to full size antibodies, is that they can be produced in different expression systems, e.g. E. coli, yeast and mammalian cells, and are no longer limited to production with the hybridoma technique. This enables large scale production at lower cost and many possibilities to genetically modulate antibody properties (Ahmad, Z. A.; Yeap, S. K.; AN, A. M.; Ho, W. Y.; Alitheen, N. B. M.; Hamid, M. ScFv antibody: Principles and clinical application. Clinical and

Developmental Immunology, 2012, 2012). Antibody fragments can be displayed on the surface of the filamentous bacteriophage, a so called phage display, which can be used to create large antibody libraries which are screened against the desired antigen. The screening procedure evaluates which antibody candidates that binds to the antigen. It is often repeated in several cycles due to unspecific binding in the first cycles. The conditions during the screening cycles can be changed in order to find the best suitable candidates for a certain environment, e.g., more stable antibodies can be selected by using a harsh environment. Another method to select antibodies with very high affinity is to perform the screening with very low concentration of antigen so that only those antibodies capable of binding during such conditions, remain. Several companies have developed their own screening technologies, and often have large antibody libraries (see e.g. Regeneron (regeneron.com), or Alligator bioscience (alligatorscience.se).

Antibody therapeutics is growing rapidly much do to the clinical success seen with several mAb therapies including Humira, Avastin, Herceptin, Yervoy and the promise of e.g. new cholesterol-lowering mAb treatments targeting PCSK9 such as Alirocumab, and Evolocumab. However, all antibodies currently on the market, and all in advanced stage clinical development are directed towards extracellular targets.

One of the main hurdles with antibody therapies is their restriction to extracellular targets. Antibodies are too large and too polar to enter through the cell membrane and are, generally, unstable in the reducing environment of the cytosol (Marschall, A. L. J.; Frenzel, A.; Schirrmann, T.; Schijngel, M.; Dubel, S. "Targeting antibodies to the cytoplasm", mAbs, 201 1 , 3, 3-16). Several techniques have been developed in order to access intracellular targets, including transport of antibodies across the cell membrane with different transport vectors, e.g., protein transfection (profection) reagents and protein transduction domains (PTDs), as well as the expression of the antibody directly within the target cell, so called intrabodies.

Delivery of genetic material, drugs and proteins through electroporation have been widely used since its discovery in the 80s. Electroporation is a method by which transient pores are introduced in the cell membrane when applying an electrical pulse, facilitating uptake of exogenous substances.

Electroporation has been used in the clinic for delivery of chemotherapeutic drugs to tumors (Gehl, J. Methods Mol. Biol. 2008, 423, 351 ) and several DNA vaccines are in clinical trials using electroporation as the delivery method (Van Drunen Littel-van den Hurk, S.; Hannaman, D. Expert Rev. Vaccines 2010, 9, 503). Electroporation of antibodies have not been as extensively studied as electroporation of chemicals and genetic material but several groups have managed to electroporate antibodies with high viability of the electroporated cells (Berglund, D. L; Starkey, J. R. Cytometry 1991 , 12, 64; Baron, S.; Poast, J.; Rizzo, D.; McFarland, E.; Kieff, E. J. Immunol. Methods 2000, 242, 1 15; Freund, G.; Sibler, A. P.; Desplancq, D.; Oulad-Abdelghani, M.; Vigneron, M.; Gannon, J.; Van Regenmortel, M. H.; Weiss, E. MAbs 2013, 5, 518). Marschall et al, recently compared electroporation of antibodies to internalization through protein transduction domains and profection agents and found electroporation to be superior (Marschall, A. L; Zhang, C; Frenzel, A.; Schirrmann, T.; Hust, M.; Perez, F.; Dubel, S. MAbs 2014, 6, 943).

To develop the technology in relation to intracellularly acting antibodies is, however, a daunting challenge, requiring new advancements both in terms of technology and methodology to discover and develop new tools to internalize the antibodies to cells in the right target organs (as well as suitable antibodies).

An approach whereby electroporation is achieved through a solution delivery device in the form of a capillary, has previously been described, and reference is made to Anal. Chem. 2001 , 73, 4469-4477. This ability was demonstrated by simultaneously porating and delivering non-cell permeable dyes into the substantia nigra in anesthetised rats placed in a stereotactic device.

This single point injection strategy used a small flexible glass (fused silica) capillary, through which a high electric potential (relative to ground) can be delivered. At the tip of the capillary the field penetrates through the cells directly at the opening. Beyond a few 10s of microns the field strength drops to a level which can no longer porate the cell membrane, limiting the rage of the effect, and enabling delivery of compounds only to the cells directly surrounding the capillary opening.

It is desired to improve the devices used for electroporating cells.

SUMMARY

According to an aspect of the invention there is provided a device for

electroporating cells, comprising:

a distal portion configured to be inserted into a tissue sample and comprising a plurality of fluid openings;

one or more networks of fluid channels, for example microfluidic channels located within the device at the distal portion and in fluid communication with the fluid openings; and optionally

one or more electrodes that optionally extend to the distal portion of the device, for generating an electric field across the tissue sample at the distal portion.

Aspects of the invention may extend to methods of using the device described above and herein.

The device for electroporating cells may be a probe, a microelectrode, a medical device, an electroporation device. Various embodiments extend to an apparatus comprising an array of devices, each of which may correspond to a device for

electroporating cells as described herein.

The device may be cylindrical, or may have an irregular shape or a variable cross- section as one moves along the length of the device. The cross-sectional area of the device may least double, triple, quadruple or quintuple as one moves along the length or longitudinal axis of the device. The device may comprise a proximal portion that may be arranged and configured to be attached to one or more surgical devices, for example a surgical manipulator and/or one or more tubes or wires. If the device forms part of an array of devices, the proximal portion may be attached to a housing for the array of devices. The proximal portion may not be configured for insertion into the tissue sample, and/or may not comprise any fluid openings. The only portion of the device comprising fluid openings may be the distal portion.

The fluid openings may be arranged and configured to transport fluid and/or particles carried in fluid to or from a tissue sample in use. The fluid openings may be arranged and configured to open into the tissue sample in use and/or may be located on the outer surface of the device.

The fluid openings may be in direct fluid communication with the tissue sample in use. By "direct fluid communication" it is meant that no other components of the device are present between the fluid openings and the tissue sample in use. The fluid openings may be in direct fluid communication with the one or more networks of fluid channels.

The one or more electrodes may be arranged and configured to generate an electric field at the distal portion of the device. The one or more electrodes may extend to the portion of the device configured to be inserted into a tissue sample in use (i.e., the distal portion).

The microfluidic channels and/or the one or more electrodes may be formed using a microfabrication technique.

The microfabrication technique may comprise one or more of micromachining, thin film deposition, chemical vapour deposition ("CVD"), physical vapour deposition ("PVD"), etching, photolithography, microforming, microstamping, microcutting.

The microfabrication technique may comprise patterning of the substrate to create the one or more networks of fluid channels and/or the one or more electrodes. The patterning may be achieved by injection moulding, embossing, reactive injection moulding, casting, photolithography, E-beam lithography, laser writing, direct machining and spark erosion. The patterning may additionally or alternatively be achieved by wet etching, dry etching, chemical developing, thermal degradation and electric degradation.

The microfabrication technique may comprise one or more of direct machining and/or drilling, ion milling, nano imprinting, sonic cavitation, laser ablation, spark erosion.

The one or more electrodes may be formed by sputtering, evaporation, chemical vapour deposition ("CVD"), electroless coating, electroplating, electrodeposition, particle sintering, low melting point flow and lamination.

The microfluidic channels and/or the one or more electrodes may be fabricated on a scale of less than 10 μηη, 50 μηη, 100 μηη, 500 μηη or 1 mm, and/or have a largest dimension of less than 10 μηη, 50 μηη, 100 μηη, 500 μηη or 1 mm. The device may comprise no fluid channels (including the microfluidic channels) greater than 10 μηη, 50 μηη, 100 μηη, 500 μηη or 1 mm in diameter or width (wherein the width is through a centre point of the channel). The number and/or density and/or cross-sectional area of the fluid channels may vary along the length of the device, for example the distal portion thereof.

The number and/or density and/or cross-sectional area of the fluid channels may change as one moves along the length or longitudinal axis of the device, for example the distal portion thereof. The variation in the number and/or density and/or cross-sectional area may be at least a two-fold, three-fold, four-fold or five-fold variation. In other words, the number and/or density and/or cross-sectional area may at least double, triple, quadruple or quintuple as one moves along the length or longitudinal axis of the device.

The one or more networks of microfluidic channels may comprise a plurality of different networks of microfluidic channels.

Each network of fluid channels may lead to a different group of fluid openings, and the different groups of fluid openings may be located at different areas on the outer surface of the device. For example, a first group of fluid openings may be located along one side of the device, and a second group of fluid openings may be located along the opposite side of the device. Alternatively, or additionally, a group of fluid openings may be located at the distal end of the device and a different group of fluid openings may be located a distance from the distal end of the device. The distance may be at least 1 μηη, 2 μηη, 3 μηη, 5 μηη, 10 μηι, 20 μηι, 30 μηι, 50 μηι, 100 μηι, 200 μηι, 300 μηι or 500 μηι.

Each of the networks of fluid channels may be in fluid communication with a different source of fluid.

For example, the device may comprise a plurality of fluid inputs that may be connectable to different sources of fluid, and each fluid input may be in fluid communication with a different network of fluid channels. A valve or flow control device may be located in each fluid input to control the delivery of fluid to each network of fluid channels. A control system may be arranged and configured to control (e.g., initiate, stop, speed up or slow down) the delivery of fluid, for example the valves or flow control devices, to each network of fluid channels.

The methods of using the device may comprise controlling the delivery of fluid to each network of fluid channels, for example using the valves or flow control devices. The methods may include varying the flow rate of the fluid delivered to each network of fluid channels whilst the device is inserted into a tissue sample.

Each of the networks of fluid channels may be in fluid communication with the same source of fluid.

For example, the device may comprise a single fluid input that may be connectable to a single source of fluid, wherein the single fluid input may be in fluid communication with the networks of fluid channels. A valve or flow control device may be located in the fluid input to control the delivery of fluid to the networks of fluid channels. A control system may be arranged and configured to control (e.g., initiate, stop, speed up or slow down) the delivery of fluid, for example the valve or flow control device, to the networks of fluid channels.

The methods of using the device may comprise controlling the delivery of fluid to the network of fluid channels, for example using the valve or flow control device. The methods may include varying the flow rate of the fluid delivered to the network of fluid channels whilst the device is inserted into a tissue sample.

A first network of microfluidic channels may comprise fluid channels having a first cross-sectional area, and a second network of microfluidic channels may comprise fluid channels having a second, different cross-sectional area.

A or the first network of microfluidic channels may comprise fluid channels having a first number or density of fluid channels, and a or the second network of microfluidic channels may comprise fluid channels having a second, different number or density of fluid channels.

Each network of microfluidic channels may extend to a different region of the device. The different regions may be spaced apart from each other, and/or may be located on different sides of the device, and/or may be located at different axial and/or radial locations on the device.

A single network of microfluidic channels may be provided, which network may be in fluid communication with a single source of fluid.

Generally, each network of microfluidic channels may comprise at least 2, 5, 10, 50, 100, 500, 1000, 5000 or 10,000 separate channels, and/or may comprise a pattern of branching or interconnecting fluid channels.

The one or more electrodes are integrated into the device, for example using a microfabrication technique, for example a microfabrication technique as described above or elsewhere herein.

The one or more electrodes may lead to an outer surface of the device, or may be positioned adjacent to an outer surface of the device. The one or more electrodes may be positioned on an outer surface of the device.

Alternatively, the one or more electrodes may be located internally within the device, such that no electrode may lead to or contact the outer surface of the device.

The one or more electrodes may comprise a plurality of electrodes.

A number and/or density of the plurality of electrodes may vary along the length of the device.

The number and/or density and/or cross-sectional area of the electrodes may change as one moves along the length or longitudinal axis of the device, for example the distal portion thereof. The variation in the number and/or density and/or cross-sectional area may be at least a two-fold, three-fold, four-fold or five-fold variation. In other words, the number and/or density and/or cross-sectional area may at least double, triple, quadruple or quintuple as one moves along the length or longitudinal axis of the device.

The variation along the length of the device could refer to any characteristic of the electrode, for example any characteristic that is able to cause a change in the electric field along the length of the device, and it not limited to number or density of electrodes. For example, the characteristic could be a material of the electrodes and the characteristic could vary along the length of the device in the same manner as described above in relation to number or density. The plurality of electrodes may comprise a plurality of separate arrays of electrodes, wherein each array of electrodes is positioned in a different region of the device.

The different regions may be spaced apart from each other, and/or may be located on different sides of the device, and/or may be located at different axial and/or radial locations on the device.

The methods of using the device may include applying a different voltage to the electrodes in each region, and/or varying the voltages applied to the electrodes in each region whilst the device is inserted in a tissue sample.

Each array of electrodes may be connectable to a different voltage supply.

For example, the device may comprise a plurality of connectors for electrical connection to separate voltage supplies, and each array of electrodes may be electrically connected to a separate connector. The different arrays of electrodes may be electrically insulated from each other.

Each array of electrodes may be connected to a common electrode for supplying a common voltage to each array of electrodes.

For example, a single connector may be provided for connection to a single voltage supply, and each array of electrodes may be electrically connected to the connector. The arrays of electrodes may be electrically connected to each other.

A first array of electrodes may comprise a first number and/or density of electrodes, and a second array of electrodes may comprise a second, different number and/or density of electrodes.

The one or more electrodes may be arranged and configured such that a strength of the electric field is variable along the length of the device.

A control system may be arranged and configured to vary the electric field of the device by controlling the voltages applied to the one or more electrodes in use. The variation in electric field strength may be achievable through the application of different voltages to the one or more electrodes, or different electrodes.

The methods of using the device may comprise varying the electric field generated by the one or more electrodes, for example by varying the voltage(s) applied to the one or more electrodes, for example prior to its insertion into a tissue sample or whilst the device is inserted into a tissue sample. In this manner, the electric field may be shaped prior to its insertion into a tissue sample, or whilst it is inserted into a tissue sample.

The device may further comprise a plurality of separate regions located at the distal portion of the device, wherein each region may comprise a separate array of electrodes and/or a separate network of fluid channels, such that an electric field strength and/or flow of fluid in each region may be separately controlled.

A first region may be located at the distal end of the device, such that upon insertion of the device into a tissue sample the first region may penetrate first or deepest into the tissue sample. A second region may be located a distance from the distal end of the device, such that upon insertion of the device into a tissue sample the second region may penetrate less deep into the tissue sample than the first region. The distance may be at least 1 μηι, 2 μηι, 3 μηι, 5 μηι, 10 μηι, 20 μηι, 30 μηι, 50 μηι, 100 μηι, 200 μηι, 300 μηη or 500 μηι.

The electrodes in different regions may differ in terms of a particular characteristic, such that each array of electrodes may be arranged and configured to provide a different electric field strength adjacent to that array of electrodes (e.g., when the same voltage is applied to each array of electrodes and/or prior to insertion into a tissue sample).

The fluid channels in different regions may differ in terms of a particular characteristic, for example cross-sectional area and/or density.

At least some of the fluid openings may be located transverse to a longitudinal axis of the device, and/or may be arranged and configured such that fluid exiting the fluid openings does so in a direction transverse to the longitudinal axis of the device.

This is distinct from conventional arrangements in which the fluid openings are typically located at the distal end of the device.

The device may further comprise one or more sensors arranged and configured to detect a characteristic of the tissue sample adjacent to the device.

The one or more sensors may comprise a thermal sensor arranged and configured to detect thermal variances within the tissue sample adjacent to the device, and/or within the device.

The one or more sensors may comprise an impedance sensor that may be arranged and configured to detect the impedance of cells in the tissue sample adjacent to the device.

According to an aspect of the invention there is provided an electroporation apparatus comprising a device as described above and herein.

The apparatus may further comprise one or more voltage supplies.

The apparatus may further comprise a or the control system and the control system may be arranged and configured to control the one or more voltage supplies to supply one or more voltages to the one or more electrodes. The control system may be arranged and adapted to supply a different voltage to different electrodes or different arrays of electrodes as described herein.

The control system may be arranged and configured to control the one or more voltage supplies to supply a voltage to the one or more electrodes that is sufficient to electroporate cells in the tissue sample.

The control system may be arranged and configured to control the one or more voltage supplies to supply a voltage to the one or more electrodes that is sufficient to reversibly electroporate cells in the tissue sample, but does not irreversibly electroporate cells in the tissue sample.

The control system may be arranged and configured to control the one or more voltage supplies to supply a voltage to the one or more electrodes that is sufficient to irreversibly electroporate and/or terminate cells in the tissue sample. The apparatus may comprise an imaging device arranged and configured to detect the location of the device within the tissue sample.

The imaging device may comprise one or more of a positron emission tomography ("PET") scanner, a computed tomography ("CT") scanner, a magnetic resonance imaging ("MRI") scanner, an ultrasound scanner and a fluorescent imaging scanner.

The methods of using the device described above may comprise the steps of: inserting the device into a tissue sample; and

electroporating cells located adjacent to the device by applying a voltage to the one or more electrodes and/or the fluid in the one or more fluid channels.

The apparatus may comprise a counter electrode or grounding device for grounding the tissue sample.

The method may further comprise delivering a biological or pharmaceutical agent (e.g., an antibody or antibodies) through the fluid openings and to the cells located adjacent the device. The biological or pharmaceutical agent maybe carried by a fluid and this may be supplied through the one or more networks of fluid channels.

The method may comprise adjusting the voltage applied to the one or more electrodes and/or the fluid in the one or more fluid channels in order to shape the electric field around the device, for example prior to, at the same time as, or after the delivery of the biological or pharmaceutical agent.

The method may comprise determining or measuring (e.g., using the one or more sensors) a characteristic of the tissue sample (e.g., extent of electroporation), and then adjusting one or more parameters of the device in response to the determined or measured characteristic. The one or more parameters might comprise supplied voltage, for example the voltage applied to the various electrodes or arrays of electrodes, and the adjustment may comprise varying, increasing or decreasing the applied voltage.

For example, if cells located at a given distance from the device are not

electroporated, due to the field strength and/or fluid flow not being sufficient, then the voltages applied to the device, and/or the flow rate of fluid through the fluid channels, may be increased. This cycle could be repeated a number of times (e.g., at least 2, 3, 5 or 10 times).

Alternatively, the voltages applied to the device, and/or the flow rate of fluid through the fluid channels may be decreased in response to the determining or measuring a characteristic of the tissue sample. For example, if it is determined that the tissue sample is not very dense adjacent a particular region, then a voltage applied to the electrodes, or array of electrodes located within that region may be reduced.

According to an aspect of the invention there is provided a method of manufacturing a device (for example a device as described above), comprising:

preparing a substrate;

microfabricating one or more networks of fluid channels on the substrate;

forming the device using the substrate, wherein the device comprises a plurality of fluid openings and the one or more networks of fluid channels are in fluid communication with the fluid openings. The fluid openings may be located at a distal portion of the device, and the distal portion may be configured to be inserted into a tissue sample.

The method may further comprise microfabricating one or more holes in the device, wherein the one or more holes form the fluid openings.

The method may further comprise microfabricating one or more electrodes on the substrate prior to the step of forming the device using the substrate.

The one or more electrodes may comprise one or more arrays of electrodes.

The one or more arrays of electrodes may comprise a uniform pattern of interconnected electrodes along the entire length of the substrate.

The one or more networks of fluid channels and/or the one or more arrays of electrodes may be microfabricated into the substrate so as to form a plurality of regions, wherein each region comprises a separate array of electrodes and/or a separate network of fluid channels.

The method may further comprise locating the one or more regions to match or correspond to a particular tissue sample.

The substrate may comprise one or more of a polymeric material, a semiconducting material, metal, silicon and glass.

The microfabricating may comprise one or more of micromachining, thin film deposition, chemical vapour deposition ("CVD"), physical vapour deposition ("PVD"), etching, photolithography.

The microfabricating may comprise one or more of microforming, microstamping or microcutting.

The microfabricating may comprise patterning of the substrate to create the one or more networks of fluid channels and/or the one or more electrodes.

The patterning may be achieved by injection moulding, embossing, reactive injection moulding, casting, photolithography, E-beam lithography, laser writing, direct machining and spark erosion. The patterning may additionally, or alternatively be achieved by wet etching, dry etching, chemical developing, thermal degradation and electric degradation.

The microfabricating may comprise post-processing the substrate.

The microfabricating may comprise one or more of direct machining/drilling, ion milling, nano imprinting, sonic cavitation, laser ablation, spark erosion.

The one or more electrodes are formed by sputtering, evaporation, chemical vapour deposition ("CVD"), electroless coating, electroplating, electrodeposition, particle sintering, low melting point flow and lamination.

The method may further comprise shaping the substrate and/or device to match or correspond to a particular tissue sample.

According to an aspect of the present invention, there is provided a microelectrode for use in electroporation, comprising a plurality of regions located at a distal portion of the microelectrode, wherein each region comprises a separate array of electrodes and/or a separate network of fluid channels, such that an electric field strength and/or flow of fluid at each region is separately controllable. The microelectrode may include any of the features described above in respect of the device for electroporating cells. The methods of using the device for electroporating cells described above extend to methods of using the microelectrode, and may involve any or all of the steps described above in this regard.

According to an aspect of the present invention, there is provided a method of fabricating a device for electroporating cells (for example a device for electroporating cells as described above), comprising:

identifying a tissue sample to be treated with the device;

obtaining image data corresponding to the tissue sample;

designing a shape or arrangement of the device based on the obtained image data; and

fabricating the device according to the designed shape or arrangement.

The method of fabricating a device may comprise any of the methods of manufacturing a device described above.

The step of designing may comprise determining an arrangement of fluid channels and/or electrodes within the device based on the obtained image data. The arrangement of fluid channels and/or electrodes may relate to the distribution of fluid channels and/or electrodes. For example, it may be determined that a portion of the device requires a high density of fluid channels and/or electrodes, and the design may include a relatively high density of fluid channels and/or electrodes at that region.

BRIEF DESCRIPTION OF THE DRAWINGS

Various embodiments will now be described, by way of example only, and with reference to the accompanying drawings in which:

Fig. 1 shows an embodiment of an electroporation device;

Fig. 2 shows a portion of the electroporation device of Fig. 1 in more detail;

Fig. 3A shows an embodiment of an electroporation device, and Fig. 3B shows a portion of the electroporation device of Fig. 3A in more detail;

Fig. 4A shows an embodiment involving insertion of an electroporation device into a tissue sample, and Fig. 4B shows an alternative embodiment to that of Fig. 4A;

Figs. 5A and 5B show the uptake of TRPV1 mediated YO-PRO after electroporation with aCOTVI in calcium free PBS, and Figs. 5C and 5D show the uptake of TRPV1 mediated YO-PRO after electroporation with aCOTV2 in the presence of 50 μΜ Ca2+; and Fig. 6 shows fluorescence images of the location of aCOTVI and aCOTV2 in the plasma membrane of cells after electroporation.

DETAILED DESCRIPTION Generally, the purpose of the technology disclosed herein is to develop a method for diagnostic and/or pharmaceutical agent delivery, using antibody delivery in some embodiments, based upon an electroporation strategy. This strategy can be optimised for temporal membrane destabilisation (electroporation). The devices described herein, which may consist of a probe or array of probes, are designed to insert compounds contained within a fluid into cell regions (such as tumor tissues) that reside in deep-lying organs as well as on the surface (e.g., skin), and shallow depth (e.g., lymph nodes).

There exist different types of electroporation in the art.

The term "reversible electroporation" may be used in cases where the pores of the cell membrane reseal after being subject to the electric field of an electroporation device.

The term "electrochemotherapy" may be used to describe the delivery of non- permeant drugs to the cell interior by reversible electroporation.

The strength of the electric field necessary to perform electroporation, e.g., reversible electroporation varies between different tissue samples. Threshold values in the range 200-400 V/cm have been reported, although this value depends on the tissue in question and may be higher (or lower).

The term "irreversible electroporation" may be used to refer to a relatively new treatment type where cells are terminated by permanent destabilisation of the cell membrane. This is distinct from electrocution which can include thermal ablation as a means for terminating the cell. In irreversible electroporation, permanent pores are created in cell membranes that ultimately lead to cell termination.

Reversible electroporation can be used to terminate a cell, through the delivery of a toxic agent during the period in which cells are permeated. The death of the cell in irreversible electroporation is not dependent, for example, on delivery of a toxic agent to the cell.

The electric field strength threshold value for irreversible pore formation leading to cell death may be in the range 450-900 V/cm. However, as stated above this value can depend of the tissue in question and may be higher.

For example, the electric field distribution obtained around an electroporation device may depend on the electrode design within the device, as well as the tissue structure adjacent the device.

The development of a meaningful therapeutic instrument based on electroporation rests on the idea that the relevant tissue, e.g., tumor tissue, is reachable by the device.

Various embodiments described herein are developed from the idea that tumor tissue can be accessed by the device, and then exposed to diagnostic and/or

pharmaceutical agent to yield tumor shrinkage. The ability to selectively electroporate different tissue, for example parts of tumors (e.g., small tumors within a lymph node), or in situ (e.g., in the pancreas), as described herein can enable targeted and/or focused delivery of the diagnostic and/or pharmaceutical agent. This can lead to improvements in treatment.

The tumor tissue that can be accessed by the device depends on many factors, including the applied electric field strength and the amount of agent delivered. For a typical tumor, any amount (including all) of the tissue can be accessed. It may also be desirable that the electroporation extends to some healthy tissue surrounding the tumor, or to only the tissue adjacent to the device when it is placed inside the tumor. The electroporation procedure requires an electric field to be generated across the sample. This may be established by coupling the electroporation device to a voltage supply, as described herein, and providing a counter electrode in the immediate vicinity of the tissue to be targeted. A counter electrode may not be needed if sufficient grounding of the tissue is already established.

The voltage may be applied in a pulsed and/or continuous manner. The current may be carried by a conductive liquid (i.e., through the fluid channels), sample solution to be delivered, electrodes patterned within the device and/or electrodes integrated into the channels (including the networks of fluid channels and secondary channels), and may extend as far as the fluid openings, as described in more detail below.

The general aim of the electroporation devices disclosed herein is to provide targeted or focused electroporation within a particular sample of tissue, so as to deliver a diagnostic or pharmaceutical agent which is to be transported through the porated cell membrane.

Probe Design

The present disclosure may generally relate to a device or probe for the

electroporation of cells in the vicinity of the device. The device may be inserted into a region of cells. The introduction of antibodies (or other diagnostic or pharmaceutical agents) into the region may be sequentially or simultaneously performed with the exposure to a pulsed or continuous electric field, which electrical field may cause transient pores to be created in the membranes of the cells, such that the antibodies (or other diagnostic or pharmaceutical agents) can be transported into the cells through the pores. The agent may also be introduced before or (shortly) after the exposure of the cells to the electric field.

Fig. 1 shows an embodiment of a device 10 (or probe) in accordance with an embodiment of the present disclosure.

The device 10 is an electroporation device (or probe) and is elongated such that it may be insertable into a sample of tissue, for example a tumor. The device 10 is connected via a tube 12 to a source of fluid, for example saline solution and/or electrolyte.

In some embodiments the device 10 may be connected to multiple sources of fluid, and this is discussed in more detail below. The device 10 is electrically connected to one or more external electrodes 14 that may be connected to one or more voltage supplies and/or signal processors.

The tube 12 and electrodes 14 connect to the device at a first, proximal end 16, which proximal end 16 is opposite a second, distal end 18. The distal end 18 is the end of the device 10 that is configured to be inserted into the sample of tissue. The proximal end

16 may remain outside of the tissue sample in use.

The device 10 may include a tip region 1 1 (or distal portion as referred to in the broadest aspects of this disclosure) that is configured to be inserted into a tissue sample.

The tip region 1 1 may be distinguished from the remainder of the device 10 by the inclusion of features located within the tip region 1 1 (which features are described below). Alternatively, or additionally, the tip region 1 1 may be distinguished from the remainder of the device 10 by a surface coating or change in dimension from the remainder of the device 10 (e.g., a reduced outer diameter).

The tube 12 is in fluid communication with a main fluid channel 20 located within the device 10. The main fluid channel 20 may run along the entire length of the device from the proximal end 16 to the distal end 18. The main fluid channel 20 may be configured to distribute fluid (e.g., from the source of fluid) to a plurality of secondary channels 22. The secondary channels 22 may have a smaller, or the same cross-section that the main channel 20. The secondary channels 22 may only be located in the tip region 1 1 of the device 10.

The flow of fluid through the main fluid channel 20 and/or the secondary channels 22 may be effectuated by any known means. For example, the fluid may be pneumatically driven, whereby the fluid within the tube 12 may include air, and a suitable pump may be used to pneumatically drive fluid through the main fluid channel 20 and/or the secondary channels 22. Other types of driving means may include electrophoretic flow.

The electrodes 14 connect to one or more internal electrodes 30 that are located within the device 10, which electrodes 30 may connect to a network of electrodes that extend to the distal end 18 of the device 10 and are located throughout the tip region 1 1 of the device 10.

A portion of the tip region 1 1 of the device 10 is shown in more detail in Fig. 2, from which it can be seen that the main fluid channel 20 extends along the longitudinal axis of the device 10 (although this is not an essential feature) and is fluidly connected to a plurality of secondary channels 22. The secondary channels 22 extend to the outer surface of the device 10, such that fluid passed through the main channel 20 (see flow direction 24) flows through each of the secondary channels 22 and out of the device (see flow direction 26) via respective outlets 27 at the end of each secondary channel 22.

In some embodiments, or modes of operation the flow of fluid may be reversed, and so the outlets 27 may be referred to as openings in the broadest aspects of this disclosure. In other embodiments, or modes of operation the fluid may be stationary, and particles may be transferred through the openings by, for example, diffusion. Particles of tissue may diffuse through the openings in this manner.

The arrangement of secondary channels 22 in the tip region 1 1 of the device 10 may be referred to as a fluid network, which fluid network is configured to deliver a fluid (e.g., saline solution) to a tissue sample contacting and/or in the region of the tip region 1 1.

The fluid may contain one or more biological or pharmaceutical agents for delivery to adjacent cells via electroporation.

Within the tip region 1 1 of the device 10 there may be greater than 2, 5, 10, 50,

100, 500, 1000 or even 10,000 secondary channels 22 that each transfer fluid from the main channel 20 and out of the device 10 via outlets 27. As such, the fluid network may comprise at least 50, 100, 500, 1000 or even 10,000 separate channels for delivering a fluid to a tissue sample contacting and/or in the region of the device 10 (e.g., the tip region 1 1 of the device 10).

In some embodiments a voltage may be applied to the fluid flowing through the main channel 20 (and secondary channels 22) in order to cause pore formation in cell membranes (i.e., electroporation) of cells in the region of the device 10 (e.g., in the region of or adjacent to the tip portion 1 1 of the device 10).

Fig. 2 also shows in more detail the electrodes 30 and their connection to a network of electrodes located at the tip region 1 1 of the device 10. The network of electrodes is shown schematically in Fig. 2 but may include a first array of electrodes 32 and a second array of electrodes 34. The first array of electrodes 32 may be connected to a first 31 of the internal electrodes 30, and the second array of electrodes may be connected to a second 33 of the internal electrodes 30.

In the embodiment shown in Fig. 2, the first array of electrodes 32 is shown as extending to a first side 40 of the device 10, and the second array of electrodes 35 is shown as extending to a second side 42 of the device 10. The electrodes may not extend to the outer surface of the device.

Through application of different voltages to the first array of electrodes 32 and the second array of electrodes 35, the electric field strength at the first side 40 of the device 10 and the second side 42 of the device 10 can be varied. This can also mean that the electric field may be shaped (e.g., in situ) according to the voltages applied to the first array of electrodes 32 and the second array of electrodes 35.

Various embodiments are envisaged in which any numbers of arrays of electrodes are included in the device 10, which arrays may be connected to separate voltage supplies and/or signal processors. The arrays of electrodes may be confined to different locations within the device in order to provide different electric field strengths adjacent to such locations. Alternatively, or additionally the arrays of electrodes may be confined to different areas of the device 10 so as to provide a shaped electric field around the device 10, which may be achieved through the application of tailored voltages to the different arrays of electrodes.

A third 35 of the internal electrodes 30 may comprise one or more instrument electrodes 35, as shown in Fig. 2. The instrument electrode 35 is connected to a sensor or other instrument 36 that is integrated into the device 10, and may be connected to a signal processor or other sensing device external to the device 10. The sensor or other instrument 36 may be configured to sense a characteristic of the environment adjacent to the sensor or other instrument 36. The signal may be analysed or processed by the signal processor or other sensing device.

For example, a thermal sensor could be used that is arranged and configured to relay a signal representative of the temperature in the immediate vicinity of the instrument 36. A parameter associated with the device 10 may be adjusted based on a change in the response of the sensor or other instrument 36. For example, the voltage applied to the fluid flowing into the main channel 20, or the electrodes 30 (e.g., the first array of electrodes 32 and/or the second array of electrodes 35) may be reduced if the temperature of the tissue surrounding the device 10 increases.

The sensor or other instrument 36 may include an impedance probe, which could be used to e.g., sense the location of the tip region 1 1 of the device 10 within a sample of tissue, for example a tumor.

The sensor or other instrument 36 may include an electrochemical sensor, which may be arranged and configured to sense or detect a particular chemical and provide an electrical response that is proportionate to the concentration of the chemical in the vicinity of the instrument 36. The response may be analysed or processed by the signal processor or other sensing device and a parameter of the device 10 may be adjusted based on the change in response of the instrument 36, as discussed above.

The main channel 20 and/or at least some or all of the secondary channels 22 may be microfluidic channels or microchannels, and may have a width (e.g., through a centre) or diameter of less than 1 μηι, 2 μηι, 3 μηι, 5 μηι, 10 μηι, 20 μηι, 30 μηι, 50 μηι, 100 μηι, 200 μηη, 300 μηη, 500 μηη, or 1 mm. The main channel 20 may be larger than the secondary channels 22 and may have a width (e.g., through a centre) or diameter of about (or less than) 1 mm, 2 mm, 5 mm, 10 mm, 15 mm or 20 mm.

The microfluidic channels or microchannels may be formed by any suitable method. For example, the microfluidic channels or microchannels may be formed by deposition or removal of a thin film onto or from a polymeric substrate. The thin film may be patterned such that the pattern represents the various microfluidic channels or microchannels.

Removal or deposition of the thin film may cause the pattern to be etched or deposited onto the polymeric substrate.

The polymeric substrate may be combined or incorporated with another substrate in order to form a device similar to the one shown and described in relation to Figs. 1 and 2. For example, the substrate incorporating the etched or deposited pattern may form the lower half of the device 100, and a similar substrate (e.g., comprising the same material) could be placed on top of the patterned substrate and bonded thereto, for example by heating. The various electrodes and networks of electrodes could be placed between the two substrates prior to bonding in order to secure them in place.

In various embodiments the microfluidic channels or microchannels may be formed by alternative methods, for example the substrate may be silicon or glass. The formation of the microfluidic channels or microchannels may include one or more of micromachining, microfabricating, thin film deposition (such as chemical vapour deposition or physical vapour deposition), etching, patterning (e.g., photolithography), microforming (e.g., microstamping or microcutting), and any other suitable method.

Microfabrication of the fluid channels (e.g., main channel 20 and/or secondary channels 22) using e.g., silicon, glass and polymeric materials opens up numerous possibilities within electroporation and fluidic device or probe design, as described in more detail herein. Designing the tip region 1 1 to include greater than 2, 5, 10, 50, 100, 500, 1000 or even 10,000 secondary channels 22 (or more), for example, can enable a large number of cells to be simultaneously porated. Only the cells located at channel exits may become porated, depending on the applied voltage.

Fig. 3A shows a microfluidic electroporation device 100 in accordance with an embodiment, and is provided to illustrate the extent to which a microfluidic electroporation device could be divided into different regions to provide targeted electroporation (including reversible or irreversible electroporation), or electric field shaping.

The device 100 comprises a plurality of pathways 102a-d, each of which includes one or more fluid channels and electrodes. Each pathway 102a-d leads to a different region 104a-d of the device 100. The fluid channels within each pathway 102a-d may be connected to separate fluid sources. Similarly, the electrodes in each pathway 102a-d may be connected to separate voltage supplies or signal processors.

The regions 104a-d of the device 100 may be set up in a similar manner to the tip region 1 1 of the device 10. For example, as shown in Fig. 3B, a main fluid channel 20a could be provided that leads to the region 104a, which main fluid channel 20a could then divide into a number of secondary channels 22a within the region 104a.

The secondary channels 22a extend to the outer surface of the device 100, such that fluid passed through the main channel 20a flows through each of the secondary channels 22a and out of the device 100 via respective outlets 27a at the end of each secondary channel 22a.

The secondary channels 22a within region 104a form a fluid network within the region 104a, which may comprise at least 2, 5, 10, 50, 100, 500, 1000 or even 10,000 separate channels (although only a few are shown in Fig. 3B) for delivering a fluid to a tissue sample contacting and/or adjacent to region 104a.

An electrode 30a is also provided that leads to the region 104a. The electrode 30a is located within the same pathway 102a through the device 100 as the main fluid channel 20a, although may not be electrically connected to the main fluid channel 20a. The electrode 30a may be connected to an array of electrodes 32a within the region 104a, all of which may be configured to generate and/or vary an electric field adjacent to the region 104a.

A voltage may be applied to the array of electrodes 32a in order to generate and/or vary an electric field adjacent the region 104a. Furthermore the electric field adjacent to region 104a may be shaped through the application of different voltages to the array of electrodes 32a.

An additional electrode 35a may be provided in the form of one or more instrument electrodes 35a. The additional electrode 35a is located within the same pathway 102a through the device 100 as the main fluid channel 20a and the electrode 30a, although may not be electrically connected to either the main fluid channel 20a or the electrode 30a.

The instrument electrode 35a may be similar to the instrument electrode 36 described above in relation to Fig. 2. For example, the instrument electrode 36a may comprise a thermal sensor arranged and configured to relay a signal representative of the temperature in the immediate vicinity of the instrument 36a, an impedance probe, or an electrochemical sensor arranged and configured to sense (e.g., oxidise) a particular chemical and provide an electrical response that is proportionate to the concentration of the chemical in the vicinity of the instrument 36a.

The main channel 20a and secondary channels 22a may be microfluidic channels or microchannels, and may have a width (e.g., through a centre) or diameter of less than 1 μηι, 2 μηι, 3 μηι, 5 μηι, 10 μηι, 20 μηι, 30 μηι, 50 μηι, 100 μηι, 200 μηι, 300 μηι, 500 μηι, or 1 mm. The main channel 20 may be larger than the secondary channels 22 and may have a width (e.g., through a centre) or diameter of about (or less than) 5 mm, 10 mm, 15 mm or 20 mm. The main channel 20a and secondary channels 22a (and the electrode 30a and array of electrodes 32a) may be formed by any suitable method, including those described above in respect of the microfluidic channels or microchannels of the device 10 shown and described in respect of Figs. 1 and 2.

It will be appreciated that the other regions 104b-d may be configured in a similar manner to region 104a as shown in Fig. 3B, wherein each region comprises a respective main channel 20b-d and a respective network of secondary channels 22b-d, as well as a respective electrode 30b-d and array of electrodes 32b-d.

In this manner, each region 104a-d may provide a different response in cells located adjacent to that region. For example, a first fluid may be provided to region 104a that has a first characteristic. A second fluid may be provided to region 104b that has a second characteristic. The characteristic may comprise conductivity, such that the first fluid may have a different conductivity to the second fluid. The first characteristic may, alternatively or additionally comprise a relatively high concentration of antibodies, and the second characteristic may comprise a relatively low concentration of antibodies.

In various embodiments, any number of regions similar to regions 104a-d may be provided, and this may enable complex fluid delivery involving multiple fluids being delivered simultaneously to different regions of a tissue sample. For example, different solutions may be directed towards different tumor sections, so that the different tumor sections may experience different responses during the electroporation procedure.

The various regions may be provided in any shape of device. For example, the elongated tube as shown in Figs. 1 and 2 may comprise different regions located in different axial positions along its length, such that a different response in cells can be exhibited at each axial position. A different electric field could be generated adjacent cells at each axial position so as to tailor the electroporation (including reversible or irreversible electroporation) of cells at each axial position.

Fig. 4A shows an embodiment of the present disclosure in a particular example, which is merely illustrative and should not be considered to be limiting to the broadest aspects of the disclosure.

A device 200 is shown and is similar to the devices shown and described in respect of Figs. 3A and 3B, except that it is in the form of an elongated tube. Located at different axial and circumferential positions on the device are regions 204a-d having the same features as the regions 104a-d described above in relation to Figs. 3A and 3B

A tumor 250 is shown and has an irregular shape, which is a common factor to many tumors and can present difficulties when attempting to target the tumor for electroporation (including reversible or irreversible electroporation), whilst trying to avoid any undesired electroporation (including reversible or irreversible electroporation) of cells in the vicinity of the tumor 250. The tumor 250 itself may have cell or other variances within its own structure, and this may present further problems if the electroporation device to be used cannot apply a different response to different parts of the tumor 250.

Once the device 200 is inserted into the tumor 250, its position can be ascertained (e.g., through the use of scanning techniques such as PET, ultrasound, etc., described above, or using appropriate sensors located within each region 204a-d).

Looking to Fig. 4A, an operator or control system may note that region 204a is not inserted very far into the tumor 250 and located adjacent a relatively small part of the tumor 250, whilst region 204d is inserted quite far into the tumor 250 and is located adjacent a relatively large part of the tumor 250. Upon noting this, the operator or control system may deduce that a relatively weak voltage should be applied to the array of electrodes in region 204a, whilst a relatively large voltage should be applied to the array of electrodes in region 204d. The operator or control system may further deduce that the amount of fluid to be transported to region 204a for distribution in the network of secondary channels therein should be relatively small, and the amount of fluid to be transported to region 204d for distribution in the network of secondary channels therein should be relatively large.

The operator or control system may then apply the determined amounts of fluid and/or voltages to the regions 204a and 204d, with the effect that the application results in a different response in different parts of the tumor 250. This means, for example, that the cells located immediately outside of the tumor 250 in the region adjacent 204a may not be affected (or less affected) by the device 200. It can also mean that the device 200 is more adaptable, for example, since conventional devices are typically limited to application of a single voltage and at a single region (typically the end of the device).

Fig. 4B shows an embodiment of the present disclosure in a particular example, which is merely illustrative and should not be considered to be limiting to the broadest aspects of the disclosure.

A device 300 is shown and comprises four regions 304a-d that are intended to provide different responses in cells adjacent to each region 304a-d. Within each region there exists a network of fluid channels similar to the secondary channels described above. These have not been shown for brevity, but comprise a network of fluid channels, for example each comprising at least 2, 5, 10, 50, 100, 500, 1000 or even 10,000 separate channels for delivering a fluid to a tissue sample contacting and/or adjacent to each region 304a-d.

A tumor 350 is shown and has an irregular shape, although slightly more regular than the tumor 250 of Fig. 4A. The device 300 is shown as inserted into the tumor 350 such that its distal end is located approximately at the centre of the tumor 350. Its position can be ascertained, for example, through the use of scanning techniques such as PET, ultrasound, etc., described above, or using appropriate sensors located within each region 304a-d. Other positioning arrangements within a tumor may be envisaged, and the location of the regions 304a-d can be adapted to positioning arrangements in which the device 300 is inserted differently, for example all the way through the target tissue, or in which the device 300 is shaped differently (e.g., such as the irregular shape of the device 100 in Fig. 3A).

Various embodiments disclosed herein relate to the shaping of an electroporation device, or the positioning of the regions (e.g., regions 304a-d) within it such that the device is customised, adapted or configured for a particular tissue sample, for example a certain tissue shape and/or density. These are described in more detail below.

A main fluid channel 310 is provided within the device that is fluidly connected to the network of fluid channels within each region. The main fluid channel 310 has a relatively large cross-sectional area, whilst the fluid channels in each network within regions 304a-d may have smaller cross-sectional areas. The main fluid channel 310 thus functions as an 'artery', with the networks of fluid channels within each region 304a-d functioning as 'capillaries'.

In order to provide different responses in cells adjacent to each region 304a-d, the networks of fluid channels within each region 304a-d may have different characteristics. This could include the fluid channels in each region 304a-d having a different cross- sectional area, or a different number and/or density of fluid channels could be provided in each region 304a-d.

In one example, the network of region 304d may have the largest number of fluid channels, and the network of region 304a may have the smallest number of fluid channels. This means that the amount of fluid delivered to the cells adjacent the region 304d may be relatively large, and the amount of fluid delivered to the cells adjacent the region 304a may be relatively small. As the amount of fluid being delivered to the cells adjacent the region 304d is relatively large, the fluid may also flow into cells in distant regions 352 of the tumor.

An electric field may be created around the distal portion of the device 300 (i.e., the portion including the regions 304a-d). This could be by applying a voltage to the fluid flowing through the main fluid channel 310.

An array of electrodes (not shown) could be present throughout the device, and a voltage could be applied to the array of electrodes to create the electric field around the device. The array of electrodes may be a network of microfabricated electrodes, for example that are positioned across a layer within the device. The voltage applied to the array of electrodes could be instead of, or in addition to a voltage applied directly to the fluid flowing through the main fluid channel 310.

The voltage (or voltages) may be applied at a level that causes cells close to the device to be irreversibly electroporated. The voltage (or voltages ) may be applied at a level such that cells in more distant regions of the tumor 350 (e.g., distant regions 352) may be reversibly electroporated. It will be appreciated that the applied voltage may be varied so as to avoid irreversible electroporation. However, the arrangement of Fig. 4B and the voltages applied can allow cells in the centre of the tumour to be irreversibly electroporated, while cells at distant regions of the tumor 350 (e.g., region 352) are reversibly electroporated.

Tailoring the device to suit a particular tissue sample

The present invention extends to methods of manufacturing or fabricating an electroporation device that is tailored or adapted to suit a particular tissue sample.

For example, a tumor may be identified that is relatively large, and an

electroporation device may be manufactured or fabricated that is, itself relatively large. More complicated situations may be envisaged. For example, a large tumor may have a highly varied cell density, and an electroporation device may be manufactured or fabricated so that more fluid channels or electrodes (for example) are located adjacent to the regions of high density, and fewer fluid channels or electrodes are located adjacent to the regions of low density, when the device is positioned inside the tumor.

The tissue sample may be imaged to ascertain what shape or arrangement of electroporation device might be suitable for the tissue sample, for example using a positron emission tomography ("PET") scanner, a computed tomography ("CT") scanner, a magnetic resonance imaging ("MRI") scanner, an ultrasound scanner or a fluorescent imaging scanner.

If a patient has multiple tumors with varying geometries, for example, a single device may not be suitable for each geometry. The method may include assessing each tumor geometry and fabricating a number of different electroporation devices, each being tailored or adapted to suit a particular tumor.

Any feature of the electroporation device may be adapted to suit a particular tissue sample. For example, the distribution of fluid channels and/or electrodes along the device, for example the different regions described above (i.e., regions 104a-d, 204a-d or 304a-d) may be tailored to the geometry of a particular tissue sample. A region having a greater number of fluid channels, for example, may be located at a portion of the device that is intended to be located adjacent a more dense region of the tissue sample.

The shape of the device may be tailored or adapted to suit a particular tissue sample. For example, it may be ascertained that a tissue sample has an irregular shape (which is typically the case when considering a tumor), and a device may be shaped so that its shape corresponds to that of the irregular shape of the tissue sample. If a tissue sample is curved, for example, then the device may be curved and may exhibit a similar curvature. Other embodiments are envisaged, see for example Fig. 3A in which the device has an irregular shape, and also selective positioning of the regions 104a-d.

In some embodiments an electroporation apparatus may comprise an array of devices, each corresponding to an electroporation device (or probe) described herein. Each device in the array may comprise one or more networks of fluid channels and/or one or more arrays of electrodes. The array may be insertable into a tissue sample.

The arrangement of devices (or probes) within the array may be tailored or adapted to suit a particular tissue sample. For example, the spacing between the devices may be adapted for a particular density of cells within the tissue sample. If a tissue comprises a relatively high density of cells, for example, then the spacing between the devices in the array may be relatively small, and vice versa.

Alternatively, or additionally, the distribution or arrangement of fluid channels and/or electrodes within each device in the array may be tailored or adapted to suit a particular tissue sample. One of the devices may comprise fluid channels having a relatively small cross-sectional area, for example, and another of the devices may comprise fluid channels having a relatively large cross-sectional area.

The embodiments described above allow a tissue sample (e.g., a tumor) to be assessed and then the manufacture or fabrication of the electroporation device to be tailored accordingly. This is considered to be an advance on conventional methods in which devices for treating certain tissue samples are constructed generically, so as to cover a broad range of such tissue samples. The methods disclosed herein allow an electroporation device to be constructed specifically for one type of, or a unique tissue sample.

The use of microfabrication techniques can further enhance these embodiments, in that the networks of fluid channels and/or arrays of electrodes can be easily tailored when fabricating the device (or array of devices), so as to suit a particular situation. The fabrication of the device itself, or the devices in the array of devices, may also be improved when using such techniques and trying to tailor the device or array as described above.

General considerations

In any of the aspects or embodiments disclosed herein, the regions adjacent the tip of the device (or probe) can terminate with at least 10 or 1000 fluid channel exits. The location of the device at this point may be accurately determined using suitable scanning methods described below, or sensors built into the device as described herein.

By using relatively small applied voltages, only the cells located adjacent to fluid channel exits may become porated. Larger applied voltages can be used to increase the amount of cells that are porated, or even irreversibly electroporate cells as described in relation to Fig. 4B and elsewhere herein.

Electrodes, or an array of electrodes may be micropatterned into any of the devices or probes described herein. This can enable additional electrode placements throughout the device. This strategy can be implemented to minimize joule heating, for example, which may be caused by passage of a large electrical current through a fluid in the fluid channels.

The electrodes (e.g., one or more arrays of electrodes) disposed within the device (or probe) may extend to the distal region of the device, which distal region is configured to be inserted into a tissue sample and comprises fluid channel outlets (for example) for the electroporation of cells adjacent to the distal region.

The fluid networks may be microfabricated into the device (or probe) and this can enable complex fluidic circuitry to be integrated into the device. This can lead to numerous examples of targeted electroporation (e.g., reversible and irreversible electroporation), as discussed herein, for example in respect of Figs. 3A-4B. For example, and with reference to e.g., Figs. 4A and 4B, this level of control can allow different tumor sections to experience different types, or amounts of electroporation.

The device may comprise a needle probe, coupled to or preloaded with, a fluid to be delivered to the tissue sample through the various fluid channels described herein.

In any of the aspects or embodiments disclosed herein, the device (e.g., device 10, 100, 200 or 300) may have a length greater than, less than or equal to about 1 mm, 5 mm, 10 mm, 50 mm, 100 mm, 150 mm or 200 mm. The device may have a width or diameter of greater than, less than or equal to about 5 μηι, 10 μηι, 50 μηι, 100 μηι, 500 μηι, 1 mm, 2 mm, 5 mm, 10 mm or 25mm.

Delivery of fluid within the device or probe to cells adjacent to the device (or the distal region of the device) may be performed though the implementation of fabricated microchannels, as described herein. These channels may have varying cross sectional dimensions (as described in e.g., Fig. 4B) to provide flow rate variances within the device and/or to balance flow resistances within the device.

The width or diameter of the microchannels may be greater than, less than or equal to about 1 μηη, 2 μηη, 5 μηη, 10 μηη, 50 μηη, 100 μηη, 500 μηη, 1 mm, 2 mm, 5 mm, 10 mm or 20mm. These channels may all be connected to the same fluid source, or separate fluid channels (e.g., separate networks of fluid channels) may be connected to separate fluid sources. The connection to separate fluid sources may be provided by the provision of separate fluid pathways within the device, or one or more valves may be provided within the device (or external to the device) to such that fluid from any of the separate fluid sources may be delivered to any of the channels, or networks of channels.

In any of the aspects and embodiments described herein, one or more sensors or instruments may be integrated into the device, which sensors or instruments may be configured to sense a characteristic of the device, environment or tissue located in the vicinity of the device, and output a signal representative of the characteristic.

The one or more sensors or instruments may comprise a thermal sensor for monitoring of tissue breakdown (e.g., the extent of irreversible electroporation, as described in Fig. 4B), and/or any thermal variances within the device and/or the tissue adjacent to the device. The one or more sensors or instruments may comprise an impedance probe and/or an electrochemical sensor, for example to sense the location of the probe within the tumor or tissue and/or to detect the extent of electroporation within tissue adjacent to the device.

The device or probe may be inserted into a tissue sample at a single point, or repeated insertions of the device into the same or different regions can be performed to increase the insertion of antibodies into the same or other regions of the tumor or regions of cells.

The device described in relation to any of the aspects or embodiments disclosed herein can be used on its own, or may be used in an array of devices having the same features, for example an array of electroporation devices as described above. The array of devices may be adjacent to (e.g., in contact with) one another. All of the devices may have the features of the devices described herein (e.g., device 10, 100, 200 and/or 300), and may be designed to target either specific locations within a tumor (see e.g., the

embodiments of Figs. 4A and 4B) or entirety of tumors or tissue sections.

An electrical field may be applied to the device that causes permanent

destabilisation of cells or cell membranes, which may lead to irreversible electroporation (leading to termination) of the cells, for example those located in the highest field regions. An example of this is described in relation to Fig. 4B.

The methods described herein, for example the combination of reversible electroporation and irreversible electroporation of cell regions within a tumor, can be combined with other cell destabilizing strategies.

For example, the tumor may be incubated with a non-cell-permeable compound, toxic to the interior of a cell and which can be diffused into the cell during electroporation, prior to the electroporation (including reversible or irreversible electroporation) of the cells within the tumor. This can restrict tumor growth and allow for compartmentalisation of the tumor, which can stop it spreading. Active components that may be prevented from being released could be components of the tumor, e.g. tumor cells, or other substances released from live tumor cells.

"Compartmentalisation" is terminology that is sometimes used in tumor staging and surgery, and describes the case where tumor growth may be restricted by the surrounding healthy tissue, that is an anatomic barrier exists.

The devices or probes described herein can be considered to be a combination of an electrical and fluidic probe. Some of the fluid channels within the probe, for example each network of fluid channels, may be connected to one or many fluids to be delivered to a local region of electroporation, for example the regions 104a-d, 204a-d or 304a-d.

The shape of the devices or probes described herein is not limited to any of those that are described, especially in relation to the embodiments. For example, the device 10 shown in respect of Figs. 1 -2 may be tapered, as shown or may be cylindrical and have the same diameter along the length of the device. The device 100 shown and described in respect of Fig. 3 may also be cylindrical as shown in Figs. 4A or 4B, as opposed to the slightly irregular shape that is illustrated. The positions of the regions 104a-d may be anywhere on the device that is configured to be inserted into a tissue sample, for example the end region as defined above.

The devices or probes described herein can be freely positioned and inserted into tissue in a manner similar to a needle. Once inserted, or during insertion, the electrical field and flow can be initialized, thus causing electroporation of cells and enabling the delivery of the antibody solution. This can be repeated at a single region, or multiple regions in order to introduce antibodies into an increased number of electroporated cells. Due to the electric field drop off away from the regions to which a voltage is applied, cells only in the immediate vicinity of (or adjacent to) the device may be electroporated, allowing a precise, controlled and focused delivery of antibodies to these cells. As described in the example of Fig. 4B, increasing the electric field to a level that electrocutes (or irreversibly electroporates) certain cells may actually be advantageous, in some circumstances, for example within large tumors and/or tissue sections. It may be desired, for example, to terminate some of the cells within the central region of a tumor.

The same insertion methods may be performed as described above. However, a larger electric field can be used that causes permanent damage to the cells adjacent to the device, permanently destabilising them and terminating these cells. As the electric field drops off away from the probe, cells in the tissue sample (e.g., a tumor) that are further away from the targeted regions will be electroporated. Increased flow rates may allow for delivery of antibody solution into these more distant cells, thus delivering to a larger region per insertion. Alternatively, or additionally, the time for which the solution is delivered could be increased, to allow the fluid to reach more distant cells.

Certain parts of the device could be coated with an substance that is arranged and configured to respond to a sample of biologic tissue (for example changes in a sample of biologic tissue). For example, at least the tip portion (or distal portion) could be coated with an anti-inflammatory agent, which may be released when the pH of tissue contacting the distal portion drops below a predetermined amount, for example 5.0 (e.g., the inflammation may be acidic)

The substance may comprise a polymer coating, that may contain a phosphate arranged and configured to remove calcium from cells that have degraded (e.g., by reacting to form insoluble calcium phosphate crystals). This may stop toxic cell content affecting healthy tissue.

The device (and more specifically the fluid channels) could be made from a polymer which dissolves in the body, for example a polylactic acid ("PLA") composite material. This may cause the device or fluid channels to break down (e.g., into harmless lactic acid) and slowly dissolve into the body. This could be used to provide a varying flow rate of fluid into the tissue over time, for example the fluid channels may widen due to the breakdown of the material, and increase the fluid flow rate into the tissue.

Other embodiments are envisaged in which the flow rate may be decreased over time. For example, a biofilm could be used as a coating for the device or liner of the fluid channels, wherein the aggregation of the biofilm decreases the width of the fluid channels and the flow rate of the fluid through the channels.

A plurality of coatings could be provided, wherein each coating may be arranged and configured to provide a different response to changes in a sample of biologic tissue. The coatings may be arranged and configured to degrade over time, so as to stagger their responses. For example, one could first provide an outer layer of an anti-inflammatory coating, which degrades over time to layer comprising a toxic material.

Detection of the device

Generally, the methods disclosed herein may be required to a have very targeted delivery, for example within small tumors only a few mm or cm in diameter. Whilst the methods are aimed at targeting different parts of the tissue, sometime multiple insertions and translations within the tissue may still be required to reach a critical permeation density and disrupt the tumor. As such, accurate determination of the location of the devices described herein within tissue can be important.

Thus, in various embodiments an imaging device may be provided and may comprise a detector configured to detect the location of the electroporation device or probe in the tissue. The detector may comprise a positron emission tomography ("PET") scanner, a computed tomography ("CT") scanner, a magnetic resonance imaging ("MRI") scanner, an ultrasound scanner, a fluorescent imaging scanner, or any other scanner that is suitable to visualise the electroporation device as it is inserted into the tissue.

The above described imaging techniques are generally available in hospitals, and the electroporation device may be tailored to maximise imaging contrast in each of the specialized techniques. For example, the electroporation device may be provided with a polymer coating and this can improve the detection of the electroporation device within the tissue. The electroporation device may also incorporate one or more thin metal films for improved detection in CT scans.

An advantage of using ultrasound is that this method can detect the electroporation device natively, due to material density differences.

When using a fluorescent imaging scanner, the electroporation device may include a fluorescent coating and/or electroporation device may comprise fluorescent particles, for example integrated into the material of the device and/or the outer layer of the device.

In various embodiments, the electroporation device may be in the form of a glass capillary coated in an insulating polyimide coating, which can render the electroporation device flexible and capable to endure handling (i.e., for strength). This polymer coating can also be functionalised, for example with a fluoropolymer that can act as a radio tracer, for example an 18F fluoropolymer or a redox responsive branching polymer. This can assist in detection of the electroporation device when using MRI.

Microfabrication

As described herein, the electroporation device can be microfabricated. This may include the use of microfabrication techniques to form the fluid channels (e.g., the networks of fluid channels) and/or the electrodes (e.g., the arrays of electrodes), which can be applied to standard polymeric, semi-conductor, metallic, silicon and glass materials, or a combination thereof. This opens up numerous possibilities and allows various

configurations for the targeted delivery of diagnostic or pharmaceutical agents.

The fluid channels and/or electrodes described herein may be formed using a substrate, for example a polymeric substrate, to which a microfabrication technique may be applied. Once the pattern of fluid channels and/or electrodes are formed on the substrate, it may be combined or incorporated with another substrate in order to form the

electroporation device. For example, as described above the substrate incorporating the pattern of fluid channels and/or electrodes may form the lower half of the device, and a similar substrate (e.g., comprising the same material) could be placed on top of the patterned substrate and bonded thereto, for example by heating, to create the fluid channels and enclosed electrodes.

In various embodiments the fluid channels and/or electrodes may be formed by alternative or additional methods. For example, the substrate may be silicon (e.g., a silicon wafer) or glass. The formation of the fluid channels and/or electrodes may include one or more of micromachining, microfabricating, thin film deposition (such as chemical vapour deposition ("CVD") or physical vapour deposition ("PVD")), etching, patterning (e.g., photolithography), microforming (e.g., microstamping or microcutting), and any other suitable method.

Patterning of the substrate and electrodes to create the fluid channels can be achieved by injection moulding, embossing, reactive injection moulding, casting, photolithography, E-beam lithography, laser writing, direct machining, and spark erosion.

Patterning can be additionally or alternatively formed by wet etching (placed in a bath to remove unprotected material), dry etching (using a reactive gas to remove the unprotected material), chemical developing (reacting or solvating unprotected or pattered masking material), thermal or electric degradation.

Alternatively, or additionally, fluid channels can be formed in the probes during initial fabrication and patterning, e.g., by photolithography. Fluid channels can also be formed by post processing a substrate using, for example, direct machining/drilling, ion milling, nano imprinting, sonic cavitation, laser ablation, spark erosion.

The electrodes, for example the arrays of microelectrodes can be formed by, for example, sputtering, evaporation, chemical vapour deposition ("CVD"), electroless coating (wet deposition), electroplating or electrodeposition (wet with an applied field), particle sintering, low melting point flow, lamination.

Introduction of fluid and other considerations

The fluid (and any diagnostic or pharmaceutical agents contained therein) may be introduced into a tissue sample through the fluid channels disposed with the device. A voltage may be applied to the fluid itself. However, in all the aspects and embodiments disclosed herein one or more arrays of electrodes may be micropatterned into the device itself. These arrays of electrodes can enable additional electrode placements, such as described in relation to Figs. 1 -4B described above.

In various embodiments the devices or probes described herein may include a single array of micropatterned electrodes dispersed throughout the device, or at least throughout the portion of the device that is configured to be inserted into a tissue sample (e.g., the distal portion as defined above) and may comprise fluid openings for the passage of a diagnostic or pharmaceutical agent into the region surrounding the device or probe. The array or arrays of micropatterned electrodes may be configured to apply a voltage to fluid flowing through, or out of at least some of the fluid channels of the device (for example the networks of fluid channels described herein, including the secondary channels or "capillary" channels, including those that lead to the fluid openings), or to tissue adjacent to the fluid channel outlets. This may improve the application of a voltage to the cells.

The electroporation probe described herein can establish liquid delivery to a targeted region through electrophoretic and/or pressure driven flow through the fluid channels. The flow rates of fluid within the fluid channels or networks of fluid channels can be balanced and modulated, for example throughout an operation to control the delivery of one or more diagnostic or pharmaceutical agents.

In various embodiments the tissue adjacent the fluid outlets may be sampled, for example by drawing fluid back through the fluid channels and into a fluid analyser, for example including an electrochemical sensor. This may be achieved through the use of a negative pressure within the probe, or by reversing the electrical polarity.

In various embodiments sensors or other instruments can be integrated into the device, for example to sense the local environment. Examples include thermal sensors for monitoring of tissue breakdown and any thermal variance within the probe, impedance probes to sense the location of the probe within the tumor or tissue, or an electrochemical sensor to detect a particular analyte in the tissue.

To aid sensing and/or imaging, agents or contrasts may be delivered with the fluid flow. For example, an agent may be used that can only enter permeabilised cells, and be detected using a sensing or imaging method described herein, and the detection of this agent may be used to monitor which areas of the tissue have been porated.

As discussed herein, usage of the device may be through insertion into a human or animal body and into a region of cells, such as a tumor. The introduction of a diagnostic or pharmaceutical agent (e.g., antibodies) into the region may either be sequentially or simultaneously performed with the exposure of the tissue to a pulsed or continuous electrical field. In various embodiments agent delivery can be before or after the exposure of the tissue to the pulsed or continuous electric field. Single or repeated insertions of the device into the same or different regions can be performed to increase the insertion of antibodies into the same or other regions of the tumor or regions of cells.

The electric field distribution around the device upon application of a voltage (or voltages) may depend on the design of the electrodes and/or fluid channels, as well as the tissue being sampled and other factors. The design of the electroporation device and the electroporation parameters, e.g., applied voltages, pulse length and how individual electrodes and/or channels are used, may be adjusted appropriately to optimise the delivery of the diagnostic or pharmaceutical agent into the tissue. Examples

A working example will now be described in which HCT1 16 xenografted mice are subjected to intratumoral electroporation experiments, after the tumor has reached a size suitable for electrode implantation. Electrodes can be implanted with a guide holder in a stereotactic device. We can use TRPV1 as well as KRAS antibodies for distribution at three different concentrations. It is believed that the protocol is generic and will work equally well for all antibodies, independent of target.

We have developed two polyclonal antibodies, aCOTVI and aCOTV2, acting on the intracellular side of the human TRPV1 ion channel. Their functional effect after

internalisation of the antibodies using electroporation was tested, by measurement of TRPV1 mediated YO-PRO uptake using laser scanning confocal microscopy.

Cells were electroporated in the presence of either 0.14 mg/ml aCOTVI or 0.27 mg/ml aCOTV2 with a Neon transfection system (Life Technologies) using a protocol suitable for antibodies. Both antibodies were tip sonicated prior to use. Control cells were electroporated in buffer, and cells electroporated with aCOTVI were then exposed to 1 μΜ YO-PRO and 100 nM capsaicin in calcium free PBS and the fluorescence signal within the cells was measured.

A 60% decrease in uptake rate could be observed during the initial 10 s of activation. The highest uptake rate for a COTV1 treated cells were observed after 18 s compared to 8 s for control.

Cells electroporated with a COTV2 were exposed to 1 μΜ YO-PRO and 100 nM capsaicin in PBS containing 50 μΜ Ca2+ relaying on desensitization through endogenous calmodulin. The fluorescence signal within the cells was measured, and an 80% increase in uptake rate could be observed after 15 s of activation. Both antibodies retained their functional effect after electroporation.

Immunohistochemistry was performed in order to visualize the antibody distribution within the cells. Cells were electroporated in either the presence of antibody or without, then fixed using 4% formaldehyde followed by 0.5% Triton-100X. A secondary goat anti rabbit antibody tagged with Alexa 488 was added and the fluorescence was visualized with laser scanning confocal microscopy. Both aCOTVI and aCOTV2 were distributed in dots across the plasma membrane. It should be noted that aCOTVI and its control were subjected to less washing steps due to less rigid binding of either aCOTVI or the secondary antibody to aCOTVI .

Figs. 5A and 5B show the uptake of TRPV1 mediated YO-PRO after electroporation with aCOTVI in calcium free PBS. Fig. 5A shows a graph of fluorescence intensity for aCOTVI , and Fig. 5B shows a graph of corresponding fluorescence intensity for aCOTVI and control.

Figs. 5B and 5C show the uptake of TRPV1 mediated YO-PRO after

electroporation with aCOTV2 in the presence of 50 μΜ Ca2+. Fig. 5C shows a graph of fluorescence intensity for aCOTV2, and Fig. 5B shows a graph of fluorescence intensity for aCOTV2 and control. Data in Figs. 5A-5D is presented as mean ± SEM. Fig. 6 shows fluorescence images of the location of aCOTVI (top left) and aCOTV2 (bottom left) in the plasma membrane of cells after electroporation, using a secondary goat anti rabbit antibody tagged with Alexa 488. Controls were electroporated in buffer.

Although the present invention has been described with reference to preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made without departing from the scope of the invention as set forth in the accompanying claims.

Claims

Claims 1 . A device for electroporating cells, comprising:

one or more networks of microfluidic channels located within said device at said distal portion and in fluid communication with said fluid openings; and

one or more electrodes that extend to the distal portion of the device, for generating an electric field across the tissue sample at said distal portion.

2. A device as claimed in claim 1 , wherein the microfluidic channels and/or the one or more electrodes are formed using a microfabrication technique.

3. A device as claimed in claim 1 or 2, wherein the number and/or density of the microfluidic channels varies along the distal portion of the device.

4. A device as claimed in claim 1 , 2 or 3, wherein the cross-sectional area of the microfluidic channels varies along the distal portion of the device.

5. A device as claimed in any preceding claim, wherein said one or more networks of microfluidic channels comprises a plurality of different networks of microfluidic channels.

6. A device as claimed in claim 5, wherein each of said networks of microfluidic channels is in fluid communication with a different source of fluid.

7. A device as claimed in claim 5, wherein each of said networks of microfluidic channels is in fluid communication with the same source of fluid.

8. A device as claimed in claim 5, 6 or 7, wherein a first network of microfluidic channels comprises fluid channels having a first cross-sectional area, and a second network of microfluidic channels comprises fluid channels having a second, different cross- sectional area.

9. A device as claimed in any of claims 5-8, wherein a first network of microfluidic channels comprises fluid channels having a first density, and a second network of microfluidic channels comprises fluid channels having a second, different density.

10. A device as claimed in any of claims 5-9, wherein each network of microfluidic channels extends to a different region of said device.

1 1 . A device as claimed in any of claims 1 -4, wherein a single network of microfluidic channels is provided, which network is in fluid communication with a single source of fluid.

12. A device as claimed in any preceding claim, wherein each network of microfluidic channels comprises at least 2, 5 or 10 separate channels.

13. A device as claimed in any preceding claim, wherein each network of microfluidic channels comprises at least 50, 100 or 500 separate channels.

14. A device as claimed in any preceding claim, wherein each network of microfluidic channels comprises at least 1000, 5000 or 10,000 separate channels.

15. A device as claimed in any preceding claim, wherein each network of microfluidic channels comprises a pattern of branching or interconnecting fluid channels.

16. A device as claimed in any preceding claim, wherein said one or more electrodes are integrated into said device.

17. A device as claimed in claim 16, wherein said one or more electrodes are integrated into said device using a microfabrication technique.

18. A device as claimed in claims 16 or 17, wherein said one or more electrodes lead to an outer surface of said device or are positioned adjacent to an outer surface of said device.

19. A device as claimed in any of claims 1 -15, wherein said one or more electrodes are positioned on an outer surface of the device.

20. A device as claimed in any preceding claim, wherein said one or more electrodes comprises a plurality of electrodes.

21 . A device as claimed in claim 20, wherein a density of said plurality of electrodes varies along the length of the device.

22. A device as claimed in claim 20 or 21 , wherein said plurality of electrodes comprises a plurality of separate arrays of electrodes, wherein each array of electrodes is positioned in a different region of said device.

23. A device as claimed in claim 22, wherein each array of electrodes is connectable to a different voltage supply.

24. A device as claimed in claim 22, wherein each array of electrodes is connected to a common electrode for supplying a common voltage to each array of electrodes.

25. A device as claimed in claim 22 or 23, wherein a first array of electrodes comprises a first density of electrodes, and a second array of electrodes comprises a second, different density of electrodes.

26. A device as claimed in any preceding claim, wherein said one or more electrodes are arranged and configured such that a strength of said electric field is variable along the length of the device.

27. A device as claimed in any preceding claim, further comprising a plurality of regions located at the distal portion of the device, wherein each region comprises a separate array of electrodes and/or a separate network of fluid channels, such that an electric field strength and/or flow of fluid in each region can be separately controlled.

28. A device as claimed in claim 27, wherein a first region is located at the distal end of the device, such that upon insertion of the device into a tissue sample the first region penetrates deepest into the tissue sample, and a second region located a distance from the distal end of the device, such that upon insertion of the device into a tissue sample the second region penetrates less deep into the tissue sample than the first region.

29. A device as claimed in claim 27 or 28, wherein the electrodes in different regions differ in terms of a particular characteristic, such that each array of electrodes is arranged and configured to provide a different electric field strength.

30. A device as claimed in claim 27, 28 or 29, wherein the fluid channels in different regions differ in terms of cross-sectional area and/or density.

31 . A device as claimed in any preceding claim, wherein at least some of said fluid openings are located transverse to a longitudinal axis of said device, and/or are arranged and configured such that fluid exiting said fluid openings does so in a direction transverse to the longitudinal axis of said device.

32. A device as claimed in any preceding claim, further comprising one or more sensors arranged and configured to detect a characteristic of said tissue sample adjacent to said device.

33. A device as claimed in claim 32, wherein said one or more sensors comprises a thermal sensor arranged and configured to detect thermal variances within the tissue sample adjacent to said device, and/or within said device.

34. A device as claimed in claim 32 or 33, wherein said one or more sensors comprises an impedance sensor arranged and configured to detect the impedance of cells in said tissue sample adjacent to said device.

35. An electroporation apparatus comprising a device as claimed in any preceding claim.

36. An apparatus as claimed in claim 35, further comprising:

one or more voltage supplies; and

a control system arranged and configured to control said one or more voltage supplies to supply one or more voltages to said one or more electrodes.

37. An apparatus as claimed in claim 36, wherein said control system is arranged and configured to control said one or more voltage supplies to supply a voltage to said one or more electrodes that is sufficient to electroporate cells in said tissue sample.

38. An apparatus as claimed in claim 36 or 37 wherein said control system is arranged and configured to control said one or more voltage supplies to supply a voltage to said one or more electrodes that is sufficient to reversibly electroporate cells in said tissue sample, but does not irreversibly electroporate cells in said tissue sample.

39. An apparatus as claimed in claim 36, 37 or 38, wherein said control system is arranged and configured to control said one or more voltage supplies to supply a voltage to said one or more electrodes that is sufficient to irreversibly electroporate and/or terminate cells in said tissue sample.

40. An apparatus as claimed in any of claims 35-39, further comprising an imaging device arranged and configured to detect the location of said device within said tissue sample.

41 . An apparatus as claimed in claim 40, wherein said imaging device comprises a positron emission tomography ("PET") scanner, a computed tomography ("CT") scanner, a magnetic resonance imaging ("MRI") scanner, an ultrasound scanner or a fluorescent imaging scanner.

42. A method of manufacturing a device for electroporating cells, comprising:

preparing a substrate;

microfabricating one or more networks of fluid channels on said substrate;

forming said device using said substrate, wherein said device comprises a plurality of fluid openings and said one or more networks of fluid channels are in fluid

communication with said fluid openings.

43. A method as claimed claim 42, wherein said fluid openings are located at a distal portion of said device, and said distal portion is configured to be inserted into a tissue sample.

44. A method as claimed in claim 42 or 43, further comprising microfabricating one or more holes in said device, wherein said one or more holes form said fluid openings.

45. A method as claimed in claim 42, 43 or 44 further comprising microfabricating one or more electrodes on said substrate prior to said step of forming said device using said substrate.

46. A method as claimed in claim 45, wherein said one or more electrodes comprises one or more arrays of electrodes.

47. A method as claimed in claim 46, wherein said one or more arrays of electrodes comprises a uniform pattern of interconnected electrodes along the entire length of the substrate.

48. A method as claimed in any of claims 42-47, wherein said one or more networks of fluid channels and/or said one or more arrays of electrodes are microfabricated into said substrate so as to form a plurality of regions, wherein each region comprises a separate array of electrodes and/or a separate network of fluid channels.

49. A method as claimed in claim 48, further comprising locating said one or more regions to match or correspond to a particular tissue sample.

50. A method as claimed in any of claims 42-49, wherein said substrate comprises one or more of a polymeric material, a semi-conducting material, metal, silicon and glass.

51 . A method as claimed in any of claims 42-50, wherein said microfabricating comprises one or more of micromachining, thin film deposition, chemical vapour deposition ("CVD"), physical vapour deposition ("PVD"), etching, photolithography

52. A method as claimed in any of claims 42-51 , wherein said microfabricating comprises one or more of microforming, microstamping or microcutting.

53. A method as claimed in any of claims 42-52, wherein said microfabricating comprises patterning of the substrate to create said one or more networks of fluid channels and/or said one or more electrodes.

54. A method as claimed in claim 53, wherein said patterning is achieved by injection moulding, embossing, reactive injection moulding, casting, photolithography, E-beam lithography, laser writing, direct machining and spark erosion.

55. A method as claimed in claim 53, wherein said patterning is achieved by wet etching, dry etching, chemical developing, thermal degradation and electric degradation.

56. A method as claimed in any of claims 42-55, wherein said microfabricating comprises post-processing said substrate.

57. A method as claimed in any of claims 42-56, wherein said microfabricating comprises one or more of direct machining/drilling, ion milling, nano imprinting, sonic cavitation, laser ablation, spark erosion.

58. A method as claimed in any of claims 42-57, wherein said one or more electrodes are formed by sputtering, evaporation, chemical vapour deposition ("CVD"), electroless coating, electroplating, electrodeposition, particle sintering, low melting point flow and lamination.

59. A method as claimed in any of claims 42-58, further comprising shaping said substrate and/or device to match or correspond to a particular tissue sample.

60. A microelectrode for use in electroporation, comprising a plurality of regions located at a distal portion of the microelectrode, wherein each region comprises a separate array of electrodes and/or a separate network of fluid channels, such that an electric field strength and/or flow of fluid at each region is separately controllable.

61 . A method of fabricating a device for electroporating cells, comprising:

identifying a tissue sample to be treated with said device;

obtaining image data corresponding to said tissue sample;

designing a shape or arrangement of said device based on said obtained image data; and

fabricating said device according to said designed shape or arrangement.

62. A method as claimed in claim 61 , wherein said designing comprises determining an arrangement of fluid channels and/or electrodes within said device based on said obtained image data.