Vacuum-assisted electroporation devices, and related systems and methods

Abstract

To provide vacuum-assisted electroporation devices, and related systems and methods.SOLUTION: A device for vacuum-assisted in vivo electroporation of tissues includes a housing that defines a chamber and at least one opening into the chamber. A port extends through the housing, is remote from the at least one opening, and is connectable to a vacuum source. The port is configured to communicate vacuum pressure from the vacuum source to the chamber. Multiple electrodes are positioned within the chamber and are configured to deliver one or more electroporation pulses to a targeted portion of a tissue extending through the opening and held in the chamber responsively to the vacuum pressure.SELECTED DRAWING: Figure 2A

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 992,513, filed March 20, 2020, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to devices for grasping and deforming tissue using vacuum pressure, injecting fluids into tissue, and electroporating tissue using electrodes, as well as systems and methods for injecting or otherwise delivering fluids to tissue and electroporating tissue. [Background technology]

[0003] In the 1970s, it was discovered that electric fields could be used to create pores in cells without causing permanent damage to the cells. This discovery, called electroporation (EP), allowed large molecules, small molecules, ions, and water to be introduced through the cell wall into the cytoplasm of the cell. In some instances, electroporation can be used to introduce chemicals and other compounds into tumors in localized treatments such as head and neck cancer. During these procedures, patients may or may not be under general anesthesia, so pain and involuntary muscle movements need to be minimized.

[0004] Skin is a popular target for EP because it is easily accessible and contains a rich variety of immune cells suitable for DNA vaccine delivery. The skin's natural immune function and its high cell turnover typically lead to a rapid and strong humoral response to EP-enhanced DNA vaccine delivery. Skin is also capable of generating a cellular immune response following EP-enhanced DNA vaccine delivery. Due to its superficial nature, skin is suitable for minimally or non-invasive EP.

[0005] Skeletal muscle is also a well-characterized target for electroporation-mediated (EP) delivery of DNA in vivo. Muscle cells are capable of producing and secreting proteins over extended periods of time, and it has been repeatedly demonstrated that EP-enhanced DNA vaccination into muscle can generate an immune response. However, the application of EP DNA delivery to muscle is complicated by the different thicknesses of subcutaneous fat, which results in different penetration depths of the needle into muscle tissue, thus preventing a "one size fits all" approach. Skeletal muscle is typically not amenable to minimally or non-invasive EP techniques, especially in large animals and humans, due to the insulating subcutaneous fat layer and the depth required to generate an electric field. Therefore, penetrating needle electrodes are most commonly used to perform EP in muscle.

[0006] Historically, adipose tissue (fat) has been considered an inert tissue used primarily to store energy in the form of lipid droplets. Therefore, it is only recently that EP-enhanced DNA procedures have been directed to the fat layer of the tissue. However, recent studies have shown that subcutaneous fat actually plays many dynamic roles. Adipose tissue contains many stem cells and immune cells, acts as an endocrine organ by secreting numerous hormones, secretes many local signals, and contains an elaborate capillary network. Attempts to achieve in vivo transfection of adipose tissue have been limited primarily to surgical techniques, which require the administrator to cut and physically remove samples of the patient's skin to allow direct contact with the adipose tissue. These procedures are highly invasive and not suitable for clinical devices. Summary of the Invention

[0007] According to one embodiment of the present disclosure, an apparatus for vacuum-assisted in vivo electroporation of tissue includes a housing defining a chamber and at least one opening into the chamber. A port extends through the housing, is remote from the at least one opening, and is connectable to a vacuum source. The port is configured to transmit vacuum pressure from the vacuum source to the chamber. A plurality of electrodes are positioned within the chamber and configured to extend through the opening in response to the vacuum pressure to deliver one or more electroporation pulses to a target portion of tissue held at least momentarily within the chamber.

[0008] According to another embodiment of the present disclosure, a method of electroporating tissue of a subject includes placing a chamber adjacent to the tissue, applying a vacuum pressure to the chamber thereby drawing the tissue through an opening in the chamber and into contact with a plurality of electrodes extending along an inner surface of the chamber, and delivering one or more electroporation pulses to the tissue through the plurality of electrodes thereby generating an electroporation field within the tissue.

[0009] According to a further embodiment of the present disclosure, an apparatus for vacuum-assisted treatment of tissue includes a housing defining a chamber and at least one opening into the chamber. A first port extends through the housing and is spaced from the at least one opening. The first port is connectable to a vacuum source such that the first port is configured to transmit vacuum pressure from the vacuum source to the chamber. The apparatus includes a jet injection device extending into the chamber through a second port. The second port is opposite the at least one opening. The jet injection device is configured to extend through the at least one opening in response to the vacuum pressure and deliver a jet injection of fluid to a target portion of tissue held within the chamber at least momentarily. [Brief description of the drawings]

[0010] This patent application contains at least one drawing executed in color. Copies of this patent application containing color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The foregoing summary, as well as the following detailed description of exemplary embodiments of the present application, will be better understood when read in conjunction with the accompanying drawings. For the purpose of explaining the structure of the present application, there are shown in the drawings exemplary embodiments. It should be understood, however, that the present application is not limited to the precise arrangements and apparatus shown.

[0011] [Figure 1] FIG. 1 is a schematic diagram of an electroporation system utilizing a vacuum-assisted electroporation cup (or "vacuum cup") in combination with a needle injection device, according to one embodiment of the present disclosure. [Figure 2A] 2 is a perspective view of the vacuum cup shown in FIG. 1 showing the vacuum chamber of the cup and an array of electrodes located within the chamber according to one embodiment of the present disclosure. [Figure 2B] FIG. 2B is a bottom view of the vacuum cup shown in FIG. 2A showing the pattern in which the electrodes are arranged. [Figure 2C] FIG. 2C is a plan elevational view of one of the electrodes shown in FIGS. 2A-2B. [Figure 2D] 2B is a cross-sectional side view of the vacuum cup shown in FIG. 2A taken along the central axis of the cup. [Figure 2E] FIG. 2E is an enlarged cross-sectional view of area 2E-2E shown in FIG. 2D. [Figure 2F] FIG. 2F is an enlarged cross-sectional view of area 2F-2F shown in FIG. 2D. [Figure 3A] FIG. 2B is a perspective view of a sleeve that carries an electrode and is insertable into the vacuum chamber shown in FIG. 2A. [Figure 3B] FIG. 3B is a bottom view of the sleeve shown in FIG. 3A. [Figure 3C-3M] 11A-11C are bottom and cross-sectional side views, respectively, of a sleeve having an alternative electrode array pattern according to an additional embodiment of the present disclosure. [Figure 4A-4F]Figures 4A-4E are cross-sectional side views of the vacuum cup shown in Figure 2A adjacent tissue, illustrating representative stages of utilizing the vacuum cup in a vacuum-assisted electroporation procedure, and Figure 4F is a cross-sectional side view of the vacuum cup shown in Figure 2A, illustrating the various degrees to which the vacuum cup can draw tissue into the vacuum chamber. [Figure 5A] FIG. 1 illustrates a perspective view of a vacuum cup having flexible electrodes according to one embodiment of the present disclosure. [Figure 5B] FIG. 5B is a cross-sectional side view of the vacuum cup shown in FIG. 5A taken along the central axis of the cup. [Figure 5C] FIG. 5C is an enlarged cross-sectional view of area 5C-5C shown in FIG. 5B, showing the electrodes in a neutral state. [Figure 5D] FIG. 5D is an enlarged cross-sectional view of area 5D-5D shown in FIG. 5B, showing the electrodes in a deflected state. [Figure 6A-6B] 1A and 1B are perspective and side elevational views, respectively, of an electrode having a contact surface defining a protrusion, according to one embodiment of the present disclosure; [Figure 6C-6D] 1A and 1B are perspective and side elevational views, respectively, of an electrode having a contact surface defining a transversely elongated protrusion, according to another embodiment of the present disclosure; [Figure 7A] FIG. 1 illustrates a perspective view of a vacuum cup having a triangular distal opening and a vacuum chamber geometry according to one embodiment of the present disclosure. [Figure 7B] FIG. 7B is a bottom view of the vacuum cup shown in FIG. 7A. [Figure 7C] FIG. 7B is a cross-sectional perspective view of the vacuum cup shown in FIG. 7A taken along the central axis of the cup. [Figure 7D] FIG. 7B is an elevated plan view of an electrode array positioned within the vacuum cup shown in FIG. 7A. [Figure 7E] FIG. 7E is a side view of the electrode array shown in FIG. 7D. [Figure 8A] FIG. 1 illustrates a perspective view of a vacuum cup having a rectangular distal opening and a vacuum chamber geometry according to one embodiment of the present disclosure. [Figure 8B] FIG. 8B is a bottom view of the vacuum cup shown in FIG. 8A. [Figure 8C] FIG. 8B is a cross-sectional perspective view of the vacuum cup shown in FIG. 8A taken along the central axis of the cup. [Figure 9A] FIG. 1 is a cross-sectional side view of an electroporation assembly including a vacuum cup having a receptacle in which a needle-free injection device is received, according to one embodiment of the present disclosure. [Figure 9B] 9B is an enlarged cross-sectional side view showing a representative step of utilizing the electroporation assembly shown in FIG. 9A to inject an agent into tissue drawn into a vacuum chamber. [Figure 10A] FIG. 13 is a cross-sectional perspective view of another embodiment of a vacuum cup having a receptacle in which a needle-free injection device is received, the vacuum cup having a cup housing defining a manifold including a plurality of ports in fluid communication with the vacuum chamber through a corresponding plurality of openings defined in the electrode. [Figure 10B] FIG. 10B is an enlarged perspective partial cross-sectional view of the corresponding ports and openings shown in FIG. 10A. [Figure 10C] 11A-11C are perspective, partial cross-sectional views of alternative configurations of corresponding ports and openings according to another embodiment of the present disclosure. [Figure 10D] FIG. 10B is an enlarged cross-sectional side view of a portion of the vacuum cup shown in FIG. 10A during use to provide electroporation treatment to adipose tissue. [Figure 11A] FIG. 2B is a visual representation of the distribution of methylene blue in subcutaneous porcine tissue without (left) and with (right) applied vacuum pressure via a vacuum cup configured similarly to the cup shown in FIG. 2A. [Figure 11B-11C] A side-by-side comparison of fluid distribution in guinea pig adipose tissue following injection of methylene blue is shown, where the injection in FIG. 11B was performed using a needleless vacuum cup similar to that shown in FIG. 9A, and the injection in FIG. 11C was performed with a subcutaneous needle method. [Figure 12]FIG. 2B is a graph showing humoral immunogenicity ELISA data in guinea pigs after electroporation in adipose tissue with pGX2013 (DNA vaccine against influenza virus nucleoprotein (NP)) at 12 weeks, specifically showing the comparative humoral immune response after electroporation using the vacuum cup shown in FIG. 2A and the caliper electrode electroporation device. [Figure 13] 2B is a graph showing 8-week humoral immunogenicity ELISA data in guinea pigs following treatment with pGX2303 (a DNA vaccine against human respiratory syncytial virus fusion glycol-protein (RSV-F)), in particular showing comparative humoral immune responses following electroporation of adipose tissue using the vacuum cup shown in FIG. 2A and following electroporation in the skin using a prior art needle array electroporation device. [Figure 14A] 1 is a graph showing six-week humoral immunogenicity ELISA data in guinea pigs following treatment with pGX2013 (a DNA vaccine against influenza virus nucleoprotein (NP)), in particular showing comparative humoral immune responses following treatment with: (1) electroporation of intradermal tissue using a prior art needle array electroporation device, (2) electroporation of adipose tissue using a needle-less vacuum cup as shown in FIG. 9A with vacuum pressure applied after electroporation, (3) injection into adipose tissue using a needle-injected vacuum cup as shown in FIG. 2A with vacuum pressure applied after electroporation but no electroporation, and (4) and electroporation of adipose tissue using a needle-injected vacuum cup as shown in FIG. 2A with vacuum pressure applied after electroporation. [Figure 14B] FIG. 14B is a chart showing the cellular immune response from the same study shown in FIG. 14A. [Figure 15A] FIG. 1 is a perspective view of a vacuum cup configured for vacuum-assisted electroporation of intradermal tissue according to one embodiment of the present disclosure. [Figure 15B] FIG. 15B is a cross-sectional side view of the vacuum cup shown in FIG. 15A. [Figure 15C] FIG. 15B is an enlarged cross-sectional view of the vacuum chamber of the cup shown in FIG. 15A during use. [Figure 16A-16B]FIG. 1B is a cross-sectional side view of a vacuum electroporation assembly with electrodes disposed on an end surface within each vacuum chamber facing a distal opening of each of the vacuum chambers, according to an embodiment of the present disclosure. [Figure 17A-17B] 16A-16B , according to an embodiment of the present disclosure. FIG. 16B is a bottom view of an electrode array pattern and a vacuum port pattern defined on each electrode support member for use with the assembly shown in FIGS. [Figure 18A] FIG. 1 is a perspective view of a vacuum electroporation device having multiple distal vacuum chambers. [Figure 18B] FIG. 18B is a cross-sectional perspective view of the vacuum electroporation device shown in FIG. 18A. [Figure 18C] FIG. 18B is a side cross-sectional view of the vacuum electroporation device shown in FIG. 18A. [Figure 18D] FIG. 18D is an enlarged cross-sectional view of area 18D-18D shown in FIG. 18C. [Figure 18E] FIG. 18B is a bottom view of the vacuum electroporation device shown in FIG. 18A. [Figure 19A] FIG. 1 is a cross-sectional side view of a vacuum electroporation device having a distal array of electrodes having vacuum ports therein according to one embodiment of the present disclosure. [Figure 19B] FIG. 19B is a bottom view of the vacuum electroporation device shown in FIG. 19A showing a top view of the electrode array pattern according to one embodiment of the present disclosure. [Figure 20] FIG. 19B is a plan view of an electrode support member having a square electrode array configured for use with a vacuum electroporation device similar to that shown in FIG. 19A. [Figure 21A] FIG. 21 is a plan view of an electrode support member having a rectangular electrode array, similar to the electrode support member shown in FIG. 20. [Figure 21B] FIG. 21B is a side cross-sectional view of the electrode support member shown in FIG. 21A. [Figure 22] FIG. 1 shows gene expression in guinea pig skin after intradermal injection of various volumes of a plasmid encoding the gene for green fluorescent protein (GFP) followed by treatment with various techniques and devices. [Figure 23]2A is a graph showing 8-week humoral immunogenicity ELISA data in guinea pigs following treatment with an HPV DNA vaccine, specifically showing comparative humoral immune responses following respective electroporation treatments in the skin using a prior art needle array electroporation device, a vacuum cup as shown in FIG. 2A, and a vacuum cup as shown in FIG. 2A with a three-fold (3x) dose of the other treatments. [Figure 24A] 2A and 2B are graphs showing humoral immunogenicity ELISA data at 11 weeks in a non-human primate model following treatment with HPV DNA vaccines (pGX3001&3002), specifically showing comparative humoral immune responses following respective electroporation treatments in the skin using a prior art needle array electroporation device, a vacuum cup as shown in FIG. 2A, and a vacuum cup as shown in FIG. 2A with three times (3x) the dosage of the other treatments. [Fig. 24B-24C] FIG. 24B is a chart showing the cellular immune response from the same study shown in FIG. 24A. [Diagram 25] FIG. 2 is a cross-sectional image showing a perspective view of a pair of blebs resulting from a prior art Mantoux injection, where the bleb shown on the left contains a drug premixed with a hyaluronidase preparation and the bleb shown on the right was injected without the hyaluronidase preparation. [Figure 26] 26 is a composite image showing a top view and a perspective view of the bleb shown in FIG. 25. [Figure 27] 2A is a plot showing 4-week humoral immune response data in terms of endpoint titers in guinea pigs following intradermal treatment with a DNA vaccine against MERS (pGX9101), specifically showing the comparative humoral immune response following respective electroporation treatments in the skin using a prior art needle array electroporation device with and without a hyaluronidase formulation and a version of the vacuum cup shown in FIG. 2A, both with and without a hyaluronidase formulation, having a 15 mm chamber diameter. [Figure 28]FIG. 2 is a graph showing six-week humoral immunogenicity data in guinea pigs in terms of endpoint titers following intradermal treatment with MERS DNA vaccine (pGX9101), specifically showing comparative humoral immune responses following electroporation treatment in the skin using a prior art needle array electroporation device and the vacuum cup version shown in FIG. 2A having chamber diameters of 15 mm and 10 mm, respectively. [Figure 29A] FIG. 2B is a graph showing six-week humoral immunogenicity data in terms of mean endpoint titers in guinea pigs following intradermal injection of a DNA vaccine against influenza nucleoprotein (pGX2013), specifically showing comparative humoral immune responses following respective electroporation treatments in the skin using a prior art needle array electroporation device and a version of the vacuum cup shown in FIG. 2A having a chamber diameter of 15 mm. [Fig. 29B-29C] 29A-C are charts showing the cellular immune response in terms of spot forming units at 2 weeks (FIG. 29B) and 4 weeks (FIG. 29C) from the same study shown in FIG. 29A. [Fig. 30A-30F] Figure 30A shows a perspective view of a circular electrode array having four electrodes spaced 90 degrees apart about a central axis for use with the circular aperture vacuum cup of the present disclosure to electroporate adipose tissue. Figures 30B-30F show the various simulated electric field strengths generated in adipose tissue according to various electrode sizes. [Fig. 31A-31F] Figure 31A shows a perspective view of the circular electrode array shown in Figure 30A for use in electroporating skin tissue. Figures 31B-31F show various simulated electric field strengths generated in skin tissue according to various electrode sizes. [Fig. 32A-32F] Figure 32A shows a perspective view of a rectangular electrode array having four planar electrodes spaced 90 degrees apart about a central axis for use with the rectangular aperture vacuum cup of the present disclosure to electroporate adipose tissue. Figures 32B-32F show the various simulated electric field strengths generated in adipose tissue according to various electrode sizes. [Fig. 33A-33F]Figure 33A shows a perspective view of the rectangular electrode array shown in Figure 32A for use in electroporating skin tissue. Figures 33B-33F show the various simulated electric field strengths generated in skin tissue according to various electrode sizes. [Fig. 34A-34B] A side-by-side comparison of cellular infiltration (blue) in guinea pig adipose tissue after vacuum-assisted treatment is shown, where the treatment in FIG. 34A was a vacuum-only treatment using a vacuum cup similar to that shown in FIG. 2A (i.e., no electroporation), and the treatment in FIG. 35B was a vacuum-assisted electroporation treatment using a vacuum cup (i.e., vacuum pressure + electroporation). [Figure 35A] FIG. 2C is a graph showing 6-week humoral immunogenicity data in guinea pigs following treatment with a DNA vaccine against MERS, specifically showing comparative humoral immune responses following each electroporation treatment in the skin using a vacuum cup similar to that shown in FIG. 2A, with various chamber diameters and the same electroporation parameters. [Figure 35B] FIG. 35B is a chart showing the cellular immune response from the same study shown in FIG. 35A. [Figure 36A] FIG. 1 shows gene expression in guinea pig skin after intradermal injection of a plasmid encoding the gene for green fluorescent protein (GFP) followed by treatment with various vacuum pressures and electroporation voltages using a vacuum cup. [Figure 36B] FIG. 36B is a graph showing measurements of skin fluorescence signal from the GDF expression results shown in FIG. 36A. [Figure 37A] FIG. 2B is a graph showing 6-week humoral immunogenicity ELISA data in guinea pigs following treatment with a DNA vaccine against MERS, specifically showing the comparative humoral immune response following respective electroporation treatments in the skin using (1) a prior art needle array electroporation device and (2) a vacuum cup similar to that shown in FIG. 2A. [Figure 37B] FIG. 37B is a chart showing the cellular immune response from the same study shown in FIG. 37A. [Figure 38]FIG. 2B is a chart showing the cellular immune response in terms of spot forming units at 6 weeks after treatment with a vaccine against recurrent respiratory papillomatosis (RRP), specifically showing the comparative response after respective electroporation treatments in the skin using (1) a prior art needle array electroporation device and (2) a vacuum cup similar to that shown in FIG. 2A. [Figure 39A] FIG. 2B is a graph showing humoral immunogenicity data in guinea pigs after treatment with a DNA vaccine against MERS over a 4-week period, specifically comparing humoral immune responses after treatment in the skin with Mantoux injection alone, vacuum pressure followed by Mantoux injection, and vacuum pressure followed by Mantoux injection and electroporation using a vacuum cup similar to that shown in FIG. 2A. [Figure 39B] FIG. 39B is a chart showing the cellular immune response from the same study shown in FIG. 39A. [Figure 40A] FIG. 2B shows a cross-sectional side view of a simulated electric field produced by an electrode array having a pair of opposing electrodes when generated in a mound of tissue drawn into a vacuum cup configured similarly to the vacuum cup shown in FIG. 2A. [Figure 40B] FIG. 15B is a cross-sectional side view of a simulated electric field produced by an electrode array having a pair of annular ring electrodes and a central concentric electrode when generated in a mound of tissue drawn into a vacuum cup configured similarly to the vacuum cup shown in FIG. 15A. [Figure 41A] 40A-40B are graphs showing 8-week humoral immunogenicity data in guinea pigs following treatment with a DNA vaccine against MERS, specifically comparing humoral immune responses following vacuum-assisted electroporation treatment in the skin based on electrode pulse patterns using the electrode array shown in FIGS. 40A-40B. [Figure 41B] FIG. 41B is a chart showing the cellular immune response from the same study shown in FIG. 41A. [Figure 42A]40A and 40B are graphs showing comparative injectate fluid distribution following a Mantoux injection with (1) no vacuum pressure applied, (2) vacuum pressure applied using a vacuum cup with an electrode array as shown in FIG. 40A, and (3) vacuum pressure applied using a vacuum cup with an electrode array as shown in FIG. 40B. [Fig. 42B-42D] FIG. 42B is a photograph showing the fluid distribution charted in FIG. 42A. [Figure 43A] FIG. 43A is a fluoroscopic image showing an injectate disposed in guinea pig skin positioned under a vacuum cup having an electrode array as shown in FIG. 40B, and FIG. 43A shows the injectate prior to application of vacuum pressure within the chamber. [Figure 43B] FIG. 43B is a fluoroscopic image showing an injectate disposed in guinea pig skin positioned under a vacuum cup with an electrode array as shown in FIG. 40B, and FIG. 43B shows the injectate during application of vacuum pressure within the chamber. [Figure 43C] 4 is a graph showing a comparison of secreted alkaline phosphatase (SEAP) expression from: (1) a single high injection volume vacuum assisted electroporation procedure using the vacuum cup shown in FIG. 43A-B and (2) a six injection, six electroporation event procedure using a prior art needle array electroporation device. [Diagram 44] 9 is a chart showing 8-week humoral immunogenicity ELISA data in guinea pigs following electroporation in skin tissue with pGX2013, specifically comparing humoral immune responses following electroporation using (1) a jet injection vacuum cup similar to that shown in FIG. 9A and (2) a prior art needle array electroporation device. [Fig. 45A-47C] 9B-9C are fluoroscopic images showing tissue deflection in a guinea pig during and after jet injection performed using a jet injection vacuum cup similar to that shown in FIG. 9A at various vacuum pressure and nozzle-skin offset distance settings. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present disclosure may be more readily understood by reference to the following detailed description, which is provided in conjunction with the accompanying figures and examples that form a part of this disclosure. It is to be understood that the present disclosure is not limited to the specific apparatus, methods, applications, conditions, or parameters described and / or illustrated herein, and that the terminology used herein is for the purpose of illustrating specific embodiments and is not intended to limit the scope of the present disclosure. Also, as used in this specification, including the appended claims, the singular forms "a," "an," and "the" include the plural, and reference to a particular numerical value includes at least that particular value unless the context clearly dictates otherwise.

[0013] As used herein, the term "plurality" means more than one. When ranges of values ​​are expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. All ranges are inclusive and combinable.

[0014] The terms "about" and "substantially" as used herein with respect to dimensions, angles, and other geometric features take into account manufacturing tolerances. Additionally, the terms "about" and "substantially" may include 10% greater or less than the recited dimension or angle. Additionally, the terms "about" and "substantially" may be equally applicable to the specific values ​​recited.

[0015] The term "agent" as used herein means a polypeptide, a polynucleotide, a small molecule, or any combination thereof. The agent may be a recombinant nucleic acid sequence encoding an antibody, a fragment thereof, a variant thereof, or a combination thereof. The agent may be formulated in water or a buffer such as, as non-limiting examples, saline-sodium citrate (SSC) or phosphate buffered saline (PBS).

[0016] As used herein, the term "intradermal" means within the layers of the skin, including the epidermis (i.e., the layer of the epidermis from the stratum corneum to the basal layer) and the dermis (i.e., the dermal layer).

[0017] As used herein, the term "adipose" refers to the layer containing adipocytes (ie, fat cells) present in the subcutaneous layer.

[0018] As used herein, the term "electroporation" refers to the use of an electric field within tissue to temporarily and reversibly increase the permeability and / or porosity of cell membranes of cells within the tissue, thereby allowing, for example, agents to be introduced into the cells.

[0019] As used herein, the term "electroporation field" refers to an electric field capable of electroporating a cell. In instances where an electric field includes a portion capable of electroporating a cell and another portion not capable of electroporating a cell, "electroporation field" specifically refers to the portion of the electric field capable of electroporating a cell. Thus, an electroporation field can be a subset of an electric field.

[0020] As used herein, the term "zone" refers to a volume of space, such as a volume of space within tissue.

[0021] As used herein, the term "transfection zone" refers to the volume of tissue where transfection occurs and may be used synonymously with the term "transfection volume."

[0022] As used herein, the term "cell infiltration" refers to the migration of cells into a volume of tissue.

[0023] As used herein, the terms "intradermal needle electrode electroporation device" and "ID needle electrode EP" each refer to a prior art electroporation device that utilizes an electrode array of three needle electrodes arranged in a triangular pattern to electroporate intradermal tissue.

[0024] The embodiments described below relate to systems and devices for vacuum-assisted electroporation of tissue, particularly target layers of tissue such as intradermal or adipose tissue. These embodiments subject a target volume of tissue (or "tissue volume") to vacuum pressure (i.e., negative pressure) to deform the tissue in a manner suitable for electroporating cells in a target zone within the tissue layer. In particular, an open end of a vacuum device, such as a vacuum cup, is placed in contact with an outer surface of the tissue (e.g., "skin") covering the tissue volume, and vacuum pressure is applied to the interior of the cup, thereby drawing the tissue volume into the vacuum cup, thereby positionally fixing the tissue volume to the cup and allowing electrodes positioned within the vacuum cup to generate a predictable and substantially uniform electroporation field within the tissue volume, thereby resulting in a predictable and substantially uniform transfection zone within the tissue volume. The vacuum pressure provided by the embodiments described below has also been observed to result in suitable redistribution of fluids within the tissue volume, including suitable in vivo dispersion of infusate within the tissue volume, as well as suitable in vivo movement of fluids into and out of the target zone. In other words, the vacuum pressure improves distribution of the injectate throughout the tissue, enlarging the transfection zone, and also draws more in vivo fluid into the target zone, increasing the amount of cells exposed to the transfected tissue. The inventors have observed that the vacuum-assisted electroporation procedures described throughout this disclosure result in an increased response of the subject to the injectate.

[0025] The inventors have also surprisingly and unexpectedly observed that applying vacuum pressure can induce transfection within a tissue volume without electroporation. Without wishing to be bound by any particular theory, the inventors believe that the vacuum pressure applied by the vacuum cup exerts mechanical stress on the cell membrane within the tissue volume, increasing cell membrane permeability and thus increasing the transfection observed within the tissue volume. The inventors also believe that the aforementioned fluid redistribution may also be at least partially responsible for the transfection observed without electroporation. The inventors further believe that the aforementioned fluid redistribution and mechanical stress likely interact with each other to generate an environment within the tissue volume that is favorable for the transfection of external agents into the cells.

[0026] Furthermore, the embodiments described below can be adapted between uses and / or between uses without mechanical reconfiguration. For example, between uses and / or between uses, the electrical parameters of the electrodes can be adjusted as needed to manipulate the electroporation field in the tissue volume to achieve a suitable treatment outcome. Additionally or alternatively, the vacuum pressure can be adjusted between uses and / or between uses as needed to physically manipulate the tissue volume to achieve a suitable treatment outcome. For example, a higher vacuum pressure can be applied to draw a larger tissue volume into the vacuum cup, and a lower vacuum pressure can be applied to draw a smaller tissue volume into the vacuum cup. In this manner, the same vacuum cup can be utilized to target different tissue layers for electroporation treatment by selectively exposing the different tissue layers to the electroporation field. Additionally, the vacuum pressure can be pulsed between uses to manipulate the mechanical behavior of the target tissue, such as to improve fluid redistribution within the tissue.

[0027] Referring now to FIG. 1, an electroporation system 100 for treating a patient according to the present disclosure includes a vacuum-assisted electroporation device 2 including a housing 4 defining an interior vacuum chamber 6 and a plurality of electrodes 8 (see, e.g., FIGS. 2A-2C) positioned within the chamber 6. The plurality of electrodes 8 are arranged in an array 9 of electrodes 8, which may also be referred to as an electrode array 9. The device 2 may also be referred to as a "vacuum cup" or simply a "cup." The housing 4 may also be referred to as a "cup housing." The vacuum cup 2 is configured to enable a physician to place a distal end 10 of the vacuum cup 2 on an exterior surface 101 of tissue 102 targeted for an electroporation procedure and apply vacuum pressure to the vacuum chamber 6 to draw, pull or otherwise direct the tissue 102 into the vacuum chamber 6 and into contact with the electrode 8 therein. The electrode 8 is configured to deliver one or more electroporation pulses to the tissue 102 drawn into and held within the chamber 6 by the vacuum pressure. Tissue 102 includes a layer targeted for treatment, such as adipose tissue 103 (also referred to herein as the "adipose layer" 103) or intradermal tissue 104 (also referred to herein as the "skin layer" 104).

[0028] The vacuum cup 2 includes one or more couplings, such as ports, for connection to one or more external components. For example, the vacuum cup 2 has a first port 12 for providing fluid communication between the vacuum chamber 6 and a vacuum source 106, such as a vacuum pump. The vacuum cup 2 can also have a second port 14 for providing access to a circuit 108 that provides electrical communication between the electrodes 8 and an energy source 110, such as a generator. The vacuum cup 2 can further include a third port 16 for providing access to the vacuum chamber 6 for an external tool, such as an injection device 18 carrying an injectate, particularly an injectate including a medicament. As illustrated, the injection device 18 can be a hypodermic infusion needle, although the vacuum cup 2 can be adapted for use with other types of injection devices 18, including jet injection devices, as described in more detail below. It should also be understood that the vacuum cup 2 can optionally be utilized after the medicament has been injected into the tissue 102.

[0029] The energy source 110 may be in electrical communication with a signal generator 112, such as a waveform generator, for generating and transmitting an electrical signal in the form of one or more electrical pulses having specific electrical parameters to the electrodes 8 to electroporate cells in the tissue 102 within the vacuum chamber 6. Such electrical parameters include electrical potential (voltage), current type (alternating current (AC) or direct current (DC)), current magnitude (amperage), pulse duration, pulse volume (i.e., number of pulses delivered), and time interval or "delay" between pulses (in case of multiple pulse delivery). The signal generator 112 may include a waveform logger for recording the electrical parameters of the delivered pulses. The signal generator 112 may be in electrical communication with a control unit 114 (also referred to herein as a "controller"), which may include a processor 116 configured to control the operation of the electroporation system 100, including the operation of the signal generator 112. The processor 116 may be in electronic communication with a computer memory 118 and may be configured to execute software and / or firmware including one or more algorithms for controlling the operation of the system 100. The processor 116 may be in electrical communication with a user interface 120, which may include a display 122 for presenting information related to the operation of the system 100, and a keypad 124 for allowing an operator, such as a physician, to input information such as commands related to the operation of the system 100. It should be understood that the display 122 may be a touch screen display, allowing an operator to input information directly at the display 122. It should also be understood that the interface 120 may be a computer interface, such as a tabletop or laptop computer, or a handheld electronic device, such as a smartphone, or the like.

[0030] 2A-2B, the distal end 10 of the vacuum cup 2 defines at least one opening 20 that leads into the vacuum chamber 6. The opening 20 may be circular as shown, although other opening shapes are within the scope of the present disclosure, as described in more detail below. The distal end 10 of the vacuum cup 2 (and thus the opening 20) may be defined by a housing 4, which may define an inner surface 22 that extends from the distal end 10 of the housing to a proximal end 24 of the chamber 6. Thus, the chamber 6 also extends from the distal end 10 to the proximal end 24. The inner surface 22 at least partially defines a boundary of the vacuum chamber 6. The inner surface 22 preferably has a bell-shaped or "bell curve" geometric shape. A distal portion 22a of the inner surface 22 that leads from the distal end 10 into the chamber 6 may have a tapered curved profile to reduce or otherwise mitigate damage, such as bruising, to tissue at the periphery of the distal end 10 during use of the vacuum cup 2. The distal portion 22a may be referred to as the "lead-in" portion 22a of the inner surface 22. The proximal portion of the inner surface 22, such as at the proximal end 24 of the vacuum chamber 6, may be referred to as the "proximal end face" or simply the "end face" of the vacuum chamber 6.

[0031] At least one and up to all of the electrodes 8 extend alongside the inner surface 22. As shown, the electrodes 8 may extend alongside the inner surface 22 between the distal end 10 and the proximal end 24 with respect to a longitudinal direction L oriented along the central axis X of the housing 4. The electrodes 8 in this embodiment are preferably substantially rigid, although in other embodiments, the electrodes 8 may have some flexibility. The electrodes 8 may include a thin layer of conductive material bonded (e.g., via coating, deposition, bonding and / or adhesion) to an associated substantially rigid non-conductive support, which may be constructed of plastic or other suitable non-conductive material. The electrodes 8 may have a surface shape that is substantially conformal with the inner surface 22. The electrodes 8 may be elongated along a direction having a directional component along the longitudinal direction L. The electrodes 8 may also extend alongside the inner surface 22 along a circumferential direction C about the central axis X. As shown in FIGS. 2B-2C, the electrodes 8 may each define a circumferential dimension C1 (or "width" C1) measured along the circumferential direction C. The electrodes 8 may be positioned at regular angular intervals A1 around the central axis X. The angular intervals A1 may be measured from the central axis 8x of each of the electrodes 8. As shown in FIG. 2B, the electrodes 8 may be positioned at angular intervals A1 of, for example, 90 degrees around the central axis X. Thus, the electrodes 8 may be said to be symmetrically spaced around the central axis X. It should be understood that other angular intervals A1 between the electrodes 8 are within the scope of the present disclosure, as described in more detail below. Furthermore, in some embodiments, the angular intervals A1 between the electrodes 8 may vary along the inner surface 22. That is, the electrodes 8 may be irregularly spaced around the central axis X. Furthermore, the electrodes 8 need not be symmetrically spaced around the central axis X.

[0032] As shown in FIG. 2C, each electrode 8 may define an electrode length L1 measured from a first end 8a to a second end 8b of the electrode 8 spaced apart from one another along a central electrode axis 8x. The electrode 8 may also have first and second side surfaces 8c, 8d spaced apart from one another to define an electrode width C1 along a circumferential direction C. Each electrode 8 may have an inner electrode surface 8z configured to contact the tissue surface 101 to deliver one or more electroporation pulses. The inner electrode surface 8z may extend from the first end 8a to the second end 8b of the electrode 8 and from the first side surface 8c to the second side surface 8d. Each electrode 8 may define a primary or "contact" portion 8e extending from the second electrode end 8b toward the first electrode end 8a of the electrode 8 and from the first side surface 8a toward the second side surface 8b. As shown, the electrode width C1 may be measured between the first and second electrode sides 8a, 8b and need not be uniform along the contact portion 8e. The electrode length L1 and width C1 may each be within a range of about 1.0 mm to about 30 mm, more particularly within a range of about 2 mm to about 25 mm, and even more particularly within a range of about 4 mm to about 20 mm. The electrode 8 may define a thickness T1 (see FIG. 2F) within a range of about 0.0005 mm to about 2.000 mm. It should be understood that the electrode length L1 may be greater than, less than, or equal to the electrode width C1.

[0033] The portion of the inner electrode surface 8z within the contact portion 8e may be referred to as the "contact surface" 8z of the electrode. The contact surface 8z may extend in an arcuate manner and concentrically (i.e., may share the same center point) with the inner surface 22 of the housing 4 in a reference plane perpendicular to the central axis X. The contact surface 8z may also have a curvilinear contour that is substantially conformal with the inner surface 22 in a direction along the central electrode axis 8x. The contact surface 8z may be smooth as shown, but in other embodiments, the contact surface 8z may be textured to improve grip against the tissue 102, such as having protrusions, dimples, indentations, micro-needles, and / or roughened surfaces, as non-limiting examples. In additional embodiments, a coating or adhesive may be applied to the contact surface 8z to improve grip and / or electrical conductivity between the electrode 8 and the tissue 102. The electrode 8 may also define a secondary portion 8f extending from the contact portion 8e to the first end 8a and configured to connect with a respective lead of the circuit 108 for delivering electroporation pulses to the electrode 8.

[0034] 2D, the housing 4 may include a housing body 26, which may be formed from a preferably somewhat flexible material, such as a polymeric material including, by way of non-limiting example, polyetheretherketone (PEEK), polyphthalamide (PPA), polyethylene, polycarbonate, polyetherimide (PEI), polyvinyl chloride (PVC), polytetrafluoroethylene (PTFE), polyamide, polyimide, polysiloxane (silicone), polyethylene terephthalate, polyurethane, crosslinked or non-crosslinked rubber (elastomer), polyester. It should be understood that other biocompatible and / or medical grade materials may be utilized for the housing body 26. The housing body 26 may optionally be a monolithic structure that defines the housing 4, but the housing body 26 need not be a monolithic structure and may instead include two or more body components bonded together to define the housing 4. The housing body 26 extends along a longitudinal direction L from a proximal end 28 to a distal end 10. The housing body 26 also defines a wall 30 that extends from the inner surface 22 to an outer surface 32 of the housing 4. The wall 30 extends circumferentially around the entire circumference of the vacuum chamber 6.

[0035] The housing body 26 defines ports 12, 14, 16. As shown, each of the first, second, and third ports 12, 14, 16 may be adjacent the proximal end 28 of the housing body 26 and spaced from the distal end 10. In other words, the ports 12, 14, 16 may be located closer to the proximal end 28 of the housing 4 than to the distal end 10. The first port 12, which may also be referred to as a "vacuum port," extends from the vacuum chamber 6 through the housing body 26 to a port coupling 34 for connection with a mounting member 36 that interconnects the vacuum port 12 with a vacuum source 106. The port coupling 34 may include a seat 38 and a tubular extension 40 extending outwardly from the seat 38 to define a receptacle such that the seat 38 defines an inner end of the receptacle. The attachment member 36 may include an attachment member coupling 42 and a cannulation stem 44 extending therefrom. The attachment member coupling 42 may be a tubular extension that interconnects with the tubular extension 40 of the port coupling 34, such as by extending in a manner that fits into a receptacle defined by the tubular extension 40. A one-way valve member 46 may be positioned on the seat 38 (which may be referred to as a "valve seat"). The valve member 46 may extend from the valve seat 38 into an interior space of the attachment member coupling 42, thereby interposing in a fluid path between the vacuum port 12 and the cannulation stem 44 of the attachment member 36. The valve member 46 may be a duckbill valve as shown, and in other embodiments, as non-limiting examples, the valve 46 may be a ball valve or an umbrella valve.

[0036] The second port 14 may extend opposite the first port 12 and may be configured to allow passage for a circuit 108, such as a wire, through the housing 4 to contact the electrode 8 in the vacuum chamber 6. The second port 14 may also be configured to allow passage through the housing 4 into the vacuum chamber 6 for one or more additional components, such as one or more tools and / or one or more sensors. While positioned within the vacuum chamber 6, such tools and / or sensors may be positionally fixed relative to tissue via vacuum pressure supplied to the chamber 6. The third port 16 may extend from the proximal end 24 of the chamber 6 along a central axis X. The housing 4 may define a mounting formation 48 at an outer end of the third port 16. The mounting formation 48 may be configured to mount a cap 50, such as a puncture stopper, onto the third port 16. The mounting formation 48 and the puncture stopper 50 may have complementary mating shapes that provide a hermetic seal between the puncture stopper 50 and the third port 16. The puncture stopper 50 may be formed of a material that can be pierced by the hypodermic injection needle 18 and allows the needle 18 to inject a drug into the tissue 102 drawn into the vacuum chamber 6 .

[0037] With continued reference to FIG. 2D , the electrode array 9 may be disposed on a sleeve-like insert 52 located within the chamber 6 and extending along the inner surface 22 of the housing 4. The sleeve 52, or at least its outer surface 53, may have substantially the same profile shape as the inner surface 22 of the housing 4. The sleeve 52 may be constructed of flexible materials such as rubber, silicone, and thermoplastic elastomers, as non-limiting examples. As shown in FIG. 2E , the outer surface 53 of the sleeve 52 may be directly attached to the inner surface 22 of the housing 4 via a friction fit, although one or more adhesives may optionally be utilized to attach the sleeve 52 to the inner surface 22. The sleeve 52 may extend from a first or proximal end 54 adjacent the proximal end 24 of the vacuum chamber 6 to a second or distal end 56 adjacent the distal end 10 of the housing 4. The distal end 56 of the sleeve 52 may extend into the lead-in portion 22a of the inner surface 22 of the housing 4. The first end 54 may define a proximal opening 52a of the sleeve 52, which may be concentric with the central axis X, and a distal opening 52b of the sleeve 52. As shown more clearly in FIG. 2F, the first end 54 of the sleeve 52 may partially occlude the first port 12 and may also partially occlude the third port 16, while allowing passage for the circuit 206 into the vacuum chamber 6. Thus, the sleeve 52 may be utilized as a mechanism for controlling or at least influencing the vacuum pressure within the chamber 6.

[0038] The vacuum chamber 6 defines a chamber volume V that may be defined from the proximal end 24 of the chamber 6 along a longitudinal direction L to the opening 10 and at least partially defined by the inner surface 22 of the housing body 26, such as along a direction substantially perpendicular to the longitudinal direction L. In the illustrated example, the direction perpendicular to the longitudinal direction L is a radial direction R that intersects with the central axis X. The chamber volume V may also be defined at least partially by the sleeve 52, such as along the radial direction R. The chamber 6 may have a depth L2 measured from the proximal end 24 of the chamber 6 relative to a reference plane that surrounds the distal end 10 of the housing 4. The chamber depth L2 may be in the range of about 1.0 mm to about 50.0 mm, more particularly in the range of about 3 mm to about 20 mm, and even more particularly in the range of about 5 mm to about 17 mm. The chamber 6 also has a base width, such as a base diameter D1, that may be measured along the radial direction R at the distal end 56 of the sleeve 52. The chamber diameter D1 may be in the range of about 1.0 mm to about 50.0 mm, particularly in the range of about 3.0 mm to about 20.0 mm, and more particularly in the range of about 6.0 mm to about 17.0 mm. In this embodiment, the chamber diameter D1 may be measured between opposing portions of the inner surface 55 of the sleeve 52 at its distal end 56. In other embodiments, the sleeve 52 may be omitted and the electrode 8 may be attached directly to the inner surface 22 of the housing 4, for example, by being embedded or at least partially embedded in the housing wall 30. In such embodiments, the vacuum chamber 6, and thus the chamber volume V, may be at least partially defined by the inner surface 22 of the housing 4 and the inner surface 8z of the electrode 8. Thus, in such embodiments, the chamber diameter D1 may be measured between opposing portions of the inner surface 22 of the housing 4 at the distal end 8b of the electrode 8.

[0039] 3A-3B, the sleeve 52 may carry each of the electrodes 8, and thus the electrode array 9. The sleeve 52 may therefore also be referred to as an electrode array base, support, or substrate. In the illustrated embodiment, the electrode array 9 defines an electrode pattern including a first electrode 8-1, a second electrode 8-2, a third electrode 8-3, and a fourth electrode 8-4, such that the first through fourth electrodes 8-1, 8-2, 8-3, 8-4 are positioned at 90 degree intervals A1 around a central axis X2 of the sleeve 52 (which is substantially coextensive with the central axis X of the cup housing 4 when the sleeve 52 is inserted therein). The electrode array 9 of this embodiment may be characterized as having a total number of electrodes 8n, which are four electrodes 8. The total number of electrodes 8n may also be referred to herein as a "total" 8n or simply as a "total" 8n. The sleeve 52 may also be interchangeable with other sleeves 52 having different electrode array 9 patterns and configurations, such as for generating electric fields with different characteristics within the tissue 102 drawn into the vacuum chamber 6. For example, as shown in FIGS. 3C-3D, the sleeve 52 may have an electrode array 9 including four electrodes 8 spaced at 90 degree intervals around the central axis X2 of the sleeve 52, as described above, but the electrodes 8 may each have a narrower circumferential dimension C1, and therefore less cumulative surface area, than the electrodes 8 in the embodiment described above. As shown in FIGS. 3E-3F, the sleeve 52 may have an electrode array 9 including six electrodes 8 spaced at 60 degree intervals around the central axis X2. As shown in FIGS. 3G-3H, the electrode array 9 may include ten electrodes 8 spaced at 36 degree intervals around the central axis X2.

[0040] 3I-3J, the electrode array 9 may include seven electrodes 8 spaced apart from one another along the longitudinal direction L, each extending around the entire circumference of the sleeve 52. The design of such an array 9 may allow the electrodes 8 to be pulsed or "fired" in a sequence that drives the resulting electroporation field "up" and / or "down" through the tissue volume grasped by the vacuum cup 2.

[0041] As shown in FIGS. 3K-3L, the electrode array 9 may include a plurality of circumferentially elongated electrodes 8, including four subsets 9a-d of circumferentially elongated electrodes 8. The electrodes 8 in each subset 9a-d may be substantially longitudinally aligned with one another, and each subset 9a-d may be circumferentially spaced apart from each adjacent subset 9a-d of electrodes 8. In this example, each subset 9a-d of electrodes 8 may include five longitudinally spaced apart electrodes 8. Thus, the sleeve 52 may include a total of 20 circumferentially elongated electrodes 8. In this embodiment, the electrodes 8 in each subset 9a-d may have an angular span A2 in the range of about 1 degree to about 90 degrees about the central axis X2, and an inter-electrode span A3 between adjacent subsets 8a-d in the range of about 1 degree to about 90 degrees. Circumferentially spacing subsets 9a-d from one another allows, among other things, subsets 9a-d to be fired (or otherwise driven by a current or voltage source) independently of one another, which may help ensure that regions within the tissue volume adjacent to and associated with subsets 9a-d (which in this embodiment may be characterized as "quadrant" of the tissue volume) each receive coverage of the electroporation field, which may result in a more symmetric electroporation field and avoid local differences in tissue conductivity within the tissue volume biasing the electroporation field away from one or more of the regions within the tissue volume.

[0042] Additionally, the circumferentially spaced subsets 9a-d shown in Figures 3K-3L may also enable the generation of uniquely directional electroporation fields within a tissue volume, such as to apply an electrical pulse to a cell from multiple angles in subsequent pulses (i.e., to expose the cell to gradients of electroporation fields along different directions), which may electropermeabilize the cell more efficiently than applying an electrical pulse to the cell from a single direction. It should be understood that such multidirectional electroporation fields may also be generated by the various array designs of Figures 3A-3H. However, the circumferentially spaced subsets 9a-d of Figures 3K-3L also enable the electrodes 8 to not only fire laterally and / or circumferentially across the tissue volume, but also "upward" and / or "downward" through the tissue volume. Increasing the amount of electrodes 8 in the array 9 may also increase the amount of unique pulse patterns possible, and also allow for more homogenous electroporation field coverage, given that the electroporation field is concentrated at the contact surface 8z of the electrodes 8.

[0043] It should be understood that the various electrode array 9 patterns described above are provided as non-limiting examples, and other electrode array 9 configurations are within the scope of the present disclosure. For example, one or more of the parameters of the electrode array 9 described above can be adjusted as needed, including, but not limited to, the amount of electrodes 8 in each array 9, the electrode length L1 and width C1, and the spacing between the electrodes. These parameters can affect the three-dimensional (3D) shape of the electric field (and thus the 3D shape of the electroporation field). In other words, the size, shape, and arrangement of the electrodes 8 can be adjusted as needed to focus the distribution of the electric field within the tissue 102 in a manner that provides an enhanced electroporation treatment.

[0044] 4A-4F, an exemplary method of using the vacuum cup 2 to provide an electroporation treatment to adipose tissue 103 will now be described.

[0045] 4A, the physician may place the distal end 10 of the vacuum cup 2 on the exterior surface 101 of the patient's skin 104 in a position that overlies a target zone 105 of adipose tissue 103. The target zone 105 may be preselected or may be the result of the placement of the vacuum cup 2. Preferably, the physician places the entire circumference of the distal end 10 in contact with the skin 104.

[0046] As shown in FIG. 4B, the physician may apply a pulse of vacuum pressure (also referred to herein as a "vacuum pulse") to the vacuum chamber 6 by, inter alia, activating the vacuum source to generate a vacuum pressure within the vacuum chamber 6 in the range of about -0.1 psi to about -14.7 psi, more preferably in the range of about -3 psi to about -14.7 psi, sufficient to draw a volume or "mound" 140 of tissue 102 into the chamber 6 and into contact with the contact surface 8z of the electrode 8. It should be understood that the magnitude of vacuum pressure included throughout this disclosure refers to such pressure relative to atmospheric pressure measured at sea level. The contact pressure between the mound of tissue 140 (particularly its skin layer 104) and the inner surface 22 of the cup 2 may be in the range of about 0.1 psi to about 200 psi. In this manner, the vacuum cup 2 draws at least a portion of the target zone 105 into the treatment zone 107 defined by the cup 2. In this example, the mound of tissue 140 includes skin 104 and adipose tissue 103. For purposes of this disclosure, the treatment zone 107 is defined as the portion of the tissue volume (such as the tissue mound 140) that extends between the electrodes 8. As shown, the bottom boundary 107a of the treatment zone 107 may be defined by an imaginary path that extends along and between the second ends 8b of the electrodes 8. Thus, after the tissue mound 140 is drawn into the vacuum chamber 6, the treatment zone 107 in this embodiment will include at least a portion of the tissue mount 140 and at least a portion of the injection site 109 (see FIG. 4C) in the treatment zone 107. In the illustrated embodiment, tissue located within the treatment zone 107 during treatment is limited to the skin layer 104 and the fat layer 103. In other embodiments, the treatment zone 107 may include a smooth muscle layer 111. Preferably, the treatment zone 107 does not include any skeletal muscle therein. As explained above, the vacuum pressure is preferably sufficient to provide the vacuum cup 2 with a firm grip on the tissue mound 140, thereby maintaining the relative position between the vacuum cup 2 and the tissue mound 140.Although the illustrated embodiment shows the skin 104 of the tissue mound 140 being placed in direct contact with the contact surface 8z of the electrode 8, it should be understood that additional substances, such as a conductive gel, may be utilized to improve electrical communication between the electrode 8 and the skin 104.

[0047] As shown in FIG. 4C, the physician may inject a medicament into the adipose tissue 103 of the mound 140. To perform the injection, the physician may penetrate the subcutaneous injection needle 18 through the puncture stopper 50, along the third port 16, through the skin 104, and into the adipose tissue 103 in the chamber volume V. The physician may then inject the medicament into the injection site in the adipose tissue 103, and subsequently withdraw the subcutaneous injection needle 18 from the vacuum cup 2. The inventors have surprisingly and unexpectedly observed that the injectate 142 expelled from the subcutaneous injection needle 18 into the adipose tissue 103 disperses towards the skin 104 in response to the vacuum pressure, rather than remaining in a pooled bolus 142a within the adipose tissue 103. Thus, a physician may inject the infusate 142 near or at the bottom of, or slightly below, the treatment zone 107 and allow the vacuum pressure to effectively draw the infusate 142 upward into, and even concentrate, the treatment zone 107. The inventors also believe, based on observations during testing, that the vacuum pressure may be manipulated to aid in mixing the infusate 142 with in vivo fluids, extracellular components, and cells, and to retain the infusate 142 within the treatment zone 107 in a manner that enhances the latency of the infusate 142 therein, thereby increasing transfection.

[0048] It should be understood that in some embodiments, the needle 18 can remain inserted into the tissue 102 after injection, and at least a portion of the needle 18 can include a sensor 152 for detecting parameters of the tissue 102, such as electrical parameters, during or after electroporation, as described in more detail below.

[0049] As shown in FIG. 4D, the physician can deliver one or more electroporation pulses to the tissue mound 140. In particular, the physician can cause the signal generator 112 to deliver an electroporation signal in the form of one or more electroporation pulses to the electrode 8, which then delivers one or more electroporation pulses to the tissue 102 in contact with the electrode 8, thereby generating an electroporation field 144 in the adipose tissue 103 in the treatment zone 107 in the illustrated embodiment. The electroporation field 144 substantially causes a reversible portion of the cell membrane of the cells (e.g., fat cells) in the treatment zone 107, causing transfection of the injectate into the temporarily porated cells. In this way, the electroporation field 144 generates a transfection zone in the treatment zone 107. The electroporation field 144 generated by the electrode 8 in this embodiment has a substantially spherical shape. It should be understood that the electroporation field 144 is a sub-region of the electric field 145 generated by the electrode 8 during pulse delivery.

[0050] The one or more electroporation pulses delivered by electrode 8 can have a potential (voltage) in the range of about 5 V to about 1000 V (1 kV).

[0051] The one or more electroporation pulses can have a current (amperage) in the range of about 0.01 Amp to about 2.0 Amp.

[0052] The one or more electroporation pulses can each have a duration within the range of about 100 microseconds to about 500 milliseconds.

[0053] The amount of electroporation pulses can range from 1 pulse to about 10 pulses.

[0054] In the case of multi-pulse delivery, each electroporation pulse can be separated in time from adjacent pulses by a pulse delay in the range of about 1 millisecond to about 1 second.

[0055] In some embodiments, the electroporation signal may include three pulses of about 200V, of about 100 millisecond duration, with a 200 millisecond delay between pulses. In other embodiments, the electroporation signal may include three pulses of about 50V, of about 100 millisecond duration, with a 200 millisecond delay between pulses. In yet other embodiments, the electroporation signal may include ten pulses of about 50V, of 100 millisecond duration, with a 1 second delay between pulses. In yet other embodiments, the electroporation signal may include eight pulses of 75V, of about 100 millisecond duration, with a 100 millisecond delay between pulses. In yet other embodiments, the electroporation signal may include three pulses of between about 500V and about 1000V, of about 10 microseconds to about 100 microseconds duration, with a delay between pulses of about 100 milliseconds to about 1 second. It should be understood that the foregoing electroporation signals are provided as non-limiting examples, particularly for reversible pore formation for DNA delivery to cells, and that the embodiments disclosed herein may be adapted to provide other types of treatments, including, by way of non-limiting examples, delivering other types of agents to cells, such as for delivery of small molecules to cells, electrochemotherapy, and for irreversible electroporation.

[0056] In a treatment involving multiple electroporation pulses, the pulses may be delivered by the electrodes 8 in the following pulse sequence or pattern, up to and including the final electroporation pulse of the treatment: a first electroporation pulse is delivered by a first positive subset of electrodes 8 through the tissue 102 to a first negative subset of electrodes 8, a second electroporation pulse is delivered by a second positive subset of electrodes 8 through the tissue 102 to a second negative subset of electrodes 8, and so on. During each electroporation pulse, each positive and negative subset of electrodes 8 may range from a single electrode 8 to any combination of at least one less than the total number of electrodes 8n of the array 9 (i.e., 8n-1). The electroporation pulse pattern may be delivered according to a programmed sequence, which may be input by a user into the controller 114 (such as via the user interface 120). Additionally, the sequence of electroporation pulses may optionally be delivered in a distributed pattern. In such a distributed pattern sequence, each pulse of the plurality of electroporation pulses may be delivered between at least two sets of electrodes 8, with each subsequent pulse of the plurality of electroporation pulses being delivered by a different set of at least two electrodes 8. A distributed electroporation pulse pattern may minimize, or preferably eliminate, the generation of electroporation-related thermal stresses on the tissue 102 being electroporated, and may improve the homogeneity of the electric field generated within the tissue 102.

[0057] The vacuum cup 2 may be configured to sense, measure or otherwise detect one or more electrical parameters of the tissue 102 during electroporation pulse delivery and relay the detected information back to the controller 114 for diagnosis and feedback. The electrical parameters detected in the tissue 102 may include, by way of non-limiting examples, voltage, current, impedance and / or resistance. One approach to detect such parameters during electroporation pulse delivery is to have at least one of the electrodes 8 measure the desired electrical parameter during the pulse. Such an electrode 8 may be characterized as a sensing electrode 8 or simply a "sensor." The sensing electrode 8 may be neutral during the pulse. As a non-limiting example of a distributed electroporation pulse pattern sequence, each pulse of the plurality of electroporation pulses may be delivered between at least two sets of electrodes 8, with at least one other electrode 8 being a sensing electrode 8 that is neutral and measures an electrical parameter of the tissue 102, such as impedance, and each subsequent pulse of the plurality of electroporation pulses is delivered by a different set of at least two electrodes 8, with at least one electrode 8 being a sensing electrode 8 that is neutral and measures an electrical parameter. The at least one neutral electrode 8 may alternate for each pulse, while the same electrode 8 (or set of electrodes 8) may remain neutral in successive pulses. Alternatively, during an electroporation pulse, at least one electrode 8 of the array 9 may actively deliver a pulse while measuring an electrical parameter of the tissue 102.

[0058] Another optional approach for detecting electrical parameters during electroporation pulse delivery is to utilize at least one separate sensor for detecting the parameters. The separate sensor may be, for example, a non-invasive sensor 150, such as a contact sensor 150, as shown in FIG. 4D. The contact sensor 150 is configured to measure a parameter and communicate information about the measured parameter to the controller 114. The contact sensor 150 may be inserted into the vacuum chamber 6 through a port, such as the second port 14. The physician may place the contact sensor 150 in contact with tissue 102, such as the skin 104, where the contact sensor 150 may measure the parameter. In other embodiments, the separate sensor may be an invasive sensor 152, such as a probe-type sensor 152. In one such example of a probe-type sensor 152, the sensor 152 may be part of the infusion needle 18, for example, its distal tip region (see FIG. 4C), and may be in electrical communication with the controller 114 to associate information about the measured parameter with the controller 114. It should be understood that multiple sensors, including one or more non-invasive sensors 150 and one or more probe-type sensors 152, may be utilized during treatment to relay information regarding a single electrical parameter or multiple electrical parameters of the tissue 102 to the controller 114.

[0059] The electrical parameter information received by the controller 114 may be utilized for performance diagnostic purposes and / or for active feedback control of the electroporation signal delivered to the electrodes 8 and thus to the tissue 102. For example, to provide active feedback control, one or more sensors 8, 150, 152 may measure one or more respective electrical parameters in the tissue 102 and communicate information about these parameters to the controller 114. The processor 116 may execute software incorporating the parameter information, such as by executing one or more algorithms incorporating the parameter information, to process or otherwise derive an output, such as a control command for controlling the electroporation pulse. The algorithm may also utilize parameter data obtained from the computer memory 118. It should be understood that the control commands derived from the algorithm may adjust the electroporation pulse in real time, such as substantially instantaneously, for electroporation purposes based on the parameter information from the sensors 8, 150, 152. In this manner, the electroporation system 100 may utilize sensors 8, 150, 152 in an active feedback loop for constant control and adjustment of electroporation pulses as needed to achieve a desired electroporation treatment outcome in the target tissue 102. Techniques and / or electronic components for performing such feedback control may be utilized as more fully disclosed in U.S. Patent No. 9,452,285, issued September 27, 2016, entitled "Electroporation Devices and Methods of Using Same for Electroporation of Cells in Mammals" ('285 Reference), and U.S. Patent Publication No. 2011 / 0009807A1, issued January 13, 2011, entitled "Variable Current Density Single Needle Electroporation System and Method" ('807 Reference), the entire disclosures of each of which are hereby incorporated by reference herein.

[0060] 4E, following one or more electroporation pulses, the physician may return the pressure in the vacuum chamber 6 to atmospheric pressure, allowing the vacuum cups 2 to release the tissue 102, allowing the tissue to return to its anatomical shape. The transfected adipocytes may define a transfection zone 105z in the adipose layer 103.

[0061] 4F, one of the key advantages of the vacuum cup 2 disclosed herein, in conjunction with the vacuum source 106, is that it allows the physician to control the volume of tissue 102 (i.e., the size of the mound 140) that is drawn into the treatment zone 107 for the electroporation procedure. Thus, if the target zone 105 is only in the skin layer 104, the physician can apply the necessary vacuum pressure to the vacuum chamber 6 to draw the skin layer 104 into the chamber volume V and into contact with the electrode 8 at its distal end 8b, etc. If the target zone 105 is in the fat layer 103, the physician can apply the increased vacuum pressure necessary to draw the fat layer 103 into the treatment zone 107. If the target zone 105 is in a muscle layer, such as a smooth muscle layer, the physician can apply an even greater increased vacuum pressure, and if the target zone 105 is in a skeletal muscle layer, an even greater increased vacuum pressure can be applied. It should be understood that the target zone 105 may be in a single layer of tissue (e.g., the skin layer 104 or the fat layer 103) or may be in multiple tissue layers including the skin layer 104, the fat layer 103, and optionally a smooth muscle layer. The vacuum pressure may be controlled as needed according to the depth of the target zone 105.

[0062] Another important advantage of the vacuum cup 2 of the present disclosure is that the vacuum cup 2, in conjunction with the vacuum source 106, allows the physician to apply multiple vacuum pulses to the tissue 102 to enhance the interaction between the tissue and the injectate, including the preferred in vivo fluid dispersion mechanism described above. Vacuum pulses may be applied before, during, and / or after injection, and may be applied before, during, and / or after electroporation. Vacuum pulses may be applied in amounts ranging from 1 pulse to 20 pulses, each pulse having a duration ranging from about 0.1 seconds to about 30 seconds. Vacuum pulses may also be applied with varying vacuum pressures and / or durations (and / or varying times between pulses) to achieve the desired results.

[0063] The inventors have made many surprising and unexpected observations in connection with the vacuum cup 2 of the present disclosure. For example, the inventors have observed an unexpected and surprising increase in immune response in test subjects treated with vacuum-assisted electroporation using the vacuum cup 2. This result was surprising and unexpected, as the inventors' original objective was to utilize the vacuum cup 2 for the primary purpose of achieving a more secure grip on the target tissue compared to the grip provided by the prior art caliper-type electroporation device. The inventors could not explain the increased immune response measured in the test subjects treated with the vacuum cup 2 simply as a result of enhanced positional stability between the vacuum cup electrode 8 and the tissue. Of note, the inventors have also observed an unexpected and surprising amount of cellular infiltration in the treatment sites treated with the vacuum cup 2. After several treatments utilizing the vacuum cup 2, the inventors observed bruising and discoloration in the skin that is characteristic of erythema and / or hematoma, the latter involving the diffusion of blood through ruptured capillaries.

[0064] Without wishing to be bound by any particular theory, the inventors believe that the surprising increase in immune response and cellular infiltration is likely related. Furthermore, the inventors believe that the increased cellular infiltration is driven, at least in part, by burst capillaries in the treatment zone and natural secretion of chemical signals from nearby leukocytes that attract and reactively migrate additional cells, such as additional leukocytes, to the treatment zone. The inventors also believe that the observed cellular infiltration may be driven, at least in part, by an inflammatory response. The inventors further believe that, based on the observed dispersion of the injectate through the tissue in response to vacuum pressure, in vivo fluids outside the treatment zone 107 are mechanically drawn into the treatment zone 107 in response to vacuum pressure.

[0065] 5A-5D, one embodiment of a vacuum cup 502 utilizing a flexible electrode 508 will now be described. The vacuum cup 502 of this embodiment is similar to the vacuum cup 2 described above. Thus, like reference numbers as used above indicate common features in this embodiment. For brevity, the following description will focus on the differences between the vacuum cup 502 of this embodiment and the vacuum cup 2 described above.

[0066] 5A-5B, the vacuum cup 502 has a housing body 526 defining an inner surface 522 that partially defines a vacuum chamber 506. The housing body 526 also defines a plurality of relief ports 560 that extend from an outer surface 532 of the housing body 526 to a plurality of channels 562 defined in the inner surface 522 of the housing body 526. The channels 562 may include a proximal channel 562 and a distal channel 562 spaced apart from one another along a longitudinal direction L. The channels 562 may each extend annularly along a full circumference about the central axis X, although in other embodiments, one or more of the channels 562 may extend annularly less than a full circumference about the central axis X.

[0067] The vacuum cup 502 resides within the vacuum chamber 506 and includes a flexible sleeve 552 carrying a plurality of electrodes 508 arranged in an electrode array 509. The flexible sleeve 552 has an outer surface 553 attached to the inner surface 522 of the housing body 526. The flexible sleeve 552 covers the channels 562 in a manner that provides a flexible barrier or membrane between the channels 562 and the vacuum chamber 6. The electrodes 508 are disposed on an inner surface 555 of the sleeve 552. The sleeve 552 may also carry circuitry, such as, for example, wired or printed circuitry, for providing electrical communication between the electrodes 508 and the controller 114. The electrodes 508 of this embodiment may be circumferentially elongated, similar to the electrodes 8 described above with reference to FIGS. 3K-3L. One or more, and up to each, of the electrodes 508 cover at least one of the channels 562 and are constructed of a flexible material. The electrode material may be a metal, such as, by way of non-limiting examples, copper, stainless steel, and gold. Alternatively or in addition, the electrode material may include, by way of non-limiting examples, a conductive polymer, or a carbon allotrope, such as graphene, which may include carbon nanotubes. In other embodiments, the electrode 508 may have a non-conductive core coated with a conductive material, such as those described above.

[0068] The electrode array 509 may include four subsets 509a-d of electrodes 508. The electrodes 508 in each subset 509a-d may be substantially aligned along a longitudinal direction L, and each subset 509a-d may be spaced apart from each adjacent subset 509a-d along a circumferential direction C. The subsets 509a-d may be regularly spaced apart from one another, such as at 90 degree intervals, about a central axis X. In the illustrated example, each subset 509a-d includes proximal and distal electrodes 508 that are longitudinally spaced apart from one another, providing the array 509 with a total of eight electrodes 508. The electrodes 508 of the array 509 may also be characterized as being arranged in a proximal annular row of electrodes 508 covering a proximal channel 562 and a distal annular row of electrodes 508 covering a distal channel 562. As explained above, the electrodes 508 are connected to circuitry that provides electrical communication between the electrodes 508 and the controller 114. It should be understood that the circuitry of the electrode array 509 can be configured such that the controller 114 can individually control parameters of the electroporation pulse for each subset 509a-d of the electrodes 508, and further can individually control parameters of the electroporation pulse for each electrode 508 within each subset 509a-d.

[0069] 5C-5D, the flexible sleeve 552 and electrode 508 are configured to deflect inwardly (towards the central axis X) into the vacuum chamber 6 upon application of vacuum pressure in the chamber 6, increasing contact between the contact surface 508z of the electrode 508 and tissue drawn into the chamber 6. The relief port 560 provides fluid communication between the channel 562 and the exterior of the vacuum cup 502, thereby allowing the pressure in the channel 562 to remain substantially at atmospheric pressure. In this manner, the vacuum pressure in the chamber provides a pressure gradient across the wall of the sleeve 552 between the chamber 6 and the channel 562, allowing the sleeve 552 and the electrode 508 thereon to deflect inwardly into the chamber 6.

[0070] 6A-6D, an electrode 608 for positioning in the vacuum chamber 6 of the vacuum cup 2, 502 may include a plurality of protrusions 664 that may be defined by a contact surface 608z of the electrode 608. Similar to the embodiments described above, the electrode 608 extends from a first end 608a to a second end 608a along a central axis 608x of the electrode 608. The electrode 608 may be elongated along a direction oriented along the central axis 608x. The electrode 608 may also extend from a first side 608c to a second side 608d along a transverse axis 608y of the electrode 608. The contact surface 608z may include a substantially smooth base portion 665, and the protrusions 664 may extend outwardly (i.e., inwardly into the chamber 6) from the base portion 665. The base portion 665 may be substantially planar as shown, although in other embodiments, the base portion 665 may have a curved contour that is substantially conformal with the inner surface 22 of the cup housing 4. By extending outwardly from the base portion 665, the protrusions 664 may increase the contact area between the contact surface 608z of the electrode 608 and tissue drawn into the chamber 6. During use, the protrusions 664 may disrupt the top layer of the skin 104 and press into the tissue 102 in an altering manner, improving the electric field distribution within the target tissue. More specifically, the protrusions 664 increase the magnitude of the electric field formed within the tissue 102 for a given input voltage, as more fully described in International (PCT) Patent Publication No. WO2018 / 057900A1, published March 29, 2018, entitled "Method and Device for Minimally Invasive In Vivo Transfection of Adipose Tissue Using Electroporation" ('900 Reference), the entire disclosure of which is incorporated herein by reference.

[0071] As shown in FIGS. 6A-6B, the protrusions 664 may have a convex, arcuate, or dome-like geometry. The protrusions 664 may be arranged in columns and rows of protrusions 664. As shown in FIG. 6C, the protrusions may be elongated along a direction oriented along a transverse axis 608y. As shown in FIGS. 6B and 6D, the protrusions 608 shown in FIGS. 6A and 6C may have similar side profiles. It should be understood that other protrusion geometries, including pointed, conical, frustum, pyramidal, and the like, are within the scope of the present disclosure. It should also be understood that the electrode 608 may be configured for positioning with the chamber 6 to be elongated along a longitudinal direction L of the vacuum cup 2, elongated along a circumferential direction C of the vacuum cup 2, or elongated along a direction oblique to the longitudinal and circumferential directions L, C.

[0072] Various parameters of the vacuum cup 2, 502 described herein, such as the chamber depth L2, chamber diameter D1, and cup shape, such as the cross-sectional shape in a reference plane perpendicular to the central axis X, and / or the shape of the opening 20, may be adjusted as necessary to achieve a desired electroporation treatment across a wide range of mammalian and skin anatomy. For example, the vacuum cup of the present disclosure may have a non-circular opening and / or chamber geometry, such as, by way of non-limiting example, a polygonal opening and / or chamber geometry. With reference to FIGS. 7A-7E, a vacuum cup 702 having a triangular opening and chamber geometry is described. With reference to FIGS. 8A-8C, a vacuum cup 802 having a rectangular opening and chamber geometry is described. In such an embodiment, the chamber dimension D1 may be referred to as a "chamber width" D1.

[0073] 7A-7C, there is shown a vacuum cup 702 having a distal end 710 that defines a triangular opening 720. The vacuum cup 702 of this embodiment is similar to the vacuum cups 2, 502 described above. Accordingly, like reference numbers as used above indicate common features in this embodiment. For brevity, the following description will focus on the differences between the vacuum cup 702 of this embodiment and the vacuum cups 2, 502 described above.

[0074] The vacuum cup 702 includes a housing body 726 that defines three side walls 730 arranged in a triangular pattern, as viewed in a reference plane orthogonal to a central axis X of the vacuum cup 702. The housing body 726 thus defines a vacuum chamber 706 that also has a triangular shape in the orthogonal reference plane. The side walls 730 intersect with each other at three corners 735 of the housing body 726, which corners 735 are preferably curved. The triangular pattern may be an equilateral triangle as shown, although other triangular patterns, including right triangles, isosceles triangles, and scalene triangles, are also within the scope of this embodiment.

[0075] The inner surface 722 of the sidewall 730 may define a main portion 722b extending from the distal lead-in portion 722a toward the proximal end 724 of the chamber 706. The main portion 722b may be planar, thereby defining a linear surface profile and may define a length L4 measured along the linear surface profile in a range of about 1 mm to about 20 mm. It should be understood that the main portion 722b may alternatively be non-planar and may have a non-linear profile. The main portion 722b preferably tapers inwardly toward the central axis X toward the proximal end 724 of the chamber 706. The main portion 722b may define a taper angle A4 measured with respect to an axis parallel to the central axis X in a range of about 0 degrees to about 80 degrees, more particularly in a range of about 0.25 degrees to about 10 degrees, and even more particularly in a range of about 0.5 degrees to about 5 degrees.

[0076] The vacuum cup 702 includes a plurality of electrodes 708 arranged in an electrode array 709, which may include three subsets 709a-c of electrodes 708. A first subset 709a of the electrodes 708 may be disposed on an inner surface 722 of a first sidewall 730, a second subset 709b of the electrodes 708 may be disposed on an inner surface 722 of a second sidewall 730, and a third subset 709c of the electrodes 708 may be disposed on an inner surface 722 of a third sidewall 730. The subsets 709a-c of the electrodes 708 may be carried by respective substrates or "pads" 752, which may be constructed of an electrically insulating material such as, by way of non-limiting examples, silicon, polyetheretherketone (PEEK), polyphthalamide (PPA), polyethylene, polycarbonate, and polyetherimide (PEI). The substrate 752 may be flexible or rigid. The substrate 752 may also carry circuitry, such as, for example, wired or printed circuitry, for providing electrical communication between the electrodes 708 and the controller 114. Thus, the substrate 752 may be a circuit board, such as a printed circuit board (PCB). As explained above, the circuitry may be configured such that the controller 114 can individually control the parameters of the electroporation pulses for each subset 709a-d of the electrodes 708 and can individually control the pulse parameters of each electrode 708 of each subset 709a-c. The outer surface 753 of the substrate 752 may be attached to the inner surface 722 of the respective sidewall 730 via an adhesive, although other fastening techniques are within the scope of the present disclosure.

[0077] Each subset 709a-c of electrodes 708 may include a single electrode 708 or preferably multiple electrodes 708. In the illustrated embodiment, each subset 709a-c has four electrodes 708. It should be understood that each subset 709a-c may have fewer or more than four electrodes 708, such as a single electrode, two, three, five, six, seven, eight, nine, ten, eleven, twelve, or more than twelve electrodes 708, for example, 100 or more electrodes 708. With advances in microelectromechanical systems (MEMS) and nanotechnology, the size of each discrete electrode 708 may be reduced to the extent that each subset 709a-c may have a virtually unlimited amount of discrete electrodes 708.

[0078] The electrodes 708 define contact surfaces 708z, which may be smooth as shown. A major portion of the contact surfaces 708z may be planar, as may the respective portions overlying the major portion 722b of the inner surface 722. However, it should be understood that the contact surfaces 708z of at least one and up to all of the electrodes 708 may be textured and / or define protrusions, similar to the embodiments described above. Additionally, the substrate 752 and electrodes 708 may be flexible and may cover channels and relief ports defined in the housing body 726 to allow the electrodes 708 to flex inwardly when vacuum pressure is applied to the chamber 706, similar to the embodiments described above with reference to FIGS. 5A-5D.

[0079] 7D, the electrodes 708 in each subset 709a-c may be parallel to one another. The electrodes 708 may define an electrode length L1, measured between the first and second ends 708a,b of the electrodes 708 along their central axis 708x. The electrode length L1 may be within the ranges described above. The electrodes 708 may also define an electrode width W1, measured between the first and second sides 708c,d of the electrodes 708. The electrode width W1 may be within the ranges described above for C1. The electrodes 708 in each subset 709a-c may also define an inter-electrode separation gap W2, which may be within the range of about 1.0 mm to about 30 mm.

[0080] 7E, the electrode 708 may extend inwardly (into the vacuum chamber 706) from the substrate 752. The electrode 708 may define an electrode thickness T1 measured from the inner surface 755 of the substrate 752 to the contact surface 708z. The electrode thickness T1 may be in the range of about 0.001 mm to about 2.000 mm. As shown, the outer surface 708w of the electrode 708 may be affixed to the inner surface 755 of the substrate 752. In other embodiments, the outer surface 708w of the electrode 708 may be partially embedded in the substrate 752. That is, the outer surface 708w of the electrode 708 may reside at a depth between the outer surface and the inner surfaces 753, 755 of the substrate 752. In further embodiments, the outer surface 708w of the electrode 708 may be fully embedded in the substrate 752. That is, the outer surface 708w of the electrode 708 may be coextensive with the outer surface 753 of the substrate 752.

[0081] In this embodiment, the electrode array 709a-c is configured such that when the vacuum pressure draws the tissue 102 into the vacuum chamber, the tissue 102 contacts the contact surface 708z and is drawn into the gap W2 between the electrodes, thereby also contacting the electrode sides 708c, d, thereby increasing the overall contact interface area between the tissue 102 and the electrodes 708.

[0082] The geometry of the vacuum chamber 706 and the configuration of the electrode arrays 709a-c of the present embodiment allow for substantially planar electrodes 708 along the sidewall 730 of the vacuum cup 702, resulting in a more columnar electroporation field (i.e., more elongated along the central axis X) in the tissue 102 compared to that of the vacuum cups 2, 502 described above. The triangular shape of the present embodiment may also advantageously confine tissue within the boundaries of the electroporation field defined by the triangular pulse pattern to a greater extent than prior art electroporation devices. In addition, the geometry of polygonal arrays, including the triangular arrays of the present embodiment, may generate a more inhomogeneous electroporation field due to the acute or "sharp" angles between adjacent electrodes on opposite corners 735 compared to other array designs, including circular designs. For example, it may be desirable to generate areas of increased electric field magnitude (resulting in increased current flow) in the target tissue, and having "sharp" or acute-angled adjacent electrode ends is one way of generating such areas of increased electric field / current flow in the tissue.

[0083] 8A-8C, there is shown a vacuum cup 802 having a distal end 810 that defines a rectangular opening 820. The vacuum cup 802 of this embodiment is similar to the vacuum cups 2, 502, 702 described above. Accordingly, like reference numbers as used above indicate common features in this embodiment. For brevity, the following description will focus on the differences between the vacuum cup 802 of this embodiment and the vacuum cups 2, 502, 708 described above, and in particular the differences from the vacuum cup 702 described above with reference to FIGS. 7A-7E.

[0084] The vacuum cup 802 includes a housing body 826 defining four walls, specifically a pair of opposing side walls 830 extending between a pair of opposing end walls 831 arranged in a rectangular pattern, thereby providing a vacuum chamber 806 that also has a rectangular shape in an orthogonal reference plane. In the illustrated embodiment, the side walls 830 are longer than the end walls 831, however, in other embodiments, the side walls 830 and the end walls 831 may be the same length such that the rectangle is a square. It should also be understood that the walls of the housing body 826 may define other quadrilateral geometric shapes (i.e., non-rectangular).

[0085] The inner surface 822 of the wall 830, 321 may define a main portion 822b extending from the distal lead-in portion 822a toward the proximal end 824 of the chamber 806. The main portion 822b of the sidewall 830 and / or end wall 831 may taper inwardly and proximally at a taper angle A4, which may be within the ranges described above. The vacuum cup 802 includes a plurality of electrodes 808 arranged in an electrode array 809, which may include two opposing subsets 809a,b of electrodes 808 disposed on the inner surface 822 of the sidewall 830. The end wall 831 may be devoid of electrodes 808 as shown, although in other embodiments, one or both of the end walls 831 may have additional subsets of electrodes 808. In still other embodiments, the end wall 831 may have electrodes and the side wall 839 may be devoid of electrodes. In yet other embodiments, one or more, and up to each, of the walls 830, 831 may have a single electrode, which may be configured according to a variety of sizes and shapes.

[0086] As mentioned above, the subsets 809a,b of electrodes 808 may be carried by respective non-conductive substrates 852 attached to the inner surface 822. Each subset 809a,b may have four electrodes 808, although each subset 809a,b may have more or less than four electrodes 808. As explained above, the electrode array 809 may include circuitry configured to enable the controller 114 to individually control parameters of electroporation pulses to each subset 809a,b of electrodes 808, and to individually control pulse parameters for each electrode 808 within each subset 809a,b.

[0087] As shown, the contact surface 808z of the electrode 808 may be smooth and a major portion thereof may be planar. However, in other embodiments, the contact surface 808z may be textured and / or define protrusions, as described above. The housing body 826, the substrate 852, and the electrode 808 may also be cooperatively configured to allow the electrode 808 to deflect inwardly in response to vacuum pressure, similar to the aspects described above with reference to FIGS. 5A-5D. The electrode 808 may have a length L1, width W1, and thickness T1 similar to those described above with reference to FIGS. 7D-7E, and may operate similarly to those described above.

[0088] The rectangular geometry of the vacuum chamber 806 and the configuration of the electrode array 809a,b of this embodiment provides a spherical electroporation field that is more elongated along a direction transverse to the central axis X (particularly along a direction perpendicular to the end wall 831) compared to that of the vacuum cup 2, 502, 702 described above. Furthermore, the rectangular array of this embodiment allows for substantially planar electrodes that are directly opposite one another and capable of firing directly opposite electrical pulses. In this regard, the array of this embodiment can be utilized in a manner similar to opposed plate or caliper type electroporation devices known in the art. For example, the rectangular array design of this embodiment may allow a physician to "pinch" elongated sections of tissue, similar to a caliper type electrode configuration. It may be advantageous to "grasp" the tissue primarily along one axis and pinch to form an elongated treatment zone within the tissue. For example, by increasing the aspect ratio, the rectangular cup 802 may be able to treat a larger overall tissue area without increasing the gap between the electrodes. This may allow, for example, the use of lower voltages and treatment of larger tissue areas than a circular array design operating at the same voltage.

[0089] Additionally, similar to that described above, polygonal array geometries, including those of the present embodiment, may generate a more inhomogeneous electroporation field due to the acute angles (which in this embodiment are nearly parallel) between adjacent electrodes on opposing side walls 830 of the cup 802. Furthermore, arrays may be utilized, as described above, to generate regions of increased electric field magnitude within tissue.

[0090] It should be understood that the vacuum cup of other embodiments may have other polygonal shaped opening and / or chamber geometries, ranging from pentagonal, hexagonal, heptagonal, octagonal, etc., to circular geometries. Furthermore, such polygonal shapes need not have equilateral configurations. In still other embodiments, the vacuum cup may have other shaped opening and / or chamber geometries, such as elliptical, oval, or irregular shapes, as non-limiting examples. It should be understood that the elliptical cup shape may provide similar benefits to the rectangular cup 802 described above, including the ability to "grab" and pinch tissue primarily along one axis, forming an elongated treatment zone within the tissue, thereby allowing for lower voltages to treat larger tissue areas.

[0091] 30A-33F, the results of simulation tests of various four-electrode arrays 9 on adipose tissue 103 and skin 104 are now described. In each of Figures 30A-33F, the counter electrodes 8 are separated from each other by a distance of 15 mm (which approximates the chamber diameter D1) and the applied voltage between the electrodes is the same. Figures 30A-33F therefore demonstrate the effect of electrode shape, size and spacing on electric field generation.

[0092] Figures 30A-31F show a circular array 9 in which the electrode contact surfaces extend along a generally circular circumference. The width of the electrodes shown in Figures 30A-31F is defined by a percentage of the chamber diameter, so a chamber that tapers inwardly toward the top will have a thinner electrode width near the top and a thicker electrode width near the bottom, while always occupying the same percentage of the chamber's circumference at any location along the chamber's inner surface. The electrodes in Figures 30B and 31B each have an angular span A2 of 15 degrees, the electrodes in Figures 30C and 31C each have an angular span A2 of 30 degrees, the electrodes in Figures 30D and 31D each have an angular span A2 of 45 degrees, the electrodes in Figures 30E and 31E each have an angular span A2 of 60 degrees, and the electrodes in Figures 30F and 31F each have an angular span A2 of 75 degrees.

[0093] Figures 32A-33F show a rectangular (specifically square) array 9, in which the contact surfaces of the electrodes define the sides of the rectangle. The electrodes in Figures 32B and 33B each have a width of 2.5 mm, the electrodes in Figures 32C and 33C each have a width of 5.0 mm, the electrodes in Figures 32D and 33D each have a width of 7.5 mm, the electrodes in Figures 32E and 33E each have a width of 10.0 mm, and the electrodes in Figures 32F and 33F each have a width of 12.5 mm. The width of the electrodes shown in Figures 32A-33F is constant from top to bottom. Thus, a chamber that tapers inwardly towards the top will cause adjacent electrodes constructed in this way to be closer to each other at the top than they are at the bottom. Depending on the dimensions of the cup and the taper angle of the walls, this is not preferred, as it may cause adjacent electrodes at the top of the chamber to be very close to each other or even to touch each other.

[0094] From these figures, it can be seen that the square arrays (FIGS. 32A-33F) create a more inhomogeneous electric field in both the adipose tissue 103 and the skin 104 than their circular array counterparts (FIGS. 30A-31F). Thus, it can be said that the circular arrays generate a more homogeneous electric field in both the adipose tissue 103 and the skin 104 than their rectangular array counterparts. These differences in the heterogeneity / homogeneity of the electric fields produced by the circular and rectangular arrays can be beneficially utilized as needed depending on the desired treatment. For example, if a particular electroporation procedure would benefit from a more homogeneous electric field, the physician can select a circular vacuum cup (and electrode array). If a particular electroporation procedure would benefit from a more heterogeneous electric field, the physician can select a polygonal vacuum cup (and electrode array). With regard to the design of the electrodes, it should be understood that it is preferable to maintain a proper separation between adjacent electrodes, especially when the vacuum chamber tapers inward at the top, such as in the design shown in FIGS. 30A-31F.

[0095] 9A, an example vacuum electroporation assembly 900 is shown, including a vacuum cup 902 configured for needle-free injection of a drug, particularly via jet injection. The vacuum cup 902 may therefore be referred to as a "needle-free" vacuum cup 902 or a "jet injection" vacuum cup 902. The vacuum cup 902 of this embodiment is similar to the vacuum cups 2, 502, 702, 802 described above, particularly the vacuum cup 2 described above with reference to Figures 1-4F. Thus, like reference numbers as used above indicate common features in this embodiment. For brevity, the following description will focus on the differences between the vacuum cup 902 of this embodiment and the vacuum cup 2 described above with reference to Figures 1-4F.

[0096] As discussed above, the vacuum cup 902 has a housing body 926 that defines a vacuum chamber 906 in which an electrode 908 is disposed, and a first port 912, a second port 914, and a third port 916 that each communicate with the vacuum chamber 906. As discussed above, the first port 912 is configured for connection to a vacuum source 106, and the second port 914 is configured for, among other things, passage of a circuit into the vacuum chamber 906. However, the third port 916 in this embodiment is configured to receive a jet injection device 970 for injecting a small stream or "jet" of injectate 142 into the tissue 902 drawn into the vacuum chamber 906. In addition, the housing body 926 defines a mounting formation 948 at an outer end of the third port 916 that is configured to provide a sealed coupling with the injection device 970. The mounting formation 948 may reside within a receptacle 953 defined in the mounting formation 948 and may carry one or more sealing members, such as a sealing O-ring 951, configured to provide a sealing engagement with the exterior of the injection set 970.

[0097] The jet injection device 970 includes an injection housing 972 that defines a fluid chamber or vessel 974 in which the injectate 142, including the medicament, is stored. An exterior surface 975 of the injection housing 972 is sized to cooperate with the third port 916 and the sealing O-ring 951 to provide a sealed connection between the injection housing 972 and the vacuum chamber 906. A distal portion 976 of the injection housing 972 defines a nozzle 978 in fluid communication with the vessel 974. The nozzle 978 is configured to eject the injectate 142 from the vessel 974 into the vacuum chamber 906. The distal portion 976 may also include a shield 980 that at least partially surrounds the nozzle 978. The shield 980 may function as a physical barrier, including against potential splash or rebound of the injectate 142 generated during jet injection. The shield 980 may also protect the circuitry of the cup 902 from exposure to the injectate 142.

[0098] A distal end 979 of the nozzle 978 preferably extends distally beyond a distal end 981 of the shield 980. The jet injection device 970 includes a plunger 982 carrying a piston 984 at its distal end. A distal tip 986 of the piston 984 and an inner surface 988 of the container 974 at its distal end 990 have complementary geometric shapes such that advancement of the piston 984 to the distal end 990 expels a jet of injectate 142 from the nozzle 978 and into the tissue 102. It should be understood that the container 974 may be configured to carry a pre-measured volume or dose of injectate. Additionally, the plunger 982 may be controlled by a firing mechanism, actuator, or the like to expel a predetermined dose of injectate through the nozzle 978 and into the patient's tissue 102. Such firing mechanisms may include, by way of non-limiting example, one or more pre-loaded or loadable springs, compressed gas canisters, and the like.

[0099] The jet injection device 970 may be constructed as more fully disclosed in any of U.S. Patent No. 10,045,911, issued August 14, 2018, entitled "Intradermal Injection Device" (the "'911 document"); U.S. Patent Publication No. 2019 / 0000489A1, published January 3, 2019, entitled "Intradermal Jet Injection Electroporation Device"; and U.S. Patent Publication No. 2009 / 0137949A1, published May 28, 2009, entitled "Needle-Free Injection Device with Nozzle Auto-Disable" (the "'949 document"), the entire disclosures of each of which are incorporated herein by reference.

[0100] The needleless vacuum cup 902 and the jet injection device 970 have complementary features that enhance the electroporation treatment provided thereby. For example, referring to FIG. 9B, as the vacuum pressure draws the mound 140 of tissue 102 into the vacuum chamber 6, the skin layer 104 over the mound 140 tightens, temporarily reducing the elasticity of the skin layer 104, thereby allowing the jet or stream of injectate exiting the nozzle 978 to more efficiently pierce the skin layer 104 with less rebound of the injectate. To assist in tightening the skin layer 104, the physician may utilize sufficient vacuum pressure to draw the skin layer 104 into contact with the distal end 979 of the nozzle 978 such that the skin layer 104 deforms around the distal end 979 of the nozzle 978, thereby forming a depression 104a in the skin layer 104 at the contact interface with the distal end 979, further stretching the skin layer 104 at the depression 104a. Such tightening in the skin layer 104, combined with the use of the jet injection device 970, allows the expelled injectate 142 to pierce the skin layer 104 during injection and penetrate the entire tissue mound 140 to a greater extent than needle injection (compare FIG. 4C) and to a greater extent than when using a jet injection device not assisted by vacuum pressure.

[0101] The penetration of the injectate throughout the tissue mound 140 can be characterized as the pressurized injectate forming thousands of minute breaks or pathways within the tissue 102, effectively rendering the tissue permeable, as the injectate is forced throughout the tissue 102, particularly the adipose tissue 103. Furthermore, by subsequently exposing the tissue 102 to vacuum pressure (i.e., during the continuation of the vacuum pulse that drew the tissue mound 140 into the chamber 906, and optionally one or more subsequent vacuum pulses, etc.), the already more widely dispersed injectate 142 can be further dispersed throughout the tissue mound 140 in response to the vacuum pressure, in accordance with the fluid dispersion mechanism described above.

[0102] 10A-10D, another embodiment of a needle-free vacuum cup 1002 is described, where the vacuum cup 1002 has a vacuum chamber 1006 configured such that tissue 102 drawn therein is at least partially drawn into a plurality of openings 1063 defined in an electrode 1008 within the vacuum chamber 1006. The vacuum cup 1002 of this embodiment is similar to the vacuum cups described above, and in particular the vacuum cup 902 described above with reference to FIGS. 9A-9B. Accordingly, like reference numbers as used above indicate common features in this embodiment. For brevity, the following description will focus on the differences between the vacuum cup 1002 of this embodiment and the vacuum cup 902 described above.

[0103] 10A-10B, the vacuum cup 1002 of this embodiment has a housing body 1026 defining a first port 1012 for connection to a vacuum source 106, a second port (not shown) for providing circuitry access to the vacuum chamber 1006, and a third port 1016 for providing a sealed connection with a jet injection device 970, as described above. In this embodiment, instead of the third port 1016 extending directly to the vacuum chamber 1006, the third port 1016 extends inwardly to an annular channel 1062 defined by the housing body 1026. The annular channel 1062 at least partially surrounds the vacuum chamber 1006 and is spaced outwardly therefrom along a radial direction R. The housing body 1026 further defines a plurality of housing ports 1060 extending inwardly from the annular channel 1062 toward the vacuum chamber 1006. The housing body 1026 may thus define a manifold that defines a plurality of housing ports 1060. As described above, the electrode 1008 may be carried by a sleeve 1052 that is affixed to the inner surface 1022 of the housing body 1026. The sleeve 1052 defines a sleeve port 1057 that provides fluid communication between the housing port 1060 and the opening 1063. The sleeve port 1057 therefore also provides fluid communication between the annular channel 1062 and the vacuum chamber 1006. Vacuum pressure is supplied to the vacuum chamber 1006 sequentially through the third port 1016, the annular channel 1062, the housing port 1060, the sleeve port 1057, and the electrode opening 1063. In embodiments in which the electrode 1008 is directly coupled to the inner surface 1022 of the housing body 1026, the electrode opening 1063 may be directly continuous with the housing port 1060. The annular channel 1062 may extend annularly around the entire circumference about the central axis, although in other embodiments the channel 1062 may extend less than the entire circumference about the central axis X.

[0104] As shown in FIG. 10A, each electrode 1008 may define a single column of apertures 1063, which may be elongated along the circumferential direction C. Each column may include five apertures 1063, which may be characterized as a "5x1" (i.e., 5 rows and 1 column) array or "aperture array" of apertures 1063, as shown. Other aperture arrays are within the scope of this disclosure. For example, as shown in FIG. 10C, the apertures 1063 of each electrode 1008 may be arranged in an aperture array having multiple rows and multiple columns of apertures 1063 aligned with corresponding rows and columns of the sleeve ports 1057 and corresponding rows and columns of the housing ports 1060. The aperture array may be a 4x5 array, although other aperture array configurations are within the scope of this disclosure.

[0105] 10D, the openings 1008 are configured such that tissue 102 drawn into the vacuum chamber 1006 may extend at least partially into one or more of the openings 1063, indicated by the highlighted areas 104a. This increases the adhesion between the skin layer 104 and the electrode 1008, and also increases the contact surface area between the tissue 102 and the electrode 1006. Furthermore, by drawing tissue 102, such as the skin layer 104, into one or more openings 1063 that transmit vacuum pressure into the chamber 1006, the vacuum cup 1002 of this embodiment may effectively stretch the skin layer 104 of the mound 140, for example, away from the central axis X, thereby allowing the jet-injected stream of infusate 142 to penetrate the tissue 102 more efficiently than in other embodiments. For example, by distributing the vacuum ports 1060 throughout the chamber 1006, the vacuum pressure in the chamber 1006 is less likely to pull the infusate out of the tissue at the injection site. Additionally, the portion of the skin layer 104 extending into the opening 1063 may disrupt and alter the upper portion of the skin layer 104, thereby improving the electric field distribution within the tissue mound 140, similar to the embodiment described above with reference to the protrusion 664 of Figures 6A-6D.

[0106] 10A-10D, it should be understood that the vacuum cup 1002 can be configured to sense the presence and / or absence of tissue within the individual openings 1063, the individual sleeve ports 1057, and / or the individual housing ports 1060. For example, one or more, and up to all, of the individual openings 1063 in each electrode 1008 can include individual sensors capable of sensing the presence and / or absence of tissue within the associated opening 1063. Such tissue sensors can include, by way of non-limiting example, separate electrodes that can be configured to sense an electrical parameter indicative of the presence / absence of tissue, such as impedance. The separate tissue sensing electrodes of the foregoing examples can be electrically isolated from the electroporation electrode 1008, or can alternatively be on a separate channel from the electroporation electrode 1008. In other embodiments, the tissue sensor may be of another type, such as, by way of non-limiting example, a force type sensor capable of detecting direct contact of tissue, or a pressure sensor capable of detecting when the individual openings 1063, sleeve ports 1057 and / or housing ports 1060 are sealed.

[0107] The tissue sensors may be connected to a circuit, such as a wired circuit or a printed circuit, in electrical communication with the controller 114. For example, such a circuit may be a printed circuit on the same printed circuit board (PCB) as the circuit for controlling the electrodes 1008. The tissue sensors may be utilized to map tissue adhesion throughout the chamber 1006. Such tissue adhesion mapping information may be utilized for data collection purposes, and may additionally or alternatively be utilized in an active pressure feedback mechanism to adjust the level of vacuum pressure upwards or downwards based on the sensor readings at each opening. In such tissue adhesion mapping embodiments, the tissue sensor circuit preferably includes individual circuits for each tissue sensor. As an alternative to tissue adhesion mapping, the tissue sensors may be collectively on a shared circuit and the controller 114 may calculate the change in the collective sensor readings compared to an initial baseline reading (i.e., an overall "delta") to provide an overall measure of how much tissue adhesion to the chamber walls has occurred.

[0108] In additional embodiments of jet injection vacuum cups, such as the cups 902, 1002 described above, the distal end 979 of the nozzle 978 may be adapted to define an array of electrodes. As a non-limiting example of such an embodiment, the distal end 979 of the nozzle 978 may be coated with or fabricated from a conductive material, such as a conductive paint, metal or polymer, and may be in electrical communication with the controller 114. In this aspect, the distal end 979 of the nozzle 978 may be utilized to deliver electroporation pulses to and / or from other electrodes on the interior surface of the vacuum chamber. In such an embodiment, the other electrodes may be annular ring-shaped electrodes or semi-annular electrodes, such as those described above with reference to Figures 3I-3L and / or described below with reference to Figures 15A-15C. Such an embodiment allows for concentric electroporation pulse patterns ("firing patterns"), including those discussed in more detail below. The inventors have successfully used such a concentric electrode array in a vacuum cup to perform intradermal jet injections of fluids in rabbits and guinea pigs, followed by electroporation of the intradermal tissue at the injection site.

[0109] As mentioned above, the inventors have discovered many beneficial results resulting from the vacuum-assisted electroporation procedure using the vacuum cup described above, including increased fluid distribution of the injectate in the subcutaneous and dermal tissues, and increased infiltration of in vivo fluids at the treatment site.

[0110] Referring now to FIG. 11A, the effect of vacuum pressure on the injection sites can be seen. In this example, equal amounts of methylene blue injectate were injected into porcine tissue at two sites using the same injection technique at the same subcutaneous depth. The injection site shown on the left was not exposed to vacuum pressure. The injection site shown on the right was exposed to a vacuum pressure of about −10.6 psi for 15 seconds using the vacuum cup 2 described above with reference to FIGS. 2A-2F, having a base diameter D1 of about 15 mm, a chamber depth L2 of about 15 mm, and an inner wall taper angle of about 4 degrees. Neither site was treated with electroporation. In this example, the vacuum pressure effectively redistributed the fluid and subsequently retained the injectate in the area under the cup. This would have resulted in a higher injectate concentration in the treatment zone of the cup if electroporation had been performed.

[0111] 11B-11C, a comparative study of vacuum pressure on fluid dispersion was performed on guinea pigs. Both subjects were administered injections of methylene blue into adipose tissue. The injection in FIG. 11B was performed using a needleless vacuum cup similar to that depicted in FIG. 9A. Notably, the tissue was in a vacuum chamber that responded to vacuum pressure during the jet injection. The injection in FIG. 11C was performed with a subcutaneous injection needle method, and no vacuum pressure was applied. None of the subjects in this study were treated with electroporation. As demonstrated, the vacuum-assisted jet injection (FIG. 11B) caused significantly more dispersion of the injectate in the adipose layer than the subcutaneous needle injection without the application of vacuum pressure (FIG. 11C).

[0112] Referring now to Figures 34A-34B, a comparative study of the cumulative effect of electroporation and vacuum pressure on cell infiltration in adipose tissue was performed on guinea pigs. A plasmid encoding the gene for green fluorescent protein (GFP) was injected into the adipose tissue via subcutaneous injection into the interscapular fat pad using a 29-gauge insulin syringe. The injection sites of both subjects were treated with the same vacuum pressure. The subject in Figure 34B was further treated via electroporation at the injection site using a vacuum cup similar to that shown in Figure 2A. The subject in Figure 34A was not treated via electroporation. Histological sections were taken at the treatment sites for comparison of GFP expression (visible as green fluorescence) and cell infiltration (visible as blue fluorescence after 4',6-diamidino-2-phenylindole (DAPI) staining). As shown, expression of GFP (green) is detectable after vacuum pressure in both subjects (i.e., whether the treatment site was electroporated or not). However, further application of electroporation in combination with vacuum pressure (FIG. 34B) increased cell infiltration (blue) compared to vacuum pressure alone (FIG. 34A). These studies demonstrate that applying vacuum pressure in combination with electroporation can enhance immunogenicity.

[0113] Now referring to Figure 12, the study data shows that over a 12-week ELISA study comparing the humoral immune response of subject guinea pigs, subjects treated with the vacuum cup 2 shown in Figure 2A (blue plot - "vacuum") showed increased humoral immunogenicity over the 12-week study compared to subjects treated with a caliper-type electroporation device (red plot - "caliper"). Subjects in both groups were injected with equal amounts of pGX2013 (a DNA vaccine against influenza virus nucleoprotein (NP)) via needle injection into the fat layer and treated with electroporation.

[0114] Now referring to FIG. 13, over an 8-week ELISA study comparing the humoral immune response of subject guinea pigs, subjects treated with injection of pGX2303 (a DNA vaccine against human respiratory syncytial virus fusion glycoprotein (RSV-F)) into adipose tissue and electroporation using the vacuum cup 2 shown in FIG. 2A (blue plot - "vacuum") showed comparable humoral immunogenicity to subjects treated with intradermal injection of the vaccine and electroporation using an intradermal needle electrode electroporation device (red plot - "ID needle electrode EP"). Injections were of equal volume (20ug). Intradermal injections were 100uL and fat injections were 300uL. The vacuum cup 2 had a chamber diameter D1 of 15mm with four electrodes.

[0115] Referring now to Figures 14A-14B, a 6-week study compares humoral immunogenicity data in guinea pigs following treatment (weeks 0, 2 and 4) with a DNA vaccine against influenza nucleoprotein (pGX2013). Figure 14A shows the ELISA data and Figure 14B shows the ELISpot data from the same study. In the graphs, the following groups are represented: (1) "ID Needle Electrode EP" (red plot) - 8ug Mantoux injection followed by intradermal electroporation using an intradermal needle electrode electroporation device, (2) "Vacuum Jet + EP" (blue plot) - 40ug jet injection into adipose tissue followed by vacuum assisted electroporation using a device similar to that shown in Figure 9A, (3) "Vacuum Needle" (green plot) - 40ug subcutaneous injection into adipose tissue followed by application of negative vacuum pressure without electroporation using the device shown in Figure 2A, and (4) "Vacuum Needle + EP" (purple plot) - 40ug subcutaneous injection into adipose tissue followed by vacuum assisted electroporation using the device shown in Figure 2A.

[0116] These studies demonstrate that the needle injection, vacuum electroporation device 2 and jet injection, vacuum electroporation device 902 described herein generate a humoral immune response when treating adipose tissue that is equivalent to the humoral response generated by treatment with an intradermal needle electrode electroporation device.

[0117] 15A-22, a vacuum cup configured to target electroporation in the skin layer 104 will now be described.

[0118] 15A-15B, an example of a vacuum cup 1502 is shown that includes an electrode array 1509 having one or more annular ring electrodes 1508 and a central electrode 1511 extending along a central axis X of the vacuum cup 1502. The vacuum cup 1502 of this embodiment is similar to the vacuum cups 2, 502, 702, 802, 902, 1002 described above, and in particular the vacuum cup 902 described above with reference to FIGS. 9A-9B. Thus, like reference numerals as used above indicate common features in this embodiment. For brevity, the following description will focus on the differences between the vacuum cup 1502 of this embodiment and the vacuum cup 902 described above.

[0119] The vacuum cup 1502 of this embodiment has a housing body 1526 defining an inner surface 1522 that at least partially defines a vacuum chamber 1506. The housing body 1526 also defines a third port 1516 extending proximally from the vacuum chamber 1506 along a central axis X. The central electrode 1511 extends through the third port 1516 from an end face portion of the inner surface 1522 into the vacuum chamber 1506. The third port 1516 also extends through a mounting formation 1548 for providing a sealing engagement with the central electrode 1511. Similar to the embodiments described above, the mounting formation 1548 may carry one or more sealing members, such as a sealing O-ring 1551, that sealingly engage the outer surface 1513 of the central electrode 1511. A distal portion 1513z of the outer surface 1513 of the central electrode 1511 is configured to contact tissue 102 drawn into the vacuum chamber 1506. The distal portion 1513z may therefore be referred to as the "contact surface" 1513z of the central electrode 1511. The contact surface 1513z may have a rounded profile, such as a hemispherical profile, with a radius R1 in the range of 0.5 mm to about 10 mm, more particularly in the range of about 1 mm to about 7 mm, and even more particularly in the range of about 1 mm to about 4 mm. The central axis X of the vacuum cup 1502 preferably extends through the apex of the contact surface 1513z. The central electrode 1511 has a proximal portion 1518, which may be narrower than the portion of the central electrode 1511 that contacts the sealing O-ring 1551. The proximal portion 1518 may therefore be referred to as the "stem" of the central electrode 1511.

[0120] 15C, the electrode array 1509 is configured such that the central electrode 1511 functions as a positive or negative electrode while one or both of the annular ring electrodes 1508 function as the other of the positive or negative electrodes (i.e., opposite polarity to the central electrode 1511) during electroporation pulse delivery. In this manner, the central electrode 1511 effectively moves the electric field 145 upward during the pulse and focuses the electroporation field 144 at the skin layer 104. The vacuum cup 1502 of this embodiment may be placed on the tissue 102 at the target zone after the agent has been injected into the skin layer 104, for example, via needle injection such as a Mantoux injection, or intradermal jet injection. It should be understood that in other embodiments, the vacuum cup 1502 may utilize a feature or "post" in place of the central electrode 1511. The post may exhibit a surface shaped similar to the surface 1513z in the vacuum chamber 1506. The post surface is configured to contact the skin drawn into the chamber 1506 via vacuum pressure. For example, the post may be configured to cause the tissue to conform or curl around a portion of the post surface during application of vacuum pressure. Such tissue-post contact has been shown to advantageously enhance fluid distribution within the tissue during application of vacuum pressure, as discussed in more detail below with reference to Figures 42A and 42D.

[0121] 16A, a version of a vacuum electroporation assembly 1600 is shown including vacuum cups 1602 each having an electrode array 1609 positioned on an end surface 1624 within a vacuum chamber 1606 opposite its distal opening 1620. The end surface 1624 may be substantially flat and may be defined by an electrode support member 1652 positioned within the chamber 1606. The support member 1652 may be referred to as an "insert" and may carry the electrodes 1608 of the array 1609. For example, the support member 1652 may be a circuit board, such as a printed circuit board (PCB), having circuitry in electrical communication with a control unit, such as the controller 114 described above. The support member 1652 also defines a number of ports 1660 extending therethrough that provide fluid communication between the vacuum chamber 1606 and an external port 1616 for connection to a vacuum source. In this manner, the ports 1660 in the support member 1652 transmit vacuum pressure into the chamber 1606, drawing the tissue 102 therein and into contact with the electrodes 1608. The electrodes 1608 may have a protruding and / or pointed geometric shape, such as a cone, pyramid, etc., to press into the skin layer 104 of the tissue mound 140 and draw it into the chamber 1606, thereby disrupting and altering the top layer of the skin 104 to improve the electric field distribution therein, as described above.

[0122] The vacuum electroporation assembly 1600 may be configured to receive an injection device, such as a needleless injection device, such as the jet injection device 970 described above. Accordingly, the vacuum cup 1602 has a housing body 1626 that may define a receptacle 1616 for receiving at least a distal portion of the jet injection device 970 such that its nozzle 978 is aligned with an injection opening 1617 defined in the support member 1652. As shown, the nozzle 978 and the injection opening may be aligned concentrically with a central axis of the vacuum cup 1602.

[0123] The housing body 1626 may also define a secondary or "standoff" chamber 1607 offset from the vacuum chamber 1606 such that the support member 1652 separates or is interposed between the vacuum chamber 1606 and the standoff chamber 1607. The standoff chamber 1607 is configured to provide a standoff distance L5 between the distal end of the nozzle 978 and the end face 1624 to facilitate suitable formation of a stream of injectate 142 between the nozzle 978 and the tissue 102 for purposes of intradermal distribution of the injectate 142. In particular, the standoff distance L5 may enable the formation of irregularities in the liquid stream as it approaches the skin 104. For example, such irregularities may include hundreds, thousands, or even a large number of micro- and / or nano-sized droplets, each of which approaches the skin 104 at a sufficient velocity to effectively enable the stream to hundreds or thousands of micro- and / or nano-sized discontinuities in the outer surface 101 of the skin 104, providing localized enhanced injectate diffusion to the skin layer 104. The standoff distance L5 may also be used as a means to control the maximum penetration depth of the injectate in combination with other factors such as the jet injector nozzle geometry and injector force. It should be understood that the standoff distance L5 may also be characterized as the minimum standoff distance between the distal end of the nozzle 978 and the outer surface 101 of the skin 104 during use. The standoff distance L5 may be in the range of about 1.0 mm to about 20 mm.

[0124] 16B, the housing body 1626 can optionally define a cannulation shield 1685 that extends through the standoff chamber 1607 and can abut the rear surface 1625 of the support member 1652 such that the cannulation of the shield 1685 is in fluid communication with the infusion opening 1617 of the support member 1652. In this manner, the cannulated shield 1685 can provide a linear, aligned, shielded pathway from the distal end of the nozzle 978 to the vacuum chamber 1606, thereby protecting the support member 1652 (and its circuitry) from inadvertent exposure to the infusate stream.

[0125] 17A-17B, different embodiments of the support member 1652 are shown in which the electrodes 1608 and ports 1660 are arranged in different patterns around the infusion opening 1617. As shown in FIG. 17A, the electrodes 1608 may be arranged in a circular or ring-like pattern along a circumferential axis C2 that is concentric with the central axis X. The electrodes 1608 may be arranged in a single concentric ring, or in multiple rings that may be concentric with the central axis X or alternatively eccentric relative to the central axis X, as shown in FIG. 17B. With continued reference to FIG. 17B, the ports 1660 may also be arranged in one or more annular rings about the central axis X. Additionally, the electrodes 1608 and / or ports 1660 may also (or alternatively) be arranged in a spoke-like pattern along respective axes R4, R5 that extend radially outward from the central axis X. The respective axes R4, R5 of adjacent electrode 1608 "spokes" and adjacent port 1660 spokes may be offset from one another at respective angular intervals A5, A6 about a central axis, which may range from about 5 degrees to about 180 degrees, and more particularly from about 15 degrees to about 120 degrees. The axes R4, R5 may be linear as shown, although in other embodiments the axes R4, R5 may be arcuate. It should be understood that the example electrode 1608 and port 1660 patterns are provided as non-limiting examples, and that other patterns, including asymmetric and / or irregular patterns, are within the scope of the present disclosure.

[0126] 18A-18B, in a further embodiment, a vacuum electroporation device 1802 can include a plurality of distal vacuum chambers 1807 for inducing tissue therein, particularly the skin. Electrodes 1808 are carried by an electrode support member 1852, which can be attached to a distal end 1810 of a vacuum housing body 1826 that defines a primary vacuum chamber 1806. In this manner, a distal end surface 1824 of the support member 1852 defines a distal end of the device 1802. The housing body 1826 can define a primary vacuum port 1812 for providing vacuum pressure to the primary vacuum chamber 1806, and can define a secondary port 1814, such as one for providing access for circuitry extending within the primary vacuum chamber 1806 and to the support member 1852.

[0127] 18B, the electrodes 1808 in this embodiment are arranged in pairs, each of which includes an outer ring electrode 1808a and an inner ring electrode 1808b that are concentric with respect to one another. The outer and inner ring electrodes 1808a,b may be tubular members that are elongated along a longitudinal direction L and extend through the support member 1852 to its rear surface 1825, and optionally further into the main vacuum chamber 1806. The outer and inner ring electrodes 1808a,b of each pair are radially spaced apart from one another by an electrically insulating annular layer 1894 of material, thereby electrically insulating the inner and outer ring electrodes 1808a,b from one another. In each concentric electrode pair, the outer ring electrode 1808a extends to the distal end surface 1824 of the support member 1852, while the inner ring electrode 1808b is recessed proximally from the distal end surface 1824. In this manner, the distal vacuum chamber 1807 is cooperatively defined by the inner surface 1808z of the outer ring electrode 1808a, the distal end of the inner ring electrode 1808b, and the distal end face of the insulating layer 1894, and optionally also the optional distal lead-in surface 1808y of the outer ring electrode 1808a.

[0128] As shown in FIGS. 18C-18D, the distal end of the inner ring electrode 1808b (and optionally the distal end of the insulating layer 1894) may be concave at an offset distance L6 from the distal surface 1824, which may be in the range of about 0.05 mm to about 5.0 mm, more particularly in the range of about 0.5 mm to about 2.0 mm, and preferably in the range of about 0.8 mm to about 1.2 mm. The device may define a device length L7 measured along the longitudinal direction L from the proximal end 1828 to the distal end surface 1824. The device length L7 may be in the range of about 15.0 mm to about 200 mm, more particularly in the range of about 20 mm to about 40 mm, although lengths L7 less than 15.0 mm and greater than 200 mm are within the scope of this embodiment.

[0129] As discussed above, the inner ring electrode 1808b may be tubular, particularly in a manner that defines a port 1860 that provides fluid communication with the main vacuum chamber 1806. In this manner, vacuum pressure applied to the main vacuum chamber 1806 is transmitted through the inner ring electrode 1808b to the distal vacuum chamber 1807, thereby enabling the device 1802 to draw a portion of the skin layer 104 into the distal vacuum chamber 1807 and into contact with the inner and outer ring electrodes 1808a,b. Such deformation of the skin layer 104 may disrupt and alter its upper portion, thus improving the electric field distribution within the skin layer 104, similar to the embodiments described above.

[0130] The inner and outer ring electrodes 1808a,b of each pair are preferably of opposite polarity, such that an electroporation pulse is delivered from one of the ring electrodes 1808a,b, through the tissue to the other of the pair of ring electrodes 1808a,b.

[0131] 18, the electrode array 1809 may be a circular array including a central electrode pair 1808a,b and a circular pattern of peripheral electrode pairs 1808a,b arranged along a circumferential axis C2 concentrically about the central electrode pair 1808a,b. It should be understood that other array patterns are within the scope of this embodiment. Peripheral adjacent electrode pairs 1808a,b may be spaced apart from one another by respective angular intervals A5, similar to the embodiments described above.

[0132] 19A-19B, in a further embodiment, a vacuum electroporation device 1902 may include an electrode support member 1952 at a distal end 1910 of a housing body 1926, similar to the device 1802 of the embodiment described above. In this embodiment, each electrode 1908 is tubular and extends through the support member 1924 from its distal end face 1924 to its rear face 1925, thereby defining a port 1960 in fluid communication with the vacuum chamber 1906 of the housing body 1926. The port 1960 of the electrode 1908 is sized such that an outer portion of the skin layer 104 may extend into the port 1960 in response to vacuum pressure within the vacuum chamber 1906. Each electrode 1908 in this embodiment is unipolar (positive or negative) during an electrical pulse. The electrodes 1908 are connected to a circuit capable of delivering an electroporation signal such that one or more of the electrodes 1908 are of one polarity (positive or negative) and one or more of the electrodes 1908 are of the opposite polarity during an electrical pulse. For example, with continued reference to FIGS. 19A-19B, the electrodes 1908 can be arranged in a circular electrode array 1908 including a central electrode 1908 and a circular pattern of peripheral electrodes 1908 arranged concentrically around the central electrode 1908. One or more, and up to each, of the electroporation pulses can be delivered between the central electrode 1908 and at least one of the peripheral electrodes 1908. The electroporation signal can include multiple electroporation pulses delivered in a sequence utilizing a distributed pattern of electrodes 1908, similar to the embodiments described above.

[0133] 20 and 21A-21B, the electrode support member 2052, 2152 can carry an electrode array 2009, 2109 having a non-circular pattern. As shown in FIG. 20, the support member 2052 can have a square electrode array 2009 that is substantially equidistant along first and second array directions AD1, AD2 that are substantially perpendicular to one another. In the illustrated embodiment, the array 2009 has a 4x4 array pattern, although other square array patterns are within the scope of the present disclosure, including, by way of non-limiting examples, 2x2, 3x3, 5x5, 6x6, 7x7, 8x8, 9x9, 10x10, etc.

[0134] 21A-21B, the support member 2152 may have a rectangular electrode array 2109 that is longer along a first array direction AD1 than along a second array direction AD2. The array 2109 may include a first column of electrodes and a corresponding second column of electrodes, such as in a 2x6 (i.e., 2 rows and 6 columns) array 2109, although other rectangular array 2109 patterns are within the scope of this disclosure, including, by way of non-limiting example, 1x2, 1x3, 1x4, 2x3, 2x4, 2x5, 2x7, 2x8, 2x9, 2x10, 3x4, 3x5, 3x6, 3x7, 3x8, 3x9, 3x10, 4x5, 4x6, 4x7, 4,8, 4x9, 4x10, 5x6, 5x7, 5,8, 5x9, 5x10 electrode arrays 2109.

[0135] It should be understood that the square and rectangular electrode arrays 2009, 2109 described above may have virtually any number of electrodes 2008, 2108 arranged in the respective patterns. Such square and rectangular electrode arrays 2009, 2109 may generate a generally rectangular electric field that may be activated in a row or column direction, allowing for a highly ordered electric field along a given axis. For example, if injectate is intentionally injected or happens to accumulate along one axial direction of tissue, additional rows or columns of electrodes 2008, 2108 may be easily activated to treat a desired tissue volume. In this regard, these electrode arrays 2009, 2109 may be characterized as providing a modular activation function. In addition, the rectangular electrode array 2109 may be placed on the skin 104 over the target zone such that a first array direction AD1 (along which the array is elongated) is substantially aligned with an axis along which the fluid was intentionally injected or along which the fluid is naturally dispersed due to the presence of anisotropic features such as fibrous tissue, thereby encapsulating the injectate within the electroporation field for a longer duration as the injectate disperses through the tissue. Furthermore, because the array 2109 also defines an array of vacuum ports that disrupt the top layer of the skin 104 during application of vacuum pressure, the rectangular array 2109 may also effectively form disruption pathways in the intradermal tissue that channel the injectate that disperses further along the electroporation field.

[0136] Referring now to FIG. 22, gene expression in guinea pig skin after intradermal injection of various volumes of plasmid encoding the gene for green fluorescent protein (GFP) followed by electroporation using various devices is shown. In this study, vacuum-assisted electroporation of intradermal tissue using the vacuum cup 2, 902 described herein significantly increased gene expression in a manner proportional to the injection volume at injection volumes of 100uL, 300uL, 500uL, and 900uL. In a similar study, we found that gene expression of GFP also increased with increasing vacuum pressure. Meanwhile, gene expression using an intradermal needle electrode electroporation device did not increase with increasing injection volume. Furthermore, increased injection volume alone, without the application of vacuum or electroporation, did not increase gene expression. These studies demonstrate that vacuum pressure and injection volume affect gene expression.

[0137] Referring now to Figures 35A-35B, a 6-week study evaluates the effect of chamber diameter D1 on immunogenicity in guinea pig skin at a given electroporation voltage and current using a vacuum-assisted electroporation device. Figure 35A compares humoral immunogenicity ELISA data after intradermal vacuum-assisted electroporation procedures using vacuum cups with chamber diameters D1 of 8mm, 10mm, 12mm and 15mm. The intradermal procedure for each vacuum cup included a 50ug Mantoux injection of MERS DNA vaccine followed by electroporation using the same voltage and current. Figure 35B shows cellular immune response ELISpot data at weeks 2 and 4 during the same study shown in Figure 35A. This study demonstrates a direct inverse correlation between humoral immune response and vacuum cup diameter at a given electroporation voltage and current limit. In terms of cellular immune responses, this study also demonstrates that vacuum cups with chamber diameters D1 between 8 mm and 12 mm yield similar results, with a decrease in cellular responses occurring when diameter D1 is increased from 12 mm to 15 mm.

[0138] Referring now to Figures 36A-36B, studies have evaluated suitable combinations of vacuum pressure and electroporation voltage parameters on gene expression in guinea pig skin. These studies utilized intradermal injection (specifically Mantoux injection) of a plasmid encoding the gene for green fluorescent protein (GFP), and then performed electroporation treatment at various vacuum pressures and voltages using a vacuum cup similar to cup 2 described above. Vacuum pressures of 0 kPa (no vacuum), 40 kPa, and 70 kPa, and voltages of 0 V (no voltage), 50 V, 100 V, and 200 V were applied to the skin. Three days after treatment, the reaction of the treatment site and expression of GFP were observed. Figure 36A shows visual observations of the treatment site at various vacuum pressures and voltages. In Figure 36B, measurements of the skin fluorescence signal are plotted for various voltages and vacuum pressures. These studies demonstrate that vacuum pressure and voltage independently increase GFP expression. These studies also demonstrate that at each voltage tested, higher vacuum pressures enhanced GFP expression.

[0139] Referring now to FIG. 23, an 8-week study compares humoral immunogenicity ELISA data in guinea pigs after intradermal treatment with HPV DNA vaccine followed by electroporation. The following groups are represented: (1) "ID Needle Electrode EP" (black plot) - treatment using an intradermal needle electrode electroporation device after a 100 uL Mantoux injection of 66.7 ug of plasmid, (2) "Vacuum Cup" (red plot) - treatment using a 15 mm vacuum cup electroporation device consisting of four wall electrodes after injection of 1 mL of the equivalent of 66.7 ug of plasmid, also prepared with 139 U / ml Hylenex (see, e.g., FIG. 2A), (3) "Vacuum Cup Triple Dose" (maroon plot) - treatment using the same device and method as the "Vacuum Cup" group, but the dose was increased to 3 times that of the previous group, i.e., 200 ug of plasmid. All groups were treated at weeks 0, 4 and 7.

[0140] Referring now to FIG. 24A, an 11-week study compares humoral immunogenicity ELISA data in non-human primates after intradermal treatment with HPV DNA vaccine (pGX3001&3002) followed by electroporation. The following groups are represented: (1) "ID Needle Electrode EP" (black plot) - treatment using the intradermal needle electrode electroporation device, (2) "Vacuum Cup" (red plot) - treatment using the vacuum cup shown in FIG. 2A after injection of a dose equivalent to "ID Needle Electrode EP", (3) "Vacuum Cup 6x Dose" (brown plot) - treatment using the same device and method as the "Vacuum Cup" group, but with the dose increased to 6x that of the previous group. All groups were treated at weeks 0, 4 and 9. Referring now to FIG. 24B-D, cellular immune response ELISpot data at weeks 2, 6 and 11 of the same study shown in FIG. 24A are shown for each group, respectively. These studies demonstrate that the larger dose of vacuum-assisted electroporation treatment (Group 3) produced more rapid humoral and cellular responses than the ID needle electrode EP device (Group 1), while at comparable doses, the vacuum-assisted electroporation treatment (Group 2) performed generally similarly to the ID needle electrode EP device (Group 1). Furthermore, the larger dose of vacuum-assisted electroporation treatment (Group 3) produced approximately 10 times (10x) greater cellular responses at 11 weeks than the ID needle electrode EP device (Group 1).

[0141] Referring now to Figures 37A-B, a 6 week study compares the performance of the vacuum assisted electroporation device with the intradermal needle electrode electroporation device in terms of immunogenicity. Figure 37A compares humoral immunogenicity ELISA data in guinea pigs after intradermal treatment with 50ug of MERS DNA vaccine with a Mantoux injection followed by electroporation. The groups represented in the graph are: (1) "ID-VEP" - treatment using a vacuum cup similar to that shown in Figure 2A and (2) "ID-EP" - treatment using an intradermal needle electrode electroporation device. Both groups were treated at weeks 0, 2 and 4. This study demonstrates that the vacuum cup generates a more rapid and robust humoral response compared to the intradermal needle electrode electroporation device. Figure 37B shows the cellular immune response data at weeks 2 and 4 during the same study shown in Figure 37A.

[0142] Figure 38 shows a 6 week study comparing guinea pig cellular immune response data following intradermal treatment with 100ug of Recurrent Respiratory Papillomatosis (RRP) DNA vaccine via Mantoux injection followed by electroporation. The groups depicted in Figure 38 include: (1) "ID-EP" treatment using an intradermal needle electrode electroporation device and (2) "ID-VEP" - treatment using a vacuum cup similar to that shown in Figure 2A.

[0143] The studies shown in Figures 37B and 38 demonstrate that the vacuum cup generates a substantially equivalent cellular immune response compared to an intradermal needle electrode electroporation device. In addition, during the studies shown in Figures 37A-38, no visible tissue damage was observed at the vacuum cup treatment site within 7-10 days after treatment.

[0144] Referring now to Figures 39A-39B, a 4-week study evaluates the cumulative effect of vacuum pressure and electroporation on immunogenicity in the skin. The study compares humoral and cellular immune responses in guinea pigs after intradermal treatment with 50ug of MERS DNA vaccine with a Mantoux injection. Figure 39A shows humoral immunogenicity ELISA data at weeks 0, 2, and 4 for the following groups: (1) "ID-VEP" - vacuum-assisted electroporation treatment using a vacuum cup similar to that shown in Figure 2A, (2) "ID-vacuum" - vacuum-pulsed non-electroporation treatment using a vacuum cup, and (3) "ID" - Mantoux injection only. Figure 39B shows cellular immune response ELISpot data at week 4 of the same study shown in Figure 39A. These studies demonstrate that while electroporation is essential for vacuum pressure to cause immunogenicity, vacuum pressure alone (i.e., without electroporation) after Mantoux injection can generate at least a partial humoral response. The inventors believe that these studies also suggest that the aforementioned immune responses are only partially explained by surface transfection of DNA vaccines.Further studies confirmed that electroporation voltage is a stronger driving force for immunogenicity than vacuum pressure.

[0145] 40A-40B, cross-sectional views of comparative electric fields generated by two different example electrode arrays within a mound of tissue drawn into a vacuum chamber are shown. FIG. 40A shows the electric field generated by an electrode array having a pair of opposing electrodes configured similarly to that of the vacuum cup 2 shown in FIG. 2A. An electroporation pulse delivery pattern (also referred to as a "firing pattern") between the opposing electrodes 8 transfers current laterally between the electrodes across the mound of tissue 140. FIG. 40B shows the electric field generated by an electrode array having two annular ring electrodes and a central electrode configured similarly to that of the vacuum cup 1502 shown in FIG. 15A. The electroporation pulse firing pattern in this example transfers current concentrically between the annular ring electrodes and the central electrode, thereby concentrating the electric field in the skin layer 104 adjacent to the central electrode. Thus, Figures 40A-40B demonstrate that an electroporation pulse firing pattern between opposing electrodes (Figure 40A) produces a more homogenous electric field through the skin layer 104 as compared to the electric field produced between an annular ring electrode and a central electrode (Figure 40B). These differences in electric field generation may be advantageously utilized based on the particular electroporation procedure desired.

[0146] Referring now to Figures 41A-41B, an 8-week study evaluates the impact of electroporation pulse firing patterns on immunogenicity. In this study, humoral and cellular immune responses of guinea pigs were examined after intradermal treatment with MERS DNA vaccine by Mantoux injection, followed by vacuum-assisted electroporation using a vacuum cup with an electrode array as shown in Figures 40A-40B. Figure 41A shows humoral immunogenicity ELISA data at weeks 0, 2, 4 and 8 for both array configurations. Both groups were treated at weeks 0, 2 and 4. Figure 41B charts cellular immune response ELISpot data at and after week 4 during the same study shown in Figure 41A. This study demonstrates that while the array configurations performed similarly in terms of humoral response, the concentric array significantly outperformed the opposing array in terms of cellular response.

[0147] Referring now to Figures 42A-42D, a study evaluates the comparative effect of a central electrode on fluid distribution in guinea pig skin. Fluid distribution of the colored injectate was measured for three groups: (1) Mantoux injection only (i.e., no vacuum pressure), (2) an array with a counter electrode without a central electrode (see Figure 40A), and (3) an array with a central electrode (see Figure 40B). Equal amounts of injectate were injected into each group via Mantoux injection. Vacuum pressure was applied to groups 2 and 3. No electroporation was performed in this study. Figures 42B, 42C, and 42D show the fluid distribution in the tissue for groups 1, 2, and 3, respectively. The results are charted in Figure 42A according to the visual aspect ratio of the colored injectate. The results demonstrate that the presence of a central electrode or feature can affect fluid distribution of the injectate when vacuum pressure is applied. These results further suggest that the internal geometry of the vacuum chamber can affect fluid distribution when vacuum pressure is applied.

[0148] It should be understood that the intradermal vacuum electroporation assemblies, devices and cups described above can be utilized with a variety of formulations to enhance the vacuum-assisted electroporation procedure. For example, the injectate 142 can include a mixture of formulations to affect the properties of the drug in a suitable manner. Non-limiting examples of such formulations include hyaluronidase and Hylenex (human recombinant hyaluronidase), which can temporarily degrade the drug matrix and allow for injection of larger drug volumes with smaller, less painful lumps or less bleeding in the skin. As shown in Figures 25-26, Mantoux injections with hyaluronidase produce smaller bleeding heights and diameters than injections without hyaluronidase (left). Hyaluronidase formulations are more fully described in U.S. Patent Publication No. 2019 / 0284263A1, published September 19, 2019, entitled "In Vivo Use of Chondroitinase and / or Hyaluronidase to Enhance Delivery of an Agent" (the "'263 document"), the entire disclosure of which is incorporated herein by reference. By utilizing hyaluronidase in injections performed with the vacuum electroporation devices disclosed herein, agents can be injected in larger volumes in intradermal tissue, allowing the physician to treat larger tissue volumes with the devices herein having larger treatment zones 107.For example, the inventors have found through numerous tests using vacuum cups with various chamber diameters D1 (e.g., 8 mm, 10 mm, and 12 mm) in combination with an injectate 142 having a formulation containing hyaluronidase (i.e., 50% Omnipaque 350 + 50% 150 U / mL hyaluronidase (75 U / mL hyaluronidase final)), that an 8 mm diameter vacuum cup can accommodate an injectate volume of 0.2 mL with substantially all of the injectate drawn into the vacuum chamber; a 10 mm diameter vacuum cup can accommodate an injectate volume of about 0.4 mL with substantially all of the injectate drawn into the vacuum chamber; and a 12 mm diameter vacuum cup can accommodate an injectate volume of 0.8 mL with substantially all of the injectate drawn into the vacuum chamber. It has been observed that the presence of hyaluronidase in the injectate formulation dramatically promotes fluid dispersion of the injectate within the skin layer. The data from these studies strongly suggest that larger volumes of injectate should utilize hyaluronidase in the injectate formulation, otherwise the injection bleb will be large and will not diffuse laterally through the skin as well.

[0149] Referring now to Figure 27, a 4 week study compares the humoral immune response of guinea pigs in terms of endpoint titers after intradermal treatment with a DNA vaccine against MERs (pGX9101) followed by electroporation. All groups received 50ug of plasmid in a 100uL Mantoux injection into the flank at weeks 0 and 2. The groups represented on the graph are as follows: (1) "ID Needle Electrode EP" (black plot) - treatment using an intradermal needle electrode electroporation device, (2) "ID Needle Electrode EP+HYA" (gray plot) - treatment using an intradermal needle electrode electroporation device as in the previous group, but the formulation contained 270 U / mL of intropharma hyaluronidase, (3) "15mm Vacuum Cup" (red plot) - treatment using vacuum cup 2 shown in Figure 2A with a chamber diameter D1 of 15 mm, (4) the same vacuum cup in the "15mm Vacuum Cup" plot, but with a formulation containing 270 U / mL of intropharma hyaluronidase.

[0150] Referring now to Figure 28, a 6-week study compares the humoral immune response of guinea pigs in terms of endpoint titers after intradermal treatment with a DNA vaccine against MERs (pGX9101) followed by electroporation at weeks 0, 2, and 4. The groups represented in the graph are: (1) "ID Needle Electrode EP" (blue plot) - 100uL injection of 50ug of plasmid followed by treatment with an intradermal needle electrode electroporation device; (2) "15mm Vacuum-500ug" (red plot) - 1mL Mantoux injection of 500ug of plasmid, where the formulation contains 270U / mL of Intropharma hyaluronidase. Electroporation was delivered by the vacuum cup shown in Figure 2A with a chamber diameter L1 of 15 mm; (4) "15mm vacuum-ID injection 500ug" (green plot) - a deep dermal injection of 1mL of 500ug of plasmid, but injected over a period of at least 2 minutes, the formulation containing 270U / mL of Intropharma hyaluronidase. Electroporation was delivered by the same vacuum cup 2 as the previous group; (4) "10mm vacuum-50ug" (purple plot) - a Mantoux injection of 100uL of 50ug of plasmid, followed by treatment with the vacuum cup 2 shown in Figure 2A with a chamber diameter L of 10mm.

[0151] Referring now to Figure 29A, a 6 week study compares the humoral immune response in terms of mean endpoint titers of guinea pigs electroporated at weeks 0, 3 and 6 after intradermal injection of a DNA vaccine against influenza nucleoprotein (pGX2013). Groups represented in the graph are: (1) "ID Needle Electrode EP 1ug" (red plot) - Mantoux injection with 1ug of plasmid and then treated with electroporation using an intradermal needle electrode electroporation device; (2) "Vacuum with HYA 10ug" (blue plot) - 1mL Mantoux injection of 10ug of plasmid in a formulation with 139.5U / mL of Hylenex was treated using a vacuum cup 2 as shown in FIG. 2A with a chamber diameter D1 of 15mm; (3) "Vacuum with HYA 1ug" (green plot) - 100uL Mantoux injection with 1ug of plasmid with 139.5U / mL of Hylenex was then electroporated using the same vacuum cup 2 as the previous group; (4) "Vacuum without HYA 1ug" (purple plot) - 100uL Mantoux injection with 1ug of plasmid was then electroporated using the same vacuum cup 2 as groups (2) and (3). Figures 29B and 29C show the cellular immune response in terms of spot forming units at weeks 2 (Figure 29B) and 4 (Figure 29C) of the study in Figure 29A.

[0152] Referring now to Figures 43A-43C, a 7-day study evaluated the comparative effectiveness of a single large-volume injection and vacuum-assisted electroporation procedure versus a multiple-injection, multiple-electroporation procedure using an intradermal needle electrode electroporation device in guinea pigs. A single high-volume vacuum-assisted electroporation procedure was performed using a 15 mm diameter vacuum cup with a pair of annular ring electrodes and a central (concentric) electrode, similar to the vacuum cup shown in Figure 15A. The single injection was a 0.8 mL Mantoux injection of a plasmid encoding secreted alkaline phosphatase (SEAP) coformulated with 135 U / mL hyaluronidase, followed by vacuum-assisted electroporation. The multiple-injection, multiple-electroporation procedure included six separate 0.1 mL Mantoux injections, each followed by electroporation using an intradermal needle electrode (for a total of 0.6 mL injection and six electroporation applications). Figure 43A shows the vacuum cup positioned over the injection prior to application of vacuum pressure. Figure 43B shows the injectate in the vacuum cup while vacuum pressure is being applied, and it can be seen that the injectate in the tissue deforms around the central electrode, thereby concentrating the injectate at the electroporation field (see Figure 40B). Figure 43C shows SEAP expression (as a measure of systemic protein production in the subject) for both treatments on days 0, 1, 2, 6 and 7. This study demonstrates that a single, high-volume, vacuum-assisted electroporation treatment using a vacuum cup performs substantially equivalent to a six-injection, six-electroporation treatment using a needle electrode device.

[0153] These studies demonstrate that vacuum-assisted electroporation using the disclosed devices and assemblies allows for large-volume delivery of DNA to the skin. Additionally, hyaluronidase formulations (e.g., Hylenex) enhance immunogenicity following vacuum-assisted electroporation of the skin. Additionally, the vacuum cups described herein are adapted to utilize the significantly higher injection volumes in intradermal tissue provided by hyaluronidase formulations including injection volumes of 1000 uL (1 mL) or more. In other words, by utilizing hyaluronidase formulations with the disclosed vacuum cups, the vacuum cups are able to treat significantly larger volumes of intradermal tissue. In addition, the disclosed devices and assemblies produce more rapid humoral responses than intradermal needle electrode electroporation devices and comparable overall humoral immune responses compared to intradermal needle electrode electroporation devices. Additionally, these studies demonstrate that the kinetics and magnitude of cellular responses can be enhanced through vacuum-assisted electroporation of intradermal tissue. The inventors have also found that the use of a hyaluronidase preparation in conjunction with vacuum-assisted electroporation of intradermal tissue effectively enables transfection of the sub-superficial dermal layers.

[0154] Referring now to FIG. 44, an 8-week study compares humoral immune response data effects of electroporation pulse firing patterns on immunogenicity. In this study, humoral and cellular immune responses of guinea pigs were examined after intradermal treatment with MERS DNA vaccine by Mantoux injection and subsequent vacuum-assisted electroporation using a vacuum cup with an electrode array as shown in FIG. 40A-B. FIG. 41A shows humoral immunogenicity ELISA data at 0, 2, 4 and 8 weeks for both array configurations. Both groups were treated at 0, 2 and 4 weeks. FIG. 41B charts cellular immune response ELISpot data at and after 4 weeks during the same study shown in FIG. 41A. This study demonstrates that the array configurations performed similarly in terms of humoral response, while the concentric array significantly outperformed the opposing array in terms of cellular response.

[0155] 45A-47C, fluoroscopic images show comparative tissue deflection in a guinea pig during jet injection at various vacuum pressures and nozzle-skin offset distances using a jet injection vacuum cup configured similarly to vacuum cup 902 shown in FIG. 9. The injectate used in these images is 50% Omnipaque 350 solution to allow radiography. In each of these images, the superimposed horizontal reference line indicates the distal end of the vacuum cup (and thus the distal end of the vacuum chamber and the initial skin-chamber interface prior to vacuum application). FIGS. 45A-45C show jet injections performed without vacuum pressure applied in the chamber. FIGS. 46A-46C show jet injections performed with vacuum pressure applied in the chamber and no nozzle-skin offset distance. FIGS. 47A-47C show jet injections performed with vacuum pressure applied in the chamber and a nozzle-skin offset distance of 3 mm. Note that Figures 45A, 46A and 47A show the tissue before injection, Figures 45B, 46B and 47B show the tissue during jet injection, and Figures 45C, 46C and 47C show the tissue after injection.

[0156] As shown in Figures 45A-45C, without the application of vacuum pressure in the chamber, injection causes a large deflection of the tissue (Figure 45B), after which the tissue is repelled towards the nozzle after injection, although the injectate remains generally below the vacuum chamber (Figure 45C).

[0157] As shown in Figures 46A-46C, when vacuum pressure is applied within the chamber during injection (Figure 46B), the tissue deflection is eliminated. However, as shown in Figure 46C, the lack of a nozzle-skin offset distance results in the injectate being below the vacuum chamber after injection.

[0158] 47A-47C, when the nozzle-skin offset distance is 3 mm and the injection is performed while vacuum pressure is applied in the chamber, tissue deflection is substantially eliminated during injection (FIG. 47B). When the skin is pulled into the vacuum chamber prior to injection, as in this study, there is intimate contact between the jet nozzle and the skin during injection and the vacuum pressure is sufficient to prevent tissue deflection. Furthermore, after injection (FIG. 47C), the injectate is present in the chamber and has a greater vertical distribution compared to the non-offset setting shown in FIG. 46C where the injectate is compressed into a smaller vertical space. These tests demonstrate the significant advantages provided by the jet infusion vacuum cup disclosed herein from the standpoint of injectate fluid distribution.

[0159] It should be understood that although the embodiments described herein are configured to target electroporation in intradermal and / or subcutaneous tissue, any of the design parameters of the vacuum cups 2, 502, 702, 802, 902, 1002, 1502, 1602 and vacuum devices 1802, 1902 may be scaled up or down in size to target more specific and / or different tissue layers, such as specific tissue layers or muscle layers within the skin, such as the smooth and skeletal muscle layers. Additionally, the design parameters of the vacuum cups 2, 502, 702, 802, 902, 1002, 1502, 1602 and vacuum devices 1802, 1902 herein may be adapted as needed to target electroporation of other types of tissue, including mucosa, organs, and the like.

[0160] Although the present disclosure has been described in detail, it should be understood that various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the present invention as defined by the appended claims. For example, features of various embodiments described herein can be incorporated into one or more, and up to all, of the other embodiments described herein. Furthermore, the scope of the present disclosure is not intended to be limited to the specific embodiments described herein. From this, as one skilled in the art will readily appreciate, any now existing or later developed process, machine, manufacture, composition of matter, means, method or step that performs substantially the same function or achieves substantially the same result as the corresponding embodiment described herein can be utilized in accordance with the present disclosure. The present disclosure also includes the following aspects. [Aspect 1] 1. An apparatus for vacuum-assisted in vivo electroporation of tissue, comprising: a housing defining a chamber and at least one opening into the chamber; at least one port extending through the housing, the at least one port being remote from the at least one opening and connectable to a vacuum source, whereby the at least one port is configured to transfer vacuum pressure from the vacuum source to the chamber; a plurality of electrodes positioned within the chamber, the plurality of electrodes configured to extend through the at least one opening in response to the vacuum pressure and deliver, at least momentarily, one or more electroporation pulses to a target portion of tissue held within the chamber. [Aspect 2] 2. The apparatus of embodiment 1, wherein the at least one opening is a single opening, and the opening is circular. [Aspect 3] The apparatus of aspect 2, wherein the housing has a wall defining an inner surface that at least partially defines the chamber, and the plurality of electrodes includes four electrodes extending along the inner surface, the four electrodes being spaced apart from one another at 90 degree intervals. Aspect 4 The device of aspect 2, further comprising a second port in addition to the at least one port, the second port configured for insertion of a jet injection device or a hypodermic injection needle into the chamber. Aspect 5 The apparatus of aspect 2, wherein the housing has an end surface opposite the at least one opening and a wall extending from the end surface to the opening, the wall defining an inner surface that at least partially defines the chamber, at least one first electrode of the plurality of electrodes extending from the end surface and at least one second electrode of the plurality of electrodes extending along the inner surface, the at least one first electrode and the at least one second electrode being concentric with one another. Aspect 6 The device of embodiment 5, wherein the at least one first electrode is a single electrode centrally positioned with respect to the end face, and the at least one second electrode extends along the entire circumference of the inner surface. Aspect 7 The apparatus of aspect 1, wherein the multiple electrodes have electrode surfaces exposed within the chamber, at least some of the electrode surfaces being one or more of textured and protruding into the chamber. Aspect 8 The device of embodiment 1, wherein the housing is constructed of a flexible material, the material including one or more of polycarbonate, polyetheretherketone, polyphthalamide, polyethylene, polyetherimide, polyvinyl chloride, polytetrafluoroethylene, polyamide, polyimide, polysiloxane (silicone), polyethylene terephthalate, polyurethane, crosslinked or non-crosslinked rubber, and polyester. Aspect 9 a signal generator in electrical communication with the plurality of electrodes and configured to deliver the one or more electroporation pulses to the plurality of electrodes; The device of aspect 1, further comprising: a processor in electrical communication with the signal generator and at least one sensor positioned within the chamber, the at least one sensor configured to sense at least one parameter of the tissue during delivery of the one or more electroporation pulses and communicate feedback data of the at least one parameter to the processor, the processor configured to execute one or more algorithms utilizing the feedback data to adjust at least one pulse parameter of the one or more pulses during delivery of the one or more pulses. Aspect 10 2. The apparatus of embodiment 1, wherein the plurality of electrodes are configured to apply pulses having a potential magnitude in the range of about 2V to about 1000V. Aspect 11 2. The apparatus of claim 1, wherein the plurality of electrodes are configured to apply pulses having a current magnitude in the range of about 0.01 amperes to about 2.0 amperes, the pulses having a pulse duration in the range of about 0.1 milliseconds to about 100 milliseconds. Aspect 12 The apparatus of embodiment 1, wherein the at least one port comprises an array of ports, each of the array of ports extending through the housing into the chamber such that the housing includes a manifold that defines the array of ports. Aspect 13 The device of embodiment 1, wherein the target portion of the tissue is at least one of skin tissue and adipose tissue. Aspect 14 1. A method of electroporating tissue in a subject, comprising: placing a chamber adjacent to tissue of the subject; applying a vacuum pressure to the chamber, thereby drawing the tissue through an opening in the chamber and into contact with a plurality of electrodes extending along an interior surface of the chamber; delivering one or more electroporation pulses to the tissue through the plurality of electrodes, thereby generating an electroporation field within the tissue. Aspect 15 15. The method of claim 14, wherein the applying step comprises applying the vacuum pressure at a level sufficient to affix the tissue to the interior surface and distribute fluid within the tissue in the electroporation field. Aspect 16 15. The method of claim 14, further comprising injecting a substance into the tissue prior to the delivering step, the delivering step comprising transporting the substance into cells of the tissue within the electroporation field through reversible pores formed in the cell membranes of the cells in response to the electroporation field. Aspect 17 17. The method of embodiment 16, wherein the agent comprises a nucleic acid that elicits an immune response in the subject. Aspect 18 15. The method of claim 14, wherein the applying step comprises applying pulses of varying vacuum pressure to the tissue and varying a duration of the pulses. Aspect 19 The method of aspect 14, further comprising injecting fluid into the tissue from an injection device extending into the chamber through a second port, wherein the injecting and adding steps are performed before the delivering step, and the injection device is one of an injection needle and a jet injection device. Aspect 20 1. An apparatus for vacuum assisted treatment of tissue, comprising: a housing defining a chamber and at least one opening into the chamber; at least one first port extending through the housing, the first port being remote from the at least one opening and connectable to a vacuum source, whereby the first port is configured to transfer vacuum pressure from the vacuum source to the chamber; and a jet injection device extending into the chamber through a second port, the second port being opposite the at least one opening, the jet injection device configured to extend through the at least one opening in response to the vacuum pressure and deliver a jet injection of fluid to a target portion of tissue held, at least momentarily, within the chamber.

Claims

1. 1. An apparatus for vacuum-assisted in vivo electroporation of tissue, comprising: a housing defining a chamber, at least one opening into the chamber at a distal end of the housing, an interior surface located within the chamber, and an end surface within the chamber opposite the opening; at least one port extending through the housing, the at least one port being remote from the at least one opening and connectable to a vacuum source, whereby the at least one port is configured to transfer vacuum pressure from the vacuum source to the chamber; and at least one electrode positioned within the chamber, the at least one electrode configured to extend through the at least one opening in response to the vacuum pressure and deliver one or more electroporation pulses to a target portion of tissue held within the chamber, at least momentarily; The at least one electrode extends along the inner surface at a spaced location between a proximal end of the chamber and the opening, the at least one electrode being elongated along a circumferential direction.

2. The apparatus of claim 1 , wherein the at least one aperture is a single aperture, the aperture being circular.

3. The device of claim 1 further comprising a second port in addition to the at least one port.

4. 2. The apparatus of claim 1, wherein the housing has an end opposite the at least one opening and a wall extending from the end to the opening, the wall defining an inner surface, the inner surface at least partially defining the chamber.

5. The device of claim 4 , wherein the at least one electrode extends around the entire circumference of the inner surface.

6. The device described in claim 1, wherein the at least one electrode includes a first electrode and a second electrode spaced apart from each other along a longitudinal direction along a central axis of the housing that intersects the proximal end of the chamber and the opening.

7. The device described in claim 6, wherein the first electrode and the second electrode are concentric with each other.

8. The device described in claim 6, wherein the first electrode and second electrode each extend along the entire circumference of the inner surface.

9. The device described in claim 1, wherein the at least one electrode includes a plurality of circumferentially elongated electrodes circumferentially spaced from one another along the inner surface.

10. The device of claim 1, wherein the at least one electrode comprises a plurality of circumferentially elongated electrodes, the plurality of circumferentially elongated electrodes comprising a plurality of subsets of circumferentially elongated electrodes, the electrodes of each subset being aligned with one another along a longitudinal direction oriented along a central axis of the housing that intersects the proximal end of the chamber and the opening.

11. 10. The apparatus of claim 1, wherein the at least one electrode has a surface exposed within the chamber, the surface being one or more of: textured and protruding into the chamber.

12. 10. The apparatus of claim 1, wherein the housing is constructed of a material that is flexible, the material including one or more of polycarbonate, polyetheretherketone, polyphthalamide, polyethylene, polyetherimide, polyvinyl chloride, polytetrafluoroethylene, polyamide, polyimide, polysiloxane (silicone), polyethylene terephthalate, polyurethane, cross-linked or non-cross-linked rubber, and polyester.

13. 10. The device of claim 1, further comprising a signal generator in electrical communication with the at least one electrode and configured to deliver the one or more electroporation pulses to the at least one electrode.

14. The device of claim 13, further comprising: a processor in electrical communication with the signal generator and at least one sensor positioned within the chamber, the at least one sensor configured to sense at least one parameter of the tissue during delivery of the one or more electroporation pulses and communicate feedback data of the at least one parameter to the processor, the processor configured to utilize the feedback data to execute one or more algorithms to adjust at least one pulse parameter of the one or more pulses during delivery of the one or more pulses.

15. The apparatus of claim 1 , wherein the at least one electrode is configured to apply the one or more pulses having a potential magnitude in the range of 2V to 1000V.

16. 10. The device of claim 1, wherein the at least one electrode is configured to apply the one or more pulses having a current magnitude in the range of 0.01 amps to 2.0 amps, and the one or more pulses have a pulse duration in the range of 0.1 milliseconds to 100 milliseconds.

17. 2. The apparatus of claim 1, wherein the at least one port comprises an array of ports, each of the array of ports extending through the housing into the chamber such that the housing includes a manifold that defines the array of ports.

18. The device of claim 1 , wherein the target portion of tissue is at least one of dermal tissue and adipose tissue.

Images