Vacuum-assisted electroporation apparatus, 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 Mar. 20, 2020, the entire content of which is incorporated herein by reference.

[0002] The present invention relates to an apparatus for gripping, deforming tissue using vacuum pressure, injecting fluid into tissue, and electroporating tissue using electrodes, as well as systems and methods for injection or other delivery of fluid to tissue and electroporation of tissue.

Background Art

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

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

[0005] Skeletal muscle is also a well-characterized target for in vivo electroporation-mediated (EP) delivery of DNA. Muscle cells are capable of producing and secreting proteins over long periods, and it has been repeatedly demonstrated that EP-enhanced DNA vaccination of muscle can generate an immune response. However, the application of EP DNA delivery to muscle is complex due to varying subcutaneous fat thicknesses, which result in different needle penetration depths into muscle tissue, thus hindering a "one size fits all" approach. Skeletal muscle is typically unsuitable for minimally invasive 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, through-needle electrodes are the most commonly used method for performing EP in muscle.

[0006] Historically, adipose tissue (fat) has been considered an inactive tissue primarily used for energy storage in the form of lipid droplets. Therefore, it is only recently that EP-enhanced DNA treatments have been directed towards the tissue's adipose layer. However, recent research has shown that subcutaneous fat actually plays many dynamic roles. Adipose tissue contains numerous stem cells and immune cells, acts as an endocrine organ by secreting a large number of hormones, secretes many local signals, and contains an intricate network of capillaries. Attempts to achieve in vivo transfection of adipose tissue have been largely limited to surgical methods, requiring administrators to physically remove a sample of the patient's skin to allow direct contact with the adipose tissue. These procedures are highly invasive and unsuitable for clinical use. [Overview of the project]

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

[0008] According to another embodiment of the present disclosure, a method for electroperforating a target tissue includes: placing a chamber adjacent to the tissue; applying vacuum pressure to the chamber to draw the tissue through an opening in the chamber and bring it into contact with a plurality of electrodes extending along the inner surface of the chamber; and delivering one or more electroperforation pulses to the tissue through the plurality of electrodes to generate an electroperforation field within the tissue.

[0009] According to further embodiments of the present disclosure, an apparatus for vacuum-assisted treatment of tissue includes a chamber and a housing defining at least one opening into the chamber. A first port extends through the housing and is away from the at least one opening. The first port is connectable to a vacuum source, thereby configuring the first port to transmit vacuum pressure from the vacuum source to the chamber. The apparatus includes a jet injector extending into the chamber through a second port. The second port is on the opposite side of the at least one opening. The jet injector is configured to respond to vacuum pressure by delivering a jet injection of fluid to a target portion of tissue extending through the at least one opening and held at least momentarily within the chamber. [Brief explanation of the drawing]

[0010] This patent application includes at least one color drawing. A copy of this patent application, including the color drawing, will be provided by the Office upon request and payment of the necessary fees. The above summary and the following detailed description of exemplary embodiments of this application will be better understood when read together with the accompanying drawings. For the purpose of illustrating the structure of this application, exemplary embodiments are shown in the drawings. However, it should be understood that this application is not limited to the exact arrangement and fixtures shown.

[0011] [Figure 1] This is a schematic diagram of an electroporation system that utilizes 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] Figure 1 is a perspective view of a vacuum cup shown in one embodiment of the present disclosure, showing the vacuum chamber of the cup and the array of electrodes located within the chamber. [Figure 2B] Figure 2A is a bottom view of the vacuum cup shown, illustrating the pattern in which the electrodes are arranged. [Figure 2C] Figures 2A and 2B show a plan view of one of the electrodes. [Figure 2D] Figure 2A is a cross-sectional side view of the vacuum cup, taken along the central axis of the cup. [Figure 2E] This is an enlarged cross-sectional view of region 2E-2E shown in Figure 2D. [Figure 2F] Figure 2D is an enlarged cross-sectional view of region 2F-2F. [Figure 3A] This is a perspective view of a sleeve that supports electrodes and can be inserted into the vacuum chamber shown in Figure 2A. [Figure 3B] Figure 3A is a bottom view of the sleeve. [Figure 3C-3M] These are a bottom view and a cross-sectional side view, respectively, of a sleeve having an alternative electrode array pattern according to an additional embodiment of the present disclosure. [Figures 4A-4F]Figures 4A-4E are cross-sectional side views of the vacuum cup shown in Figure 2A, adjacent to the tissue, illustrating typical stages of using the vacuum cup in vacuum-assisted electroporation. Figure 4F is a cross-sectional side view of the vacuum cup shown in Figure 2A, illustrating the varying degrees to which the vacuum cup can draw tissue into the vacuum chamber. [Figure 5A] This is a perspective view of a vacuum cup having a flexible electrode according to one embodiment of the present disclosure. [Figure 5B] Figure 5A is a cross-sectional side view of the vacuum cup, taken along the central axis of the cup. [Figure 5C] This is an enlarged cross-sectional view of region 5C-5C shown in Figure 5B, which indicates the electrode in a neutral state. [Figure 5D] This is an enlarged cross-sectional view of region 5D-5D shown in Figure 5B, illustrating the electrode in a bent state. [Figure 6A-6B] These are a perspective view and a side elevation view, respectively, of an electrode having a contact surface defining a protruding portion, according to one embodiment of the present disclosure. [Figure 6C-6D] These are perspective views and side elevation views, respectively, of an electrode having a contact surface defining a laterally elongated projection, according to another embodiment of the present disclosure. [Figure 7A] This is a perspective view of a vacuum cup having a triangular distal opening and a vacuum chamber geometric shape according to one embodiment of the present disclosure. [Figure 7B] Figure 7A is a bottom view of the vacuum cup shown. [Figure 7C] Figure 7A is a cross-sectional perspective view of the vacuum cup, taken along the central axis of the cup. [Figure 7D] Figure 7A is an elevation view and plan view of the electrode array positioned inside the vacuum cup shown. [Figure 7E] Figure 7D is a side view of the electrode array shown. [Figure 8A] This is a perspective view of a vacuum cup having a rectangular distal opening and a vacuum chamber geometric shape according to one embodiment of the present disclosure. [Figure 8B] Figure 8A is a bottom view of the vacuum cup shown. [Figure 8C] It is a cross-sectional perspective view of a vacuum cup shown in FIG. 8A taken along the central axis of the cup. [Figure 9A] It is a cross-sectional side view of an electroporation assembly including a vacuum cup having a receptacle for receiving a needleless injection device according to an embodiment of the present disclosure. [Figure 9B] It is an enlarged cross-sectional side view showing a representative step of using the electroporation assembly shown in FIG. 9A to inject a drug into tissue drawn into a vacuum chamber. [Figure 10A] It is a cross-sectional perspective view of another embodiment of a vacuum cup having a receptacle for receiving a needleless injection device, the vacuum cup having a cup housing defining a manifold including a plurality of ports in fluid communication with a vacuum chamber through corresponding plural openings defined in electrodes. [Figure 10B] It is an enlarged perspective partial cross-sectional view of corresponding ports and openings shown in FIG. 10A. [Figure 10C] It is a perspective partial cross-sectional view of an alternative configuration of corresponding ports and openings according to another embodiment of the present disclosure. [Figure 10D] It is an enlarged cross-sectional side view of a part of the vacuum cup shown in FIG. 10A during use for providing an electroporation treatment to adipose tissue. [Figure 11A] It is a visual representation of the distribution of methylene blue in subcutaneous pig tissue without (left) and with (right) application of vacuum pressure through a vacuum cup configured similarly to the cup shown in FIG. 2A. [Figure 11B-11C] It shows a juxtaposed comparison of fluid distributions in guinea pig adipose tissue after injection of methylene blue, the injection in FIG. 11B being performed using a needleless vacuum cup similar to that shown in FIG. 9A, and the injection in FIG. 11C being performed by a subcutaneous injection method. [Figure 12]This graph shows 12-week humoral immunogenicity ELISA data in guinea pigs after electroporation of adipose tissue with pGX2013 (a DNA vaccine against influenza virus nucleoprotein (NP)). In particular, it shows a comparative humoral immune response between electroporation using a vacuum cup (as shown in Figure 2A) and electroporation using a caliper electrode electroporation device. [Figure 13] This graph shows 8-week humoral immunogenicity ELISA data in guinea pigs after treatment with pGX2303 (a DNA vaccine against human respiratory syncytial virus fusion glycol-protein (RSV-F)), and in particular, it shows a comparative humoral immune response after electroporation of adipose tissue using a vacuum cup as shown in Figure 2A, and after electroporation of skin using a prior art needle array electroporation device. [Figure 14A] This graph shows ELISA data for humoral immunogenicity in guinea pigs after 6 weeks of treatment with pGX2013 (a DNA vaccine against influenza virus nucleoprotein (NP)), and in particular shows comparative humoral immune responses after treatment with the following: (1) electroporation of intradermal tissue using a prior art needle array electroporation device, (2) electroporation of adipose tissue using a needleless vacuum cup shown in Figure 9A, with vacuum pressure applied after electroporation, (3) injection into adipose tissue using a needle-injection vacuum cup shown in Figure 2A, with vacuum pressure applied after electroporation but no electroporation, and (4) electroporation of adipose tissue using a needle-injection vacuum cup shown in Figure 2A, with vacuum pressure applied after electroporation. [Figure 14B] Figure 14A is a chart showing the cellular immune response from the same study. [Figure 15A] This 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] Figure 15A is a cross-sectional side view of the vacuum cup shown. [Figure 15C] Figure 15A shows an enlarged cross-sectional view of the vacuum chamber of the cup shown during use. [Figures 16A-16B]This is a cross-sectional side view of a vacuum electroporation assembly according to an embodiment of the present disclosure, in which electrodes are disposed on the end faces inside each vacuum chamber facing each distal opening of the vacuum chamber. [Figures 17A-17B] These are bottom views of the electrode array patterns and vacuum port patterns defined on each electrode support member for use with the assemblies shown in Figures 16A to 16B according to embodiments of the present disclosure. [Figure 18A] This is a perspective view of a vacuum electroporation apparatus having multiple distal vacuum chambers. [Figure 18B] Figure 18A is a cross-sectional perspective view of the vacuum electroporation apparatus shown. [Figure 18C] Figure 18A is a side cross-sectional view of the vacuum electroporation apparatus shown. [Figure 18D] This is an enlarged cross-sectional view of region 18D-18D shown in Figure 18C. [Figure 18E] Figure 18A is a bottom view of the vacuum electroporation apparatus shown. [Figure 19A] This is a cross-sectional side view of a vacuum electroporation apparatus having a distal array of electrodes with vacuum ports, according to one embodiment of the present disclosure. [Figure 19B] Figure 19A is a bottom view of the vacuum electroporation apparatus shown, which is a plan view of an electrode array pattern according to one embodiment of the present disclosure. [Figure 20] This is a plan view of an electrode support member having a square electrode array configured for use with a vacuum electroporation apparatus similar to that shown in Figure 19A. [Figure 21A] This is a plan view of an electrode support member having a rectangular electrode array, similar to the electrode support member shown in Figure 20. [Figure 21B] Figure 21A is a side cross-sectional view of the electrode support member. [Figure 22] This figure shows gene expression in guinea pig skin after intradermal injection of plasmids encoding the green fluorescent protein (GFP) gene in various volumes, followed by treatment using various techniques and devices. [Figure 23]This graph shows 8-week humoral immunogenicity ELISA data in guinea pigs treated with HPV DNA vaccine, and in particular, comparative humoral immune responses after each electroporation treatment in the skin using a prior art needle array electroporation device, the vacuum cup shown in Figure 2A, and the vacuum cup shown in Figure 2A with a dose three times (3x) higher than that of other treatments. [Figure 24A] This graph shows 11-week humoral immunogenicity ELISA data in non-human primate models after treatment with HPV DNA vaccines (pGX3001 & 3002), and in particular, comparative humoral immune responses after each electroporation treatment in the skin using a prior art needle array electroporation device, the vacuum cup shown in Figure 2A, and the vacuum cup shown in Figure 2A with a dose three times (3x) higher than that of other treatments. [Figures 24B-24C] Figure 24A is a chart showing the cellular immune response from the same study. [Figure 25] These are tomographic images showing a pair of blebs resulting from prior art mantou injections. The bleb shown on the left contains a drug pre-mixed with a hyaluronidase preparation, while the bleb shown on the right was injected without the hyaluronidase preparation. [Figure 26] Figure 25 is a composite image showing a top view and a perspective view of the bleb. [Figure 27] This plot shows 4-week humoral immune response data in guinea pigs after intradermal treatment with a DNA vaccine (pGX9101) against MERS, in terms of endpoint titer, and specifically compares humoral immune responses after electroporation treatment in the skin using a prior art needle array electroporation device with and without hyaluronidase preparation, and a vacuum cup version shown in Figure 2A, with and without hyaluronidase preparation, both having a 15 mm chamber diameter. [Figure 28]This graph shows 6-week humoral immunogenicity data in guinea pigs in terms of endpoint titer after intradermal treatment with the MERS DNA vaccine (pGX9101), and in particular, comparative humoral immune responses after electroporation treatment in the skin using the prior art needle array electroporation device and the vacuum cup version shown in Figure 2A, which has chamber diameters of 15 mm and 10 mm. [Figure 29A] This graph shows 6-week humoral immunogenicity data in guinea pigs after intradermal injection of a DNA vaccine against influenza nucleoprotein (pGX2013) in terms of mean endpoint titer, and in particular, comparative humoral immune responses after each electroporation treatment in the skin using a prior art needle array electroporation device and a vacuum cup version shown in Figure 2A with a chamber diameter of 15 mm. [Figures 29B-29C] Figure 29A shows a chart illustrating the cellular immune response from the perspective of spot-forming units in the second week (Figure 29B) and fourth week (Figure 29C) of the same study. [Figures 30A-30F] Figure 30A shows a perspective view of a circular electrode array having four electrodes spaced 90 degrees apart around a central axis, for use with the circular opening vacuum cup of the present disclosure for electroporation of adipose tissue. Figures 30B–30F show various simulated electric field intensities generated in adipose tissue according to various electrode sizes. [Figures 31A-31F] Figure 31A shows a perspective view of the circular electrode array shown in Figure 30A for use in electroporation of skin tissue. Figures 31B–31F show various simulated electric field intensities generated in skin tissue according to different electrode sizes. [Figures 32A-32F] Figure 32A shows a perspective view of a rectangular electrode array having four planar electrodes spaced 90 degrees apart around a central axis, for use with the rectangular opening vacuum cup of the present disclosure for electroporation of adipose tissue. Figures 32B–32F show various simulated electric field intensities generated in adipose tissue according to various electrode sizes. [Figures 33A-33F]Figure 33A shows a perspective view of the rectangular electrode array shown in Figure 32A for use in electroporation of skin tissue. Figures 33B–33F show various simulated electric field intensities generated in skin tissue according to different electrode sizes. [Figures 34A-34B] Figure 34A shows a side-by-side comparison of cell infiltration (blue) in guinea pig adipose tissue after vacuum-assisted treatment. The treatment in Figure 34A was a vacuum-only treatment using a vacuum cup, similar to that shown in Figure 2A (i.e., no electroporation), while the treatment in Figure 35B was a vacuum-assisted electroporation treatment using a vacuum cup (i.e., vacuum pressure + electroporation). [Figure 35A] This graph shows humoral immunogenicity data over 6 weeks in guinea pigs treated with a DNA vaccine against MERS, and in particular, it shows comparative humoral immune responses after each electroporation treatment in the skin, using vacuum cups similar to those shown in Figure 2A, with various chamber diameters and the same electroporation parameters. [Figure 35B] Figure 35A is a chart showing the cellular immune response from the same study. [Figure 36A] This figure shows gene expression in guinea pig skin after intradermal injection of a plasmid encoding the green fluorescent protein (GFP) gene, followed by treatment with various vacuum pressures and electroporation voltages using a vacuum cup. [Figure 36B] Figure 36A is a graph showing the measured values ​​of skin fluorescence signals from the results of GDF expression. [Figure 37A] This graph shows 6-week humoral immunogenicity ELISA data in guinea pigs treated with a DNA vaccine against MERS, and in particular, comparative humoral immune responses after electroporation treatment in the skin using (1) a prior art needle array electroporation device and (2) a vacuum cup similar to that shown in Figure 2A. [Figure 37B] Figure 37A is a chart showing the cellular immune response from the same study. [Figure 38]This chart shows the cellular immune response in terms of spot-forming units at 6 weeks after vaccine treatment for recurrent respiratory papillomatosis (RRP), and in particular, it shows a comparative response in the skin after each electroporation treatment using (1) a prior art needle array electroporation device and (2) a vacuum cup similar to that shown in Figure 2A. [Figure 39A] This graph shows humoral immunogenicity data over 4 weeks in guinea pigs treated with a DNA vaccine against MERS, specifically comparing the humoral immune response after treatment with Mantou injection alone, Mantou injection followed by vacuum pressure, and Mantou injection followed by vacuum pressure and electroporation using a vacuum cup similar to that shown in Figure 2A. [Figure 39B] Figure 39A is a chart showing the cellular immune response from the same study. [Figure 40A] This figure shows a cross-sectional side view of the simulated electric field generated by an electrode array with a pair of opposing electrodes in a mound of tissue drawn into a vacuum cup, similar to the vacuum cup shown in Figure 2A. [Figure 40B] This is a cross-sectional side view of the simulated electric field generated by an electrode array having a pair of annular ring electrodes and a central concentric electrode when it is generated in a mound of tissue drawn into a vacuum cup configured similarly to the vacuum cup shown in Figure 15A. [Figure 41A] This graph shows humoral immunogenicity data over 8 weeks in guinea pigs treated with a DNA vaccine against MERS, and in particular, it compares the humoral immune response after vacuum-assisted electroporation treatment in the skin based on electrode pulse patterns using the electrode arrays shown in Figures 40A-40B. [Figure 41B] Figure 41A is a chart showing the cellular immune response from the same study. [Figure 42A]This graph shows a comparative fluid dispersion of the injected material after mantou injection, comparing (1) no vacuum pressure applied, (2) vacuum pressure applied using a vacuum cup with the electrode array shown in Figure 40A, and (3) vacuum pressure applied using a vacuum cup with the electrode array shown in Figure 40B. [Figures 42B-42D] Figure 42A is a photograph illustrating the fluid dispersion shown in the chart. [Figure 43A] Figure 40B is a fluorescence fluoroscopy image showing an injector placed in guinea pig skin positioned beneath a vacuum cup with an electrode array, and Figure 43A shows the injector before vacuum pressure is applied to the chamber. [Figure 43B] Figure 40B is a fluorescence fluoroscopy image showing an injector placed in guinea pig skin positioned beneath a vacuum cup with an electrode array, and Figure 43B shows the injector while vacuum pressure is being applied to the chamber. [Figure 43C] The following graphs compare the expression of secreted alkaline phosphatase (SEAP): (1) a single high-injection-volume vacuum-assisted electroporation procedure using the vacuum cup shown in Figures 43A-B, and (2) six injections, six electroporation events using a prior art needle array electroporation device. [Figure 44] This chart shows 8-week humoral immunogenicity ELISA data in guinea pigs after electroporation of skin tissue using pGX2013, and specifically compares humoral immune responses after electroporation using (1) a jet-injection vacuum cup similar to that shown in Figure 9A and (2) a prior art needle array electroporation device. [Figure 45A-47C] These are fluorescence fluoroscopic images showing tissue flexure in guinea pigs during and after jet injection using a jet injection vacuum cup similar to the one shown in Figure 9A, with various vacuum pressure settings and nozzle-skin offset distance settings. [Modes for carrying out the invention]

[0012] This disclosure may be more readily understood by referring to the following detailed description provided in connection with the accompanying figures and examples that form part of this disclosure. It should be understood that this disclosure is not limited to any specific apparatus, method, application, condition or parameter described and / or shown herein, and that the terms used herein are for illustrative purposes of illustrating specific embodiments and are not intended to limit the scope of this disclosure. Also, as used herein, including in the accompanying claims, the singular forms “a,” “an,” and “the” include the plural forms, and references to specific numerical values ​​include at least that specific value unless the context clearly indicates otherwise.

[0013] As used herein, the term "plural" means more than one. Where a range of values ​​is expressed, an alternative embodiment includes a certain value and / or other specific values. Similarly, the use of the antecedent "about" will be understood to mean that when a value is expressed as an approximation, a certain value forms an alternative embodiment. All ranges are inclusive and combinable.

[0014] The terms “approximately” and “substantially” used herein with respect to dimensions, angles, and other geometric shapes take into account manufacturing tolerances. Furthermore, the terms “approximately” and “substantially” may include values ​​that are 10% greater or less than the stated dimensions or angles. Furthermore, the terms “approximately” and “substantially” may be applied equally to specific values ​​stated.

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

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

[0017] As used herein, the term "fat" refers to the layer containing adipocytes (i.e., fat cells) located in the subcutaneous layer.

[0018] As used herein, the term "electropermeation" means utilizing an electric field within a tissue to temporarily and reversibly increase the permeability and / or porosity of the cell membrane of cells within the tissue, thereby enabling, for example, the introduction of a drug into the cell.

[0019] As used herein, the term “electroporation field” means an electric field capable of electroporating cells. In examples where an electric field includes a portion capable of electroporating cells and another portion that is not, “electroporation field” specifically refers to the portion of the electric field capable of electroporating cells. 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 an organization.

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

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

[0023] As used herein, the terms “intradermal needle electrode electroporation device” and “ID needle electrode EP” refer to prior art electroporation devices that utilize an electrode array of three needle electrodes arranged in a triangular pattern for electroporating intradermal tissue, respectively.

[0024] The embodiments described below relate to systems and apparatus for vacuum-assisted electroporation of tissue, particularly target layers of tissue such as intradermal or adipose tissue. These embodiments expose 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 within a target zone within the tissue layer. In particular, the open end of a vacuum device, such as a vacuum cup, is placed in contact with the outer surface of the tissue (e.g., "skin") covering the tissue volume, and vacuum pressure is applied inside the cup, thereby drawing the tissue volume into the vacuum cup, thereby positionally fixing the tissue volume in the cup, 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 favorable redistribution of fluid within the tissue volume, including favorable in vivo dispersion of injectors within the tissue volume, as well as favorable in vivo inflow and outflow of fluid into and from the target zone. In other words, vacuum pressure improves the dispersion of the injector throughout the tissue, expanding the transfection zone and drawing more in vivo fluid into the target zone, increasing the amount of cells exposed to the transfected cells. The inventors have observed that the vacuum-assisted electroporation procedure described throughout this disclosure results in an increased response of the target to the injector.

[0025] The inventors also observed, surprisingly and unexpectedly, that applying vacuum pressure can induce transfection within tissue volume without electroporation. While not wishing to be bound by any particular theory, the inventors believe that the vacuum pressure applied by the vacuum cup imparts mechanical stress to 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 involved in the transfection observed without electroporation. The inventors further believe that the aforementioned fluid redistribution and mechanical stress likely interact with each other to create a favorable environment within the tissue volume for the transfection of external drugs into 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 necessary to manipulate the electroporation field in the tissue volume to achieve a favorable treatment outcome. In addition to or alternatively, the vacuum pressure can be adjusted as necessary between uses and / or between uses to physically manipulate the tissue volume to achieve a favorable 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 way, the same vacuum cup can be used to target different tissue layers for electroporation treatment by selectively exposing different tissue layers to the electroporation field. In addition, 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 here to Figure 1, the electropermeabilization system 100 for treating a patient according to this disclosure includes a vacuum-assisted electropermeabilization device 2, which includes a housing 4 defining an internal vacuum chamber 6 and a plurality of electrodes 8 (see, for example, Figures 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 the electrode array 9. The device 2 may also be referred to as the “vacuum cup” or simply the “cup”. The housing 4 may also be referred to as the “cup housing”. The vacuum cup 2 is configured such that a physician places the distal end 10 of the vacuum cup 2 on the outer surface 101 of the tissue 102 to be targeted for electropermeabilization and applies vacuum pressure to the vacuum chamber 6 to draw, pull or otherwise guide the tissue 102 into the vacuum chamber 6 so that it comes into contact with the electrodes 8 therein. The electrodes 8 are configured to deliver one or more electropermeabilization pulses to the tissue 102 that is drawn into the chamber 6 by the vacuum pressure and held therein. The tissue 102 includes the layer targeted by the treatment, such as adipose tissue 103 (also referred to herein as “fat layer” 103) or intradermal tissue 104 (also referred to herein as “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 may also have a second port 14 for providing access to a circuit 108 that provides electrical communication between the electrode 8 and an energy source 110, such as a generator. The vacuum cup 2 may further include a third port 16 for providing access to the vacuum chamber 6 to an external tool, such as an injector 18 that carries an injector, particularly one containing a drug. As shown in the figure, the injector 18 may be a subcutaneous injection needle, but the vacuum cup 2 may be adapted for use with other types of injectors 18, including jet injectors, as will be described in more detail below. It should also be understood that the vacuum cup 2 may optionally be used after the drug has been injected into the tissue 102.

[0029] The energy source 110 can be electrically connected to a signal generator 112, such as a waveform generator, for generating an electrical signal in the form of one or more electrical pulses having specific electrical parameters for electroperforating cells in tissue 102 within a vacuum chamber 6, and transmitting it to an electrode 8. Such electrical parameters include potential (voltage), current type (alternating current (AC) or direct current (DC)), current magnitude (amperes), pulse duration, pulse quantity (i.e., the number of pulses delivered), and time interval or "delay" between pulses (in the 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 can be electrically connected to a control unit 114 (also referred to herein as the "controller"), which may include a processor 116 configured to control the operation of the electroperforation system 100, including the operation of the signal generator 112. The processor 116 can be electronically connected to computer memory 118 and may be configured to run software and / or firmware containing one or more algorithms for controlling the operation of the system 100. The processor 116 can be electrically connected to 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 that allows 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 touchscreen display that allows an operator to directly input information on the display 122. It should also be understood that the interface 120 may be a computer interface such as a tabletop computer or laptop computer, or a handheld electronic device such as a smartphone.

[0030] Referring here to Figures 2A-2B, the distal end 10 of the vacuum cup 2 defines at least one opening 20 leading to the vacuum chamber 6. The opening 20 may be circular as shown, but other opening shapes are also within the scope of this disclosure, as will be described in more detail below. The distal end 10 of the vacuum cup 2 (and therefore the opening 20 as well) may be defined by the housing 4, which may define an inner surface 22 extending from the distal end 10 of the housing to the 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 defines at least partially the boundary of the vacuum chamber 6. The inner surface 22 preferably has a bell-shaped or "bell curve" geometric shape. The distal portion 22a of the inner surface 22 extending from the distal end 10 into the chamber 6 may have a tapered curved contour to reduce or otherwise mitigate injuries 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 parallel to the inner surface 22. As shown, the electrodes 8 may extend parallel to the inner surface 22 between the distal end 10 and the proximal end 24 with respect to the longitudinal direction L oriented along the central axis X of the housing 4. The electrodes 8 in this embodiment are preferably substantially rigid, but 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 nonconductive support, which may be constructed of plastic or other suitable nonconductive material. The electrodes 8 may have a surface shape substantially conformal to the inner surface 22. The electrodes 8 may be elongated in a direction having a directional component along the longitudinal direction L. The electrodes 8 may also extend parallel to the inner surface 22 along the circumferential direction C about the central axis X. As shown in Figures 2B-2C, each electrode 8 may define a circumferential dimension C1 (or "width" C1) measured along the circumferential direction C. The electrodes 8 can be positioned at constant angular intervals A1 around the central axis X. The angular interval A1 can be measured from each of the central axes 8x of the electrodes 8. As shown in Figure 2B, the electrodes 8 can be positioned, for example, at 90-degree angular intervals A1 around the central axis X. Thus, it can be said that the electrodes 8 are placed at symmetrical intervals around the central axis X. It should be understood that other angular intervals A1 between the electrodes 8 are also within the scope of this disclosure, as will be described in more detail below. Furthermore, in some embodiments, the angular interval A1 between the electrodes 8 can vary along the inner surface 22. That is, the electrodes 8 can be placed at irregular intervals around the central axis X. Furthermore, the electrodes 8 do not need to be placed at symmetrical intervals around the central axis X.

[0032] As shown in Figure 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 each other along the central electrode axis 8x. The electrode 8 may also have first and second sides 8c, 8d spaced apart from each other to define an electrode width C1 along the circumferential direction C. Each electrode 8 may have an internal electrode surface 8z configured to contact the tissue surface 101 to deliver one or more electroporation pulses. The internal electrode surface 8z may extend from the first end 8a to the second end 8b and from the first side 8c to the second side 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 and from the first side 8a toward the second side 8b. As shown in the figure, the electrode width C1 may be measured between the first and second electrode sides 8a, 8b and does not need to be uniform along the contact portion 8e. The electrode length L1 and width C1 may each be in the range of approximately 1.0 mm to approximately 30 mm, more specifically in the range of approximately 2 mm to approximately 25 mm, and more specifically in the range of approximately 4 mm to approximately 20 mm. The electrode 8 may have a thickness T1 (see Figure 2F) in the range of approximately 0.0005 mm to approximately 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 internal 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 arc and concentrically with the inner surface 22 of the housing 4 in a reference plane perpendicular to the central axis X (i.e., they may share the same center point). The contact surface 8z may also have a curved contour substantially conformal to the inner surface 22 in the 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 on the tissue 102, such as having protrusions, dimples, notches, microneedles and / or rough 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 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, which may be configured to connect to the respective leads of the circuit 108 for transmitting electroporation pulses to the electrode 8.

[0034] Referring here to Figure 2D, the housing 4 may include a housing body 26, which may be formed from a polymer material, preferably with some flexibility, such as, in non-limiting examples, 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), and polyester. It should be understood that other biocompatible and / or medical materials may be used for the housing body 26. The housing body 26 may optionally be a monolithic structure defining the housing 4, but the housing body 26 does not have to be a monolithic structure, and instead may include two or more body components bonded together to define the housing 4. The housing body 26 extends along the longitudinal direction L from the proximal end 28 to the distal end 10. The housing body 26 also defines a wall 30 that extends from the inner surface 22 to the 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, and 16. As shown in the figure, each of the first, second, and third ports 12, 14, and 16 may be adjacent to the proximal end 28 of the housing body 26 and away from the distal end 10. In other words, ports 12, 14, and 16 may be located closer to the proximal end 28 than to the distal end 10 of the housing 4. The first port 12, which may also be called the "vacuum port," extends from the vacuum chamber 6 through the housing body 26 to a port coupling 34 for connecting the vacuum port 12 to a mounting member 36 that interconnects the vacuum port 12 with the vacuum source 106. The port coupling 34 may include a seat 38 and a tubular extension 40 extending outward from the seat 38, defining a receptacle such that the seat 38 defines the inner end of the receptacle. The mounting member 36 may include a mounting member coupling 42 and an insertion stem 44 extending therefrom. The mounting member coupling 42 may be a tubular extension that interconnects with the tubular extension 40 of the port coupling 34, for example, 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 a seat 38 (which may be referred to as the "valve seat"). The valve member 46 may extend from the valve seat 38 into the internal space of the mounting member coupling 42 and thereby interpose in the fluid path between the vacuum port 12 and the insertion stem 44 of the mounting member 36. The valve member 46 may be a duckbill valve as shown, and in other embodiments, as an unspecified example, the valve 46 may be a ball valve or an umbrella valve.

[0036] A second port 14 may extend on the opposite side of the first port 12 and may be configured to allow a passage for a wire-like circuit 108 that passes through the housing 4 into contact with an electrode 8 in the vacuum chamber 6. The second port 14 may also be configured to allow a 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 in the vacuum chamber 6, such tools and / or sensors may be fixed in position relative to the tissue via the vacuum pressure supplied to the chamber 6. A third port 16 may extend from the proximal end 24 of the chamber 6 along the central axis X. The housing 4 may define a mounting portion 48 at the outer end of the third port 16. The mounting portion 48 may be configured to mount a cap 50, such as a puncture stopper, onto the third port 16. The mounting portion 48 and the puncture stopper 50 may have complementary mating shapes that provide an hermetically sealed environment between the puncture stopper 50 and the third port 16. The puncture stopper 50 may be formed of a material that can be punctured by the subcutaneous injection needle 18, allowing the needle 18 to inject a drug into the tissue 102 that has been drawn into the vacuum chamber 6.

[0037] Continuing to refer to Figure 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 a flexible material such as rubber, silicone, and thermoplastic elastomer, as non-limiting examples. As shown in Figure 2E, the outer surface 53 of the sleeve 52 may adhere directly to the inner surface 22 of the housing 4 via a friction fit, although one or more adhesives may optionally be used to attach the sleeve 52 to the inner surface 22. The sleeve 52 may extend from a first or proximal end 54 adjacent to the proximal end 24 of the vacuum chamber 6 to a second or distal end 56 adjacent to 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 can define a proximal opening 52a and a distal opening 52b of the sleeve 52, which may be concentric with the central axis X. As clearly shown in Figure 2F, the first end 54 of the sleeve 52 can partially close the first port 12 and also partially close the third port 16, while allowing a passage for the circuit 206 into the vacuum chamber 6. Thus, the sleeve 52 can be used as a mechanism to control or at least influence the vacuum pressure in the chamber 6.

[0038] The vacuum chamber 6 defines a chamber volume V that is defined from the proximal end 24 of the chamber 6 along the longitudinal direction L to the opening 10, and is 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 the radial direction R intersecting the central axis X. The chamber volume V can also be at least partially defined 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 with respect to a reference plane surrounding 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 specifically in the range of about 3 mm to about 20 mm, and more specifically 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, which can 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 approximately 1.0 mm to approximately 50.0 mm, more specifically in the range of approximately 3.0 mm to approximately 20.0 mm, and more specifically in the range of approximately 6.0 mm to approximately 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 mounted directly to the inner surface 22 of the housing 4, for example, by being embedded in or at least partially embedded within the housing wall 30. In such embodiments, the vacuum chamber 6, and therefore the chamber volume V, may be defined at least partially by the inner surface 22 of the housing 4 and the inner surface 8z of the electrode 8. Therefore, 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] Referring here to Figures 3A-3B, the sleeve 52 can support each of the electrodes 8, and thus the electrode array 9. Thus, the sleeve 52 may also be referred to as the 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 to fourth electrodes 8-1, 8-2, 8-3, and 8-4 are positioned at 90-degree intervals A1 around the central axis X2 of the sleeve 52 (which is substantially coherent 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 is four electrodes 8. The total number of electrodes 8n may also be referred to herein as “total” 8n or simply “whole” 8n. 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 tissue 102 drawn into the vacuum chamber 6. For example, as shown in Figures 3C-3D, sleeve 52 may have an electrode array 9 including four electrodes 8 spaced 90 degrees apart around the central axis X2 of sleeve 52, as described above, but each electrode 8 may also have a narrower circumferential dimension C1 than the electrode 8 in the embodiments described above, and therefore a smaller cumulative surface area. As shown in Figures 3E-3F, sleeve 52 may have an electrode array 9 including six electrodes 8 spaced 60 degrees apart around the central axis X2. As shown in Figures 3G-3H, the electrode array 9 may include ten electrodes 8 spaced 36 degrees apart around the central axis X2.

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

[0041] As shown in Figures 3K to 3L, the electrode array 9 may include multiple circumferentially elongated electrodes 8, each comprising four subsets 9a to d of circumferentially elongated electrodes 8. The electrodes 8 within each subset 9a to d may be substantially aligned longitudinally with respect to each other, and each subset 9a to d may be circumferentially spaced apart from each adjacent subset 9a to d of electrode 8. In this example, each subset 9a to d of electrode 8 may contain five longitudinally spaced electrodes 8. Thus, the sleeve 52 may contain a total of 20 circumferentially elongated electrodes 8. In this embodiment, the electrodes 8 within each subset 9a to d may have an angular span A2 in the range of approximately 1 to approximately 90 degrees around the central axis X2, and an inter-electrode span A3 in the range of approximately 1 to approximately 90 degrees between adjacent subsets 8a to d. Separating subsets 9a-d circumferentially from each other allows, among many other possibilities, that subsets 9a-d be able to be fired independently of each other (or driven separately by current or voltage sources). This helps ensure that each of the regions within the tissue volume adjacent to and associated with subsets 9a-d (in this embodiment, such tissue regions may be characterized as "quadrants" of the tissue volume) accepts the scope of application of the electroporation field, which results in a more symmetrical electroporation field and avoids a situation where local differences in the conductivity of the tissue within the tissue volume bias the electroporation field away from one or more of the regions within the tissue volume.

[0042] Furthermore, the circumferentially spaced subsets 9a-d shown in Figures 3K-3L can also generate a uniquely directional electroporation field within the tissue volume, such as for applying electrical pulses to cells from multiple angles in subsequent pulses (i.e., exposing cells to gradients of electroporation fields along different directions), which can electrically permeate cells more efficiently than applying electrical pulses to cells from a single direction. It should be understood that such multidirectional electroporation fields can also be generated by the various array designs in Figures 3A-3H. However, the circumferentially spaced subsets 9a-d in Figures 3K-3L also allow the electrodes 8 to emit not only 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 can also increase the amount of possible unique pulse patterns, and a more homogeneous electroporation field application range is also possible, considering that the electroporation field is concentrated on the contact surface 8z of the electrodes 8.

[0043] The various electrode array 9 patterns described above are provided as non-limiting examples, and it should be understood that other configurations of electrode array 9 are also within the scope of this disclosure. For example, one or more of the parameters of the electrode array 9 described above can be adjusted as needed, and these parameters include, but are not limited to, the quantity of electrodes 8 in each array 9, the electrode length L1 and width C1, and the spacing between electrodes. These parameters can affect the three-dimensional (3D) shape of the electric field (and therefore 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 concentrate the distribution of the electric field within the tissue 102 in a manner that provides an enhanced electroporation procedure.

[0044] Referring to Figures 4A-4F, an exemplary method of using the vacuum cup 2 to provide electroporation treatment to adipose tissue 103 is described here.

[0045] As shown in Figure 4A, the physician may position the distal end 10 of the vacuum cup 2 on the outer surface 101 of the patient's skin 104, with the distal end 10 positioned to cover a target zone 105 of the adipose tissue 103. The target zone 105 may be pre-selected or may be a result of the positioning 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 Figure 4B, a physician may apply a vacuum pulse (also referred to herein as a "vacuum pulse") to the vacuum chamber 6 by generating a vacuum pressure within the vacuum chamber 6, in particular by operating a vacuum source, that is sufficient to draw the volume of tissue 102 or "mound" 140 into the chamber 6 and bring it into contact with the contact surface 8z of the electrode 8, within a range of about -0.1 psi to about -14.7 psi. 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 tissue mound 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 way, 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 tissue mound 140 includes skin 104 and adipose tissue 103. For the purposes of this disclosure, the treatment zone 107 is defined as the portion of tissue volume (such as a tissue mound 140) extending between the electrodes 8. As illustrated, the bottom boundary 107a of the treatment zone 107 may be defined by a hypothetical path extending 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 of this embodiment will include at least a portion of the tissue mound 140 and at least a portion of the injection site 109 (see Figure 4C). In the illustrated embodiment, the tissue located within the treatment zone 107 during the 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 contain any skeletal muscle. As described 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.The illustrated embodiment shows that the skin 104 of the tissue mound 140 is placed in direct contact with the contact surface 8z of the electrode 8, but it should be understood that additional materials such as conductive gel may be used to improve electrical communication between the electrode 8 and the skin 104.

[0047] As shown in Figure 4C, a physician can inject a drug into the adipose tissue 103 of the mound 140. To perform the injection, the physician can puncture 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 within the chamber volume V. The physician can then inject the drug into the injection site in the adipose tissue 103 and subsequently withdraw the subcutaneous injection needle 18 from the vacuum cup 2. Surprisingly and unexpectedly, the inventors observed that the injectable material 142 discharged from the subcutaneous injection needle 18 into the adipose tissue 103 did not remain in the pooled bolus 142a within the adipose tissue 103, but rather dispersed toward the skin 104 in response to the vacuum pressure. Therefore, the physician may inject the injector 142 near or at the bottom of the treatment zone 107, or slightly below the treatment zone 107, allowing the vacuum pressure to effectively draw the injector 142 upward into the treatment zone 107, and even allowing the injector 142 to be concentrated within the treatment zone 107. Based on observations during testing, the inventors also believe that the vacuum pressure may be manipulated to assist in mixing the injector 142 with the in vivo fluid, extracellular components, and cells, and to hold the injector 142 within the treatment zone 107 in a manner that increases the latency of the injector 142 there, thereby increasing transfection.

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

[0049] As shown in Figure 4D, the physician may deliver one or more electroporation pulses to the tissue mound 140. In particular, the physician may 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 within the adipose tissue 103 in the treatment zone 107, in the illustrated embodiment. The electroporation field 144 substantially creates reversible portions in the cell membranes of cells (e.g., adipocytes) in the treatment zone 107, causing transfection of the injector into the temporarily porous cells. In this way, the electroporation field 144 generates a transfection zone within 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 subregion of the electric field 145 generated by the electrode 8 during pulse delivery.

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

[0051] One or more electroporation pulses may have a current (amperage) in the range of approximately 0.01 Amp to approximately 2.0 Amp.

[0052] Each of the one or more electroporation pulses may have a duration ranging from approximately 100 microseconds to approximately 500 milliseconds.

[0053] The number of electroporation pulses can range from 1 pulse to approximately 10 pulses.

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

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

[0056] In a procedure involving multiple electroporation pulses, the pulses may be delivered by electrode 8 in the following pulse sequence or pattern, including and including the last electroporation pulse of the procedure: a first electroporation pulse delivered through tissue 102 by a first positive subset of electrode 8 to a first negative subset of electrode 8, a second electroporation pulse delivered through tissue 102 by a second positive subset of electrode 8 to a second negative subset of electrode 8, and so on. Between each electroporation pulse, each positive and negative subset of electrode 8 may range from a single electrode 8 to any combination of electrodes 8 at least one less than the total number of electrodes 8n in array 9 (i.e., 8n-1 electrodes). The electroporation pulse pattern may be delivered according to a programmed sequence, which may be input by the user to controller 114 (e.g., via user interface 120). Furthermore, the sequence of electroporation pulses may be delivered in a distributed pattern, optionally. In such a dispersed pattern sequence, each pulse of the multiple electroporation pulses may be delivered between at least two sets of electrodes 8, and each subsequent pulse of the multiple electroporation pulses may be delivered by at least two different sets of electrodes 8. The dispersed electroporation pulse pattern may minimize, or preferably eliminate, the generation of thermal stress associated with electroporation in 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. Electrical parameters detected in the tissue 102 may include, in non-limiting examples, voltage, current, impedance, and / or resistance. One technique for detecting 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 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 a plurality of electroporation pulses may be delivered between at least two sets of electrodes 8, where at least one other electrode 8 is neutral and a sensing electrode 8 that measures electrical parameters of tissue 102 such as impedance, and each subsequent pulse of a plurality of electroporation pulses may be delivered by a different set of at least two electrodes 8, where at least one electrode 8 is neutral and a sensing electrode 8 that measures electrical parameters. At least one neutral electrode 8 may alternate with each pulse, but the same electrode 8 (or set of electrodes 8) may remain neutral in consecutive pulses. Alternatively, during electroporation pulses, at least one electrode 8 of the array 9 may actively deliver pulses while measuring electrical parameters of tissue 102.

[0058] Another optional technique 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 a non-invasive sensor 150, such as a contact sensor 150, as shown in Figure 4D. The contact sensor 150 is configured to measure a parameter and transmit 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 a second port 14. The physician may position the contact sensor 150 in contact with a tissue 102, such as skin 104, where the contact sensor 150 can measure a 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 a part of the injection needle 18, for example, its distal tip region (see Figure 4C), and may be electrically connected to 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 used during the procedure to relay information about a single or multiple electrical parameters of tissue 102 to the controller 114.

[0059] The electrical parameter information received by the controller 114 may be used for performance diagnostic purposes and / or for active feedback control of the electroporation signal delivered to the electrode 8 and therefore 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 transmit 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 that incorporate the parameter information to process or otherwise derive outputs such as control commands for controlling the electroporation pulses. The algorithms may also utilize parameter data obtained from computer memory 118. It should be understood that the control commands derived from the algorithms may adjust the electroporation pulses in real time, such as substantially instantaneously, for electroporation purposes, based on the parameter information from sensors 8, 150, 152. In this manner, the electroporation system 100 may utilize sensors 8, 150, and 152 in an active feedback loop for constant control and adjustment of the electroporation pulses as needed to achieve the desired electroporation treatment results in the target tissue 102. Techniques and / or electronic components for performing such feedback control may be available as fully disclosed in U.S. Patent No. 9,452,285, published September 27, 2016, entitled "Electroporation Devices and Methods of Using Same for Electroporation of Cells in Mammals" (Reference 285), and U.S. Patent Publication No. 2011 / 0009807A1, published January 13, 2011, entitled "Variable Current Density Single Needle Electroporation System and Method" (Reference 807), the full disclosures of each of these are incorporated herein by reference.

[0060] As shown in Figure 4E, following one or more electroporation pulses, the physician may return the pressure in the vacuum chamber 6 to atmospheric pressure, allowing the vacuum cup 2 to separate the tissue 102, which can then return to its anatomical shape. The transfected adipocytes may define a transfection zone 105z in the fat layer 103.

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

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

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

[0064] While we do not wish to be bound by any particular theory, we believe that the remarkable increase in immune response and cell infiltration is likely related. Furthermore, we believe that the increased cell infiltration is driven, at least in part, by the spontaneous secretion of chemical signals from ruptured capillaries and nearby leukocytes in the treatment zone, which reactively attract and migrate additional cells, such as additional leukocytes, into the treatment zone. We also believe that the observed cell infiltration may be driven, at least in part, by an inflammatory response. We further believe, based on the observed dispersion of the injector through the tissue in response to vacuum pressure, that the in vivo fluid outside the treatment zone 107 is mechanically drawn into the treatment zone 107 in response to vacuum pressure.

[0065] Here, with reference to Figures 5A to 5D, an embodiment of the vacuum cup 502 utilizing the flexible electrode 508 will be described. The vacuum cup 502 of this embodiment is the same as the vacuum cup 2 described above. Therefore, the same reference numerals used above indicate common features in this embodiment. For the sake of 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] As shown in Figures 5A-5B, the vacuum cup 502 has a housing body 526 that defines an inner surface 522 that partially defines the vacuum chamber 506. The housing body 526 also defines a plurality of relief ports 560 that extend from the 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 proximal channels 562 and distal channels 562 that are spaced apart from each other along the longitudinal direction L. Each channel 562 may extend annularly along the entire circumference around the central axis X, but in other embodiments, one or more channels 562 may extend annularly over a shorter range than the entire circumference around the central axis X.

[0067] The vacuum cup 502 includes a flexible sleeve 552 that resides within the vacuum chamber 506 and supports a plurality of electrodes 508 arranged in the 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 channel 562 in a manner that provides a flexible barrier or membrane between the channel 562 and the vacuum chamber 6. The electrodes 508 are disposed on the inner surface 555 of the sleeve 552. The sleeve 552 may also support a circuit, such as a wired circuit or a printed circuit, for providing electrical communication between the electrodes 508 and the controller 114. The electrodes 508 in this embodiment may be elongated in the circumferential direction, similar to the electrodes 8 described above with reference to Figures 3K-3L. One or more electrodes 508, and up to each, cover at least one of the channels 562 and are constructed of a flexible material. The electrode material may be a metal such as copper, stainless steel, and gold, in non-limiting examples. Alternatively, or in addition, the electrode material may include, in non-limiting examples, a conductive polymer or a carbon allotrope such as graphene, which may contain 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 to d of electrodes 508. The electrodes 508 within each subset 509a to d may be substantially aligned along the longitudinal direction L, and each subset 509a to d may be separated from each adjacent subset 509a to d along the circumferential direction C. The subsets 509a to d may be regularly separated from each other, such as at 90-degree intervals around the central axis X. In the illustrated example, each subset 509a to d includes a proximal electrode 508 and a distal electrode 508 separated longitudinally from each other, 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 described above, the electrode 508 is connected to a circuit that provides electrical communication between the electrode 508 and the controller 114. It should be understood that the circuit of the electrode array 509 can be configured such that the controller 114 can individually control the parameters of the electroporation pulse for each subset 509a to d of the electrode 508, and further, individually control the parameters of the electroporation pulse for each electrode 508 within each subset 509a to d.

[0069] Referring here to Figures 5C-5D, the flexible sleeve 552 and electrode 508 are configured to bend inward (towards the central axis X) into the vacuum chamber 6 when vacuum pressure is applied within the chamber 6, increasing contact between the contact surface 508z of electrode 508 and the tissue drawn into the chamber 6. The relief port 560 provides fluid communication between the channel 562 and the outside of the vacuum cup 502, thereby allowing the pressure within the channel 562 to remain substantially at atmospheric pressure. In this way, the vacuum pressure within the chamber creates a pressure gradient on the wall of the sleeve 552 between the chamber 6 and the channel 562, allowing the sleeve 552 and the electrode 508 on it to bend inward into the chamber 6.

[0070] Referring here to Figures 6A-6D, the electrode 608 for positioning the vacuum cup 2, 502 within the vacuum chamber 6 may include a plurality of projections 664 that can be defined by the contact surface 608z of the electrode 608. Similar to the embodiments described above, the electrode 608 extends along the central axis 608x of the electrode 608 from a first end 608a to a second end 608. The electrode 608 may be elongated along the direction oriented along the central axis 608x. The electrode 608 may also extend along the transverse axis 608y of the electrode 608 from a first side surface 608c to a second side surface 608d. The contact surface 608z may include a substantially smooth base portion 665, and the projections 664 may extend outward (i.e., inward into the chamber 6) from the base portion 665. The base portion 665 may be substantially planar as shown, but in other embodiments, the base portion 665 may have a curved contour substantially conformal to the inner surface 22 of the cup housing 4. By extending outward from the base portion 665, the projection 664 can increase the contact area between the contact surface 608z of the electrode 608 and the tissue drawn into the chamber 6. During use, the projection 664 can be pushed into the tissue 102 in a manner that breaks and alters the uppermost layer of skin 104, thereby improving the electric field distribution within the target tissue. More specifically, the projection 664 increases the magnitude of the electric field formed within the tissue 102 in response to a given input voltage, as further and more fully described in International (PCT) Patent Publication WO2018 / 057900A1 (Reference 900), published on March 29, 2018, entitled "Method and Device for Minimally Invasive In Vivo Transfection of Adipose Tissue Using Electroporation," and the entire disclosure is incorporated herein by reference.

[0071] As shown in Figures 6A-6B, the projections 664 may have convex, arcuate, or dome-shaped geometric forms. The projections 664 may be arranged in columns and rows. As shown in Figure 6C, the projections may be elongated along a direction oriented along the transverse axis 608y. As shown in Figures 6B and 6D, the projections 608 shown in Figures 6A and 6C may have similar side profiles. It should be understood that other geometric forms of projections, including pointed, conical, frustoconical, and pyramidal shapes, are also within the scope of this disclosure. It should also be understood that the electrodes 608 may be configured for positioning with respect to the chamber 6 such that they are elongated along the longitudinal direction L of the vacuum cup 2, elongated along the circumferential direction C of the vacuum cup 2, or elongated along directions oblique to the longitudinal and circumferential directions L and C.

[0072] Various parameters of the vacuum cups 2, 502 described herein, such as the chamber depth L2, chamber diameter D1, and cup shape, including the cross-sectional shape on a reference plane perpendicular to the central axis X, and / or the shape of the opening 20, can be adjusted as necessary to achieve a desired electroporation procedure, such as across a wide range of mammalian and skin biostructures. For example, the vacuum cups of this disclosure may have non-circular opening and / or chamber geometric shapes, such as polygonal opening and / or chamber geometric shapes, as a non-limiting example. A vacuum cup 702 having a triangular opening and chamber geometric shape is described with reference to Figures 7A-7E. A vacuum cup 802 having a rectangular opening and chamber geometric shape is described with reference to Figures 8A-8C. In such embodiments, the chamber dimension D1 may be referred to as the "chamber width" D1.

[0073] Referring here to Figures 7A-7C, a vacuum cup 702 is shown having a distal end 710 defining a triangular opening 720. The vacuum cup 702 of this embodiment is similar to the vacuum cups 2 and 502 described above. Therefore, similar reference numbers 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 and 502 described above.

[0074] The vacuum cup 702 includes a housing body 726 that defines three side walls 730 arranged in a triangular pattern when viewed in a reference plane perpendicular to the central axis X of the vacuum cup 702. Thus, the housing body 726 defines a vacuum chamber 706, which also has a triangular shape in the perpendicular reference plane. The side walls 730 intersect each other at three angles 735 of the housing body 726, and these angles 735 are preferably curved. The triangular pattern may be equilateral triangles as shown, but 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 side wall 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 when measured along this linear surface profile, it may define a length L4 in the 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 is preferably tapered inward toward the central axis X toward the proximal end 724 of the chamber 706. The taper angle A4 of the main portion 722b, measured with respect to an axis parallel to the central axis X, may be defined in the range of about 0 to about 80 degrees, more specifically in the range of about 0.25 to about 10 degrees, and more specifically in the range of about 0.5 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 to c of the electrodes 708. The first subset 709a of electrodes 708 may be disposed on the inner surface 722 of the first side wall 730, the second subset 709b of electrodes 708 may be disposed on the inner surface 722 of the second side wall 730, and the third subset 709c of electrodes 708 may be disposed on the inner surface 722 of the third side wall 730. The subsets 709a to c of electrodes 708 may be supported by their respective substrates or "pads" 752, which may be constructed of electrical insulating materials such as silicon, polyetheretherketone (PEEK), polyphthalamide (PPA), polyethylene, polycarbonate, and polyetherimide (PEI) as non-limiting examples. The substrate 752 may be flexible or rigid. The substrate 752 may also carry circuits, such as wired or printed circuits, 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 described above, the circuits 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 also individually control the pulse parameters of each electrode 708 in each subset 709a-c. The outer surface 753 of the substrate 752 may be attached to the inner surface 722 of each side wall 730 via adhesive, but other fastening techniques are also within the scope of this disclosure.

[0077] Each subset 709a-c of electrode 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 electrodes 708 than four, such as a single electrode, two, three, five, six, seven, eight, nine, ten, eleven, twelve, or more than twelve electrodes 708, for example, more than 100 electrodes 708. Advances in micro-electromechanical systems (MEMS) technology and nanotechnology can reduce the size of each discrete electrode 708 to such an extent that each subset 709a-c may have a virtually unlimited number of discrete electrodes 708.

[0078] The electrode 708 defines a contact surface 708z which may be smooth as shown. The main portion of the contact surface 708z may be planar, as is the portion covering the main portion 722b of the inner surface 722. However, it should be understood that the contact surface 708z of at least one and up to all of the electrodes 708 may be textured and / or have protrusions defined, as in the embodiments described above. Furthermore, the substrate 752 and the electrode 708 may be flexible and may cover channels and relief ports defined in the housing body 726 to allow the electrode 708 to flex inward when vacuum pressure is applied to the chamber 706, as in the embodiments described above with reference to Figures 5A-5D.

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

[0080] Referring here to Figure 7E, the electrode 708 may extend inward from the substrate 752 (into the vacuum chamber 706). 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 attached 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 exist at a depth between the outer and 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 extend co-existing with the outer surface 753 of the substrate 752.

[0081] In this embodiment, the electrode arrays 709a to c are configured such that when vacuum pressure draws the tissue 102 into the vacuum chamber, the tissue 102 comes into contact with the contact surface 708z and is drawn into the gap W2 between the electrodes, thereby also coming into contact with the sides 708c and d of the electrodes, thereby increasing the overall contact interface area between the tissue 102 and the electrode 708.

[0082] The geometric shape of the vacuum chamber 706 and the configuration of the electrode arrays 709a-c in this embodiment allow for substantially planar electrodes 708 along the side walls 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 those of the vacuum cups 2,502 described above. The triangular shape of this embodiment also advantageously allows for the confinement of the tissue within the boundaries of the electroporation field defined by the triangular pulse pattern to a greater extent than in prior art electroporation devices. In addition, the geometric shapes of polygonal arrays, including the triangular arrays of this embodiment, can generate a more heterogeneous electroporation field compared to other array designs, including circular designs, due to the acute angles, or "sharp" angles, between adjacent electrodes opposite the corners 735. For example, it may be desirable to generate regions of increased electric field magnitude (resulting in increased current) within the target tissue, and having "sharp," or acute-angled, adjacent electrode ends is one way to generate such regions of increased electric field / current within the tissue.

[0083] Referring here to Figures 8A-8C, a vacuum cup 802 is shown having a distal end 810 defining a rectangular opening 820. The vacuum cup 802 of this embodiment is similar to the vacuum cups 2, 502, and 702 described above. Therefore, similar reference numbers 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, and 708 described above, in particular the differences between the vacuum cup 702 described above with reference to Figures 7A-7E.

[0084] The vacuum cup 802 includes a housing body 826 defining four walls, in particular 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 which 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, but in other embodiments, the side walls 830 and end walls 831 may be of the same length so that the rectangle becomes a square. It should also be understood that the walls of the housing body 826 may define other quadrangular geometric shapes (i.e., non-rectangular).

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

[0086] As described above, the subsets 809a and 809b of electrode 808 may be supported by their respective non-conductive substrates 852 attached to the inner surface 822. Each subset 809a and 809b may have four electrodes 808, but each subset 809a and 809b may have more or fewer electrodes 808 than four. As described above, the electrode array 809 may include a circuit configured so that the controller 114 can individually control the parameters of the electroporation pulses to each subset 809a and 809b of electrode 808, and also individually control the pulse parameters of each electrode 808 within each subset 809a and 809b.

[0087] As shown in the figures, the contact surface 808z of the electrode 808 may be smooth, and its main portion may be planar. However, in other embodiments, the contact surface 808z may be textured and / or have protrusions defined, as described above. The housing body 826, the substrate 852 and the electrode 808 may also be configured to cooperate so as to allow the electrode 808 to flex inward in response to vacuum pressure, similar to the embodiments described above with reference to Figures 5A-5D. The electrode 808 may have the same length L1, width W1 and thickness T1 as described above with reference to Figures 7D-7E, and may operate in the same manner as described above.

[0088] The rectangular geometric shape of the vacuum chamber 806 and the configuration of the electrode arrays 809a and 809b in this embodiment provide a spherical electroporation field that is more elongated along the direction across the central axis X (particularly along the direction perpendicular to the end wall 831) compared to those of the vacuum cups 2, 502, and 702 described above. Furthermore, the rectangular array in this embodiment allows for substantially planar electrodes that face each other directly and are capable of emitting directly opposing electrical pulses. In this regard, the array in this embodiment can be used in a manner similar to opposing plate type or caliper type electroporation devices known in the art. For example, the rectangular array design in this embodiment may allow a physician to "pinch" elongated sections of tissue, similar to caliper type electrode configurations. It may be advantageous to "grasp" the tissue mainly along one axis and pinch in a way that forms an elongated treatment zone within the tissue. For example, by increasing the aspect ratio, the rectangular cup 802 can treat larger tissue areas without increasing the gaps between electrodes. This could, for example, allow for the use of lower voltages and treatment of larger tissue areas compared to a circular array design operating at the same voltage.

[0089] In addition, as described above, the polygonal array shape including the array of this embodiment can generate a more heterogeneous electroporation field due to the acute angles (approximately parallel in this embodiment) between adjacent electrodes on the opposing side walls 830 of the cup 802. Furthermore, as described above, the array can be used to generate regions of increased electric field magnitude within the tissue.

[0090] It should be understood that vacuum cups of other embodiments may have geometric shapes of openings and / or chambers of other polygonal shapes, ranging from pentagonal, hexagonal, heptagonal, octagonal, and so on, to circular geometric shapes. Furthermore, such polygonal shapes do not need to have equilateral configurations. In yet another embodiment, the vacuum cup may have geometric shapes of openings and / or chambers of other shapes, such as elliptical, oblong, or irregular shapes, as non-limiting examples. It should be understood that an elliptical cup shape may offer similar advantages to the rectangular cup 802 described above, including the ability to "grasp" tissue mainly along one axis, pinch it, and form an elongated treatment zone within the tissue, thereby enabling lower voltage for treating larger tissue areas.

[0091] Here, with reference to Figures 30A to 33F, the results of various simulated tests of four-electrode arrays 9 on adipose tissue 103 and skin 104 will be explained. In each of Figures 30A to 33F, the counter electrodes 8 are separated from each other by a distance of 15 mm (this distance is close to the chamber diameter D1), and the applied voltage between the electrodes is the same. Therefore, Figures 30A to 33F demonstrate the effects of electrode shape, size, and separation on electric field generation.

[0092] Figures 30A to 31F show a circular array 9 in which the electrode contact surface extends along a generally circular outer circumference. The electrode widths shown in Figures 30A to 31F are defined by a ratio to the chamber diameter. Thus, a chamber that tapers inward towards the top will have a thinner electrode width near the top and a thicker electrode width near the bottom, while always occupying the same ratio to the circumference of the chamber at any point along the inner surface of the chamber. 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. Therefore, a chamber that tapers inward towards the top causes adjacent electrodes constructed in this way to be closer to each other at the top than at the bottom. Depending on the dimensions of the cup and the taper angle of the wall, this is undesirable as it can cause adjacent electrodes to be very close to each other or even in contact with each other at the top of the chamber.

[0094] These figures show that square arrays (Figures 32A-33F) create a more heterogeneous electric field in both adipose tissue 103 and skin 104 than their circular array counterparts (Figures 30A-31F). Therefore, it can also be said that circular arrays generate a more homogeneous electric field in both adipose tissue 103 and skin 104 than their rectangular array counterparts. These differences in the heterogeneity / homogeneity of the electric field produced by circular and rectangular arrays can be utilized to their advantage as needed, depending on the desired procedure. For example, if a particular electroperforation procedure benefits from a more homogeneous electric field, the physician may choose a circular vacuum cup (and electrode array). If a particular electroperforation procedure benefits from a more heterogeneous electric field, the physician may choose a polygonal vacuum cup (and electrode array). Regarding electrode design, it should be understood that maintaining adequate spacing between adjacent electrodes is preferable, especially when the vacuum chamber tapers inward at the top, as in the designs shown in Figures 30A-31F.

[0095] Referring here to Figure 9A, an example of a vacuum electroporation assembly 900 is shown, which includes a vacuum cup 902 configured for needleless injection of a drug, particularly via jet injection. Thus, the vacuum cup 902 may be referred to as the “needleless” vacuum cup 902 or the “jet injection” vacuum cup 902. The vacuum cup 902 of this embodiment is similar to the vacuum cups 2, 502, 702, and 802 described above, particularly the vacuum cup 2 described above with reference to Figures 1-4F. Thus, similar reference numbers 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 described above, the vacuum cup 902 has a vacuum chamber 906 in which electrodes 908 are disposed, and a housing body 926 defining a first port 912, a second port 914, and a third port 916, each communicating with the vacuum chamber 906. As described above, the first port 912 is configured for connection to the vacuum source 106, and the second port 914 is configured for a circuit passage into the vacuum chamber 906, among others. 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 an injector 142 into the tissue 902 drawn into the vacuum chamber 906. In addition, the housing body 926 defines a mounting portion 948 at the outer end of the third port 916, which is configured to provide a sealed connection with the injection device 970. The mounting portion 948 may be located within the receptacle 953 defined in the mounting portion 948 and may carry one or more sealing members, such as a sealing O-ring 951, which is configured to provide a sealing engagement with the outside of the injection device 970.

[0097] The jet injection device 970 includes an injection housing 972 that defines a fluid chamber or container 974 in which an injection material 142 containing a drug is stored. The outer surface 975 of the injection housing 972 is sized to cooperate with a third port 916 and a sealing O-ring 951 to provide a sealed connection between the injection housing 972 and the vacuum chamber 906. The distal portion 976 of the injection housing 972 defines a nozzle 978 that is in fluid communication with the container 974. The nozzle 978 is configured to discharge the injection material 142 from the container 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 potential splashing or rebound of the injection material 142 generated during jet injection. The shield 980 can also protect the circuitry of the cup 902 from exposure to the injection material 142.

[0098] The distal end 979 of the nozzle 978 preferably extends distally beyond the distal end 981 of the shield 980. The jet injector 970 includes a plunger 982 carrying a piston 984 at its distal end. The inner surface 988 of the container 974 at the distal tip 986 of the piston 984 and its distal end 990 has a complementary geometric shape such that the advance of the piston 984 toward the distal end 990 discharges a jet of injector 142 from the nozzle 978 into the tissue 102. It should be understood that the container 974 may be configured to carry a pre-measured volume or dose of injector. Furthermore, the plunger 982 may be controlled by a launching mechanism or actuator, etc., to discharge a predetermined dose of injector through the nozzle 978 into the patient's tissue 102. Such a launching mechanism may include, in non-limiting examples, one or more pre-loaded or loadable springs, compressed gas canisters, and similar.

[0099] The jet injection device 970 may be constructed to be further fully disclosed in any of the following: U.S. Patent Publication No. 10,045,911, issued on 14 August 2018, entitled “Intradermal Injection Device” (the “'911 Document”), U.S. Patent Publication No. 2019 / 0000489A1, published on 3 January 2019, entitled “Intradermal Jet Injection Electroporation Device” and U.S. Patent Publication No. 2009 / 0137949A1, published on 28 May 2009, entitled “Needle-Free Injection Device with Nozzle Auto-Disable” (the “'949 Document”), each of which is incorporated herein by reference in its entirety.

[0100] The needleless vacuum cup 902 and the jet injector 970 have complementary features that enhance the electroperforation procedure provided thereby. For example, referring to Figure 9B, when vacuum pressure draws the mound 140 of tissue 102 into the vacuum chamber 6, the skin layer 104 on the mound 140 tightens, temporarily reducing the elasticity of the skin layer 104, thereby allowing the jet or stream of injector exiting the nozzle 978 to puncture the skin layer 104 more efficiently with less injector rebound. To assist in tightening the skin layer 104, the physician may use sufficient vacuum pressure to draw the skin layer 104 into contact with the distal end 979 of the nozzle 978 so 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, and further stretching the skin layer 104 in the depression 104a. Combined with the use of the jet injector 970, such tightening in the skin layer 104 allows the discharged injector 142 to penetrate the entire tissue mound 140 to a greater extent than with needle injection (compared to Figure 4C) and to a greater extent than when using a jet injector that is not assisted by vacuum pressure, by puncturing the skin layer 104 at the time of injection.

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

[0102] With reference to Figures 10A-10D, another embodiment of the needleless vacuum cup 1002 is described, in which the vacuum cup 1002 has a vacuum chamber 1006 configured such that the tissue 102 drawn therein is at least partially drawn into a plurality of openings 1063 defined by an electrode 1008 in the vacuum chamber 1006. The vacuum cup 1002 of this embodiment is similar to the vacuum cups described above, in particular the vacuum cup 902 described above with reference to Figures 9A-9B. Thus, similar reference numbers 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] Referring here to Figures 10A-10B, the vacuum cup 1002 of this embodiment has a housing body 1026 defining a first port 1012 for connection to the vacuum source 106, a second port (not shown) for providing the circuit with access to the vacuum chamber 1006, and a third port 1016 for providing a sealing connection to the jet injection device 970. In this embodiment, instead of the third port 1016 extending directly to the vacuum chamber 1006, the third port 1016 extends inward to an annular channel 1062 defined by the housing body 1026. The annular channel 1062 at least partially surrounds the vacuum chamber 1006 and moves outward from there along the radial direction R. The housing body 1026 further defines a plurality of housing ports 1060 extending inward from the annular channel 1062 toward the vacuum chamber 1006. Therefore, the housing body 1026 can define a manifold defining a plurality of housing ports 1060. As described above, the electrode 1008 can be supported by a sleeve 1052 attached 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. Therefore, the sleeve port 1057 also provides fluid communication between the annular channel 1062 and the vacuum chamber 1006. Vacuum pressure is supplied to the vacuum chamber 1006 through the third port 1016, the annular channel 1062, the housing port 1060, the sleeve port 1057, and the electrode opening 1063 in sequence. In embodiments in which the electrode 1008 is directly coupled to the inner surface 1022 of the housing body 1026, the electrode opening 1063 can be directly continuous with the housing port 1060. The annular channel 1062 may extend in an annular manner along the entire circumference around the central axis, but in other embodiments, the channel 1062 may extend over a smaller area than the entire circumference around the central axis X.

[0104] As shown in Figure 10A, each electrode 1008 may define a single row of apertures 1063 that may be elongated along the circumferential direction C. Each row may contain five apertures 1063, which may be characterized as a “5x1” (i.e., 5 rows and 1 column) array of apertures 1063 or an “aperture array,” as shown. Other aperture arrays are also within the scope of this disclosure. For example, as shown in Figure 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 the corresponding rows and columns of the sleeve port 1057 and the corresponding rows and columns of the housing port 1060. The aperture array may be a 4x5 array, but other aperture array configurations are also within the scope of this disclosure.

[0105] As shown in Figure 10D, the opening 1008 is configured such that the tissue 102 drawn into the vacuum chamber 1006 can extend at least partially into one or more of the openings 1063 indicated by the highlighted region 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 can 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 the injector 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 likelihood of the vacuum pressure in the chamber 1006 pulling the injector out of the tissue at the injection site is reduced. Furthermore, the portion of the skin layer 104 extending into the opening 1063 can be altered by destroying the upper part of the skin layer 104, thereby improving the electric field distribution within the tissue mound 140, similar to the embodiments described above with reference to the protrusions 664 in Figures 6A-6D.

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

[0107] The tissue sensor may be connected to a wired circuit or a printed circuit that is electrically connected to 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 electrode 1008. The tissue sensor may be used to map tissue adhesion throughout the chamber 1006. Such tissue adhesion mapping information may be used for data acquisition purposes, or in addition to or alternatively, in an active pressure feedback mechanism to adjust the vacuum pressure level upward or downward based on sensor readings at each opening. In such embodiments of tissue adhesion mapping, the tissue sensor circuit preferably includes a separate circuit 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 (i.e., the overall "delta") compared to the initial baseline measurement to provide an overall measure of the extent to which tissue adhesion has occurred to the chamber wall.

[0108] In additional embodiments of the jet-injection vacuum cups, such as cups 902 and 1002 described above, the distal end 979 of the nozzle 978 may be adapted to define the electrodes of the array. In a non-limiting example of such an embodiment, the distal end 979 of the nozzle 978 may be coated with or manufactured from a conductive material such as conductive paint, metal, or polymer, and may be electrically connected to the controller 114. In this embodiment, the distal end 979 of the nozzle 978 may be used to deliver electroporation pulses to and / or from other electrodes on the inner surface of the vacuum chamber. In such an embodiment, the other electrodes may be annular 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 embodiments enable concentric electroporation pulse patterns ("ejection patterns"), including those discussed in more detail below. The inventors successfully used such a concentric electrode array in a vacuum cup to perform intradermal jet injection of fluid into rabbits and guinea pigs, and subsequently to perform electroporation of intradermal tissue at the injection site.

[0109] As described above, the inventors have discovered many beneficial results resulting from vacuum-assisted electroporation procedures using the vacuum cup described above. These beneficial results include increased fluid dispersion of the injector in subcutaneous and cutaneous tissue, as well as increased in vivo fluid infiltration at the treatment site.

[0110] Referring to Figure 11A, the effect of vacuum pressure on the injection site can be seen. In this example, equal volumes of methylene blue 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 subjected to vacuum pressure. The injection site shown on the right was subjected to a vacuum pressure of approximately -10.6 psi for 15 seconds using vacuum cup 2, described above with reference to Figures 2A-2F, which has a base diameter D1 of approximately 15 mm, a chamber depth L2 of approximately 15 mm, and an inner wall taper angle of approximately 4 degrees. Neither site was treated with electroporation. In this example, the vacuum pressure effectively redistributed the fluid and subsequently retained the injector within the area below the cup. This would have resulted in a higher injector concentration within the treatment zone of the cup if electroporation had been performed.

[0111] Referring to Figures 11B-11C, a comparative study of vacuum pressure and fluid dispersion was conducted on guinea pigs. Both subjects received injections of methylene blue into adipose tissue. The injection in Figure 11B was performed using a needleless vacuum cup similar to that illustrated in Figure 9A. In particular, the tissue was in a vacuum chamber that responded to vacuum pressure during the jet injection. The injection in Figure 11C was performed using a subcutaneous needle injection method, and no vacuum pressure was applied. Neither subject in this study was treated with electroporation. As demonstrated, vacuum-assisted jet injection (Figure 11B) caused significantly greater dispersion of the injected material in the adipose layer than subcutaneous needle injection without vacuum pressure (Figure 11C).

[0112] Referring here to Figures 34A-34B, a comparative study of the cumulative effects 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 adipose pad using a 29-gauge insulin syringe. The injection sites of both subjects were treated with the same vacuum pressure. The subjects in Figure 34B were further treated via electroporation at the injection site using a vacuum cup similar to that shown in Figure 2A. The subjects in Figure 34A were not treated via electroporation. Histological sections were taken from the treatment sites for comparison of GFP expression (appearing as green fluorescence) and cell infiltration (appearing as blue fluorescence after 4',6-diamidino-2-phenylindole (DAPI) staining). As shown, GFP expression (green) is detectable after vacuum pressure in both subjects (i.e., regardless of whether the treatment site was electroporated or not). However, compared to applying vacuum pressure alone (Figure 34A), further application of electroporation combined with vacuum pressure (Figure 34B) increased cell infiltration (blue). These studies demonstrate that applying vacuum pressure in combination with electroporation can enhance immunogenicity.

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

[0114] Referring to Figure 13, over an 8-week ELISA study comparing the humoral immune response of 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 vacuum cup 2 shown in Figure 2A (blue plots - "vacuum") showed comparable humoral immunogenicity to subjects treated with intradermal injection of the vaccine and electroporation using an intradermal needle electrode electroporation device (red plots - "ID needle electrode EP"). The injection volume was equal (20 ug). Intradermal injection was 100 uL, and fat injection was 300 uL. Vacuum cup 2 had a chamber diameter D1 of 15 mm with four electrodes.

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

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

[0117] A vacuum cup configured to target electroporation in the skin layer 104 is described here with reference to Figures 15A-22.

[0118] Referring here to Figures 15A-15B, an example of a vacuum cup 1502 is shown, which includes an electrode array 1509 having one or more annular ring electrodes 1508 and a central electrode 1511 extending along the 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, and 1002 described above, and in particular the vacuum cup 902 described above with reference to Figures 9A-9B. Thus, similar reference numbers 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 that defines an inner surface 1522 that at least partially defines the vacuum chamber 1506. The housing body 1526 also defines a third port 1516 that extends proximal to the vacuum chamber 1506 along a central axis X. The central electrode 1511 extends into the vacuum chamber 1506 from the end face portion of the inner surface 1522 through the third port 1516. The third port 1516 also extends through a mounting forming portion 1548 for providing a sealing engagement with the central electrode 1511. Similar to the embodiments described above, the mounting forming portion 1548 may carry one or more sealing members, such as a sealing O-ring 1551, which seals to the outer surface 1513 of the central electrode 1511. The distal portion 1513z of the outer surface 1513 of the central electrode 1511 is configured to contact the tissue 102 drawn into the vacuum chamber 1506. Therefore, the distal portion 1513z can 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 specifically in the range of about 1 mm to about 7 mm, and more specifically 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. Therefore, the proximal portion 1518 can be referred to as the “stem” of the central electrode 1511.

[0120] Referring here to Figure 15C, the electrode array 1509 is configured such that, during electroporation pulse delivery, the central electrode 1511 functions as either a positive or negative electrode, while one or both of the annular ring electrodes 1508 function as the other (i.e., opposite polarity to the central electrode 1511). In this way, the central electrode 1511 effectively moves the electric field 145 upward during the pulse, concentrating the electroporation field 144 on the skin layer 104. The vacuum cup 1502 of this embodiment may be placed on the tissue 102 in the target zone after the drug has been injected into the skin layer 104, for example, via needle injection such as mantou injection, or intradermal jet injection. In other embodiments, it should be understood that the vacuum cup 1502 may utilize a feature or “post” instead of the central electrode 1511. The post may have a surface with a shape similar to the surface 1513z in the vacuum chamber 1506. The post surface is configured to contact skin drawn into the chamber 1506 via vacuum pressure. For example, the post may be configured to conform the tissue around a portion of the post surface or to curve it around that portion while vacuum pressure is applied. Such tissue-post contact has been shown to favorably enhance fluid dispersion within the tissue while vacuum pressure is applied, as will be discussed in more detail below with reference to Figures 42A and 42D.

[0121] Referring here to Figure 16A, a version of the vacuum electroporation assembly 1600 is shown, including a vacuum cup 1602 having an electrode array 1609 positioned on an end face 1624 in the vacuum chamber 1606 opposite its distal opening 1620. The end face 1624 may be substantially flat and may be defined by an electrode support member 1652 positioned in 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 a circuit that is electrically in communication with a control unit such as the controller 114 described above. The support member 1652 also defines a number of ports 1660 extending through the member, providing fluid communication between the vacuum chamber 1606 and an external port 1616 for connection to a vacuum source. In this way, the port 1660 in the support member 1652 transmits vacuum pressure into the chamber 1606, drawing the tissue 102 therein and bringing it into contact with the electrode 1608. The electrode 1608 may have a geometric shape with protruding and / or pointed ends, such as a cone or pyramidal shape, to press into the skin layer 104 of the tissue mound 140 and draw it into the chamber 1606, thereby destroying and altering the uppermost layer of skin 104 and improving 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. Thus, the vacuum cup 1602 has a housing body 1626 that can define a receptacle 1616 for receiving at least the distal portion of the jet injection device 970, such that its nozzle 978 is aligned with an injection opening 1617 defined on a support member 1652. As shown in the figure, the nozzle 978 and the injection opening may be aligned concentrically with the 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 a support member 1652 separates or interposes 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 allow for the favorable formation of a stream of the injector 142 between the nozzle 978 and the tissue 102 for the purpose of intradermal dispersion of the injector 142. In particular, the standoff distance L5 may allow for the formation of irregularities in the liquid stream as the liquid stream approaches the skin 104. For example, such irregularities may contain hundreds, thousands, or even more micro and / or nano-sized droplets, each approaching the skin 104 at a speed sufficient to effectively allow a stream to hundreds or thousands of micro and / or nano-sized gaps in the outer surface 101 of the skin 104, providing enhanced injector diffusion localized to the skin layer 104. The standoff distance L5 can also be used as a means of controlling the maximum penetration depth of the injector in combination with other factors such as the nozzle shape of the jet injector and the force of the injector. It should be understood that the standoff distance L5 may also be characterized during use as the minimum standoff distance between the distal end of the nozzle 978 and the outer surface 101 of the skin 104. The standoff distance L5 may be in the range of about 1.0 mm to about 20 mm.

[0124] As shown in Figure 16B, the housing body 1626 can optionally define an insertion shield 1685 that extends through the standoff chamber 1607 and can abut against the rear surface 1625 of the support member 1652 so that the insertion of the shield 1685 is in fluid communication with the injection opening 1617 of the support member 1652. In this way, the insertion shield 1685 can provide a linear, aligned, shielded passage from the distal end of the nozzle 978 to the vacuum chamber 1606, thereby protecting the support member 1652 (and its circuitry) from accidental exposure to the injector stream.

[0125] Referring here to Figures 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 injection opening 1617. As shown in Figure 17A, the electrodes 1608 may be arranged in a circular or ring-shaped pattern along a circumferential axis C2 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 with respect to the central axis X, as shown in Figure 17B. Referring again to Figure 17B, the ports 1660 may also be arranged in one or more annular rings around the central axis X. Furthermore, the electrodes 1608 and / or ports 1660 may also (or alternatively) be arranged in a spoke-shaped pattern along their respective axes R4, R5 extending radially outward from the central axis X. The axes R4 and R5 of adjacent electrode 1608 "spokes" and adjacent port 1660 spokes may be offset from each other by their respective angular intervals A5 and A6 around the central axis, which may range from about 5 degrees to about 180 degrees, and more specifically from about 15 degrees to about 120 degrees. The axes R4 and R5 may be linear as shown, but in other embodiments, the axes R4 and R5 may be arc-shaped. The examples of patterns of electrode 1608 and port 1660 are provided as non-limiting examples, and it should be understood that other patterns, including asymmetric and / or irregular patterns, are also within the scope of this disclosure.

[0126] Referring here to Figures 18A-18B, in a further embodiment, the vacuum electroporation apparatus 1802 may include a plurality of distal vacuum chambers 1807 for inducing tissue, particularly skin, within. An electrode 1808 is supported by an electrode support member 1852, which can be attached to the distal end 1810 of a vacuum housing body 1826 defining a main vacuum chamber 1806. In this way, the distal end face 1824 of the support member 1852 defines the distal end of the apparatus 1802. The housing body 1826 may define a main vacuum port 1812 for providing vacuum pressure to the main vacuum chamber 1806 and a second port 1814, such as one for providing access for circuits extending into the main vacuum chamber 1806 and the support member 1852.

[0127] Referring here to Figure 18B, the electrodes 1808 in this embodiment are arranged in pairs, each including an outer ring electrode 1808a and an inner ring electrode 1808b that are concentric with respect to each other. The outer and inner ring electrodes 1808a,b may be tubular members that are elongated along the longitudinal direction L, extending through the support member 1852 to its rear surface 1825, and optionally further extending into the main vacuum chamber 1806. The outer and inner ring electrodes 1808a,b of each pair are radially separated from each other by an electrically insulating annular layer 1894 of the material, thereby electrically insulating the inner and outer ring electrodes 1808a,b from each other. 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 concave proximally from the distal end surface 1824. In this way, the distal vacuum chamber 1807 is collaboratively defined by the inner surface 1808z of the outer ring electrode 1808a, the distal end of the inner ring electrode 1808b, the distal end face of the insulating layer 1894, and optionally any distal retraction surface 1808y of the outer ring electrode 1808a.

[0128] As shown in Figures 18C to 18D, the distal end of the inner ring electrode 1808b (and optionally the distal end of the insulating layer 1894 as well) 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 specifically in the range of about 0.5 mm to about 2.0 mm, preferably in the range of about 0.8 mm to about 1.2 mm. The apparatus may define an apparatus length L7 measured along the longitudinal direction L from the proximal end 1828 to the distal end face 1824. The apparatus length L7 may be in the range of about 15.0 mm to about 200 mm, more specifically in the range of about 20 mm to about 40 mm, but lengths L7 smaller than 15.0 mm and larger than 200 mm are also within the range of this embodiment.

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

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

[0131] As shown in Figure 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 concentrically around the central electrode pair 1808a,b along the circumferential axis C2. It should be understood that other array patterns are also within the scope of this embodiment. Adjacent peripheral electrode pairs 1808a,b may be separated from each other by their respective angular spacings A5, as in the embodiments described above.

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

[0133] Referring here to Figures 20 and 21A-21B, electrode support members 2052, 2152 can support electrode arrays 2009, 2109 having a non-circular pattern. As shown in Figure 20, support member 2052 may have a square electrode array 2009 that is substantially equidistant along first and second array directions AD1, AD2 which are substantially perpendicular to each other. In the illustrated embodiment, the array 2009 has a 4x4 array pattern, but other square array patterns, including 2x2, 3x3, 5x5, 6x6, 7x7, 8x8, 9x9, 10x10, etc., are also within the scope of this disclosure as non-limiting examples.

[0134] As shown in Figures 21A-21B, the support member 2152 may have a rectangular electrode array 2109 that is longer along the first array direction AD1 than along the second array direction AD2. The array 2109 may include a first row of electrodes and a corresponding second row of electrodes, for example, in a 2x6 (i.e., 2 rows x 6 columns) array 2109, but other rectangular array 2109 patterns, including 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, are also within the scope of this disclosure.

[0135] It should be understood that the square and rectangular electrode arrays 2009, 2109 described above may have substantially any number of electrodes 2008, 2108 arranged in each pattern. Such square and rectangular electrode arrays 2009, 2109 may generate a roughly rectangular electric field that can be activated in the row or column direction, and may enable a highly ordered electric field along a given axis. For example, if an injector is intentionally injected or incidentally accumulates along one axial direction of tissue, additional rows or columns of electrodes 2008, 2108 may be readily 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 is positioned on the skin 104 over the target zone such that the first array direction AD1 (along which the array is elongated) substantially aligns with the axis to which the fluid is intentionally injected, or with the axis to which the fluid naturally disperses due to the presence of anisotropic features such as fibrous tissue, thereby allowing the injector to be encapsulated within the electroporation field for a longer duration as the injector disperses through the tissue. Furthermore, since the array 2109 also defines an array of vacuum ports that disrupt the uppermost layer of skin 104 while vacuum pressure is applied, the rectangular array 2109 can also effectively form disruption pathways within the intradermal tissue that allow the injector to flow further along the electroporation field.

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

[0137] Referring here to Figures 35A-35B, a 6-week study evaluated the effect of chamber diameter D1 on immunogenicity in guinea pig skin at given electroporation voltages and currents using a vacuum-assisted electroporation device. Figure 35A compares humoral immunogenicity ELISA data after intradermal vacuum-assisted electroporation treatment using vacuum cups with chamber diameters D1 of 8 mm, 10 mm, 12 mm, and 15 mm. Intradermal treatment for each vacuum cup included a mantou injection of 50 ug of MERS DNA vaccine, followed by electroporation using the same voltages and currents. Figure 35B shows cellular immune response ELISA 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 given electroporation voltage and current limits. From the perspective of cellular immune response, this study also demonstrates that vacuum cups with a chamber diameter D1 of 8 mm to 12 mm yield similar results, and that a decrease in cellular response occurs when the diameter D1 increases from 12 mm to 15 mm.

[0138] Referring here to Figures 36A-36B, studies evaluated preferred combinations of vacuum pressure and electroporation voltage parameters for gene expression in guinea pig skin. These studies utilized intradermal injection (specifically mantou injection) of a plasmid encoding the green fluorescent protein (GFP) gene, followed by electroporation 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 response of the treatment site and GFP expression were observed. Figure 36A shows visual observations of the treatment site at various vacuum pressures and voltages. In Figure 36B, measured skin fluorescence signals 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 higher vacuum pressures enhanced GFP expression at each voltage tested.

[0139] Referring to Figure 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 100 μL Mantou injection of 66.7 μg plasmid; (2) "Vacuum Cup" (red plot) - treatment using a 15 mm vacuum cup electroporation device with four wall electrodes after injecting 1 mL of an equivalent amount of 66.7 μg plasmid prepared using 139 U / ml Hylenex (see, e.g., Figure 2A); (3) "Vacuum Cup Triple Dosage" (chestnut plot) - treatment using the same apparatus and method as the "Vacuum Cup" group, but with a dose tripled to 200 μg plasmid. All groups were treated at weeks 0, 4, and 7.

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

[0141] Referring to Figures 37A and 37B, a 6-week study compares the performance of a vacuum-assisted electroporation device (VACU) with that of an intradermal needle electroporation device (EPH) from an immunogenicity perspective. Figure 37A compares humoral immunogenicity ELISA data from guinea pigs after intradermal treatment with 50 ug of MERS DNA vaccine via mantou 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 EEP intradermal needle electroporation device. Both groups were treated at weeks 0, 2, and 4. This study demonstrates that the vacuum cup produces a more rapid and stronger humoral response compared to the EEP intradermal needle electroporation device. Figure 37B shows 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 cellular immune response data in guinea pigs after intradermal administration of 100 ug of recurrent respiratory papillomavirus (RRP) DNA vaccine via electroporation following Mantou injection. The groups shown in Figure 38 include: (1) administration using the "ID-EP" intradermal needle electrode electroporation device and (2) administration using the "ID-VEP" vacuum cup similar to that shown in Figure 2A.

[0143] The studies shown in Figures 37B and 38 demonstrate that vacuum cups generate substantially equivalent cellular immune responses compared to intradermal needle electroporation devices. 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 post-treatment.

[0144] Referring here to Figures 39A-39B, a 4-week study evaluated the cumulative effects of vacuum pressure and electroporation on immunogenicity in the skin. This study compared humoral and cellular immune responses in guinea pigs after intradermal treatment with 50ug of MERS DNA vaccine via Mantou 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" - Mantou injection only. Figure 39B shows cellular immune response ELISA data at week 4 for the same study shown in Figure 39A. These studies demonstrate that while electroporation is essential for vacuum pressure to induce immunogenicity, vacuum pressure alone (i.e., without electroporation) after Mantou injection can produce at least a partial humoral response. The inventors believe these investigations also suggest that the aforementioned immune response is only partially explained by surface transfection of DNA vaccines. Further investigations confirmed that electroporation voltage is a stronger driving force than vacuum pressure in terms of immunogenicity.

[0145] Referring here to Figures 40A and 40B, cross-sectional views of the comparative electric fields generated by two different electrode arrays in a tissue mound drawn into a vacuum chamber are shown. Figure 40A shows the electric field generated by an electrode array having a pair of counter electrodes configured similarly to that of vacuum cup 2 shown in Figure 2A. The electroporation pulse delivery pattern (also called the "emission pattern") between the counter electrodes 8 moves the current laterally across the tissue mound 140 between the electrodes. Figure 40B shows the electric field generated by an electrode array having two annular ring electrodes and a central electrode configured similarly to that of vacuum cup 1502 shown in Figure 15A. In this example, the electroporation pulse emission pattern moves the 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 and 40B demonstrate that the electroporation pulse emission pattern between opposing electrodes (Figure 40A) generates a more homogeneous electric field through the skin layer 104 compared to the electric field generated between the annular ring electrode and the central electrode (Figure 40B). These differences in electric field generation can be advantageously utilized based on the desired specific electroporation procedure.

[0146] Referring to Figures 41A–41B, an 8-week study evaluated the effect of electroporation pulse firing patterns on immunogenicity. In this study, the humoral and cellular immune responses of guinea pigs were tested after intradermal treatment with MERS DNA vaccine via mantou injection and subsequent vacuum-assisted electroporation using a vacuum cup with electrode arrays 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 ELISA data at week 4 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 here to Figures 42A–42D, the study evaluates the comparative effect of a central electrode on fluid dispersion in guinea pig skin. Fluid dispersion of colored injectors was measured for three groups: (1) Mantou injection only (i.e., no vacuum pressure), (2) an array with counter electrodes but no central electrode (see Figure 40A), and (3) an array with a central electrode (see Figure 40B). Equal volumes of injectors were injected into each group via Mantou injection. Vacuum pressure was applied to groups 2 and 3. Electroporation was not performed in this study. Figures 42B, 42C, and 42D show the fluid dispersion 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 injectors. These results demonstrate that the presence of a central electrode or feature can affect the fluid dispersion of injectors when vacuum pressure is applied. These results further suggest that the internal geometry of the vacuum chamber can affect fluid dispersion when vacuum pressure is applied.

[0148] It should be understood that the intradermal vacuum electroporation assemblies, apparatus, and cups described above can be used with various formulations to enhance vacuum-assisted electroporation procedures. For example, the injectable 142 may include mixtures with formulations to influence the properties of the drug in a preferred manner. Non-limiting examples of such formulations include hyaluronidase and Hylenex (human recombinant hyaluronidase), which can temporarily break down the drug matrix, allowing for the injection of larger drug volumes with only small, less painful lumps or less bleeding in the skin. As shown in Figures 25-26, Mantou injections containing hyaluronidase result in less bleeding in both height and diameter than injections without hyaluronidase (left figure). Hyaluronidase preparations are more fully described in U.S. Patent Publication No. 2019 / 0284263A1, published on September 19, 2019, entitled “In Vivo Use of Chondroitinase and / or Hyaluronidase to Enhance Delivery of an Agent” (“'263 Document”), the entire disclosure of which is incorporated herein by reference. By utilizing hyaluronidase in injections performed using the vacuum electroporation apparatus disclosed herein, the agent can be injected in a larger volume in the intradermal tissue, enabling physicians to treat larger tissue volumes using the apparatus herein which has a larger treatment zone 107.For example, through numerous tests using vacuum cups having various chamber diameters D1 (e.g., 8 mm, 10 mm, and 12 mm) in combination with an injectable 142 having a hyaluronidase-containing formulation (i.e., 50% Omnipaque 350 + 50% 150 U / mL hyaluronidase (ultimately 75 U / mL hyaluronidase)), the inventors found that an 8 mm diameter vacuum cup could accommodate an injectable volume of 0.2 mL with substantially all of the injectable drawn into the vacuum chamber; a 10 mm diameter vacuum cup could accommodate an injectable volume of approximately 0.4 mL with substantially all of the injectable drawn into the vacuum chamber; and a 12 mm diameter vacuum cup could accommodate an injectable volume of 0.8 mL with substantially all of the injectable drawn into the vacuum chamber. The presence of hyaluronidase in the injectable formulation was observed to dramatically promote the fluid dispersion of the injectable within the skin layer. The data obtained from these tests strongly suggest that the larger the volume of the injectable, the more hyaluronidase should be utilized within the injectable formulation; otherwise, the injectable blebs will become larger and will not diffuse properly laterally through the skin.

[0149] Referring to Figure 27, a 4-week study compares the humoral immune response in guinea pigs in terms of endpoint titer after intradermal administration of a DNA vaccine against MERs (pGX9101) followed by electroporation. All groups received 50 ug of plasmid via 100 uL mantou infusion in the flank at weeks 0 and 2. The groups shown in the graph are as follows: (1) "ID Needle Electrode EP" (black plot) - procedure using an intradermal needle electrode electroporation device, (2) "ID Needle Electrode EP + HYA" (gray plot) - procedure using an intradermal needle electrode electroporation device similar to the previous group, but the formulation contains 270 U / mL of intropharma hyaluronidase, (3) "15mm Vacuum Cup" (red plot) - procedure 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, using a formulation containing 270 U / mL of intropharma hyaluronidase.

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

[0151] Referring to Figure 29A, a 6-week study compares the humoral immune response of guinea pigs that underwent electroporation at weeks 0, 3, and 6 after intradermal injection of a DNA vaccine against influenza nucleoprotein (pGX2013) in terms of mean endpoint titer. The groups shown in the graph are as follows: (1) "ID needle electrode EP 1ug" (red plot) - Mantou injection containing 1ug of plasmid, followed by electroporation using an intradermal needle electrode electroporation device; (2) "Vacuum with HYA 10ug" (blue plot) - 1 mL Mantou injection of 10ug of plasmid in a formulation containing 139.5 U / mL of Hylenex, treated using vacuum cup 2 shown in Figure 2A with a chamber diameter D1 of 15 mm; (3) "Vacuum with HYA 1ug" (green plot) - 100 μL Mantou injection with 1ug of plasmid containing 139.5 U / mL of Hylenex, followed by electroporation using the same vacuum cup 2 as the previous group; (4) "Vacuum without HYA 1ug" (purple plot) - 100 μL Mantou injection with 1ug of plasmid, followed by electroporation using the same vacuum cup 2 as groups (2) and (3). Figures 29B and 29C show the cellular immune response from the perspective of spot-forming units during the second week (Figure 29B) and fourth week (Figure 29C) of the study in Figure 29A.

[0152] Referring to Figures 43A-43C, a 7-day study evaluated the comparative effects of a single high-volume injection and vacuum-assisted electroporation procedure with multiple injections and electroporation procedures 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 Mantou injection of a plasmid encoding secreted alkaline phosphatase (SEAP) co-compounded with 135 U / mL hyaluronidase, followed by vacuum-assisted electroporation. The multiple injection and electroporation procedure included six individual 0.1 mL Mantou injections, each followed by electroporation using an intradermal needle electrode (a total of 0.6 mL of injection and six electroporations). Figure 43A shows the vacuum cup positioned over the injection before vacuum pressure is applied. Figure 43B shows the injector in the vacuum cup while vacuum pressure is applied, illustrating how the injector in the tissue deforms around the central electrode, thereby concentrating the injector in the electroporation field (see Figure 40B). Figure 43C shows SEAP expression (as a measure of systemic protein production in the subjects) 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 acts substantially equivalently to six injections and six electroporation treatments using a needle electrode device.

[0153] These studies demonstrate that vacuum-assisted electroporation using the apparatus and assemblies of this disclosure enables high-volume delivery of DNA to the skin. Furthermore, hyaluronidase preparations (e.g., Hylenex) enhance the immunogenicity following vacuum-assisted electroporation of the skin. Moreover, the vacuum cup described herein is adapted to utilize the significantly higher injection volumes in intradermal tissue provided by hyaluronidase preparations containing injection volumes of 1000 uL (1 mL) or more. In other words, by utilizing hyaluronidase preparations with the vacuum cup of this disclosure, the vacuum cup can treat a considerable volume of intradermal tissue. In addition, the apparatus and assemblies of this disclosure produce a faster humoral response than intradermal needle electroporation devices and a comparable overall humoral immune response compared to intradermal needle electroporation devices. Furthermore, these studies demonstrate that the dynamics and magnitude of the cellular response can be enhanced through vacuum-assisted electroporation of intradermal tissue. The inventors have also found that using a hyaluronidase preparation in conjunction with vacuum-assisted electroporation of intradermal tissue effectively enables transfection of the dermis layer below the surface.

[0154] Referring here to Figure 44, an 8-week study compares the effect of electroporation pulse firing patterns on humoral immune response data on immunogenicity. In this study, the humoral and cellular immune responses of guinea pigs were tested after intradermal treatment with MERS DNA vaccine by mantou injection and vacuum-assisted electroporation using a vacuum cup with electrode arrays shown in subsequent 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 ELISA data at week 4 and after week 4 during the same study shown in Figure 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] Referring here to Figures 45A–47C, fluorescence fluoroscopic images show comparative tissue deflection in guinea pigs between jet injections at various vacuum pressures and nozzle-skin offset distances, using a jet injection vacuum cup configured similarly to the vacuum cup 902 shown in Figure 9. The injector used in these images is a 50% Omnipaque 350 solution to enable radiography. In each of these images, superimposed transverse reference lines indicate the distal end of the vacuum cup (and therefore the distal end of the vacuum chamber and the initial skin-chamber interface before vacuum application). Figures 45A–45C show jet injections performed without vacuum pressure applied to the chamber. Figures 46A–46C show jet injections performed with vacuum pressure applied to the chamber and no nozzle-skin offset distance. Figures 47A–47C show jet injections performed with vacuum pressure applied to the chamber and a nozzle-skin offset distance of 3 mm. Please 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, if no vacuum pressure is applied to the chamber, the injection causes significant tissue deformation (Figure 45B), and although the injected material remains largely below the vacuum chamber, the tissue is repelled towards the nozzle after injection (Figure 45C).

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

[0158] Referring here to Figures 47A-47C, when the nozzle-skin offset distance is 3 mm and injection is performed while vacuum pressure is applied within the chamber, tissue flexure is substantially eliminated during injection (Figure 47B). As in this study, when the skin is retracted into the vacuum chamber before injection, a tight seal exists between the jet nozzle and the skin during injection, and the vacuum pressure is sufficient to prevent tissue flexure. Furthermore, after injection (Figure 47C), the injector is present within the chamber and has a greater vertical distribution compared to the non-offset setting shown in Figure 46C, where the injector is compressed into a smaller vertical space. These tests demonstrate the significant advantages provided by the jet injection vacuum cup disclosed herein in terms of injector fluid distribution.

[0159] While the embodiments described herein are configured to target electroperforation in intradermal and / or subcutaneous tissue, it should be understood that any of the design parameters of vacuum cups 2, 502, 702, 802, 902, 1002, 1502, 1602 and vacuum devices 1802, 1902 can be scaled up or down to target more specific and / or different tissue layers, such as certain tissue layers or muscle layers within the skin, such as the smooth muscle layer and the skeletal muscle layer. Furthermore, the design parameters of vacuum cups 2, 502, 702, 802, 902, 1002, 1502, 1602 and vacuum devices 1802, 1902 in this specification can be adapted as needed to target electroperforation of other types of tissue, including mucous membranes, organs, etc.

[0160] Although this disclosure has been described in detail, it should be understood that various modifications, substitutions, and alternatives can be made herein without departing from the spirit and scope of the 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 this disclosure is not intended to be limited to any specific embodiment described herein. As will be readily apparent to those skilled in the art, existing or subsequently developed processes, machines, manufactures, compositions of materials, means, methods, or steps that perform substantially the same function or achieve substantially the same results as the corresponding embodiments described herein can be utilized in accordance with this disclosure. This disclosure also includes the following aspects: [Aspect 1] Apparatus for vacuum-assisted in vivo electroporation of tissue, A chamber and a housing defining at least one opening into the chamber, At least one port extending through the housing, wherein the at least one port is separated from the at least one opening and is connectable to a vacuum source, thereby configured to transmit vacuum pressure from the vacuum source to the chamber, An apparatus comprising: a plurality of electrodes positioned within the chamber, each electrode configured to deliver one or more electroporation pulses to a target portion of tissue held within the chamber at least momentarily, extending through the at least one opening in response to the vacuum pressure. [Aspect 2] The apparatus according to embodiment 1, wherein the at least one opening is a single opening, and the opening is circular. [Aspect 3] The apparatus according to embodiment 2, wherein the housing has a wall defining an inner surface that defines at least partially the chamber, and the plurality of electrodes include four electrodes extending along the inner surface, the four electrodes being spaced 90 degrees apart from each other. [Aspect 4] The apparatus according to embodiment 2, further comprising a second port in addition to the at least one port, wherein the second port is configured for the insertion of a jet injection device or a subcutaneous injection needle into the chamber. [Aspect 5] The apparatus according to embodiment 2, wherein the housing has an end face opposite to the at least one opening and a wall extending from the end face to the opening, the wall defining an inner surface that at least partially defines up to the chamber, at least one first electrode of the plurality of electrodes extending from the end face, at least one second electrode of the plurality of electrodes extending along the inner surface, and the at least one first electrode and the at least one second electrode being concentric with each other. [Aspect 6] The apparatus according to embodiment 5, wherein the at least one first electrode is a single electrode positioned centrally 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 according to embodiment 1, wherein the plurality of electrodes have electrode surfaces exposed within the chamber, and at least some of the electrode surfaces are textured and protrude into the chamber. [Aspect 8] The apparatus according to embodiment 1, wherein the housing is constructed of a flexible material, the material comprising one or more of the following: 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 configured to be electrically connected to the plurality of electrodes and to transmit one or more electroporation pulses to the plurality of electrodes, The apparatus according to embodiment 1, further comprising: a processor electrically communicating with the signal generator and at least one sensor located within the chamber, wherein the at least one sensor is configured to sense at least one parameter of the tissue during the delivery of one or more electroporation pulses and to communicate feedback data of the at least one parameter to the processor, and the processor is configured to use the feedback data to execute one or more algorithms and to adjust at least one pulse parameter of the one or more pulses during the delivery of the one or more pulses. [Aspect 10] The apparatus according to embodiment 1, wherein the plurality of electrodes are configured to have pulses applied to them having a potential magnitude in the range of approximately 2V to approximately 1000V. [Aspect 11] The apparatus according to embodiment 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, and the pulses have a pulse duration in the range of about 0.1 milliseconds to about 100 milliseconds. [Aspect 12] The apparatus according to Embodiment 1, wherein the at least one port includes an array of ports, each of which extends through the housing into the chamber such that the housing includes a manifold defining the array of ports. [Aspect 13] The apparatus according to embodiment 1, wherein the target portion of the tissue is at least one of skin tissue and adipose tissue. [Aspect 14] A method of electroporating the target tissue, Placing the chamber adjacent to the aforementioned target organization, By applying vacuum pressure to the chamber, the tissue is drawn in through the opening of the chamber and brought into contact with a plurality of electrodes extending along the inner surface of the chamber. A method comprising delivering one or more electropermeating pulses to the tissue through the plurality of electrodes, thereby generating an electropermeating field within the tissue. [Aspect 15] The method according to embodiment 14, wherein the additional step includes attaching the tissue to the inner surface and applying the vacuum pressure at a level sufficient to disperse the fluid within the tissue of the electroporation chamber. [Aspect 16] The method according to embodiment 14, further comprising injecting a substance into the tissue prior to the delivery step, wherein the delivery step comprises transferring 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] The method according to embodiment 16, wherein the substance comprises nucleic acids that induce an immune response in the subject. [Aspect 18] The method according to embodiment 14, wherein the adding step includes applying pulses of fluctuating vacuum pressure to the tissue and varying the duration of the pulses. [Aspect 19] The method according to embodiment 14, further comprising injecting fluid into the tissue from an injection device extending into the chamber through a second port, wherein the injecting step and the adding step are performed prior to the delivering step, and the injection device is one of an injection needle and a jet injection device. [Aspect 20] A device for vacuum-assisted treatment of tissue, A chamber and a housing defining at least one opening into the chamber, A first port extending through the housing, the first port being separate from the at least one opening and connectable to a vacuum source, thereby configured to transmit vacuum pressure from the vacuum source to the chamber, and at least one other port, A device comprising: a jet injection device extending into the chamber through a second port, the second port being opposite to the at least one opening, and the jet injection device being configured to respond to the vacuum pressure to deliver a jet injection of fluid to a target portion of tissue extending through the at least one opening and held at least momentarily within the chamber.

Claims

1. Apparatus for vacuum-assisted in vivo electroporation of tissue, A housing comprising: a chamber; at least one opening into the chamber located at the distal end of the housing; an inner surface located within the chamber; and an end face within the chamber located opposite the opening; At least one port extending through the housing, wherein the at least one port is separated from the at least one opening and is connectable to a vacuum source, and is configured to transmit vacuum pressure from the vacuum source to the chamber, The device comprises at least one electrode positioned within the chamber, wherein the at least one electrode is configured to respond to the vacuum pressure by extending through the at least one opening and delivering one or more electroporation pulses to a target portion of tissue held at least momentarily within the chamber, The at least one electrode extends along the inner surface at a position spaced between the proximal end of the chamber and the opening, and the at least one electrode is elongated in the circumferential direction. The apparatus wherein the at least one electrode includes at least a first electrode and a second electrode, which are spaced apart from each other along the longitudinal direction of the housing along the central axis that intersects the proximal end of the chamber and the opening.

2. The apparatus according to claim 1, wherein the at least one opening is a single opening, and the opening is circular.

3. The apparatus according to claim 1, further comprising a second port in addition to the at least one port.

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

5. The apparatus according to claim 4, wherein the at least one electrode extends along the entire circumference of the inner surface.

6. The apparatus according to claim 1, wherein the first electrode and the second electrode are concentric with respect to each other.

7. The apparatus according to claim 1, wherein the first electrode and the second electrode each extend along the entire circumference of the inner surface.

8. The apparatus according to claim 1, wherein the at least one electrode includes a plurality of circumferentially elongated electrodes arranged along the inner surface at circumferential intervals from one another.

9. The apparatus according to claim 1, wherein the at least one electrode comprises a plurality of circumferentially elongated electrodes, the plurality of circumferentially elongated electrodes comprises a plurality of subsets of circumferentially elongated electrodes, the electrodes of each subset being aligned with each other along a longitudinal direction oriented along the central axis of the housing intersecting the proximal end of the chamber and the opening.

10. The apparatus according to claim 1, wherein the at least one electrode has a surface exposed in the chamber, and the surface is one or more of the following: textured and protruding into the chamber.

11. The apparatus according to claim 1, wherein the housing is constructed of a flexible material, the material comprising one or more of the following: polycarbonate, polyetheretherketone, polyphthalamide, polyethylene, polyetherimide, polyvinyl chloride, polytetrafluoroethylene, polyamide, polyimide, polysiloxane (silicone), polyethylene terephthalate, polyurethane, crosslinked or non-crosslinked rubber, and polyester.

12. The apparatus according to claim 1, further comprising a signal generator that is electrically in communication with the at least one electrode and configured to transmit one or more electroporation pulses to the at least one electrode.

13. The apparatus according to claim 12, further comprising: a processor electrically communicating with the signal generator and at least one sensor located within the chamber, wherein the at least one sensor is configured to sense at least one parameter of the tissue during the delivery of one or more electroporation pulses and to communicate feedback data of the at least one parameter to the processor, and the processor is configured to use the feedback data to execute one or more algorithms and to adjust at least one pulse parameter of the one or more pulses during the delivery of the one or more pulses.

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

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

16. The apparatus according to claim 1, wherein the at least one port includes an array of ports, each of which extends through the housing into the chamber such that the housing includes a manifold defining the array of ports.

17. The apparatus according to claim 1, wherein the target portion of the tissue is at least one of skin tissue and adipose tissue.

Images