Devices, systems and methods for enhancing tissue delivery of drugs

Abstract

Provided is a device and method for administering a drug to a biological tissue of a patient, for example, by intracellular and / or dermal delivery. The device comprises: a piezoelectric pulse generator; and a microneedle electrode array electrically coupled to the piezoelectric pulse generator, wherein the device is configured to generate one or more electrical pulses and deliver the one or more electrical pulses through the microneedle electrode after the microneedle electrode is inserted into the biological tissue, the electrical pulses being effective to electroporate cells in the biological tissue, and being configured to enable drug delivery to the electroporated cells.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 031,767, filed May 29, 2020, which is incorporated herein by reference.

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This invention was made with government support under Grant No. R01AI143844 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention. Background Art

[0005] The present invention relates generally to the field of devices and methods for administering therapeutic and prophylactic agents to persons in need thereof, and more specifically to methods and devices for introducing therapeutic agents, vaccines, nucleic acids, etc. into biological tissues and / or across tissue barriers, particularly when the site of action of the drug is intracellular.

[0006] Many formulations and devices have been developed for delivering drugs, nucleic acids, biological agents (e.g., vaccines and therapeutic proteins) through the skin and into cells. The stratum corneum of mammalian skin is an effective barrier, especially for molecules greater than 500 Daltons, even when penetration enhancers, ultrasound, electroporation, or microneedles are used. Conventional devices using ultrasound and electroporation generally require expensive technology and in all cases require access to a continuous power source such as an AC outlet or batteries.

[0007] The plasma membrane is another effective barrier, especially for molecules that are not usually taken up by active transport processes, even when using nanoparticles, lipids, polymers and other formulations. This is especially important for drugs based on genetic material (e.g., DNA and RNA). The site of action of DNA-based drugs is usually in the cell nucleus. The site of action of RNA-based drugs is usually in the cell cytosol.

[0008] Therefore, there is a pressing need for an inexpensive device for dermal and intracellular drug delivery that does not require auxiliary technology such as an ultrasound generator and a continuous power source (eg, an AC power outlet or batteries) and does not require chemical formulations that may produce adverse side effects. Summary of the invention

[0009] Electroporation-based drug delivery devices and methods are provided that may overcome one or more of the aforementioned problems associated with conventional devices and methods for administering drugs to patients.

[0010] In one aspect, a device is provided for administering a drug into or through a biological tissue of a patient. In an embodiment, the device comprises: (i) a piezoelectric pulse generator; and (ii) a microneedle electrode array electrically coupled to the piezoelectric pulse generator, wherein the device is configured to generate one or more electrical pulses after the microneedle electrode is inserted into the biological tissue and deliver the one or more electrical pulses generated by the piezoelectric pulse generator through the microneedle electrode, the electrical pulses being effective for electroporating cells in the biological tissue, and being configured to enable drug delivery to the electroporated cells. In some embodiments, the device comprises a drug and is configured to release the drug into the biological tissue. In some other embodiments, the drug is not provided as part of the device, and the drug is administered to the biological tissue from a separate supply source. The drug may be a therapeutic or prophylactic agent having a molecule greater than 500 Daltons, such as those containing nucleic acids. In some embodiments, the drug is a vaccine, such as an RNA vaccine.

[0011] In another aspect, a method for delivering a drug into or through a biological tissue is provided. In an embodiment, the method comprises: (i) positioning the device adjacent to a target tissue site in the biological tissue, the device comprising an array of microneedle electrodes electrically coupled to a piezoelectric pulse generator; (ii) inserting the microneedle electrodes into the target tissue site; (iii) activating the device to deliver one or more electrical pulses through the microneedle electrodes and into the target tissue site, the electrical pulses being effective for electroporating cells at the target tissue site; (iv) and delivering the drug to the tissue at the target tissue site. In some embodiments, the biological tissue is mammalian skin or mucosa. In some embodiments, the target tissue site comprises dermis or epidermis. In some embodiments, the drug is first applied to the target tissue site and then the electrical pulse is delivered, or the electrical pulse is delivered while the drug is applied to the target tissue site. In some embodiments, the drug is applied to the target tissue site from or through the microneedle electrodes. In some other embodiments, the drug is applied to the biological tissue from another part of the device or from a separate supply source. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1A is a plan view of a housing and a piezoelectric pulse generator of a drug delivery system according to one embodiment of the present disclosure.

[0013] Figure 1B is an exploded view of a piezoelectric pulse generator according to one embodiment of the present disclosure.

[0014] Figure 2A and 2Bis when the microneedle electrode array and its associated metal plate according to an embodiment of the present disclosure are located within the cartridge housing ( Figure 2A ) or when there is no cartridge housing ( Figure 2B ) perspective view.

[0015] Figure 3 is a perspective view of a drug delivery system according to an embodiment of the present disclosure, the drug delivery system comprising (i) a housing containing a piezoelectric pulse generator and (ii) a cartridge including a microneedle electrode array.

[0016] Figure 4 is a schematic diagram of a cartridge having a solid microneedle electrode array according to one embodiment of the present disclosure.

[0017] Figure 5 is a schematic diagram of a cartridge having a hollow microneedle electrode array according to another embodiment of the present disclosure.

[0018] Figures 6A-6D is a schematic diagram of electrical connections of a microneedle electrode array according to some embodiments of the present disclosure.

[0019] Figures 7A-7F is a schematic diagram of an electroporator according to one embodiment of the present disclosure.

[0020] Figures 8A-8C is a graph of a representative electrical output profile of a piezoelectric pulse generator for electroporation according to one embodiment of the present disclosure.

[0021] Fig. 9 is a graph showing GFP expression in rat skin after electroporation using a microneedle electrode array (MEA) or clamp electrodes.

[0022] Figures 10A-10B Figure 2 shows the humoral immune response of mice after vaccination with SARS-CoV-2 DNA vaccine ( Fig. 10A ) and virus neutralization ( Fig. 10B ) curve graph.

[0023] Figures 11A-11D Depicts the measurement of voltage and current during the application of electrical pulses according to an embodiment of the present disclosure. Fig.11A ) and current ( Fig. 11B ) is a schematic illustration of a circuit. Fig. 11C and 11D Representative graphs of voltage and current, respectively, after pulse modulation using a commercial benchtop electroporator.

[0024] Fig.12 is a computer simulation result showing the electric field intensity distribution in the skin produced by one embodiment of an electroporation device as described herein. DETAILED DESCRIPTION

[0025] A new and improved electroporation device and method for administering drugs to a patient has been developed. The method and device are particularly suitable for delivering drugs greater than 500 Da, such as those including nucleic acids or other biological agents.

[0026] As used herein, the term "peak voltage" refers to the maximum voltage drop achieved across an electrode when the electrode is applied to biological tissue; the term "peak-to-peak voltage" refers to the voltage difference between the peak positive voltage and the peak negative voltage when the electrode is applied to biological tissue; the term "peak static voltage" refers to the maximum voltage drop achieved across an electrode when the electrode is applied directly to an oscilloscope lead; the term "peak-to-peak static voltage" refers to the voltage difference between the peak positive voltage and the peak negative voltage when the electrode is applied directly to an oscilloscope lead; the term "peak current" refers to the maximum current passing through biological tissue; the term "nominal electric field strength" refers to the voltage across an electrode divided by the spacing between oppositely charged electrodes; the term "initial pulse length" refers to the time elapsed between the start of a pulse and the time when the voltage first returns to the voltage before the start of the pulse when the electrode is applied to biological tissue; and the term "total pulse length" refers to the time elapsed between the start of a pulse and the time when the voltage no longer reaches an absolute value greater than 10% of the absolute value of the peak voltage when the electrode is applied to biological tissue.

[0027] Device and method

[0028] The device comprises: a piezoelectric pulse generator; and a microneedle electrode array electrically coupled to the piezoelectric pulse generator, wherein the device is configured to generate an electrical pulse and deliver the electrical pulse through the microneedle electrode after the microneedle electrode is inserted into the biological tissue, the electrical pulse being effective to electroporate cells in the biological tissue, and being configured to enable drug delivery to the electroporated cells. In a preferred embodiment, the device is sized and shaped to be handheld and manually operable, preferably with the same hand. In some embodiments, the device further comprises a base from which the microneedle electrode array extends. The base and the electrode array may be part of a cartridge configured to be releasably coupled to a housing containing the piezoelectric pulse generator.

[0029] In some embodiments, the device further comprises a drug to be administered, and comprises any suitable means for storing the drug and releasing the drug to a biological tissue site. In some embodiments, the drug is in the form of a coating on the microneedle electrode. In some embodiments, the drug is stored in one or more reservoirs in the device, such as in a cartridge, and / or at least a portion of the microneedle electrode each comprises a hollow hole and a conduit in fluid communication with one or more reservoirs, so that the hollow hole of the microneedle electrode enables the drug to be transferred from the reservoir and into the biological tissue before, after, or simultaneously with the insertion of the microneedle electrode into the biological tissue. In another variation, the cartridge body comprises an orifice adjacent to the microneedle electrode, which may be solid (i.e., without a hollow hole), wherein the orifice is in fluid communication with the reservoir and enables the release of the drug from the reservoir through the orifice at the junction of the biological tissue and the cartridge body, wherein the drug can flow into the puncture site formed by the microneedle electrode. In another embodiment, the microneedles may have grooves or other non-planar surface features that provide pathways for fluid flow along the microneedle surface (ie, as opposed to hollow holes that enable fluid flow inside the microneedle), thereby facilitating drug delivery into biological tissue.

[0030] In some embodiments, the drug is provided separately from the device. For example, the device can be administered to a target tissue site of a biological tissue by conventional means, such as a hypodermic needle, jet injection, iontophoresis, or a separate microneedle device. The microneedles used to administer the drug can be part of the electroporation device or can be a separate device.

[0031] In an embodiment, the method provided herein for delivering a drug into or through a biological tissue comprises: (i) positioning an electroporator device at a target tissue site in the biological tissue; inserting a microneedle electrode into the target tissue site; and (ii) activating the device to deliver one or more electrical pulses through the microneedle electrode and into the target tissue site, the electrical pulses being effective to electroporate cells at the target tissue site; and (iii) delivering the drug into the tissue at the target tissue site. The drug may be administered to the target tissue site before, after, while, or some combination thereof, the electrical pulse is delivered. The drug may be administered to the target tissue site from or through the microneedle electrode or from a separate delivery member.

[0032] The method may include manually applying force to the electroporator device to effectively penetrate the tissue surface with the microneedle electrode array, followed by manually pressing a button or switch on the device, which triggers a mechanical force on the piezoelectric device, thereby generating an electrical pulse that can effectively induce pore formation in the cell. Pressing the same button or switch or a different button or switch can activate the release of a drug, for example, from an onboard reservoir.

[0033] In a preferred embodiment, the electroporator device has no stored electrical energy (e.g., no batteries) and no wired or wireless connection to an energy source (e.g., not plugged into an electrical outlet). The energy to generate the voltage across the electrodes of the device is provided by mechanical action of a user of the device. For example, energy associated with manually pressing a button or switch on the device provides the energy to generate the voltage across the electrodes. The piezoelectric assembly can convert the mechanical energy input by the user into electrical energy that generates the voltage.

[0034] Advantageously, the device and method provide an ultra-low cost (<$2, e.g., less than about $1), handheld, battery-free electroporation system that is inexpensive enough for single use and stable enough for repeated use if desired. In some embodiments, a system or kit is provided that includes the device and a blister package of drug / microneedle electrode array cartridges. In addition, the device of the present invention is easily portable and suitable for handheld operation. For example, the device may weigh less than 300 grams (preferably less than 150 grams, preferably less than 100 grams, preferably less than 50 grams) and may be less than 100 cm in size. 3 , preferably less than 50cm 3 , preferably less than 20cm 3 , and requires no batteries or power source other than the piezoelectric crystal, which in turn can be powered by mechanical input from the user.

[0035] The devices and methods can be applied to any suitable biological tissue of a patient. That is, electrical pulses and drugs can be delivered to cells at or near physically accessible sites of the patient's body. The patient can be a human or other mammal. Electrical pulses and drugs can be delivered to cells in the skin, cells in the epithelium of the body, or cells inside the body that can be accessed, for example, by laparoscope or due to surgical treatment. In some embodiments, the biological tissue includes mammalian skin. In some preferred embodiments, the target tissue site includes the dermis or epidermis. In some other embodiments, the biological tissue includes a mucosa. In some embodiments, the target tissue site can be in the mouth, nose, eye, gastrointestinal tract, or vagina.

[0036] In certain methods, the method advantageously uses microneedle electrodes for targeted delivery to the skin, which has been shown to provide greater immunogenicity for DNA, RNA, and other vaccines compared to vaccines delivered into muscle. The microneedles are short, in some embodiments, may be only 650 μm long, which may focus the electric field in the epidermis, and may also focus the electric field in the dermis. The epidermis is particularly rich in antigen presenting cells and is therefore an ideal delivery location. In addition, delivery to the epidermis beneficially prevents the electric field from stimulating sensory and motor nerves deeper in the underlying dermis, subcutaneous, or muscle tissue.

[0037] The device and method can be used to administer any suitable drug, specifically, a drug in which electroporation can promote the cellular uptake of the drug. The drug can be substantially any therapeutic agent or preventive known in the art or developed. In an embodiment, the drug is a small molecule, a biological agent or a vaccine. Non-limiting examples include therapeutic proteins (e.g., antibodies, enzymes, growth factors, hormones, interferons, interleukins, engineered proteins and vaccines), RNA (e.g., messenger RNA (mRNA), RNA interference (RNAi) comprising short interfering RNA (siRNA) and microRNA (miRNA), antisense RNA (asRNA) or short hairpin RNA (shRNA) or RNA aptamers), DNA (e.g., plasmids, oligonucleotides, DNA aptamers, DNA ribozymes), anticancer drugs, antibacterial drugs, (e.g., protein synthesis, cell wall synthesis, enzyme activity, biochemical pathways) inhibitors, drugs that affect intracellular or intercellular signaling, drugs that affect gene regulation. The device and method can be used to deliver two or more different drugs. In a preferred embodiment, the drug includes nucleic acids. In a preferred embodiment, the drug is a vaccine. In various embodiments, the drug is an RNA or DNA vaccine, such as an mRNA vaccine. The vaccine can be selected to be effective against any of a variety of viruses, bacteria, or other pathogens (including but not limited to SARS-CoV-2, Ebola, influenza, etc.). The vaccine can be composed of mRNA and / or DNA encoding one or more antigens, or can be a vaccine effective against multiple variants of the disease.

[0038] As mentioned above, the device includes a piezoelectric pulse generator; and a microneedle electrode array electrically coupled to the piezoelectric pulse generator. The piezoelectric pulse generator and the coupled microneedle electrode array are sometimes referred to herein as an "ePatch" device or an electroporation device. The microneedle electrode array is sometimes referred to herein as a "microneedle electrode array" or MEA. The piezoelectric pulse generator may include any mechanism that can effectively generate a suitable electrical pulse from a piezoelectric crystal. In some embodiments, the piezoelectric pulse generator includes (i) a piezoelectric crystal, (ii) a spring-locked hammer mechanism configured to strike a surface of the piezoelectric crystal, the spring-locked hammer mechanism being effective to generate the electrical pulse, and (iii) an electrical connector for conducting the electrical pulse to the microneedle electrode.

[0039] The device may include any suitable piezoelectric crystal. In some embodiments, the piezoelectric crystal includes lead zirconate titanate (PZT). It may include various dopants known in the art. In some embodiments, the piezoelectric crystal includes lead zirconate titanate, silicon nitride, barium titanate, quartz, zinc oxide and / or sodium tungstate. A piezoelectric crystal is a material that exhibits a large amplitude piezoelectric effect, i.e., a large voltage output with a short time constant that produces an electric field and thereby a pulse. This may occur through mechanical force, pressure or compression applied to the surface of the crystal, thereby producing a temporary deformation, thereby forming an electric charge that is then released in the form of an electrical pulse.

[0040] The device may include a housing for a piezoelectric crystal including electrical connections for relaying pulses to the microneedle electrodes. The housing may include a lower electrode and a side electrode extending from the housing. The device may include a cartridge including an array of microneedle electrodes and including a first bracket for mating engagement with the lower electrode and a second bracket for mating engagement with the side electrode, the first bracket and the second bracket being in electrical communication with the microneedle electrodes.

[0041] The device may include a toggle switch having a wedge-shaped control latch configured to release a hammer driven by spring decompression. A pin (e.g., a metal pin) may be disposed between the hammer and the piezoelectric crystal. In one embodiment, the device may operate as follows: after the microneedle electrode is inserted into the skin, mucosa, or other biological tissue, the user applies a force to the toggle switch (e.g., a thumb toggle switch) that compresses the lower spring and pushes the wedge toward the hammer locked in the latch. As the user pushes all the way, the wedge forces the hammer out of the latch, which then strikes the pin next to the crystal to gather the force. The voltage output may then be directed to the electrodes on the device. The upper spring may then decompress to reset the hammer and latch to the original locked state. The user may repeat the operation as needed to generate additional pulses, thereby providing effective electroporation of the tissue to deliver the drug into the cell.

[0042] The microneedle electrodes are sized, shaped and spaced apart in the array in a manner that allows them to effectively penetrate into the target tissue (e.g., through the stratum corneum) and to achieve electroporation of cells using electric pulses generated by piezoelectric crystals. The microneedles are mechanically stable to prevent the microneedle electrodes from breaking or otherwise being damaged during insertion or removal of the microneedles. The microneedle electrodes may be made of stainless steel, nickel, iron or other metals or alloys or other appropriately conductive biocompatible materials. The microneedle electrodes may include a straight or tapered body, which may be cylindrical or square in shape, and may include a tapered tip portion. The microneedle electrodes may be manufactured using any suitable method known in the art, such as photolithography, 3D printing, micromolding and laser cutting. The microneedle electrodes may be solid, with a hollow hole extending from the base to the tip, or may have one or more grooves in the side of the microneedle.

[0043] In some embodiments, the microneedle electrodes extend from a series of metal plates configured to conduct electrical pulses from a piezoelectric pulse generator to the microneedle electrodes. In some embodiments, a linear array of microneedle electrodes extends from one side of each metal plate. It should be understood that on the plate, some microneedle electrodes may be positive and some microneedle electrodes may be negative. All microneedle electrodes on the plate may not be connected to each other. The connection may be selective among the electrodes, and there may be multiple connection / electrode groupings. In some embodiments, the plates are parallel to each other and spaced apart from each other. In some embodiments, the plate is fixed in an insulating holder, such as an insulating holder made of a polymeric material. For example, the insulating holder may be a cartridge body having a series of grooves, and the plate can be fixed to the grooves using microneedle electrodes extending from the surface of the cartridge body.

[0044] In some embodiments, two or more microneedles of the same polarity (i.e., cathode or anode) are part of the same length of conductive material (e.g., metal), which is formed, for example, from a single length of material and cut, etched, or otherwise processed to achieve the microneedle array geometry. The material can be a sheet and the microneedles can be in the same plane as the sheet, or they can be at a non-zero angle, such as about 90 degrees, to the plane of the sheet.

[0045] In some embodiments, the microneedle electrode may include a non-conductive microneedle array in which a conductive material (e.g., metal) is patterned on the surface of the microneedle array in such a way that all anodes are electrically connected and all cathodes are electrically connected, but the anodes and cathodes are electrically insulated from each other on the microneedle array.

[0046] In some embodiments, the microneedle electrodes are made of a conductive material and connected to a non-conductive substrate (i.e., the microneedle electrodes and the substrate include a microneedle array). The microneedle electrodes are electrically connected to each other by patterning the conductive material on one or more surfaces of the substrate and / or by electrical connections located in portions of the ePatch other than the microneedle array, which may involve wires electrically connected to the microneedle electrodes via through-holes in the microneedle array surface and / or the substrate.

[0047] In some embodiments, microneedle electrodes with the same polarity are all electrically connected to each other on the microneedle array, or in other cases, the microneedle electrodes are not all electrically connected to each other on the microneedle array, but are connected to each other through electrical connectors that are not on the microneedle array but are located elsewhere in the ePatch device.

[0048] In some embodiments, the microneedle electrode array is prepared by the method described in "Intracellular protein delivery and gene transfection by electroporation using a microneedle electrode array" by SO Choi, YC Kim, JW Lee, JH Park, MR Prausnitz, MG Allen, Small, 8, 1081-1091 (2012), which is incorporated herein by reference.

[0049] In some embodiments, the microneedle electrode array is arranged to be inserted into the 1 mm 2 Up to 10cm 2 in the tissue area.

[0050] The microneedle electrode array may have any suitable number of microneedle electrodes. In some embodiments, the array has 2 to 10,000 microneedle electrodes, such as 50 to 5000, 100 to 1000, or 20 to 200 microneedle electrodes. It is also contemplated that the device has other numbers of microneedles per array. The microneedle electrodes may be arranged in various patterns. The cathode and anode may be in alternating rows, may be in a checkerboard pattern, or may be arranged in other patterns.

[0051] In some embodiments, the microneedle electrode has a length of 10 μm to 2 mm. In some embodiments, the microneedle electrode has a length between 50 μm and 1.5 mm or between 100 μm and 1 mm. In some preferred embodiments, the length is in the range of 10 μm to 1 mm, for example, in the range of 650 μm or 750 μm. In some embodiments, the microneedle may have a base width or diameter of 50 μm to 600 μm. Other microneedle electrode sizes are also contemplated.

[0052] In some embodiments, a cartridge having a microneedle electrode array is configured to be replaceable and disposable, and a piezoelectric pulse generator is configured to be reusable with a series of such cartridges.

[0053] The electroporation portion of the device described herein provides electrical pulses that can enhance the delivery of molecules into cells. Using a piezoelectric crystal as an electrical pulse source induces membrane permeation through high voltage, short constant pulses (e.g., microseconds), which is believed to induce temporary changes in the cell membranes of certain cells in the electric field generated by the microneedle electrodes during the electrical pulse, including cells in contact with or near the microneedle electrodes, thereby allowing molecules to enter the cells and produce the desired effect.

[0054] In some preferred embodiments, the piezoelectric pulse generator generates bipolar oscillating pulses that can more effectively electroporate cells than conventional unipolar or exponentially decaying or square wave pulses.

[0055] Microneedle electrodes with smaller spacing are needed to achieve the high field strength required for microsecond pulses to be effectively used to electroporate cells in biological tissues. Since piezoelectric pulses have a microsecond duration, effective electroporation benefits from field strengths>500V / cm. In order to achieve this high field strength, the microneedle electrodes need to be appropriately spaced apart at a smaller spacing, for example, less than 1mm apart from each other (i.e., the closest adjacent electrodes in the array). This close spacing allows piezoelectric pulses of hundreds of volts to achieve the extremely large required field strength. In contrast, if conventional clamp electrodes with spacings of multiple millimeters or centimeters are used in some embodiments, a much larger voltage may be required to achieve this field strength, and the microneedle electrode arrays of the apparatus and method of the present invention are spaced between 0.1mm and 10mm in the array, preferably between 0.2mm and 5mm, and more preferably between 0.3mm and 2mm. In some embodiments, the spacing is in the range of 0.5mm to 1.5mm.

[0056] Conventional electroporators produce millisecond pulses. In contrast, the devices and methods of the present invention typically produce shorter pulses of about a few microseconds, such as less than 100 μs, such as about 10 μs. As the pulse length becomes shorter, the electric field needs to become higher. Greater electric field strength is achieved with greater voltage and shorter spacing between electrodes. Conventional electroporators use longer medium voltage pulses; however, these voltage pulses may often cause burns and / or leave marks on the skin. The short duration pulses of the system of the present disclosure provide the additional advantage of enhanced safety features. The field strength may not be constant, depending on the location in the microneedle array. However, in general, the field strength can be determined using the voltage divided by the electrode spacing. Therefore, when the electrode spacing is small, only microsecond pulses can be used to generate an electric field sufficient to electroporate the tissue, as described herein.

[0057] In some embodiments, the device is configured to generate a nominal electric field strength between 100 V / cm and 30,000 V / cm, preferably between 200 V / cm and 10,000 V / cm, and more preferably between 300 V / cm and 5,000 V / cm. For example, the nominal electric field strength may be in the range of 500 V / cm to 3,500 V / cm.

[0058] In some embodiments, the peak voltage absolute value of one or more electrical pulses is between 10 V and 10,000 V, preferably between 50 V and 5,000 V, and more preferably between 100 V and 1,000 V. For example, the peak voltage absolute value may be in the range of 200 V to 500 V. In some embodiments, the peak static voltage absolute value of one or more electrical pulses is between 100 V and 35,000 V, preferably between 1,000 V and 30,000 V. For example, the peak static voltage absolute value is in the range of 15,000 V to 27,500 V.

[0059] In some embodiments, the peak-to-peak voltage absolute value of the one or more electrical pulses is between 20 V and 20,000 V, preferably between 100 V and 10,000 V, and more preferably between 200 V and 2,000 V. For example, the peak-to-peak voltage absolute value may be in the range of 400 V to 1,000 V. In some embodiments, the peak-to-peak quiescent voltage absolute value of the one or more electrical pulses is between 200 V and 70,000 V, preferably between 2,000 V and 60,000 V, and more preferably between 25,000 V and 50,000 V.

[0060] In some embodiments, the ratio between the absolute value of the peak voltage and the absolute value of the peak-to-peak voltage of one or more electrical pulses is between 0.1 and 10, preferably between 0.3 and 5, and more preferably between 0.5 and 2.

[0061] In some embodiments, the ratio between the absolute value of the peak quiescent voltage and the absolute value of the peak voltage of one or more electrical pulses is greater than 2, or greater than 5, or greater than 10, or greater than 100.

[0062] In some embodiments, the peak current absolute value of one or more electrical pulses is between 1A and 1,000A, more preferably between 5A and 500A, and more preferably between 10A and 100A. For example, the peak current absolute value may be in the range of 20A to 50A.

[0063] In some embodiments, the initial pulse length of each of the one or more electrical pulses is between 1 μs and 1,000 μs, preferably between 3 μs and 100 μs, and more preferably between 5 μs and 50 μs. For example, the initial pulse length may be in the range of 10 μs to 30 μs.

[0064] In some embodiments, the total pulse length of each of the one or more electrical pulses is between 5 μs and 5,000 μs, preferably between 10 μs and 1,000 μs, and more preferably between 20 μs and 500 μs. For example, the total pulse length may be in the range of 30 μs to 200 μs.

[0065] In some embodiments, the ratio between the initial pulse length and the total pulse length of each of the one or more electrical pulses is between 1.5 and 100, preferably between 2 and 50, and more preferably between 3 and 20.

[0066] Unless otherwise indicated as a static voltage (i.e., peak static voltage, peak-to-peak static voltage), the aforementioned pulse values ​​refer to pulses in biological tissue (e.g., skin). It should be noted that similar conventional piezoelectric devices are designed to produce sparks in air. In contrast, the piezoelectric device described herein is coupled to a microneedle electrode and is used to pass current through a conductive medium (i.e., no sparks).

[0067] In a preferred embodiment of the apparatus and method of the present invention, a bipolar oscillating pulse is used for electroporation rather than a unipolar pulse, typically in the form of an exponentially decaying or square wave pulse conventionally used for electroporation. This bipolar oscillating pulse may be a natural result of the compression and extension of the piezoelectric crystal caused by the impact of a spring in the housing containing the piezoelectric crystal. Compared with conventional unipolar pulses, the bipolar oscillating pulse can not only produce dielectric breakdown of the cell membrane, but also produce sonication motion in the cell membrane, thereby inducing more effective cell poration. In addition, the oscillating pulse can provide better cell viability by avoiding polarizing the cell membrane beyond a critical potential for a long period of time, thereby preventing irreversible rupture of the cell membrane.

[0068] exist Figure 7A-7B One embodiment of an electroporation device is depicted in The system includes a manual toggle switch, a piezoelectric crystal, a housing surrounding the crystal, copper wires, and an MEA.

[0069] In an embodiment, the microneedle electrodes are arranged on the base in a square or rectangular arrangement, where rows or columns can alternately serve as positive and negative electrodes. Alternatingly, the positive and negative electrodes can be in a checkerboard pattern. In an embodiment, the cartridge includes an array of microneedles. Other arrangements such as a circular arrangement are possible; the microneedles can be arranged in any suitable shape.

[0070] In one embodiment, the MEA has an array of 54 microneedle electrodes, 650 μm in length, with 0.9 mm spacing between rows of oppositely charged electrodes that focus the electric field onto the epidermis. Electroporation requires approximately 10 3 The tight spacing of electrodes also reduces the depth of penetration of the electric field into the skin, which helps with epidermal targeting and reduces nerve stimulation.

[0071] The pulse length (or decay constant) is on the order of microseconds (e.g., 1-1000 microseconds), and may be on the order of nanoseconds (e.g., 1-1000 nanoseconds), depending on the piezoelectric crystal utilized. For example, the pulse length may be controlled by selecting different dopants utilized during crystal fabrication, selecting different dopants and different piezoelectric materials, and other methods known in the art. The pulse length may be in the range of 1 ns to 1 ms, preferably in the range of 1 to 100 μs.

[0072] Although for non-oscillating pulses, the initial pulse length and the total pulse length are the same, the pulses of the devices and methods described herein can be oscillating, so that the initial pulse length and the total pulse length can be different. In addition, although conventional electroporators produce generally monopolar pulses, the devices and methods described herein preferably use bipolar pulses, where each pulse alternates between positive and negative values.

[0073] Figure 1A-1B An example of the housing and piezoelectric pulse generator of the electroporation device described herein is depicted. Here, the user toggle switch 2 can be compressed, thereby compressing the upper spring 4, so that the spring 4 is closer to the hammer 6. When the toggle switch 2 is pushed all the way, the attached wedge 126 pushes the hammer 6 out of its latch, and the upper spring 4 is decompressed. When the toggle switch 2 is pressed, this spring 4 is also compressed to store elastic potential energy. Once the wedge 126 pushes the hammer 6 out of its latch, this spring 4 is decompressed to make the hammer 6 protrude. The hammer 6, which can be made of a metal material, protrudes onto the metal pin 10 in contact with the piezoelectric crystal 12 to induce mechanical deformation and generate an electric pulse. When the toggle switch 2 is compressed, this spring 8 (lower compression spring) is also compressed below and around the hammer 6. After the latch release stage, when the hammer 6 protrudes onto the metal pin 10 and thus onto the crystal 12, this spring 8 is decompressed, thereby resetting the toggle switch 2, the hammer 6 and the upper compression spring 4. This pin 10 rests on the piezoelectric crystal 12 and contacts it after the hammer 6 protrudes. The metal pin 10 concentrates the force on the crystal 12. When the hammer 6 hits the metal pin 10, the crystal 12 generates an electrical pulse. The device also includes a crystal housing 14 with electrode contacts. This housing 14 houses the piezoelectric crystal 12 and has a pin extending from the bottom to form a first electrical connection 16 and also has a conductive piece extending to a second electrical connection on the side 18. The metal piece protruding from the crystal housing 14 acts as an electrical connection, here the lower electrical connection 16. The metal piece protruding from the side mechanism housing 24 also acts as an electrode, here the side electrode 18. Threads can be used to screw the optional housing on the top. Both the lower thread 20 and the upper thread 22 can be used. The spring lock hammer mechanism housing 24 houses the elements of the mechanism. A wedge piece 126 is attached to the toggle switch 2 for pushing the hammer 6 out of the latch and causing it to protrude onto the metal pin 10 and the crystal 12.

[0074] Figure 2A-2B One embodiment of a microneedle electrode array and a holder or cartridge therefor is depicted. Figure 2A A microneedle electrode housing (cartridge housing) 30 is shown, which serves as a housing for a series of six metal plates 36, such as Figure 2A and Figure 2B As shown in both, these metal plates have a linear array of microneedle electrodes extending from one edge surface 34 of the plate 36, which is adjacent to the surface 32 of the housing 30. Wire connectors 26, 28 are directly or indirectly connected to the metal plate 36 within the housing and extend from the housing 30 for connection to the piezoelectric pulse generator. A first wire 38 and a second wire 40 connect the metal conductive plate 36 to transmit electrical pulses from the piezoelectric pulse generator to the microneedle electrodes. The wires 38, 40 can serve as, for example, positive and negative electrical connectors, respectively.

[0075] Figure 3 One embodiment of a device is depicted, comprising: (i) a housing comprising a piezoelectric pulse generator, and (ii) a cartridge comprising a microneedle electrode array. The piezoelectric pulse generator 42 may be similar to Figure 1A-1B The piezoelectric pulse generator shown in FIG. Figure 3 , the device further includes a microneedle electrode array 50 extending from the cartridge 48. The cartridge includes electrode insertion points 56 and 58, depicted here as slots, to cooperate with electrical connections from the piezoelectric pulse generator 42 to electrically connect with the metal conductors 52, 54, which in turn are electrically connected to the microneedle electrodes 50. The device may further include an optional support handle 44, which may assist the user in depressing a toggle switch on the piezoelectric pulse generator 42. The support handle 44 may include a threaded aperture 46 to mate with threads on the housing of the piezoelectric pulse generator 42.

[0076] Figure 4-5 Two embodiments of microneedle electrode arrays and housings / cartridges are shown. Figure 4 As shown in FIG. 1 , the end portion of the device (e.g., a cartridge) includes an array of solid microneedle electrodes 62. The microneedles 62 are connected to / contained within a housing 60. Here, the drug 64 is coated on the surface of one or more of the microneedle electrodes 62. Figure 5 As shown in FIG, the end portion of a device 66 (e.g., a cartridge) includes an array of hollow microneedle electrodes 68 and a plurality of reservoirs 72 containing a fluid drug formulation 74. In other embodiments (not shown), the cartridge may have a single shared reservoir that supplies drug to the hollow microneedle electrodes. A conduit 70 provides fluid communication between the reservoir 72 and the pores of the hollow microneedle electrodes 68. The reservoir 74 may be used to store the fluid drug formulation 74 prior to release and may contain more drug than may be contained in a coating on a solid microneedle.

[0077] Figures 6A-6DFour embodiments of electrical connectors for microneedle arrays are shown. Fig. 6A As shown in FIG. 1 , the conductive microneedle electrodes 200 are connected to an electrically non-conductive substrate 202. Electrical connectors 204 (e.g., wires) on the upper surface of the substrate 202 (i.e., the same side of the substrate 202 that the microneedle electrodes 200 are connected to) selectively provide electrical connections between microneedle electrodes 200 of the same polarity.

[0078] like Figure 6B As shown in FIG. 1 , the conductive microneedle electrodes 200 are connected to an electrically non-conductive substrate 202. Electrical connectors 206 (e.g., wires) on the lower surface of the substrate 202 (i.e., the same side of the substrate 202 that is connected to the microneedle electrodes 200) selectively provide electrical connections between microneedle electrodes 200 of the same polarity.

[0079] like Figure 6C As shown in FIG. 1 , the conductive microneedle electrode 200 is connected to an electrically non-conductive substrate 202. An electrical connector 208 extends from the upper surface of the substrate 202 (i.e., the same side of the substrate 202 to which the microneedle electrode 200 is connected) to the lower surface of the substrate 202. The electrical connector 210 selectively provides electrical connection between microneedle electrodes 200 of the same polarity.

[0080] like Fig.6D As shown in FIG. 2 , electrically non-conductive microneedles 214 are connected to electrically non-conductive substrates 202. Conductive material 212 at least partially covers the surface of microneedles 214 to form microneedle electrodes. Electrical connectors 216 (e.g., wires) on the upper surface of substrate 202 (i.e., the same side where substrate 202 and microneedles 214 are connected) selectively provide electrical connections between conductive materials 212 forming electrodes of the same polarity.

[0081] Application of the device and method

[0082] The methods described herein are used to administer an agent, typically a drug, to a patient by means of electroporation. As used herein, the term "electroporation" includes related phenomena known in the art, such as electrofusion, and may include viruses, virus-like particles, liposomes, and any other particulate objects containing a lipid bilayer membrane. The patient may be a human or other mammal in need thereof.

[0083] The method can be advantageously used to deliver macromolecular drugs to the patient's body tissues using electroporation without the use of batteries, capacitors or other conventional power storage devices, and without the use of electricity from an external power source (e.g., not plugged into an electrical outlet). However, a variety of drugs, including small molecules, proteins, nucleic acid-based compounds and biologics, may facilitate their delivery. The scope of drugs includes biologics, which may include antigens (derived from or mimicking foreign substances to induce immune responses when introduced) synthesized in the form of nucleic acids or proteins or naturally obtained from related sources; nucleic acids in plasmid or linear form, such as DNA, RNA or other polynucleotide compounds; peptides (amino acid sequences); viral vectors or viruses; any type of vaccine; gene therapy, such as CAR-T cell therapy for cancer treatment; immunotherapy; chemically or biologically synthesized proteins; and / or any combination of these, or drug-loaded particles, such as lipid nanoparticles, polymer nanoparticles.

[0084] In some embodiments, the electric field is concentrated to the skin, especially the epidermis, where concentration to the epidermis means that most of the cells undergoing electroporation conditions are located in the epidermis rather than the dermis or deeper tissues. This concentration is facilitated by limiting the penetration of the electric field into deeper tissues and the fact that the cell density in the viable epidermis is much greater than that in the dermis. Targeting the electric field and the electroporation caused to the skin and especially the epidermis improves immunogenicity and reduces side effects. Unlike the substantially acellular dermis, the epidermis is densely populated with cells, including keratinocytes and effective antigen presenting cells, such as dendritic cells (including Langerhans cells). Targeting antigens to these epidermal cells has been shown to improve immune responses compared to IM injections. Although the value of dermal electroporation is low, it is beneficial for the antigens produced in epidermal cells to diffuse to the upper dermis because the presence of dermal dendritic cells and a rich vascular system enables drainage to lymph nodes, which also enhances immunogenicity. In addition, concentrating the electric field to the epidermis can reduce nerve stimulation, thereby making electroporation more tolerable. Of particular concern is the stimulation of motor nerves and muscle cells beneath the skin, which can result in the violent convulsions reported in other cases as a result of skin or muscle electroporation. In fact, this is shown in the examples disclosed herein, where conventional electroporation using clamp electrodes induces strong muscle contractions at the electroporation site after each pulse is applied. The animals were anesthetized, suggesting that action potentials in muscle cells and / or motor nerves are directly stimulated. These contractions are not visible when electroporation is performed with MEAs, which focus the electric field at the surface, away from the muscle.

[0085] The electric field concentration method is somewhat different from other methods. Other methods propose to use penetrating electrodes that are much longer than existing microneedle electrodes. These other penetrating electrodes penetrate completely through the epidermis and dermis, and penetrate into the subdermal tissue, usually deeper. Because the microneedle electrodes of the present invention penetrate into the epidermis and possibly a portion of the dermis, the microneedle electrodes do not completely penetrate the dermis and do not contact the tissue below the dermis. Because the electric field generated when these microneedle electrodes are pulsed is strongest in the tissue between the oppositely charged electrodes, tissue deeper than the penetration depth of the microneedle electrodes receives a weaker electric field and is less likely to cause electroporation.

[0086] Another method of electroporating tissue involves the use of surface electrodes that contact the surface of the skin but do not penetrate into the skin. They may involve treating the skin surface to facilitate contact, such as applying a conductive gel or other material or using sandpaper or other methods to at least partially remove the stratum corneum. In this case, the depth to which the electric field penetrates the skin is similar to the spacing between the electrodes. This spacing in a typical system is at least a millimeter or more. Therefore, the electric field will not be concentrated in the dermis, and certainly not in the epidermis, but will reach the tissue below the dermis. It is possible to have surface electrodes spaced at very small spacings (e.g., less than 1 mm) to better limit the exposure of the subdermal tissue to the electric field. However, compared with penetrating microneedle electrode arrays, they produce more uneven electric field strengths in the skin.

[0087] The present invention may be further understood by reference to the following non-limiting examples.

[0088] Examples

[0089] Experiments were performed to compare the devices and methods described herein with commercially available devices and methods.

[0090] The microneedle electrode array (MEA) described in the following example was made by assembling 6 rows of stainless steel microneedles (electrodes) with a length of 650 μm and a cross section of 200 μm × 50 μm, tapering to a sharp tip, mounted in a 3D printed insulating holder made of polylactic acid. Each row of electrodes with the same electrical polarity consists of 9 microneedles, each microneedle is separated by a spacing of 0.8 mm within each row (i.e., microneedle electrodes with the same polarity), and each row is separated by a spacing of 0.9 mm (i.e., separation of microneedle electrodes with opposite polarities). Fig. 7E (Right) is an enlarged view of the electrode row according to one embodiment. This close spacing is used to achieve the larger electric field strength required for electroporation using microsecond pulses. The piezoelectric pulse generator is connected to the MEA using wires for the positive and negative terminals (see, e.g. Fig.7D In use, the MEA is pressed against the skin, allowing the microneedles to penetrate the skin's stratum corneum barrier into the viable epidermis and superficial dermis, after which the thumb toggle switch is pressed to apply the pulses ( Fig. 7A ). Fig. 7A Electroporator 80 is shown held in position by hand 82 prior to activation. Electroporator 80 includes housing 86 having toggle switch 84 that can be activated, for example, by a user's thumb. Electroporator 80 further includes MEA cartridge 88. Figure 7B is an exploded view of an electroporator 90, referred to as an ePatch, comprising a piezoelectric pulse generator and a metal MEA. The piezoelectric pulse generator comprises a crystal housing 96 housing a piezoelectric crystal 94. An MEA cartridge 102 comprising microneedles 100 is connected to the piezoelectric pulse generator via copper wires 98. The electroporator 90 further comprises a hand-operated toggle switch 92. Figure 7C A row of microneedle electrodes (left) and a single electrode 120 (right) are shown. Fig.7D is a diagram showing an electrode configuration in one embodiment of an MEA, Fig. 7E is an enlarged view of the section of the MEA relative to the finger 132. Fig. 7E In FIG. 1 , the MEA cartridge 130 holds the microneedles 134 . Figure 7F is a plan view of a polymeric holder / housing 110 for a plate of microneedle electrodes that may be connected to the holder 110 via slots 112 .

[0091] Examples herein show that methods and systems including piezoelectric pulse generators and metal MEAs can selectively deliver molecules to cells in the epidermis using microsecond pulses without obvious signs of damage to the skin. In contrast, electroporation using millisecond pulses from conventional electroporation devices may show significant damage at each site where a microneedle electrode penetrates the skin. The methods and systems of the present invention can deliver DNA vaccines and produce stable humoral immune responses and virus neutralization, showing at least a 10-fold dose saving compared to intradermal (ID) or intramuscular (IM) injections without electroporation.

[0092] Example 1 - Electroporation studies in animals using a piezoelectric pulse generator and microneedle electrode arrays

[0093] Animals and plasmids

[0094] All animal experiments were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines of Emory University and Georgia Institute of Technology. Adult female Wistar rats (250-300 g) and 5-6 week old female BALB / c mice were used. Animals were maintained on a 12 h / 12 ​​h light / dark cycle in the animal care facility with free access to food and water and were acclimated for at least 7 days before the experiments. A high expression reporter plasmid gWiz-GFP was used. The DNA of the SARS-CoV-2 surface glycoprotein without the transmembrane domain was cloned into the pCAGGS vector using fusion cloning technology.

[0095] Design of piezoelectric pulse generator and microneedle electrode array

[0096] The device including the piezoelectric pulse generator and the electrically coupled MEA is referred to herein as an "ePatch". The electrical pulses are generated by a device derived from a common household piezoelectric stove lighter ( Figure 7B ). 3D printed cylindrical chamber to hold piezoelectric crystals collected from commercial lighters. The chamber has a wire connected to the piezoelectric crystal and exits the chamber through its base. A hand-activated toggle switch is attached to the top to provide the same force as a regular lighter when pressed down. The holder is 3D printed using poly(lactic acid) through a 3D printer.

[0097] The MEA was fabricated by assembling 6 rows of solid metal microneedles in an insulating holder. Each row had 9 microneedles with a tip-to-tip spacing of 0.79 mm for each microneedle. Microneedles with opposite electrical polarity were positioned adjacent to each other with a distance of 0.90 mm between rows. Figures 11A-11B The circuit shown in ) measures the pulse distribution from the ePatch device.

[0098] Numerical simulation of electric fields for electroporation

[0099] The electric field intensity distribution was analyzed by numerical modeling using commercial modeling software. The parameters used for the numerical simulation of the electric field in the skin are shown in Table 1 below.

[0100] Skin anatomy Rat Mouse Resistance(S / m) Stratum corneum 20μm 10μm 0.0005 Epidermis and Dermis 650μm 150μm 0.2 Subcutaneous layer 1300μm 300μm 0.05

[0101] Table 1

[0102] To simplify the model, the conductivity change of the permeabilized tissue during electroporation was not considered, so as to capture the peak electric field intensity when the pulse was first applied to the previously untreated skin. The electric field simulation was performed in an electrostatic mode, in which multiple rows of metal needle electrodes were alternately set to a static high potential and a static low potential so that the voltage between adjacent rows met the target voltage value. The medium between the needle tip segments was set using skin parameters to simulate the situation when the microneedles penetrate the skin. The results of using this model are given in Examples 5 and 7.

[0103] Differentiation of non-viable cells from electroporated cells by confocal microscopy

[0104] To study the effect of electroporation on cell viability and cell permeability in skin, a cell-impermeable probe, SYTOX Green, was used to identify transient cell membrane permeability uptake caused by electroporation. SYTOX Green was coated on the microneedles before electroporation and used as an indicator of transient permeability caused by electroporation. Another cell-impermeable probe, ethidium bromide (EB), was used as an indicator of non-viable cells caused by electroporation. BALB / c mouse skin was used as a tissue model.

[0105] Under anesthesia, the back dermis hair was removed with a scraper, and then a depilatory was applied and waited for 3 minutes. The skin was cleaned with wet gauze to remove the depilatory. Three days after depilation, the mice were anesthetized with isoflurane, the MEA was pressed into the skin, and 5 or 20 piezoelectric pulses were applied. After 10 minutes of electroporation of the skin, the mice were euthanized with carbon dioxide. The skin was collected and immersed in phosphate-buffered saline (PBS) containing EB (50 μg / ml), incubated at 4 ° C and shaken for 1.5 h. The skin was washed 3 times with fresh PBS and imaged using a laser scanning confocal microscope (20 times objective) (not shown). SYTOX Green and EB were excited in sequence using argon lasers of 488 nm and 514 nm, respectively. Under a confocal microscope, non-viable cells had red fluorescence from EB, while electroporated cells had green fluorescence from SYTOX Green (not shown).

[0106] Live imaging of GFP expression and histological examination of skin

[0107] One day before the DNA delivery study, prepare the rats under anesthesia by removing the hair on their back skin using a trimmer, then apply a depilatory cream, wait 4 minutes, and wipe clean with water. 2 Animals were anesthetized in an induction chamber and then fitted with a standard rodent mask to maintain general anesthesia during the procedure.

[0108] Twenty microliters of PBS (2.5 μg / μl) containing GFP plasmid was injected into the ID to form visible blisters in the skin. After 1 min of DNA injection, electroporation pulses were applied to the injection site with MEA or clamp electrodes. The ePatch device generates a specified number of microsecond pulses (1, 5, 10, 20 pulses) to study the effect of the number of pulses on gene expression. A commercial desktop electroporator with programmable pulse voltage was also used to study the effect of millisecond pulse voltage on gene expression. The fluorescence intensity of GFP in the skin was monitored on different days by the IVIS Spectrum CT in vivo imaging system.

[0109] For histological examination studies, the skin of mice was electroporated with 20 pulses by the ePatch device and imaged under a stereomicroscope immediately after electroporation and again after 3h. After collecting the skin 12 hours after electroporation, the tissue was embedded in Tissue-Plus OCT embedding medium and frozen overnight at -20°C, after which it was sliced ​​at a thickness of 20μm using a freezing microtome. The tissue sections were imaged by a laser scanning confocal microscope (not shown). For H&E staining, the tissue was fixed in 10% formalin buffer overnight and then dehydrated by an automatic tissue dehydration system. The dehydrated tissue was embedded in paraffin, sliced ​​at a thickness of 5μm using a rotary microtome, and stained. The tissue was imaged by an inverted microscope (not shown).

[0110] Mouse Immunity Studies

[0111] For mouse immunization studies, the same electroporation parameters used in rats have been shown to also produce strong GFP expression in mouse skin. BALB / c mice were randomly divided into five groups (n=5 mice per group) and received injections of 10 μl of PBS solution containing SARS-CoV-2 S protein DNA vaccine: (i) 10 μg DNA vaccine IM injection, (ii) 100 μg DNA vaccine IM injection, (iii) 10 μg DNA vaccine ID injection, (iv) 10 μg DNA vaccine ID injection followed by 20 pulses via the ePatch device, and (V) PBS ID injection as a negative control. Mice were anesthetized with isoflurane during surgery. Each animal received a second administration 4 weeks later via the same procedure as the first administration. At week 7, blood was drawn by orbital sinus puncture and serum was separated.

[0112] ELISA for SARS-CoV-2 Spike Protein Antibody Analysis

[0113] IgG titers against SARS-CoV-2 spike surface protein in mouse sera were measured using enzyme-linked immunosorbent assay (ELISA). ELISA plates were coated with purified spike protein and then blocked with 5% bovine serum albumin. Serum samples were diluted 80-fold with PBS containing 0.1% Tween 20 (PBST) and then added to the ELISA plate, incubated for 1 h at room temperature (20-25°C), and then washed three times with PBST. Horseradish peroxidase-conjugated goat anti-mouse antibody was added and incubated for 1 h. The plate was washed again, followed by the addition of 3,3′,5,5′-tetramethylbenzidine substrate for color development. The reaction was terminated by commercial stop solution. The absorbance was read at 450 nm by an ELISA plate reader. The optical density (OD) values ​​were recorded and used as relative antibody expression levels in mice.

[0114] Pseudovirus neutralization assay

[0115] SARS-CoV-2 spike protein pseudotyped viruses were used in the neutralization assay. Pseudoviruses were produced by co-transfecting 293T cells with env-deficient HIV-1 backbone plasmid DNA and a DNA plasmid expressing the full-length SARS-CoV-2 spike protein flow-through protocol. Pseudoviruses were produced and self-packaged in 293T cells. Pseudoviruses secreted into the supernatant of 293T cells were collected.

[0116] To analyze serum neutralization activity, 293T cells expressing angiotensin-converting enzyme 2 (ACE2) were inoculated in 96-well plates and grown overnight. Mouse serum samples were diluted 100 times, 300 times, and 900 times with Dulbecco's Modified Eagle Medium. Each diluted sample (50 μl) was mixed with an equal volume of virus suspension (50 μl) and then incubated at 37 ° C for 1 h. Then, samples containing serum pseudovirus mixtures were added in triplicate to the wells in 96-well plates inoculated with 293T cells expressing ACE2, which were grown to 50% confluence. Six hours after infection, the suspension was centrifuged at 1500 x g for 5 minutes, the supernatant was removed and replaced with DMEM containing 5% fetal bovine serum. After 48 hours, the cells in each well were lysed and the luciferase activity was determined. The neutralizing activity of immune sera was determined by the following formula: [(pseudovirus only)-(pseudovirus+serum)] / [pseudovirus only]×100%.

[0117] Example 2 - Preliminary porcine skin testing

[0118] First, preliminary fluorescence studies were performed using pig skin to compare a conventional microneedle injection platform with a delivery system consisting of a piezoelectric pulse generator operably coupled to a microneedle electrode array. Specifically, SYTOX Green was used to monitor expression. A red dye, ethidium bromide, was also used to indicate cells killed by injection. Pig skin was injected using a conventional microneedle platform and the cells were imaged (not shown). Pig skin was also injected using a new delivery system with a piezoelectric pulse generator and a microneedle electrode array. Images were taken at 0 pulses, 5 pulses, and 10 pulses (not shown). Not only was expression significantly higher using the new delivery system compared to the conventional microneedle platform, there was also no significant loss of cell viability due to injection.

[0119] Example 3 - Gene transfection in the epidermis

[0120] To assess targeting of gene transfection to the epidermis, histological analysis was performed 1 day after DNA delivery. Electroporation with MEA using microsecond pulses from an "ePatch" device consisting of a piezoelectric pulse generator and a metal MEA, or using millisecond pulses from a commercial benchtop electroporator (BTX Electro Cell Manipulator 600, Harvard Apparatus, Cambridge, MA) produced strong green fluorescence that was evident in the skin surface exposed to the MEA and throughout the viable epidermis when viewed head-on from the skin surface, with little evidence of GFP expression in the dermis or stratum corneum when viewed as frozen tissue sections. Images showed that transfected cells were labeled almost exclusively within the epidermal layer below the stratum corneum (not shown). In comparison, GFP fluorescence intensity was less when electroporation was performed using clamp electrodes (compared to Fig. 9 In addition, although the majority of transfected cells were in the epidermis, evidence of GFP transfection was seen in the deep dermis, especially in the hair follicles. These findings confirm the ability of MEA to focus electroporation to the epidermis.

[0121] Example 4 - Analysis of high voltage pulses and electric fields

[0122] Using a high voltage probe and oscilloscope, the voltage output of the ePatch sparking through air without skin penetration was determined. Figures 8A-8C Representative electrical output profiles of a piezoelectric pulse generator used for electroporation are shown. Electroporation was performed by directly connecting the electrode leads to an oscilloscope probe (a, voltage profile) and by pulse modulation in vitro in pig skin (b, voltage profile). Figure 8B , voltage distribution; Figure 8C , current distribution) to activate the piezoelectric pulse generator. Fig. 8A In FIG. 1 , multiple replicate voltage distributions (n=20) are shown. The peak positive static voltage and peak negative static voltage of the output pulses are 24.4±1.3 kV and 7.0±0.8 kV, respectively. Fig. 8A ). Given an electrode spacing of 0.9 mm, this corresponds to a peak positive static field strength of 1200 kV / cm, which is higher than the dielectric strength of air (i.e., about 30 kV / cm). The peak positive static voltage is reached after 10.6 ± 0.7 μs, and the subsequent oscillating voltage output decays within about 100 μs ( Fig. 8A ). This extremely high static voltage is caused by the extremely high electrical impedance and insufficient current.

[0123] When the MEA was used as an electrode and applied to pig skin in vitro, the peak positive voltage output and peak negative voltage output were 283±49V and -345±54V, respectively. Figure 8B ).use Fig.11AVoltage measurements were made using the device shown in . The time to reach peak voltage was 12.6±1.1μs. Here, due to the lower impedance of the skin compared to when connected to an oscilloscope, the voltage was much lower, which allowed the current to pass for the entire pulse duration. The electrical pulses were in the form of a bipolar oscillatory decaying waveform, which is characteristic of piezoelectric pulses. For comparative analysis, electrical pulses were also generated using a commercial bench electroporator commonly used for laboratory transfection and coupled in vitro to the MEA in pig skin. This pulse generator generated unipolar exponentially decaying pulses of 31±3V or 100±5V, with a pulse duration (i.e., exponential decay time constant) of 59.2±7.8ms or 55.4±2.3ms, respectively ( Fig. 11C ). These millisecond monopolar pulses are more typically used in conventional electroporation, in contrast to the microsecond oscillatory pulses produced by the ePatch.

[0124] The current through the skin during the pulse from the ePatch device was also measured, which showed an oscillatory decaying waveform similar in shape to the voltage waveform, and a peak positive current of 24.8 ± 1.1 A was obtained ( Figure 8C ).use Fig. 11B For a commercial desktop electroporator, the highest currents passing through the skin were 0.28 ± 0.03 A and 1.33 ± 0.16 A, respectively, when pulses of 32 ± 3 V and 105 ± 4 V were applied. Fig.11D The apparent electrical impedance (i.e., characterized as peak voltage divided by peak current) was 11.4Ω during ePatch pulsing and 114Ω or 78.9Ω during pulsing with a conventional electroporator (32V or 105V, respectively). The much lower apparent electrical impedance measured using the microsecond pulses from the ePatch compared to the millisecond pulses from the conventional pulse generator may be due to the known impedance reduction that occurs when skin and other tissues are exposed to electric fields with complex waveforms having higher frequency components.

[0125] To better understand the electric field distribution in the skin when pulses are applied using MEAs, Fig.12 As shown, the electric field intensity in the skin during electroporation was modeled. Fig.12 The electric field intensity distribution in the skin determined by computer simulation is shown. When a 300V pulse is applied using (a) MEA or (b) clamp electrodes, similar to the pulse from the ePatch, the peak electric field intensity is shown. The field intensity distribution is shown from above the MEA (top left) and from the side view (bottom and right). The dermis-epidermis junction is indicated by a dotted line. The scale bar is 1 mm.

[0126] For MEA, the electric field strength is highest around each microneedle electrode, especially near the tip, where electrode curvature is known to increase the electric field strength ( Fig.12). The electric field strength is weakest between electrodes of the same polarity. The electric field also does not penetrate deeply into the tissue beneath the electrodes, and therefore falls off over a length scale of hundreds of microns. In this way, the electric field is concentrated in the epidermis and upper dermis, which are rich in antigen presenting cells (e.g., epidermal Langerhans cells and dermal dendritic cells) and have efficient drainage to lymph nodes, all of which may enhance vaccine immunogenicity.

[0127] The threshold of reversible electroporation depends on the duration of exposure to the electric field. For millisecond long pulses, the electroporation threshold is expected to be about 400-600V / cm, and for microsecond pulse duration, the threshold is increased to 1.0-1.5kV / cm. When using a piezoelectric electroporator to apply a 300V pulse, the highest electric field strength in the tissue next to the electrode is 15kV / cm, but the field strength experienced by most tissues is 2-3kV / cm, which is higher than the threshold required for successful electroporation, but still low enough to avoid extensive cell killing. In this way, it is expected that the highly concentrated cell death adjacent to the electrode and the smaller area where electroporation does not occur between the electrodes of the same polarity, but most tissues experience the field strength that is expected to cause reversible electroporation.

[0128] These findings were further compared to the field strength in the skin produced using conventional clamp electrodes at the same voltage (300 V); it was found that the larger spacing of the clamp electrodes (i.e., 3.9 mm) produced much weaker electric field strengths than the MEA. The electric field strength exceeded 1 kV / cm only in a portion of the space between the electrodes and exceeded 1.5 kV / cm only at the extreme edges of the electrodes.

[0129] Also studied the field intensity in the skin during the representative pulse (30V and 100V) from a conventional electroporator applied using clamp electrodes or MEA. The 30V pulse using clamp electrodes produces very low field intensity, mostly below 300V / cm, which does not reach the expected electroporation threshold of millisecond pulses. The pulse of 30V applied by MEA with small spacing intervals makes the tissue next to the electrode reach 400-600V / cm, but most of the tissues experience much weaker electric field. When using a 100V pulse, clamp electrodes reach the field intensity expected to be electroporated in some tissues, and MEA produces an electric field strong enough to be electroporated in most tissues.

[0130] Example 5—Stable reporter gene transfection of ePatch

[0131] To evaluate the effect of the ePatch device on plasmid delivery and transfection, DNA plasmids encoding green fluorescent protein (GFP) were delivered in vivo to rat skin. Gene expression levels were measured by in vivo imaging of GFP fluorescence over time.

[0132] The effects of high field strength microsecond pulses using the ePatch have been tested and a single pulse has been found to produce visible GFP expression ( Fig. 9 ). Fig. 9 Figure 2 shows the radiative efficiency of GFP fluorescence in the skin at different days after delivery of a GFP reporter plasmid using an ePatch (waveform shown in Figure 2) that delivers 1-20 pulses of approximately 300 V. Figure 8B Electroporation was performed using a conventional exponential decay electroporation pulse generator at a controlled peak voltage (10-100 V) with a decay time constant (tau = 49-57 ms). Pulses were applied using a microneedle electrode array (MEA) or clamp electrodes. Data represent mean ± standard deviation (n = 5-6 replicates in each case). (***p < 0.001).

[0133] More pulses increased GFP expression up to 10 pulses (p=0.001); increasing to 20 pulses did not further increase GFP expression (p>0.05). Three days after electroporation, GFP expression decreased over time (p=0.002). After 5 days, GFP fluorescence could not be detected, probably due to GFP protein degradation in the skin.

[0134] As a negative control, GFP plasmid ID was injected into the skin without electroporation, which resulted in almost undetectable GFP transfection ( Fig. 9 ). Compared with ID injection alone, ePatch increased GFP expression 416-fold (p<0.001).

[0135] To better explain these results, additional experiments were performed using a cell-impermeable green marker compound (SYTOX Green) present during electroporation to identify permeable cells, followed by the addition of a red viability dye to identify non-viable cells. Examination of the skin by microscopy showed that there was a loss of cell viability at the site where the microneedles pierced the skin, independent of electroporation, as indicated by the presence of red fluorescent cells (not shown). This may be due to damage caused by mechanical puncture of the microneedles. The application of 5 or 10 electroporation pulses from the ePatch did not appear to increase cell viability loss, but did result in increased cell permeability as the green marker compound was taken up into viable cells around the non-viable core at the site of each microneedle penetration. Microscopic examination of the skin surface showed only faint and transient signs of skin damage at the site of each microneedle electrode penetration, as discussed below.

[0136] Next, the effect of medium field strength millisecond pulses using the MEA coupled to a commercial electroporator was tested. Electroporation under these conditions resulted in GFP expression, which peaked at 30 V (p = 0.02) ( Fig. 9). Peak GFP expression at 30 V was not significantly different from that produced by the ePatch using 10 pulses (p=0.055), while GFP expression at other voltages was significantly lower (p<0.05). Similar to the ePatch, cells transfected using millisecond pulse electroporation also showed a decrease in GFP fluorescence within 3 days after electroporation (p=0.008).

[0137] The dependence of GFP expression on voltage can be explained by the lower extent of electroporation at 10V compared to 30V, resulting in less transfection. Above 30V, the loss of cell viability caused by irreversible electroporation and tissue heating increases within milliseconds of long-term exposure to high electric field strengths, which may offset the increase in DNA delivery to cells. Additional skin imaging after delivery of green labeled compounds and application of red vitality dye (not shown) supports this explanation. As the voltage increases, the loss of cell viability increases, and tissue heating at the site of microneedle electrode placement also increases, reaching a peak of up to 50°C. Microscopic examination of the skin surface showed that the discoloration of the site penetrated by each microneedle electrode lasted for at least two days, which is consistent with the extensive cell death observed at higher voltages using millisecond pulses (not shown).

[0138] To address current skin electroporation methods, clamp electrodes (with a 3.9 mm gap) were used instead of MEAs. When pulsed with a microsecond piezoelectric pulse generator, a single pulse did not result in detectable GFP expression, but application of 10, 20, or 30 pulses produced GFP expression independent of the number of pulses (p>0.05). Using clamp electrodes with millisecond pulses from a commercial electroporator, detectable GFP expression was found at 60V, which also increased slightly when the voltage was increased to 100V (p>0.05). With the ePatch or commercial electroporator, GFP expression using clamp electrodes was significantly reduced compared to GFP expression when using MEAs (p<0.001).

[0139] In summary, these results demonstrate that (i) the ePatch achieves high levels of DNA transfection and expression, (ii) the use of MEAs produces sufficiently high electric field strengths to enable microsecond pulses from a piezoelectric pulse generator to be effective and (iii) enables conventional electroporators to be effective at much lower voltages than clamp electrodes, and (iv) microsecond pulse modulation minimizes tissue heating that appears to damage tissue when millisecond pulses are used.

[0140] Example 6—Stable immune response and virus neutralization after SARS-CoV-2 DNA vaccination in mice

[0141] After confirming that the ePatch could significantly enhance gene expression in vivo, the immunogenicity of the SARS-CoV-2 DNA vaccine delivered by the ePatch, ID injection without electroporation, and IM injection without electroporation (including IM vaccination) was evaluated at two different doses (10 μg and 100 μg DNA). IM vaccination produced a higher humoral immune response at the 100 μg dose than at the 10 μg dose, as measured by antigen-specific IgG titers (p=0.04) and virus neutralization assays (p<0.001) ( Figures 10A-10B Specifically, mice were immunized at weeks 0 and 4; blood samples were drawn at week 7. The IgG titer against the SARS-CoV-2 spike surface protein in mouse serum was expressed as the absorbance at 450 nm ( Fig. 10A Neutralization of IgG against pseudovirus was analyzed at different serum dilutions and expressed as the percentage of neutralization for each dilution ( Fig. 10B ). As a control, mice were immunized with PBS. IM_10μg and IM_100μg: mice were immunized with 10μg and 100μg DNA vaccine by IM injection, respectively. ID_10μg: mice were immunized with 10μg DNA vaccine by ID injection. ID_10μg_ePatch: mice were immunized with 10μg DNA vaccine by ID injection followed by electroporation with 20 pulses by ePatch. n=5 mice per group. (*p<0.05, **p<0.01, ***p<0.001).

[0142] ID injection of 10 μg DNA vaccine produced results similar to those produced by IM vaccination at the same dose (p>0.05). When ID vaccination was performed using electroporation with ePatch, the immune response was significantly higher than ID or IM vaccination without electroporation at the same DNA dose (p=0.01 and 0.004, respectively). In addition, there was no significant difference between ePatch vaccination with 10 μg DNA and IM vaccination with 100 μg DNA (p=0.47), indicating that ePatch vaccination can save at least 10 times the dose. Finally, it is worth noting that when the serum was diluted 100 times, 90% antibody neutralization against SARS-CoV-2 pseudovirus was found for the low-dose ePatch and high-dose IM injection groups, while only 20% neutralization was found for the low-dose IM and ID injection groups without electroporation ( Fig. 10B ). In conclusion, this study demonstrated that the ePatch significantly improved the immune response to SARS-CoV-2 compared with IM or ID injection alone.

[0143] Clinical and histological examinations showed that vaccination using the ePatch was very well tolerated. Imaging of the skin surface immediately after electroporation showed signs of microneedle punctures and / or focal electroporation when viewed with a magnifying glass (not shown). Follow-up imaging 3 hours later showed no residual signs of the vaccination process. Histological examination of the skin 12 hours after ePatch vaccination showed no inflammatory markers. In contrast, high voltage (100V) millisecond pulse modulation caused extensive infiltration of inflammatory cells visible in the skin 12h after electroporation. Clinical examination of the animals in the weeks following vaccination did not produce significant findings. These data indicate that ePatch vaccination causes only mild, transient effects on the skin and does not generate safety signals.

[0144] In summary, the delivery systems and methods of the present disclosure can provide a DNA vaccination approach that benefits from the combination of two innovations: a piezoelectric-based power source for electroporation and a MEA that generates a large electric field targeted to the epidermis. This combination in the form of an ePatch is demonstrated to enable DNA vaccination using a simple, ultra-low-cost system, thereby expanding the scope and speed of vaccination against COVID-19 and future pandemics.

[0145] The delivery systems and methods of the present disclosure are capable of providing expression levels that are orders of magnitude higher than conventional injection without electroporation.

[0146] Modifications and variations of the methods and systems described herein will be apparent to those skilled in the art from the foregoing detailed description. Such modifications and variations are intended to fall within the scope of the appended claims.

Claims

1. A device for administering a drug into or through a biological tissue of a patient, wherein include: Piezoelectric pulse generator; as well as a microneedle electrode array electrically coupled to the piezoelectric pulse generator; Wherein, the piezoelectric pulse generator comprises: - piezoelectric crystals, - a mechanism configured to impinge upon a surface of the piezoelectric crystal, the mechanism being effective to generate an electrical pulse, and - electrical connections for conducting said electrical pulses to the microneedle electrodes; and Wherein, after the microneedle electrode is inserted into the biological tissue, the device is configured to generate one or more electric pulses and deliver the one or more electric pulses through the microneedle electrode, wherein the one or more electric pulses can effectively electroporate cells in the biological tissue and are configured to enable the delivery of drugs to the electroporated cells.

2. The device of claim 1, further comprising (i) a base from which the microneedle electrode array extends, and (ii) a housing connected to the base and containing the piezoelectric pulse generator.

3. The device according to claim 1, wherein the device further comprises the drug and is configured to release the drug into the biological tissue. The device according to claim 3 , wherein the drug is coated on the microneedle electrode.

5. The device according to claim 3, in: The drug is stored in one or more reservoirs in the device, At least a portion of the microneedle electrodes each comprises a hollow hole or a groove on its surface, and The device comprises one or more conduits in fluid communication with the one or more reservoirs and the hollow holes or grooves of the microneedle electrodes for delivering the drug. The device according to claim 1 , wherein the drug is in the form of particles containing the drug.

7. The device of claim 1, wherein the drug comprises a nucleic acid.

8. The device of claim 1, wherein the drug is a vaccine.

9. The device of claim 8, wherein the vaccine is an RNA vaccine or a DNA vaccine.

10. The device of claim 1, wherein the microneedle electrodes are made of stainless steel.

11. The device of claim 1, wherein the microneedle electrodes extend from one or more metal plates configured to conduct the electrical pulses from the piezoelectric pulse generator to the microneedle electrodes.

12. The device of claim 11, wherein the linear array of microneedle electrodes extends from one edge of each of the metal plates.

13. The device of claim 11, wherein the plates are parallel to each other and spaced apart from each other.

14. The device of claim 1, wherein the microneedle electrodes extend from a single metal plate and the array is a two-dimensional array.

15. The device of claim 1, wherein the microneedle electrodes extend from at least one non-conductive plate, and wherein electrical connectors are disposed between the microneedle electrodes and are configured to conduct the one or more electrical pulses from the piezoelectric pulse generator to the microneedle electrodes.

16. The device of claim 15, wherein the electrical connector is positioned on a surface of the at least one non-conductive plate.

17. The apparatus of claim 15, wherein the electrical connector spans from a first side of the at least one non-conductive plate through a hole in the at least one non-conductive plate to an opposing second side of the plate.

18. The device of claim 1, wherein the microneedle electrodes each comprise a non-conductive core and a conductive electrode material covering at least a portion of a surface of the microneedle core.

19. The device according to any one of claims 1 to 18, in, The mechanism includes a spring-lock hammer mechanism.

20. The device of claim 19, wherein the piezoelectric crystal comprises lead zirconate titanate, silicon nitride, barium titanate, quartz, zinc oxide, or sodium tungstate.

21. The apparatus of claim 19, further comprising a toggle switch having a wedge-shaped control latch configured to release a hammer driven by spring decompression.

22. The device of claim 21, further comprising a metal pin disposed between the hammer and the piezoelectric crystal.

23. The device of claim 19, further comprising a housing for the piezoelectric crystal, wherein the electrical connection consists of a lower electrode and side electrodes extending from the housing.

24. The device according to claim 23 further comprises a cartridge, wherein the cartridge contains the microneedle electrode array and comprises a first bracket for mating with the lower electrode and a second bracket for mating with the side electrode, the first bracket and the second bracket being electrically connected to the microneedle electrodes.

25. The device according to any one of claims 1 to 18, wherein the microneedle electrode array is arranged to be inserted into 1 mm 2 Up to 10cm 2 in the tissue area.

26. The device of any one of claims 1 to 18 having 2 to 1000 microneedle electrodes.

27. The device according to any one of claims 1 to 18, wherein the microneedle electrode has a length of 10 μm to 2 mm.

28. The device according to any one of claims 1 to 18, wherein the microneedle electrode array is configured to be replaceable and disposable, and the piezoelectric pulse generator is configured to be reusable with a series of the arrays.

29. The device of any one of claims 1 to 18, configured to generate one or more electrical pulses having a peak voltage absolute value between 10V and 10,000V.

30. The device of any one of claims 1 to 18, configured to generate one or more electrical pulses having a ratio between the absolute value of the peak voltage and the absolute value of the peak-to-peak voltage of between 0.1 and 10.

31. The device of any one of claims 1 to 18, configured to generate one or more electrical pulses having a peak current absolute value between 1A and 1,000A.

32. The apparatus of any one of claims 1 to 18, configured to generate one or more electrical pulses having a peak quiescent voltage absolute value of between 100V and 35,000V.

33. The apparatus of any one of claims 1 to 18, configured to produce a nominal electric field strength of between 100 V / cm and 30,000 V / cm.

34. The apparatus of any one of claims 1 to 18, configured to generate one or more electrical pulses having an initial pulse length of between 1 μs and 1,000 μs.

35. The device of any one of claims 1 to 18, configured to generate one or more electrical pulses having a ratio between an initial pulse length and a total pulse length of between 1.5 and 100.

36. The device of any one of claims 1 to 18, wherein the microneedle electrodes are spaced between 0.1 mm and 10 mm apart in the array.

37. The device according to claim 6, in, The drug-containing particles include drug-loaded lipid nanoparticles or drug-loaded polymer nanoparticles.

38. The device of any one of claims 1 to 18, configured to generate one or more electrical pulses having a peak voltage absolute value between 50V and 5,000V.

39. The device of any one of claims 1 to 18, configured to generate one or more electrical pulses having a peak voltage absolute value between 100V and 1,000V.

40. The device according to any one of claims 1 to 18, configured to generate one or more electrical pulses having a peak voltage absolute value of between 200V and 500V.

41. The device of any one of claims 1 to 18, configured to generate one or more electrical pulses having a ratio between the absolute value of the peak voltage and the absolute value of the peak-to-peak voltage of between 0.3 and 5.

42. The device of any one of claims 1 to 18, configured to generate one or more electrical pulses having a ratio between the absolute value of the peak voltage and the absolute value of the peak-to-peak voltage of between 0.5 and 2.

43. The device according to any one of claims 1 to 18, configured to generate one or more electrical pulses, the peak current absolute value of the electrical pulses being between 5A and 500A.

44. The device of any one of claims 1 to 18, configured to generate one or more electrical pulses having a peak current absolute value between 10A and 100A.

45. The device of any one of claims 1 to 18, configured to generate one or more electrical pulses having a peak current absolute value between 20A and 50A.

46. ​​The device of any one of claims 1 to 18, configured to generate one or more electrical pulses having a peak quiescent voltage absolute value between 1,000V and 30,000V.

47. The apparatus of any one of claims 1 to 18, configured to generate one or more electrical pulses having a peak quiescent voltage absolute value of between 15,000V and 27,500V.

48. The device of any one of claims 1 to 18, configured to produce a nominal electric field strength of between 200 V / cm and 10,000 V / cm.

49. The device of any one of claims 1 to 18, configured to produce a nominal electric field strength of between 300 V / cm and 5,000 V / cm.

50. The apparatus of any one of claims 1 to 18, configured to produce a nominal electric field strength of between 500 V / cm and 3,500 V / cm.

51. The apparatus of any one of claims 1 to 18, configured to generate one or more electrical pulses having an initial pulse length of between 3 μs and 100 μs.

52. The apparatus of any one of claims 1 to 18, configured to generate one or more electrical pulses having an initial pulse length of between 5 μs and 50 μs.

53. The apparatus of any one of claims 1 to 18, configured to generate one or more electrical pulses having an initial pulse length of between 10 and 30 μs.

54. The device of any one of claims 1 to 18, configured to generate one or more electrical pulses having a ratio between an initial pulse length and a total pulse length of between 2 and 50.

55. The device of any one of claims 1 to 18, configured to generate one or more electrical pulses having a ratio between an initial pulse length and a total pulse length of between 3 and 20.

56. The device of any one of claims 1 to 18, wherein the microneedle electrodes are spaced between 0.2 mm and 5 mm in the array.

57. The device of any one of claims 1 to 18, wherein the microneedle electrodes are spaced between 0.3 mm and 2 mm in the array.

58. The device of any one of claims 1 to 18, wherein the microneedle electrodes are spaced between 0.5 mm and 1.5 mm apart in the array.

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