Apparatus for improving transfection efficiency and / or protein expression and methods of use thereof

Abstract

A method and apparatus for improving transfection efficiency in eukaryotic cells is provided. The method includes providing a transfection mixture containing a reagent associated with at least one amphiphilic structure suitable for transfection. Adding the transfection mixture to one or more eukaryotic cells to form transfection complexes and allowing the transfection complexes to undergo a transfection process to form one or more transfected cells. The method also includes directing a pulsed electromagnetic signal, supplied at a given frequency, a given pulse rate, a given power, or any combination thereof, to the transfection mixture in step a) prior to generating the transfection complexes, to the transfection complexes in step b), to the transfection complexes in step c), and / or to the complexes of transfected cells after step c).

Description

[Technical Field]

[0001] The present invention relates to an apparatus and method for achieving improved transfection efficiency and / or protein expression. The apparatus can also be used to improve protein expression in cells. R .

[0002] Although the following description refers to how the device allowing for improved transfection of cells can be used for gene therapy purposes, those skilled in the art will understand that the present invention may be used for any purpose or application requiring transfection of cells, such as in the generation of viral vectors, gene therapy or modification, protein expression, autologous cell therapy, etc. It will also be understood that the device and method of the present invention may be performed on in vitro cells, ex vivo cells and / or in vivo cells. [Background technology]

[0003] Transfection is the process of introducing nucleic acids into eukaryotic cells. Transfection can be stable, in that the transfected nucleic acid continues to be expressed and is inherited by daughter cells. Alternatively, transfection can be transient, in that the transfected nucleic acid is expressed only for a short period of time after transfection and is not inherited by daughter cells. The use of both types of transfection is well known in the field of gene therapy [1], which focuses on the therapeutic delivery of nucleic acids to patient cells to act as therapeutic agents for disease. For example, the goal may be to replace a defective gene in a patient that, if untreated, would lead the patient to suffer from a genetically linked or inherited condition. In laboratory settings, exogenous genes, typically in the form of a plasmid, are often transfected into immortal cell lines. After transfection, successfully transfected cells will express the exogenous gene.

[0004] In transient transfection, an exogenous gene (typically encapsulated in a carrier such as polyethyleneimine (PEI)) is introduced into a population of cells. A portion of these cells will be successfully transfected and will begin to express the exogenous gene. After a short period of time, expression levels will decline, at which point the cells are typically processed or otherwise disposed of.

[0005] In stable transfection, cells are transfected as described above. A portion of the cells will stably incorporate the exogenous gene. Stably transfected cells can be isolated and selected from a cell population based on expression of the exogenous gene, and these cells can be expanded to create immortal cell lines that express the exogenous gene for longer periods of time.

[0006] Transfection efficiency (i.e., the rate at which exogenous genes are successfully transfected into cells) is typically low with prior art methods. Several strategies have been employed in an attempt to increase the transfection efficiency of cell lines (e.g., electroporation, specialized transfection reagents, etc.). What is needed are devices and methods that further enhance transfection protocols to increase the transfection efficiency of any given transfection protocol.

[0007] A recent development involves manipulating the genetic sequence of a patient's own immune cells to transform them into cells that will recognize and attack specific cancerous cells within the patient's body. [2] The process by which a patient's cells are genetically engineered is known as "gene therapy." One promising avenue of gene therapy for the treatment of cancer is to genetically engineer T cells to express chimeric antigen receptors, which allow them to more effectively target cancerous tissue growths. Such cells are known as chimeric antigen receptor T cells ("CAR-T" cells), and this therapy is known as "CAR-T cell therapy." Immune cells are first removed from the patient's body and then undergo an ex vivo transfection process, which converts them into cancer-seeking killer cells. The transfected cells are then readministered back into the patient to treat the cancer. Transfection of these cells is typically achieved using a technique that involves associating exogenous genetic material with a carrier molecule, such as a nanoparticle or liposome carrier.

[0008] One example of a conventional transfection process involves encapsulating target DNA in phospholipid bilayer vesicles, or liposomes, and then administering them to eukaryotic cells. [3] Because liposomes are made of phospholipids, they have an affinity for eukaryotic cell membranes, which also contain phospholipid bilayers, allowing for fusion between these systems. Therefore, external DNA can be transferred into eukaryotic cells via this fusion mechanism and become extrachromosomal genetic information for the cell. A simple conventional transfection process involves encapsulating exogenous nucleic acids (e.g., DNA plasmids carrying genes of interest) in cationic polymers (PEI). [6] While such processes have significant potential advantages, they are slow and have low transfection efficiency. These methods are wasteful, time-consuming, and expensive due to their low transfection efficiency. Summary of the Invention [Problem to be solved by the invention]

[0009] It is therefore an object of the present invention to provide an apparatus for improving transfection efficiency and / or protein expression in eukaryotic cells that overcomes the above-mentioned problems.

[0010] It is a further object of the present invention to provide methods for improving transfection efficiency and / or protein expression in eukaryotic cells.

[0011] It is a further object of the present invention to provide a transfection-enhancing device and / or method of use thereof.

[0012] It is yet a further object of the present invention to provide an apparatus for improving the effectiveness of gene therapy and / or therapeutic treatment in animals or humans.

[0013] It is yet a further object of the present invention to provide methods for improving the effectiveness of gene therapy and / or therapeutic treatment in animals or humans.

[0014] It is yet a further object of the present invention to provide an apparatus and / or method of use that improves viral vector production.

[0015] It is yet a further object of the present invention to provide an apparatus and / or method of use for enhancing protein expression in human and / or animal cells.

[0016] A further object of the present invention is to allow for an increase in the speed of preparation and / or application of transfection agents, and a still further object is to allow for an increase in the yield of transfected cells.

[0017] It is a still further object of the present invention to provide devices and / or methods of use that allow for the delivery of reagents, drugs and / or therapeutic treatments through a patient's skin in a more targeted and efficient manner at a lower cost.

[0018] It is a further object of the present invention to provide a device and / or method of use that allows for a device that can be used to provide delivery of reagents, drugs and / or therapeutic treatments to a patient that is portable and / or easy to use in the patient's home. [Means for solving the problem]

[0019] According to a first aspect of the present invention there is provided a method of improving transfection efficiency in a eukaryotic cell, the method comprising: a) providing a transfection mixture comprising a reagent associated with at least one amphiphilic structure suitable for transfection; b) introducing the transfection mixture into one or more eukaryotic cells to form transfection complexes; c) allowing the transfection complex to undergo a transfection process to form one or more transfected cells; Including, The method comprises the step of directing a pulsed electromagnetic signal supplied at any one or any combination of a given frequency, a given pulse rate, and a given power to the transfection mixture in step a) prior to producing the transfection complexes, to the transfection complexes in step b), to the transfection complexes in step c), and / or to the complexes of transfected cells after transfection step c).

[0020] Applicants have surprisingly discovered that administering a pulsed electromagnetic (PEM) signal before, during, and / or after transfection significantly increases the transfection efficiency and / or protein expression yield produced by the transfection process. Transfection rates are significantly improved, allowing for an increased frequency of transfected cells harboring reagents and / or exogenous nucleic acids. Thus, the present invention provides a non-invasive, non-chemical approach to improving cell viability, gene transfer, transfection rates, and / or protein production. The present invention enhances the transport of extracellular materials or reagents from an environment external to the cell to the internal environment inside the cell.

[0021] As used herein, the term "pulsed electromagnetic signal" is preferably defined as a sequence or pattern of signals in the electromagnetic spectrum that change in amplitude from a baseline to a higher or lower value, followed by a return to or substantially return to the baseline. More preferably, the signal amplitude changes are fast, transient, and appear as a repetitive sequence. In one example, the baseline corresponds to a state in which there is no electromagnetic signal emitted by the electromagnetic signal source or transmission means. Preferably, the baseline is considered a rest or relaxation period for the cells and / or the pulsed electromagnetic signal.

[0022] Preferably, the method may be performed entirely in vitro, entirely in vivo, or partially in vitro and partially in vivo. For example, eukaryotic cells may be transfected in vitro and used in vitro for one or more purposes or applications. In a further example, eukaryotic cells may be extracted from a patient, transfected in vitro, and then reintroduced back into the patient (this is interchangeably referred to as an "ex vivo" method). ) Alternatively, the transfection complex may be injected or otherwise delivered to the patient, and the patient's cells may be transfected in vivo.

[0023] Preferably, the reagents in the transfection mixture are any reagents and / or nucleic acids suitable for transfection, pharmaceutical and / or therapeutic reagents or compounds, reagents of therapeutic and / or pharmaceutical benefit, small molecules or small molecular materials of less than 5 kilodaltons, large molecules or large molecular materials of about 5 kilodaltons or more, one or more proteins, vaccines, one or more antibodies, organic agents, and the like, or any combination thereof.

[0024] The term "pharmaceutical and / or therapeutic reagent or compound" preferably refers to a compound that has been deployed or is under development for deployment in the clinic and has proven medicinal value.

[0025] The term "agents of therapeutic and / or pharmaceutical interest" preferably refers to compounds that have been developed for and / or are under investigation for use in research and / or clinical applications. These agents or compounds may have known mechanisms of action, but may not be demonstrated or investigated for clinical suitability and relevance. In some embodiments, the mechanisms of action of these agents or compounds may not yet be clear. Nevertheless, the mechanisms underlying the present invention allow for superior intracellular delivery of these agents or compounds.

[0026] In one aspect, a method of intracellular delivery is provided, said method comprising: a) providing a first mixture comprising a reagent associated with at least one amphiphilic structure suitable for intracellular delivery; b) introducing the first mixture into one or more eukaryotic cells to form a second mixture comprising the first mixture and the one or more eukaryotic cells; c) allowing said second mixture to undergo a process by which said reagent associated with at least one amphiphilic structure is delivered to said one or more eukaryotic cells; Including, The method includes providing a pulsed electromagnetic signal at either or any combination of a given frequency, a given pulse rate, or a given power; to the first mixture in step a) prior to producing the second mixture; to the second mixture in step b); to the second mixture in step c); and / or the one or more eukaryotic cells after step c) The method further comprises a step of directing.

[0027] In some embodiments, the reagent is or comprises a nucleic acid, and transfection refers to a process by which a nucleic acid is introduced into one or more eukaryotic cells, thereby causing expression of the exogenous nucleic acid (e.g., exogenous RNA, DNA, RNA / DNA hybrid, or a gene encoded by the nucleic acid, or a protein expressed therefrom) by the cell or cells. In other embodiments, when the reagent does not comprise a nucleic acid, the term transfection is used synonymously with the term "intracellular delivery," which in this context means that the reagent is delivered across the cell membrane of one or more eukaryotic cells into the cytoplasm. In some embodiments, the method can therefore be considered a transmembrane delivery method, particularly when the reagent does not comprise a nucleic acid.

[0028] In any aspect or embodiment described herein, unless otherwise clear, the method may be considered to be a transfection, intracellular delivery or transmembrane delivery method.

[0029] In any aspect or embodiment described herein, unless otherwise clear, the transfection mixture may be described as a first mixture and / or the transfection complex may be described as a second mixture. Delivery to a cell may be considered to be transmembrane, intracellular, or cytoplasmic delivery.

[0030] Preferably, the reagent is associated with at least the amphiphilic structure, such as by being contained within the amphiphilic structure, by being complexed with the amphiphilic structure, by being contained on the amphiphilic structure, by being bound to the amphiphilic structure, etc.

[0031] In one embodiment, the eukaryotic cells may include any one or any combination of adherent cells, suspension cells, blood cells, lymphocytes, granulocytes, T cells, and the like.

[0032] In some embodiments, the eukaryotic cells are suspended in solution, attached to a substrate, or a mixture of both suspended and attached cells.

[0033] In some embodiments, the eukaryotic cell is an immortal cell or a cell obtained from an immortal cell line, such as a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, a human colon tumor (HCT) 116 cell, or a Jurkat E6 cell.

[0034] In some embodiments, the eukaryotic cells are cells found in or obtained from the tissue of a human or animal subject. For example, the cells may be extracted from the subject, transfected, and then reintroduced into the subject. In some embodiments, the eukaryotic cells are obtained from the subject's blood. In some embodiments, the eukaryotic cells are T cells, lymphocytes, granulocytes, macrophages, and / or other white blood cells. In some embodiments, the T cells are either helper T cells or cytotoxic T cells, or any combination thereof. In some embodiments, the T cells include CD4+ cytotoxic T lymphocytes and / or CD8+ cytotoxic T lymphocytes.

[0035] One exemplary use of the devices and methods of the present invention is in adoptive T cell therapy (ACT), which involves the generation of so-called "CAR-T" cells. In such techniques, the devices and / or methods are used on T cells obtained from a subject. These cells are cultured and transfected in vitro to express a chimeric antigen receptor, then expanded in vitro before being reintroduced into the patient. The devices and / or methods improve transfection efficiency, which in turn results in a higher yield of CAR-T cells.

[0036] In some embodiments, the method may not be a procedure or surgical method performed on the human or animal body, hi some embodiments, the method may not be a method for modifying the genetic identity of a human germline.

[0037] Preferably, the method comprises the step of mixing the nucleic acid or reagent with at least one amphiphilic structure to form a transfection mixture, wherein the nucleic acid or reagent associates with the amphiphilic structure to form the transfection mixture.

[0038] In one embodiment, the at least one amphiphilic structure may comprise or consist of any or any combination of at least one liposomal substance or vehicle, at least one PEGylated liposomal substance or vehicle, a micelle, a structure having a phospholipid bilayer, a cationic polymer, polyethyleneimine (PEI), and the like.

[0039] For example, the cationic polymer can be Turbofect™.

[0040] Preferably, the nucleic acid is deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or includes a combination of DNA and RNA (e.g., a DNA / RNA hybrid oligonucleotide). When the nucleic acid is RNA, it can be mRNA, tRNA, siRNA, miRNA, etc.

[0041] In one embodiment, the nucleic acid is or comprises one or more expression vectors. For example, the one or more expression vectors may be one or more DNA plasmids containing one or more exogenous genes intended for expression in one or more eukaryotic cells.

[0042] In one embodiment, when the reagent is a nucleic acid, the transfection process results in stable expression, in that the transfected nucleic acid continues to be expressed in the transfected cell and is passed on to daughter cells.

[0043] In one embodiment, when the reagent is a nucleic acid, the transfection process results in transient expression, where the transfected nucleic acid is expressed for only a relatively short period of time and is not inherited by daughter cells.

[0044] Preferably, the step of directing a pulsed electromagnetic signal is performed at room temperature (e.g., 20°C), or in an incubator that can be set to a temperature above room temperature or at the patient's body temperature (e.g., 37°C).

[0045] In one embodiment, the step of directing the pulsed electromagnetic signal is carried out for a given period of time. In one example, the time during which the cells receive the pulsed electromagnetic signal when directed to the transfection mixture in step a) prior to producing the transfection complexes is about 15 minutes or up to 15 minutes. However, it will be understood that longer or shorter periods may be used as needed.

[0046] In one embodiment, the period of time during which the cells receive the pulsed electromagnetic signal is about 1-4 hours or thereabouts, more preferably about 3-4 hours, when directed to the transfection complex to form the transfected cell in step c) and / or after the transfection step. However, it will be understood that longer or shorter periods may be used as needed. For example, in one embodiment, the period of time may be up to 16 hours, or up to 24 hours.

[0047] In one embodiment, the transfection is reverse transfection (ie, a eukaryotic cell is introduced into the transfection mixture).

[0048] In one embodiment, the transfection is a forward transfection (ie, the transfection mixture is introduced into a eukaryotic cell).

[0049] Preferably, the pulsed electromagnetic signal is generated by one or more electronic devices.

[0050] Preferably, the one or more electronic devices include transmission means for generating and / or transmitting a pulsed electromagnetic signal in use.

[0051] Preferably, the transmission means comprises one or more electronic transmission chips arranged to generate, emit and / or transmit one or more pulsed electromagnetic signals in use.

[0052] In one embodiment, reference to a transmitting means or one or more electronic transmitting chips may include one or more transmitters, one or more transmitters and at least one receiver, or one or more transceivers. Thus, in one example, a pulsed electromagnetic signal may be transmitted from a central location or master transmitter and received by and subsequently retransmitted or emitted from one or more remote and / or slave receivers and / or transceivers.

[0053] In one embodiment, the electronic device includes a single transmitting means or electronic transmitting chip. Such a single transmitting means or electronic transmitting chip is sufficient to provide a pulsed electromagnetic signal, in one example, to a tissue culture plate. In one exemplary embodiment, the single transmitting means or electronic transmitting chip is provided, attached to, or incorporated into a bioreactor containing one or more suspension cells. Such bioreactors operate by stirring the suspension, so that the suspended cells, typically in a medium, pass by the transmitting means or electronic transmitting chip and are thus exposed to the pulsed electromagnetic signal of the present invention.

[0054] In one embodiment, the electronic device has two or more transmission means or electronic transmission chips, which are preferably arranged at a given spatial distance from each other in the electronic device.

[0055] Preferably, the given spatial separation is such that it provides one or more items or substances pulsed with the electromagnetic pulsing signal with sufficient signal strength to achieve the desired effect (i.e., the effect of increasing transfection efficiency) and / or to provide an even or substantially even distribution of electromagnetic radiation / signal during use.

[0056] Preferably, the electronic device comprises a plurality of transmission means or electronic transmission chips arranged in a given pattern and / or array.

[0057] While a single transmission means or electronic transmission tip is sufficient to provide the advantageous properties of the present invention, it has been found that having multiple transmission means or electronic transmission tips allows for delivery of the pulsed electromagnetic signal over a wider surface area while still maintaining maximum effectiveness. 2 It has been found that having the transmission means or electronic transmission chips evenly distributed, with at least one chip per chip, provides sufficient coverage for optimum effect.

[0058] In some embodiments, the device includes one or more transmission means or electronic transmission chips. In some embodiments, the device includes 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more transmission means or electronic transmission chips.

[0059] In some embodiments, about 105-115 cm of the surface of the housing of the device or the surface of an article as defined herein. 2 and preferably within about 110 cm of the surface of the housing of the device or the surface of the article as defined herein. 2 There is one transmission means or electronic transmission chip per device.

[0060] In some embodiments, about 50-60 cm of the surface of the housing of the device or the surface of an article as defined herein. 2 and preferably within about 55 cm of the surface of the housing of the device or the surface of the article as defined herein. 2 There is one transmission means or electronic transmission chip per device.

[0061] In some embodiments, about 25-30 cm of the surface of the housing of the device or the surface of an article as defined herein. 2 and preferably within about 27.5 cm of the surface of the housing of the device or the surface of the article as defined herein. 2 There is one transmission means or electronic transmission chip per device.

[0062] In some embodiments, about 15-20 cm of the surface of the housing of the device or the surface of an article as defined herein. 2 and preferably within about 18.5 cm of the surface of the housing of the device or the surface of the article as defined herein. 2 There is one transmission means or electronic transmission chip per device.

[0063] In some embodiments, about 10-15 cm of the surface of the housing of the device or the surface of an article as defined herein. 2and preferably within about 12.2 cm of the surface of the housing of the device or the surface of the article as defined herein. 2 There is one transmission means or electronic transmission chip per device.

[0064] Articles as defined herein preferably include cell culture plates, flasks, roller bottles, and other containers known to those skilled in the art, such as standard laboratory microplates, T25, T75, T125, T175, T225, and larger cell culture plates as defined below. One or more transmitting means or electronic transmitting chips are set apart at a given distance depending on the surface area of ​​such containers placed on the instrument in use and / or based on the surface of the device housing.

[0065] In an exemplary embodiment, the device is provided with six transmission means or electronic transmission chips, and standard laboratory microplates are placed on top of the device. These standard laboratory microplates come in 6-well, 12-well, 24-well, 48-well, 96-well, 384-well, and 1536-well plates (and more). These microplates are generally standardized sizes, measuring approximately 128 mm long by 85 mm wide, so the plate is approximately 110 cm long. 2 1000 Hz to provide a surface area of ​​1000 sq m. Thus, in an exemplary embodiment, six transmitting means or electronic transmitting chips may be evenly spaced to provide a given spacing that is optimal for providing a pulsed electromagnetic signal according to the present invention to any of these plate types. In one example, the electronic device includes six transmitting means or electronic transmitting chips. Preferably, the six transmitting means or electronic transmitting chips are disposed a given distance apart such that when a 24-well plate is positioned on, above, or against the electronic device in use, each transmitting means or chip is capable of emitting and / or directing an electromagnetic signal of sufficient strength to four wells of the plate.

[0066] More preferably, the transmitting means or transmitting chip is located adjacent to the center or substantially the center of four wells of a 24-well plate.

[0067] In one embodiment, when more than one transmission means or electronic transmission chip is required, the spacing between the multiple transmission means or electronic transmission chips must be optimized. To achieve the optimal spacing between each transmission means or electronic transmission chip, the transmission means or electronic transmission chips must be located at a distance equal to or substantially equal to half the wavelength of the electromagnetic radiation frequency being used. Preferably, this distance must be considered relative to any plane orientation, or when two or more transmission means or electronic transmission chips are used together as part of a device. For example, if the wavelength is 12.4 cm, the transmission chips must be spaced approximately 6.2 cm apart to produce an optimal electromagnetic field when in use.

[0068] In one example, given spatial distance = wavelength / 2.

[0069] In one example, a given spatial distance in the X and / or Y axis is half a wavelength between each transmission means or electronic transmission chip in an evenly spaced grid, such an arrangement minimizing the risk of destructive interference.

[0070] In one embodiment, the electronic device includes a housing, and one or more transmission means or transmission chips are located in the housing.

[0071] Preferably, the housing includes at least one flat or planar surface that allows the housing to be positioned stably relative to another item that receives the pulsed electromagnetic signal in use. Alternatively, the housing may include one or more curved or non-planar surfaces that allow the housing to be positioned stably relative to one or more items that receive the pulsed electromagnetic signal in use.

[0072] In one example, at least one surface of the housing includes one or more recesses for positioning one or more items that receive the pulsed electromagnetic signal during use.

[0073] In one example, the electronic device is referred to as a transfection plate for laboratory use.

[0074] In one embodiment, the housing includes a base surface that allows the housing to be supported directly or indirectly on a surface during use. More preferably, the housing includes a top surface opposite the base surface. Preferably, the top surface is a surface upon which one or more items that receive the pulsed electromagnetic signal may be placed during use.

[0075] In one example, one or more of the articles may be cell culture plates or flasks known to those skilled in the art in which eukaryotic cells may be cultured.

[0076] In one embodiment, the electronics and / or housing are attachable to the exterior of a vessel, reaction vessel, etc. For example, the electronics and / or housing may be attachable by one or more attachment means or devices including any or any combination of one or more screws, nuts and bolts, magnets, strings, clips, straps, interengaging members, adhesives (stickers), welding, etc.

[0077] Preferably, the distance between the transmission means and the top surface of the housing and / or one or more items receiving the pulsed electromagnetic signal when positioned on, at or against the housing or electronic device in use is about 25 cm or less, 20 cm or less, 15 cm or less, 10 cm or less, or 5 cm or less. More preferably, the distance is about 1 cm.

[0078] Preferably, the pulsed electromagnetic signal is provided in a given pulse sequence.

[0079] In one embodiment, the electronics is arranged to transmit a pulsed electromagnetic signal at a frequency in the range of about 2.2-2.6 GHz, and more preferably, the pulsed electromagnetic signal is transmitted at a frequency of about 2.4 GHz ± 50 MHz or more preferably 2.45 GHz ± 50 MHz.

[0080] In one embodiment, the electronics is arranged to transmit pulsed electromagnetic signals at frequencies within the Industrial, Scientific and Medical Radio Frequency Bandwidth (ISM band) of 2.4 to 2.4835 GHz, preferably 2.45 GHz ±50 MHz.

[0081] Preferably, the pulsed electromagnetic signal is pulsed at a frequency of about 50 Hz or less, more preferably about 25 Hz or less, and even more preferably about 15 Hz or less.

[0082] Preferably, each pulse of the pulsed electromagnetic signal lasts between about 1 ms and 20 ms, and more preferably, each pulse lasts about 1 ms.

[0083] Preferably, the time period between pulses (also called the "rest period" or "relaxation period") is about 66 ms or less.

[0084] Preferably, the pulsed electromagnetic signal has a duty cycle of less than 2%.

[0085] In one embodiment, the transmission power provided by each transmission means or chip of the electronic device is 2 dBm to +4 dBm, about 1 mW, about 2 mW or about 2.5119 mW.

[0086] In one embodiment, the given frequency of the pulsed electromagnetic signal is about 2.2 to 2.6 GHz, 2.4 GHz ± 50 MHz, or 2.45 GHz ± 50 MHz, the given pulse rate is about 15 Hz or has a duty cycle of less than 2%, and the given power is +2 dBm to +4 dBm, 1 mW, 2 mW, or 2.5119 mW.

[0087] Without wishing to be bound by theory, it is believed that the use of electromagnetic waves or signals employed in the device or method of the present invention is sufficient to cause HO to periodically rotate around its dipole with relatively long rest or relaxation periods. The periodic rotation of HO is believed to disrupt hydrogen bonds in the phospholipid bilayer and / or amphiphilic structures. This periodic or intermittent low-energy perturbation of the cell membrane is therefore believed to stimulate increased interactions between certain molecules, the cell membrane and / or amphiphilic structures, and their environment, such as nucleic acids or reagents associated with the amphiphilic structures. This is believed to enhance the transport of reagents across the cell membrane, leading to increased uptake of one or more reagents, such as nucleic acids, peptides, small molecules, and other reagents, by one or more eukaryotic cells. It can thus be seen that the transfection and / or intracellular delivery process of the present invention can be significantly improved using extremely low-energy electromagnetic waves or signals. The relatively long rest or relaxation periods between pulses of the pulsed electromagnetic signal are believed to be sufficient to maintain cellular integrity. Thus, in the context of the present invention, the use of pulsed electromagnetic signals, waves or fields is believed to provide improved transport of molecules across cell membranes, leading to more efficient transfection and / or intracellular delivery of reagents as defined above.

[0088] Preferably, the pulsed electromagnetic signal is transmitted using Gaussian frequency shift keying (GFSK) between 0.45 and 0.55. Preferably, the pulsed electromagnetic signal is a radio frequency (RF) data signal. Preferably, the pulsed electromagnetic signal is a digital sequence of pulsed electromagnetic signals.

[0089] Preferably, the radio frequency signals utilize the advertising feature of the Bluetooth LE (BLE) protocol. Preferably, the advertising RF signals are on channels 37, 38, and 39, corresponding to frequencies 2402 MHz, 2426 MHz, and 2480 MHz, respectively.

[0090] Preferably, the pulsed electromagnetic signal is directed at an aqueous medium consisting of or containing the transfection mixture, transfection complexes and / or post-transfection complexes.

[0091] In one embodiment, the electronic device includes a power supply means for powering the device in use. Preferably, the power supply means includes a mains power supply, one or more batteries, power cells, one or more rechargeable batteries, generator means, or the like.

[0092] In one embodiment the electronic device includes control means for controlling the operation of the electronic device and / or the transmission means in use.

[0093] In one embodiment, the electronic device comprises one or more circuit boards, preferably in the form of integrated circuits, on which transmission means may be provided and / or on which other components, such as storage means, are located.

[0094] In one embodiment, the electronic device includes a storage means such as a memory device, a data storage device, or the like.

[0095] Preferably, other components of the electronic device include one or more components necessary for the selective operation of the device to generate a pulsed electromagnetic signal and for its controlled operation when activated. For example, user selection means may be provided on the device to allow user selection of one or more conditions, operations and / or one or more parameters of the device during use; display means may be provided on the device to display one or more settings, selection options, etc.

[0096] In one embodiment, the further component or power supply means comprises one or more power cells, which may all be contained within the housing.

[0097] In one embodiment, the housing of the electronic device is provided in a configuration that allows it to engage with and / or be positioned based on a container in which one or more materials and / or items that will be exposed to electromagnetic signals during use are located.

[0098] In one embodiment, the control means includes an option that allows a user to select any or any combination of the signal frequency, signal strength, signal power, signal pulse rate, signal pulsing time duration, etc. of said pulsed electromagnetic signal. In one embodiment, the selection of frequency, strength, power, pulse rate, pulsing time duration, etc. may be based on the particular form of the material and / or one or more articles that will be exposed to the pulsed electromagnetic signal in use, the quantity of said material, the dimensions of a container within which the device will be placed in use, and / or other parameters.

[0099] Cells exposed to pulsed signals such as those of the present invention have been found to provide a uniform or substantially uniform distribution or dispersion of cells upon in vitro transfection, in contrast to transfections in which non-pulsed techniques are used and cell clumping has been observed.

[0100] According to one aspect of the present invention there is provided an apparatus for improving transfection efficiency in eukaryotic cells, said apparatus comprising a housing, transmitting means located in said housing and arranged in use to transmit a pulsed electromagnetic signal supplied at any or any combination of a given frequency, a given pulse rate or a given power, control means for controlling operation of at least said transmitting means in use, and power supply means for supplying power to said transmitting means and / or control means in use.

[0101] Preferably, one or more given parameters of the device may be pre-set by the device manufacturer and / or may be user-selectable depending on the user's requirements.

[0102] Preferably, the control means is used to allow user selection of one or more of the given user selectable parameters.

[0103] In one embodiment, the device is arranged to be worn directly or indirectly on or adjacent to a person's skin to allow for the directing of pulsed electromagnetic signals to an area of ​​the body in use to enhance the transfection process in the body, in this embodiment the device is preferably a wearable device.

[0104] In one embodiment, to enhance the transfection process taking place in the body, attachment means may be provided on and / or associated with the device to allow for removably attachment to or to the skin or exterior of the user's body, the interior and / or exterior of clothing or articles worn by the user in use, etc.

[0105] In an exemplary embodiment, the device is a wearable device, such as an armband, that is placed directly over the injection site of, for example, a DNA or RNA vaccine to be administered to the patient.

[0106] In an exemplary embodiment, there is a method of administering a vaccine, comprising the steps of injecting a vaccine into a subject, and then positioning a device of the present invention at the injection site, and providing a pulsed electromagnetic signal of the present invention to the injection site.

[0107] Preferably, the device and / or transmission means or one or more electronic transmission chips are arranged on the device such that, in use, the pulsed electromagnetic signal is directed towards the skin or body of a user. For example, the pulsed electromagnetic signal may be directed through a first surface of the housing, said first surface being arranged to be in direct or indirect contact with the skin of a user.

[0108] In one embodiment, the device is arranged to be implantable in the body. For example, the device may be implanted in a region of the body requiring treatment. In this embodiment, the device is preferably an implant.

[0109] Preferably, at least the exterior of the device is coated with and / or formed of a material suitable for implantation in the body.

[0110] Preferably, the attachment means comprises any one or combination of one or more straps, strings, necklaces, pendants, belts, bracelets, clips, key rings, lanyards, VELCRO® (hook and loop fasteners), hooks, buttons, buttonholes, adhesives, bandages, sutures, clips, biocompatible adhesives, and the like.

[0111] In one embodiment, the device is provided with at least one holding means or reservoir for holding or containing, respectively, the transfection mixture that will transfect a person in use.

[0112] Preferably, the holding means or reservoir is disposed on the device such that, in use, it can be positioned on and / or adjacent to a person's skin. Directing a pulsed electromagnetic signal to one or more parts of the body can assist in enhancing absorption and / or transfection of the reagent through the person's skin and into one or more cells of the person.

[0113] In one embodiment, directing a pulsed electromagnetic signal to a user's skin is believed to improve the permeability of the user's skin, allowing for increased and / or enhanced uptake of the transfection mixture during use. Typically, the improvement in skin permeability occurs over at least the period of time that the pulsed electromagnetic signal is directed to the user's skin. Typically, the improvement in the permeability of the user's skin continues after transmission of the pulsed electromagnetic signal has ceased, but diminishes over time.

[0114] In one embodiment, the strength and range of the pulsed electromagnetic signal is sufficient such that when the housing of the electronic device is positioned relative to a portion of a user's skin, the pulsed electromagnetic signal passes through the skin and enters the user's body, preferably at least adjacent to an internal area directly adjacent to the portion of the user's skin.

[0115] According to one aspect of the present invention, there is provided a method for increasing transfection efficiency in eukaryotic cells and / or an apparatus for increasing transfection efficiency in eukaryotic cells.

[0116] According to a further aspect of the present invention there is provided a method for increasing protein expression in a transfected eukaryotic cell and / or an apparatus for increasing protein expression in a transfected eukaryotic cell.

[0117] According to one aspect of the present invention, there is provided a method of providing gene therapy in vivo, the method comprising: a) providing a transfection mixture comprising a reagent associated with at least one amphiphilic structure suitable for transfection; b) introducing or injecting the transfection mixture into the patient to allow transfection of one or more cells of the patient with the transfection mixture in vivo; Including, The method comprises the step of directing a supplied pulsed electromagnetic signal at any one or any combination of a given frequency, a given pulse rate, and a given power to a transfection reagent in step a) before directing or injecting the transfection mixture, to a patient during directing or injecting the transfection mixture to a patient in step b), and / or to a patient after transfection step b).

[0118] Preferably, the method of introducing the transfection mixture into the patient includes orally, transdermally, subcutaneously, and the like.

[0119] According to one aspect of the present invention, there is provided a method of providing gene therapy in vitro, the method comprising: a) providing a transfection mixture comprising a reagent associated with at least one amphiphilic structure suitable for transfection; b) adding said transfection mixture to one or more eukaryotic cells obtained from the patient prior to said method to form said transfection complex; c) transfecting the transfection complex to form one or more transfected cells; Including, The method is characterized in that it comprises the step of directing a pulsed electromagnetic signal supplied at any one or any combination of a given frequency, a given pulse rate, and a given power to the transfection mixture in step a) prior to producing the transfection complexes, to the transfection complexes in step b), to the transfection complexes in step c), and / or to the transfected cell complexes after transfection step c).

[0120] According to a further aspect of the present invention there is provided a method for improving transfection efficiency in a eukaryotic cell, the method comprising: a) providing a transfection mixture comprising nucleic acid associated with at least one amphiphilic structure suitable for transfection; b) adding the transfection mixture to one or more eukaryotic cells to form transfection complexes; c) allowing the transfection complex to undergo a transfection process to form one or more transfected cells; Including, The method includes directing a pulsed electromagnetic signal supplied at any or any combination of a given frequency, a given pulse rate, and a given power to the transfection mixture in step a) prior to producing the transfection complexes, to the transfection complexes in step b), to the transfection complexes in step c), and / or to the complexes of transfected cells after transfection step c).

[0121] After the patient's cells have been transfected by this method, the cells can then optionally be reintroduced back into the patient or another patient as needed.

[0122] According to one aspect of the present invention there is provided an apparatus for assisting in the delivery of gene therapy in eukaryotic cells, said apparatus comprising a housing; transmission means located in said housing and arranged in use to transmit a pulsed electromagnetic signal supplied at any or any combination of a given frequency, a given pulse rate or a given power; control means for controlling the operation of at least said transmission means in use; and power supply means for supplying power to said transmission means and / or control means in use.

[0123] According to a further aspect of the present invention there is provided a method of altering gene expression and / or protein expression comprising the steps of: - providing one or more eukaryotic cells; The method comprises the step of directing a pulsed electromagnetic signal, provided at any or any combination of a given frequency, a given pulse rate, and a given power, to one or more eukaryotic cells to alter the gene expression and / or protein expression in the eukaryotic cells.

[0124] In one embodiment, the method kills cancer cells and increases DNA repair in healthy cells and tissues.

[0125] In one embodiment, the device is implantable in a patient, for example, in an area at or adjacent to cancerous tissue, to treat the cancerous tissue. This method can be useful when the cancerous tissue is further away from the patient's skin.

[0126] In one embodiment, the device is worn by a patient on or adjacent to the patient's skin and may be used to deliver one or more pharmaceutical reagents or agents to cancerous tissue, such as, for example, located near a subcutaneous tumor, such as a melanoma, and / or to treat a virus.

[0127] Thus, in one embodiment, the device can be used to deliver pulsed electromagnetic signals through a patient's skin to promote apoptosis of cancerous cells by directly interacting with the cells' DNA, and / or to help produce healthy cells so that DNA damage is repaired.

[0128] In one embodiment, the device is used to deliver a pulsed electromagnetic signal through the patient's skin to provide an antiviral effect.

[0129] In one aspect of the invention there is provided a cell or progeny thereof produced using any one of the methods defined herein.

[0130] It should be noted that when referring to improved transfection efficiency herein, it refers to an increase in the number of transfected cells and an increase or maintenance of cell viability after the transfection process.

[0131] It will be appreciated that the present invention can be used in a laboratory-based environment or can be scaled up for use in an industrial-level environment.

[0132] Specific embodiments of the present invention will be described below with reference to the accompanying drawings. [Brief explanation of the drawings]

[0133] [Figure 1a] 1 illustrates an apparatus according to one embodiment of the present invention, where the electronics includes one transmitter chip. [Figure 1b] 1 illustrates an apparatus according to one embodiment of the present invention, where the electronics includes one transmitter chip. [Figure 2] 1a and 1b in use for carrying out a method according to the present invention in one embodiment. [Figure 3] An apparatus in one embodiment of the present invention is shown in conjunction with an example of a 24-well plate that may be used with the electronics, where the electronics includes an array of six transmitter chips. [Figure 4a] We show the results of transfection of adherent CHO K1 cells using DNA plasmids associated with Turbofect amphiphilic constructs, where a pulsing technique involving a single electronic transmitter chip was used, according to one embodiment of the present invention. [Figure 4b] We show the results of transfection of adherent CHO K1 cells using a DNA plasmid associated with Turbofect amphiphilic constructs, where a pulsing technique involving six electronic transmitter chips was used, according to one embodiment of the present invention. [Figure 4c] The results of transfection of adherent HCT 116 cells using DNA plasmids associated with Turbofect amphiphilic constructs are shown, where a pulsing technique was used, according to one embodiment of the present invention. [Figure 5a] 1 shows the results of transfection in HCT 116 cells with a DNA plasmid containing the IGFBP3 promoter and associated with a PEI amphiphilic structure using the pulsing technique of the present invention. [Figure 5b] 1 shows the results of transfection of HCT 116 cells with a DNA plasmid containing an SV40 promoter and associated with a PEI amphiphilic structure using the pulsing technique of the present invention. [Figure 6]1 is a graph showing the results of transfection in suspension HEK 293 Freestyle cells using a green fluorescent protein (GFP)-containing plasmid associated with PEI amphiphilic structures using the pulsing technique of the present invention. [Figure 7] 10 is a graph showing further results of transfection in suspension HEK 293 Freestyle cells using a green fluorescent protein (GFP)-containing plasmid associated with PEI amphiphilic structures using the pulsing technique of the present invention. [Figure 8] 1 is a graph showing the results of transfection in suspension Jurkat E6 cells with DNA plasmids associated with TransIT2020 amphiphilic constructs using the pulsing technique of the present invention. [Figure 9a] 1 shows a diagram of an apparatus according to an embodiment of the present invention; [Figure 9b] 1 shows a diagram of an apparatus according to an embodiment of the present invention; [Figure 10a] 1 shows a diagram of an apparatus according to a further embodiment of the present invention; [Figure 10b] 1 shows a diagram of an apparatus according to a further embodiment of the present invention; [Figure 11] 2 illustrates a further embodiment of the present invention. [Figure 12a] 1 shows an elevation view of a still further embodiment of the present invention; [Figure 12b] 1 shows an elevation view of a still further embodiment of the present invention; [Figure 13] 1 shows Western blots from experiments in applicant's co-pending patent application that provide support for the claims of the present invention. [Figure 14] 1 shows additional Western blots from experiments in applicant's co-pending patent application that provide support for the claims of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0134] Referring to Figures 1a, 1b and 2, there is shown, in one embodiment, an apparatus 2 for carrying out the method of the present invention for improving transfection efficiency in eukaryotic cells.

[0135] The device 2 is in the form of an electronic device capable of emitting a pulsed electromagnetic signal at a given frequency, at a given pulse rate, at a given power level, and for a given period of time. The given parameters may be pre-set by the manufacturer or may be user-selectable as required. The technique used in this device is hereinafter referred to as the "pulsing technique of the present invention."

[0136] Apparatus 2 includes a housing 4. In this particular example, housing 4 is in the form of a laboratory transfection plate and includes a base surface 5, a top surface 7 opposite base surface 5, and one or more side walls 9 located between top surface 5 and base surface 7.

[0137] Within the interior confines of the housing 4 is provided a circuit board 6 having integrated circuits 8 arranged and interconnected thereon so as to generate a pulsed electromagnetic signal in operation. Control means in the form of a control unit 10 is provided to allow selective operation of the device 2. A memory device 12 is provided for storing data, one or more operating parameters, software etc. to allow retrieval when required. The control unit preferably includes microprocessing means to allow processing of data etc.

[0138] The device 2 also typically includes one or more power cells 14 that power the device. Optionally, a rechargeable facility can also be provided to allow the power cells 14 to be charged from a remote power source rather than having to be replaced.

[0139] It will be understood that the housing 4 may be provided in any form suitable for its intended use, and may, for example, be provided with engagement means that allow it to be positioned with the interior or exterior of a container in which the cells to be processed are located. Alternatively, the housing may be formed as part of the container in which the cells to be processed are located. Furthermore, the upper surface 7 may provide a planar or flat surface on which the container in which the cells are to be processed or located can be placed. Furthermore, the upper surface 7 of the housing may define a recess 17 for stably supporting the placement of a container 16, for example, in the form of a culture flask, Petri dish, or other culture vessel, such that the housing 4 is positioned below the container 16 and the container 16 is supported in the recess 17.

[0140] The integrated circuit 8 includes an electronic transmission chip that is arranged, in use, to emit a pulsed electromagnetic signal from the device 2. More particularly, in one embodiment of the invention, the electronic transmission chip is arranged, in use, to be spaced less than 5 cm, preferably about 1 cm, from the container 16 located in the recess 17. This allows the electromagnetic signal emitted by the chip, in use, to be directed towards the cells located in the container 16.

[0141] The device of the present invention is designed to be used at room temperature (i.e., about 20°C), in temperatures below room temperature, such as in a refrigeration unit, and / or can be used at temperatures above room temperature, such as in an incubator unit.

[0142] In one embodiment, the control unit 10 is programmed to control the transmitter chip to allow it to emit a pulsed electromagnetic signal at a frequency of 2.45 GHz ± 50 MHz, at a pulsing frequency of 15 Hz, and at a power output of approximately 2 mW. It will be appreciated that parameters associated with the pulsed electromagnetic signal may be adjusted as needed and / or may be user-selectable. For example, the user may select the duration for which the pulsed electromagnetic signal is emitted, if desired. Additionally, the power output may be adjusted, although it is typically kept in the milliwatt range to avoid excessive energy delivery to cells contained within the vessel 16 during use. In one example, the pulsed signal lasts for 1 ms, with a rest period of 66 ms between signals. This provides a duty cycle of less than 2%.

[0143] In one example, the electromagnetic signal is an RF signal using the advertising function of the Bluetooth LE protocol and is transmitted using GFSK between 0.45 and 0.55.

[0144] However, it should be noted that in use the electronic device may be capable of transmitting any frequency in the industrial, medical and scientific frequency band (ie 2.4 to 2.4835 GHz, preferably 2.45 GHz ±50 MHz).

[0145] Referring to Figure 3, a further example of an apparatus 102 for providing a pulsed electromagnetic signal according to a further embodiment is shown. While Figures 1a-1b show an apparatus including a single electronic chip for transmitting the pulsed electromagnetic signal, Figure 3 shows an apparatus 102 having an array of six electronic chips 104 for transmitting the pulsed electromagnetic signal. The same reference numerals are used to describe the same features as in Figures 1a-1b. Although Figure 3 shows the electronic chips 104 as being on the apparatus 102, this is shown this way merely for clarity, as the chips 104 actually reside within the confines of the apparatus 102.

[0146] Six electronic chips 104 are provided on the device 102 at a spatial distance apart. The spacing between the chips can be any desired distance, but in one example, the chips are spaced apart such that, in use, when a 24-well cell plate 106 is positioned on the top surface 7 of the device, one transmitting chip 104 is located at the center of four of the wells. Thus, each electronic chip 102 directs a pulsed electromagnetic signal to four wells per 24-well cell plate. An on / off operating switch 108 is provided on the device 102 to move the device between an on state and an off state in use.

[0147] In the present invention, the device described above can be used to provide a pulsed electromagnetic signal directed to reagents and / or cells involved in one or more different steps of the transfection process. The device can also be used to direct a pulsed electromagnetic signal to transfected or untransfected cells to enhance cellular protein expression. As previously mentioned, the pulsing technology of the present invention has a wide variety of applications, including gene therapy and cell transfection.

[0148] The applicants have undertaken experiments which show that when reagents in the form of nucleic acids, such as DNA, RNA, DNA plasmids, etc., are provided in association with amphiphilic structures, such as liposome vehicles, and transfected into different types of eukaryotic cells, the use of the pulsing techniques of the present invention at various stages of the transfection process can significantly increase the transfection efficiency process and protein expression yields.

[0149] As a brief overview, in one example, a material comprising a dispersion of eukaryotic cells combined with a liposomal formulation of nucleic acid (DNA, RNA or small fragments of either) is placed in a suitable container such as a culture vessel, flask or dish, which in one embodiment is located on the device 2, 102, and a pulsed electromagnetic signal is emitted from the device and directed through the wall of the container 16 into the material 20.

[0150] The pulsing techniques of the present invention can be used on transfection mixtures prior to transfection, e.g., on nucleic acids and / or amphiphilic structures. The pulsing techniques of the present invention can also or alternatively be used on transfection complexes comprising the transfection mixture and eukaryotic cells. Additionally, or alternatively, the pulsing techniques of the present invention can be used on cells after transfection and / or on untransfected eukaryotic cells to increase protein expression in those cells.

[0151] In the following experiments used to illustrate the present invention, the same pulsing technique of the present invention was used on the transfection mixture prior to mixing with various eukaryotic cell lines and / or on the eukaryotic cell lines mixed with the transfection mixture during the transfection process.

[0152] The nucleic acids used in the experiments were those derived from the arginine vasopressin (AVP) promoter, the simian virus 40 (SV40) promoter, or the insulin-like growth factor binding promoter. protein DNA plasmid material containing the IGFBP3 promoter was included. Cytomegalovirus (Adluc) plasmid, luciferase control vector (Renilla) plasmid, or green fluorescent protein (GFP) plasmid was also used.

[0153] The amphiphilic structures used in the experiments were either cationic polymers (Turbofect®) (Thermo Fisher, USA), polyethyleneimine (PEI) (Fisher Scientific, USA), or transfection reagents containing TransIT2020 (Mirus Bio, USA).

[0154] The cell lines used in the experiments were Chinese Hamster Ovary-K1 (CHO) cells (adherent cells) (ATCC, USA - ATCC® CCL-61™), human embryonic kidney (HEK) 293 freestyle cells (suspension cells) (Thermo Fisher, USA), human colon tumor (HCT) 116 cells (adherent cells) (ATCC, USA - ATCC® CCL-247™) or Jurkat E6 (suspension T cells) (ECACC, UK).

[0155] To determine the efficiency of the cell transfection process using the above components, the luciferase activity or the amount of green fluorescent protein was measured using a suitable device.

[0156] A transfection mixture was formed by complexing a selected DNA plasmid material with an amphiphilic structure using known techniques. In some experiments, this transfection mixture was subjected to the pulsing technique of the present invention. The transfection mixture (with or without exposure to the pulsing technique) was then mixed into a suspension of one of the mammalian cell lines in a suitable cell culture vessel to form a transfection complex. The cell culture vessel was then placed in the device housing of the present invention and subjected to the pulsing technique as described above for a given period of time. The pulsed electromagnetic signal was then stopped, allowing the material to come to equilibrium. In addition, a control experiment was also performed in exactly the same way using the same materials and mixing requirements, but in the absence of the pulsing technique of the present invention.

[0157] A more detailed description of the methodology used in the experiments, results and findings are provided below. [Example]

[0158] <<Methodology>> Experiment 1 - Transfection of adherent CHO K1 and HCT116 cells with Adluc and Renilla plasmids and with either PEI or Turbofect as amphiphilic constructs. This experiment was conducted to investigate the effect of the pulsing technique of the present invention on the process of transfection in adherent Chinese hamster ovary (CHO) K1 cells (ATCC, USA) and HCT116 (human colon carcinoma cell line) (ATCC, USA) using Adluc and Renilla plasmids in either PEI (Fisher Scientific, USA) or Turbofect (Thermo Fisher, USA) amphiphilic structures. The pulsing technique was applied to a) the cells and transfection mixture (transfection complex) only during the transfection process; and b) the transfection mixture before forming the transfection complex with the cells and then the transfection complex during the transfection process.

[0159] <Consumables> Opti-MEM™ I low serum medium (Thermo Fisher, USA) Dulbecco's modified Eagle's medium (DMEM) (Thermo Fisher, USA) Fetal calf serum (FCS) (Hyclone, USA) 2 x 24-well Nunc plates (1.9 cm2 / well) (Thermo Fisher, USA) 200 ng of AdLuc plasmid / well (luciferase expression plasmid / DNA) (prepared by the University of Dundee, UK) 2ng Renilla plasmid / well (luciferase expression plasmid / DNA) (prepared by the University of Dundee, UK) Alfa Aesar™ Polyethyleneimine, Linear, Molecular Weight 25.00 0 (PEI) (Fisher Scientific, USA) Turbofect (Thermo Fisher, USA)

[0160] <Method Steps> <Control - PEI used> 1.650 μL of Opti-MEM medium was mixed with 2.6 μg of AdLuc plasmid and 26 ng of Renilla plasmid in the first tube; 2.650 μL of Opti-MEM medium was mixed with 7.88 μg of PEI in a second tube; 3. Mix the contents of the second tube dropwise into the first tube by gently vortexing using a Vortex-Genie2, model G560E (Scientific Industries, USA) to achieve a final volume of 1.3 mL of mixture; 4. The transfection mixture was incubated at room temperature (approximately 20°C) for 15 minutes; 5. 100 μL of this incubated transfection mixture was then dispensed into wells labeled A1 to A6 in each of two 24-well plates (Plates 1 and 2), forming the transfection mixture.

[0161] <Invention - Pulsing technique for transfection mixture before transfection complex is produced, using PEI> 1. Steps 1-3 above were then repeated, except that in step 4—the mixture forming the transfection mixture was incubated for 15 minutes at room temperature (approximately 20° C.) by placing the first tube on a pulsed electromagnetic signal device according to the present invention, the pulsed device operating as described above (i.e., the pulsed device was operated at 2.45 GHz±50 MHz, with a power output of 2 mW and a pulsing frequency of 15 Hz). 2. 100 μL of this cultured pulsed transfection mixture was dispensed into wells designated B1 to B6 in each of two 24-well plates (plates 1 and 2).

[0162] <Control - Turbofect used> 1.650 μL of Opti-MEM medium was mixed with 2.6 μg of AdLuc plasmid and 26 ng of Renilla plasmid in the first tube; 2.13 μL of Turbofect was added and mixed by vortexing using a Vortex-Genie 2, model G560E, (Scientific Industries, USA); 3. The transfection mixture was incubated at room temperature (approximately 20°C) for 15 minutes. 4. 100 μL of this incubated transfection mixture was dispensed into wells designated C1 to C6 in each of two 24-well plates (plates 1 and 2).

[0163] <Invention - Pulsing technique for the transfection mixture before the transfection complex is produced, using Turbofect> 1. Steps 1-2 above for the Turbofect control were repeated. In step 3 - the transfection mixture was incubated for 15 minutes at room temperature (approximately 20°C) by placing the first tube on a pulsed electromagnetic signal device according to the present invention. The pulsed device was operated at 2.45 GHz ± 50 MHz, with a power output of 2 mW and a pulsing frequency of 15 Hz. 2. 100 μL of this cultured pulsed transfection mixture was dispensed into wells designated D1 to D6 in each of two 24-well plates (plates 1 and 2).

[0164] <Cell lines were added to plates 1 and 2> - For plates 1 and 2, 2 x 10 cells of either CHO K1 or HCT116 cells were added to each well of two 24-well plates. 4 Transfection complexes were generated by adding 100 cells / well, and then the final volume was adjusted to 600 μL of Dulbecco's modified Eagle's medium (DMEM) + 10% fetal calf serum (FCS). Specifically, CHO K1 cells were added to A1-A3, B1-B3, C1-C3, and D1-D3; HCT 116 cells were added to A4-A6, B4-B6, C4-C6, and D4-D6. - Plates 1 and 2 were incubated in an incubator at 37°C and 5% CO 2 and incubated for 3 hours; - In plate 1, the transfection complexes were not subjected to the pulsing technique during the 3-hour incubation phase, while in plate 2, they were subjected to the pulsing technique according to the invention during the incubation phase for 3 hours. After 4 hours, the wells were topped up with DMEM containing Turbofect transfection reagent. - The average value of three wells for each experimental condition was measured and recorded.

[0165] In some cases, the above experiments were performed using a first type of pulsing technique in which only a single transmitter was provided on the pulsing instrument (Technique 1 pulsing technique). In some cases, the above experiments were performed using a second type of pulsing technique in which an array of multiple transmitters was used on the pulsing instrument (Technique 2 pulsing technique). Specifically, in experiments using Type 2 pulsing technique, six transmitters were provided, each disposed at or substantially at the center of four wells of a 24-well plate when the plate was placed on the pulsing instrument.

[0166] <<Luciferase assay protocol - using the Dual Luciferase Reporter Assay System (Promega, USA)>> The protocol used is shown in the link below, but the protocol is outlined below: https: / / www.promega.co.uk / products / luciferase-assays / reporter-assays / dual_luciferase-reporter-assay-system / ?catNum=E1910#protocols

[0167] <Method Steps> 1. 24 hours after the transfection experiment, the medium was removed from the cells. 2. The cells were washed twice with phosphate-buffered saline (PBS). 3. 100 μL of 1× passive lysis buffer (Promega, USA) was added to the cells. 4. The cells were incubated for 15 minutes at 37°C with gentle rocking on a Belly Dancer® orbital shaker (Sigma, Aldrich). 5. 10 μL of cells were taken from each well and placed into a white 96-well plate. 6. Cells were analyzed in a microplate luminometer LB 96V (EG & G Berthold, Germany) using the Dual Luciferase Assay System protocol (Promega, USA). 7. The analysis was performed by injecting 30 μL Luciferase Assay Reagent II (Promega, USA) to measure firefly luciferase activity, then injecting 30 μL Stop&Glo™ reagent to block firefly luciferase and measure Renilla luciferase activity. 8. The lysed extracts were then kept at -20°C for Western blotting if required. 9. The transfection efficiency of cells was determined by placing the cells in an Incucyte® live cell analysis system (Sartorius, Germany) for 72-96 hours, and measuring their fluorescence every hour. Data were collected and analyzed in Excel®.

[0168] <<Experimental Results 1>> Table 1 shows the results of experiments with CHO K1 cells using Technique 1 pulsing technology in conjunction with Turbofect amphiphilic constructs and associated methodology. <Table 1 (Technique 1: Pulsing Technology)> [Table 1] % increase in pulsed technique compared to control - 178% Mean fold increase in pulsed technique compared to control -1.8 t-test -0.024

[0169] Table 2 shows the results of experiments with CHO K1 cells using the Technique 2 pulsing technique in conjunction with Turbofect amphiphilic constructs and associated methodology. <Table 2 (Technique 2 Pulsing Technology)> [Table 2] % increase in pulsed technique compared to control - 232.7% Mean fold increase in pulsed technique compared to control - 2.3 t-test - 0.044

[0170] Table 3 shows the results of HCT 116 cell experiments using the pulsing technique in conjunction with Turbofect amphiphilic structures and related methodologies. [Table 3] % increase in pulsed technique compared to control - 138.5% Mean fold increase in pulsed technique compared to control - 1.4 t-test - 0.044

[0171] Referring to Tables 1 and 2 and Figures 4a and 4b, the transfection efficiency in CHO K1 cells associated with Turbofect amphiphilic structures is shown for the control and the pulsing technique of the present invention (Pulzar). Three replicates were included for each condition. Luminescence was measured for all cells as a measure of luciferase activity (i.e., transfection).

[0172] It was seen that the transfection efficiency in CHO K1 cells using the Technique 1 pulsing technique was significantly improved compared to control cells, with a t-test value of 0.024, a mean fold increase of 1.8, and a % increase of 178.0.

[0173] It was also observed that the transfection efficiency in CHO K1 cells using the Technique 2 pulsing technique was significantly improved compared to the control cells, with a t-test value of 0.044, a mean fold increase of 2.3, and a % increase of 232.7.

[0174] Furthermore, it can be seen that experiments performed with the Technique 2 pulsing technique (i.e., the six electronic transmitter chip array) produced significantly better results than experiments performed using the Technique 1 pulsing technique.

[0175] Referring to Table 3 and Figure 4c, the transfection efficiency in HCT 116 cells associated with Turbofect amphiphilic constructs is shown for the control and the pulsing technique of the present invention (Pulzar). Three replicates were included for each condition. Luminescence was measured for all cells as a measure of luciferase activity.

[0176] It was seen that the transfection efficiency in HCT 116 cells using the pulsed technique was significantly improved compared to the control cells, with a t-test value of 0.044, a fold increase of 1.4, and a % increase of 138.5.

[0177] Therefore, it can be concluded that the pulsing technique of the present invention significantly increased the transfection efficiency in adherent CHO K1 and HCT 116 cells compared to when no pulsing technique was used. Furthermore, the use of six electron transmitters resulted in a further increase in transfection efficiency compared to when only a single electron transmitter was used.

[0178] Experiment 2 - Transfection of adherent HCT cells using either a plasmid containing the IGFBP3 promoter or a plasmid containing the SV40 promoter and PEI as the amphiphilic construct. Experiment 2 was conducted to investigate the effect of the pulsing technique of the present invention on the process of transfection of adherent HCT116 (human colon carcinoma cell line) (ATCC, USA) using Adluc and Renilla plasmids harboring either the IGFBP3 promoter or the SV40 promoter in a PEI (Fisher Scientific, USA) amphiphilic construct. For Experiment 2, the methodology of Experiment 1 was followed.

[0179] <<Results of Experiment 2>> Table 4 shows the results of HCT 116 cell experiments on the IGFBP3 promoter using PEI amphiphilic constructs and associated methodology. [Table 4] % Increase Factor = 168.4991974 t-test p<0.004154274

[0180] Table 5 shows the results of HCT 116 cell experiments on the SV40 promoter using PEI amphiphilic constructs and associated methodology. [Table 5] % Increase Factor = 155.2371016 t-test p<0.026953884

[0181] Referring to Tables 4 and 5 and Figures 5a and 5b, the transfection efficiency in HCT 116 cells of DNA plasmids containing either the IGFBP3 promoter or the SV40 promoter and associated with PEI amphiphilic structures is shown for the control and the pulsing technique of the present invention (Pulzar). Each graph contains two experiments with three replicates. Luminescence was measured for all cells as a measure of luciferase activity.

[0182] It was found that the transfection efficiency (indicated by the IGFBP3 promoter) in HCT 116 cells using the pulsing technique was significantly improved compared to control cells, with a t-test value of 0.004 and a % increase of 168.5.

[0183] It was found that the transfection efficiency (indicated by the SV40 promoter) in HCT 116 cells using the pulsing technique was significantly improved compared to control cells, with a t-test value of 0.027 and a % increase of 155.2.

[0184] Therefore, it can be concluded that the pulsing technique of the present invention significantly increased the transfection efficiency in adherent HCT 116 cells compared to without the pulsing technique.

[0185] Experiment 3 - Transfection of suspension HEK 293 Freestyle cells using GFP plasmid and PEI as amphiphilic construct This experiment was conducted to investigate the effect of the pulsing technique of the present invention on the process of transfection of human embryonic kidney (HEK) suspension cells 293 Freestyle with a green fluorescent protein (GFP) plasmid in a PEI amphiphile construct. The pulsing technique was applied only to the cells and transfection reagent during the transfection process.

[0186] <Consumables> Opti-MEM™ I low serum medium (Thermo Fisher, USA) Green fluorescent protein (GFP) plasmid (created by the University of Dundee, UK) 293-Freestyle suspension cells (Thermo Fisher, USA) 293-Free Expression Medium (Sigma-Aldrich, USA) Alfa Aesar™ Polyethyleneimine, Linear, Molecular Weight 25.00 0 (PEI) (Fisher Scientific, USA)

[0187] Method steps when pulsing technique is used only for reagent and cell mixtures 1. The day before transfection, 6 x 10 5 ~7×10 5 Seed 293-F cells / mL. 2. On the day of transfection, count the cells and, if necessary, reduce the cell density to 1 x 10 6 Dilute cells to 100 cells / mL. Transfect 3.15 µg of green fluorescent protein (GFP) plasmid / flask with 30 µL of 293-Free expression medium / flask. A DNA:PEI ratio of 4.1:2 is used. 5. Use 293-Free Expression Medium according to the manufacturer's instructions (https: / / www.sigmaaldrich.com / content / dam / sigma-aldrich / docs / SAJ / Brochure / 2TB5515.pdf) - User Protocol TB515 Rev.B 0411JN[4] 6. To prepare the DNA-transfection mixture: - Add 2.4 mL of Opti-MEM to the flask. - Add 30 μg of GFP plasmid to the flask. Add 60 μL of 293-Free Expression Medium. - Divide the resulting mixture volume into two 125 mL Erlenmeyer flasks, each containing 1 x 10 cells in 28.8 mL of 293 Expression Medium. 6 Contain cells / mL; - The two flasks were incubated in two separate incubators on a Bellydancer orbital shaker (Sigma, Aldrich) at 125 rpm and 8% CO 2 The flasks are cultured at 37°C for 3 hours. One of the flasks is pulsed for 3 hours using the pulsing technique of the present invention in one of the incubators, while the other flask is cultured in a second incubator without any pulsing technique. After 3 hours, both flasks are placed in the same incubator without any pulsing technique, allowing for measurement of transfection efficiency over time for as long as required (120 hours in this experiment).

[0188] <Experiment 3 Results> FIG. 6 shows the maximum increase in transfection efficiency achieved in these experiments using the pulsing technique methodology described herein.

[0189] FIG. 7 shows the mean improvement in transfection efficiency using the pulsing technique of the present invention.

[0190] 6 and 7, the transfection efficiency of GFP plasmids associated with PEI amphiphilic structures in HEK 293 Freestyle suspension cells is shown for the control and the pulsing technique of the present invention (Pulzar). Growth curves after Pulzar exposure and without Pulzar exposure (control) are visualized in the graphs of FIGS. 6 and 7.

[0191] Transfection efficiency (as measured by the mean green fluorescence intensity) in HEK 293 Freestyle suspension cells using the pulsed technique was found to be significantly improved compared to control cells, with a t-test value of less than 0.05 and a peak increase in GFP expression of 2.3-fold higher, as shown in Figure 6.

[0192] Transfection efficiency (as measured by the mean green fluorescence intensity) in HEK 293 Freestyle suspension cells using the pulsed technique was found to be significantly improved compared to control cells, with a t-test value of less than 0.05 and an increase in GFP expression of more than 50%, as shown in Figure 7. Delta was calculated to mark the % increase in GFP expression over the entire experimental period.

[0193] Therefore, it can be concluded that the pulsing technique of the present invention significantly increased the transfection efficiency in HEK 293 Freestyle suspension cells compared to without the pulsing technique.

[0194] Experiment 4 - Transfection of Suspension Jurkat E6 Cells with Adluc and Renilla Plasmids and Using Either PEI or TransIT2020 as the Amphiphilic Structure This experiment was conducted to investigate the effect of the pulsing technique of the present invention on the process of transfection in Jurkat E6 cells (human leukemia T-cell lymphoblastoid cells) (European Collection of Authenticated Cell Cultures (ECACC), UK) with Adluc and Renilla plasmids in either PEI (Fisher Scientific, USA) or TransIT2020 (Mirus Bio, USA) amphiphilic structures. The pulsing technique was applied to a) the cells and transfection mixture (transfection complex) only during the transfection process; and b) the transfection mixture before forming the transfection complex with the cells and then the transfection complex during the transfection process.

[0195] <Consumables> Opti-MEM™ I low serum medium (Thermo Fisher, USA) Fetal calf serum (FCS) (Hyclone, USA) RPMI medium (Sigma-Aldrich, UK) 2 x 24-well Nunc plates (1.9 cm2 / well) (Thermo Fisher, USA) 1 μg of AdLuc plasmid / well (luciferase expression plasmid / DNA) (prepared by the University of Dundee, UK) 80ng Renilla plasmid / well (luciferase expression plasmid / DNA) (prepared by the University of Dundee, UK) Alfa Aesar™ Polyethyleneimine, Linear, Molecular Weight 25.00 0 (PEI) (Fisher Scientific, USA) TransIT2020 (Mirus Bio, USA)

[0196] <Method Steps> <Control - PEI used> 1. 650 μL of Opti-MEM medium was mixed with 13 μg of AdLuc plasmid and 1 μg of Renilla plasmid in the first tube; 2. 650 μL of Opti-MEM medium was mixed with 42 μg of PEI in a second tube; 3. Mix the contents of the second tube dropwise into the first tube using a Vortex-Genie2, model G560E, (Scientific Industries, USA) with gentle vortexing to achieve a final volume of 1.3 mL of mixture; 4. The transfection mixture was incubated at room temperature (approximately 20°C) for 15 minutes; 5. 100 μL of this incubated transfection mixture was then dispensed into wells labeled A1 to A6 in each of two 24-well plates (Plates 1 and 2), forming the transfection mixture.

[0197] <Invention - Pulsing technique for transfection mixture before transfection complex is produced, using PEI> 1. Steps 1-3 above were then repeated, except in step 4—the mixture forming the transfection mixture was incubated for 15 minutes at room temperature (approximately 20° C.) by placing the first tube on a pulsed electromagnetic signal device according to the present invention, the pulsed device operating as described above (i.e., the pulsed device was operated at 2.45 GHz±50 MHz, with a power output of 2 mW and a pulsing frequency of 15 Hz). 2. 100 μL of this cultured pulsed transfection mixture was dispensed into wells designated B1 to B6 in each of two 24-well plates (plates 1 and 2).

[0198] <Control - TransIT2020 used> 1 700 μL of Opti-MEM medium was mixed with 13 μg of AdLuc plasmid and 1 μg of Renilla plasmid in the first tube; 2.42 μL of TransIT2020 was added and mixed by vortexing using a Vortex-Genie 2, model G560E, (Scientific Industries, USA); 3 The transfection mixture was incubated at room temperature (approximately 20°C) for 15 minutes. 4 0.50 μL of this incubated transfection mixture was dispensed into wells designated C1-C6 in each of two 24-well plates (plates 1 and 2).

[0199] <Invention - Pulsing technique for transfection mixture before transfection complex is generated, using TransIT2020> 3. Steps 1-2 above for the TransIT2020 control were repeated. In step 3 - the transfection mixture was incubated for 15 minutes at room temperature (approximately 20°C) by placing the first tube on a pulsed electromagnetic signal device according to the present invention. The pulsed device was operated at 2.45 GHz ± 50 MHz, with a power output of 2 mW and a pulsing frequency of 15 Hz. 4.50 μL of this cultured pulsed transfection mixture was dispensed into wells designated D1 to D6 in each of two 24-well plates (plates 1 and 2).

[0200] <Cell lines were added to plates 1 and 2> - For plates 1 and 2, Jurkat E6 cells in RPMI and 10% FCS were plated at 2 x 10 in each well of two 24-well plates. 5 Transfection complexes were generated by adding cells / well and then made up to a final volume of 600 μL. - Plates 1 and 2 were incubated in an incubator at 37°C and 5% CO 2 were incubated overnight at ; In plate 1, the transfection complexes were not subjected to the pulsing technique during the overnight culture phase, whereas in plate 2, the pulsing technique according to the invention was applied for 3 hours during the overnight culture phase. - The average value of three wells for each experimental condition was measured and recorded.

[0201] <<Luciferase assay protocol - using the Dual Luciferase Reporter Assay System (Promega, USA)>> <Method Steps - as explained above>

[0202] <<Results of Experiment 4>> <> Table 6 shows the results of Jurkat E6 cell experiments with AdLuc and Renilla plasmids using PEI or TransIT2020 amphiphilic constructs and associated methodologies. [Table 6] Experiment A - Here, the pulsing technique was applied only to the transfection complex (ie, after the transfection mixture had been added to the cells, during the culture). Experiment B - Here, the pulsing technique was applied only to the transfection mixture (before adding Jurkat E6 cells). Experiment C - Here, the pulsing technique was applied to the transfection mixture (before adding Jurkat E6 cells) and then also to the transfection complex (i.e., after adding the transfection mixture to the cells, during the incubation).

[0203] Results of Experiment 4 Referring to Table 6 and Figure 8, each bar on the graph represents the average of three replicates. When only the transfection complex was subjected to the pulsing technique, a 1.7-fold increase in transfection efficiency was observed. When only the transfection mixture was subjected to the pulsing technique, a 2.0-fold increase in transfection efficiency was observed. When both the transfection mixture and the transfection complex were subjected to the pulsing technique, a 2.3-fold increase in transfection efficiency was observed. Therefore, it can be concluded that the pulsing technique of the present invention significantly increased transfection efficiency when used with either the transfection mixture or the transfection complex alone, but a further increase in transfection efficiency was observed when the pulsing technique was applied to both the transfection mixture and the transfection complex.

[0204] In one embodiment of the present invention, as shown in Figures 9a and 9b, there is provided a device 301 in the form of an electronic device that can be used to improve transfection efficiency and / or intracellular delivery of one or more reagents, to provide one or more therapeutic treatment methods to a patient, to increase delivery of pharmaceutical and / or therapeutic reagents to a patient, to increase and / or decrease gene expression, protein expression, etc.

[0205] As previously described, the device 301 has the capability of emitting a pulsed electromagnetic signal at a given frequency, at a given pulse rate, at a given power level, and for a given period of time. However, the device 301 may be worn adjacent to a patient's body, thereby allowing the pulsed electromagnetic signal to be directed toward the patient's body during use. The given parameters may be pre-set by the manufacturer or may be user-selectable as desired.

[0206] The device 301 includes a housing 302 which contains a pulsed signal transmission system. In particular, in this example the pulsed signal transmission system includes a circuit board 307 which includes transmission means in the form of an electronic transmission chip 304, typically provided as part of an integrated circuit, which allows the transmission of a pulsed electromagnetic signal when the device is in an operational state in use.

[0207] In one example, each housing includes a base surface 303, a top surface 311 opposite the base surface, and one or more side walls 313 located between the top surface 311 and the base surface 303.

[0208] Control means in the form of a control unit 310 may be provided to allow selective operation of the apparatus 301. A memory device 306 is provided to store data, one or more operating parameters, software etc. to allow retrieval when required. The control unit preferably includes microprocessing means to allow processing of data etc.

[0209] The device 301 may also include one or more power cells 310 that power the device. Optionally, a rechargeable facility may also be provided to allow the power cells to be charged from a remote power source rather than having to be replaced.

[0210] The electronic transmission chip 304 is disposed in the housing 302 so as to, during use, emit a pulsed electromagnetic signal from the device 301 in one or more particular directions. The direction of transmission of the pulsed electromagnetic signal will typically depend on the purpose for which the device 1 is intended. If the device is intended to be worn by a user, the signal is typically directed through the base surface 303 toward the user.

[0211] In one embodiment of the present invention, the electronic transmission chip is disposed in the housing 302 less than 5 cm, preferably about 1 cm, from the surface of the housing 302 that will come into contact with the user's skin in use, allowing the electromagnetic signal emitted by the chip to be directed towards the patient in use.

[0212] The devices of the present invention are designed for use at room temperature (i.e., about 20° C.), in temperatures below room temperature, and / or can be used at temperatures above room temperature, for example, the body temperature of a patient.

[0213] In one embodiment, the control unit 310 is programmed to control the transmitter chip to allow it to emit a pulsed electromagnetic signal at a frequency of 2.45 GHz ± 50 MHz, at a pulsing frequency of 15 Hz, and at a power output of approximately 2 mW. It will be appreciated that parameters associated with the pulsed electromagnetic signal may be adjusted as needed and / or may be user-selectable. For example, the user may select the duration for which the pulsed electromagnetic signal is emitted, if desired. Additionally, the power output may be adjusted, although it is typically kept in the milliwatt range to avoid excessive current flow to cells contained within the vessel 16 during use. In one example, the pulsed signal lasts for 1 ms, with a rest period of 66 ms between signals. This provides a duty cycle of less than 2%.

[0214] However, it should be noted that in use the electronic device may be capable of transmitting any frequency in the industrial, medical and scientific frequency band (ie 2.4 to 2.4835 GHz, preferably 2.45 GHz ±50 MHz).

[0215] In one example, the electromagnetic signal is an RF signal using the advertising function of the Bluetooth LE protocol and is transmitted using GFSK between 0.45 and 0.55, however it should be noted that in use the electronic device may be capable of transmitting at any frequency in the industrial, medical and scientific frequency bands.

[0216] In the example shown in Figure 9a, selection means 305 are provided that allow selection of a particular pulse sequence, frequency, timing and / or intensity of pulses to allow the device to be configured according to the user's requirements.

[0217] 9a and 9b, the device 301 is illustrated as being positioned directly on the surface of a patient's skin 312. In this example, attachment means in the form of a band 314 is provided for removably attaching the device 301 to a user's body. More particularly, the band 314 is wrapped around the patient's arm or limb, thereby securing the housing 302 in a desired position relative to a portion of the patient's skin. Alternatively, the base surface 303 of the housing that will come into contact with the skin may be provided with an adhesive thereon to allow it to be attached to the patient's skin in a desired position. In use, as the device 301 is operated, a pulsed electromagnetic signal 322 emitted from the housing 302 penetrates at least a portion of the patient's skin, and potentially further penetrates the tissues 324 and cells of the patient's body.

[0218] In another embodiment of the invention, as shown in Figures 10a and 10b, the device housing 302 is positioned over a drug delivery "patch" 325 (sometimes referred to as a "transdermal patch"), which is in turn adhered to a portion of the user's skin 312. In this embodiment, a pulsed electromagnetic signal 322 is emitted from the housing 302 and directed into the patch 325, through a portion of the patch containing a reagent or drug 326, and onto the skin 312. The drug is delivered to the user's tissues and cells 324 by passing through the user's skin. The use of a pulsed electromagnetic signal enhances absorption and uptake of the drug through the user's skin. Reference to a "drug" can refer to any reagent, pharmaceutical and / or therapeutic agent, as appropriate.

[0219] In another embodiment of the invention, the device is provided as an implantable device, as shown in Figure 11. More specifically, the device's housing 302 provides a sterile exterior that is implanted subcutaneously beneath a user's skin 312 and / or into the user's tissue 324. Once implanted, the device emits a pulsed electromagnetic signal 322 therefrom. The implant is positioned to emit the signal 322 in a desired direction, for example, toward a cancerous tumor 328.

[0220] In a still further embodiment of the invention, the device is provided in the form of a pendant 336, as shown in Figures 12a and 12b. In this figure, the pendant is attached to a chain 337 and arranged so that the pendant is located at the level of the throat / upper rib cage 338 of a patient or person 339. A pulsed electromagnetic signal 322 is then directed from the pendant towards the body of the wearer, as indicated by arrow 341 in Figure 12a. A surface 343 of the pendant 336 is arranged so that the pendant can be located closest to the person when worn in the required position.

[0221] In one example, the device of the present invention may be worn to minimize viral growth and as a means of providing the wearer with greater immunological protection. Thus, in this embodiment, when the pendant 336 is worn at throat / upper thorax level, it enhances immunity in this critical respiratory area of ​​the wearer.

[0222] Typically, in either embodiment, the device of the present invention is provided on or adjacent to a selected portion of the user's skin to provide localized and focused treatment to a given location.

[0223] For example, if the purpose of the device is to provide treatment for a cancerous tumor in a patient, the device may be located adjacent to or implanted in a recognized cancerous tumor, such as may be present in the liver, kidney, breast, or bone. Alternatively, if the device is to provide a therapeutic benefit or to limit or prevent the possibility of infection, the device may be located external to the patient, adjacent to a portion of the patient's body where the therapeutic or prophylactic effect is believed to be most beneficial, such as the throat area of ​​the patient or person.

[0224] Thus, when the device is placed directly on the patient's skin 312, the pulsed electromagnetic signal is emitted through the skin into the tumor, causing a change in the state of the tumor cells. When the device is used in conjunction with a patch or other drug-carrying article, such as those shown in FIGS. 10a and 10b, the drug can then pass through the patient's skin more easily than previously possible. It is believed that the pulsed electromagnetic signal increases the size of the skin's pores, allowing more space for the drug to pass through. Thus, pharmaceutical drugs or other reagents can be delivered more efficiently and effectively using the present invention. Additionally, pharmaceutical drugs or other reagents that cannot currently be delivered transdermally can now be delivered into the body using the methods of the present invention. The provision of the device of the present invention enhances drug delivery through increased skin permeability while providing the benefits of direct treatment.

[0225] While the above examples demonstrate that transfection of reagents in the form of nucleic acids associated with amphiphilic structures in various types of eukaryotic cells is significantly enhanced after exposure to the pulsing techniques of the present invention at various stages of the transfection process, Applicants fully expect and anticipate that the transfection and / or intracellular delivery of one or more pharmaceutical and / or therapeutic reagents or compounds, small molecules or small molecular materials of less than 5 kilodaltons, large molecules or large molecular materials of about 5 kilodaltons or more, one or more proteins, vaccines, organic agents, and / or one or more antibodies when associated with amphiphilic structures will be significantly enhanced in one or more eukaryotic cells upon exposure thereof to the pulsing techniques of the present invention. These predictions and expectations are based on data already collected by the applicants in their co-pending applications claiming priority from UK Patent Applications GB2004411.1, GB2009297.9, GB20044112.9, and GB2009296.1 (the contents of which are incorporated herein by reference), which demonstrate that intracellular delivery of a "naked" reagent (i.e., not associated with an amphiphilic structure) in the form of doxorubicin in eukaryotic cells is significantly enhanced when exposed to the pulsing techniques of the present invention. The data from these experiments are reproduced below to provide support for the breadth of the claims of this application. The applicants anticipate the same or similar mechanisms for the enhancement of transfection efficiency and / or intracellular delivery when the reagent is associated with an amphiphilic structure as when a "naked" reagent (i.e., not associated with an amphiphilic structure) is used. This is because the pulsed electromagnetic waves or signals of the present invention are believed to be sufficient to cause HO to periodically rotate around its dipole with relatively long rest or relaxation periods. The periodic rotation of HO is believed to disrupt hydrogen bonds in the phospholipid bilayer or cell membrane of eukaryotic cells. Therefore, this periodic or intermittent low-energy perturbation of the cell membrane is believed to stimulate increased interactions between reagents, some molecules and / or cell membranes and their environment, such as, for example, nucleic acids or reagents and cell membranes.It is believed that the relatively long rest or relaxation periods between pulses of the pulsed electromagnetic signal are sufficient to maintain cellular integrity.

[0226] In the following experiment, taken from the applicant's co-pending patent application, the same pulsing technique of the present invention was used on a "naked reagent" in the form of doxorubicin when added to a eukaryotic cell line.

[0227] 24 hours before treatment, two CELLSTAR® 6-well plates (9.6 cm 2 3 × 10 human colon tumor (HCT) 116 cells (adherent cells) (ATCC, USA - ATCC® CCL-247™) were plated per well in a final volume of 5 mL of Dulbecco's modified Eagle's medium (DMEM) (Thermo Fisher, USA) + 10% fetal bovine serum (FBS) (Hyclone, USA) in a 1000 ml plate. 5 The cells were seeded at a density of 1000×.

[0228] The naked reagent used was doxorubicin (0.25 μM) (Sigma Aldrich) in absolute ethanol, which was given to the cells for a 1-hour treatment period, in 5% CO 2 The cells were cultured at 37°C.

[0229] After treatment, the medium was removed and fresh medium was added to the cells. One of the plates was incubated in 5% CO 2 The second plate was placed in a separate incubator and incubated directly at 37°C in 5% CO using the pulsed technique of the present invention. 2 The cells were pulse-treated at 37°C.

[0230] Protein extracts were collected after 3, 6, 9, 16 or 24 hours of treatment and analyzed by SDS-PAGE.

[0231] The following Western blot protocol is presented in reference [5].

[0232] Preparation of protein extracts for Western blotting 1. For protein extraction, cells were washed twice with ice-cold PBS and then lysed in NP-40 extraction buffer (50 mM Tris pH 7.5; 10% glycerol; 0.1% "NP-40 Alternative" (Merck Millipore, USA); 100 mM NaCl; 0.2 mM EDTA) supplemented with 1x Complete™ protease inhibitor cocktail (Roche, Switzerland). Extracts were sonicated (20 seconds, 20% amplitude), and protein concentrations were determined using the BCA™ Protein Assay Kit (ThermoFisher Scientific, USA) according to the manufacturer's recommendations.

[0233] Western Blot Protocol 1. Protein extracts (15 / 20 μg depending on the experiment) were supplemented with 0.1 M dithiothreitol (DTT) and 1× LDS buffer (Invitrogen, USA), heated at 95°C for 10 min, and then loaded onto NuPAGE 10% Bis-Tris polyacrylamide gels (Invitrogen, USA). Protein samples were separated by electrophoresis (100 V) using 2.1x MOPS running buffer. Protein transfer to a nitrocellulose membrane (Protran 0.1 μm from GE Healthcare, USA) was carried out overnight at 12 V in 1x transfer buffer supplemented with 20% methanol. 1x transfer buffer is prepared from 10x Western blot solution containing 144 g glycine and 30 g Tris base in a final volume of 1 L Milli-Q water. 3. The membranes were blocked with 5% BSA diluted in PBS-0.1% Tween 20 for 30 minutes and then incubated overnight with the primary antibody (mouse monoclonal antibody DO1). After a 15-minute wash with PBS-Tween 20, the membranes were incubated for 1 hour with the corresponding secondary antibody (HRP-conjugated donkey anti-mouse). All horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Jackson ImmunoResearch Lab and used at a 1:10,000 / 1:15,000 dilution (depending on the antibody) in 5% BSA-PBS-Tween 20.

[0234] At the end of the incubation, the membrane was washed twice with PBS-Tween 20 for 15 min, followed by a final wash with PBS for 10 min. Chemiluminescent signals were detected on Hyperfilm™ ECL (Cytiva, USA) using the Amersham ECL Western Blotting Detection System (Cytiva, USA).

[0235] result Referring to Figure 13, the Western blot shows that p53α—the major isoform of p53 protein—was upregulated after treatment with the pulsing technique of the present invention. This effect was observed as soon as 3 hours after drug addition and was most evident 24 hours after treatment. Other isoforms of p53, namely, d133p53α, d133p53β, and d160p53β, were also upregulated following doxorubicin treatment with the pulsing technique of the present invention. In Western blots, γH2AX was used as a marker to ensure that any effects observed were not due to ionizing radiation. γH2AX expression changes in the presence of ionizing radiation, and since no changes were observed between the inventive pulse technique and the control arm, we concluded that the inventive pulse technique did not emit ionizing radiation.

[0236] Ku80 was used as a loading control to ensure that equal concentrations of each sample were loaded into each well, which results in comparable residual bands on the Western blot.

[0237] Referring to Figure 14, in another experiment, some cells were treated with the pulsing technique of the present invention, and some cells served as a control without the pulsing technique of the present invention and without doxorubicin for 5 days. No change in p53α expression was observed. When 0.25 μM doxorubicin was added to the cells for 1 hour, the cells affected by the pulsing technique of the present invention showed significant overproduction of p53α after 16 hours compared to the control.

[0238] In conclusion, there is clear evidence that treating cells with the pulse technique of the present invention increases their ability to take up doxorubicin from the culture medium, as various p53 isoforms were more upregulated in the pulse technique arm compared to the control arm. It can be concluded that this effect is not caused by ionizing radiation, as the radiological marker gH2AX remained unchanged between the pulse technique arm and the control arm.

[0239] Thus, the combined effect of enhanced delivery of anti-cancer drugs and direct application of the pulsing technique of the present invention will beneficially influence the regulation of p53 oncogene-mediated replication, improving cancer treatment. Furthermore, the effect of the pulsing technique of the present invention on non-mutated p53 in healthy cells will increase the repair of those cells.

[0240] <<References>> [1]- Gene Therapy-An Industry Coming Of Age-The Cell Culture Dish Inc.2020 pages 1-49 [2]- Global Manufacturing of CAR T Cell Therapy-Bruce Levine et al;Molecular Therapy:Methods and Clinical Development,Vol.4,March 207;92-101;2017 Novartis Pharmaceuticals Corp. [3] Efficient Lipid-Mediated Transfection Of DNA Into Primary RatHepatocytes-Sheri L.Holmes et al;In Vitro Cell.Dev.Biol.30;347-351-May 1995-1995 Society for In Vitro Biology. [4] Novagen - User Protocol TB515 Rev.B0411JN - pages 1 - 4 - 293 - Free Transfection Reagent(2011 (Copyright) EM Chemicals Inc). [5] Bourdon et al., Genes Dev. 2005, PMID 16131611. [6] Longo PA, Kavran JM, Kim MS, Leahy DJ. “Transient Mammalian Cell Transfection With Polyethylenimine(PEI). Methods Enzymol. 2013;529 - 227 - 240. Doi:10.1016 / B978 - 0 - 12 - 418687 - 3.00018 - 5.

Claims

1. 1. An in vitro method for improving transfection efficiency in eukaryotic cells, the method comprising: a) providing a transfection mixture comprising a reagent associated with at least one amphiphilic structure suitable for transfection; b) introducing the transfection mixture into one or more eukaryotic cells to form transfection complexes; c) allowing the transfection complex to undergo a transfection process to form one or more transfected cells; Including, 10. The method of claim 9, further comprising the step of directing a pulsed electromagnetic signal at a given frequency between 2.2 and 2.6 GHz and at a given power of +2 dBm to +4 dBm, 1 mW to 2.5119 mW, 1 mW, 2 mW, or 2.5119 mW, or any combination thereof, to the transfection mixture in step a) prior to producing the transfection complexes, to the transfection complexes in step b), to the transfection complexes in step c), and / or to the complexes of transfected cells after step c).

2. 10. The method of claim 1, wherein the eukaryotic cells are suspended in solution and / or attached to a substrate and / or are immortal cells.

3. 2. The method of claim 1, wherein the reagents in the transfection mixture are any reagent suitable for transfection and / or any combination of nucleic acids, i.e., deoxyribonucleic acid (DNA), ribonucleic acid (RNA), mRNA, tRNA, siRNA, or miRNA, pharmaceutical reagents, therapeutic reagents, small molecules or small molecular materials of less than 5 kilodaltons, large molecules or large molecular materials of 5 kilodaltons or more, one or more proteins, vaccines, organic agents, one or more antibodies, or one or more expression vectors.

4. 2. The method of claim 1, wherein the reagent associated with the at least one amphiphilic structure is contained within the amphiphilic structure, the reagent is complexed with the amphiphilic structure, the reagent is contained on the amphiphilic structure, or the reagent is bound to the amphiphilic structure.

5. 10. The method of claim 1, wherein the amphiphilic structure comprises or consists of at least one liposomal substance or vehicle, at least one PEGylated liposomal substance or vehicle, a micelle, a structure having a phospholipid bilayer, a cationic polymer, or polyethyleneimine (PEI), or any combination thereof.

6. 2. The method of claim 1, wherein when the reagent is a nucleic acid, the transfection process results in transient expression, or when the reagent is a nucleic acid, the transfection process results in stable expression, the method further comprising the steps of isolating one or more of the eukaryotic cells after the transfection process, testing the level of expression of one or more peptides encoded by the reagent in the one or more isolated eukaryotic cells or their progeny, and selecting the one or more isolated eukaryotic cells or their progeny based on the expression level.

7. 10. The method of claim 1, wherein the step of directing a pulsed electromagnetic signal is carried out for a given period of time, the given period being 15 minutes when the pulsed electromagnetic signal is directed at the reagent and / or 1 to 4 hours when the pulsed electromagnetic signal is directed at a transfection mixture during or after transfection.

8. 2. The method of claim 1, wherein the pulsed electromagnetic signal is generated by one or more electronic devices, the one or more electronic devices including transmission means or one or more electronic transmission chips that, in use, generate and / or transmit the pulsed electromagnetic signal from, each electronic device including a single transmission means or electronic chip.

9. 9. The method of claim 8, wherein the distance between the transmission means and the transfection mixture and / or the transfection complex that receives the pulsed electromagnetic signal in use is 25 cm or less, 20 cm or less, 15 cm or less, 10 cm or less, 5 cm or less, or equal to or less than 1 cm.

10. 2. The method of claim 1, wherein the given frequency of the pulsed electromagnetic signal is 2.4 GHz ± 50 MHz or 2.45 GHz ± 50 MHz.

11. 10. The method of claim 1, wherein the given pulse rate of the pulsed electromagnetic signal is 50 Hz or less, 25 Hz or less, 15 Hz or less, and / or has a duty cycle of less than 2%.

12. The method of claim 1, wherein each pulse of the pulsed electromagnetic signal lasts between 1 ms and 20 ms, or is less than 1 ms, and optionally the pause between each pulse of the pulsed electromagnetic signal lasts less than 66 ms.

13. The method of claim 1, wherein the pulsed electromagnetic signal is transmitted using Gaussian frequency shift keying (GFSK) between 0.45 and 0.

55.

14. 9. The method of claim 8, wherein the one or more electronic devices include any or any combination of control means for controlling the operation or one or more parameters of the electronic device or transmission means, power supply means for providing power to the one or more electronic devices in use, one or more circuit boards, memory means for storing data, user selection means for allowing a user to select the operation of the device, the one or more parameters, or display means for displaying one or more settings.

15. 15. The method of claim 14, wherein the one or more user-selectable parameters of the electronic device include any or any combination of signal frequency, signal strength, signal or transmission power, time duration of each pulse or rest period between signal pulses, and signal pulse rate of the pulsed electromagnetic signal.

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