Transfection methods

Abstract

In order to provide a novel means for safely and efficiently introducing a nucleic acid, protein, or other target substance into a T-cell or other immune cell, the present invention provides a system for delivering a target substance into an immune cell, the system comprising a combination of an ultrasonic wave generating device and an ultrafine-bubble aqueous solution or ultrafine-bubble water including ultrafine bubbles which have an average diameter of 200 nm or less and do not include a phospholipid. The present invention provides, inter alia, a method for increasing deliverability of a nucleic acid or a protein into an immune cell using ultrasonic waves and the aforementioned ultrafine-bubble water, etc.

Description

[Technical field]

[0001] The present invention relates to a system for delivering a target substance into immune cells, which comprises a combination of ultra-fine bubble water or an ultra-fine bubble aqueous solution containing ultra-fine bubbles and an ultrasound generator; a method for increasing the intracellular delivery of nucleic acids or proteins using the ultra-fine bubble water or aqueous solution and ultrasound; a preparation for delivering nucleic acids or proteins into immune cells in combination with ultrasound irradiation, which comprises a combination of nucleic acids or proteins and the ultra-fine bubble water or aqueous solution; and a method for delivering nucleic acids or proteins into immune cells by contacting immune cells with the preparation and treating them with ultrasound.

[0002] BACKGROUND OF THEINVENTION Ultrasound has been used primarily as an ultrasound contrast device in the medical field. Microbubbles, an ultrasound contrast agent, have brought about revolutionary advances in ultrasound diagnosis. Recently, ultrasound has become available for purposes other than diagnosis. For example, non-invasive cancer hyperthermia, which focuses ultrasound energy on the affected area to heat only the affected area, has been clinically applied to uterine fibroids and prostate cancer. In addition, focusing on its non-invasive nature and ease of spatial and temporal control, research is also being conducted on the use of ultrasound irradiation as a means of drug delivery system (DDS) to deliver genes and drugs to target cells (sonoporation).

[0003] So far, bubble liposomes, which are polyethylene glycol (PEG)-modified liposomes encapsulating perfluoropropane, have been reported as gene delivery systems to skeletal muscles that combine bubble liposomes and ultrasound technology (Non-Patent Document 1). In addition, a drug for treating Duchenne muscular dystrophy has been reported in which a PEG-modified bubble liposome (bubble lipopolyplex) with an average particle size of 50 to 500 nm, which has a specific morpholino oligomer bound to its surface and a perfluorohydrocarbon encapsulated inside, is administered into muscle tissue or blood vessels, and then the muscle tissue is irradiated with ultrasound from outside the body, thereby introducing the morpholino oligomer into muscle cells with high efficiency (Patent Document 1). Furthermore, it has been reported that gas-encapsulated microbubbles and ultrasound can be used to deliver drugs and genes to the brain (Non-Patent Document 2).

[0004] It has been reported that by using perfluoropropane gas as the encapsulating gas, bubble liposomes smaller in size can be obtained than those using perfluorobutane gas or nitrogen gas (Non-Patent Document 3). In addition, it has been reported that when bubble lipopolyplex, which combines nanobubbles and plasmids, was administered to ddY mice and the brain tissue was irradiated with ultrasound, the plasmid was introduced into the vascular endothelium or extravascular site, and the introduction site differed depending on the encapsulating efficiency of the gas (Non-Patent Document 4).

[0005] However, the use of bubble liposomes raises concerns about the antigenicity of lipids. In addition, ultrasonic irradiation requires 1.5 to 2.5 W / cm 2 The output intensity used in these ultrasound diagnostic devices raises safety concerns, making them unsuitable for practical use.

[0006] The inventors have used 2.0 × 10 8 It has been revealed that nanobubble water containing phospholipid-free nanobubbles at a concentration of 1 / mL or more has an excellent antibacterial effect (Patent Document 2). However, there have been no reports of the introduction of target substances such as nucleic acids and proteins into cells by combining the nanobubble water with ultrasound.

[0007] Meanwhile, research and development of cancer immunotherapy using CAR-T cells or TCR-T cells transfected with chimeric antigen receptors (CAR) or T cell receptors (TCR) derived from cancer antigen-specific killer T cells is progressing rapidly. Current CAR-T cell therapy, such as Kymriah (trade name) and Yescarta (trade name) approved in the United States, generally involves the ex vivo transduction of CAR genes into T cells collected from a patient using a viral vector such as a lentivirus vector to produce CAR-T cells, and then administering the CAR-T cells to the patient. However, this method has the problem that the manufacturing cost is high due to the costs involved in cell culture and preparation of viral vectors, etc. If CAR or exogenous TCR could be selectively introduced into immune cells such as T cells in vivo, ex vivo preparation would be unnecessary, making it possible to provide CAR- or TCR-immune cell therapy with low manufacturing costs. Furthermore, if it were possible to selectively introduce CAR or exogenous TCR into immune cells such as T cells ex vivo without using viral vectors, which have high manufacturing costs, costs associated with virus residual tests, etc. would be unnecessary, making it possible to provide CAR- or TCR-immune cell therapy with low manufacturing costs.

[0008] However, immune cells such as T cells undergo low cell division and are floating cells, so they are known to have poor transfection efficiency compared to other cells, and a more efficient method for introducing nucleic acids into immune cells is needed. To date, there have been no reports of selectively introducing nucleic acids (e.g., mRNA, DNA) encoding CAR or exogenous TCR into immune cells such as T cells using sonoporation. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] WO2012 / 153635 [Patent Document 2] WO2015 / 182647 [Non-patent literature]

[0010] [Non-Patent Document 1] YAKUGAKU ZASSHI 130(11) 1489-1496 (2010) [Non-Patent Document 2] NATURE REVIEWS 12 161-174 (2016) [Non-Patent Document 3] Drug Delivery 24(1) 320-327 (2017) [Non-Patent Document 4] "Evaluation of pharmacokinetics in the brain for brain-directed DDS using ultrasound-responsive nanobubbles," Yuki Fuchigami et al., Nagasaki University, Abstract of the 137th Annual Meeting of the Pharmaceutical Society of Japan (March 2017) Summary of the Invention [Problem to be solved by the invention]

[0011] An object of the present invention is to provide a novel means for safely and efficiently introducing a target substance such as a nucleic acid or a protein into immune cells such as T cells. [Means for solving the problem]

[0012] In order to achieve the above object, the present inventors have focused on the fact that the nanobubble water with excellent antibacterial properties that they previously developed (see Patent Document 2 above; the International Organization for Standardization (ISO) defines bubbles less than 1 μm (1000 nm) as "ultrafine bubbles" (ISO 20480-1), and therefore in this specification, the term "ultrafine bubbles" is used instead of "nanobubbles") does not contain phospholipids. The ultrafine bubbles contained in this ultrafine bubble water have an average diameter of 200 nm or less, which is smaller than conventional bubbles. Conventionally, it has been thought that ultrafine bubbles cause more severe damage to cell membranes than microbubbles when crushed, but the present inventors have dared to combine this ultrafine bubble water with ultrasonic irradiation to study the introduction of nucleic acids and proteins into T cells. As a result, it was unexpectedly shown that the ultrafine bubble water can dramatically increase the introduction efficiency of nucleic acids by sonoporation, and that the combination of the ultrafine bubble water and ultrasonic waves can also increase the introduction efficiency of proteins more significantly than either treatment alone. Based on these findings, the present inventors conducted further investigations and have now completed the present invention.

[0013] That is, the present invention provides [1] A system for delivering a target substance into immune cells, comprising ultra-fine bubble water or ultra-fine bubble aqueous solution containing phospholipid-free ultra-fine bubbles with an average diameter of 200 nm or less, in combination with an ultrasonic generator; [2] The system according to [1], wherein the ultra-fine bubble aqueous solution contains one or more components selected from one or more surfactants selected from anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants, a hydrophilic resin, and a buffer solution; [3] The system according to [1] or [2], wherein the ultra-fine bubble aqueous solution comprises a nonionic surfactant and / or a hydrophilic resin; [3-1] The system according to [1] or [2], wherein the ultra-fine bubble aqueous solution comprises an ionic surfactant and / or a hydrophilic resin; [4] The system according to any one of [1] to [3], wherein the ultrafine bubbles are composed of perfluorohydrocarbons or air; [5] The system according to any one of [1] to [4], wherein the average diameter of the ultra-fine bubbles is 50 nm to 200 nm; [6] The system according to any one of [1] to [5], wherein the d90 / d10 ratio of the ultrafine bubbles is 5 or less; [7] The density of ultra-fine bubbles in ultra-fine bubble water or ultra-fine bubble aqueous solution is 1.0×10 8 The system according to any one of [1] to [6], wherein the number of particles per mL is equal to or more than one. [8] The ultrasonic output intensity of the ultrasonic generator is 720mW / cm 2 A system according to any one of [1] to [7], [9] The ultrasonic output intensity of the ultrasonic generator is 50 to 500 mW / cm 2 and the ultrasonic frequency is 0.5 to 10 MHz;

[10] The system according to any one of [1] to [9], wherein the target substance is a nucleic acid or a protein;

[11] The system of

[10] , wherein the nucleic acid encodes a chimeric antigen receptor or an exogenous T cell receptor;

[12] The system according to any one of [1] to

[11] , wherein the immune cell is a T cell.

[13] The system according to

[11] or

[12] for delivering nucleic acids into immune cells;

[14] A method for enhancing intracellular delivery of a nucleic acid or a protein to immune cells by using ultra-fine bubble water or an ultra-fine bubble aqueous solution containing phospholipid-free ultra-fine bubbles having an average diameter of 200 nm or less, and ultrasound;

[15] A preparation for delivering a nucleic acid or a protein into immune cells, comprising a combination of a nucleic acid or a protein and ultra-fine bubble water or an ultra-fine bubble aqueous solution containing ultra-fine bubbles not containing phospholipids and having an average diameter of 200 nm or less, wherein an effective amount of the nucleic acid or the protein is delivered into immune cells in combination with ultrasonic irradiation;

[16] A method for delivering a nucleic acid or protein into an immune cell, comprising contacting the cell with the formulation according to

[15] and treating the cell with ultrasound; etc. Effect of the Invention

[0014] According to the system for delivering a target substance into immune cells of the present invention, a target substance such as a nucleic acid or a protein can be selectively delivered non-invasively into immune cells such as T cells by irradiating ultrasonic waves with an output intensity that does not adversely affect the living body, so that a highly safe delivery system can be provided. Since the system does not require the use of liposomes, it does not require the addition of special additives such as phospholipids, can be manufactured at low cost, and does not cause the problem of antigenicity caused by phospholipids. Furthermore, conventionally, ultrafine bubbles were thought to be unsuitable for cell transfection because they cause more severe damage to cell membranes when crushed than microbubbles. However, surprisingly, according to the present invention, the use of ultrafine bubbles can significantly increase the efficiency of introducing target substances into cells compared to the use of microbubbles. Furthermore, the ultra-fine bubbles with an average diameter of 200 nm or less are stable for a long period of time in ultra-fine bubble water or an aqueous solution of ultra-fine bubbles. [Brief description of the drawings]

[0015] [Figure 1]This is a diagram showing that the efficiency of plasmid transfer into T cells was improved by combining an ultra-fine bubble solution with ultrasound. The vertical axis indicates the fluorescence intensity of GFP per viable cell. US (ultrasound irradiation): - (none), + (present); Bubble (ultra-fine bubble): Cat (positive charge), An (negative charge), - (none) [Diagram 2] This is a diagram showing that the efficiency of protein delivery into T cells was improved by combining an ultra-fine bubble solution with ultrasound. The vertical axis shows the fluorescence intensity of GFP per viable cell. US (ultrasound irradiation): - (none), + (present); Bubble (ultra-fine bubble): Cat (positive charge), An (negative charge), - (none)

[0016] Detailed Description of the Invention I. The System of the Present Invention The present invention provides a system for delivering a target substance into immune cells (hereinafter also referred to as "the system of the present invention") which comprises a combination of ultra-fine bubble water or ultra-fine bubble aqueous solution containing phospholipid-free ultra-fine bubbles with an average diameter of 200 nm or less, and an ultrasonic generator.

[0017] As used herein, "delivery of a target substance into a cell" means that a substance that is inherently difficult to pass through a cell membrane (e.g., a water-soluble compound that is difficult to passively diffuse, a compound with a large molecular weight, or a compound that does not have a selective transporter or receptor) passes through the cell membrane and migrates into the cell. Therefore, the system of the present invention may deliver a target substance into a cell by any mechanism, such as, but not limited to, temporarily opening a hole in the cell membrane.

[0018] As used herein, "immune cells" refers to cells involved in immune responses, including, for example, lymphocytes such as T cells, B cells, natural killer (NK) cells, and natural killer T (NKT) cells, granulocytes such as neutrophils, eosinophils, and basophils, monocytes, macrophages, and dendritic cells, as well as unipotent or multipotent stem cells or progenitor cells that can be terminally differentiated into these (excluding embryonic and other totipotent or pluripotent stem cells). "Immune cells" may be a cell population containing any of the above cells, and may be an isolated specific immune cell or a heterogeneous cell population containing multiple types of immune cells, such as lymphocytes. The lymphocytes can be collected from, for example, peripheral blood, bone marrow, or umbilical cord blood of a human or non-human mammal, or can be produced by inducing differentiation from stem cells such as iPS cells using a method known per se. When isolated immune cells (e.g., T cells) into which a substance of interest has been introduced according to the present invention are used for the treatment of a disease such as cancer, the cells are preferably collected from the subject of treatment or a donor whose MHC type matches that of the subject of treatment. Alternatively, the lymphocytes may be lymphocytes present in an animal individual.

[0019] In a preferred embodiment, the "immune cells" are isolated and purified T cells. In another preferred embodiment, the immune cells may be a cell population containing T cells, such as T cells present in an animal. As used herein, "T cells" refers to a type of white blood cell found in lymphoid organs or peripheral blood, etc., and refers to a classification of lymphocytes characterized by differentiating and maturing mainly in the thymus and expressing TCR. Examples of T cells that can be used in various embodiments of the present invention include cytotoxic T cells (CTLs) that are CD8 positive cells, helper T cells, regulatory T cells, effector T cells, etc. that are CD4 positive cells, and are preferably cytotoxic T cells.

[0020] The target substance delivered into the immune cell using the system of the present invention is not particularly limited as long as it is a substance that can impart a desired physiological activity to the immune cell by being delivered into the immune cell, and examples thereof include, but are not limited to, polymer compounds [e.g., nucleic acids, such as small RNA / DNA such as siRNA (e.g., FAM-siRNA, manufactured by Nippon Gene), ssRNA, shRNA, miRNA, S-oligo DNA (phosphorothioate, etc.), genes (e.g., plasmid DNA, mRNA, etc.), proteins (including peptides) (e.g., antibodies (e.g., IgG (e.g., Alexa-IgG, manufactured by Invitrogen)), etc.), polysaccharides (e.g., dextran, fluorescein isothiocyanate dextran, etc.)], low molecular weight compounds (e.g., fluorescein, sodium fluorescein, etc.), etc. Among these, polymer compounds and low molecular weight compounds are preferred. Polymer compounds are more preferred. Nucleic acids and proteins are even more preferred. Nucleic acids and antibodies are particularly preferred.

[0021] In this specification, "ultra-fine bubbles" may contain gas at normal atmospheric pressure or higher, or the inside of the ultra-fine bubbles may be a vacuum. Here, "vacuum" refers to a state in which the space is filled with gas at a pressure lower than normal atmospheric pressure. In the present invention, "ultra-fine bubbles not containing phospholipids" refer to ultra-fine bubbles whose bubble shells do not form a phospholipid bilayer structure. In the present invention, "ultra-fine bubble water" refers to water containing ultra-fine bubbles. In the present invention, the term "ultra-fine bubble aqueous solution" refers to an aqueous solution containing ultra-fine bubbles. The aqueous solution constituting the ultra-fine bubble aqueous solution is, for example, 1) one or more surfactants selected from anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants; 2) Hydrophilic resin and 3) Buffer solution It contains one or more substances selected from the above as ingredients.

[0022] In the present invention, examples of the "anionic surfactant" include sodium lauryl sulfate. In the present invention, examples of the "nonionic surfactant" include glycerin fatty acid esters (e.g., glycerin monostearate, etc.), sucrose fatty acid esters, sorbitan fatty acid esters (e.g., sorbitan monostearate, sorbitan monolaurate, etc.), polyglycerin fatty acid esters, polyoxyethylene (hydrogenated) castor oil, polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan laurate esters (e.g., polysorbate 20, etc.), polyoxyethylene sorbitan oleate esters (e.g., polysorbate 80, etc.), etc. ), polyethylene glycol fatty acid ester, polyoxyethylene alkyl ether (e.g., polyoxyethylene lauryl ether, etc.), polyoxyethylene polyoxypropylene alkyl ether (e.g., polyoxyethylene polyoxypropylene cetyl ether, etc.), polyoxyethylene alkyl phenyl ether (e.g., polyoxyethylene nonyl phenyl ether, etc.), macrogols, polyoxyethylene polyoxypropylene glycol (e.g., poloxamer 407, poloxamer 235, poloxamer 188, poloxamine, etc.). Among them, polyoxyethylene sorbitan laurate (e.g., polysorbate 20, etc.) and polyoxyethylene sorbitan oleate (e.g., polysorbate 80, etc.) are preferred. Polysorbate 20 or polysorbate 80 is more preferred. Polysorbate 80 is particularly preferred. In the present invention, examples of the "cationic surfactant" include benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, hexadecyltrimethylammonium bromide, dequalinium chloride, and the like. In the present invention, examples of the "amphoteric surfactant" include cocamidopropyl betaine, cocamidopropyl hydroxysultaine, and the like. The above surfactants may be used alone or in combination of two or more kinds.

[0023] In the present invention, examples of the "hydrophilic resin" include acrylic resins (e.g., polyacrylamide, polyacrylic acid, polymethyl methacrylate), vinyl resins (e.g., polyvinylpyrrolidone, polyvinyl alcohol (PVA), polyvinyl ethyl ether); and polysaccharides (e.g., tragacanth gum, karaya gum, methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, hyaluronic acid, agarose, curdlan, etc.). Among these, polyvinyl alcohol and hydroxypropyl cellulose are preferred. Polyvinyl alcohol is more preferred. The above hydrophilic resins may be used alone or in combination of two or more kinds.

[0024] In the present invention, examples of the "buffer solution" include acidic buffer solutions (e.g., acetate buffer solution, citrate buffer solution, diluted McLivaine's buffer solution) and neutral buffer solutions (e.g., 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution, tris(hydroxymethyl)aminomethane (Tris) buffer solution, phosphate buffer solution, phosphate buffered saline (PBS)). Dilute McLivaine's buffer solution is preferred as the buffer solution.

[0025] The aqueous solution constituting the "ultra-fine bubble aqueous solution" in the present invention is preferably an aqueous solution composed of 1) one or more surfactants selected from an anionic surfactant, a nonionic surfactant, a cationic surfactant, and an amphoteric surfactant, and / or 2) a hydrophilic resin. Among them, an aqueous solution composed of a nonionic surfactant and / or a hydrophilic resin is preferable. Alternatively, an aqueous solution composed of a nonionic surfactant is also preferable. More preferably, an aqueous solution composed of 1) one or two surfactants selected from polysorbate 80 and polysorbate 20, and / or 2) polyvinyl alcohol is preferable. More preferably, an aqueous solution composed of polysorbate 80 and / or polyvinyl alcohol is preferable. In particular, an aqueous solution composed of polysorbate 80 is preferable. In another preferred embodiment, the aqueous solution constituting the "ultra-fine bubble aqueous solution" may be an aqueous solution containing an ionic (anionic or cationic) surfactant and / or a hydrophilic resin.

[0026] In the present invention, examples of the "gas" constituting ultrafine bubbles include, but are not limited to, one or a mixture of two or more selected from perfluorohydrocarbons (e.g., perfluoropropane (C3F8), perfluorobutane, etc.), air, nitrogen, ozone, oxygen, argon, carbon dioxide, helium, etc. Among these, preferred are perfluorohydrocarbons (e.g., perfluoropropane, perfluorobutane, etc.), air, nitrogen, ozone, oxygen, and argon. More preferred are perfluorohydrocarbons (e.g., perfluoropropane, perfluorobutane, etc.) and air. When air is used, ultrafine bubbles can be produced cheaply and easily. More preferred is air.

[0027] The "ultra-fine bubble aqueous solution" in the present invention is preferably an ultra-fine bubble aqueous solution consisting of (A) an aqueous solution of a nonionic surfactant and / or a hydrophilic resin, and (B) ultra-fine bubbles composed of one or more types of gas selected from perfluorohydrocarbons, air, etc. More preferably, there is an ultra-fine bubble aqueous solution consisting of (A) an aqueous solution of 1) one or two types selected from polysorbate 80 and polysorbate 20 and / or 2) polyvinyl alcohol, and (B) ultra-fine bubbles composed of one or two types of gas selected from perfluorohydrocarbons and air. More preferably, there is an ultra-fine bubble aqueous solution consisting of (A) an aqueous solution of polysorbate 80 and / or polyvinyl alcohol, and (B) ultra-fine bubbles composed of perfluorohydrocarbons and / or air. In particular, there is an ultra-fine bubble aqueous solution consisting of an aqueous solution of polysorbate 80 and ultra-fine bubbles composed of air.

[0028] The "ultra-fine bubbles" of the present invention do not contain amphipathic phospholipids such as liposomes, and therefore do not exhibit antigenicity, making it possible to provide a safer formulation.

[0029] In the present invention, the average diameter of the "ultra-fine bubbles" is about 200 nm or less. The preferred average diameter is, for example, 10 nm to 200 nm, more preferably 50 nm to 200 nm, and even more preferably 100 nm to 180 nm. In this specification, the term "average diameter" refers to the particle diameter (mode diameter) corresponding to the mode of the distribution (maximum value in terms of number percentage). In this specification, "ultra-fine bubble water" or "ultra-fine bubble aqueous solution" refers to water or an aqueous solution in which gas particles (ultra-fine bubbles) with a diameter of 1000 nm or less exist stably. The ultra-fine bubble water or ultra-fine bubble aqueous solution of the present invention (hereinafter also referred to as "ultra-fine bubble water, etc. of the present invention") is characterized by containing ultra-fine bubbles with an average diameter of approximately 200 nm or less.

[0030] In the present invention, it is desirable that the size of ultra-fine bubbles is uniform. For example, when the diameters of ultra-fine bubbles corresponding to the cumulative 10% and cumulative 90% from the small diameter side of the ultra-fine bubble number-based distribution are defined as d10 and d90, respectively, the "d90 / d10 ratio" is preferably 5 or less, and more preferably 4.5 or less.

[0031] In the present invention, the number of ultra-fine bubbles contained in ultra-fine bubble water, etc. means the number of ultra-fine bubbles present in 1 mL of ultra-fine bubble water or ultra-fine bubble aqueous solution, and is sometimes referred to as "ultra-fine bubble density" in this specification. There is no particular limit to the number of ultra-fine bubbles contained in ultra-fine bubble water, etc. in the present invention. The lower limit of the "ultra-fine bubble density" is, for example, 1.0 x 10 8 / mL or more, preferably 2.0 × 10 8 / mL or more, more preferably 2.5 × 10 8 The upper limit of "ultra-fine bubble density" is, for example, 2.0 x 10 9 / mL or less, preferably 1.0 × 10 9 The "ultra-fine bubble density" in the present invention is, for example, 1.0 × 10 8 ~2.0×10 9 / mL, preferably 2.0 × 10 8 ~1.0×10 9 / mL, more preferably 2.5 × 10 8 ~1.0×10 9pieces / mL.

[0032] The ultrafine bubble diameter (including the average diameter of ultrafine bubbles; the same applies below), the ultrafine bubble number distribution (including the d90 / d10 ratio; the same applies below), and the number of ultrafine bubbles can be measured using a method that uses laser light scattering based on Brownian motion (e.g., Nanosite, LM20 or LM10, etc.), a method that uses electrical resistance changes (e.g., Beckman Coulter, Multisizer4, etc.), a method that uses laser diffraction scattering (e.g., Shimadzu, SALD-7100H, etc.), a method that uses Mie scattering (e.g., Nippon Denshoku Industries, NP-500T, etc.), etc. In the present invention, the ultrafine bubble diameter and the ultrafine bubble number distribution are measured using a tracking method that uses laser light scattering using Nanosite (instrument name LM10) manufactured by Nanosite, or a method measured in accordance therewith. The values ​​of the ultra-fine bubble diameter, the standard distribution of the number of ultra-fine bubbles and the number of ultra-fine bubbles may usually be measured immediately after production of ultra-fine bubble water, etc., or may be measured after long-term storage. Since the ultra-fine bubble diameter, the standard distribution of the number of ultra-fine bubbles and the number of ultra-fine bubbles in the ultra-fine bubble water of the present invention are stably maintained for an extremely long period of time (for example, about 6 months to 2 years), the ultra-fine bubble diameter, the standard distribution of the number of ultra-fine bubbles and the number of ultra-fine bubbles may be values ​​measured after the ultra-fine bubble water is produced and stored in a sealed state for a certain period of time, and immediately before use.

[0033] In this specification, the "water" containing ultra-fine bubbles (and the "water" used in the "aqueous solution" containing ultra-fine bubbles) is not particularly limited, and examples of the water that can be used include tap water, deionized water, distilled water, sterile distilled water, purified water for injection, and ultrapure water. When the water is used as an injection, sterile distilled water, purified water for injection, and the like are preferred.

[0034] In the present specification, the "aqueous solution" containing ultra-fine bubbles includes water containing one or more surfactants selected from the above-mentioned anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants, hydrophilic resins, and one or more components selected from buffers, as well as any additives that may be commonly used in the field of pharmaceutical formulations. Examples of the "additives" include electrolytes, excipients, lubricants, binders, disintegrants, solubilizers, suspending agents, dispersing agents, isotonicity agents, soothing agents, preservatives, antioxidants, colorants, sweeteners, pH adjusters, stabilizers, acidulants, flavorings, and fluidizing agents. Pharmacologically acceptable additives are preferred, and one or more additives selected from suspending agents, stabilizers, dispersing agents, isotonicity agents, and the like are more preferred. The above-mentioned additives may be used in a mixture of two or more kinds in an appropriate ratio. These additives (including one or more surfactants selected from anionic surfactants, nonionic surfactants, cationic surfactants and amphoteric surfactants, hydrophilic resins and buffers) can be dissolved in water in advance to directly prepare an ultra-fine bubble aqueous solution, as long as they do not affect the generation and stability of ultra-fine bubbles. Alternatively, ultra-fine bubbles can be generated in additive-free water to prepare ultra-fine bubble water, and then the additives can be dissolved therein at the time of use to prepare an ultra-fine bubble aqueous solution. As the aqueous solution, any of uncharged ultra-fine bubble aqueous solution, positively charged ultra-fine bubble aqueous solution, and negatively charged ultra-fine bubble aqueous solution can be used, and can be appropriately selected according to the type of target immune cells, the surrounding microenvironment, the type of target substance, etc. In the method of the present invention for delivering a target substance into immune cells using an ultra-fine bubble aqueous solution and ultrasound, for example, when the target substance is a polymeric compound (e.g., nucleic acid, protein, etc.), the ultra-fine bubble aqueous solution may be preferably negatively charged. In another aspect, in the method of the present invention for delivering a target substance into immune cells using an ultra-fine bubble aqueous solution and ultrasound, when the target substance is a polymeric compound, the ultra-fine bubble aqueous solution may be preferably positively charged. The charge of the ultra-fine bubble aqueous solution can be adjusted as appropriate, for example, by adjusting the pH of the buffer solution used. For example, when the ultra-fine bubble aqueous solution is to be positively charged, the pH of the ultra-fine bubble aqueous solution is preferably, for example, 1 to 4. On the other hand, when the ultra-fine bubble aqueous solution is to be negatively charged, the pH of the ultra-fine bubble aqueous solution is preferably, for example, 7 to 14.

[0035] Methods for producing ultra-fine bubble water are roughly divided into two types: one in which microbubbles (gas particles with a diameter of about 1 to 60 μm) and ultrafine bubbles are simultaneously generated in water, and then the microbubbles are floated and separated, leaving only the ultrafine bubbles, and the other in which ultrafine bubbles are generated directly. The former type is currently the mainstream. Examples of the former type include a high-speed swirling liquid flow type in which gas is crushed by high-speed swirling to generate a large number of microbubbles, which are then floated and separated, leaving the ultrafine bubbles in the water, and a pressurized dissolution type in which gas is pressurized and dissolved in a supersaturated liquid, which is rapidly depressurized to generate microbubbles and ultrafine bubbles, which are then floated and separated, leaving the ultrafine bubbles in the water. The method for producing an aqueous solution of ultrafine bubbles is similar to that described above.

[0036] The method for producing ultra-fine bubble water or ultra-fine bubble aqueous solution in the present invention is preferably a pressurized dissolution method. For example, the following steps 1) to 3) can be used; 1) in a pressurized container pressurized to about 0.2 to 0.5 MPa by a pressure pump, gas is forcibly dissolved in a liquid in which (i) one or more surfactants selected from anionic surfactants, nonionic surfactants, cationic surfactants and amphoteric surfactants, (ii) a hydrophilic resin and / or (iii) a buffer solution are dissolved; 2) the gas is flushed into water through a nozzle, and the reduced pressure and supersaturated gas is released as microbubbles or ultrafine bubbles into the wastewater, producing a mixture of microbubble water and ultrafine bubble water; 3) the aeration is stopped and the mixture is left to stand, allowing the microbubbles to rise and separate naturally. This produces clear ultra-fine bubble water in which only ultrafine bubbles remain.

[0037] The ultra-fine bubble generator used in the production of ultra-fine bubble water or ultra-fine bubble aqueous solution in the present invention is, for example, a pressurized dissolution type device (e.g., IDEC's nanoGALF TM , OM4-MD5-045 manufactured by Auratec, Microbubble Generator manufactured by Nikuni, etc.), high-speed swirling liquid flow type device (e.g., YJ manufactured by Bayclean, Microbubble Generator manufactured by Aquaair, Microblade manufactured by Royal Electric, etc.), etc. As the ultra-fine bubble generator, a pressurized dissolution type device (e.g., nanoGALF manufactured by IDEC, TM ).

[0038] In the present invention, the number of ultra-fine bubbles in the ultra-fine bubble water or ultra-fine bubble aqueous solution can be increased by using one or more surfactants selected from anionic surfactants, nonionic surfactants, cationic surfactants and amphoteric surfactants, a hydrophilic resin and / or a buffer solution in the production of the ultra-fine bubble water or ultra-fine bubble aqueous solution.

[0039] The content of one or more surfactants selected from anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants, hydrophilic resins, and / or buffers in water used in the present invention is not particularly limited. The upper limit is preferably 50% (W / V) or less, more preferably 20% (W / V) or less, and even more preferably 10% (W / V) or less. The lower limit is preferably 0.01% (W / V) or more, more preferably 0.05% (W / V) or more, and even more preferably 0.1% (W / V) or more. Here, (W / V) means g / mL. When two or more of the surfactants, hydrophilic resins and buffers are used in combination, the total amount is defined as the content in water.

[0040] The ultra-fine bubble water or the like of the present invention produced as described above is stored in a sealed vial or ampoule, preferably under light-shielded conditions, at a storage temperature of preferably room temperature or lower, more preferably 10°C or lower.

[0041] The ultra-fine bubble water or ultra-fine bubble aqueous solution of the present invention is preferably produced in the presence of one or more surfactants selected from anionic surfactants, nonionic surfactants, cationic surfactants and amphoteric surfactants, a hydrophilic resin and / or a buffer solution, and therefore the number of ultra-fine bubbles in the ultra-fine bubble water, etc. of the present invention can be maintained for a period during which the action and effect of the ultra-fine bubble water, etc. of the present invention (e.g., the cell membrane perforation effect of immune cells when ultra-fine bubble water, etc. is used in combination with ultrasonic treatment, the effect of increasing the delivery of a target substance into immune cells by using ultra-fine bubble water, etc. and ultrasonic waves) is required to be maintained (for example, when used as a base for a target substance that is desired to be delivered through a cell membrane, the effective period (e.g., 3 months or more, more preferably 6 months or more, and even more preferably 1 year or more)). 8 It can be maintained at more than 100 cells / mL. The ultra-fine bubble water of the present invention can be heat sterilized, and even after heat sterilization, the number of ultra-fine bubbles remains below 1.0 x 10 8 It can be maintained at more than 100 cells / mL.

[0042] The ultra-fine bubble water of the present invention has a bubble density of 1.0×10 8 When the number of ultra-fine bubbles is more than 1 / mL, the liquid pharmaceutical preparation exhibits excellent antibacterial activity and therefore preservative effectiveness, and is therefore also useful as a base for multiple-dose liquid pharmaceutical preparations that are used repeatedly from the same container over a certain period of time, such as injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, infusions, intracerebral injections, cerebrospinal fluid injections, intraocular injections, etc.).

[0043] The ultra-fine bubble water, etc. of the present invention is preferably produced in the presence of one or more surfactants selected from anionic surfactants, nonionic surfactants, cationic surfactants and amphoteric surfactants (preferably polysorbate 80 and / or polysorbate 20, more preferably polysorbate 80). In the ultra-fine bubble water, etc. produced, the average diameter of the ultra-fine bubbles is smaller and the size is more uniform (i.e., the d90 / d10 ratio is smaller), so that the system of the present invention can achieve a safer delivery effect of the target substance into immune cells.

[0044] Any "ultrasonic generator" can be used in the system of the present invention, as long as it can generate ultrasonic waves sufficient for delivering a target substance into immune cells when used in combination with the ultra-fine bubble water of the present invention, etc. For example, any device that has been conventionally used for ultrasonic diagnosis in medical settings, or a commercially available ultrasonic gene transfer device (e.g., Sonitron GTS (manufactured by Nepa Gene Co., Ltd.), etc.) can be appropriately used.

[0045] The conditions of the "ultrasonic wave generator" in the present invention include, for example, the output strength of the ultrasonic wave (per unit area (cm 2) per unit time) is 10 mW or more, preferably 30 mW or more, and more preferably 50 mW or more. There is no particular upper limit to the output intensity, but when the system of the present invention is intended for in vivo transfection of immune cells in mammals, including humans, it is preferable to set the upper limit at a range that does not have adverse effects (e.g., cytotoxicity) on the animal. For example, the third-party certification standard (2005) of the revised Pharmaceutical Affairs Law states that "720 mW / cm 2 (the same as the upper limit of the US FDA Track 3), and ultrasound diagnostic devices in Japan are controlled so as not to exceed this upper limit. Therefore, preferably, the upper limit of the ultrasound output intensity in the system of the present invention is 720 mW / cm 2 The output intensity of the ultrasonic wave is preferably 50 to 720 mW / cm. 2 , and more preferably 50 to 500 mW / cm 2 The output intensity of ultrasound used for conventional gene transfer is significantly higher than the above-mentioned standard for ultrasound diagnostic use (e.g., 1.5 to 2.5 W / cm 2 ) There is a large risk to safety, but in the system of the present invention, a small output intensity (preferably 50 to 500 mW / cm 2 ) is an extremely safe delivery system that can efficiently deliver target substances into immune cells regardless of the molecular weight of the target substance.

[0046] As a condition of the "ultrasonic generator" in the present invention, the frequency of the ultrasonic waves is not particularly limited and can be appropriately selected within the range of, for example, 0.5 to 10 MHz. The currently widely adopted frequency is about 1 MHz, but since higher frequencies are thought to have less adverse effects on living organisms, the frequency can be appropriately selected within the range of preferably 1 to 5 MHz, more preferably 1 to 3 MHz.

[0047] As a condition for using the "ultrasonic generator" of the present invention, the ultrasonic irradiation time is not particularly limited as long as it is a time sufficient to deliver the target substance into immune cells, and may vary depending on the output intensity of the ultrasonic wave. For example, the output intensity is 50 mW / cm 2Even if the ultrasonic irradiation time is 10 seconds, the target substance can be delivered into the immune cells. The ultrasonic irradiation time can be, for example, 1 to 60 seconds, preferably 1 to 30 seconds, and more preferably 1 to 20 seconds. The conditions for using the "ultrasonic generator" of the present invention include, for example, (i) an ultrasonic output intensity of 50 to 720 mW / cm 2 (Preferably 50 to 500 mW / cm 2 ), (ii) an ultrasonic frequency of 0.5 to 10 MHz, and (iii) an ultrasonic irradiation time of 1 to 60 seconds. Among these, the combination of (i) an ultrasonic output intensity of 50 to 500 mW / cm 2 (ii) an ultrasonic frequency of 1 to 5 MHz and (iii) an ultrasonic irradiation time of 1 to 60 seconds.

[0048] When the system of the present invention is used for transfection of immune cells isolated from an animal or immune cells induced to differentiate from stem cells such as iPS cells using a method known per se, the system may include a step of culturing the immune cells after the transfection treatment. Therefore, the system of the present invention may further include a means for culturing the immune cells (e.g., a culture vessel (e.g., a dish, a flask, etc.), a medium, a culture device (e.g., a CO2 incubator, etc.)). Since immune cells grow in a suspended state, the culture vessel is preferably one coated with a non-adhesive or low-adhesive substrate. The above culture step also includes a subculture step. When cells are subcultured by the system of the present invention, the expression intensity of the target substance introduced into the cells can be maintained.

[0049] II. The Deliverability Enhancement Method of the Present Invention The present invention also provides a method for enhancing delivery of nucleic acids or proteins into immune cells using ultra-fine bubble water or an ultra-fine bubble solution containing phospholipid-free ultra-fine bubbles with an average diameter of 200 nm or less, and ultrasound (hereinafter also referred to as the "delivery enhancement method of the present invention"). The method comprises administering the ultra-fine bubble water of the present invention to a subject to deliver it to the vicinity of immune cells, and irradiating the immune cells with ultrasound, thereby delivering the nucleic acid or protein into the immune cells. Here, "immune cells" has the same meaning as defined in the above-mentioned system of the present invention.

[0050] The ultra-fine bubble water or ultra-fine bubble aqueous solution used in the "method for enhancing delivery of the present invention" may be the ultra-fine bubble water, etc., of the present invention described above. Furthermore, ultrasonic irradiation in the "method for enhancing delivery of the present invention" may be carried out under the same conditions as those for use of the ultrasonic generator in the system of the present invention described above.

[0051] The nucleic acid or protein whose deliverability into immune cells is increased by the delivery enhancing method of the present invention is not particularly limited, and may be, for example, a substance that can impart a desired physiological activity to the immune cell by being delivered into the immune cell. For example, examples of the nucleic acid include small RNA / DNA such as siRNA (e.g., FAM-siRNA, manufactured by Nippon Gene), ssRNA, shRNA, miRNA, S-oligo DNA (phosphorothioate, etc.), and genes (e.g., plasmid DNA, mRNA, etc.). Examples of the protein include, but are not limited to, antibodies (e.g., IgG (e.g., Alexa-IgG, manufactured by Invitrogen), etc.), peptides, etc. Among them, preferred examples of the nucleic acid include nucleic acids encoding chimeric antigen receptors (CAR) or exogenous T cell receptors (TCR), and examples of the protein include antibodies, etc.

[0052] The number of nucleotides in the nucleic acid and the molecular weight of the protein are not particularly limited, but for example, when the nucleic acid is a small RNA / DNA such as siRNA or antisense oligonucleic acid, it can be 10mer to 30mer, preferably 15mer to 25mer, when the nucleic acid is a nucleic acid (plasmid DNA) encoding CAR or TCR, it can be 1 to 10kb, preferably 2 to 8kb, and when the protein is a protein, it can be 25kDa to 900kDa, preferably 25kDa to 320kDa.

[0053] In a preferred embodiment, the nucleic acid is a nucleic acid encoding a CAR or an exogenous TCR. The nucleic acid encoding a CAR or an exogenous TCR is described in detail below.

[0054] (a) a nucleic acid encoding a CAR CAR is an artificially constructed hybrid protein that contains an antigen-binding domain of an antibody (e.g., scFv) linked to a T cell signaling domain. The characteristics of CAR include the ability to utilize the antigen-binding properties of monoclonal antibodies to redirect the specificity and reactivity of T cells to selected targets in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition gives CAR-expressing T cells the ability to recognize antigens independent of antigen processing, thereby bypassing a major mechanism of tumor escape. Moreover, when expressed in T cells, CAR advantageously does not dimerize with endogenous TCR α and β chains.

[0055] The CAR used in the present invention comprises an antigen-binding domain of an antibody capable of specifically recognizing a surface antigen (e.g., a cancer antigen peptide, a surface receptor whose expression is enhanced in cancer cells, etc.) to be recognized by a target immune cell (e.g., T cell, NK cell, NKT cell, monocyte, macrophage, dendritic cell, etc.), an extracellular hinge domain, a transmembrane domain, and an intracellular T cell signaling domain.

[0056] Examples of surface antigens that the antigen-binding domain specifically recognizes include various cancers (e.g., acute lymphocytic carcinoma, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal, anal canal, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical, gallbladder, or pleural cancer, nasal, nasal cavity, or middle ear cancer, oral cancer, vulva cancer, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia (e.g., acute lymphoblastic leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myelogenous ... leukemia), liquid tumors, liver cancer, lung cancer (e.g., non-small cell lung cancer), lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma), malignant mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer; peritoneal, omental and mesenteric cancer; pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumors, gastric cancer, testicular cancer, thyroid cancer, ureteral cancer, etc.) surface receptors with increased expression, such as CD19, EGF receptor, BCMA, CD30, Her2, ROR1, MUC16, CD20, mesothelin, B-cell Examples of antigens include, but are not limited to, cancer antigen peptides (e.g., peptides derived from WT1, GPC3, MART-1, gp100, NY-ESO-1, MAGE-A4, etc.), and the like.

[0057] The antigen-binding domain used in the present invention is not particularly limited as long as it is an antibody fragment capable of specifically recognizing a target antigen, but considering the ease of CAR production, it is preferable to use a single-chain antibody (scFv) in which a light chain variable region and a heavy chain variable region are linked via a linker peptide. The arrangement of the light chain variable region and the heavy chain variable region in a single-chain antibody is not particularly limited as long as both can reconstitute a functional antigen-binding domain, but can usually be designed in the order of light chain variable region-linker peptide-heavy chain variable region from the N-terminus. As the linker peptide, a publicly known linker peptide that is usually used in the production of single-chain antibodies can be used. The DNA encoding the light chain variable region and the DNA encoding the heavy chain variable region can be prepared, for example, by cloning the light chain gene and the heavy chain gene from an antibody-producing cell, respectively, and performing PCR using them as a template, or can be chemically synthesized from the sequence information of an existing antibody. The DNA encoding the single-chain antibody can be obtained by ligating each of the obtained DNA fragments and the DNA encoding the linker peptide by an appropriate method. It is preferable that a leader sequence is further added to the N-terminus of the antigen-binding domain in order to present the CAR on the surface of immune cells.

[0058] As the extracellular hinge domain and transmembrane domain, any domain derived from a T cell surface molecule commonly used in the art can be used as appropriate, including, but not limited to, domains derived from CD8α and CD28.

[0059] Examples of intracellular signaling domains include those having a CD3ζ chain, those having an additional costimulatory signaling motif such as CD28, CD134, CD137, Lck, DAP10, ICOS, or 4-1BB between the transmembrane domain and the CD3ζ chain, and those having two or more costimulatory signaling motifs, but are not limited to these, and any domain commonly used in the art can be used in combination.

[0060] Nucleic acid sequence information encoding the extracellular hinge domain, transmembrane domain, and intracellular signaling domain is well known in the art, and a person skilled in the art can easily obtain DNA fragments encoding each domain from T cells based on that information. The DNA fragments thus obtained, each encoding the antigen-binding domain, the extracellular hinge domain, the transmembrane domain, and the intracellular signaling domain, can be ligated by standard methods to obtain DNA encoding a CAR.

[0061] The obtained DNA encoding CAR can be inserted into an expression vector, preferably a plasmid vector, containing a promoter functional in T cells, either as is or after adding an appropriate linker and / or nuclear localization signal, etc. Examples of promoters functional in T cells include, but are not limited to, constitutive SRα promoters in mammalian cells, SV40 promoters, LTR promoters, CMV (cytomegalovirus) promoters, RSV (Rous sarcoma virus) promoters, MoMuLV (Moloney murine leukemia virus) LTRs, and HSV-TK (herpes simplex virus thymidine kinase) promoters. Gene promoters such as CD3, CD4, and CD8 that are specifically expressed in T cells can also be used.

[0062] RNA encoding a CAR, preferably mRNA, can be prepared by transcribing an expression vector containing DNA encoding the CAR as a template into mRNA in an in vitro transcription system known per se.

[0063] (b) a nucleic acid encoding an exogenous TCR As used herein, the term "T cell receptor (TCR)" refers to a receptor that is composed of a dimer of TCR chains (α chain, β chain) and recognizes an antigen or the antigen-HLA (human leukocyte antigen) (MHC; major histocompatibility complex) complex to transmit a stimulatory signal to a T cell. Each TCR chain is composed of a variable region and a constant region, and the variable region has three complementarity determining regions (CDR1, CDR2, CDR3). The TCR used in the present invention includes not only TCRs in which the α chain and β chain of TCR constitute a heterodimer, but also TCRs in which the α chain and β chain constitute a homodimer. Furthermore, the TCR also includes TCRs in which a part or all of the constant region is deleted, TCRs in which the amino acid sequence is modified, and TCRs in which the constant region is soluble. The term "exogenous TCR" means exogenous to the T cell, which is the target cell in the present invention. The amino acid sequence of the exogenous TCR may be the same as or different from the endogenous TCR expressed by the T cell, which is the target cell in the present invention.

[0064] The nucleic acid encoding a TCR used in the present invention is a nucleic acid encoding the α and β chains of a TCR capable of specifically recognizing a surface antigen (e.g., a cancer antigen peptide, etc.) to be recognized by a target T cell. The nucleic acid can be prepared by a method known per se. When the amino acid sequence or nucleic acid sequence of the target TCR is known, it is possible to construct a DNA encoding the full length or a part of the TCR of the present invention based on the sequence, for example, by chemically synthesizing a DNA strand or an RNA strand, or by connecting synthesized short oligo-DNA strands that overlap each other using the PCR method or the Gibson Assembly method.

[0065] When the sequence of the TCR of interest is not known, for example, the T cells of interest can be isolated from a cell population containing T cells expressing the TCR of interest, and a nucleic acid encoding the TCR can be obtained from the T cells. Specifically, a cell population (e.g., PBMC) containing T cells is collected from a living body (e.g., human), and the cell population is cultured while being stimulated in the presence of an epitope of a cell surface antigen recognized by the TCR of interest. From the cell population, T cells that specifically recognize cells expressing the cell surface antigen can be selected by a known method using the specificity for cells expressing the cell surface antigen and cell surface antigens such as CD8 and CD4 as indicators. The specificity of T cells for cells expressing the surface antigen can be measured, for example, using a dextromer assay, an ELISPOT assay, or a cytotoxicity assay. The cell population containing the T cells is preferably collected, for example, from a living body having a large number of cells expressing a cell surface antigen recognized by the target TCR (e.g., a patient with a disease such as cancer, or a T cell-containing population contacted with an epitope of the antigen or dendritic cells pulsed with the epitope).

[0066] The nucleic acid of the present invention can be obtained by extracting DNA from the isolated T cells by a conventional method, amplifying the TCR gene based on the nucleic acid sequence of the constant region of the TCR using the DNA as a template, and cloning it. Alternatively, RNA can be extracted from the cells by a conventional method, synthesizing cDNA, and using the cDNA as a template to perform 5'-RACE (Rapid amplification of cDNA ends) using antisense primers complementary to the nucleic acids encoding the constant regions of the TCR α chain and β chain, respectively. 5'-RACE may be performed by a known method, for example, using a commercially available kit such as SMART PCR cDNA Synthesis Kit (manufactured by Clontech). The obtained DNAs encoding the α chain and β chain of TCR can be inserted into an appropriate expression vector, similar to the DNA encoding the CAR. The DNA encoding the α chain and the DNA encoding the β chain may be inserted into the same vector or into separate vectors. When inserted into the same vector, the expression vector may express both chains polycistronically or monocistronically. In the former case, an intervening sequence allowing polycistronic expression, such as an IRES or FMV 2A, is inserted between the DNA encoding both strands. Furthermore, RNA, preferably mRNA, encoding each chain of the TCR can be prepared in the same manner as the RNA encoding the CAR, for example, using the expression vector as a template.

[0067] III. Formulations of the Invention The present invention also provides a preparation for delivering a nucleic acid or a protein into immune cells, comprising a combination of a nucleic acid or a protein with ultra-fine bubble water or an ultra-fine bubble aqueous solution containing phospholipid-free ultra-fine bubbles with an average diameter of 200 nm or less, the preparation being characterized in that an effective amount (an amount sufficient for immune cells to exert a desired effect) of the nucleic acid or the protein is delivered into immune cells by combining with ultrasonic irradiation (hereinafter also referred to as the "preparation of the present invention"). Here, "immune cells" has the same meaning as defined in the above-mentioned system of the present invention.

[0068] In the formulation of the present invention, the nucleic acid or protein may be any of the nucleic acids or proteins exemplified in the above "Method of Enhancing Delivery of the Present Invention." Furthermore, the ultra-fine bubbled water or ultra-fine bubbled aqueous solution in the formulation of the present invention is the ultra-fine bubbled water or ultra-fine bubbled aqueous solution described in the above "System of the Present Invention."

[0069] In the formulations of the present invention, the nucleic acid or protein may be mixed with the ultra-fine bubble water or the like of the present invention and administered to a subject as a single formulation, or, as long as both can coexist in the vicinity of immune cells at the same time, they may be formulated separately and administered simultaneously or at different times via the same or different routes.

[0070] When a nucleic acid or a protein and the ultra-fine bubble water or the like of the present invention are formulated separately, the nucleic acid or protein can be blended with a pharma- ceutically acceptable carrier in an amount that is tolerable to humans or other mammals. Examples of pharma- ceutically acceptable carriers include pH adjusters such as monosodium phosphate, dipotassium phosphate, disodium phosphate, monopotassium phosphate, sodium hydroxide, and hydrochloric acid; antibiotics such as kanamycin sulfate, erythromycin lactobionate, and potassium penicillin G; stabilizers such as lactose, potassium glutamate, D-sorbitol, aminoacetic acid, and human serum albumin; colorants such as phenol red; and isotonic agents such as sodium chloride and potassium chloride.

[0071] The content of the nucleic acid or protein in the formulation of the present invention is not particularly limited as long as it is an amount that can impart desirable physical properties such as a desired physiological activity to the immune cells when they are delivered into the immune cells by ultrasonic irradiation. For example, when the formulation of the present invention contains a nucleic acid encoding a CAR or an exogenous TCR as a nucleic acid and is administered to a mammal including a human, the amount of the nucleic acid encoding the CAR or exogenous TCR is administered in the range of 0.001 mg to 10 mg per kg of body weight per dose. For example, when administered to a human patient, the amount is administered in the range of 0.001 to 50 mg for a patient weighing 60 kg. The above dosage is an example, and the dosage can be appropriately selected depending on the type of nucleic acid used, the administration route, the age, body weight, symptoms, etc. of the subject or patient. On the other hand, the amount of ultra-fine bubble water, etc. is not particularly limited as long as it is an amount sufficient to deliver the nucleic acid or protein into the immune cells by ultrasonic irradiation. For example, when the ultra-fine bubble density in the vicinity of the immune cells is 1×10 8 ~10×10 8 / mL, preferably 2 x 10 8 ~5×10 8 Any amount that can deliver ultra-fine bubbles so that the number of bubbles per mL is sufficient.

[0072] IV. Delivery Methods of the Invention The present invention also provides a method for delivering a nucleic acid or protein into an immune cell, comprising contacting the immune cell with the preparation of the present invention and treating the cell with ultrasound. Here, the term "immune cell" has the same meaning as defined in the system of the present invention.

[0073] The subject to which the "delivery enhancing method of the present invention" and the "delivery method of the present invention" can be applied is not particularly limited as long as it is an immune cell collected from a living body (also referred to as "ex vivo immune cell" in this specification) or a tissue containing the same, or an immune cell in an animal body (also referred to as "in vivo immune cell" in this specification), and examples thereof include humans and other mammals (e.g., mice, rats, rabbits, dogs, cats, cows, horses, pigs, monkeys, etc.), or immune cells derived therefrom or tissues containing the same. There is no particular limit to the means for administering the ultra-fine bubble water, etc. of the present invention to a subject, as long as the administration route can deliver ultra-fine bubbles to the vicinity of immune cells. Hereinafter, the case where a nucleic acid encoding a CAR or an exogenous TCR is delivered as a nucleic acid will be described separately as ex vivo immune cells and in vivo immune cells.

[0074] (a) Delivery of nucleic acid encoding a CAR or an exogenous TCR to immune cells ex vivo According to the delivery method of the present invention, the ultra-fine bubble water or the like of the present invention can be combined with ultrasound to deliver a nucleic acid encoding a CAR or an exogenous TCR to an ex vivo immune cell, thereby producing an ex vivo immune cell expressing the CAR or the exogenous TCR. Therefore, the present invention also provides an ex vivo immune cell obtained by the method. The "immune cell" here is not particularly limited as long as it is a cell (so-called immune effector cell) that has the ability to damage a target cell (pathogenic cell) such as a cancer cell by some mechanism of action among those defined in the above-mentioned system of the present invention, and examples thereof include T cells that are responsible for cellular immunity among acquired immunity, NK cells, monocytes, macrophages, dendritic cells, etc. that are responsible for natural immunity, and NKT cells, which are T cells having the properties of NK cells. In a preferred embodiment, the immune cell may be a T cell. T cells collected from a living body are also referred to as "ex vivo T cells" in this specification. On the other hand, in another preferred embodiment, the immune cell may be a cell that is responsible for natural immunity, such as a NK cell, a macrophage, or a dendritic cell. Although T cells have a considerable risk of causing GVHD through allogeneic (allo) transplantation even when the MHC type is the same, allogeneic NK cells and the like are thought not to cause GVHD. Therefore, if allogeneic ex vivo immune cells of various MHC types are prepared, they can be used off-the-shelf. CAR-NK cells are described, for example, in US2016 / 0096892, Mol Ther. 25(8): 1769-1781 (2017), and CAR-dendritic cells, CAR-macrophages, and the like are described, for example, in WO2017 / 019848, eLIFE. 2018 e36688, and the like.

[0075] The ex vivo immune cells to which the nucleic acid encoding a CAR or exogenous TCR is introduced by the formulation of the present invention may be any cell population containing the above-mentioned effector immune cells, or unipotent or multipotent stem cells or progenitor cells thereof that can be terminally differentiated into the effector immune cells (excluding embryonic and other totipotent or pluripotent stem cells), and may be isolated and purified specific immune cells (e.g., T cells, NK cells, monocytes, macrophages, dendritic cells, etc., NKT cells or their stem / progenitor cells), or may be a heterogeneous cell population containing multiple types of immune cells such as lymphocytes. Examples of isolated and purified T cells include cytotoxic T cells (CTLs) that are CD8 positive cells, helper T cells, regulatory T cells, effector T cells, etc. that are CD4 positive cells, and are preferably cytotoxic T cells.

[0076] The lymphocytes can be collected from, for example, peripheral blood, bone marrow, and umbilical cord blood of a human or non-human mammal, or can be produced from stem cells such as iPS cells. When ex vivo immune cells (e.g., ex vivo T cells) into which a nucleic acid encoding a CAR or an exogenous TCR has been introduced by the formulation of the present invention are used to treat a disease such as cancer, the cell population is preferably collected from the subject to be treated or from a donor whose MHC type matches that of the subject to be treated.

[0077] Examples of unipotent or multipotent stem or progenitor cells capable of terminal differentiation into immune cells include hematopoietic stem cells, common myelo-lymphoid progenitors (MLPs), myeloid progenitors (MPs), granulocyte mononuclear progenitors (GMPs), macrophage-dendritic cell progenitors (MDPs), dendritic cell progenitors (DCPs), etc. These stem / progenitor cells can be differentiated into various immune cells, such as T cells, by methods known per se.

[0078] There is no particular limitation on the method of contacting the preparation of the present invention with ex vivo immune cells, but for example, the preparation of the present invention may be added to a normal immune cell culture medium. Since immune cells grow in a floating state without adhering to a culture vessel, it is preferable to pre-culture the ex vivo immune cells and contact them with the preparation of the present invention in a culture vessel coated with a non-adhesive or low-adhesive substrate. Ultrasonic irradiation can be performed on the ex vivo immune cells using the ultrasonic generator and irradiation conditions described in the above-mentioned system of the present invention.

[0079] When the formulation of the present invention contains a nucleic acid encoding an exogenous TCR as an active ingredient, the expression of the endogenous TCRα chain and TCRβ chain that the T cell naturally expresses may be suppressed by siRNA, from the viewpoint of increasing the expression of the exogenous TCR, suppressing the appearance of mispaired TCR, or suppressing autoreactivity. When the nucleic acid is applied to the method, it is preferable that the base sequence of the nucleic acid encoding the TCR is a different sequence (codon-converted sequence) from the base sequence corresponding to the RNA on which the siRNA that suppresses the expression of the endogenous TCRα chain and TCRβ chain acts, in order to avoid the effect of the siRNA on the exogenous TCR. These methods are described, for example, in International Publication No. 2008 / 153029. The base sequence can be prepared by introducing silent mutations into a nucleic acid encoding a TCR obtained from nature, or by chemically synthesizing an artificially designed nucleic acid. Alternatively, in order to avoid mispairing with the endogenous TCR chain, a part or all of the constant region of the nucleic acid encoding the exogenous TCR may be replaced with a constant region derived from an animal other than human, for example, a mouse.

[0080] (b) Delivery of a nucleic acid encoding a CAR or an exogenous TCR to immune cells in vivo. According to the delivery method of the present invention, the formulation of the present invention is administered to a mammal (human or other mammal (e.g., mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey), preferably human) and tissues and organs containing immune cells (e.g., spleen, thymus, etc.) are irradiated with ultrasound, whereby the nucleic acid is introduced into immune cells in the animal body, for example, T cells (also referred to as "in vivo T cells" in the present specification), and expression of CAR or exogenous TCR can be induced. The in vivo immune cells specifically recognize cancer cells, etc. expressing the surface antigen targeted by CAR or exogenous TCR, and kill the diseased cells, thereby exerting a preventive or therapeutic effect against the disease.

[0081] The preparation of the present invention can be used, for example, in the form of an injection, to deliver the nucleic acid and ultra-fine bubble water, etc., to the vicinity of in vivo immune cells by subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection, drip infusion, intracerebral injection, intracerebrospinal fluid injection, intraocular injection, etc. In addition, ultrasound irradiation can be performed by performing the procedure conventionally used in ultrasound diagnosis, except that the target site is a tissue or organ containing in vivo immune cells.

[0082] When the "delivery enhancing method of the present invention" and the "delivery method of the present invention" are applied to ex vivo immune cells, i.e., immune cells isolated from an animal or immune cells induced to differentiate from stem cells such as iPS cells using a method known per se, a step of culturing the immune cells after transfection treatment may be included. The culturing step can be performed in a medium that is usually used for maintaining and culturing terminally differentiated immune cells or for inducing differentiation from less differentiated stem / progenitor cells to terminally differentiated immune cells, using a culture vessel (e.g., dish, flask, etc.) or a culture device (e.g., CO2 incubator, etc.) known per se. Since immune cells grow in a floating state without adhering to a culture vessel, it is preferable to culture ex vivo immune cells in a culture vessel coated with a non-adhesive or low-adhesive substrate. The above-mentioned culturing step also includes a subculture step. When cells are subcultured by the "delivery enhancing method of the present invention" and the "delivery method of the present invention", the expression intensity of the target substance introduced into the cells can be maintained.

[0083] V. Medicines containing ex vivo immune cells into which a substance of interest has been introduced according to the present invention The ex vivo immune cells into which a target substance has been introduced using the system of the present invention or the delivery method of the present invention exert a desired effect (e.g., acquire a new physiological activity) due to the action of the target substance, and can be prepared as a pharmaceutical composition as is or by mixing with a known pharma- ceutically acceptable carrier (including excipients, diluents, bulking agents, binders, lubricants, flow aids, disintegrants, surfactants, etc.) or conventional additives. Excipients are well known to those skilled in the art, and adjuvants such as wetting agents or emulsifiers, and pH buffers can also be used. Furthermore, formulation adjuvants such as suspending agents, preservatives, stabilizers, and dispersants may also be used. The pharmaceutical composition may also be in a dry form for reconstitution with an appropriate sterile liquid before use. The pharmaceutical composition can be administered orally or parenterally, systemically or locally, depending on the form to be prepared (oral administration agents such as tablets, pills, capsules, powders, granules, syrups, emulsions, and suspensions; parenteral administration agents such as injections, drips, external preparations, and suppositories), etc. When administered parenterally, it can be administered intravenously, intradermally, subcutaneously, rectally, transdermally, etc. When used in the form of an injection, acceptable buffers, solubilizing agents, isotonic agents, etc. can also be added.

[0084] For example, when the target substance is a nucleic acid encoding a CAR or an exogenous TCR, the ex vivo immune cells into which the target substance has been introduced can specifically recognize and kill (e.g., induce apoptosis) cells expressing a surface antigen specifically recognized by the CAR or exogenous TCR. Thus, by containing, as an active ingredient, a nucleic acid encoding a CAR or an exogenous TCR that recognizes a surface molecule that is specifically expressed or whose expression is enhanced in diseased cells such as cancer cells as a surface antigen, the ex vivo immune cells into which the nucleic acid has been introduced and which express the CAR or exogenous TCR can be used for the prevention or treatment of diseases such as cancer, and can be safely administered to humans or other mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys), preferably humans. In a preferred embodiment, the medicament comprising the ex vivo immune cells into which a target substance has been introduced by the present invention may be a preventive or therapeutic drug for cancer. There is no particular limitation on the cancer to which the medicament is applied, and examples of the cancer that the medicament is applied to include acute lymphocytic cancer, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer (e.g., medulloblastoma), breast cancer, anal, anal canal or anorectal cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, cervical, gallbladder or pleural cancer, nasal, nasal cavity or middle ear cancer, oral cancer, vulva cancer, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, head and neck cancer (e.g., head and neck squamous cell carcinoma), hypopharyngeal cancer, kidney cancer, laryngeal cancer, leukemia (e.g., acute lymphoblastic leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia), liquid tumors, liver cancer, lung cancer (e.g., non-small cell lung cancer), lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma), malignant mesothelioma, mast cell tumor, melanoma, multiple myeloma, nasopharyngeal cancer, ovarian cancer, pancreatic cancer; peritoneal, omental and mesenteric cancer; pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer, etc., are not limited thereto.

[0085] The dosage of the pharmaceutical containing ex vivo immune cells into which a substance of interest has been introduced according to the present invention is, for example, 0.001 mg to 10 mg of nucleic acid encoding CAR or exogenous TCR per kg of body weight per administration. For example, when administered to a human patient, the dosage is 0.001 to 50 mg for a patient weighing 60 kg. The dosages mentioned above are merely examples, and can be appropriately selected depending on the type of nucleic acid used, the administration route, and the age, body weight, symptoms, etc. of the subject or patient. The pharmaceutical containing the ex vivo immune cells into which a target substance has been introduced according to the present invention is preferably administered parenterally to a subject. Parenteral administration methods include intravenous, intraarterial, intramuscular, intraperitoneal, and subcutaneous administration. The dosage is appropriately selected depending on the condition, weight, age, etc. of the subject, but usually, the number of cells is 1×10 per administration for a subject weighing 60 kg. 6 ~1×10 10Preferably 1×10 7 ~1×10 9 10, more preferably 5×10 7 ~5×10 8 The dose may be administered once or multiple times.

[0086] The present invention will be further described below with reference to Reference Examples and Examples, but the present invention is not limited to these in any sense. In the following Reference Examples and Examples, "%" indicates weight / volume % unless otherwise specified. EXAMPLES

[0087] Reference Example 1: Preparation of ultra-fine bubble water 2 g of polysorbate 80 was dissolved in 2 L of water for injection or 2 L of diluted McLivaine buffer (pH 3.0) (0.1% polysorbate 80), and the solution was injected into an ultra-fine bubble generator (nanoGALF, manufactured by IDEC). TM Using a pH adjuster (FZ1N-02), an ultra-fine bubble solution was prepared under the following settings. Using an acidic buffer solution (diluted McLivaine buffer solution: pH 3.0) produces positively charged ultra-fine bubbles, and using water for injection produces negatively charged ultra-fine bubbles. Gas used in preparation: Air -Bubble water flow rate: Approx. 4.0L / min Dissolution pressure: 300KPa±5%(sd) The prepared ultra-fine bubble aqueous solution was sterilized by high-pressure steam at 121 to 124°C for 30 minutes using an autoclave. The average diameter of ultra-fine bubbles, the density of ultra-fine bubbles, and the d90 / d10 ratio were measured using a tracking method that utilizes laser light scattering using an LM10 (manufactured by Epson Corporation).

[0088] The results are shown below. Ultra-fine bubble average diameter: 120nm±16nm Ultra-fine bubble density: 4 x 10 8 pieces / mL d90 / d10 ratio: 3.3

[0089] Example 1 Gene transfer into T cells Jurkat E6.1 (3 × 10 4 Cells (0.5 mL) were seeded in 48 wells. After removing the medium, 0.5 mL of ultra-fine bubble / RPMI medium containing 5 μg of pGFP was added. The ultra-fine bubble / RPMI medium was prepared by adding 1 L of the ultra-fine bubble solution prepared in Reference Example 1 to the RPMI medium powder, and adding 2 g / L NaHCO3 and 10% FBS to the medium for transfection.

[0090] After adding the pGFP-containing medium, the mixture was sonicated using an ultrasonic generator (NEPAGENE) at a frequency of 1 MHz and an output intensity of 0.5 W / cm. 2 After irradiating with ultrasonic waves for 10 seconds at 400 rpm, the cells were cultured for 2 hours, after which the transfection medium was removed and the cells were cultured in RPMI medium (culture medium) for 48 hours. The cells were then collected and completely dissolved in 0.15 mL of cell lysis solution, after which the amount of GFP fluorescence was measured using a spectrofluorophotometer. The number of viable cells was also measured, and the results were converted to the amount of fluorescence per viable cell for comparison. The results are shown in Figure 1.

[0091] Example 2 Protein transduction into T cells Jurkat E6.1 (3 × 10 4 Cells, 0.5 mL) were seeded in 48 wells. After removing the medium, 0.5 mL of ultra-fine bubble / RPMI medium containing IgG-FITC (4 mL of ultra-fine bubble / RPMI medium was added to 40 μL of IgG-FITC) was added. Ultra-fine bubble / RPMI medium was prepared by adding 1 L of the ultra-fine bubble solution prepared in Reference Example 1 to the RPMI medium powder, and adding 2 g / L NaHCO3 and 10% FBS to the medium for transfection.

[0092] After adding the IgG-FITC-containing medium, the mixture was sonicated using an ultrasonic generator (NEPAGENE) at a frequency of 1 MHz and an output intensity of 0.5 W / cm. 2 After 10 seconds of ultrasonic irradiation at 400 rpm, the cells were cultured for 2 hours, after which the transfection medium was removed and the cells were cultured in RPMI medium (culture medium) for 48 hours. The fluorescence intensity of IgG-FITC was then measured using a spectrofluorophotometer. The number of viable cells was also measured and converted to the fluorescence intensity per viable cell for comparison. The results are shown in Figure 2. [Industrial Applicability]

[0093] The system of the present invention can efficiently deliver target substances such as nucleic acids and proteins into immune cells by irradiating ultrasonic waves with low output intensity, and therefore can provide a highly safe drug delivery system into immune cells. Furthermore, compared with the use of conventional microbubbles, the use of ultrafine bubbles can significantly increase the efficiency of introducing target substances into cells. Furthermore, since there is no need to use liposomes, the system can be produced at low cost and is highly safe. From the above, the system of the present invention is extremely useful as a novel DDS into immune cells.

[0094] This application is based on a patent application filed in Japan, Patent Application No. 2019-119164 (filing date: June 26, 2019), the contents of which are incorporated in their entirety into this specification.

Claims

1. A system for delivering a target substance into immune cells, comprising an ultrasonic generator and an ultra-fine bubble solution containing phospholipid-free ultra-fine bubbles having an average diameter of 200 nm or less, the ultra-fine bubble solution containing a non-ionic surfactant, and The ultra-fine bubbles have a negative or positive charge, the nonionic surfactant is polyoxyethylene sorbitan laurate or polyoxyethylene sorbitan oleate; The concentration of the nonionic surfactant contained in the ultra-fine bubble aqueous solution is 0.01 to 10% (W / V); and, A system in which the density of ultra-fine bubbles in the ultra-fine bubble aqueous solution is 1.0 x 10 8 to 2.0 x 10 9 bubbles / mL.

2. 2. The system of claim 1, wherein the ultra-fine bubbles are composed of perfluorinated hydrocarbons or air.

3. The system according to claim 1, wherein the average diameter of the ultra-fine bubbles is from 50 nm to 200 nm.

4. 2. The system of claim 1, wherein the ultra-fine bubbles have a d90 / d10 ratio of 5 or less.

5. The ultrasonic output intensity of the ultrasonic generator is 720 mW / cm ２ 2. The system of claim 1, wherein:

6. The ultrasonic output intensity of the ultrasonic generator is 50 to 500 mW / cm ２ and the frequency of the ultrasound is 0.5 to 10 MHz.

7. The system of claim 1 , wherein the target substance is a nucleic acid or a protein.

8. The system of claim 7 , wherein the nucleic acid encodes a chimeric antigen receptor or an exogenous T cell receptor.

9. The system of claim 1 , wherein the immune cell is a T cell.

10. The system of claim 8 for delivering a nucleic acid into an immune cell.

11. A method for enhancing ex vivo intracellular delivery of a nucleic acid or a protein into immune cells by using an aqueous solution of ultra-fine bubbles containing phospholipid-free ultra-fine bubbles having an average diameter of 200 nm or less and ultrasound, the method comprising the steps of: The ultra-fine bubbles have a negative or positive charge, the nonionic surfactant is polyoxyethylene sorbitan laurate or polyoxyethylene sorbitan oleate; The concentration of the nonionic surfactant contained in the ultra-fine bubble aqueous solution is 0.01 to 10% (W / V), and The method comprises the step of: providing an aqueous ultra-fine bubble solution having a density of ultra-fine bubbles of 1.0 x 10 8 to 2.0 x 10 9 bubbles / mL.

12. A preparation for delivering a nucleic acid or a protein into immune cells, comprising a combination of a nucleic acid or a protein and an aqueous ultra-fine bubble solution containing ultra-fine bubbles having an average diameter of 200 nm or less and containing no phospholipids, the preparation being characterized in that an effective amount of the nucleic acid or the protein is delivered into immune cells by combined use with ultrasonic irradiation, the aqueous ultra-fine bubble solution containing a non-ionic surfactant, and The ultra-fine bubbles have a negative or positive charge, the nonionic surfactant is polyoxyethylene sorbitan laurate or polyoxyethylene sorbitan oleate; The concentration of the nonionic surfactant contained in the ultra-fine bubble aqueous solution is 0.01 to 10% (W / V), and The density of ultra-fine bubbles in the ultra-fine bubble aqueous solution is 1.0 x 10 8 to 2.0 x 10 9 bubbles / mL. formulation.

13. 13. A method for ex vivo delivery of a nucleic acid or protein into an immune cell, comprising contacting said cell with the formulation of claim 12 and treating said cell with ultrasound.

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