Lipid nanoparticles with improved genetic material delivery efficiency through regulation of extracellular matrix and ph control of intracellular organelles and use thereof

Abstract

The present invention relates to: lipid nanoparticles with improved genetic material delivery efficiency through the regulation of an extracellular matrix and pH control of intracellular organelles; and use thereof. A composition for delivering a genetic material, according to the present invention, comprises lipid nanoparticles and a proton pump inhibitor, whereby the genetic material can be effectively delivered by overcoming various intracellular and extracellular barriers and the limitations of existing delivery systems.

Description

Lipid nanoparticles with improved genetic material transfer efficiency through extracellular matrix regulation and pH regulation of intracellular organelles, and their uses

[0001] The present invention relates to lipid nanoparticles with improved genetic material delivery efficiency through the regulation of the extracellular matrix and the pH of intracellular organelles, and to the uses thereof. More specifically, the invention relates to a composition for delivering genetic material capable of increasing genetic material delivery efficiency by including a hydrogen pump inhibitor to increase the cellular uptake rate of lipid nanoparticles through the regulation of the extracellular matrix and inducing effective endosome / lysosome escape of genetic material through the regulation of the function of acidic organelles in the cell.

[0002] Genetic materials such as mRNA and DNA are biomolecules that present limitations: they are easily degraded by hydrolytic enzymes when injected into the body, and their negative charge makes delivery into cells difficult. Furthermore, once delivered into the cell, genetic material must escape from acidic intracellular organelles—such as endosomes and lysosomes—and migrate to the nucleus or cytoplasm to ultimately lead to the production of target proteins. Consequently, delivery systems utilizing various materials are being developed to protect genetic material from hydrolytic enzymes while simultaneously inducing effective protein expression at desired sites.

[0003] Among genetic material carriers, lipid nanoparticles are a mixture of four components: ionized lipids, phospholipids (helper lipids), cholesterol (structure-maintaining lipids), and lipid-PEG conjugates. They are the most actively researched particles currently being developed due to their high delivery efficiency among developed gene carriers. Nevertheless, it is known that the amount of genetic material that escapes from endosomes / lysosomes after being introduced into cells via lipid nanoparticles is a small amount, approximately 1–2%.

[0004] Accordingly, the inventors have completed the present invention by developing a composition for delivering genetic material that can increase the efficiency of genetic material delivery by increasing the cellular absorption of lipid nanoparticles and inducing the effective escape of genetic material from endosomes / lysosomes.

[0005] The present invention aims to provide a composition for delivering genetic material that can increase the efficiency of genetic material delivery by increasing the cellular absorption of lipid nanoparticles and inducing the effective escape of genetic material from endosomes / lysosomes.

[0006] In addition, the present invention aims to provide a composition for delivering genetic material capable of improving the immune response through enhanced antigen delivery in a cancer vaccine using mRNA.

[0007] In addition, the present invention aims to provide a composition for delivering genetic material applicable to various fields, such as genetic material-based vaccines and genetic material-based therapeutic agents targeting various diseases.

[0008] The present invention is capable of various modifications and may have various embodiments, and specific embodiments are to be illustrated and described in detail. However, this is not intended to limit the invention to specific embodiments, and it should be understood that the invention includes all modifications, equivalents, and substitutions that fall within the spirit and scope of the invention.

[0009] The terms used in this application are used merely to describe specific embodiments and are not intended to limit the invention. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this application, terms such as "comprising" or "having" are intended to specify the presence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0010] Where in this specification, when a quantity, concentration, or other value or parameter is given as an enumeration of a range, a preferred range, a preferred upper limit, and a preferred lower limit, it should be understood that any pair of any upper range limit or preferred value and any lower range limit or preferred value are specifically disclosed, regardless of whether the range is disclosed separately.

[0011] Where a range of numerical values ​​is mentioned in this specification, unless otherwise stated, the range and the scope of the parent invention within that range are not intended to be limited to the specific value mentioned when defining the range.

[0012] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the present invention pertains. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this application. Specific details for the implementation of the above invention are described below.

[0013] The present invention relates to lipid nanoparticles with improved genetic material transfer efficiency through the regulation of the extracellular matrix and the pH of intracellular organelles, and to the uses thereof.

[0014] Specifically, the present invention provides a composition for genetic material delivery comprising: lipid nanoparticles comprising ionized lipids, phospholipids, cholesterol, and lipid-PEG (polyethylene glycol) conjugates; and a hydrogen pump inhibitor.

[0015] Proton pump inhibitors are known to inhibit the activity of hydrogen / potassium pumps in acidic organelles, such as endosomes and lysosomes, thereby inducing membrane permeability by causing dysfunction of these organelles through an increase in internal pH. Additionally, proton pump inhibitors are known to downregulate transforming growth factor beta (TGF-β)-induced fibrillation. Utilizing this, enhanced gene expression was induced by in vivo co-administering lipid nanoparticles encapsulating nucleic acid drugs with proton pump inhibitors. By utilizing the various effects of these hydrogen pump inhibitors (proton pump inhibitors), it is possible to increase the penetration of lipid nanoparticles into tissues through the regulation of the extracellular matrix and to induce the effective escape of genetic material from endosomes and lysosomes by regulating the function of acidic organelles, thereby increasing the efficiency of genetic material delivery.

[0016] In addition, the composition for transferring genetic material according to the present invention may further include genetic material.

[0017] More specifically, the genetic material may be encapsulated within the lipid nanoparticles, and the genetic material may be encapsulated stably and with high efficiency to exhibit an excellent expression effect through the delivery composition. In addition, there is an advantage in that the type of genetic material encapsulated within the lipid nanoparticles can be varied according to the purpose.

[0018] The genetic material may be DNA and / or RNA, and may include an antisense nucleotide, mRNA, siRNA (small interfering RNA), miRNA (microRNA), ssRNA (Single-Stranded RNA), pDNA (plasmid DNA), cDNA (complementary), etc. associated with the genetic material, and preferably may include mRNA or pDNA.

[0019] In this specification, "mRNA" refers to synthetic mRNA capable of gene expression (in vitro transcribed mRNA).

[0020] The above hydrogen pump inhibitor may include one or more selected from the group consisting of esomeprazole, pantoprazole, omeprazole, lansoprazole, rabeprazole, ilaprazole, and dexlansoprazole, and preferably may include one or more selected from the group consisting of esomeprazole, pantoprazole, omeprazole, and rabeprazole.

[0021] The present invention aimed to increase more effective expression in the delivery of genetic material through lipid nanoparticles, and for this purpose, a hydrogen pump inhibitor, specifically esomeprazole (or pantoprazole, omeprazole, rabeprazole), was administered in combination. Enhanced delivery of genetic material is possible through increased cell uptake due to the action of the hydrogen pump inhibitor, along with increased expression of genetic material (mRNA) through the induction of endosome / lysosome dysfunction, and furthermore, increased efficacy of genetic material therapeutic agents is possible.

[0022] In addition, the composition for transferring genetic material according to the present invention may have the hydrogen pump inhibitor encapsulated inside the lipid nanoparticle.

[0023] More specifically, the hydrogen pump inhibitor may be encapsulated inside the lipid nanoparticles together with the genetic material, and this enables enhanced delivery of the genetic material through increased genetic material expression, similar to the mixed administration of lipid nanoparticles containing the genetic material and the hydrogen pump inhibitor.

[0024] Among the components of lipid nanoparticles according to one example, the ionized lipid is an ionizable compound having properties similar to lipids, and can play a role in encapsulating the dielectric material within the lipid nanoparticles with high efficiency through electrostatic interaction with the dielectric material.

[0025] The above ionized lipids are 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP), 8-[2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM-102), (6Z, 9Z, 28Z, 31Z)-hephtatriaconta 6,9,28,31-tetraene-19-yl4-(dimethylamino)butanoate (DLin-MC3-DMA), [(4-hydroxybutyl)azandyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyl Carbamoyloxy-3-dimethylaminopropane (DLin-CDAP), 1,2-dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-(2,3-dioleyloxy)propylamine (DODMA), dioctadecylamidoglycyl spermine (DOGS), spermine cholesteryl carbamate (GL-67), Bis-guanidinium-spermidine-cholesterol (BGTC), 3β-(N(N',N'-dimethylaminoethane)-carbamoyl) cholesterol (DC-Chol), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydecyl)amino)ethyl)(2-hydroxydecyl)amino)ethyl)piperazine-1-yl)ethylazandyl)dododecane-2-ol (C12-200), Nt-butyl-N'-tetradecylamino-propionamidine (diC14-amidine), dimethyldioctadecylammonium bromide (DDAB), N(1,2-dimyristyl oxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N,N-dioleylN,N-dimethylammonium Chloride (DODAC), Dioleyl oxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE), N-(1-(2,It may include one or more selected from the group consisting of 3-dioleyl oxy)propyl)-N-2-(sperminecarboxamide)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), N-(1-(2,3-dioleyl oxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), aminopropyl-dimethyl-bis(dodecyloxy)-propane aluminum bromide (GAP-DLRIE), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dipalmityl-3-trimethylammonium propane (DPTAP), 1,2-distearyl-3-trimethylammonium propane (DSTAP), and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), and preferably It may include one or more selected from the group consisting of 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP), 8-[2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM-102), and (6Z, 9Z, 28Z, 31Z)-hephtatriaconta 6,9,28,31-tetraene-19-yl4-(dimethylamino)butanoate (DLin-MC3-DMA).

[0026] According to one example, the phospholipid, among the components of the lipid nanoparticle, plays a role in protecting the core formed by the interaction of ionized lipids and genetic material (mRNA) within the lipid nanoparticle by enveloping it, and binds to the phospholipid bilayer of target cells to facilitate cell membrane passage and endosomal escape during intracellular drug delivery.

[0027] The above phosphatidyl ethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylethanolamine (DSPE), It may include one or more selected from the group consisting of phosphatidylethanolamine (PE), dipalmitoylphosphatidylethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS), and 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine], and preferably one or more selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE) and distearoylphosphatidylcholine (DSPC).

[0028] In this specification, "lipid-PEG (polyethyleneglycol) conjugate," "lipid-PEG," "PEG-lipid," or "lipid-PEG" refers to a form in which lipids and PEG are conjugated, meaning a lipid to which a polyethylene glycol (PEG) polymer, a hydrophilic polymer, is attached to one end. The lipid-PEG conjugate contributes to the stability of the nanoparticles within the lipid nanoparticles in serum and prevents aggregation between nanoparticles. Additionally, the lipid-PEG conjugate can enhance the in vivo stability of nucleic acids by protecting them from degrading enzymes during in vivo delivery.

[0029] The above lipid-PEG conjugate may include one or more selected from the group consisting of PEG-DMG, PEG conjugated to dialkyloxypropyl (PEG-DAA), PEG conjugated to diacylglycerol (PEG-DAG), PEG conjugated to phospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramide (PEG-CER), PEG conjugated to cholesterol or a derivative thereof, PEG-c-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, and PEG-DSPE, and preferably may include PEG-DMG.

[0030] The average molecular weight of the above lipid-PEG conjugate is 100 to 10,000 daltons, 200 to 10,000 daltons, 500 to 10,000 daltons, 1,000 to 10,000 daltons, 1,500 to 10,000 daltons, 2,000 to 10,000 daltons, 100 to 7,500 daltons, 200 to 7,500 daltons, 500 to 7,500 daltons, 1,000 to 7,500 daltons, 1,500 to 7,500 daltons, 2,000 to 7,500 daltons, 100 to 5,000 daltons, 200 to 5,000 daltons, 500 to 5,000 daltons, 1,000 to 5,000 daltons, 1,500 to 5,000 daltons, 2,000 to 5,000 daltons, 100 to 3,000 daltons, 200 to 3,000 daltons, 500 to 3,000 daltons, 1,000 to 3,000 daltons, 1,500 to 3,000 daltons, 2,000 to 3,000 daltons, 100 to 2,600 daltons, 200 to 2,600 daltons, 500 to 2,600 daltons, 1,000 to 2,600 daltons, 1,500 to 2,600 daltons, 2,000 to 2,600 daltons, 100 to 2,500 daltons, 200 to It may be 2,500 daltons, 500 to 2,500 daltons, 1,000 to 2,500 daltons, 1,500 to 2,500 daltons, or 2,000 to 2,500 daltons.

[0031] In the above composition, the concentration of the hydrogen pump inhibitor may be 1 to 250 μg / mL, preferably 3 to 225 μg / mL, 20 to 200 μg / mL, 50 to 175 μg / mL, 100 to 150 μg / mL, more preferably 110 to 140 μg / mL, and even more preferably 120 to 130 μg / mL. If the concentration of the hydrogen pump inhibitor is less than 1 μg / mL or exceeds 250 μg / mL, the effect (increase in mRNA expression within cells) resulting from the composition containing the hydrogen pump inhibitor is negligible and is therefore undesirable.

[0032] The lipid nanoparticles may contain ionized lipids : phospholipids : cholesterol : lipid-PEG conjugates in a molar ratio of 10 to 60 : 5 to 50 : 30 to 50 : 0.5 to 5. Preferably, the lipid nanoparticles may contain ionized lipids : phospholipids : cholesterol : lipid-PEG conjugates in a molar ratio of 15 to 55 : 8 to 42 : 35 to 45 : 0.8 to 3, and more preferably in a molar ratio of 20 to 50 : 10 to 40 : 37 to 40 : 1 to 2. The molar ratio of the components can be maintained by decreasing the molar amount of phospholipids by the amount by which the molar amount of ionized lipids is increased, while keeping the sum of the molar amounts of ionized lipids and phospholipids among the components included in the lipid nanoparticles constant.

[0033] In this specification, the molar ratio refers to the mole ratio.

[0034] The lipid nanoparticles may have an average diameter of 50 to 200 nm, preferably 60 nm to 180 nm, 70 nm to 150 nm, 80 nm to 120 nm, and more preferably 90 nm to 100 nm.

[0035] If the size of the lipid nanoparticles is smaller than the lower limit of the above range, (i) the binding of lipid nanoparticles to apolipoproteins (e.g., ApoE (e.g., ApoE3)) present in the blood during systemic circulation may be reduced, thereby reducing the number of lipid nanoparticles entering the cell, and / or (ii) the surface area of ​​the lipid nanoparticles may be excessively increased, making it difficult to maintain stability, and consequently, the efficiency of genetic material delivery to target tissues (or target cells) and / or the therapeutic effect of the genetic material carried by the lipid nanoparticles may be reduced.

[0036] Lipid nanoparticles having a diameter within the above range have superior drug delivery efficiency to target organs and / or cells compared to lipid nanoparticles having a diameter exceeding the upper limit of the above range.

[0037] In this specification, "encapsulation" refers to encapsulating a delivery substance to efficiently incorporate it into the body, genetic material encapsulation efficiency (encapsulation efficiency) refers to the content of genetic material encapsulated within lipid nanoparticles relative to the total content of genetic material used in preparation, and hydrogen pump inhibitor encapsulation efficiency refers to the content of hydrogen pump inhibitor encapsulated within lipid nanoparticles relative to the total content of hydrogen pump inhibitor used in preparation.

[0038] The dielectric material encapsulation efficiency of the above-mentioned dielectric material delivery composition may be 70% or more, 75% or more, 80% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, greater than 80% to 99% or less, greater than 80% to 97% or less, greater than 80% to 95% or less, 85% or more to 95% or less, 87% or more to 95% or less, 90% or more to 95% or less, 91% or more to 95% or less, or 91% or more to 94% or less.

[0039] When a hydrogen pump inhibitor is encapsulated inside the above lipid nanoparticles, the encapsulation efficiency of the hydrogen pump inhibitor may be 60% or more, 65% or more, 70% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, greater than 70% to 99% or less, greater than 70% to 97% or less, greater than 70% to 95% or less, greater than 75% to 95% or less, 77% or more to 95% or less, 80% or more to 95% or less, 81% or more to 95% or less, or 81% or more to 90% or less.

[0040] According to one example, the encapsulation efficiency can be calculated by a commonly used method, for example, the dielectric material encapsulation efficiency and the hydrogen pump inhibitor encapsulation efficiency may be measured through RiboGreen analysis and a UV / Vis spectrophotometer, respectively.

[0041] A composition for delivering genetic material according to one example can contain genetic material, particularly mRNA, with high encapsulation efficiency. Previously known compositions for delivering mRNA contained mRNA at a low rate, which limited their use as compositions for delivering mRNA. In contrast, lipid nanoparticles according to one example can contain mRNA with high encapsulation efficiency, specifically with an encapsulation efficiency of 85% or more.

[0042] In the present invention, the composition can be used to deliver genetic material into biological tissue or blood. The composition can be delivered through cells or intercellular junctions constituting the biological tissue, but there are no restrictions on the delivery method.

[0043] The above biological tissues refer to one or more epithelial tissues, muscle tissues, nerve tissues, and connective tissues. Since each organ may be composed of one or more tissues, various biological organs such as mucosa, skin, brain, lungs, liver, kidneys, spleen, heart, stomach, large intestine, digestive tract, bladder, ureter, urethra, ovary, testis, genitals, muscle, blood, blood vessels, lymphatic vessels, lymph nodes, thymus, pancreas, adrenal glands, thyroid gland, parathyroid glands, larynx, tonsils, bronchi, and alveoli may be included, but are not limited thereto.

[0044] The composition of the present invention can be applied to a method for delivering genetic material into cells in vivo. This method can be achieved by parenteral administration or direct injection into tissues, organs, or systems.

[0045] That is, the composition according to the present invention can be used in mammals, preferably humans, and can deliver genetic material into cells by administering it via routes such as intravein, intraperitoneal, intramuscular, subcutaneous, intradermal, nasal, mucosal, inhalation, and oral.

[0046] The above composition may be provided in a suitable form. The above formulation may be used in the form of oral formulations such as powders, granules, tablets, capsules, ointments, suspensions, emulsions, syrups, aerosols, etc., or parenteral formulations such as transdermal formulations, suppositories, and sterile injectable solutions, etc., according to conventional methods.

[0047] In addition, the above formulation may further contain auxiliary agents such as pharmaceutically suitable and physiologically acceptable carriers, excipients, and diluents. Carriers, excipients, and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulating, commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be used.

[0048] More specifically, the above formulation may include a carrier for formulation in addition to the above composition. The carrier may use a binder, a lubricant, a suspending agent, a solubilizer, a buffer, a preservative, a lubricant, an isotonic agent, an excipient, a stabilizer, a dispersant, a suspending agent, a colorant, a fragrance, etc.

[0049] In a specific embodiment of the above composition, the composition may be administered alone, but may also be administered mixed with a pharmaceutical carrier selected in consideration of the method of administration and standard pharmaceutical practice.

[0050] For example, when the above formulation is provided for parenteral use, it may be a liquid formulation such as a liquid, gel, cleansing composition, insertable tablet, suppository, cream, ointment, dressing solution, spray, or other topical agent, or a liquid formulation such as a solution, suspension, or emulsion, and may include a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized preparation, a suppository, cream, ointment, jelly, foam, cleanser, or insert, preferably a liquid, gel, cleansing composition, insertable tablet, etc. The above formulation may be prepared, for example, by adding a solubilizing agent, an emulsifier, a buffer for pH adjustment, etc. to sterile water. As the non-aqueous solvent or suspension solvent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. may be used.

[0051] In addition, when the above preparation is provided for oral use, it may be administered orally, intraorally, or under the tongue, for example, in the form of a tablet containing starch or lactose, or in the form of a capsule containing the same or an excipient, or in the form of an elixir or suspension containing a chemical agent that gives flavor or color.

[0052] Preferably, the vaccine composition or pharmaceutical composition may be delivered to the lungs via pulmonary delivery, which may be achieved by different approaches including the use of nebulized, aerosolized, micellular, or dry powder-based formulations. As an example, the composition is formulated to be administered via a liquid nebulizer, an aerosol-based inhaler, and / or a dry powder dispersant. Most preferably, the composition may be applied via aerosol spray into the nasal cavity.

[0053] The dosage of the above preparation may vary depending on the patient's age, body weight, gender, form of administration, health condition, and severity of disease, and may be divided into one to several doses per day at regular intervals at the discretion of a doctor or pharmacist. For example, based on the content of the active ingredient, the daily dosage is 0.001 to 10,000 mg / kg, 0.01 to 10,000 mg / kg, 0.1 to 10,000 mg / kg, 0.5 to 10,000 mg / kg, 0.001 to 1,000 mg / kg, 0.01 to 1,000 mg / kg, 0.1 to 1,000 mg / kg, 0.5 to 1,000 mg / kg, 0.001 to 500 mg / kg, 0.01 to 500 mg / kg, 0.1 to 500 mg / kg, 0.5 to 500 mg / kg, 0.001 to 300 mg / kg, 0.01 to 300 mg / kg, 0.1 to 300 mg / kg, Or it may be 0.5 to 300 mg / kg. The above dosage is an example of an average case, and the dosage may be higher or lower depending on individual differences.

[0054] If the daily dosage of the above preparation is less than the above dosage, a significant effect cannot be obtained, and if it exceeds that, it is not only uneconomical but also outside the range of the usual dosage, so there is a risk of undesirable side effects, so it is better to keep it within the above range.

[0055] The subjects of administration of the above preparation may be mammals such as humans, cells, tissues, body fluids isolated from mammals, or cultures thereof.

[0056] The present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising: lipid nanoparticles comprising ionized lipids, phospholipids, cholesterol, and lipid-PEG (polyethylene glycol) conjugates; a hydrogen pump inhibitor; and a genetic material.

[0057] The present invention provides a pharmaceutical composition for a cancer vaccine comprising: lipid nanoparticles comprising ionized lipids, phospholipids, cholesterol, and lipid-PEG (polyethylene glycol) conjugates; a hydrogen pump inhibitor; and a genetic material.

[0058] The lipid nanoparticles included in the above-mentioned pharmaceutical composition for cancer prevention or treatment and / or pharmaceutical composition for cancer vaccine are identical to the lipid nanoparticles included in the aforementioned composition for genetic material delivery.

[0059] The hydrogen pump inhibitor included in the above pharmaceutical composition for cancer prevention or treatment and / or pharmaceutical composition for cancer vaccine is the same as the hydrogen pump inhibitor included in the aforementioned composition for genetic material delivery.

[0060] The genetic material included in the above pharmaceutical composition for cancer prevention or treatment and / or pharmaceutical composition for cancer vaccine may include one or more selected from the group consisting of DNA, mRNA, and siRNA, preferably mRNA, more preferably mRNA suitable for cancer prevention or treatment.

[0061] The above pharmaceutical composition for cancer prevention or treatment may include lipid nanoparticles with genetic material enclosed inside.

[0062] The term "prevention" as used in this invention refers to any act of inhibiting cancer formation or delaying its onset through the administration of a composition.

[0063] In the present invention, "treatment" refers to any act in which the symptoms of the said disease are improved or beneficially altered by the administration of the composition.

[0064] In the present invention, the cancer may be skin or intraocular melanoma, breast cancer, brain cancer, neurocancer, colorectal cancer, lung cancer, small cell lung cancer, stomach cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, uterine cancer, ovarian cancer, rectal cancer, pro-anal cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma and pituitary adenoma, etc.

[0065] In one embodiment of the present invention, a composition comprising lipid nanoparticles containing ionized lipids, phospholipids, cholesterol, and lipid-PEG (polyethylene glycol) conjugates, a hydrogen pump inhibitor, and tumor antigen mRNA (OVA mRNA) was administered to the muscle of a mouse, and subsequently, tumor cells (B16-OVA) expressing the tumor antigen were inoculated subcutaneously, and it was confirmed that the antitumor effect (inhibition of cancer cell proliferation and metastasis) was significantly superior (Fig. 10).

[0066] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier and may be formulated according to conventional methods into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as topical preparations, suppositories, and sterile injectable solutions.

[0067] The above-mentioned pharmaceutically acceptable carriers include, but are not limited to, those commonly used in the art, such as lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Additionally, the pharmaceutical composition of the present invention may include, but is not limited to, fillers, extenders, binders, wetting agents, disintegrants, diluents or excipients such as surfactants, and other pharmaceutically acceptable additives.

[0068] When the pharmaceutical composition of the present invention is formulated into an oral solid dosage form, it includes tablets, pills, powders, granules, capsules, etc., and such solid dosage forms may include at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., and may include, but are not limited to, lubricants such as magnesium stearate, talc, etc.

[0069] When the pharmaceutical composition of the present invention is formulated into an oral liquid form, it includes suspensions, liquid formulations, emulsions, syrups, etc., and includes diluents such as water and liquid paraffin, humectants, sweeteners, flavorings, preservatives, etc., but is not limited thereto.

[0070] When the pharmaceutical composition of the present invention is formulated for parenteral administration, it comprises a sterile aqueous solution, a non-aqueous solvent, a suspension agent, an emulsion, a lyophilized formulation, and a suppository. The non-aqueous solvent and suspension agent may include, but are not limited to, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. The base of the suppository may include, but is not limited to, Witepsol, Macrogol, Tween 61, cocoa gluten, laurin gluten, glycerogelatin, etc.

[0071] The above composition may be administered as a single or multiple doses in pharmaceutically effective amounts. The term "pharmaceutically effective amount" in this invention means an amount sufficient to prevent or treat a disease with a reasonable benefit / risk ratio applicable to medical prevention or treatment, and the effective dose level may be determined based on factors including the severity of the disease, the activity of the drug, the patient's age, weight, health, gender, the patient's sensitivity to the drug, the time of administration of the composition of this invention used, the route of administration and elimination rate, the duration of treatment, drugs combined with or used concurrently with the composition of this invention used, and other factors well known in the medical field.

[0072] The pharmaceutical composition of the present invention may be administered to mammals such as rats, mice, livestock, and humans by various routes, for example, oral administration, intradural, inner ear, abdominal cavity or vein, muscle, subcutaneous, intrauterine dura mater, sublingual or intracerebrovascular injection, but is not limited thereto.

[0073] According to one embodiment, the pharmaceutical composition may be administered in a dose of 0.1 to 100 mg / kg, 0.1 to 50 mg / kg, 1 to 10 mg / kg, or 1 to 5 mg / kg based on the concentration of the drug (genetic material) included in the pharmaceutical composition.

[0074] Another aspect of the present invention provides a method for preventing or treating cancer comprising the step of administering to an individual in need a pharmaceutical composition comprising: lipid nanoparticles comprising therapeutically effective amounts of ionized lipids, phospholipids, cholesterol, and lipid-PEG (polyethylene glycol) conjugates; a hydrogen pump inhibitor; and genetic material.

[0075] The term "individual" in the present invention refers to any animal that has developed or may develop cancer, and may be an animal capable of exhibiting beneficial effects through treatment using a pharmaceutical composition comprising lipid nanoparticles comprising the ionized lipid, phospholipid, cholesterol, and lipid-PEG (polyethylene glycol) conjugate; a hydrogen pump inhibitor; and genetic material; however, any individual having symptoms of cancer or having the potential to have such symptoms is included without limitation. As described above, the cancer can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to an individual.

[0076] Another aspect of the present invention provides the use of a pharmaceutical composition comprising: lipid nanoparticles comprising ionized lipids, phospholipids, cholesterol, and lipid-PEG (polyethylene glycol) conjugates for manufacturing a medicine for the prevention or treatment of cancer; a hydrogen pump inhibitor; and a genetic material.

[0077] Another aspect of the present invention provides a pharmaceutical composition for use in the prevention or treatment of cancer, comprising lipid nanoparticles comprising ionized lipids, phospholipids, cholesterol, and lipid-PEG (polyethylene glycol) conjugates; a hydrogen pump inhibitor; and a genetic material.

[0078] Conventional genetic material delivery systems faced difficulties in delivering genetic material due to limitations in particle uptake caused by the structure of the extracellular matrix and low endosome / lysosome escape ability. The present invention can effectively deliver genetic material by overcoming the limitations of existing delivery systems and various intracellular and extracellular barriers through the combined administration of lipid nanoparticles and a hydrogen pump inhibitor, or by using lipid nanoparticles encapsulated with a hydrogen pump inhibitor.

[0079] In addition, the present invention is applicable to various fields, such as genetic material-based vaccines and genetic material-based therapeutic agents targeting various diseases.

[0080] Figure 1 shows the results of measuring the increase in intracellular mRNA expression after simultaneously treating Hela cells with lipid nanoparticles (SM-102 LNP, MC3 LNP, DOTAP LNP) and a hydrogen pump inhibitor (esomeprazole; ESO).

[0081] Figure 2 shows the results of measuring the increase in intracellular mRNA expression after simultaneously treating Hela cells with mRNA-encapsulated lipid nanoparticles (SM-102 LNP, MC3 LNP, DOTAP LNP) and various hydrogen pump inhibitors (omeprazole; OME, esomeprazole; ESO, pantoprazole; PAN, rabeprazole; RAB).

[0082] Figure 3 shows the results of measuring the increase in intracellular pDNA expression after simultaneously treating Hela cells with pDNA-encapsulated lipid nanoparticles (SM-102 LNP, MC3 LNP) and various hydrogen pump inhibitors (omeprazole; OME, esomeprazole; ESO, pantoprazole; PAN, rabeprazole; RAB).

[0083] Figure 4 shows the results of measuring the increase in intracellular pDNA expression after simultaneously treating natural killer cells (NK cells), which are immune cells, with lipid nanoparticles (SM-102 LNP) encapsulating pDNA encoding NKG2D and various hydrogen pump inhibitors (omeprazole; OME, esomeprazole; ESO, pantoprazole; PAN, rabeprazole; RAB).

[0084] Figure 5 shows the results of measuring the increase in intracellular mRNA expression after treating Hela cells with lipid nanoparticles co-encapsulated with various hydrogen pump inhibitors (omeprazole; OME, esomeprazole; ESO, pantoprazole; PAN, rabeprazole; RAB).

[0085] Figure 6 shows the results of measuring the increase in mRNA expression after administering a mixture of lipid nanoparticles (SM-102 LNP) and a proton pump inhibitor (PPI) to the subcutaneous and muscle of animals.

[0086] Figure 7 shows the results of measuring the increase in mRNA expression after administering a mixture of lipid nanoparticles (MC3 LNP) and a hydrogen pump inhibitor to the subcutaneous and muscle of animals.

[0087] Figure 8 shows the results of measuring the increase in mRNA expression after administering a mixture of lipid nanoparticles (DOTAP LNP) and a hydrogen pump inhibitor to the subcutaneous and muscle of animals.

[0088] Figure 9 confirms the increase in the antitumor effect of a cancer vaccine depending on whether lipid nanoparticles and a hydrogen pump inhibitor were simultaneously treated to the muscles of animals.

[0089] Examples are provided to aid in understanding the present invention. The following examples are provided merely to facilitate a better understanding of the invention, and the scope of the invention is not limited by these examples.

[0090] <Preparation Example: Preparation of lipid nanoparticles>

[0091] Preparation Example 1 (SM-102_mRNA)

[0092] A total of four lipids were dispersed in ethanol and used as the organic phase: 350.5 μg of 8-[2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM-102), 78 μg of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 146.8 μg of cholesterol, and 37.3 μg of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000).

[0093] For the aqueous phase, each mRNA was dispersed in a pH 5.2 25 mM sodium acetate buffer solution. Lipid nanoparticles were fabricated using a microfluidic mixing method with each organic phase and aqueous phase. After particle fabrication, organic solvents such as ethanol were removed by dialysis in phosphate-buffered saline.

[0094] Preparation Example 2 (MC3_mRNA)

[0095] Lipid nanoparticles were prepared using the same method as in Preparation Example 1, except that a total of four lipids were dispersed in ethanol and used as the organic phase: (6Z, 9Z, 28Z, 31Z)-heptatriaconta-6,9,28,31-tetraene-19-yl-(dimethylamino)butanoate (D-Lin-MC3-DMA, hereinafter MC3) 350.5 μg, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) 78 μg, cholesterol 146.8 μg, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000) 37.3 μg.

[0096] Preparation Example 3 (DOTAP_mRNA)

[0097] Lipid nanoparticles were prepared using the same method as in Preparation Example 1, except that a total of four lipids—1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP) 159.4 μg, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) 338.8 μg, cholesterol 169.2 μg, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000) 42.7 μg—were dispersed in ethanol and used as the organic phase.

[0098] Preparation Example 4 (SM-102_pDNA)

[0099] A total of four lipids were dispersed in ethanol and used as the organic phase: 350.5 μg of 8-[2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM-102), 78 μg of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 146.8 μg of cholesterol, and 37.3 μg of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000).

[0100] For the aqueous phase, each pDNA was dispersed in a pH 5.2 25 mM sodium acetate buffer solution. Lipid nanoparticles were fabricated using a microfluidic mixing method with each organic phase and aqueous phase. After particle fabrication, organic solvents such as ethanol were removed by dialysis in phosphate-buffered saline.

[0101] Preparation Example 5 (MC3_pDNA)

[0102] Lipid nanoparticles were prepared using the same method as in Preparation Example 4, except that a total of four lipids were dispersed in ethanol and used as the organic phase: (6Z, 9Z, 28Z, 31Z)-heptatriaconta-6,9,28,31-tetraene-19-yl-(dimethylamino)butanoate (D-Lin-MC3-DMA, hereinafter MC3) 350.5 μg, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) 78 μg, cholesterol 146.8 μg, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000) 37.3 μg.

[0103] Preparation Example 6 (DOTAP_pDNA)

[0104] Lipid nanoparticles were prepared using the same method as in Preparation Example 4, except that a total of four lipids—1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP) 159.4 μg, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) 338.8 μg, cholesterol 169.2 μg, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000) 42.7 μg—were dispersed in ethanol and used as the organic phase.

[0105]

[0106] Table 1 below lists the constituent lipids, their ratios, and the encapsulated genetic material of the lipid nanoparticles according to Preparation Examples 1 to 6.

[0107]

[0108]

[0109] <Example: Preparation of a composition for genetic material delivery>

[0110] Example 1: Use of lipid nanoparticles (SM-102_mRNA) from Preparation Example 1 + hydrogen pump inhibitor

[0111] Example 1-1 (SM-102_mRNA + ESO)

[0112] Esomeprazole (ESO) magnesium trihydrate was dissolved in ethanol, then diluted to the desired concentration in phosphate-buffered physiological saline and used. A composition for genetic material delivery was prepared by simply mixing the diluted solution of esomeprazole magnesium trihydrate with the diluted solution containing the lipid nanoparticles of Preparation Example 1.

[0113] Examples 1-2 (SM-102_mRNA + OME), Examples 1-3 (SM-102_mRNA + PAN), and Examples 1-4 (SM-102_mRNA + RAB)

[0114] The genetic material transfer compositions of Examples 1-2 to 1-4 were prepared in the same manner as in Example 1-1, except that instead of the diluted solution of Esomeprazole (ESO) magnesium trihydrate, the diluted solutions of Omeprazole (OME), Pantoprazole (PAN) sodium hydrate, and Rabeprazole (RAB) sodium, respectively, were dissolved in ethanol and then diluted to the desired concentration in phosphate-buffered physiological saline.

[0115] Example 2: Use of lipid nanoparticles (MC3_mRNA) from Preparation Example 2 + hydrogen pump inhibitor

[0116] Examples 2-1 (MC3_mRNA + ESO), 2-2 (MC3_mRNA + OME), 2-3 (MC3_mRNA + PAN), and 2-4 (MC3_mRNA + RAB)

[0117] The compositions for genetic material delivery of Examples 2-1 to 2-4 were prepared in the same manner as in Example 1-1, except that the lipid nanoparticles of Preparation Example 2 were used instead of the lipid nanoparticles of Preparation Example 1, and the diluents were each used as Esomeprazole (ESO) magnesium trihydrate, Omeprazole (OME), Pantoprazole (PAN) sodium hydrate, and Rabeprazole (RAB) sodium.

[0118] Example 3: Use of lipid nanoparticles (DOTAP_mRNA) from Preparation Example 3 + hydrogen pump inhibitor

[0119] Examples 3-1 (DOTAP_mRNA + ESO), 3-2 (DOTAP_mRNA + OME), 3-3 (DOTAP_mRNA + PAN), and 3-4 (DOTAP_mRNA + RAB)

[0120] The genetic material transfer compositions of Examples 3-1 to 3-4 were prepared in the same manner as in Example 1-1, except that the lipid nanoparticles of Preparation Example 3 were used instead of the lipid nanoparticles of Preparation Example 1, and the diluents were each used as Esomeprazole (ESO) magnesium trihydrate, Omeprazole (OME), Pantoprazole (PAN) sodium hydrate, and Rabeprazole (RAB) sodium.

[0121] Example 4: Use of lipid nanoparticles (SM-102_pDNA) from Preparation Example 4 + hydrogen pump inhibitor

[0122] Example 4-1 (SM-102_pDNA + ESO), Example 4-2 (SM-102_pDNA + OME), Example 4-3 (SM-102_pDNA + PAN) and Example 4-4 (SM-102_pDNA + RAB)

[0123] The genetic material transfer compositions of Examples 4-1 to 4-4 were prepared in the same manner as in Example 1-1, except that the lipid nanoparticles of Preparation Example 4 were used instead of the lipid nanoparticles of Preparation Example 1, and the diluents were each used as Esomeprazole (ESO) magnesium trihydrate, Omeprazole (OME), Pantoprazole (PAN) sodium hydrate, and Rabeprazole (RAB) sodium.

[0124] Example 5: Use of lipid nanoparticles (MC3_pDNA) from Preparation Example 5 + hydrogen pump inhibitor

[0125] Examples 5-1 (MC3_pDNA + ESO), 5-2 (MC3_pDNA + OME), 5-3 (MC3_pDNA + PAN), and 5-4 (MC3_pDNA + RAB)

[0126] The genetic material delivery compositions of Examples 5-1 to 5-4 were prepared in the same manner as in Example 1-1, except that the lipid nanoparticles of Preparation Example 5 were used instead of the lipid nanoparticles of Preparation Example 1, and the diluents were each used as Esomeprazole (ESO) magnesium trihydrate, Omeprazole (OME), Pantoprazole (PAN) sodium hydrate, and Rabeprazole (RAB) sodium.

[0127] Example 6: Use of lipid nanoparticles (DOTAP_pDNA) from Preparation Example 6 + hydrogen pump inhibitor

[0128] Example 6-1 (DOTAP_pDNA + ESO), Example 6-2 (DOTAP_pDNA + OME), Example 6-3 (DOTAP_pDNA + PAN) and Example 6-4 (DOTAP_pDNA + RAB)

[0129] The genetic material transfer compositions of Examples 6-1 to 6-4 were prepared in the same manner as in Example 1-1, except that the lipid nanoparticles of Preparation Example 6 were used instead of the lipid nanoparticles of Preparation Example 1, and the diluents were each used as Esomeprazole (ESO) magnesium trihydrate, Omeprazole (OME), Pantoprazole (PAN) sodium hydrate, and Rabeprazole (RAB) sodium.

[0130] Example 7: Lipid nanoparticles (PPI@SM LNP) encapsulated with a hydrogen pump inhibitor and dielectric material

[0131] Example 7-1 (ESO@SM LNP)

[0132] 350.5 μg of 8-[2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM-102), 78 μg of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 146.8 μg of cholesterol, 37.3 μg of 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000), and 36.3 μg of a hydrogen pump inhibitor (Esomeprazole (ESO) magnesium trihydrate) were dispersed in ethanol and used as the organic phase.

[0133] For the aqueous phase, each mRNA was dispersed in a pH 5.2 25 mM sodium acetate buffer solution. Lipid nanoparticles (compositions for genetic material delivery) containing both the hydrogen pump inhibitor and mRNA were prepared by microfluidically mixing each organic phase and aqueous phase. After particle preparation, organic solvents such as ethanol were removed by dialysis in phosphate-buffered saline.

[0134] Examples 7-2 (OME@SM LNP), 7-3 (PAN@SM LNP), and 7-4 (RAB@SM LNP)

[0135] The compositions for genetic material transfer of Examples 7-2 to 7-4 were prepared in the same manner as in Example 7-1, except that Omeprazole (OME), Pantoprazole (PAN) sodium hydrate, and Rabeprazole (RAB) sodium were used instead of Esomeprazole (ESO) magnesium trihydrate as hydrogen pump inhibitors.

[0136]

[0137] Experimental Example 1. Confirmation of particle characteristics after lipid nanoparticle fabrication

[0138] The size and charge of the lipid nanoparticles prepared in Preparation Examples 1 to 3 above were observed via dynamic light scattering (DLS), and the mRNA encapsulation efficiency of each lipid nanoparticle was measured via RiboGreen analysis. The respective measurement results are shown in Table 2 below.

[0139]

[0140]

[0141] According to Table 2, lipid nanoparticles prepared according to Preparation Examples 1 to 3 were observed to have sizes of 90 nm, 102 nm, and 121 nm, respectively, and neutral zeta potentials, and were confirmed to have encapsulation efficiencies of approximately 91%, 85%, and 91%, respectively.

[0142] Experimental Example 2. Confirmation of particle characteristics after fabrication of lipid nanoparticles encapsulated with a hydrogen pump inhibitor and genetic material

[0143] The size and charge of lipid nanoparticles containing the hydrogen pump inhibitor prepared in Example 7 were observed via dynamic light scattering (DLS), and the mRNA encapsulation efficiency and hydrogen pump inhibitor encapsulation efficiency of each lipid nanoparticle were measured using RiboGreen analysis and UV / Vis spectrophotometry. The respective measurement results are shown in Table 3 below.

[0144]

[0145]

[0146] According to Table 3, lipid nanoparticles prepared according to Examples 7-1 to 7-4 were observed to have sizes of 147 nm, 138 nm, 144 nm, and 147 nm, respectively, and neutral zeta potentials, and were found to have mRNA encapsulation efficiencies of approximately 87%, 89%, 90%, and 86%, respectively, and hydrogen pump inhibitor encapsulation efficiencies of approximately 82%, 77%, 81%, and 74%, respectively.

[0147] Experimental Example 3. Confirmation of increased intracellular mRNA expression after simultaneous treatment of cells with lipid nanoparticles and a hydrogen pump inhibitor

[0148] To confirm the increase in genetic material expression efficiency of esomeprazole (hereinafter ESO) at the cellular level, a luminescence measurement method was used. Specifically, after treating Hela cells with the compositions of Example 1-1 (SM-102_mRNA + ESO), Example 2-1 (MC3_mRNA + ESO), and Example 3-1 (DOTAP_mRNA + ESO), substrates were added to the plate 24 hours later. Immediately after the addition of the substrates, the luminescence intensity of the plate was measured using an instrument, and the results are shown in Figure 1.

[0149] At this time, the compositions of Examples 1-1, 2-1, and 3-1 were each prepared to contain various concentrations of esomeprazole (ESO) (1.95 μg / mL, 3.91 μg / mL, 7.61 μg / mL, 15.63 μg / mL, 31.25 μg / mL, 62.50 μg / mL, 125.00 μg / mL, 500.00 μg / mL, and 1000.00 μg / mL).

[0150] As shown in Figure 1, when cells were simultaneously treated with each of the three lipid nanoparticles and esomeprazole, it was confirmed that the effect showed the most distinct trend at a certain concentration (125.00 μg / mL) for SM-102 LNP and MC3 LNP, and at a certain concentration (7.81 μg / mL) for DOTAP LNP, although it varied depending on the lipid nanoparticle.

[0151] Experimental Example 4. Confirmation of increased intracellular mRNA expression after simultaneous treatment of cells with lipid nanoparticles and various hydrogen pump inhibitors

[0152] Luminescence measurement was used to confirm the increase in genetic material expression efficiency of hydrogen pump inhibitors at the cellular level. Specifically, after treating Hela cells with the compositions of Examples 1 to 3, a substrate was added to the plate 24 hours later. Immediately after adding the substrate, the luminescence level of the plate was measured using an instrument, and the results are shown in Figure 2.

[0153] To determine whether similar efficacy is observed in other hydrogen pump inhibitors as well as in esomeprazole, the expression-enhancing effects were examined using representative hydrogen pump inhibitors such as omeprazole (OME), pantoprazole (PTZ), and rabeprazole (RAB).

[0154] Specifically, in FIG. 2, "Only LNP" refers to Hela cells treated with lipid nanoparticles (SM-102_mRNA, MC3_mRNA, DOTAP_mRNA) of Preparation Examples 1 to 3, and "+ OME" refers to Hela cells treated with genetic material delivery compositions of Example 1-2 (SM-102_mRNA + OME), Example 2-2 (MC3_mRNA + OME), and Example 3-2 (DOTAP_mRNA + OME). "+ ESO" refers to Hela cells treated with the genetic material delivery compositions of Example 1-1 (SM-102_mRNA + ESO), Example 2-1 (MC3_mRNA + ESO), and Example 3-1 (DOTAP_mRNA + ESO); "+ PAN" refers to Hela cells treated with the genetic material delivery compositions of Example 1-3 (SM-102_mRNA + PAN), Example 2-3 (MC3_mRNA + PAN), and Example 3-3 (DOTAP_mRNA + PAN); and "+ RAB" refers to Hela cells treated with the genetic material delivery compositions of Example 1-4 (SM-102_mRNA + RAB), Example 2-4 (MC3_mRNA + RAB), and Example 3-4 (DOTAP_mRNA + RAB).

[0155] At this time, the compositions of Examples 1-1 to 1-4, 2-1 to 2-4 and 3-1 to 3-4 were each prepared to contain 125.00 μg / mL of a hydrogen pump inhibitor.

[0156] As shown in Figure 2, it was confirmed that mRNA expression efficiency increased when cells were simultaneously treated with three types of lipid nanoparticles and hydrogen pump inhibitors, including esomeprazole.

[0157] In addition, to confirm whether similar efficacy is observed in other genetic material carriers as well as mRNA, a comparison was made using pDNA, one of the representative genetic materials, and the results are shown in Figure 3.

[0158] Specifically, in FIG. 3, "NC" is a Hela cell that has not been treated with anything, "SM-102 LNP" is a Hela cell treated with the lipid nanoparticles (SM-102_pDNA) of Preparation Example 4, and "MC3 LNP" is a Hela cell treated with the lipid nanoparticles (MC3_pDNA) of Preparation Example 5. "+ OME" is a Hela cell treated with the genetic material delivery compositions of Example 4-2 (SM-102_pDNA + OME) and Example 5-2 (MC3_pDNA + OME). "+ ESO" refers to Hela cells treated with the genetic material delivery compositions of Example 4-1 (SM-102_pDNA + ESO) and Example 5-1 (MC3_pDNA + ESO), "+ PAN" refers to Hela cells treated with the genetic material delivery compositions of Example 4-3 (SM-102_pDNA + PAN) and Example 5-3 (MC3_pDNA + PAN), and "+ RAB" refers to Hela cells treated with the genetic material delivery compositions of Example 4-4 (SM-102_pDNA + RAB) and Example 5-4 (MC3_pDNA + RAB).

[0159] At this time, the compositions of Examples 4-1 to 4-4 and 5-1 to 5-4 were each prepared to contain 125.00 μg / mL of hydrogen pump inhibitor.

[0160] As shown in Figure 3, it was confirmed that pDNA expression efficiency increased when cells were simultaneously treated with two types of lipid nanoparticles and hydrogen pump inhibitors, including esomeprazole.

[0161] In addition, to confirm whether similar efficacy is observed in various cells such as suspension cells and immune cells, LNP containing pDNA and a hydrogen pump inhibitor were treated and compared in representative suspension cells and immune cells, such as natural killer cells (NK cells), and the results are shown in Figure 4.

[0162] Specifically, in FIG. 4, SF is an NK cell that has not been treated with anything, pDNA is an NK cell treated directly with pDNA, LNP is an NK cell treated with the lipid nanoparticles (SM-102_pDNA) of Preparation Example 4, LNP + OME is an NK cell treated with the genetic material delivery composition of Example 4-2 (SM-102_pDNA + OME), LNP + ESO is an NK cell treated with the genetic material delivery composition of Example 4-1 (SM-102_pDNA + ESO), LNP + PAN is an NK cell treated with the genetic material delivery composition of Example 4-3 (SM-102_pDNA + PAN), and LNP + RAB is an NK cell treated with the genetic material delivery composition of Example 4-4 (SM-102_pDNA + RAB).

[0163] At this time, the compositions of Examples 4-1 to 4-4 were each prepared to contain 125.00 μg / mL of hydrogen pump inhibitor.

[0164] As shown in Figure 4, it was confirmed that the pDNA expression efficiency in natural killer cells increased when cells were simultaneously treated with hydrogen pump inhibitors, including esomeprazole.

[0165] Experimental Example 5. Confirmation of increased intracellular mRNA expression after treating cells with lipid nanoparticles co-encapsulating mRNA and various hydrogen pump inhibitors

[0166] Luminescence measurement was used to confirm the increase in genetic material expression efficiency of a formulation containing mRNA and a hydrogen pump inhibitor at the cellular level. Specifically, after treating Hela cells with the composition of Example 7, a substrate was added to the plate 24 hours later. Immediately after adding the substrate, the luminescence level of the plate was measured using an instrument, and the results are shown in Fig. 5.

[0167] To determine whether similar efficacy is observed in other hydrogen pump inhibitors as well as in esomeprazole, the expression-enhancing effects were examined using representative hydrogen pump inhibitors such as omeprazole (OME), pantoprazole (PTZ), and rabeprazole (RAB).

[0168] Specifically, in FIG. 5, SM LNP is Hela cells treated with the lipid nanoparticles (SM-102_mRNA) of Preparation Example 1, OME@SM LNP is Hela cells treated with the lipid nanoparticles (OME@SM LNP) of Example 7-2, ESO@SM LNP is Hela cells treated with the lipid nanoparticles (ESO@SM LNP) of Example 7-1, PAN@SM LNP is Hela cells treated with the lipid nanoparticles (PAN@SM LNP) of Example 7-3, and RAB@SM LNP is Hela cells treated with the lipid nanoparticles (RAB@SM LNP) of Example 7-4.

[0169] As shown in Figure 5, it was confirmed that mRNA expression efficiency increased when lipid nanoparticles encapsulating each of the four hydrogen pump inhibitors and mRNA were treated to cells.

[0170] Experimental Example 6. Measurement of increased mRNA expression after administering a mixture of lipid nanoparticles and a hydrogen pump inhibitor subcutaneously to animals

[0171] To confirm the increase in genetic material expression efficiency of proton pump inhibitors (PPIs) at the biological level, the compositions of Examples 1-1, 2-1, and 3-1 were administered subcutaneously or intramuscularly to mice, and images of luminescent protein expression were measured using an instrument (LUCI, CST science, Korea).

[0172] Figure 6 shows the increase in mRNA expression measured after administering the composition of Example 1-1 subcutaneously to a mouse, Figure 7 shows the increase in mRNA expression measured after administering the composition of Example 2-1 subcutaneously to a mouse, and Figure 8 shows the increase in mRNA expression measured after administering the composition of Example 3-1 subcutaneously to a mouse.

[0173] As shown in Figures 6 to 8, when the composition according to the present invention (lipid nanoparticles + PPI) was used, a higher degree of mRNA expression was observed compared to when only lipid nanoparticles (SM-102, MC3, DOTAP) were administered.

[0174] In addition, the composition for delivering genetic material according to the present invention exhibited a higher level of fluorescence expression compared to a control group using other mRNA carriers (PBS-treated group, mRNA-treated group, lipid nanoparticles (SM-102, MC3, DOTAP)).

[0175] The above results indicate that when the composition for genetic material delivery according to the present invention is used, there is a tendency for the efficiency of genetic material expression in vivo to increase.

[0176] Experimental Example 7. Confirmation of increased antitumor effect of cancer vaccine following increased tumor antigen mRNA expression after mixed administration of lipid nanoparticles and a hydrogen pump inhibitor to animal muscles.

[0177] To confirm the increase in antitumor effect of proton pump inhibitors (PPIs) as a result of increased expression efficiency of genetic material for tumor antigens at the biological level, the compositions of Examples 1-1, 2-1, and 3-1, prepared using tumor antigen mRNA (OVA mRNA), were administered to the muscles of mice a total of three times (each dose being 2 μg OVA mRNA), and subsequently, tumor cells (B16-OVA) expressing the corresponding tumor antigen were inoculated subcutaneously to compare the growth trends of the tumors.

[0178] As shown in Figure 9, a higher antitumor effect was observed when using the composition according to the present invention (lipid nanoparticles + PPI) compared to when lipid nanoparticles were administered alone.

[0179] These results indicate that when using the composition for genetic material delivery according to the present invention, the efficiency of genetic material expression can be increased through the activity of the hydrogen pump inhibitor.

[0180]

[0181] The specification omits detailed descriptions of matters that can be sufficiently recognized and inferred by those skilled in the art of the present invention, and various modifications are possible within the scope of not altering the technical concept or essential configurations of the present invention, in addition to the specific examples described in this specification. Accordingly, the present invention may be implemented in a manner different from that specifically described and exemplified in this specification, and this is a matter that can be understood by those skilled in the art.

Claims

1. Lipid nanoparticles comprising ionized lipids, phospholipids, cholesterol, and lipid-PEG (polyethylene glycol) conjugates; and hydrogen pump inhibitor; A composition for delivering genetic material comprising 2. In Paragraph 1, A composition wherein the above composition further comprises a dielectric material enclosed within the lipid nanoparticles.

3. In Paragraph 1, A composition in which the above hydrogen pump inhibitor is enclosed inside the above lipid nanoparticles.

4. In Paragraph 1, A composition comprising one or more hydrogen pump inhibitors selected from the group consisting of esomeprazole, pantoprazole, omeprazole, lansoprazole, rabeprazole, ilaprazole, and dexlansoprazole.

5. In Paragraph 1, The above ionized lipids are 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt) (DOTAP), 8-[2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octylnonyl ester (SM-102), (6Z, 9Z, 28Z, 31Z)-hephtatriaconta 6,9,28,31-tetraene-19-yl4-(dimethylamino)butanoate (DLin-MC3-DMA), [(4-hydroxybutyl)azandyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyl Carbamoyloxy-3-dimethylaminopropane (DLin-CDAP), 1,2-dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-(2,3-dioleyloxy)propylamine (DODMA), dioctadecylamidoglycyl spermine (DOGS), spermine cholesteryl carbamate (GL-67), Bis-guanidinium-spermidine-cholesterol (BGTC), 3β-(N(N',N'-dimethylaminoethane)-carbamoyl) cholesterol (DC-Chol), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydecyl)amino)ethyl)(2-hydroxydecyl)amino)ethyl)piperazine-1-yl)ethylazandyl)dododecane-2-ol (C12-200), Nt-butyl-N'-tetradecylamino-propionamidine (diC14-amidine), dimethyldioctadecylammonium bromide (DDAB), N(1,2-dimyristyl oxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N,N-dioleylN,N-dimethylammonium Chloride (DODAC), Dioleyl oxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE), N-(1-(2,A composition comprising one or more selected from the group consisting of 3-dioleyl oxy)propyl)-N-2-(spermine carbokixamide)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), N-(1-(2,3-dioleyl oxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), aminopropyl-dimethyl-bis(dodecyloxy)-propane aluminum bromide (GAP-DLRIE), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dipalmityl-3-trimethylammonium propane (DPTAP), 1,2-distearyl-3-trimethylammonium propane (DSTAP), and 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC).

6. In Paragraph 1, The above phosphatidyl ethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylethanolamine (DSPE), A composition comprising one or more selected from the group consisting of phosphatidylethanolamine (PE), dipalmitoylphosphatidylethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS), and 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine].

7. In Paragraph 1, A composition comprising one or more selected from the group consisting of the lipid-PEG conjugate, PEG-DMG, PEG-DAA conjugated to dialkyloxypropyl, PEG-DAG conjugated to diacylglycerol, PEG-PE conjugated to a phospholipid such as phosphatidylethanolamine, PEG-CER conjugated to ceramide, PEG conjugated to cholesterol or a derivative thereof, PEG-c-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, and PEG-DSPE.

8. In Paragraph 1, A composition in which the concentration of the hydrogen pump inhibitor in the above composition is 1 to 250 μg / mL.

9. In Paragraph 1, A composition comprising the above lipid nanoparticles in a molar ratio of ionized lipid : phospholipid : cholesterol : lipid-PEG conjugate of 10 to 60 : 5 to 50 : 30 to 50 : 0.5 to 5.

10. A pharmaceutical composition for the prevention or treatment of cancer comprising: lipid nanoparticles comprising ionized lipids, phospholipids, cholesterol, and lipid-PEG (polyethylene glycol) conjugates; a hydrogen pump inhibitor; and a genetic material.

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