Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
  • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
  • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
  • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
  • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
  • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle

Definitions

• Carrier composition for nucleic acid delivery is a composition for nucleic acid delivery
• the present invention provides a low-toxicity and high-safety nucleic acid delivery carrier capable of protecting nucleic acids from degradation and efficiently delivering them into cells when the nucleic acids are administered to animal-derived cells or organisms.
• the present invention relates to a composition and a composition for nucleic acid delivery.
• siRNA small interfering RNA
• RNA interference RNA interference
• This target gene expression inhibition function by RNA interference is useful for reducing or treating disease symptoms caused by abnormal expression of a specific gene or group of genes, and siRNA can be developed as a therapeutic agent.
• siRNA can be developed as a therapeutic agent.
• the nucleic acid is efficiently delivered into the target cell. It is essential to establish technology.
• Patent Document 1 has reported a cationic lipid having a specific structure as a carrier for non-viral nucleic acid delivery that facilitates the delivery of nucleic acids such as siRNA into cells.
• the cationic lipid reported in Patent Document 1 has a drawback that it exhibits toxicity when administered to cultured cells or living bodies.
• Patent Document 2 discloses an amphiphilic composition as a carrier composition having relatively low toxicity and capable of delivering siRNA into cells. A composition comprising a compound and a polycation is disclosed.
• Patent Document 2 when a sufficient amount of siRNA was introduced into the cell, there was still a problem in terms of toxicity to the cell.
• Patent Document 1 Japanese Translation of Special Publication 2002-529439
• Patent Document 2 Special Publication 2005-508394
• an object of the present invention is to solve the above-described problems of the prior art. Specifically, the object of the present invention is that when a nucleic acid such as siRNA is administered to an animal-derived cell or organism, the nucleic acid can be efficiently delivered into the cell while protecting its degradability and is low in toxicity.
• An object of the present invention is to provide a nucleic acid delivery carrier composition exhibiting high safety, and a nucleic acid delivery composition containing the carrier composition and a nucleic acid.
• a carrier composition for nucleic acid delivery comprising (A) a cationic lipid having a steroid nucleus, and (B) a quaternary ammonium salt type cationic lipid.
• the carrier composition for nucleic acid delivery according to Item 1 further comprising (C) an oily substrate.
• the component (A) is 3 ⁇ -[ ⁇ - ( ⁇ ,, ⁇ , -dimethylaminoethane) -force rubermoyl] cholesterol, and / or 3 ⁇ -[ ⁇ ,, ⁇ ', ⁇ ,- Trimethylaminoethane] cholesterol iodide Item 2.
• Item 4 Component strength Dimethyldioctadecylammo-umbromide salt, dioleoyltrimethylammonium propane, and N- (1- (2,3-bis (oleoyloxy) propyl) - ⁇ , ⁇ , ⁇ -trimethylammo- Item 2.
• Item 5 The carrier composition for nucleic acid delivery according to Item 1, wherein component (ii) is contained in an amount of 10 to 200 parts by weight per 100 parts by weight of component (ii).
• Item 6 The carrier composition for nucleic acid delivery according to Item 1, which is a carrier for delivering siRNA.
• Item 7 A nucleic acid delivery composition comprising the nucleic acid and the nucleic acid delivery carrier composition according to any one of Items 1 to 5.
• Item 8 The nucleic acid delivery composition according to Item 7, which is RNA.
• Item 9 A method for introducing a nucleic acid, wherein the nucleic acid is introduced into a cell by bringing the composition for nucleic acid delivery according to Item 7 into contact with the cell.
• Item 10 Use of (A) a cationic lipid having a steroid nucleus and (B) a quaternary ammonium salt type cationic lipid for the production of a carrier composition for nucleic acid delivery.
• nucleic acid delivery carrier composition and nucleic acid delivery composition of the present invention According to the nucleic acid delivery carrier composition and nucleic acid delivery composition of the present invention, the following excellent effects can be obtained.
• the nucleic acid can be efficiently delivered into the cell and the function of the nucleic acid can be effectively exhibited in the cell.
• the carrier composition for nucleic acid delivery and the composition for nucleic acid delivery are useful for the treatment of various diseases by introduction of nucleic acid, particularly for the treatment of intractable diseases that are difficult to treat with low molecular weight compounds.
• nucleic acid delivery composition of the present invention can be lyophilized, it can be stored in a lyophilized state.
• FIG. 1 is a diagram showing the results of observation of fluorescence images derived from nucleic acids of cells using a fluorescence microscope after treating cells with various test samples in Test Example 1.
• FIG. 2 shows the results of measuring the luciferase activity of cells after treating the cells with various test samples in Test Example 2.
• FIG. 3 shows the results of measuring neprilysin (NEP) activity in rat lungs in Test Example 3.
• FIG. 4 is a graph showing the results of measurement of the number of viable cells after treatment with various test samples in Test Example 4.
• FIG. 5 is a diagram showing the results of staining rat lung tissue sections with hematoxylin-eosin in Test Example 5.
• hematoxylin stains nuclei and ribosomes in blue-blue
• eosin stains cytoplasm, fibers and erythrocytes in red.
• the carrier composition for nucleic acid delivery of the present invention comprises (A) a cationic lipid having a steroid nucleus, and (B) a quaternary ammonium salt type cationic lipid. is there.
• the nucleic acid delivery carrier composition of the present invention is used as a nucleic acid carrier for delivering (introducing) a nucleic acid into a cell.
• the nucleic acid to which the carrier composition for nucleic acid delivery of the present invention is applied is not particularly limited as long as it is required to be delivered into cells.
• Specific examples of the nucleic acid include siRNA, mRNA, tRNA, rRNA, cDNA, miRNA (microRNA), ribosome, antisense oligo, decoy oligonucleotide, plasmid DNA, peptide nucleo acid, triplex forming oligonucleotide (Triplex Forming Oligonucleotide) , TFO) and genes.
• the carrier composition for nucleic acid delivery of the present invention is particularly useful for delivery of siRNA into cells.
• the nucleic acid to which the carrier for nucleic acid delivery of the present invention is applied may be derived from humans, animals, plants, bacteria, viruses, etc., or may be produced by chemical synthesis. In addition, these nucleic acids are single-stranded, double-stranded, 3 There are no particular restrictions on the molecular weight of any of the strands. In the present invention, the nucleic acid may be modified with chemistry, an enzyme, or a peptide. In the present invention, one kind of nucleic acid may be used alone, or two or more kinds of nucleic acids may be used in appropriate combination.
• the cationic lipid having a steroid nucleus used in the carrier composition for nucleic acid delivery of the present invention (hereinafter sometimes referred to as “component (A)”) is a steroid nucleus (perhydrocyclopentaphenanthrene). It is a lipid having a ring; Used in the present invention
• the cationic lipid having a steroid nucleus is not particularly limited as long as it is pharmacologically acceptable. Specific examples include 3
• the cationic lipid having a steroid nucleus is preferably a cholesterol derivative (cationic lipid having a cholesterol skeleton), more preferably 3
• the quaternary ammonium salt type cationic lipid (hereinafter also referred to as “component (B)”) used in the carrier composition for nucleic acid delivery of the present invention is a drug. There is no particular limit as long as it is physically acceptable.
• DDAB dimethyldioctadecylmomum bromide salt
• DOTAP 1,2-dimyristoyl-3-trimethylammonium propane
• DOTAP 1,2-dioleoyl-3-trimethylammonium propanemethyl sulfate
• 1,2-dipalmitoyl-3-trimethylammonium propane 1,2-distearoyl-3-trimethylammonium propane, N- ( 1- (2,3-bis (oleoyloxy) propyl) - ⁇ , ⁇ , ⁇ -trimethylammonium hydrochloride (DOTMA),
• DDAB dioleotrimethylammonium propane
• DOTAP dioleotrimethylammonium propane
• DOTMA N- (l- (2,3-bis (oleyloxy) propyl pill)- ⁇ , ⁇ , ⁇ -trimethylammonium hydrochloride
• the mixing ratio of the component (i) and the component (i) is not particularly limited, but the nucleic acid is more efficiently delivered into the cell.
• the component (ii) is 10 to 200 parts by weight, preferably 30 -150 weight part, More preferably, 75-125 weight part is illustrated.
• the total amount of the component (A) and the component (B) relative to the total amount of the nucleic acid delivery carrier of the present invention is, for example, 10 to 100% by weight, preferably 20 to 80% by weight, more preferably 40 to 70% by weight. %
• the carrier composition for nucleic acid delivery of the present invention may further contain an oily base (hereinafter also referred to as component (C)) in addition to the components (A) and (B). .
• component (C) oily base
• component (C) oily base
• the specific gravity of the carrier composition for nucleic acid delivery by blending an oily base, the contact efficiency between the cell and the carrier composition for nucleic acid delivery can be controlled and the introduction efficiency in vitro can be improved. It becomes possible.
• the nucleic acid carrier core is disrupted under a predetermined temperature condition to induce fluctuations on the cell surface and improve the efficiency of nucleic acid introduction. It becomes possible to make it.
• the core of the nucleic acid carrier composition is disrupted by external stimuli to induce fluctuations on the cell surface and improve nucleic acid introduction efficiency. It becomes possible to make it.
• Examples of the oily base material to be blended in the carrier composition for nucleic acid delivery of the present invention include, for example, perphnore mouth carbon, perfoleo mouth pentane, perfanolol octyl bromide, perfluoro mouth hexane, perfluoro Tributylamine, soybean oil, refined soybean oil, soybean hardened oil, soybean oil unsaponifiable oil, squalene, castor oil, thiodi oil, sorbitan trioleate, turpentine oil, safflower oil, safflower oil fatty acid, oleic acid, coconut oil, Rapeseed oil, fusel oil, olive oil, linseed oil, sesame oil, chlorophyll oil, haze oil, bergamot oil, cedar oil, orange oil, wikial oil, eucalyptus oil, corn oil, lavender oil, cypress oil, lemon oil , Cottonseed oil, palm oil
• perfluoropentane is temperature sensitive and has the property of gasifying by boiling at 29.5 ° C.
• Perfluoro oral hexane, perfluorooctyl bromide, and perfluorotributylamine have external stimulatory disintegration properties, and the core of the carrier composition is stimulated by external force stimulation such as stimulation by ultrasonic irradiation. It has the characteristic that it generates and collapses the bite.
• the ratio of the oily base is not particularly limited as long as the effect of the present invention is not hindered, but the total amount of the component (A) and the component (B) is 100 parts by weight.
• the ratio of the oily base material is 0.1 to 50 parts by weight, preferably 1 to 30 parts by weight, and more preferably 5 to 20 parts by weight.
• the carrier composition for nucleic acid delivery of the present invention may contain a membrane-fusible lipid (helper lipid) as necessary. By containing such a membrane-fusible lipid, it becomes possible to further improve the efficiency of nucleic acid delivery into the cell.
• a membrane-fusible lipid helper lipid
• membrane-fusible lipids examples include dioleoylphosphatidylethanolamine, dioleoylphosphatidylcholine, phosphatidylphosphatidylethanolamine, 1,2-bis (10,12- (Tricosadinol) -phosphoethanolamine, 1,2-gerideylphosphoethanolamine, 1,2-dihexadecylphosphoethanolamine, 1,2-dihexanoylphosphoethanol 1,2-dilauroylphosphoethanolamine, 1,2-dilinoleoylphosphoethanolamine, 1,2-dimyristoylphosphoethanolamine, 1,2-dioleoylphosphoethanol Ethanolamine, 1,2-Dipalmitooleoylphosphoethanol Ethanolamine, 1,2-Dipalmitoylphosphoethanolamine, 1,2-Difilter Nylphosphoethanolamine, 1,2-distearoylphosphoethanolamine, 1-palmitoyl-2-oleoylphosphoethanolamine, 1-palmitoyl-2- (10,12-tricos
• the ratio of the membrane-fusible lipid is not particularly limited as long as the effect of the present invention is not hindered.
• the total amount of the above (ii) component and (ii) component is 100.
• the carrier composition for nucleic acid delivery of the present invention has various additions such as tonicity agents, excipients, diluents, thickeners, stabilizers, buffers, preservatives, etc., depending on the form of use.
• An agent can be contained.
• the blending amount of these additives can be appropriately set according to the usage form of the carrier for nucleic acid delivery.
• the carrier composition for nucleic acid delivery of the present invention is produced by mixing the component (ii), the component (ii), and other components as necessary.
• the nucleic acid delivery composition of the present invention comprises the above-described nucleic acid delivery carrier composition and a nucleic acid, and in the nucleic acid delivery composition, the nucleic acid is ionically bonded to the components of the nucleic acid delivery carrier composition.
• a complex is formed by hydrophobic bonding, and the delivery rate of nucleic acids into cells is improved.
• the nucleic acid delivery composition of the present invention is a mixture of the above-described nucleic acid delivery carrier composition and a nucleic acid, or a mixture of nucleic acids and the above-mentioned nucleic acid delivery carrier composition in any order. Manufactured by doing.
• the binding ratio between the nucleic acid and the nucleic acid delivery carrier composition includes the type of nucleic acid to be used, the type of nucleic acid delivery carrier, the type of cells to which nucleic acid is delivered, and the like.
• the ratio is 2.5 to 50 parts by weight.
• the total amount of the components (A) and (B) contained in the nucleic acid delivery composition is, for example, 10 to 90% by weight, preferably 30 to 80% by weight, based on the total amount of the composition. More preferred is 50 to 70% by weight.
• the nucleic acid delivery composition of the present invention comprises various additives such as isotonic agents, excipients, diluents, thickeners, stabilizers, buffers, preservatives, etc., depending on the form of use. Can be contained. The blending amount of these additives can be appropriately set according to the usage form of the nucleic acid delivery composition.
• examples of cells to which nucleic acids are delivered include cultured cells, cells extracted from living organisms (including established cells), cells existing in vivo, and the like.
• the usage mode of the nucleic acid delivery composition of the present invention is not particularly limited as long as the nucleic acid delivery composition is applied so as to come into contact with a cell into which a nucleic acid is to be introduced.
• nucleic acid delivery composition for example, direct injection into tissues; injection into veins, subcutaneous muscles, abdominal cavity, intraocular, digestive organs, endodontics, etc .; nasal cavity, oral cavity And inhalation administration to the lung, etc .; oral administration; transdermal administration via the skin; and transmucosal administration via the oral mucosa, vaginal mucosa, ocular mucosa, rectal mucosa, uterine mucosa and the like.
• nucleic acids are to be delivered to cultured cells or cells extracted from living organisms, the nucleic acid delivery composition should be selected at the time of cultivation.
• a method for culturing cells in the existing state is exemplified.
• nucleic acids can be delivered into cells even in the presence of serum.
• a carrier composition for nucleic acid delivery having the following composition was prepared.
• nucleic acid-containing solution having the following composition was prepared.
• a carrier composition for nucleic acid delivery having the following composition was prepared.
• a carrier composition for nucleic acid delivery having the following composition was prepared.
• A549 cells adjusted to a concentration of 1.2 ⁇ 10 5 cells / ml using DMEM medium (Dulbecco-Minimum Essential Medium) were transferred to a 24-well plate at 6.0 ⁇ 10 4 cells per well. Cells were seeded to enter.
• the test sample 5001 shown in Table 1 was added to each well and incubated at 37 ° C under 5% CO for 24 hours. Then derived from the nucleic acid of the cell
• the light image was observed using a light microscope (Olympus IX 71 fluorescence microscope; Olympus, Tokyo, Japan), and the delivery of siRNA into the cells was evaluated.
• LFA2000 Li po fec tami ne2000; manufactured by Invitrogen (1.0 mg / m,
• NeoPhec tin (NeoPharm) (1. O mg / mL), siRNA (20 pmol / ml)
• a plasmid encoding the luciferase gene is transiently introduced into the cell, and then the nucleic acid delivery composition of Example 2 above is added to express luciferase. The amount was evaluated.
• GL3-siRNA siRNA against firefly luciferase; Dharmacon, Boulder, CO, USA; sense: 5′-CUUACGCUGAGUACUUCGAdTdT, antisense: 5′-UCGAAG UACUCAGCGUAAGdTdT
• siRNA was used as the siRNA in this test.
• the cells in the well are lysed according to a conventional method to obtain a cell lysate, and the activity of the noreiferase in the cell lysate is determined using the Dua Luciferase Reporter Assay System (Promega, Madison, WI, USA). And evaluated. Luciferase activity was determined by calculating the ratio of firefly luciferase activity to relative luciferase (relative activity:%).
• siRNA for Rat Neprilysin Rat Neprilysin (Rat Neprilysin (NM_012608); RNA—TEC NV, Belgium; Sense: 5′-GCUCCAAAGCCGAAGAAGAdTdT, Antisense: 5′-UCUUCUUCGGCUUUGGAGCdTdT) was used as the siRNA in this test.
• the functionality and delivery of siRNA in lung tissue cells of the carrier composition for nucleic acid delivery were evaluated.
• a nucleic acid delivery composition was prepared by mixing the nucleic acid delivery carrier composition of Example 3 and siRNA in a 1: 1 weight ratio.
• test solution obtained by diluting the nucleic acid delivery composition in an appropriate carrier (8.89 w / v% sucrose aqueous solution) was added to a male SD rat (SLC, Tokyo, Japan) weighing 250-320 g.
• the IB inhalation device (PENNCENTURY, Philad elphia, PA, USA) was used for transpulmonary administration under intoxication with the inhalation anesthetic agent isoflurane (Dainippon Pharmaceutical Co., Ltd.).
• the test solution was prepared by appropriately diluting the nucleic acid delivery composition so that the siRNA dosage was 0.04 to 0.12 mg / kg (rat).
• ether was anesthetized and the rat was fixed in the dorsal position. Rats were laparotomized along the median gland and exsanguinated from the inferior vena cava. The lungs were then removed from the rats and washed with ice-cold physiological saline. Using the isolated lung, the mRNA expression level of the model target gene NEP (neutral endopeptidase) and the mRNA expression level of the housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were measured. Furthermore, rat neprilys in (NEP) activity in the isolated lung was measured. These specific measuring methods and results are as follows. As a control, the same test was performed when only the carrier was administered under the same conditions. For comparison, instead of Rat Neprilysin, EGFP (enhanced green fluorescent protein siRNA (Takara, Japan; Sense: 5
• Rat neprilysin (NEP) activity was measured in the presence or absence of phosp horamidon (SIGMA), a NEP-specific inhibitor, in the presence or absence of NEP substrate DAGNPG (N-Dansyl-D-Ala-Gly).
• DAGNPG N-Dansyl-D-Ala-Gly
• SIGMA phosp horamidon
• the rat lung homogenate used at this time was 50 mL, the concentration of the substrate DAGPNG was 1 mM, and in the presence of an inhibitor, 10 mM phosphoramidon was added, and the reaction was carried out in a total volume of 100 mL.
• the reaction is set at 37 ° C for 10 minutes, and the reaction is stopped by incubating at 90 ° C for 10 minutes, and the amount of degradation product DAG (Dansy ⁇ D-Ala-Gly) generated at this time is measured. NEP activity was calculated.
• the amount of degradation product DAG was determined by measuring the fluorescence of the degradation product, and the fluorescence was measured by emitting at 535 nm after excitation at 360 nm.
• the cytotoxicity of the carrier composition for nucleic acid delivery obtained in Example 1 use the Premix W3 ⁇ 4 ⁇ -1 cell proliferation assay system (Takara, biga, Japan). Specifically, A549 cells (human lung cancer-derived cell line; manufactured by Dainippon Pharmaceutical Co., Ltd.) were placed on a 96-well plate using DMEM medium (Dul becco-Minimum Essential Medium) (1 X 10 ”) to adjust the concentration of the individual / ml, and seeded as the number of cells per each Ueru becomes 10 4 then the test samples [nucleic acid delivery carrier in example 1, LFA2000.
• DMEM medium Dul becco-Minimum Essential Medium
• the rat was laparotomized along the median gland and exsanguinated from the inferior vena cava.
• the lungs were then removed from the rats and washed with ice-cold physiological saline.
• the excised lung tissue sections were prepared, stained with hematoxylin-eosin, and observed under a microscope to evaluate the toxicity of the carrier composition for nucleic acid delivery to the lung tissue.
• LFA2000 Lipofectamine2000; manufactured by Invitrogen
• NeoPhectin manufactured by NeoPharm

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