Lipid nanoparticles comprising long-chain ceramide and composition for apoptosis comprising same

Abstract

Long-chain ceramides, such as exogenous C18-ceramides, have very low solubility in aqueous solutions and thus are limited in use, even if used as medicaments, with poor cell delivery. In this aspect, the invention provides lipid nanoparticles comprising long chain ceramides and methods of making the same. According to the present invention, by delivering a long-chain ceramide, such as C18-ceramide, to a target in vivo through lipid nanoparticles, cell death can be effectively induced according to a purpose, and utilization as a cell therapy product having high water solubility and high water dispersibility can be increased.

Description

[Technical field]

[0001] The present invention relates to lipid nanoparticles containing long-chain ceramide, and a nanoparticle delivery composition for cell apoptosis having excellent water solubility and water dispersibility comprising the lipid nanoparticles.

[0002] The present invention also relates to a method for preparing lipid nanoparticles containing long-chain ceramides with high in vivo targeted delivery, and a method for inducing cell apoptosis using the nanoparticles. [Background technology]

[0003] Ceramide is a sphingolipid composed of sphingosine and fatty acids of various chain lengths. The endogenous form of ceramide is usually composed of a long carbon chain of C16 to C24 in animal cells. There are six types of ceramide synthase in animal cells, which are responsible for attaching fatty acyl-CoA of specific length to the long chain base to determine the acyl chain length of sphingolipids (including ceramide, sphingomyelin and glycosphingolipids).

[0004] C16-C24-ceramides classified according to carbon chain length can function as active signaling molecules that regulate various cellular processes. In particular, C18-ceramide can cause cell death and cell proliferation inhibition by catalysis by ceramide synthases 1 and 4 in the de novo synthesis pathway. However, since these long-chain ceramides have extremely low solubility in aqueous solutions, their use is extremely limited despite their efficacy when they can only be dissolved in organic solvents such as ethanol. In addition, since most C16-C20-ceramides exhibit antiproliferative and apoptotic effects, they can cause side effects such as cell death in normal cells if they are not delivered to specific cells such as cancer cells. In this regard, a method for dispersing and utilizing long-chain ceramides with extremely low water solubility is urgently needed.

[0005] [Prior art literature]

[0006] [Patent Literature]

[0007] (Patent Document 1) Korean Patent No. 10-2325570 (Cosmetic composition for moisturizing and improving skin barrier containing nanoliposomes including oxygen, water and ceramide and preparation method thereof)

[0008] (Patent Document 2) Korean Patent No. 10-0713555 (Hair protection composition containing nanoliposomes stabilized by encapsulating ceramide as an active ingredient)

[0009] (Patent Document 3) Korean Patent No. 10-2267378 (a pharmaceutical composition for preventing or treating breast cancer containing C12, C16 or C18-ceramide as an active ingredient)

[0010] (Patent Document 4) Korean Patent No. 10-1796036 (Nanoliposome delivery carrier composition of a complex of encapsulated Cas9 protein, guide RNA for inhibiting KRAS gene expression, and cationic polymer, or a colorectal cancer therapeutic agent for treating anticancer drug resistance caused by KRAS gene mutation containing the composition)

[0011] (Patent Document 5) Korean Patent No. 10-1710026 (Nanoliposome delivery carrier composition containing Cas9 protein and guide RNA hybrid)

[0012] (Patent Document 6) Korean Patent No. 10-2328197 (Nanoliposome delivery carrier composition with KRAS and p53 gene editing function)

[0013] (Patent Document 7) Japanese Patent No. 6944061 (Nanoliposome-microbubble complex encapsulating a complex of Cas9 protein, guide RNA for inhibiting SRD5A2 gene expression, and a cationic polymer, and a composition for treating or preventing hair loss containing the complex)

[0014] (Patent Document 8) Japanese Patent No. 6876202 (Nanoliposome-microbubble complex encapsulating a drug for hair loss treatment and a composition for alleviating or treating hair loss containing the complex)

[0015] (Patent Document 9) Japanese Patent No. 3920330 (Polyethylene glycol-modified ceramide lipids and their use in liposomes)

[0016] [Detailed description of the invention]

[0017]

Technical issues

[0018] The object of the present invention is to prepare a lipid nanoparticle (LNP) composition by incorporating exogenous long-chain ceramide into a lipid layer, thereby providing lipid nanoparticles containing long-chain ceramide having excellent water solubility and water dispersibility.

[0019] Furthermore, the present invention provides an excellent apoptosis composition comprising lipid nanoparticles containing long-chain ceramide having excellent water solubility and water dispersibility.

[0020] Furthermore, the present invention provides an anticancer composition having high targeted delivery in vivo, the composition comprising lipid nanoparticles containing long-chain ceramide having excellent water solubility and water dispersibility.

[0021] [Technical solution to the problem]

[0022] The present invention relates to lipid nanoparticles comprising a long-chain ceramide having a structure shown in the following Chemical Formula 1:

[0023]

Chemical formula 1

[0024]

[0025] (In the above Chemical Formula 1, R1 is a saturated or unsaturated aliphatic chain having 17 carbon atoms, and R2 is one selected from the following: phytosphingosine, sphingosine and dihydrosphingosine, and the number of carbon atoms is 10 to 16).

[0026] The long-chain ceramide having the structure of Chemical Formula 1 may be a compound selected from the following: an N-oleoyl-phytosphingosine-based compound or a derivative thereof; an N-stearoyl-erythro-sphingosine-based compound or a derivative thereof; an N-oleoyl-erythro-sphingosine-based compound or a derivative thereof; an N-stearoyl-erythro-dihydrosphingosine-based compound or a derivative thereof; and an N-oleoyl-erythro-dihydrosphingosine-based compound or a derivative thereof.

[0027] The N-oleoyl-phytosphingosine-based compound or a derivative thereof may be a compound of the following Chemical Formula 2 or a derivative thereof.

[0028]

Chemical formula 2

[0029]

[0030] The N-stearoyl-erythro-sphingosine-based compound or a derivative thereof may be a compound selected from the following Chemical Formulas 3 and 4 or a derivative thereof.

[0031]

Chemical formula 3

[0032]

[0033]

Chemical formula 4

[0034]

[0035] The N-oleoyl-erythro-sphingosine-based compound or a derivative thereof may be a compound selected from the following Chemical Formulas 5 and 6, or a derivative thereof.

[0036]

Chemical formula 5

[0037]

[0038]

Chemical formula 6

[0039]

[0040] The N-stearoyl-erythro-dihydrosphinganine-based compound or a derivative thereof may be a compound of the following Chemical Formula 7 or a derivative thereof.

[0041]

Chemical formula 7

[0042]

[0043] The N-oleoyl-erythro-dihydrosphinganine-based compound or a derivative thereof may be a compound of the following Chemical Formula 8 or a derivative thereof.

[0044]

Chemical formula 8

[0045]

[0046] The lipid nanoparticle is characterized by comprising a long-chain ceramide of Chemical Formula 1, and

[0047] The composition is characterized in that it comprises at least one lipid selected from the group consisting of phosphatidylcholine, cholesterol, non-cationic lipids, and PEG-modified lipids. Preferably, it may comprise all of the following: phosphatidylcholine, cholesterol, non-cationic lipids, and PEG-modified lipids.

[0048] Lecithin is widely distributed in the animal / plant kingdom, so it has excellent biocompatibility and its stability has been verified, so it is widely used in food and drug delivery technology. In addition, it can also be used as a material to facilitate the size control and modification of lipid nanoparticles.

[0049] Cholesterol provides morphological rigidity to the lipid packing in lipid nanoparticles and plays a role in improving the stability of the nanoparticles by being dispersed in the core and surface of the nanoparticles.

[0050] The non-cationic lipid may include at least one lipid selected from the group consisting of a neutral lipid and an anionic lipid.

[0051] Neutral lipids refer to lipids that are uncharged or have a neutral zwitterionic form within a pH range of 4.0 to 8.0. In addition, the neutral lipids may include any neutral lipids known to those skilled in the art. For example, the neutral lipids may be HSPC (hydrogenated soybean phosphatidylcholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), POP (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine). The invention also includes but is not limited to phosphocholine, sphingolipids, diacylglycerol, succinyl-sn-glycero-3-phosphoethanolamine, phosphatidylcholine, phosphatidylcholine, sphingolipids, diacylglycerol, phosphatidylethanolamine, tetraether lipids, sphingolipids, diacryloylglycerol and glycerides, or combinations thereof.

[0052] Anionic lipids refer to any amphipathic lipids having at least one negative charge within the range of pH 4.0 to pH 8.0. In addition, anionic lipids may include any anionic lipids known to those skilled in the art. For example, anionic lipids may be DOPS (dioleoylphosphatidylserine), DMPG (dimyristoylphosphatidylglycerol), DPPG (dipalmitoylphosphatidylglycerol), DTPA (diethylenetriaminepentaacetic acid), DPTGA (1,4-dipalmitoyl-tartrate-2,3-diglutaric acid), DSTSA (1,4-distearoyl-tartrate-2,3-disuccinic acid), CHHDA (2-carboxy heptadecayl heptadecanamide), DMPS (dimyristoylphosphatidylserine) Acid), DPPS (dipalmitoylphosphatidylserine), POPS (palmitoyloleoylphosphatidylserine), DOPG (dioleoylphosphatidylglycerol), POPG (palmitoyloleoylphosphatidylglycerol), DMPA (dimyristoylphosphatidic acid), DPPA (dipalmitoylphosphatidic acid), DOPA (dioleoylphosphatidic acid), POPA (palmitoyl-oleoylphosphatidic acid), CetylP (cetyl phosphate) and CHEMS (cholesterol hemisuccinate), or a combination thereof.

[0053] As long as it is a lipid that can bind to polyethylene glycol, the lipid of the PEG-modified lipid can be used without limitation. Preferably, it can be a non-cationic phospholipid that can bind to polyethylene glycol (PEG).

[0054] The PEG in the above-mentioned PEG-modified lipids is a hydrophilic polymer that has the ability to inhibit plasma protein adsorption, thereby increasing the circulation time of lipid nanoparticles in the body and preventing aggregation between nanoparticles. In addition, PEG-modified lipids exhibit a stealth function in the body, thereby preventing the degradation of nanoparticles.

[0055] The PEG may be a functionalized PEG having a functional group bound to one side not bonded to the lipid. In this case, the functional group may be one or more selected from the following: succinyl, carboxylic acid, maleimide, amine, biotin, cyano, folate, etc.

[0056] The above-mentioned PEG refers to typical polyethylene glycol, and in the present invention, POZ [poly(2-oxazoline)] can be used as a substitute thereof.

[0057] The particle size of the lipid nanoparticles may be 10 to 2,000 nm. If the size of the lipid nanoparticles is less than 10 nm or greater than 2,000 nm, the particle stability may be reduced, which is not preferred.

[0058] The lipid nanoparticles preferably contain 2-5 mol of a long-chain ceramide having a structure of Chemical Formula 1, 0.5-5 mol of phosphatidylcholine, 0.1-1 mol of cholesterol, 0.01-0.5 mol of a non-cationic lipid (such as DSPC) and 0.05-0.5 mol of a PEG-modified lipid (such as DSPE-PEG (2000) amine) in the following ratios.

[0059] The present invention relates to a nanoparticle delivery composition for cell apoptosis, comprising lipid nanoparticles. The lipid nanoparticles comprise a long-chain ceramide having the structure of the above Chemical Formula 1.

[0060] The composition may contain 1×10 9 ~1× 12 A composition of particles.

[0061] The cell may be a cancer cell. The cancer cell may be derived from a cancer tissue selected from the group consisting of hemangioma, angiofibroma, lung cancer, non-small cell lung cancer, liver cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, melanoma, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, anal cancer, colon cancer, breast cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, 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, bladder cancer, kidney cancer or ureter cancer, renal cell carcinoma, renal pelvis cancer, central nervous system tumors, primary central nervous system lymphomas, spinal cord tumors, brain stem gliomas, and pituitary adenomas.

[0062] The present invention also relates to a method for preparing lipid nanoparticles, wherein the lipid nanoparticles contain the long-chain ceramide of Chemical Formula 1.

[0063] Preferably, it may include:

[0064] (Step 1) preparing a mixed composition by mixing at least one lipid selected from a neutral lipid, cholesterol, a non-cationic lipid and a PEG-modified lipid with the long-chain ceramide of Chemical Formula 1 in an organic solvent;

[0065] (Step 2) preparing a mixed composition in a dispersed state and performing column purification; and

[0066] (Step 3) The purified product was purified by sterile filtration column.

[0067] In step 1, the concentration of the long-chain ceramide having the structure of Chemical Formula 1 is preferably 1 to 5 mol, more preferably 2 to 5 mol. In this case, if the concentration of the long-chain ceramide exceeds 5 mol, the size of the finally manufactured nanoparticles is uneven or the number of manufactured particles is reduced, making it unsuitable for use as a drug delivery carrier.

[0068] The lipids may include lecithin, cholesterol, non-cationic lipids and PEG-modified lipids. Preferably, the lipids include: 0.1-10 mol lecithin, 0.01-1 mol cholesterol, 0.005-1 mol non-cationic lipids and 0.01-1 mol PEG-modified lipids, more preferably: 0.5-5 mol lecithin, 0.1-1 mol cholesterol, 0.01-0.5 mol non-cationic lipids and 0.05-0.5 mol PEG-modified lipids.

[0069] The organic solvent can be selected from: methanol, ethanol, benzene, butanol, butyl acetate, carbon tetrachloride, chloroform, cyclohexane, ethylene dichloride, dichloromethane, ethyl ether, isopropyl ether, ethyl acetate, heptane, hexane, isooctane, methyl ethyl ketone (MEK), methyl tert-butyl ether (MTBE), pentane, toluene, trichloroethylene, xylene and mixed solvents thereof.

[0070] In this case, in step 2, the dispersion can be prepared by ultrasonic treatment or freeze-thaw. When the process of freezing or thawing the mixed composition is performed, it is best to repeat 1 to 12 times. By repeating the freeze-thaw step of the mixed composition, lipid nanoparticles with more uniform sizes can be formed, and the formation efficiency of lipid nanoparticles can be improved. If it exceeds 12 times, the formation of lipid nanoparticles of uniform size may be reduced, so it is best to be within 12 times.

[0071] In this case, the dispersion may be prepared in a separate reactor in step 2. The lipid nanoparticles may be formed by repeatedly freezing or thawing the mixed composition in the reactor.

[0072] As an alternative approach, in step 2, the dispersion can be prepared using a droplet-based microfluidic system.

[0073] The filter used for the sterile filtration in step 3 is characterized in that it has a pore size of 0.25 to 0.4 μm.

[0074] The composition of the present invention may be provided as a pharmaceutical composition or a cosmetic composition.

[0075] Pharmaceutical composition can be prepared and used in the following forms according to conventional methods respectively: oral preparations (such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc.), external preparations, suppositories and sterile injections. The carrier, excipient and diluent that can be included in the pharmaceutical composition can include: lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talcum powder, magnesium stearate and mineral oil. In the case of formula, it is prepared by using diluent or excipient (such as filler, filler, adhesive, wetting agent, disintegrant, surfactant, etc.). Solid preparations for oral administration include: tablets, pills, powders, granules, capsules, etc., and such solid preparations are prepared by mixing the composition of the present invention with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, they may also contain various excipients such as wetting agents, sweeteners, spices, preservatives, etc. Preparations for parenteral administration include: sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations and suppositories. As non-aqueous solvents and suspending agents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. can be used. As the base of the suppository, Witepsol, polyethylene glycol, Tween 61, cocoa butter, laurel oil, glycerin gelatin, etc. can be used.

[0076] The dosage of the pharmaceutical composition of the present invention will vary according to the age, sex and weight of the subject being treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the route of administration and the judgment of the prescriber. The dosage determination based on these factors is within the level of those skilled in the art, and the dosage is generally in the range of about 0.01 mg / kg / day to 2000 mg / kg / day. A more preferred dosage is 1 mg / kg / day to 500 mg / kg / day. Administration can be once a day or divided into several times. The above dosage does not limit the scope of the present invention in any way.

[0077] The pharmaceutical composition of the present invention can be administered to mammals such as mice, livestock and humans by various routes. All administration methods are conceivable, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine or intracerebroventricular injections.

[0078] The formulation of the cosmetic composition of the present invention can be formulated in any formulation commonly used in the art, and can be selected from: essence, lotion, emulsion, facial mask, hand cream, foot cream, lip balm, lipstick, eye shadow, eyeliner, eyebrow pencil, blush, highlighter, general lotion, skin cream, essence, beauty soap, softening lotion, medicinal lotion, whole body detergent, cleansing foam, cleansing lotion, gel, makeup remover oil, cleansing cream, shampoo, conditioner, hair conditioner, hair styling water, facial cleansing paper and makeup remover.

[0079] The cosmetic composition may include any suitable auxiliary material composition. Preferably, they may include: oil components, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, pH regulators, alcohols, colorants, flavoring agents, blood circulation promoters, cooling agents, restrictors, purified water, etc.

[0080] Furthermore, the cosmetic composition may contain another active ingredient, and preferably may contain a wrinkle improving agent, a skin aging inhibitor, a skin whitening agent, an antioxidant, a skin anti-inflammatory agent, a moisturizing agent, or a hair growth agent.

[0081] As the above-mentioned wrinkle improving agent, substances that inhibit MMP-1 (an extracellular matrix (ECM) protein decomposing enzyme) such as silicic acid, N-methyl-L-serine, isoflavone compounds, dehydroepiandrosterone and paeoniflorin can be selected, and substances that prevent skin aging by removing active oxygen that promotes ECM breakdown can be used, such as bezastatin and coenzyme Q10. In addition, adenosine, ascorbyl glucoside, kinetin, auxin, peptides, retinol, retinyl palmitate, polyethoxylated retinoic acid amide, α-hydroxy acid, etc., which are known to have various wrinkle improving effects, can be used.

[0082] The whitening agent may include: arbutin, niacinamide, ascorbic acid, magnesium ascorbyl phosphate, ascorbic acid-2-glucoside, mulberry extract, ascorbic acid ethyl ether (oil-soluble licorice extract), etc.

[0083] As the moisturizing agent, one or more selected from the group consisting of hydroxyproline, glycerin, glycerol, urea, amino acid, lactic acid, and pyroglutamic acid can be used.

[0084] In addition, if necessary, metal chelating lipids can be further included in the lipid nanoparticles of the present invention. Preferably, one or more selected from the following can be used: DOGS-NTA-Ni lipids (*1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl) iminodiacetic acid) succinyl] (nickel salt)), DMPE-DTPA-Gd lipids (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid (gadolinium salt)), DMPE-DTPA-Cu lipids (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-diethylenetriaminepentaacetic acid (copper salt)).

[0085] In another sense, metal chelating lipids can be referred to as ionizable lipids. Ionizable lipids are ionizable compounds with properties similar to lipids, which can be used to efficiently encapsulate drugs in lipid nanoparticles through electrostatic interactions with drugs (e.g., anionic drugs and / or nucleic acids). Ionizable lipids can be protonated (positively charged) at pH values ​​lower than the pKa of cationic lipids, and are substantially neutral at pH values ​​higher than the pKa. In an example, lipid nanoparticles can include protonated ionizable lipids and / or ionizable lipids that exhibit neutrality.

[0086] If necessary, an antibody can be combined with the lipid nanoparticle. The antibody is an antibody that can specifically bind to a receptor or antigen on the cell surface, which can improve the efficiency of substance delivery to the target cell nucleus. For example, preferably, a polyclonal antibody or a monoclonal antibody directed against biological components such as cell membrane protein receptors specifically expressed in the target tissue or organ is disposed on the surface of the lipid nanoparticle.

[0087] Antibodies can be easily prepared using techniques known in the art. Polyclonal antibodies can be obtained from serum collected from animals injected with a protein specifically expressed on the surface of target cells. As animals, any animal host can be used, such as goats, rabbits or pigs. Monoclonal antibodies can use hybridoma methods (Kohler G. and Milstein C.) or phage antibody libraries (Clackson et al.; Marks et al.), as is well known in the technical field involved in the present invention. In order to perform the hybridoma method, immunologically suitable host animals (such as mice) and cells of cancer or myeloma cell lines can be used. Thereafter, these two types of cells can be fused by methods using polyethylene glycol, etc. (i.e., methods known in the art involved in the present invention), and cells producing antibodies can be propagated using standard tissue culture methods. Subsequently, after obtaining a uniform cell population by subcloning using a limiting dilution technique, hybridomas capable of producing a protein specifically expressed on the surface of target cells with specific antibodies can be cultured in vitro or in vivo on a large scale according to standard techniques. The phage antibody library method can be performed in the following manner: obtaining an antibody gene for a protein specifically expressed on the surface of a target cell, expressing the antibody gene as a fusion protein on the surface of a phage, preparing an antibody library in vitro, and isolating and preparing a monoclonal antibody that binds to a protein specifically expressed on the surface of a target cell. The antibodies prepared by the above method can be separated by electrophoresis, dialysis, ion exchange chromatography, affinity chromatography, etc.

[0088] The antibody may include a complete form having two full-length light chains and two full-length heavy chains and a functional fragment of an antibody molecule. A functional fragment of an antibody molecule refers to a fragment having at least antigen binding function, including: Fab, F(ab'), F(ab')2, F(ab)2, Fv, etc.

[0089] The antibody can be conjugated by using one or more cross-linking agents selected from the following as linkers: 1,4-bis-maleimidobutane, 1,11-bis-maleimidotetraethylene glycol, 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride, succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxy-[6-amidohexanoate] and its sulfonate (sulfo-SMCC), succinimidyl 6-[3-(2-pyridyldithio)-propionylamino]hexanoate] and its sulfonate (sulfo-SPDP), m-maleimidobenzoyl-N-hydroxysuccinimide ester and its sulfonate (sulfo-MBS), succinimidyl [4-(p-maleimidophenyl)butyrate] and its sulfonate (sulfo-SMPB). In this case, the linker is characterized in that it connects the PEG-modified lipid of the lipid nanoparticle to the antibody.

[0090] [Effects of the invention]

[0091] Long-chain ceramides, such as exogenous C18-ceramide, have very low solubility in aqueous solution, so they are limited in use, and even if used as drugs, cell delivery is also low. Therefore, the present invention provides a lipid nanoparticle comprising long-chain ceramide and a method for preparing the same. According to the present invention, long-chain ceramides (such as C18-ceramide) can be delivered to targets in vivo via lipid nanoparticles, thereby effectively inducing cell death as needed, and can enhance the practicality of cell therapy products as having high water solubility and water dispersibility.

Brief Description of the Figures

[0093] Figure 1 A schematic diagram of the structure of lipid nanoparticles containing C18-ceramide is shown.

[0094] Figure 2 The C18-ceramide used in the present invention is shown 1 H NMR results.

[0095] Figure 3 DLS data results of lipid nanoparticles prepared except C18-ceramide are shown (a: number, b: intensity).

[0096] Figure 4 DLS data results of lipid nanoparticles containing 0.1 mM C18-ceramide are shown (a: number, b: intensity).

[0097] Figure 5 DLS data results of lipid nanoparticles containing 0.5 mM C18-ceramide are shown (a: number, b: intensity).

[0098] Figure 6 DLS data results of lipid nanoparticles containing 1 mM C18-ceramide are shown (a: quantity, b: intensity).

[0099] Figure 7 DLS data results of lipid nanoparticles containing 2 mM C18-ceramide are shown (a: number, b: intensity).

[0100] Figure 8 DLS data results of lipid nanoparticles containing 5 mM C18-ceramide are shown (a: number, b: intensity).

[0101] Fig. 9 The results of confirming the cell viability by treating MDA-MB-231 cells and MDA-MB-468 cells with lipid nanoparticles containing C18 ceramide are shown (a: MDA-MB-231, b: MDA-MB-468).

[0102] Fig.10 The results of confirming the expression of apoptosis-related proteins by Western blotting by treating MDA-MB-468 cells with lipid nanoparticles containing C18-ceramide are shown. [Example]

[0103] The preferred embodiments of the present invention will be described in detail below. However, the present invention is not limited to the embodiments described herein, and may also be implemented in other forms. On the contrary, it is intended to fully convey the spirit of the present invention to those skilled in the art so that the content introduced here is thorough and complete.

[0104] [Example 1: Synthesis of ceramide-containing lipid nanoparticles (LNPs) using thin film hydration method]

[0105] Five different lipids were added to 1 mL of chloroform at the following concentrations:

[0106] 1 mM phosphatidylcholine;

[0107] 0.2 mM cholesterol;

[0108] 0.05mM DSPC:

[0109] 0.1 mM DSPE-PEG(2000) amine; and

[0110] C18-ceramide (N-oleoyl-phytosphingosine, chemical formula 2) was added at concentrations of 0, 0.1, 0.5, 1, 2, and 5 mM (the purity of C18-ceramide was 95.3%, and the 1H NMR results were as follows Figure 2 shown).

[0111] Afterwards, when each raw material is dissolved, the chloroform solvent is evaporated to form a lipid film. Next, the lipid film is hydrated with 1 mL of phosphate buffered saline (PBS) (pH 7.2). After the hydrated liquid phase is ultrasonically treated, a freeze-thaw procedure of repeating the hydrated lipid film freeze-thaw procedure five times with liquid nitrogen is performed, and then the hydrated liquid phase is ultrasonically treated again to obtain a dispersed liquid phase, and then the LNP dispersion is purified with a Sepharose CL-4B size extraction column and filtered through a 0.2 μm syringe filter to finally obtain lipid nanoparticles (LNP).

[0112] [Example 2: Synthesis of ceramide-containing lipid nanoparticles (LNPs) using a microfluidic method]

[0113] Five different lipids were added to 1 mL of ethanol at the following concentrations:

[0114] 1 mM phosphatidylcholine;

[0115] 0.2 mM cholesterol;

[0116] 0.05 mM DSPC;

[0117] 0.1 mM DSPE-PEG(2000) amine; and

[0118] C18-ceramide (N-oleoyl-phytosphingosine, chemical formula 2) at concentrations of 0, 0.1, 0.5, 1, 2, and 5 mM, respectively.

[0119] Thereafter, when each raw material was dissolved, the solution flowed through the organic solvent line of the microfluidic instrument (which is a droplet-based microfluidic system) to prepare a dispersion of lipid nanoparticles, and water flowed through the water solvent line. In this case, the solvents were mixed in a volume ratio of 1:3 ethanol: water. After obtaining a liquid phase in a dispersed state forming lipid nanoparticles, the LNP dispersion was purified with a Sepharose CL-4B size extraction column and then filtered through a 0.2 μm syringe filter to finally obtain lipid nanoparticles (LNP). Finally, a solvent replacement agent was used to remove ethanol.

[0120] [Example 3: Characterization of lipid nanoparticles (LNPs) containing C18-ceramide]

[0121] The lipid nanoparticles prepared with each concentration of C18-ceramide were analyzed by dynamic light scattering (DLS) analysis and zeta potential measurement to analyze the dispersibility and surface charge. The results are shown in Tables 1 and Figure 3 In this case, since the results of the lipid nanoparticles obtained in Example 1 and Example 2 were almost similar to each other, all subsequent results were performed for each sample, but the results of the lipid nanoparticles obtained in Example 2 were shown as a representative.

[0122]

Table 1

[0123] C18-ceramide concentration of NPs DLS data (PDI) Zeta potential (mV) 0mM 0.13 -13.2±0.93 0.1mM 0.16 -11.8±1.05 0.5mM 0.15 -11.15±1.08 1mM 0.19 -11.1±0.75 2mM 0.16 -10.54±0.94 5mM 0.19 -11.73±2.16

[0124] According to Table 1 and Figure 3 The results showed that even when the C18-ceramide concentration in the lipid nanoparticles was high, the dispersibility (PDI; polydispersity index) of the nanoparticles was less than 0.2, and thus the dispersibility in water was very excellent.

[0125] In addition, the particle size and particle concentration were evaluated by NTA (Nanoparticle Tracking Analysis), and the results are shown in Table 2.

[0126]

Table 2

[0127]

[0128] Referring to the results in Table 2, it is confirmed that the lipid nanoparticles prepared with each concentration of C18-ceramide are all around 100-150 nm, and the number of particles synthesized is 10 12 More than 100 particles / mL.

[0129] [Example 4: Confirmation of cell viability after treatment with lipid nanoparticles (LNP) containing C18-ceramide]

[0130] MDA-MB-231 and MDA-MB-468 cells were cultured in ATCC modified RPMI medium (Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37° C., 5% CO 2 .

[0131] In the experiment, the inoculum (1.5×10 3 MDA-MB-231 and MDA-MB-468 cells (number of particles: 4.5 × 10 cells / well) in 96-well plates were treated with lipid nanoparticles prepared with each concentration of C18-ceramide. 8 ±2.05×10 8 ). After 24 h of incubation, WST-8 reagent (Dojindo) was added to the culture medium for 1 h at 37 °C. The absorbance was measured at 450 nm using a microplate reader (BioTek). Thereafter, cell viability was confirmed by measuring the absorbance every 24 h. When lipid nanoparticles prepared at 2 mM and 5 mM C18-ceramide concentrations were treated, the cell viability of MDA-MB-231 cells was reduced to 74% on average ( Fig. 9 a).

[0132] Furthermore, when lipid nanoparticles prepared at a C18-ceramide concentration of 5 mM were treated, the cell viability of MDA-MB-468 cells was reduced to 53% ( Fig. 9 b).

[0133] [Example 5: Lipid Nanoparticles (LNP) Containing C18-Ceramide Confirm the Expression of Anti-tumor Related Proteins]

[0134] MDA-MB-468 cells were cultured in ATCC modified RPMI medium (Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37° C., 5% CO 2 .

[0135] In the experiment, MDA-MB-468 cells were seeded into 60 mm 2 On cell culture plate (1.3×10 6After 24 hours, LNPs prepared with different concentrations of C18-ceramide were added, so that each well of 2.5 mL of opti-MEM medium contained 2.48×10 11 ±2.75×10 10 The medium containing LNPs was treated and incubated at 37°C for 24 hours.

[0136] After that, cells were collected and western blotted. To this end, MDA-MB-468 cells treated with LNPs were lysed in RIPA buffer containing protease inhibitors and phosphatase inhibitors (Sigma). Total cell lysates were quantified using a BCA protein assay kit (Thermo Scientific). Next, cell lysates (50 μg) were loaded onto a 4-15% gradient SDS-PAGE gel (Biorad) under denaturing conditions and transferred to a PVDF membrane (iBlot2 Dry BlottingSystem, Thermo Scientific). The membrane was blocked with 5% skim milk for 1 hour at room temperature and the first antibodies for BAX, BAD, BCL2, actin (Santa Cruz) and cleaved caspase 3 (cell signaling) were reacted overnight at 4°C.

[0137] The secondary antibody coupled with HRP was reacted at room temperature for 2 hours, and then the membrane was treated with Western ECL, and the luminescence image was analyzed using LAS500 (GE Healthcare). Fig.10 shown.

[0138] pass Fig.10 The expression levels of apoptosis-related proteins (BAX, BAD, BCL2, and cleaved caspase 3) were confirmed. When lipid nanoparticles containing high concentrations of C18-ceramide were treated, the expression level of the anti-apoptotic protein BCL2 tended to decrease, but the expression levels of the pro-apoptotic proteins BAX and BAD tended to increase. In addition, the cleaved form of caspase 3, which is the lowest stage of apoptotic signaling, was identified, indicating that apoptosis is induced by C18-ceramide.

[0139] Therefore, it can be seen that LNPs containing C18-ceramide have an excellent killing effect on cancer cells.

[0140] Furthermore, when ceramide was dispersed alone in an aqueous solution at the same content as in Examples 1 and 2 and column purified to perform the cell viability test of Example 4 and the anti-tumor-related protein expression test of Example 5, it was confirmed that the results were no different compared to the LNP containing ceramide. Since C18-ceramide itself is insoluble in an aqueous solution, C18-ceramide as an active ingredient was not present in the aqueous solution in the cell viability and anti-tumor protein expression tests, it was found that the effect of C18-ceramide itself did not appear.

Claims

1. A lipid nanoparticle comprising a long-chain ceramide having the structure of the following chemical formula 1: 【Chemical formula 1】 In the chemical formula 1, R1 is a saturated or unsaturated aliphatic chain having 17 carbon atoms, R2 is one selected from the group consisting of phytosphingosine, sphingosine and dihydrosphingosine, and The number of carbon atoms is 10 to 16.

2. The lipid nanoparticle according to claim 1, wherein The long-chain ceramide having the structure of Chemical Formula 1 is a compound selected from the following: N-oleoyl-phytosphingosine-based compounds or derivatives thereof; N-stearoyl-erythro-sphingosine-based compounds or derivatives thereof; N-oleoyl-erythro-sphingosine-based compounds or derivatives thereof; N-stearoyl-erythro-dihydrosphingosine-based compounds or derivatives thereof; and N-oleoyl-erythro-dihydrosphingosine-based compounds or derivatives thereof.

3. The lipid nanoparticle according to claim 1, wherein The lipid nanoparticles comprise at least one lipid selected from the group consisting of phosphatidylcholine, cholesterol, non-cationic lipids and polyethylene glycol (PEG)-modified lipids.

4. The lipid nanoparticle according to claim 3, wherein The non-cationic lipid is a lipid comprising at least one selected from the group consisting of a neutral lipid and an anionic lipid.

5. The lipid nanoparticle according to claim 4, wherein The neutral lipid is a lipid comprising at least one selected from the group consisting of HSPC (hydrogenated soybean phosphatidylcholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), SM (N-palmitoyl-D-erythro-sphingosylphosphocholine), DLPE (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine), DPPE (1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine), phosphatidylcholine, dimyristoylglycerol, succinyl-diacylglycerol, phosphatidylethanolamine, tetraether lipids, sphingolipids, diacryloylglycerol and glycerides or combinations thereof.

6. The lipid nanoparticle according to claim 4, wherein The anionic lipid is a lipid comprising at least one selected from the group consisting of DOPS (dioleoylphosphatidylserine), DMPG (dimyristoylphosphatidylglycerol), DPPG (dipalmitoylphosphatidylglycerol), DTPA (diethylenetriaminepentaacetic acid), DPTGA (1,4-dipalmitoyl-tartrate-2,3-diglutaric acid), DSTSA (1,4-distearoyl-tartrate-2,3-disuccinic acid), CHHDA (2-carboxyheptadecanoylheptadecanamide), DMPS (dimyristoylphosphatidylglycerol), DPPG (dipalmitoylphosphatidylglycerol), DTPA (diethylenetriaminepentaacetic acid), DPTGA (1,4-dipalmitoyl-tartrate-2,3-diglutaric acid), DSTSA (1,4-distearoyl-tartrate-2,3-disuccinic acid), CHHDA (2-carboxyheptadecanoylheptadecanamide), phosphatidylserine), DPPS (dipalmitoylphosphatidylserine), POPS (palmitoyloleoylphosphatidylserine), DOPG (dioleoylphosphatidylglycerol), POPG (palmitoyloleoylphosphatidylglycerol), DMPA (dimyristoylphosphatidic acid), DPPA (dipalmitoylphosphatidic acid), DOPA (dioleoylphosphatidic acid), POPA (palmitoyl-oleoylphosphatidic acid), CetylP (cetyl phosphate) and CHEMS (cholesterol hemisuccinate) or a combination thereof.

7. The lipid nanoparticle according to claim 3, wherein PEG-modified lipids are a combination of non-cationic lipids and polyethylene glycol (PEG).

8. The lipid nanoparticle according to claim 3, wherein The particle size of the lipid nanoparticles is 10 to 2,000 nm.

9. A nanoparticle delivery composition for cell apoptosis, comprising the lipid nanoparticle according to any one of claims 1 to 8.

10. The nanoparticle delivery composition according to claim 9, wherein The cell is a cancer cell.

11. The nanoparticle delivery composition according to claim 9, wherein The cancer cells are derived from a cancer tissue selected from the group consisting of hemangioma, angiofibroma, lung cancer, non-small cell lung cancer, liver cancer, colon cancer, bone cancer, pancreatic cancer, skin cancer, head cancer, neck cancer, melanoma, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, anal cancer, colon cancer, breast cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, 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, bladder cancer, kidney cancer or ureter cancer, renal cell carcinoma, renal pelvis cancer, central nervous system tumors, primary central nervous system lymphomas, spinal cord tumors, brain stem gliomas, and pituitary adenomas.

12. An anticancer pharmaceutical composition comprising the nanoparticle delivery composition according to claim 9.

13. A method for preparing lipid nanoparticles, comprising: In step 1, a mixed composition is prepared by mixing a lipid selected from lecithin, cholesterol, a non-cationic lipid and a lipid modified with polyethylene glycol (PEG) and a long-chain ceramide having a structure of the following Chemical Formula 1 in an organic solvent: 【Chemical formula 1】 In Chemical Formula 1, R1 is a saturated or unsaturated aliphatic chain having 17 carbon atoms, R2 is one selected from the group consisting of phytosphingosine, sphingosine and dihydrosphingosine, and The number of carbon atoms is 10 to 16; Step 2, preparing a mixed composition in a dispersed state and performing column purification; and Step 3: Purify the obtained product by sterile filtration column.

14. The method according to claim 13, wherein: The long-chain ceramide having the structure of Chemical Formula 1 is a compound selected from the following: N-oleoyl-phytosphingosine-based compounds or derivatives thereof; N-stearoyl-erythro-sphingosine-based compounds or derivatives thereof; N-oleoyl-erythro-sphingosine-based compounds or derivatives thereof; N-stearoyl-erythro-dihydrosphingosine-based compounds or derivatives thereof; and N-oleoyl-erythro-dihydrosphingosine-based compounds or derivatives thereof.

15. The method according to claim 13, wherein: The non-cationic lipid is a lipid comprising any one or more selected from the group consisting of a neutral lipid and an anionic lipid.

16. The method according to claim 15, wherein: The neutral lipids are selected from any one or more of the following lipids: HSPC (hydrogenated soybean phosphatidylcholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine), palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), SM (N-palmitoyl-D-erythro-sphingosylphosphocholine), DLPE (1,2-dilauroyl-sn-glycero-3-phosphoethanolamine), DPPE (1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine), phosphatidylcholine, dimyristoylglycerol, succinyl-diacylglycerol, phosphatidylethanolamine, tetraether lipids, sphingolipids, diacryloylglycerol and glycerides, or combinations thereof.

17. The method according to claim 15, wherein: The anionic lipids are selected from any one or more of the following lipids: DOPS (dioleoylphosphatidylserine), DMPG (dimyristoylphosphatidylglycerol), DPPG (dipalmitoylphosphatidylglycerol), DTPA (diethylenetriaminepentaacetic acid), DPTGA (1,4-dipalmitoyl-tartrate-2,3-diglutaric acid), DSTSA (1,4-distearoyl-tartrate-2,3-disuccinic acid), CHHDA (2-carboxyheptadecanoylheptadecanamide), DMPS (dimyristoylphosphatidylglycerol), DPPG (dipalmitoylphosphatidylglycerol), DTPA (diethylenetriaminepentaacetic acid), DPTGA (1,4-dipalmitoyl-tartrate-2,3-diglutaric acid), DSTSA (1,4-distearoyl-tartrate-2,3-disuccinic acid), CHHDA (2-carboxyheptadecanoylheptadecanamide), phosphatidylserine), DPPS (dipalmitoylphosphatidylserine), POPS (palmitoyloleoylphosphatidylserine), DOPG (dioleoylphosphatidylglycerol), POPG (palmitoyloleoylphosphatidylglycerol), DMPA (dimyristoylphosphatidic acid), DPPA (dipalmitoylphosphatidic acid), DOPA (dioleoylphosphatidic acid), POPA (palmitoyl-oleoylphosphatidic acid), CetylP (cetyl phosphate), CHEMS (cholesterol hemisuccinate), or a combination thereof.

18. The method according to claim 13, wherein: The PEG-modified lipid is a combination of a non-cationic lipid and polyethylene glycol (PEG).

19. The method according to claim 13, wherein: The organic solvent is selected from the group consisting of methanol, ethanol, benzene, butanol, butyl acetate, carbon tetrachloride, chloroform, cyclohexane, ethylene dichloride, dichloromethane, ether, isopropyl ether, ethyl acetate, heptane, hexane, isooctane, methyl ethyl ketone (MEK), methyl tert-butyl ether (MTBE), pentane, toluene, trichloroethylene, xylene and a mixed solvent thereof.

20. The method according to claim 13, wherein: In step 2, a dispersion is prepared by sonication or freeze-thaw.

21. The method according to claim 13, wherein: In step 2, the dispersion is prepared using a droplet-based microfluidic system.

22. The method according to claim 13, wherein: The filter used for sterile filtration in step 3 has a pore size of 0.25 to 0.4 μm.

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