WO2018174325A1 - Peptide surface-modified nanoparticle for controlling freezing - Google Patents

Peptide surface-modified nanoparticle for controlling freezing Download PDF

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Publication number
WO2018174325A1
WO2018174325A1 PCT/KR2017/003238 KR2017003238W WO2018174325A1 WO 2018174325 A1 WO2018174325 A1 WO 2018174325A1 KR 2017003238 W KR2017003238 W KR 2017003238W WO 2018174325 A1 WO2018174325 A1 WO 2018174325A1
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Prior art keywords
nanoparticles
present
peptide
functional
freezing
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PCT/KR2017/003238
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French (fr)
Korean (ko)
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곽민석
김학준
안동준
임동권
Original Assignee
부경대학교 산학협력단
고려대학교 산학협력단
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Publication of WO2018174325A1 publication Critical patent/WO2018174325A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3526—Organic compounds containing nitrogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K3/00—Materials not provided for elsewhere
    • C09K3/18—Materials not provided for elsewhere for application to surfaces to minimize adherence of ice, mist or water thereto; Thawing or antifreeze materials for application to surfaces
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/20—Ingredients acting on or related to the structure
    • A23V2200/25—Nanoparticles, nanostructures
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif

Definitions

  • the present invention relates to functional nanoparticles capable of controlling the formation and decomposition of ice crystals.
  • AFPs antifreeze proteins
  • the present invention aims to provide functional nanoparticles and compositions that can be easily synthesized, genetically engineered to increase the composition of specific amino acids, and do not cause precipitation when used at high concentrations.
  • the present invention aims to provide functional nanoparticles and compositions that can interact with ice crystals.
  • An object of the present invention is to provide a functional nanoparticle composition that can minimize the damage of cells by slowing the rate of ice production during freezing.
  • An object of the present invention is to provide a functional nanoparticles and compositions capable of absorbing visible or near infrared rays to control the rate of dissolution of ice crystals from inside the cell, thereby minimizing cell death during cold thawing.
  • nanoparticles And amino acids or peptides bonded to the surface of the nanoparticles.
  • nanoparticles are colloidal nanoparticles
  • functional nanoparticles are colloidal nanoparticles
  • amino acid is at least one selected from the group consisting of threonine, valine and serine, functional nanoparticles.
  • the functional nanoparticles further comprises a cell permeable peptide, functional nanoparticles.
  • composition for controlling ice formation comprising the functional nanoparticles of any one of the above 1 to 7.
  • the synthesis is simple, genetic engineering can increase the composition of a specific amino acid, it can provide functional nanoparticles and compositions that do not occur precipitation at high concentrations.
  • the functional nanoparticles and the composition of the present invention when added to the cells, it is possible to dissolve ice crystals from inside the cell by irradiating visible or near infrared rays, thereby minimizing apoptosis during cold thawing.
  • Functional nanoparticles and compositions can be provided.
  • FIG. 1 is a schematic diagram showing an embodiment of the functional nanoparticles of the present invention.
  • Figure 2 shows that in the presence of the functional nanoparticles of the present invention the growth of ice crystals is suppressed and the ice freezing temperature is lowered.
  • Figure 3 shows the change in the temperature hysteresis (TH) according to the number and arrangement of amino acids interacting with the ice crystals in the freezing protein.
  • Figure 4 shows that recrystallization of the ice is prevented during thawing.
  • Figure 5 shows that the cell-permeable amino acid sequence may affect the freezing and thawing of ice in and out of cells when present on the surface of the nanoparticles
  • the photograph is an electron micrograph showing the functional nanoparticles of the present invention into the cell to be.
  • Figure 6 shows the results of synthesizing the gold nanoparticles surface-modified with threonine according to an embodiment of the present invention.
  • Figure 7 shows the results of ice crystal growth experiments with the functional nanoparticles synthesized according to an embodiment of the present invention, the results showed that the functional nanoparticles of the present invention hinder the growth of ice crystals.
  • FIG. 8 illustrates a cell experiment of the effect of freezing control of nanoparticles. Specifically, when 20 nm gold nanoparticles or 40 nm gold nanoparticles bound with DMSO or threonine are added to mESC cells, Apoptotic effect, showing apoptosis effect under the condition of thawing by irradiation with light.
  • the present invention relates to a peptide surface-modified nanoparticle for freezing control, the nanoparticle; And amino acids or peptides bound to the surface of the nanoparticles, do not have toxicity, can be applied to biological samples, easy to synthesize, genetically engineered to increase the composition of specific amino acids, precipitation occurs when using high concentrations It can interact with ice crystals to control freezing and thawing, slow down ice formation to minimize cell damage, and minimize cell death during freeze-thawing process. Peptide surface-modified nanoparticles that can minimize damage during thawing.
  • the present invention provides functional nanoparticles comprising nanoparticles and amino acids or peptides bound to the nanoparticle surface.
  • the nanoparticles mean particles having an average particle diameter of 90% or more of 5 ⁇ m or less, preferably 2 ⁇ m or less, more preferably 1 ⁇ m or less, even more preferably 0.5 ⁇ m or less, and may be plasmon nanoparticles. This is not restrictive.
  • Nanoparticles may be prepared by reducing a dilute solution of a metal complex salt, and specifically, may be prepared a process such as a reduction reaction, nucleation initiation reaction, nuclear growth reaction, but is not limited thereto.
  • the nanoparticles may be colloidal nanoparticles, but is not limited thereto.
  • the nanoparticles may be circular or rod-shaped, as shown in the schematic diagram of Figure 1, but is not limited thereto.
  • the nanoparticles may be at least one of gold or silver or an alloy thereof, and specifically, the nanoparticles may be gold, but is not limited thereto.
  • the functional nanoparticles of the present invention can absorb light in the visible or near-infrared region, thereby controlling the melting process of ice from inside the cells by irradiating light upon thawing of the cells. This can minimize cell damage during cell thawing.
  • Amino acids are, for example, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, pyrrolysine, proline, glutamine, arginine, serine, threonine, seleno Cysteine, valine, tryptophan, tyrosine, and the like, but is not limited thereto.
  • the amino acid may be at least one of threonine, valine and serine, but is not limited thereto.
  • Threonine, valine and serine are the major amino acid components of the antifreeze peptides, which make ice interaction more effective, and ice interaction is better than other amino acids, making ice crystals more detailed and effective. Regulation (especially prevention).
  • the bonding strength with the nanoparticles is also excellent, making it easy to manufacture functional nanoparticles.
  • the surface area that can interact with ice increases exponentially to grow, form, and thaw ice crystals. And the like's control ability may be significantly increased compared to the case where the amino acid is used alone.
  • the amino acid may be threonine, but is not limited thereto.
  • the functional complex of the present invention can slow down ice formation in cells when frozen, thereby minimizing cell damage.
  • the peptide may comprise at least one amino acid selected from the group consisting of threonine, valine and serine, the number of threonine, valine and serine included in the peptide is limited There is no For example, it may include 1 to thousands of at least one of threonine, valine and serine, but is not limited thereto.
  • the peptide may include at least one of threonine, valine and serine continuously or separately, but is not limited thereto.
  • the peptide may be a mixed sequence including at least two or all of threonine, valine and serine, but is not limited thereto.
  • the peptide may be a peptide comprising threonine and cysteine.
  • Cysteine included in the peptide also includes a case where the cysteine is linked to a spacer material such as polyethylene glycol.
  • the linkage may be when polyethylene glycol is positioned between the peptide and cysteine, but is not limited thereto.
  • five threonine may be continuously coupled, and cysteine may be bound to the terminals of the five threonine, but is not limited thereto.
  • the peptide may be a peptide including the compound represented by Formula 1, but is not limited thereto.
  • the peptide containing the compound represented by the formula (1) may induce anisotropic growth of ice crystals, it may interfere with the growth of ice crystals (Figs. 6 and 7). In addition, it is possible to minimize the damage during thawing of frozen cells (Fig. 8).
  • the peptide refers to a molecule found in any animal, plant, insect, microorganism, or the like, or an artificially and naturally synthesized molecule, and is a peptide that non-uniformly reduces the freezing point of water.
  • the peptide is a protein that is characterized by binding to ice to prevent the growth of ice crystals, anti-freezing peptides (AFPs), anti-freezing glycoproteins, anti-freezing glycopeptides (anti-freezing glycopeptides, AFGP), thermal history polypeptide, and the like.
  • AFPs anti-freezing peptides
  • AFGP anti-freezing glycoproteins
  • thermal history polypeptide thermal history polypeptide
  • the peptide is an anti-freeze peptide produced in cold blooded aquatic polar fish species that live in water of the polar regions of the earth, including fats in the Arctic and Antarctic rings; Antifreeze peptides produced in various higher plants, including polar diatoms, carrots and dandelions; Antifreeze peptides produced by various species, such as insects, bacteria, fish; may include, but are not limited thereto.
  • the peptide may be an antifreezing protein such as FfIBP, NglIBP, Type III AFP, but is not limited thereto.
  • the peptide may have a primary or secondary structure.
  • it may have a structure of alpha helix, beta sheet, a repeat or combination thereof, but is not limited thereto.
  • the peptide is an amino acid sequence of the anti-freezing peptides (AFPs), anti-freezing glycoproteins, anti-freezing glycopeptides (AFGP) or heat history polypeptide Modification may be made in part or in whole, or by additional binding of other amino acids at the beginning, middle or end of the amino acid sequence, and the modification may be made within the scope of interaction with ice.
  • AFPs anti-freezing peptides
  • AFGP anti-freezing glycoproteins
  • AFGP anti-freezing glycopeptides
  • Heat history polypeptide Modification may be made in part or in whole, or by additional binding of other amino acids at the beginning, middle or end of the amino acid sequence, and the modification may be made within the scope of interaction with ice.
  • the peptide may be fused to an affinity tag.
  • Affinity tags including but not limited to His-tag, polypeptide A tag, avidin / streptavidin, polypeptide G, glutathione-S-transferase, dihydrofolate reductase (DHFR), green fluorescent polypeptide (GFP ), Polyarginine, polycysteine, c-myc, calmodulin binding polypeptide, influenza virus hemagglutinin, maltose binding protein (MBP), hyaluronic acid (HA), and the like.
  • the peptide may be a peptide in which one or more amino acid residues in the peptide are modified.
  • One or more modification (s) may include chemical derivatization in vivo or in vitro, for example acetylation or carboxylation; Glycosylation; Deglycosylation; Phosphorylation such as phosphotyrosine, phosphoserine and phosphothreonine; Etc., but is not limited thereto.
  • the peptide may be, but is not limited to, naturally occurring L-amino acids, naturally occurring L-amino acids and non-naturally occurring synthetic amino acids.
  • the peptide may be a peptide having a blocking group introduced at the N-terminus and / or the C-terminus, but is not limited thereto.
  • Blocking groups are chemical substituents suitable for protecting and stabilizing the N-terminus and / or C-terminus, including branched or unbranched alkyl groups and acyl groups introduced by alkylation or acylation of the N-terminus; C-terminal blockade groups comprising ester or ketone-forming alkyl groups, for example, lower (C1 to C6) alkyl groups such as methyl, ethyl and propyl, and amide-forming amino groups; Do not.
  • Amide-forming amino groups include primary amines (-NH2), mono- and di-alkylamino groups, and the like, and mono- and di-alkylamino groups include methylamino, ethylamino, dimethylamino, diethylamino, Methylethylamino and the like, but are not limited thereto.
  • Suitable concentrations of peptide bound to the nanoparticles can vary depending on the application and can range from, for example, about 1 ppb to about 1 ppt (ie, about 1 ⁇ g / L to about 1 mg / L).
  • the peptides also have excellent binding ability with nanoparticles, so when used as functional nanoparticles, the surface area that can interact with ice increases exponentially, so that the ability to control the growth, formation, thawing of crystals, etc. is used when the peptides are used alone. It can be remarkably superior in comparison.
  • the binding of the nanoparticles with amino acids or peptides may comprise chemical bonding of the nanoparticle surfaces with amino acids or peptides.
  • the nanoparticles may have a modified surface.
  • the binding of the nanoparticles to amino acids or peptides may be to directly bind amino acids or peptides to the nanoparticles by modifying them with carboxylic acids and then reducing them to amino acids or peptides.
  • the binding of the nanoparticles to amino acids or peptides may be binding by a linker.
  • the binding by the linker may bind the linker to the nanoparticles, and then bind the nanoparticles to amino acids or peptides by binding the linker to amino acids or peptides, but is not limited thereto.
  • the linker may be a cysteine, an o-linker, but is not limited thereto.
  • the functional nanoparticles of the present invention may interact with ice by combining amino acids or peptides on the surface of the nanoparticles, thereby performing functions such as inhibiting, delaying, and preventing ice crystal formation.
  • the temperature history phenomenon was increased according to the number and arrangement of amino acids or peptides included in the functional nanoparticles of the present invention (FIG. 3).
  • the functional nanoparticles of the present invention do not use chemicals and do not cause toxicity problems for biological samples of cryopreservatives (antifreezes, DMSO, etc.). Therefore, it can be used without limitation in various biological samples such as cells of insects, mammals, reptiles, fish and the like.
  • the functional nanoparticles of the present invention can enter the cell (Fig. 5), it is possible to control the thawing of the ice from the inside of the biological sample by irradiating light to the light during thawing of the cell, thereby thawing after freezing storage Damage to biological samples that can occur can be minimized (FIG. 8).
  • the biological sample may be, but is not limited to, mammalian cells, insect cells, fish cells, plant cells, eukaryotic cells or prokaryotic cells.
  • the functional nanoparticles of the present invention can substantially reduce cellular damage due to ice crystal growth, thereby allowing sensitive biological organisms such as blood and blood products, therapeutic agents, polypeptide drugs, bioassay reagents and vaccines, etc. It may be suitable for storage of the sample.
  • the functional nanoparticles may further comprise a cell permeable peptide.
  • the functional nanoparticles can be delivered into the cells, thereby controlling freezing of the cells by affecting water-ice behavior such as ice crystallization of the inner cells.
  • Cell-penetrating peptides are a type of signal peptide, e.g. from the 11 amino acid sequence b-galactosidase (120 kDa) protein present in the TAT protein, from Antennapedia (Penetratin) and HSV-1 virus from Drosophila protein VP22, Pep-1 derived from Simian Virus 40 large antigen T, and the like, but are not limited thereto.
  • signal peptide e.g. from the 11 amino acid sequence b-galactosidase (120 kDa) protein present in the TAT protein, from Antennapedia (Penetratin) and HSV-1 virus from Drosophila protein VP22, Pep-1 derived from Simian Virus 40 large antigen T, and the like, but are not limited thereto.
  • it may include a peptide in which a plurality of cationic amino acids such as Arginine and Lysine are repeatedly connected, such as poly Arginine and poly Lysine, but are not limited thereto.
  • Hph-1, Vectocell, Lactoferrin, Sim-2, LPIN3, 2IL-1a, dNP2, etc. may be mentioned as cell permeable peptides, but are not limited thereto.
  • Functional nanoparticles of the present invention can be introduced into or in contact with food products to reduce or inhibit ice crystal growth and / or formation, thereby allowing the texture, taste, and effectiveness of frozen food products, including vegetables. It can improve the shelf life.
  • the functional nanoparticles of the present invention can inhibit and / or reduce the formation of ice crystals associated with freezing or subcooling of objects or materials including foodstuffs.
  • the present invention provides a functional food comprising functional nanoparticles in another aspect.
  • the functional food of the present invention may contain such functional nanoparticles such that this ice crystal growth process may be reduced, or even completely prevented, or at least minimized.
  • Foods are low-fat spreads, mayonnaise, yogurt, bakery, margarine, reconstituted fruit, jams, fruit products, fruit cows, ripple, fruit sauces, fruit stew, coffee bleach, instant fruit desserts, sweets (e.g. marshmallows) , Potato-based foods (eg chips, french fries and croquettes), processed foods (eg casserole and stews) and fine foods (eg dressings including salad dressings; ketchup, vinaigrette dressings and soups) Etc., but is not limited thereto.
  • sweets e.g. marshmallows
  • Potato-based foods eg chips, french fries and croquettes
  • processed foods eg casserole and stews
  • fine foods eg dressings including salad dressings; ketchup, vinaigrette dressings and soups
  • food products may be prepared from raw, processed or pasteurized foods, including beverages, raw meats, cooked meats, raw poultry products, cooked poultry products, raw seafood products, cooked seafood products [raw or cooked meat, poultry and Seafood products], sausages, frankfurters, ready-to-eat foods, pasta sauces, pasteurized soups, marinades, oil-in-water emulsions, water-in-oil emulsions, cheese spreads, processed cheeses, dairy desserts, flavored milk, cream, Filled with fermented dairy products, cheeses, butters, condensed milk products, cheese spreads, pasteurized liquid eggs, ice cream mixes, soy products, pasteurized liquid eggs, sugar products, fruit products, and fat-based or water-containing cows Food, etc., but is not limited thereto.
  • the food may be, but is not limited to, confectionery products such as bread, cakes, fine bakery and dough.
  • the functional food of the present invention may be in a form in which functional nanoparticles are incorporated into the food as a whole and / or in a form applied to a surface of a food product, but is not limited thereto.
  • the functional food of the present invention may be prepared in various forms such as tablets, capsules, powders, granules, liquids, pills, powders, flakes, pastes, syrups, gels, jellies, bars, and the like. It may also be prepared in form.
  • Functional food of the present invention may further include ingredients that are commonly added in the manufacture of food within the scope of the present invention, for example, may further include proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents. .
  • Such carbohydrates are monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as, but are not limited to, conventional sugars such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.
  • the flavoring agent may further include natural flavoring agents such as taumartin, stevia extract (for example, Rebaudioside A, glycyrgin, etc.) and synthetic flavoring agents such as saccharin and aspartame, but are not limited thereto. .
  • natural flavoring agents such as taumartin, stevia extract (for example, Rebaudioside A, glycyrgin, etc.) and synthetic flavoring agents such as saccharin and aspartame, but are not limited thereto. .
  • the functional food of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols. And carbonation agents used in carbonated beverages.
  • the functional food of the present invention may contain fruit flesh for the production of natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination.
  • the proportion of such additives is not critical, but may be added in the range of 0.01 to 0.20 parts by weight per 100 parts by weight of the functional food composition of the present invention.
  • the mixed amount of the active ingredient can be suitably determined according to the purpose of use (prevention, health treatment). In general, in the manufacture of foods or beverages, it can be added in an amount of 0.0001 to 30% by weight, preferably 0.0001 to 10% by weight, more preferably 0.1 to 5% by weight relative to the total weight of the raw material. However, in the case of prolonged ingestion for health and hygiene purposes or for health control purposes, the amount may be adjusted below the above range.
  • Functional food of the present invention may further comprise a stabilizer.
  • Stabilizers include polypeptides such as gelatin; Plant extracts such as gum arabic, gatti gum, karaya gum, tragacanth gum; Seed gums such as locust bean gum, guar gum, tara gum, picillium seed gum, quince seed gum or tamarind seed gum; Konjac met; Seaweed extracts such as agar, alganate, carrageenan or purselane; Pectin, for example lower methoxyl or higher methoxyl-type pectin; Cellulose derivatives such as sodium carboxymethyl cellulose, microcrystalline cellulose, methyl and methylethyl cellulose, or hydroxylpropyl and hydroxypropylmethyl cellulose; And microbial gums such as dextran, xanthan or ⁇ -1,3-glucan; And the like, but is not limited thereto.
  • the functional food of the present invention is a sugar such as sucrose, fructose, dextrose, lactose, corn syrup, sugar alcohol; Or other raw materials such as pigments and flavorings.
  • the present invention provides a composition for controlling freezing comprising functional nanoparticles effective for cryopreservation of biological samples (eg, biological cells or extracts thereof).
  • the biological sample may be, but is not limited to, bacterial cells, yeast cells, plant cells, animal cells, insect cells, reptile cells, fish cells, mammalian cells, blast samples and cell extracts.
  • the biological sample may be a mammalian cell, a wide range of types of cells such as oocytes, embryos, leukocytes, erythrocytes, platelets, pancreatic islets and hepatocytes; Skin tissue, bone marrow tissue, corneal tissue and other broad types of tissue; And various types of organs such as liver, kidney, heart, brain, lung, pancreas, spleen, ovary, and stomach; but are not limited thereto.
  • types of cells such as oocytes, embryos, leukocytes, erythrocytes, platelets, pancreatic islets and hepatocytes
  • Skin tissue bone marrow tissue, corneal tissue and other broad types of tissue
  • organs such as liver, kidney, heart, brain, lung, pancreas, spleen, ovary, and stomach; but are not limited thereto.
  • microorganisms such as bacteria, soft tissues such as animals, plants, insects, etc. may exhibit less damage when frozen or thawed in the presence of the composition of the invention, the addition of the composition before and after freezing Cell integrity in later thawing may be useful in situations where important or desirable (eg, tissue culture deposits). In other words, it is possible to minimize the loss of inherent properties or intrinsic form due to the freeze-thaw process.
  • the composition comprising the functional nanoparticles can inhibit the growth of ice crystals, thereby greatly improving the survival rate during freezing and thawing of biological samples (eg, cells). (FIG. 8).
  • compositions of the present invention may be in various forms, such as solids, semisolids, fluids, gases, and the like.
  • it may be a fluid, but is not limited thereto.
  • composition of the present invention may further contain any of a broad mixture of salts, sugars, ions and other nutrients contained in an electrolyte solution known to be useful for preserving biological agents. These include tissue culture medium, organ perfusate and the like.
  • the present invention also provides a cosmetic composition or a dermatological preparation comprising the functional nanoparticles of the present invention.
  • the optimum temperature of the cellular enzymes is lost due to a pronounced climate- and weather-induced temperature drop resulting in changes in cell physiology in the cell and extracellular space. Prevents or improves skin structure and cell damage from damaged cold
  • the skin structure and cell damage is caused by cold, wind and / or UV light induced skin damage, skin erythema and skin pulling feeling and increased sensory sensitivity, temperature-sensitive skin, environment It includes, but is not limited to, changes in skin, lips and nose and oral mucosa and skin appendages due to stress (due to temperature changes and UV light, smoking, smog, reactive oxygen species, free radicals).
  • compositions, cosmetic compositions and topical dermatological preparations of the present invention may further comprise at least one of stabilizers, emulsifiers, surfactants and other additives known to those skilled in the art.
  • additives include, but are not limited to, anti-corruption agents, antioxidants, discoloration inhibitors, antimicrobial agents, emulsion stabilizers, and the like.
  • Ice crystal growth experiments showed anisotropic ice crystal growth patterns. This is a result of the interfering effect of ice crystal growth by nanoparticles (Result: FIG. 7).
  • threonine peptide peptide with cysteine bound at five ends of threonine
  • the analysis results of the synthesized nanoparticles are shown in FIG. 6.
  • Ice crystal growth experiments showed anisotropic ice crystal growth patterns. This is a result of the interfering effect of ice crystal growth by nanoparticles (Result: FIG. 7).
  • the freezing point and the melting point were the same at 0 ° C., but in the water containing the functional nanoparticles, the freezing point and the melting point were different because the nanoparticles were combined with ice crystals and interfered with the crystals. That is, the temperature history phenomenon was observed in the experimental group, from which it was confirmed that the functional nanoparticles of the present invention hinder the growth of ice crystals.
  • mESC cells were DMEM / high glucose, 200 mM L- glutamine, 1% Penicillin / Streptomycin, 15% FBS, HEPES (pH 7.3), MEM Nonessential Amino Acids, 1000 U / ml media and passaged every 2 days. .
  • 0.1% Gelatin was coated in a 60cm culture dish at 37 ° C., 5% CO 2 , for at least 3 hours.
  • TC1 cells were cultured in DMEM-High glucose, 10% FBS, 1% Penicillin / Streptomycin and passaged every 3 days.
  • mESC was washed with 1 ml PBS and 0.4 ml of 0.25% Trypsin was added. After 1 minute, 3.6 ml of culture was added to neutralize. After centrifugation at 1,200 rpm for 5 minutes, the media was carefully removed.
  • 0.5 ml freezing media (70% DMEM / high glucose, 20% FBS, 10% DMSO) were mixed in the cell pellet and transferred to cryovial. Cryovial was placed in a freezing container and stored in a -80 ° C deep freezer for 2 days. After 2 days, the cell stock vial was taken out of the deep freezer, rapidly thawed at 37 ° C. for 1 minute, and then 1 ml of media was added. After centrifugation at 1,200 rpm for 5 minutes, the culture was removed. The cells were mixed with 4 ml of media, added to a 60 cm culture dish and incubated.
  • TC1 cells were washed with 1ml PBS and 2ml of 0.25% Trypsin was added. After 1 minute, 2 ml of culture was added to neutralize. After centrifugation at 1,200 rpm for 5 minutes, the media was carefully removed. 0.5 ml freezing media (90% FBS, 10% DMSO) was mixed in the cell pellet (2x10 5 ), and then transferred to cryovial. Cryovial was placed in a freezing container and stored in a -80 ° C deep freezer for 2 days. After 2 days, the cell stock vial was taken out of the deep freezer, rapidly thawed at 37 ° C. for 1 minute, and 4.5 ml of media was added thereto.
  • freezing media 90% FBS, 10% DMSO
  • 520 nm laser (10 mw / cm 2) was added slowly at 0 ° C. and 4.5 ml of media was added. After centrifugation at 1,200 rpm for 5 minutes, the culture was removed. The cells were mixed with 2 ml of media, added to 6 well culture plates, and cultured (result: FIG. 8).
  • mESC cells were incubated for 2 days, and then the media was removed. The cell was washed 1 or 3 times using PBS. After making 0.2% or 0.1% using Trypan blue and PBS, and added to 60 cm culture dish, the cells were confirmed by a microscope.
  • TC1 cells were incubated for 2 days, and then the media was removed. The cells were washed using PBS. The cells were fixed for 10 minutes with 4% Paraformaldehyde / sucrose, and then stained with 0.5% crystal violet solution for 30 minutes. After 30 minutes, the dye solution was removed using water flowing through a 6 well culture plate. Cell state was confirmed using a microscope. As a result of comparing the number of living cells, it was confirmed that more cells survived when 20 nm or 40 nm gold nanoparticles were added as compared to the case where the cells were frozen and thawed using DMSO (Result: FIG. 8).

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Abstract

The present invention relates to a peptide surface-modified nanoparticle for controlling freezing, the peptide surface-modified nanoparticle comprising: a nanoparticle; and an amino acid or peptide bound to the surface of the nanoparticle. Thus the peptide surface-modified nanoparticle is not toxic and thus can be applied to a bio-sample, is conveniently synthesized, can increase the production of a specific amino acid through genetic engineering, does not precipitate when used in a high concentration, can adjust freezing and thawing by interacting with an ice crystal, can minimize damage to cells by slowing down the rate at which ice is formed during freezing, can minimize apoptosis during freezing and thawing, and can minimize damage during the thawing of a bio-sample.

Description

결빙 제어용 펩타이드 표면 개질된 나노입자 Peptide Surface Modified Nanoparticles for Freezing Control
본 발명은 얼음 결정의 형성 및 분해를 조절할 수 있는 기능성 나노입자에 관한 것이다.The present invention relates to functional nanoparticles capable of controlling the formation and decomposition of ice crystals.
얼음의 결정은 일정 크기 이상이 되면 생체 시료에 손상을 주기 때문에, 저온 환경이 요구되는 생체 시료 보관 산업에서는 얼음 결정 형성에 의한 피해를 막기 위한 다양한 시도들이 이루어지고 있다.Since ice crystals damage biological samples when they are above a certain size, various attempts have been made to prevent damage caused by ice crystal formation in the biological sample storage industry requiring a low temperature environment.
화학약품을 사용하는 동결보존제 (부동액, DMSO 등)을 많이 사용하고 있으나, 이는 생체 내에서 독성을 나타내므로 생체 시료에 적용하기 어렵다.Cryopreservatives (antifreezes, DMSO, etc.) that use chemicals are used a lot, but since they are toxic in vivo, they are difficult to apply to biological samples.
동결보존제를 대체하기 위해 다양한 결빙방지 단백질 (Antifreeze protein, 이하 AFP)이 개발되고 있으나, 결빙방지 단백질은 미생물, 어류, 곤충 등 다양한 생물이 생체 내에서 발현되는 것으로, 특정 아미노산의 조성을 높이면 구조의 변형을 초래할 수 있다는 문제점이 있다. 또한, 고농도의 단백질을 생체 시료에 사용 시 단백질이 침전될 수 있는 문제점이 있다.Various antifreeze proteins (AFPs) have been developed to replace cryopreservatives, but antifreeze proteins are expressed in various organisms such as microorganisms, fish, and insects. There is a problem that can cause. In addition, there is a problem that the protein can be precipitated when using a high concentration of protein in a biological sample.
따라서, 보다 효과적이고 전술한 문제점이 발생하지 않는 얼음 결빙을 조절할 수 있는 물질의 개발이 절실히 필요한 실정이다.Therefore, there is an urgent need for the development of a material that can control ice freezing that is more effective and does not cause the aforementioned problems.
본 발명은 화학약품을 사용하는 동결보존제의 생체 시료에 대한 독성 발생 문제가 없는 기능성 나노입자 및 조성물의 제공을 목적으로 한다.It is an object of the present invention to provide functional nanoparticles and compositions which do not have a problem of generating toxicity against a biological sample of a cryopreservative using a chemical.
본 발명은 합성이 간편하고, 유전공학적으로 특정 아미노산의 조성을 높일 수 있으며, 고농도 사용 시 침전이 발생하지 않는 기능성 나노입자 및 조성물의 제공을 목적으로 한다.The present invention aims to provide functional nanoparticles and compositions that can be easily synthesized, genetically engineered to increase the composition of specific amino acids, and do not cause precipitation when used at high concentrations.
본 발명은 얼음 결정과 상호작용할 수 있는 기능성 나노입자 및 조성물의 제공을 목적으로 한다.The present invention aims to provide functional nanoparticles and compositions that can interact with ice crystals.
본 발명은 냉동시 얼음의 생성속도를 느리게하여 세포의 손상을 최소화시킬 수 있는 기능성 나노입자 조성물의 제공을 목적으로 한다.An object of the present invention is to provide a functional nanoparticle composition that can minimize the damage of cells by slowing the rate of ice production during freezing.
본 발명은 가시광선 또는 근적외선의 흡수가 가능하여 세포 내부에서부터 얼음 결정의 용해속도 조절이 가능하여, 냉해동과정에서의 세포사멸을 최소화시킬 수 있는 기능성 나노입자 및 조성물을 제공을 목적으로 한다.An object of the present invention is to provide a functional nanoparticles and compositions capable of absorbing visible or near infrared rays to control the rate of dissolution of ice crystals from inside the cell, thereby minimizing cell death during cold thawing.
본 발명은 저온보관 시료의 해빙 시 생체 시료의 손상을 최소화할 수 있는 기능성 나노입자 및 조성물의 제공을 목적으로 한다.It is an object of the present invention to provide functional nanoparticles and compositions capable of minimizing damage to biological samples during thawing of cryopreserved samples.
1. 나노입자; 및 상기 나노입자 표면에 결합되는 아미노산 또는 펩타이드;를 포함하는, 기능성 나노입자.1. nanoparticles; And amino acids or peptides bonded to the surface of the nanoparticles.
2. 위 1에 있어서, 상기 나노입자는 콜로이드성 나노입자인, 기능성 나노입자.2. In the above 1, wherein the nanoparticles are colloidal nanoparticles, functional nanoparticles.
3. 위 1에 있어서, 상기 나노입자는 금인, 기능성 나노입자.3. In the above 1, wherein the nanoparticles are gold, functional nanoparticles.
4. 위 1에 있어서, 상기 아미노산은 쓰레오닌, 발린 및 세린으로 이루어진 군으로부터 선택되는 적어도 하나인, 기능성 나노입자.4. According to the above 1, wherein the amino acid is at least one selected from the group consisting of threonine, valine and serine, functional nanoparticles.
5. 위 1에 있어서, 상기 아미노산은 쓰레오닌인, 기능성 나노입자.5. The functional nanoparticles of 1 above, wherein the amino acid is threonine.
6. 위 1에 있어서, 상기 펩타이드는 화학식 1로 표시되는 화합물을 포함하는, 기능성 나노입자:6. In the above 1, wherein the peptide comprises a compound represented by Formula 1, functional nanoparticles:
[화학식 1][Formula 1]
Figure PCTKR2017003238-appb-I000001
Figure PCTKR2017003238-appb-I000001
7. 위 1에 있어서, 상기 기능성 나노입자는 세포투과성 펩타이드를 더 포함하는, 기능성 나노입자.7. In the above 1, wherein the functional nanoparticles further comprises a cell permeable peptide, functional nanoparticles.
8. 위 1 내지 7 중 어느 한 항의 기능성 나노입자를 포함하는 기능성 식품.8. Functional food comprising the functional nanoparticles of any one of the above 1 to 7.
9. 위 1 내지 7 중 어느 한 항의 기능성 나노입자를 포함하는 결빙 조절용 조성물.9. Composition for controlling ice formation comprising the functional nanoparticles of any one of the above 1 to 7.
본 발명의 일 실시예에 따르면, 화학약품을 사용하는 동결보존제의 생체 시료에 대한 독성 문제가 발생하지 않는 기능성 나노입자 및 조성물을 제공할 수 있다.According to one embodiment of the present invention, it is possible to provide functional nanoparticles and compositions that do not cause toxicity problems for biological samples of cryopreservatives using chemicals.
또한, 본 발명의 일 실시예에 따르면, 합성이 간편하고, 유전공학적으로 특정 아미노산의 조성을 높일 수 있으며, 고농도 사용 시 침전이 발생하지 않는 기능성 나노입자 및 조성물을 제공할 수 있다.In addition, according to one embodiment of the present invention, the synthesis is simple, genetic engineering can increase the composition of a specific amino acid, it can provide functional nanoparticles and compositions that do not occur precipitation at high concentrations.
또한, 본 발명의 일 실시예에 따르면, 얼음 결정과 상호작용할 수 있는 기능성 나노입자 및 조성물을 제공할 수 있다.In addition, according to one embodiment of the present invention, it is possible to provide functional nanoparticles and compositions that can interact with ice crystals.
또한, 본 발명의 일 실시예에 따르면, 냉동시 얼음의 생성속도를 느리게하여 세포의 손상을 최소화시킬 수 있는 기능성 나노입자 및 조성물을 제공할 수 있다.In addition, according to one embodiment of the present invention, it is possible to provide a functional nanoparticles and compositions that can minimize the damage to the cells by slowing the rate of ice production during freezing.
또한, 본 발명의 일 실시예에 따르면, 본 발명의 기능성 나노입자 및 조성물을 세포에 가하면, 가시광선 또는 근적외선을 조사하여 세포 내부에서부터 얼음 결정의 용해가 가능하므로, 냉해동 과정에서 세포사멸을 최소화시킬 수 있는 기능성 나노입자 및 조성물을 제공할 수 있다.In addition, according to an embodiment of the present invention, when the functional nanoparticles and the composition of the present invention is added to the cells, it is possible to dissolve ice crystals from inside the cell by irradiating visible or near infrared rays, thereby minimizing apoptosis during cold thawing. Functional nanoparticles and compositions can be provided.
또한, 본 발명의 일 실시예에 따르면, 저온보관 시료의 해빙 시 생체 시료의 손상을 최소화할 수 있는 기능성 나노입자 및 조성물을 제공할 수 있다.In addition, according to an embodiment of the present invention, it is possible to provide functional nanoparticles and compositions that can minimize damage to biological samples during thawing of low temperature storage samples.
도 1은 본 발명의 기능성 나노입자의 일 실시예를 나타낸 모식도이다.1 is a schematic diagram showing an embodiment of the functional nanoparticles of the present invention.
도 2는 본 발명의 기능성 나노입자가 있는 경우 얼음 결정의 성장이 억제되고 얼음의 결빙 온도가 낮아지는 것을 나타낸 것이다.Figure 2 shows that in the presence of the functional nanoparticles of the present invention the growth of ice crystals is suppressed and the ice freezing temperature is lowered.
도 3은 결빙방지 단백질에서 얼음 결정과 상호작용하는 아미노산의 개수와 배열에 따라 온도이력 (Thermal hysteresis, TH)이 변화하는 것을 나타낸 것이다.Figure 3 shows the change in the temperature hysteresis (TH) according to the number and arrangement of amino acids interacting with the ice crystals in the freezing protein.
도 4는 해빙 시 얼음의 재결정화가 방지된 것을 나타낸 것이다.Figure 4 shows that recrystallization of the ice is prevented during thawing.
도 5는 세포 투과성 아미노산서열이 나노입자 표면에 존재하는 경우 세포 안팎으로 얼음의 결빙 및 해빙에 영향을 줄 수 있다는 것을 나타낸 것으로, 사진은 세포 내로 본 발명의 기능성 나노입자가 들어간 것을 나타낸 전자현미경 사진이다.Figure 5 shows that the cell-permeable amino acid sequence may affect the freezing and thawing of ice in and out of cells when present on the surface of the nanoparticles, the photograph is an electron micrograph showing the functional nanoparticles of the present invention into the cell to be.
도 6은 본 발명의 일 실시예에 따라 쓰레오닌으로 표면이 개질된 금 나노입자를 합성한 결과를 나타낸 것이다.Figure 6 shows the results of synthesizing the gold nanoparticles surface-modified with threonine according to an embodiment of the present invention.
도 7은 본 발명의 일 실시에 따라 합성한 기능성 나노입자로 얼음 결정 성장 실험한 결과를 나타낸 것으로, 그 결과 본 발명의 기능성 나노입자가 얼음 결정의 성장을 방해하는 것을 나타났다.Figure 7 shows the results of ice crystal growth experiments with the functional nanoparticles synthesized according to an embodiment of the present invention, the results showed that the functional nanoparticles of the present invention hinder the growth of ice crystals.
도 8은 나노입자의 결빙 제어 효과의 세포 실험을 나타낸 것으로, 구체적으로, mESC cell에 DMSO 또는 쓰레오닌이 결합된 20 nm 금 나노입자 또는 40 nm 금 나노입자을 가하였을 때, 급속 해동 과정에서의 세포사멸효과, 빛을 조사하여 해동한 조건에서의 세포사멸효과를 나타낸 것이다.FIG. 8 illustrates a cell experiment of the effect of freezing control of nanoparticles. Specifically, when 20 nm gold nanoparticles or 40 nm gold nanoparticles bound with DMSO or threonine are added to mESC cells, Apoptotic effect, showing apoptosis effect under the condition of thawing by irradiation with light.
본 발명은 결빙 제어용 펩타이드 표면 개질된 나노입자에 관한 것으로, 나노입자; 및 상기 나노입자 표면에 결합되는 아미노산 또는 펩타이드;를 포함함으로써, 독성을 가지지 않아 생체시료에 적용할 수 있고, 합성이 간편하고, 유전공학적으로 특정 아미노산의 조성을 높일 수 있으며, 고농도 사용 시 침전이 발생하지 않으며, 얼음 결정과 상호작용하여 빙결 및 해빙을 조절할 수 있고, 냉동시 얼음의 생성속도를 느리게하여 세포의 손상을 최소화시킬 수 있고, 냉해동과정에서 세포사멸을 최소화시킬 수 있으며, 생체 시료의 해빙 시 손상을 최소화시킬 수 있는, 결빙 제어용 펩타이드 표면 개질된 나노입자에 관한 것이다.The present invention relates to a peptide surface-modified nanoparticle for freezing control, the nanoparticle; And amino acids or peptides bound to the surface of the nanoparticles, do not have toxicity, can be applied to biological samples, easy to synthesize, genetically engineered to increase the composition of specific amino acids, precipitation occurs when using high concentrations It can interact with ice crystals to control freezing and thawing, slow down ice formation to minimize cell damage, and minimize cell death during freeze-thawing process. Peptide surface-modified nanoparticles that can minimize damage during thawing.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 나노입자 및 상기 나노입자 표면에 결합되는 아미노산 또는 펩타이드를 포함하는 기능성 나노입자를 제공한다.The present invention provides functional nanoparticles comprising nanoparticles and amino acids or peptides bound to the nanoparticle surface.
나노입자는 90% 이상이 5㎛ 이하, 바람직하게는 2㎛ 이하, 보다 바람직하게는 1㎛ 이하, 더욱 더 바람직하게는 0.5㎛ 이하의 평균입경을 가지는 입자를 의미하며, 플라즈몬 나노입자일 수 있으나, 이에 제한되지 않는다.The nanoparticles mean particles having an average particle diameter of 90% or more of 5 μm or less, preferably 2 μm or less, more preferably 1 μm or less, even more preferably 0.5 μm or less, and may be plasmon nanoparticles. This is not restrictive.
나노입자는 금속 착염의 묽은 용액을 환원시켜 제조할 수 있으며, 구체적으로 환원반응, 핵형성개시 반응, 핵성장 반응 등의 과정을 제조할 수 있으나, 이에 제한되지 않는다.Nanoparticles may be prepared by reducing a dilute solution of a metal complex salt, and specifically, may be prepared a process such as a reduction reaction, nucleation initiation reaction, nuclear growth reaction, but is not limited thereto.
본 발명의 일 실시예에 따르면, 나노입자는 콜로이드성 나노입자일 수 있으나, 이에 제한되지 않는다.According to one embodiment of the invention, the nanoparticles may be colloidal nanoparticles, but is not limited thereto.
본 발명의 일 실시예에 따르면, 나노입자는 도 1의 모식도에 나타나 있는 바와 같이, 원형 또는 막대형일 수 있으나, 이에 제한되지 않는다.According to one embodiment of the invention, the nanoparticles may be circular or rod-shaped, as shown in the schematic diagram of Figure 1, but is not limited thereto.
본 발명의 일 실시예에 따르면, 나노입자는 금 또는 은 중 적어도 하나 또는 이들의 합금일 수 있으며, 구체적으로, 나노입자는 금일 수 있으나, 이에 제한되지 않는다.According to an embodiment of the present invention, the nanoparticles may be at least one of gold or silver or an alloy thereof, and specifically, the nanoparticles may be gold, but is not limited thereto.
상기 나노입자가 금일 경우, 본 발명의 기능성 나노입자는 가시광선, 또는 근적외선 영역의 빛의 흡수가 가능하기 때문에 세포의 해동 시 빛을 조사하여 세포내부에서부터 얼음이 녹는 과정을 조절할 수 있다. 이를 통해 세포의 해동과정에서 세포의 손상을 최소화시킬 수 있다.When the nanoparticles are gold, the functional nanoparticles of the present invention can absorb light in the visible or near-infrared region, thereby controlling the melting process of ice from inside the cells by irradiating light upon thawing of the cells. This can minimize cell damage during cell thawing.
아미노산은, 예를 들어, 알라닌, 시스테인, 아스파르트산, 글루탐산, 페닐알라닌, 글라이신, 히스티딘, 이소류신, 라이신, 류신, 메티오닌, 아스파라긴, 피롤라이신, 프롤린, 글루타민, 아르기닌, 세린, 쓰레오닌, 셀레노시스테인, 발린, 트립토판, 티로신 등일 수 있으나, 이에 제한되지 않는다.Amino acids are, for example, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, pyrrolysine, proline, glutamine, arginine, serine, threonine, seleno Cysteine, valine, tryptophan, tyrosine, and the like, but is not limited thereto.
본 발명의 일 실시예에 따르면, 아미노산은 쓰레오닌, 발린 및 세린 중 적어도 하나일 수 있으나, 이에 제한되지 않는다.According to one embodiment of the invention, the amino acid may be at least one of threonine, valine and serine, but is not limited thereto.
쓰레오닌, 발린 및 세린은 결빙 방지 펩타이드의 주요 아미노산 성분으로, 얼음과의 상호작용이 더욱 효과적으로 일어나게 되며, 얼음과의 상호작용이 다른 아미노산에 비해 더 뛰어나 얼음 결정이 성장하는 것을 더욱 세밀하고 효과적으로 조절 (특히, 방지)할 수 있다.Threonine, valine and serine are the major amino acid components of the antifreeze peptides, which make ice interaction more effective, and ice interaction is better than other amino acids, making ice crystals more detailed and effective. Regulation (especially prevention).
또한, 나노입자와의 결합력 또한 우수하여 기능성 나노입자로 제조가 용이하다.In addition, the bonding strength with the nanoparticles is also excellent, making it easy to manufacture functional nanoparticles.
또한, 본 발명의 일 실시예에 따라 상기 아미노산이 나노입자 표면에 개질되어 본 발명의 기능성 나노입자로 사용되면, 얼음과 상호작용할 수 있는 표면적이 기하급수적으로 증가하여 얼음 결정의 성장, 형성, 해빙 등의 조절 능력이 상기 아미노산을 단독으로 사용한 경우에 비하여 현저히 증가할 수 있다.In addition, when the amino acid is modified on the surface of the nanoparticles and used as the functional nanoparticles of the present invention according to an embodiment of the present invention, the surface area that can interact with ice increases exponentially to grow, form, and thaw ice crystals. And the like's control ability may be significantly increased compared to the case where the amino acid is used alone.
본 발명의 일 실시예에 따르면, 아미노산은 쓰레오닌일 수 있으나, 이에 제한되지 않는다.According to one embodiment of the invention, the amino acid may be threonine, but is not limited thereto.
쓰레오닌이 결합된 본 발명의 기능성 복합체는 냉동 시 세포 내에서 얼음 생성속도를 느리게 하여 세포의 손상을 최소화시킬 수 있다.The functional complex of the present invention, combined with threonine, can slow down ice formation in cells when frozen, thereby minimizing cell damage.
본 발명의 일 실시예에 따르면, 펩타이드는 쓰레오닌, 발린 및 세린으로 이루어진 군으로부터 선택되는 적어도 하나의 아미노산을 포함할 수 있으며, 상기 펩타이드에 포함되는 쓰레오닌, 발린 및 세린의 개수에는 제한이 없다. 예컨대, 쓰레오닌, 발린 및 세린 중 적어도 하나를 1 내지 수천개로 포함할 수 있으나, 이에 제한되지 않는다.According to one embodiment of the invention, the peptide may comprise at least one amino acid selected from the group consisting of threonine, valine and serine, the number of threonine, valine and serine included in the peptide is limited There is no For example, it may include 1 to thousands of at least one of threonine, valine and serine, but is not limited thereto.
본 발명의 일 실시예에 따르면, 펩타이드는 쓰레오닌, 발린 및 세린 중 적어도 하나를 연속으로 또는 이격적으로 포함할 수 있으나, 이에 제한되지 않는다. According to one embodiment of the present invention, the peptide may include at least one of threonine, valine and serine continuously or separately, but is not limited thereto.
본 발명의 일 실시예에 따르면, 펩타이드는 쓰레오닌, 발린 및 세린의 적어도 2개 또는 전부를 포함하는 혼합서열일 수 있으나, 이에 제한되지 않는다.According to one embodiment of the present invention, the peptide may be a mixed sequence including at least two or all of threonine, valine and serine, but is not limited thereto.
본 발명의 일 실시예에 따르면, 펩타이드는 쓰레오닌 및 시스테인을 포함하는 펩타이드일 수 있다. 펩타이드에 포함되는 시스테인은 폴리에틸렌 글라이콜 등의 스페이서 (spacer) 물질로 연결되는 경우도 포함한다. 예컨대, 상기 연결은 펩타이드와 시스테인 사이에 폴리에틸렌 글라이콜이 위치하는 경우일 수 있으나, 이에 제한되지 않는다.According to one embodiment of the invention, the peptide may be a peptide comprising threonine and cysteine. Cysteine included in the peptide also includes a case where the cysteine is linked to a spacer material such as polyethylene glycol. For example, the linkage may be when polyethylene glycol is positioned between the peptide and cysteine, but is not limited thereto.
본 발명의 구체적인 일 실시예에 따르면, 5개의 쓰레오닌이 연속으로 결합되어 있고, 상기 5개의 쓰레오닌의 말단에 시스테인이 결합되어 있는 것일 수 있으나, 이에 제한되지 않는다.According to a specific embodiment of the present invention, five threonine may be continuously coupled, and cysteine may be bound to the terminals of the five threonine, but is not limited thereto.
본 발명의 일 실시예에 따르면, 펩타이드는 화학식 1로 표시되는 화합물을 포함하는 펩타이드일 수 있으나, 이에 제한되지 않는다.According to one embodiment of the present invention, the peptide may be a peptide including the compound represented by Formula 1, but is not limited thereto.
화학식 1로 표시되는 화합물을 포함하는 펩타이드의 경우 얼음 결정의 비등방성 성장을 유도하여, 얼음 결정의 성장을 방해할 수 있다(도 6 및 7). 또한, 냉동 보관된 세포의 해동 시 손상을 최소화시킬 수 있다 (도 8).In the case of the peptide containing the compound represented by the formula (1) may induce anisotropic growth of ice crystals, it may interfere with the growth of ice crystals (Figs. 6 and 7). In addition, it is possible to minimize the damage during thawing of frozen cells (Fig. 8).
[화학식 1][Formula 1]
Figure PCTKR2017003238-appb-I000002
Figure PCTKR2017003238-appb-I000002
또한, 본 발명에서, 펩타이드는 어떤 동물, 식물, 곤충, 미생물 등에서 발견되는 분자 또는 인위적으로, 자연적으로 합성된 분자를 의미하며, 물의 결빙점을 비속일적으로 감소시키는 펩타이드이다.In addition, in the present invention, the peptide refers to a molecule found in any animal, plant, insect, microorganism, or the like, or an artificially and naturally synthesized molecule, and is a peptide that non-uniformly reduces the freezing point of water.
본 발명에서, 펩타이드는 얼음과 결합하여 얼음 결정의 성장을 막는 특징을 가진 단백질로, 결빙방지 폴리펩타이드 (anti-freezing peptides, AFPs), 결빙방지 당단백질, 결빙방지 글리코펩타이드 (anti-freezing glycopeptides, AFGP), 열 이력 폴리펩타이드 등일 수 있다.In the present invention, the peptide is a protein that is characterized by binding to ice to prevent the growth of ice crystals, anti-freezing peptides (AFPs), anti-freezing glycoproteins, anti-freezing glycopeptides (anti-freezing glycopeptides, AFGP), thermal history polypeptide, and the like.
본 발명의 일 실시예에 따르면, 펩타이드는 북극 및 남극환 내의 지방을 포함해서 지구의 극지 지방의 물에서 사는 냉혈 수생 극지 어류 종에서 생산되는 결빙 방지 펩타이드; 극지 규조, 당근 및 민들레를 비롯한 다양한 고등 식물에서 생산되는 결빙 방지 펩타이드; 곤충, 세균, 어류 등 다양한 종에서 생산되는 결빙 방지 펩타이드;를 포함할 수 있으나, 이에 제한되지 않는다.According to one embodiment of the invention, the peptide is an anti-freeze peptide produced in cold blooded aquatic polar fish species that live in water of the polar regions of the earth, including fats in the Arctic and Antarctic rings; Antifreeze peptides produced in various higher plants, including polar diatoms, carrots and dandelions; Antifreeze peptides produced by various species, such as insects, bacteria, fish; may include, but are not limited thereto.
본 발명의 일 실시예에 따르면, 펩타이드는 FfIBP, NglIBP, Type III AFP 등의 결빙방지 단백질일 수 있으나, 이에 제한되지 않는다.According to one embodiment of the present invention, the peptide may be an antifreezing protein such as FfIBP, NglIBP, Type III AFP, but is not limited thereto.
본 발명의 일 실시예에 따르면, 펩타이드는 1차 또는 2차 구조를 가지는 것일 수 있다. 예컨대, 알파 헬릭스, 베타 시트, 이들의 반복 또는 조합의 구조를 가질 수 있으나, 이에 제한되지 않는다.According to one embodiment of the invention, the peptide may have a primary or secondary structure. For example, it may have a structure of alpha helix, beta sheet, a repeat or combination thereof, but is not limited thereto.
본 발명의 일 실시예에 따르면, 펩타이드는 상기 결빙방지 폴리펩타이드 (anti-freezing peptides, AFPs), 결빙방지 당단백질, 결빙방지 글리코펩타이드 (anti-freezing glycopeptides, AFGP) 또는 열 이력 폴리펩타이드의 아미노산 서열 일부 또는 전부를 변형하거나, 상기 아미노산 서열의 처음, 중간 또는 말단에 다른 아미노산을 추가로 결합하는 등을 통해 변형될 수 있으며, 상기 변형은 얼음과의 상호작용 목적 범위 내에서 이루어질 수 있다.According to one embodiment of the invention, the peptide is an amino acid sequence of the anti-freezing peptides (AFPs), anti-freezing glycoproteins, anti-freezing glycopeptides (AFGP) or heat history polypeptide Modification may be made in part or in whole, or by additional binding of other amino acids at the beginning, middle or end of the amino acid sequence, and the modification may be made within the scope of interaction with ice.
본 발명의 일 실시예에 따르면, 펩타이드는 친화성 택에 융합될 수 있다. 친화성 택으로, 이로 제한되지 않으나, His-tag, 폴리펩티드 A 택, 아비딘/스트렙타비딘, 폴리펩티드 G, 글루타티온-S-트랜스페라제, 디히드로폴레이트 환원효소(DHFR), 녹색 형광 폴리펩티드(GFP), 폴리아르기닌, 폴리시스테인, c-myc, 칼모듈린 결합 폴리펩티드, 인플루엔자바이러스 헤마글루티닌, 말토스 결합 단백질(MBP), 히알루론산 (HA) 등을 들 수 있으나, 이에 제한되지 않는다.According to one embodiment of the invention, the peptide may be fused to an affinity tag. Affinity tags, including but not limited to His-tag, polypeptide A tag, avidin / streptavidin, polypeptide G, glutathione-S-transferase, dihydrofolate reductase (DHFR), green fluorescent polypeptide (GFP ), Polyarginine, polycysteine, c-myc, calmodulin binding polypeptide, influenza virus hemagglutinin, maltose binding protein (MBP), hyaluronic acid (HA), and the like.
본 발명의 일 실시예에 따르면, 펩타이드는 상기 펩타이드 내 하나 이상의 아미노산 잔기가 변형된 펩타이드일 수 있다. 하나 이상의 변형(들)은 생체내 또는 시험관내 화학적 유도체화, 예를 들어 아세틸화 또는 카르복실화; 글리코실화; 탈글리코실화; 인산화, 예를 들어 포스포티로신, 포스포세린 및 포스포쓰레오닌; 등일 수 있으나, 이에 제한되지 않는다.According to an embodiment of the present invention, the peptide may be a peptide in which one or more amino acid residues in the peptide are modified. One or more modification (s) may include chemical derivatization in vivo or in vitro, for example acetylation or carboxylation; Glycosylation; Deglycosylation; Phosphorylation such as phosphotyrosine, phosphoserine and phosphothreonine; Etc., but is not limited thereto.
본 발명의 일 실시예에 따르면, 펩타이드는 자연발생 L-아미노산, 자연발생 L-아미노산 및 비-자연발생 합성 아미노산 등일 수 있으나, 이에 제한되지 않는다.According to one embodiment of the invention, the peptide may be, but is not limited to, naturally occurring L-amino acids, naturally occurring L-amino acids and non-naturally occurring synthetic amino acids.
본 발명의 일 실시예에 따르면, 펩타이드는 N-말단 및/또는 C-말단에 봉쇄기가 도입된 펩타이드일 수 있으나, 이에 제한되지 않는다. 봉쇄기는 N-말단 및/또는 C-말단을 보호하고 안정화하는데 적합한 화학 치환체로, N-말단의 알킬화 또는 아실화로 도입된 분지 또는 비분지 알킬기 및 아실기; 에스테르 또는 케톤-형성 알킬기를 포함하는 C-말단 봉쇄기로, 예를 들어 저급(C1 내지 C6) 알킬기, 예를 들어 메틸, 에틸 및 프로필, 및 아미드-형성 아미노기;등을 들 수 있으나, 이에 제한되지 않는다.According to an embodiment of the present invention, the peptide may be a peptide having a blocking group introduced at the N-terminus and / or the C-terminus, but is not limited thereto. Blocking groups are chemical substituents suitable for protecting and stabilizing the N-terminus and / or C-terminus, including branched or unbranched alkyl groups and acyl groups introduced by alkylation or acylation of the N-terminus; C-terminal blockade groups comprising ester or ketone-forming alkyl groups, for example, lower (C1 to C6) alkyl groups such as methyl, ethyl and propyl, and amide-forming amino groups; Do not.
아미드-형성 아미노기로는 1차 아민(-NH2), 및 모노- 및 디-알킬아미노기 등을 들 수 있으며, 모노- 및 디-알킬아미노기로는 메틸아미노, 에틸아미노, 디메틸아미노, 디에틸아미노, 메틸에틸아미노 등을 들 수 있으나, 이에 제한되지 않는다.Amide-forming amino groups include primary amines (-NH2), mono- and di-alkylamino groups, and the like, and mono- and di-alkylamino groups include methylamino, ethylamino, dimethylamino, diethylamino, Methylethylamino and the like, but are not limited thereto.
나노입자와 결합되는 펩타이드의 적합한 농도는 용도에 따라 변할 수 있으며, 예컨대 약 1 ppb 내지 약 1 ppt(즉, 약 1 μg/L 내지 약 1 mg/L)의 범위일 수 있다.Suitable concentrations of peptide bound to the nanoparticles can vary depending on the application and can range from, for example, about 1 ppb to about 1 ppt (ie, about 1 μg / L to about 1 mg / L).
상기 펩타이드들은 나노입자와의 결합력 또한 우수하여 기능성 나노입자로 사용될 경우 얼음과 상호작용할 수 있는 표면적이 기하급수적으로 증가하여 결정의 성장, 형성, 해빙 등의 조절 능력이 상기 펩타이드들을 단독으로 사용한 경우에 비하여 현저히 우수해질 수 있다.The peptides also have excellent binding ability with nanoparticles, so when used as functional nanoparticles, the surface area that can interact with ice increases exponentially, so that the ability to control the growth, formation, thawing of crystals, etc. is used when the peptides are used alone. It can be remarkably superior in comparison.
본 발명의 일 실시예에 따르면, 나노입자와 아미노산 또는 펩타이드와의 결합은 나노입자 표면과 아미노산 또는 펩타이드와의 화학적 결합을 포함할 수 있다. 상기 나노입자는 표면이 개질된 것일 수 있다. 예컨대, 나노입자와 아미노산 또는 펩타이드와의 결합은 카르복시산으로 개질한 다음 아미노산 또는 펩타이드로 환원시켜 직접 나노입자에 아미노산 또는 펩타이드를 결합시키는 것일 수 있다.According to one embodiment of the invention, the binding of the nanoparticles with amino acids or peptides may comprise chemical bonding of the nanoparticle surfaces with amino acids or peptides. The nanoparticles may have a modified surface. For example, the binding of the nanoparticles to amino acids or peptides may be to directly bind amino acids or peptides to the nanoparticles by modifying them with carboxylic acids and then reducing them to amino acids or peptides.
본 발명의 일 실시예에 따르면, 나노입자와 아미노산 또는 펩타이드와의 결합은 링커에 의한 결합일 수 있다. 링커에 의한 결합은 링커를 나노입자에 결합시킨 다음, 상기 링커와 아미노산 또는 펩타이드를 결합시킴으로써 나노입자와 아미노산 또는 펩타이드를 결합시킬 수 있으나, 이에 제한되지 않는다. 예컨대, 링커는 시스테인, o-linker일 수 있으나, 이에 제한되지 않는다.According to an embodiment of the present invention, the binding of the nanoparticles to amino acids or peptides may be binding by a linker. The binding by the linker may bind the linker to the nanoparticles, and then bind the nanoparticles to amino acids or peptides by binding the linker to amino acids or peptides, but is not limited thereto. For example, the linker may be a cysteine, an o-linker, but is not limited thereto.
본 발명의 기능성 나노입자는 나노입자 표면에 아미노산 또는 펩타이드가 결합됨으로써 얼음과 상호작용이 가능하게 되어 얼음 결정의 형성을 억제, 지연, 방지 등의 기능을 수행할 수 있다.The functional nanoparticles of the present invention may interact with ice by combining amino acids or peptides on the surface of the nanoparticles, thereby performing functions such as inhibiting, delaying, and preventing ice crystal formation.
본 발명의 일 실시예에 따르면, 본 발명의 기능성 나노입자에 포함되는 아미노산 또는 펩타이드의 개수 및 배열에 따라 온도이력현상이 증가하는 것으로 나타났다 (도 3).According to one embodiment of the present invention, the temperature history phenomenon was increased according to the number and arrangement of amino acids or peptides included in the functional nanoparticles of the present invention (FIG. 3).
따라서, 나노입자가 형성되는 과정에서 아미노산 또는 펩타이드의 수를 조절하여 기능성 나노입자의 면적, 크기, 모양 등을 조절함으로써 얼음과의 결합력, 결합 면적 등을 조절하여 얼음 결정 형성 시간, 방지, 해빙 시간 등을 보다 더 세밀하게 조절할 수 있다.Therefore, by adjusting the number of amino acids or peptides in the process of forming nanoparticles, by adjusting the area, size, shape, etc. of functional nanoparticles, ice crystal formation time, prevention time, thawing time You can fine tune your back.
또한, 본 발명의 기능성 나노입자는 화학약품을 사용하지 않아 동결보존제 (부동액, DMSO 등)의 생체 시료에 대한 독성문제를 발생시키지 않는다. 따라서, 곤충, 포유류, 파충류, 어류 등의 세포 등 다양한 생체 시료에 제한 없이 사용될 수 있다.In addition, the functional nanoparticles of the present invention do not use chemicals and do not cause toxicity problems for biological samples of cryopreservatives (antifreezes, DMSO, etc.). Therefore, it can be used without limitation in various biological samples such as cells of insects, mammals, reptiles, fish and the like.
또한, 저온 환경에서 보관된 생체 시료는 해빙 시 얼음 재결정화로 인하여 세포 손상의 위험이 있으나, 본 발명의 기능성 나노입자를 생체 시료에 첨가하여 저온 환경에 보관 후 세포를 해빙시킬 때 발생하는 얼음의 재결정화를 방지하여 생체 시료의 손상을 최소화할 수 있다.In addition, although biological samples stored in a low temperature environment may risk cell damage due to ice recrystallization during thawing, recrystallization of ice generated when thawing cells after storage in a low temperature environment by adding functional nanoparticles of the present invention to a biological sample. Prevention of ignition can minimize damage to biological samples.
본 발명의 일 실시예에 따르면, 본 발명의 기능성 나노입자를 사용할 경우, 얼음의 재결정화가 방지되는 것으로 나타났다 (도 4).According to one embodiment of the present invention, it was shown that when using the functional nanoparticles of the present invention, recrystallization of ice is prevented (FIG. 4).
또한, 본 발명의 기능성 나노입자는 세포 내부로 진입할 수 있어 (도 5), 세포의 해동 시 빛을 빛을 조사하여 생체 시료 내부에서부터 얼음의 해동을 조절할 수 있고, 이를 통해 냉동 보관 후 해동 시 발생할 수 있는 생체 시료의 손상을 최소화시킬 수 있다 (도 8).In addition, the functional nanoparticles of the present invention can enter the cell (Fig. 5), it is possible to control the thawing of the ice from the inside of the biological sample by irradiating light to the light during thawing of the cell, thereby thawing after freezing storage Damage to biological samples that can occur can be minimized (FIG. 8).
생체 시료는 포유동물세포, 곤충세포, 어류세포, 식물세포, 진핵세포 또는 원핵세포 등일 수 있으나, 이에 제한되지 않는다.The biological sample may be, but is not limited to, mammalian cells, insect cells, fish cells, plant cells, eukaryotic cells or prokaryotic cells.
본 발명의 일 실시예에 따르면, 본 발명의 기능성 나노입자는 얼음 결정 성장으로 인한 세포 손상을 실질적으로 감소시킬 수 있어, 혈액 및 혈액 제품, 치료제, 폴리펩티드 약물, 바이오어세이 시약 및 백신 등 민감한 생체 시료의 보관에 적합할 수 있다.According to one embodiment of the present invention, the functional nanoparticles of the present invention can substantially reduce cellular damage due to ice crystal growth, thereby allowing sensitive biological organisms such as blood and blood products, therapeutic agents, polypeptide drugs, bioassay reagents and vaccines, etc. It may be suitable for storage of the sample.
본 발명의 일 실시예에 따르면, 기능성 나노입자는 세포투과성 펩타이드를 더 포함할 수 있다.According to one embodiment of the invention, the functional nanoparticles may further comprise a cell permeable peptide.
세포투과성 펩타이드가 본 발명의 기능성 나노입자에 더 포함됨으로써, 세포 내로 기능성 나노입자를 전달하여 세포 안팍의 얼음 결정화 등 물-얼음 거동에 영향을 미침으로써 세포의 결빙을 조절할 수 있다.As the cell permeable peptide is further included in the functional nanoparticles of the present invention, the functional nanoparticles can be delivered into the cells, thereby controlling freezing of the cells by affecting water-ice behavior such as ice crystallization of the inner cells.
세포 투과성 펩타이드는 일종의 신호 펩타이드 (Signal Peptide)이며, 예로 TAT 단백질에 존재하는 11개 아미노산 서열의 b-galactosidase (120 kDa) 단백질, 초파리의 단백질에서 유래한 Antennapedia (Penetratin), HSV-1 바이러스로부터 유래된 VP22, Simian Virus 40 large antigen T 로부터 유래한 Pep-1 등을 들 수 있으나, 이에 제한되지 않는다.Cell-penetrating peptides are a type of signal peptide, e.g. from the 11 amino acid sequence b-galactosidase (120 kDa) protein present in the TAT protein, from Antennapedia (Penetratin) and HSV-1 virus from Drosophila protein VP22, Pep-1 derived from Simian Virus 40 large antigen T, and the like, but are not limited thereto.
또한, poly Arginine, poly Lysine 과 같이 단순히 Arginine, Lysine 과 같은 양이온성 아미노산이 반복적으로 여러 개가 연결된 펩타이드가 포함될 수 있으나, 이제 제한되지 않는다.In addition, it may include a peptide in which a plurality of cationic amino acids such as Arginine and Lysine are repeatedly connected, such as poly Arginine and poly Lysine, but are not limited thereto.
예컨대, 세포투과성 펩타이드로 Hph-1, Vectocell, Lactoferrin, Sim-2, LPIN3, 2IL-1a, dNP2 등을 들 수 있으나, 이에 제한되지 않는다.For example, Hph-1, Vectocell, Lactoferrin, Sim-2, LPIN3, 2IL-1a, dNP2, etc. may be mentioned as cell permeable peptides, but are not limited thereto.
본 발명의 기능성 나노입자는 식용품에 도입되거나, 또는 식용품과 접촉되어 얼음 결정 성장 및/또는 형성을 감소 또는 억제할 수 있고, 이를 통해 야채류를 포함하는 냉동 식용품의 질감, 맛, 및 유효 저장수명을 향상시킬 수 있다. 그리고 본 발명의 기능성 나노입자는 식용품을 포함하는 물체나 물질의 결빙 또는 과냉각과 관련된 얼음 결정 형성을 억제 및/또는 감소시킬 수 있다.Functional nanoparticles of the present invention can be introduced into or in contact with food products to reduce or inhibit ice crystal growth and / or formation, thereby allowing the texture, taste, and effectiveness of frozen food products, including vegetables. It can improve the shelf life. In addition, the functional nanoparticles of the present invention can inhibit and / or reduce the formation of ice crystals associated with freezing or subcooling of objects or materials including foodstuffs.
따라서, 본 발명은 다른 양태로 기능성 나노입자를 포함하는 기능성 식품을 제공한다.Accordingly, the present invention provides a functional food comprising functional nanoparticles in another aspect.
본 발명의 기능성 식품은 상기 기능성 나노입자를 포함함으로써 이런 얼음 결정 성장 과정이 감소되거나, 또는 심지어 완전히 방지되거나, 또는 적어도 최소화될 수 있다.The functional food of the present invention may contain such functional nanoparticles such that this ice crystal growth process may be reduced, or even completely prevented, or at least minimized.
식품은 저지방 스프레드, 마요네즈, 요구르트, 제과소, 마가린, 재구성 과일, 잼, 과일 제조물, 과일소, 리플, 과일 소스, 과일 스튜, 커피 표백제, 인스턴트 과일 디저트, 당과(예를 들어, 마쉬멜로우), 감자 베이스 식품(예를 들어, 칩, 프렌치 프라이 및 크로켓), 가공 식품(예를 들어, 캐서롤 및 스튜) 및 파인푸드(예를 들어, 샐러드 드레싱을 비롯한 드레싱; 케찹, 비네그레트 드레싱 및 수프) 등일 수 있으나, 이에 제한되지 않는다.Foods are low-fat spreads, mayonnaise, yogurt, bakery, margarine, reconstituted fruit, jams, fruit products, fruit cows, ripple, fruit sauces, fruit stew, coffee bleach, instant fruit desserts, sweets (e.g. marshmallows) , Potato-based foods (eg chips, french fries and croquettes), processed foods (eg casserole and stews) and fine foods (eg dressings including salad dressings; ketchup, vinaigrette dressings and soups) Etc., but is not limited thereto.
또한, 식품은 음료, 생고기, 요리된 고기, 생 가금류 제품, 요리된 가금류 제품, 생 해산물 제품, 요리된 해산물 제품을 포함하는 생, 가공된 또는 저온살균된 식품[생 또는 요리된 고기, 가금류 및 해산물 제품], 소시지, 프랑크프루트, 바로 먹을 수 있는 음식, 파스타 소스, 저온살균된 수프, 매리네이드, 수중유 에멀젼, 유중수 에멀젼, 치즈 스프레드, 가공 치즈, 유제품 디저트, 향을 가한 밀크, 크림, 발효된 유제품, 치즈, 버터, 응축 밀크 제품, 치즈 스프레드, 저온살균된 액체 난, 아이스크림 믹스, 대두 제품, 저온살균된 액체 난, 당과 제품, 과일 제품, 및 지방 베이스 또는 물-함유 소를 채운 식품 등일 수 있으나, 이에 제한되지 않는다.In addition, food products may be prepared from raw, processed or pasteurized foods, including beverages, raw meats, cooked meats, raw poultry products, cooked poultry products, raw seafood products, cooked seafood products [raw or cooked meat, poultry and Seafood products], sausages, frankfurters, ready-to-eat foods, pasta sauces, pasteurized soups, marinades, oil-in-water emulsions, water-in-oil emulsions, cheese spreads, processed cheeses, dairy desserts, flavored milk, cream, Filled with fermented dairy products, cheeses, butters, condensed milk products, cheese spreads, pasteurized liquid eggs, ice cream mixes, soy products, pasteurized liquid eggs, sugar products, fruit products, and fat-based or water-containing cows Food, etc., but is not limited thereto.
또한, 식품은 빵, 케이크, 파인 베이커리 및 도우와 같은 제과 제품일 수 있으나, 이에 제한되지 않는다.In addition, the food may be, but is not limited to, confectionery products such as bread, cakes, fine bakery and dough.
본 발명의 기능성 식품은 기능성 나노입자가 상기 식품 전체에 혼입된 형태 및/또는 식용품의 표면에 적용된 형태일 수 있으나, 이에 제한되지 않는다.The functional food of the present invention may be in a form in which functional nanoparticles are incorporated into the food as a whole and / or in a form applied to a surface of a food product, but is not limited thereto.
본 발명의 기능성 식품은 정제, 캡슐, 분말, 과립, 액상, 환, 분말, 편상, 페이스트상, 시럽, 겔, 젤리, 바 등 다양한 형태로 제조될 수 있으며, 가공 식품뿐만 아니라 가공되지 않은 식품의 형태로도 제조될 수 있다.The functional food of the present invention may be prepared in various forms such as tablets, capsules, powders, granules, liquids, pills, powders, flakes, pastes, syrups, gels, jellies, bars, and the like. It may also be prepared in form.
본 발명의 기능성 식품은 본 발명의 목적 범위 내에서 식품 제조 시 통상적으로 첨가되는 성분을 더 포함할 수 있으며, 예컨대, 단백질, 탄수화물, 지방, 영양소, 조미제 및 향미제 등을 더 포함할 수 있다.Functional food of the present invention may further include ingredients that are commonly added in the manufacture of food within the scope of the present invention, for example, may further include proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents. .
상기 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스, 올리고당 등; 및 폴리사카라이드, 예를 들어 덱스트린, 사이클로덱스트린 등과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜일 수 있으나, 이에 제한되는 것은 아니다.Examples of such carbohydrates are monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as, but are not limited to, conventional sugars such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.
상기 향미제로서 타우마틴, 스테비아 추출물 (예를 들어 레바우디오시드A, 글리시르히진 등) 등의 천연 향미제 및 사카린, 아스파탐 등의 합성 향미제를 더 포함할 수도 있으나, 이에 제한되는 것은 아니다. The flavoring agent may further include natural flavoring agents such as taumartin, stevia extract (for example, Rebaudioside A, glycyrgin, etc.) and synthetic flavoring agents such as saccharin and aspartame, but are not limited thereto. .
상기 외에 본 발명의 기능성 식품은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다.In addition to the above, the functional food of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols. And carbonation agents used in carbonated beverages.
그 밖에 본 발명의 기능성 식품은 천연 과일쥬스, 과일쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨 가제의 비율은 크게 중요하진 않지만 본 발명의 기능성 식품 조성물 100 중량부 당 0.01~0.20 중량부의 범위로 첨가될 수 있다.In addition, the functional food of the present invention may contain fruit flesh for the production of natural fruit juice, fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not critical, but may be added in the range of 0.01 to 0.20 parts by weight per 100 parts by weight of the functional food composition of the present invention.
그 외의 통상적인 방법에 따라 적절하게 사용할 수 있다. 유효 성분의 혼합양은 그의 사용 목적(예방, 건강적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조 시에는, 원료의 전체중량에 대하여 0.0001 내지 30중량%, 바람직하게는 0.0001 내지 10 중량%, 더욱 바람직하게는 0.1 내지 5 중량%의 양으로 첨가될 수 있다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하로 조절할 수 있다.It can use suitably according to other conventional methods. The mixed amount of the active ingredient can be suitably determined according to the purpose of use (prevention, health treatment). In general, in the manufacture of foods or beverages, it can be added in an amount of 0.0001 to 30% by weight, preferably 0.0001 to 10% by weight, more preferably 0.1 to 5% by weight relative to the total weight of the raw material. However, in the case of prolonged ingestion for health and hygiene purposes or for health control purposes, the amount may be adjusted below the above range.
본 발명의 기능성 식품은 안정제를 더 포함할 수 있다. 안정제로는 폴리펩티드, 예를 들어 젤라틴; 식물 추출물, 예를 들어 아라비아 검, 가티 검, 카라야 검, 트래거캔스 검; 종자 검, 예를 들어 로커스트빈 검, 구아 검, 타라검, 피실리움씨 검, 모과씨 검 또는 타마린드씨 검; 곤약 만난; 해초 추출물, 예를 들어 아가, 알가네이트, 카라게난 또는 푸르셀레란; 펙틴, 예를 들어 저급 메톡실 또는 고급 메톡실-타입 펙틴; 셀룰로오스 유도체, 예를 들어 나트륨 카르복시메틸 셀룰로오스, 미세결정 셀룰로오스, 메틸 및 메틸에틸 셀룰로오스, 또는 히드록실프로필 및 히드록시프로필메틸 셀룰로오스; 및 미생물 검, 예를 들어 덱스트란, 크산탄 또는 β-1,3-글루칸; 등을 들 수 있으나, 이에 제한되지 않는다.Functional food of the present invention may further comprise a stabilizer. Stabilizers include polypeptides such as gelatin; Plant extracts such as gum arabic, gatti gum, karaya gum, tragacanth gum; Seed gums such as locust bean gum, guar gum, tara gum, picillium seed gum, quince seed gum or tamarind seed gum; Konjac met; Seaweed extracts such as agar, alganate, carrageenan or purselane; Pectin, for example lower methoxyl or higher methoxyl-type pectin; Cellulose derivatives such as sodium carboxymethyl cellulose, microcrystalline cellulose, methyl and methylethyl cellulose, or hydroxylpropyl and hydroxypropylmethyl cellulose; And microbial gums such as dextran, xanthan or β-1,3-glucan; And the like, but is not limited thereto.
또한, 본 발명의 기능성 식품은 당분, 예를 들어 수크로오스, 프럭토스, 덱스트로스, 락토스, 옥수수시럽, 당 알코올; 또는 다른 원료들, 예를 들어 색소와 향료 등을 함유할 수 있다.In addition, the functional food of the present invention is a sugar such as sucrose, fructose, dextrose, lactose, corn syrup, sugar alcohol; Or other raw materials such as pigments and flavorings.
본 발명의 기능성 나노입자는 DMSO를 사용하여 세포를 냉동 및 해동한 경우와 비교할 때 더욱 많은 수의 세포가 해동 후 살아있는 것으로 나타났다 (도 8). 따라서, 본 발명은 또 다른 양태로 생체 시료 (예컨대, 생물학적 세포 또는 이의 추출물 등)의 냉동 보관에 효과적인 기능성 나노입자를 포함하는 결빙 조절용 조성물을 제공한다.The functional nanoparticles of the present invention were found to be alive after thawing as compared to the case of freezing and thawing cells using DMSO (FIG. 8). therefore, In another aspect, the present invention provides a composition for controlling freezing comprising functional nanoparticles effective for cryopreservation of biological samples (eg, biological cells or extracts thereof).
생체 시료는 박테리아 세포, 효모 세포, 식물 세포, 동물 세포, 곤충 세포, 파충류 세포, 어류 세포, 포유동물 세포, 아세포 샘플 및 세포 추출물 등일 수 있으나, 이에 제한되지 않는다.The biological sample may be, but is not limited to, bacterial cells, yeast cells, plant cells, animal cells, insect cells, reptile cells, fish cells, mammalian cells, blast samples and cell extracts.
본 발명의 일 실시예에 따르면, 생체 시료는 포유동물 세포일 수 있으며, 난모세포, 배아, 백혈구, 적혈구, 혈소판, 췌장섬 및 간세포 등의 광범위한 타입의 세포; 피부 조직, 골수 조직, 각막 조직 및 다른 광범한 타입의 조직; 그리고 간, 신장, 심장, 뇌, 폐, 췌장, 비장, 난소 및 위 등 광범위한 타입의 장기;를 포함할 수 있으나, 이에 제한되지 않는다.According to one embodiment of the invention, the biological sample may be a mammalian cell, a wide range of types of cells such as oocytes, embryos, leukocytes, erythrocytes, platelets, pancreatic islets and hepatocytes; Skin tissue, bone marrow tissue, corneal tissue and other broad types of tissue; And various types of organs such as liver, kidney, heart, brain, lung, pancreas, spleen, ovary, and stomach; but are not limited thereto.
본 발명의 일 실시예에 따르면, 박테리아 등의 미생물, 동물, 식물, 곤충 등의 연조직은 본 발명의 조성물의 존재 하에 냉동 또는 해동될 때 더 적은 손상을 나타낼 수 있으며, 상기 조성물의 첨가는 냉동 전과 후의 해동시 세포 완전성이 중요하거나 바람직한 상황 (예를 들어, 조직 배양물 기탁)에서 유용할 수 있다. 즉, 냉동-해동 과정으로 인한 고유 특성 또는 고유 형태의 손실을 최소화시킬 수 있다.According to one embodiment of the invention, microorganisms such as bacteria, soft tissues such as animals, plants, insects, etc. may exhibit less damage when frozen or thawed in the presence of the composition of the invention, the addition of the composition before and after freezing Cell integrity in later thawing may be useful in situations where important or desirable (eg, tissue culture deposits). In other words, it is possible to minimize the loss of inherent properties or intrinsic form due to the freeze-thaw process.
또한, 본 발명의 일 실시예에 따를 때, 기능성 나노입자를 포함하는 조성물은 얼음 결정의 성장을 억제할 수 있고, 이를 통해 생체 시료 (예컨대, 세포)의 냉동 및 해동 시 생존률을 크게 향상시킬 수 있다 (도 8).In addition, according to one embodiment of the present invention, the composition comprising the functional nanoparticles can inhibit the growth of ice crystals, thereby greatly improving the survival rate during freezing and thawing of biological samples (eg, cells). (FIG. 8).
본 발명의 조성물은 고체, 반고체, 유체, 기체 등 다양한 형태일 수 있다. 예컨대, 유체일 수 있으나, 이에 제한되지 않는다.The compositions of the present invention may be in various forms, such as solids, semisolids, fluids, gases, and the like. For example, it may be a fluid, but is not limited thereto.
본 발명의 조성물은 생물학적 제제를 보존하는데 유용하다고 공지된 전해질 용액에 포함되는 염, 당, 이온 및 다른 영양물의 광범한 혼합물 중 어느 것을 더 함유할 수 있다. 이들은 조직 배양 배지, 장기 관류액 등을 포함한다.The composition of the present invention may further contain any of a broad mixture of salts, sugars, ions and other nutrients contained in an electrolyte solution known to be useful for preserving biological agents. These include tissue culture medium, organ perfusate and the like.
또한, 본 발명은 본 발명의 기능성 나노입자를 포함하는 화장료 조성물 또는 피부과용 제제를 제공한다.The present invention also provides a cosmetic composition or a dermatological preparation comprising the functional nanoparticles of the present invention.
화장료 조성물이나 국소 피부과용 제제에 본 발명의 기능성 나노입자를 포함시킴으로써, 극명한 기후- 및 날씨-유도 온도 강하에 의해 세포 효소의 최적 온도가 손실됨으로써 세포 및 세포외 공간에서 세포 생리학에 변화가 야기되어 손상되는 추위로 인한 피부 구조 및 세포 손상을 예방 또는 개선시킬 수 있다By incorporating the functional nanoparticles of the present invention in cosmetic compositions or topical dermatological preparations, the optimum temperature of the cellular enzymes is lost due to a pronounced climate- and weather-induced temperature drop resulting in changes in cell physiology in the cell and extracellular space. Prevents or improves skin structure and cell damage from damaged cold
본 발명의 일 실시예에 따르면, 상기 피부 구조 및 세포 손상은 추위, 바람 및/또는 UV 광에 의해 유도된 피부 손상, 피부 홍반 및 피부가 당기는 느낌 및 증가된 감각 감수성, 온도-민감성 피부, 환경 스트레스(온도 변화 및 UV 빛, 흡연, 스모그, 반응성 산소 종, 자유 라디칼로 인한)로 인한 피부, 입술 및 코와 구강 점막 그리고 피부 부속기관들의 부정적인 변화를 포함하나, 이에 제한되지 않는다.According to one embodiment of the invention, the skin structure and cell damage is caused by cold, wind and / or UV light induced skin damage, skin erythema and skin pulling feeling and increased sensory sensitivity, temperature-sensitive skin, environment It includes, but is not limited to, changes in skin, lips and nose and oral mucosa and skin appendages due to stress (due to temperature changes and UV light, smoking, smog, reactive oxygen species, free radicals).
또한, 본 발명의 조성물, 화장료 조성물 및 국소 피부과용 제제는 당업자에게 공지된 안정제, 유화제, 계면활성제 및 다른 첨가제 중 적어도 하나를 더 포함할 수 있다.In addition, the compositions, cosmetic compositions and topical dermatological preparations of the present invention may further comprise at least one of stabilizers, emulsifiers, surfactants and other additives known to those skilled in the art.
다른 첨가제로 항부패제, 항산화제, 변색 방지제, 항미생물제, 에멀젼 안정화제 등을 들 수 있으나, 이에 제한되지 않는다.Other additives include, but are not limited to, anti-corruption agents, antioxidants, discoloration inhibitors, antimicrobial agents, emulsion stabilizers, and the like.
이하, 본 발명을 하기 실시예에 의하여 더욱 상세히 설명한다. 하기 실시예는 본 발명을 설명하기 위한 것일 뿐, 본 발명의 범위를 제한하는 것은 아니다.Hereinafter, the present invention will be described in more detail by the following examples. The following examples are only intended to illustrate the present invention, but not to limit the scope of the present invention.
실시예Example
실시예 1 쓰레오닌이 표면개질된 금 나노입자 (20 nm)의 합성Example 1 Synthesis of Gold Nanoparticles (20 nm) Surface-Modified with Threonine
20 nm 직경의 금 나노입자 용액 (Abs = 1.0 at 520 nm) 1 ml에 쓰레오닌 펩타이드 (쓰레오닌 5개의 말단에 시스테인이 결합되어 있는 펩타이드; 화학식 1) 80 uL (1.0 mg/ml)를 가하여 상온에서 12시간 동안 shake 한다. 원심분리 (12000 rpm/15분)를 통해 상층액을 제거하고, 바닥에 가라앉은 침전물에 증류수 1ml를 가하여 나노입자를 재분산 시킨다. 합성된 나노입자의 분석결과는 도 6에 나타냈다.To 1 ml of a 20 nm diameter gold nanoparticle solution (Abs = 1.0 at 520 nm) threonine peptide (peptide with cysteine at the five ends of threonine; Formula 1) 80 uL (1.0 mg / ml) Shake for 12 hours at room temperature. The supernatant is removed by centrifugation (12000 rpm / 15 min), and 1 ml of distilled water is added to the precipitate settled to the bottom to redisperse the nanoparticles. The analysis results of the synthesized nanoparticles are shown in FIG. 6.
얼음 결정 성장 실험을 실시한 결과 비등방성의 얼음 결정 성장 형태가 관찰되었다. 이는 나노입자에 의한 얼음 결정 성장의 방해효과로 인한 결과이다 (결과: 도 7).Ice crystal growth experiments showed anisotropic ice crystal growth patterns. This is a result of the interfering effect of ice crystal growth by nanoparticles (Result: FIG. 7).
실시예 2 쓰레오닌이 표면개질된 금 나노입자 (50 nm)의 합성Example 2 Synthesis of Gold Nanoparticles (50 nm) Surface-Modified with Threonine
50 nm 직경의 금 나노입자 용액 (Abs = 1.0 at 530 nm) 1 ml에 쓰레오닌 펩타이드 (쓰레오닌 5개의 말단에 시스테인이 결합되어 있는 펩타이드) 80 ㎕ (1.0 mg/ml)를 가하여 상온에서 12시간 동안 shake 한다. 원심분리 (12000 rpm/15분)를 통해 상층액을 제거하고, 바닥에 가라앉은 침전물에 증류수 1ml를 가하여 나노입자를 재분산 시킨다. 합성된 나노입자의 분석결과는 도 6 에 나타내었다. 얼음 결정 성장 실험을 실시한 결과 비등방성의 얼음 결정 성장 형태가 관찰되었다. 이는 나노입자에 의한 얼음 결정 성장의 방해효과로 인한 결과이다 (결과: 도 7).To 1 ml of a 50 nm diameter gold nanoparticle solution (Abs = 1.0 at 530 nm), 80 μl (1.0 mg / ml) of threonine peptide (peptide with cysteine at the five ends of threonine) was added at room temperature. Shake for 12 hours. The supernatant is removed by centrifugation (12000 rpm / 15 min), and 1 ml of distilled water is added to the precipitate settled to the bottom to redisperse the nanoparticles. The analysis results of the synthesized nanoparticles are shown in FIG. 6. Ice crystal growth experiments showed anisotropic ice crystal growth patterns. This is a result of the interfering effect of ice crystal growth by nanoparticles (Result: FIG. 7).
실시예 3 쓰레오닌이 표면개질된 막대형 금 나노입자 (폭 10 nm , 길이 35 nm)의 합성 Example 3 Threonine Synthesis of Surface-Modified Rod-Gold Nanoparticles ( 10 nm wide , 35 nm long )
막대형 금 나노입자 용액 (Abs = 1.0 at 780 nm) 1 ml에 쓰레오닌 펩타이드 (쓰레오닌 5개의 말단에 시스테인이 결합되어 있는 펩타이드) 80 ㎕ (1.0 mg/ml)를 가하여 상온에서 12시간 동안 shake 한다. 원심분리 (12000 rpm/15분)를 통해 상층액을 제거하고, 바닥에 가라앉은 침전물에 증류수 1ml를 가하여 나노입자를 재분산 시킨다. 합성된 나노입자의 분석결과는 도 6에 나타내었다.To 80 ml (1.0 mg / ml) of threonine peptide (peptide with cysteine bound at five ends of threonine) was added to 1 ml of rod-type gold nanoparticle solution (Abs = 1.0 at 780 nm) at room temperature for 12 hours. Should shake while. The supernatant is removed by centrifugation (12000 rpm / 15 min), and 1 ml of distilled water is added to the precipitate settled to the bottom to redisperse the nanoparticles. The analysis results of the synthesized nanoparticles are shown in FIG. 6.
얼음 결정 성장 실험을 실시한 결과 비등방성의 얼음 결정 성장 형태가 관찰되었다. 이는 나노입자에 의한 얼음 결정 성장의 방해효과로 인한 결과이다 (결과: 도 7).Ice crystal growth experiments showed anisotropic ice crystal growth patterns. This is a result of the interfering effect of ice crystal growth by nanoparticles (Result: FIG. 7).
실시예 4. 온도이력현상 확인Example 4. Checking the temperature history phenomenon
실시예 1 내지 3에서 제작한 기능성 나노입자를 물에 넣은 실험군과 상기 나노입자를 물에 넣지 않은 대조군을 0℃이하의 온도 조건으로 얼렸다.The experimental group in which the functional nanoparticles prepared in Examples 1 to 3 were put in water and the control group in which the nanoparticles were not in water were frozen at a temperature of 0 ° C. or lower.
그 결과, 대조군에서는 어는점과 녹는점이 0℃로 동일하였으나, 기능성 나노입자가 포함된 물에서는 상기 나노입자가 얼음의 결정과 결합하여 결정의 설장을 방해함으로써 어는점과 녹는점이 달라지는 것을 확인하였다. 즉, 실험군에서 온도이력현상이 관찰되었으며, 이로부터 본 발명의 기능성 나노입자가 얼음 결정의 성장을 방해하는 것을 확인할 수 있었다.As a result, in the control group, the freezing point and the melting point were the same at 0 ° C., but in the water containing the functional nanoparticles, the freezing point and the melting point were different because the nanoparticles were combined with ice crystals and interfered with the crystals. That is, the temperature history phenomenon was observed in the experimental group, from which it was confirmed that the functional nanoparticles of the present invention hinder the growth of ice crystals.
실시예 5. 나노입자의 결빙 제어 효과 (세포 실험) Example 5. Freezing Control Effects of Nanoparticles (Cell Experiments)
1. 세포 배양 방법1. Cell Culture Method
mESC cell은 DMEM/high glucose, 200 mM L-glutamine, 1 % Penicillin/Streptomycin, 15 % FBS, HEPES (pH 7.3), MEM Nonessential Amino Acids, 1000 U/ml media를 사용하였으며, 2일마다 계대 배양하였다. mESC cell을 계대 배양하기 위해서 0.1 % Gelatin을 60cm culture dish에 37℃, 5% CO2, 3시간 이상 코팅한 후 사용하였다. TC1 cells은 DMEM-High glucose, 10 % FBS, 1 % Penicillin/Streptomycin에서 배양하였으며, 3일마다 계대 배양하였다.mESC cells were DMEM / high glucose, 200 mM L- glutamine, 1% Penicillin / Streptomycin, 15% FBS, HEPES (pH 7.3), MEM Nonessential Amino Acids, 1000 U / ml media and passaged every 2 days. . To passaging mESC cells, 0.1% Gelatin was coated in a 60cm culture dish at 37 ° C., 5% CO 2 , for at least 3 hours. TC1 cells were cultured in DMEM-High glucose, 10% FBS, 1% Penicillin / Streptomycin and passaged every 3 days.
2. Cell Freezing and Thawing 실험2. Cell Freezing and Thawing Experiment
mESC은 1ml PBS로 washing 한 후, 0.4 ml의 0.25% Trypsin을 첨가하였다. 1분 후 배양액 3.6 ml을 첨가하여 중화하였다. 1,200 rpm, 5 분 동안 원심분리 후, media를 조심스레 제거하였다. 0.5 ml Freezing media (70 % DMEM/high glucose, 20% FBS, 10% DMSO)로 cell pellet에 섞은 후, cryovial로 옮겼다. Cryovial은 Freezing container에 넣은 후, -80℃ Deep freezer에서 2일간 저장하였다. 2일 후, Deep freezer에서 cell stock vial을 꺼낸 후, 37℃에서 1분간 급속 해동시킨 후, 1 ml의 media을 첨가하였다. 1,200 rpm, 5 분 동안 원심분리 후, 배양액을 제거하였다. 4 ml의 media로 cell을 섞은 후, 60 cm culture dish에서 첨가한 후 배양하였다.mESC was washed with 1 ml PBS and 0.4 ml of 0.25% Trypsin was added. After 1 minute, 3.6 ml of culture was added to neutralize. After centrifugation at 1,200 rpm for 5 minutes, the media was carefully removed. 0.5 ml freezing media (70% DMEM / high glucose, 20% FBS, 10% DMSO) were mixed in the cell pellet and transferred to cryovial. Cryovial was placed in a freezing container and stored in a -80 ° C deep freezer for 2 days. After 2 days, the cell stock vial was taken out of the deep freezer, rapidly thawed at 37 ° C. for 1 minute, and then 1 ml of media was added. After centrifugation at 1,200 rpm for 5 minutes, the culture was removed. The cells were mixed with 4 ml of media, added to a 60 cm culture dish and incubated.
TC1 cell은 1ml PBS로 washing 한 후, 2 ml의 0.25% Trypsin을 첨가하였다. 1분 후 배양액 2 ml을 첨가하여 중화하였다. 1,200 rpm, 5 분 동안 원심분리 후, media를 조심스레 제거하였다. 0.5 ml Freezing media (90% FBS, 10% DMSO)로 cell pellet (2x105)에 섞은 후, cryovial로 옮겼다. Cryovial은 Freezing container에 넣은 후, -80℃ Deep freezer에서 2일간 저장하였다. 2일 후, Deep freezer에서 cell stock vial을 꺼낸 후, 37℃에서 1분간 급속 해동시킨 후, 4.5 ml의 media을 첨가하였다. 1,200 rpm, 5 분 동안 원심분리 후, 배양액을 제거하였다. 2 ml의 media로 cell을 섞은 후, 6 well culture plate에서 첨가한 후 배양하였다. 상기 실험에서 10% DMSO를 대신하여 10% 20 nm (또는 10% 40 nm) Poly threonine5-AuNP로 Cell Freezing을 하였다 (도 5). 도 5에 나타난 바와 같이, Freezing 시 세포 내부로 Poly threonine5-AuNP가 침투된 것을 확인할 수 있다.TC1 cells were washed with 1ml PBS and 2ml of 0.25% Trypsin was added. After 1 minute, 2 ml of culture was added to neutralize. After centrifugation at 1,200 rpm for 5 minutes, the media was carefully removed. 0.5 ml freezing media (90% FBS, 10% DMSO) was mixed in the cell pellet (2x10 5 ), and then transferred to cryovial. Cryovial was placed in a freezing container and stored in a -80 ° C deep freezer for 2 days. After 2 days, the cell stock vial was taken out of the deep freezer, rapidly thawed at 37 ° C. for 1 minute, and 4.5 ml of media was added thereto. After centrifugation at 1,200 rpm for 5 minutes, the culture was removed. After mixing the cells in 2 ml of media, and added in 6 well culture plate and incubated. Cell freezing was performed with 10% 20 nm (or 10% 40 nm) Poly threonine5-AuNP in place of 10% DMSO (FIG. 5). As shown in Figure 5, it can be confirmed that the poly threonine5-AuNP penetrated into the cell during freezing.
3. 빛을 이용한 해동과정3. Defrost process using light
37℃에서 1분간 급속 해동시키는 방법이 아닌, 0℃에서 520 nm laser(10 mw/cm2)을 가하여 서서히 해동시킨 후, 4.5 ml의 media을 첨가하였다. 1,200 rpm, 5 분 동안 원심분리 후, 배양액을 제거하였다. 2 ml의 media로 cell을 섞은 후, 6 well culture plate에서 첨가한 후 배양하였다 (결과: 도 8).Rather than rapidly thaw at 37 ° C. for 1 minute, 520 nm laser (10 mw / cm 2) was added slowly at 0 ° C. and 4.5 ml of media was added. After centrifugation at 1,200 rpm for 5 minutes, the culture was removed. The cells were mixed with 2 ml of media, added to 6 well culture plates, and cultured (result: FIG. 8).
4. Cell Staining 실험4. Cell Staining Experiment
mESC cell은 2일간 배양한 후, media를 제거하였다. PBS를 이용하여 1번 또는 3번 cell을 washing 하였다. Trypan blue와 PBS를 이용하여 0.2 % 또는 0.1%로 만든 후, 60 cm culture dish에 첨가한 후, 현미경을 이용하여 cell을 확인하였다.mESC cells were incubated for 2 days, and then the media was removed. The cell was washed 1 or 3 times using PBS. After making 0.2% or 0.1% using Trypan blue and PBS, and added to 60 cm culture dish, the cells were confirmed by a microscope.
TC1 cell은 2일간 배양한 후, media를 제거하였다. PBS를 이용하여 cell을 washing 하였다. 4 % Paraformaldehyde/sucrose로 10분간 cell을 고정시킨 후, 0.5 % crystal violet용액으로 30분간 염색하였다. 30분 후, 6 well culture plate를 흐르는 물을 이용하여 염색용액을 제거하였다. Cell 상태는 현미경을 이용하여 확인하였다. 살아있는 세포들의 군수를 비교한 결과 DMSO를 사용하여 세포를 냉동 해동한 경우와 비교하여 20nm 또는 40 nm 금 나노입자를 가한 경우에 더욱 많은 수의 세포가 살아 있음을 확인하였다 (결과: 도 8).TC1 cells were incubated for 2 days, and then the media was removed. The cells were washed using PBS. The cells were fixed for 10 minutes with 4% Paraformaldehyde / sucrose, and then stained with 0.5% crystal violet solution for 30 minutes. After 30 minutes, the dye solution was removed using water flowing through a 6 well culture plate. Cell state was confirmed using a microscope. As a result of comparing the number of living cells, it was confirmed that more cells survived when 20 nm or 40 nm gold nanoparticles were added as compared to the case where the cells were frozen and thawed using DMSO (Result: FIG. 8).

Claims (9)

  1. 나노입자; 및 상기 나노입자 표면에 결합되는 아미노산 또는 펩타이드;를 포함하는, 기능성 나노입자.Nanoparticles; And amino acids or peptides bonded to the surface of the nanoparticles.
  2. 청구항 1에 있어서, 상기 나노입자는 콜로이드성 나노입자인, 기능성 나노입자.The functional nanoparticle of claim 1, wherein the nanoparticles are colloidal nanoparticles.
  3. 청구항 1에 있어서, 상기 나노입자는 금인, 기능성 나노입자.The functional nanoparticle of claim 1, wherein the nanoparticle is gold.
  4. 청구항 1에 있어서, 상기 아미노산은 쓰레오닌, 발린 및 세린으로 이루어진 군으로부터 선택되는 적어도 하나인, 기능성 나노입자.The functional nanoparticle of claim 1, wherein the amino acid is at least one selected from the group consisting of threonine, valine, and serine.
  5. 청구항 1에 있어서, 상기 아미노산은 쓰레오닌인, 기능성 나노입자.The functional nanoparticle of claim 1, wherein the amino acid is threonine.
  6. 청구항 1에 있어서, 상기 펩타이드는 화학식 1로 표시되는 화합물을 포함하는, 기능성 나노입자:The functional nanoparticle of claim 1, wherein the peptide comprises a compound represented by Formula 1:
    [화학식 1][Formula 1]
    Figure PCTKR2017003238-appb-I000003
    Figure PCTKR2017003238-appb-I000003
  7. 청구항 1에 있어서, 상기 기능성 나노입자는 세포투과성 펩타이드를 더 포함하는, 기능성 나노입자.The functional nanoparticle of claim 1, wherein the functional nanoparticle further comprises a cell permeable peptide.
  8. 청구항 1 내지 7 중 어느 한 항의 기능성 나노입자를 포함하는 기능성 식품.A functional food comprising the functional nanoparticle of any one of claims 1 to 7.
  9. 청구항 1 내지 7 중 어느 한 항의 기능성 나노입자를 포함하는 결빙 조절용 조성물.A composition for controlling freezing comprising the functional nanoparticles of any one of claims 1 to 7.
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