KR102332124B1 - Surface-modified nanoparticles with freezing control peptides - Google Patents

Abstract

The present invention relates to a peptide surface-modified nanoparticles for controlling freezing, nanoparticles; and amino acids or peptides bound to the surface of the nanoparticles; by including, they do not have toxicity and thus can be applied to biological samples, can be easily synthesized, can be genetically engineered to increase the composition of specific amino acids, and precipitate when used at high concentrations Freezing and thawing can be controlled by interacting with ice crystals, and damage to cells can be minimized by slowing the production rate of ice during freezing, apoptosis can be minimized during freezing and thawing, and It relates to a peptide surface-modified nanoparticles for controlling freezing, which can minimize damage during thawing.

Description

Surface-modified nanoparticles with freezing control peptides {Surface-modified nanoparticles with freezing control peptides}

The present invention relates to functional nanoparticles capable of controlling the formation and decomposition of ice crystals.

Since ice crystals damage biological samples when they exceed a certain size, various attempts are being made to prevent damage caused by ice crystal formation in the biological sample storage industry that requires a low-temperature environment.

Many cryopreservatives (antifreeze, DMSO, etc.) using chemicals are used, but they are toxic in vivo, so it is difficult to apply to biological samples.

Various antifreeze proteins (AFPs) are being developed to replace cryopreservatives. However, antifreeze proteins are expressed in various organisms such as microorganisms, fish, and insects in vivo. There is a problem that can lead to In addition, when a high concentration of protein is used in a biological sample, there is a problem that the protein may be precipitated.

Therefore, there is an urgent need to develop a material capable of controlling ice freezing that is more effective and does not cause the aforementioned problems.

Korean Patent Publication No. 2014-49634

An object of the present invention is to provide functional nanoparticles and compositions that do not have a problem of toxicity to biological samples of cryopreservatives using chemicals.

An object of the present invention is to provide functional nanoparticles and compositions that are easy to synthesize, can increase the composition of specific amino acids by genetic engineering, and do not cause precipitation when used at high concentrations.

The present invention aims to provide functional nanoparticles and compositions capable of interacting with ice crystals.

An object of the present invention is to provide a functional nanoparticle composition capable of minimizing damage to cells by slowing the generation rate of ice during freezing.

An object of the present invention is to provide functional nanoparticles and compositions capable of absorbing visible light or near-infrared rays, thereby controlling the dissolution rate of ice crystals from inside the cell, thereby minimizing cell death in the freeze-thaw process.

An object of the present invention is to provide functional nanoparticles and compositions capable of minimizing damage to a biological sample when thawing a low-temperature storage sample.

1. nanoparticles; And amino acids or peptides bound to the surface of the nanoparticles; Containing, functional nanoparticles.

2. The functional nanoparticles according to the above 1, wherein the nanoparticles are colloidal nanoparticles.

3. The functional nanoparticles according to the above 1, wherein the nanoparticles are gold.

4. The functional nanoparticles according to the above 1, wherein the amino acid is at least one selected from the group consisting of threonine, valine and serine.

5. The functional nanoparticles according to the above 1, wherein the amino acid is threonine.

6. The functional nanoparticles according to the above 1, wherein the peptide comprises a compound represented by Formula 1:

[Formula 1]

7. The method of 1 above, wherein the functional nanoparticles further comprise a cell-permeable peptide, functional nanoparticles.

8. Functional food comprising the functional nanoparticles of any one of 1 to 7 above.

9. A composition for controlling freezing comprising the functional nanoparticles of any one of 1 to 7 above.

According to an embodiment of the present invention, it is possible to provide functional nanoparticles and compositions that do not cause toxicity to biological samples of cryopreservatives using chemicals.

In addition, according to an embodiment of the present invention, it is possible to provide functional nanoparticles and compositions that are easy to synthesize, can increase the composition of specific amino acids through genetic engineering, and do not cause precipitation when used at high concentrations.

In addition, according to an embodiment of the present invention, it is possible to provide functional nanoparticles and compositions capable of interacting with ice crystals.

In addition, according to an embodiment of the present invention, it is possible to provide functional nanoparticles and compositions capable of minimizing damage to cells by slowing the production rate of ice during freezing.

In addition, according to an embodiment of the present invention, when the functional nanoparticles and composition of the present invention are added to the cells, it is possible to dissolve the ice crystals from the inside of the cells by irradiating visible or near infrared rays, thereby minimizing apoptosis in the process of freezing and thawing. It is possible to provide functional nanoparticles and compositions that can

In addition, according to an embodiment of the present invention, it is possible to provide functional nanoparticles and compositions capable of minimizing damage to a biological sample during thawing of a low-temperature storage sample.

1 is a schematic diagram showing an embodiment of the functional nanoparticles of the present invention.
2 shows that the growth of ice crystals is suppressed and the freezing temperature of ice is lowered when the functional nanoparticles of the present invention are present.
3 shows the change in thermal hysteresis (TH) according to the number and arrangement of amino acids interacting with ice crystals in the anti-freezing protein.
4 shows that recrystallization of ice is prevented during thawing.
Figure 5 shows that when the cell-permeable amino acid sequence is present on the surface of the nanoparticles, it can affect the freezing and thawing of ice inside and outside the cell. am.
6 shows the results of synthesizing gold nanoparticles whose surface is modified with threonine according to an embodiment of the present invention.
7 shows the results of an ice crystal growth experiment using functional nanoparticles synthesized according to an embodiment of the present invention, and as a result, it was shown that the functional nanoparticles of the present invention interfered with the growth of ice crystals.
8 shows a cell experiment of the effect of controlling the freezing of nanoparticles. Specifically, when DMSO or threonine-conjugated 20 nm gold nanoparticles or 40 nm gold nanoparticles were added to mESC cells, the rapid thawing process was performed. It shows the apoptosis effect and the apoptosis effect under the conditions of thawing by irradiation with light.

The present invention relates to a peptide surface-modified nanoparticles for controlling freezing, nanoparticles; and amino acids or peptides bound to the surface of the nanoparticles; by including, they do not have toxicity and thus can be applied to biological samples, can be easily synthesized, can be genetically engineered to increase the composition of specific amino acids, and precipitate when used at high concentrations Freezing and thawing can be controlled by interacting with ice crystals, and damage to cells can be minimized by slowing the production rate of ice during freezing, apoptosis can be minimized during freezing and thawing, and It relates to a peptide surface-modified nanoparticles for controlling freezing, which can minimize damage during thawing.

Hereinafter, the present invention will be described in detail.

The present invention provides functional nanoparticles comprising nanoparticles and amino acids or peptides bound to the surface of the nanoparticles.

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, and still more preferably 0.5 μm or less, and may be plasmonic nanoparticles, but , but not limited thereto.

Nanoparticles may be prepared by reducing a dilute solution of a metal complex salt, and specifically, processes such as a reduction reaction, a nucleation initiation reaction, and a nuclear growth reaction may be prepared, but the present invention is not limited thereto.

According to an embodiment of the present invention, the nanoparticles may be colloidal nanoparticles, but is not limited thereto.

According to an embodiment of the present invention, the nanoparticles may be circular or rod-shaped, as shown in the schematic diagram of FIG. 1 , but is not limited thereto.

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.

When the nanoparticles are gold, the functional nanoparticles of the present invention can absorb light in the visible or near-infrared region, so that when the cells are thawed, they are irradiated with light to control the melting process of ice from the inside of the cells. In this way, damage to cells can be minimized 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. It may be cysteine, valine, tryptophan, tyrosine, or the like, but is not limited thereto.

According to an embodiment of the present invention, the amino acid may be at least one of threonine, valine, and serine, but is not limited thereto.

Threonine, valine, and serine are major amino acid components of the anti-freeze peptide, and the interaction with ice occurs more effectively, and the interaction with ice is superior to other amino acids, so the growth of ice crystals is more precisely and effectively can be controlled (especially prevented).

In addition, the binding force with the nanoparticles is also excellent, so that it is easy to manufacture them as functional nanoparticles.

In addition, according to an embodiment of the present invention, when the amino acid is modified on the surface of the nanoparticles and used as the functional nanoparticles of the present invention, the surface area that can interact with ice increases exponentially, resulting in growth, formation, and thawing of ice crystals. The control ability of the back can be significantly increased compared to the case of using the amino acid alone.

According to an embodiment of the present invention, the amino acid may be threonine, but is not limited thereto.

The functional complex of the present invention to which threonine is bound can minimize damage to cells by slowing the rate of ice formation in cells during freezing.

According to an embodiment of the present invention, the peptide may include at least one amino acid selected from the group consisting of threonine, valine and serine, and the number of threonine, valine and serine included in the peptide is limited. there is no For example, it may include at least one of threonine, valine, and serine in 1 to thousands, but is not limited thereto.

According to an embodiment of the present invention, the peptide may include at least one of threonine, valine, and serine consecutively or spaced apart, but is not limited thereto.

According to an 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.

According to an embodiment of the present invention, the peptide may be a peptide including threonine and cysteine. Cysteine included in the peptide includes a case where it is linked with a spacer material such as polyethylene glycol. For example, the linkage may be a case in which polyethylene glycol is positioned between the peptide and the cysteine, but is not limited thereto.

According to a specific embodiment of the present invention, five threonines may be continuously bonded and a cysteine may be bonded to the ends of the five threonines, but is not limited thereto.

According to an embodiment of the present invention, the peptide may be a peptide including the compound represented by Formula 1, but is not limited thereto.

In the case of a peptide containing the compound represented by Formula 1, anisotropic growth of ice crystals may be induced, thereby hindering the growth of ice crystals ( FIGS. 6 and 7 ). In addition, damage upon thawing of frozen cells can be minimized ( FIG. 8 ).

[Formula 1]

In addition, in the present invention, a peptide refers to a molecule found in any animal, plant, insect, microorganism, etc. or an artificially or naturally synthesized molecule, and is a peptide that non-unilaterally reduces the freezing point of water.

In the present invention, the peptide is a protein having the characteristics of preventing the growth of ice crystals by binding to ice, and includes anti-freezing peptides (AFPs), anti-freezing glycoproteins, anti-freezing glycopeptides, AFGP), a thermal history polypeptide, and the like.

According to one embodiment of the present invention, the peptide is an anti-freeze peptide produced in cold-blooded aquatic polar fish species living in the waters of the polar regions of the Earth, including fats in the Arctic and Antarctic rings; anti-freeze peptides produced by various higher plants including polar diatoms, carrots and dandelions; It may include, but is not limited to; anti-freezing peptides produced in various species such as insects, bacteria, and fish.

According to an embodiment of the present invention, the peptide may be an anti-freeze protein such as FfIBP, NglIBP, Type III AFP, but is not limited thereto.

According to an embodiment of the present invention, the peptide may have a primary or secondary structure. For example, it may have a structure of an alpha helix, a beta sheet, a repeating or a combination thereof, but is not limited thereto.

According to one embodiment of the present invention, the peptide is the amino acid sequence of the anti-freezing peptides (AFPs), anti-freezing glycoprotein, anti-freezing glycopeptides (AFGP) or heat history polypeptide It may be modified by modifying some or all of it, or by additionally attaching other amino acids to the beginning, middle or terminus of the amino acid sequence, and the modification may be made within the scope of the purpose of interaction with ice.

According to an embodiment of the present 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, but are not limited thereto.

According to one embodiment of the present invention, the peptide may be a peptide in which one or more amino acid residues in the peptide are modified. The one or more modification(s) may include in vivo or in vitro chemical derivatization, eg, acetylation or carboxylation; glycosylation; deglycosylation; phosphorylation such as phosphotyrosine, phosphoserine and phosphothreonine; and the like, but is not limited thereto.

According to an embodiment of the present invention, the peptide may be a naturally occurring L-amino acid, a naturally occurring L-amino acid, a non-naturally occurring synthetic amino acid, or the like, but is not limited thereto.

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 C-terminus, but is not limited thereto. A blocking group is a chemical substituent suitable for protecting and stabilizing the N-terminus and/or C-terminus, including branched or unbranched alkyl and acyl groups introduced by alkylation or acylation of the N-terminus; C-terminal blocking groups, including ester or ketone-forming alkyl groups, include, but are not limited to, lower (C1 to C6) alkyl groups such as methyl, ethyl and propyl, and amide-forming amino groups; does not

Amide-forming amino groups include primary amines (-NH2), and mono- and di-alkylamino groups, and mono- and di-alkylamino groups include methylamino, ethylamino, dimethylamino, diethylamino, methylethylamino and the like, but are not limited thereto.

Suitable concentrations of peptides bound to nanoparticles may vary depending on the application, for example, may range from about 1 ppb to about 1 ppt (ie, from about 1 μg/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 when the peptides are used alone, the ability to control crystal growth, formation, thawing, etc. It can be significantly better than that.

According to an embodiment of the present invention, the binding between the nanoparticles and the amino acid or peptide may include a chemical bond between the nanoparticle surface and the amino acid or peptide. The nanoparticles may have a modified surface. For example, the binding of nanoparticles to amino acids or peptides may be to directly bind amino acids or peptides to nanoparticles by modifying them with carboxylic acids and then reducing them with amino acids or peptides.

According to an embodiment of the present invention, the binding between the nanoparticles and amino acids or peptides may be binding by a linker. Binding by the linker may bind the nanoparticles and amino acids or peptides by binding the linker to the nanoparticles and then binding the linker and amino acids or peptides, but is not limited thereto. For example, the linker may be a cysteine or an o-linker, but is not limited thereto.

The functional nanoparticles of the present invention can interact with ice by binding amino acids or peptides to the surface of the nanoparticles, thereby inhibiting, delaying, and preventing the formation of ice crystals.

According to an embodiment of the present invention, it was found that the temperature hysteresis increases according to the number and arrangement of amino acids or peptides included in the functional nanoparticles of the present invention (FIG. 3).

Therefore, in the process of nanoparticle formation, by controlling the number of amino acids or peptides to control the area, size, shape, etc. of functional nanoparticles, the ice crystal formation time, prevention, and thawing time are controlled by controlling the binding force with ice, the binding area, etc. The back can be adjusted more precisely.

In addition, since the functional nanoparticles of the present invention do not use chemicals, the toxicity of cryopreservatives (antifreeze, DMSO, etc.) to biological samples does not occur. Therefore, it can be used without limitation in various biological samples such as cells of insects, mammals, reptiles, fish, and the like.

In addition, although there is a risk of cell damage due to ice recrystallization when thawing a biological sample stored in a low-temperature environment, ice recrystallization occurs when the functional nanoparticles of the present invention are added to the biological sample and stored in a low-temperature environment and then thawed. It is possible to minimize damage to the biological sample by preventing oxidization.

According to an embodiment of the present invention, it was found that when the functional nanoparticles of the present invention were used, recrystallization of ice was prevented ( FIG. 4 ).

In addition, the functional nanoparticles of the present invention can enter the cell (FIG. 5), so that when the cells are thawed, light is irradiated to control the thawing of ice from the inside of the biological sample, and through this, when thawing after freezing It is possible to minimize damage to the biological sample that may occur ( FIG. 8 ).

The biological sample may be a mammalian cell, an insect cell, a fish cell, a plant cell, a eukaryotic cell, or a prokaryotic cell, but is not limited thereto.

According to an embodiment of the present invention, the functional nanoparticles of the present invention can substantially reduce cell damage caused by ice crystal growth, and thus sensitive living organisms such as blood and blood products, therapeutic agents, polypeptide drugs, bioassay reagents and vaccines. It may be suitable for storage of a sample.

According to an embodiment of the present invention, the functional nanoparticles may further include a cell-permeable peptide.

By further including the cell-permeable peptide in the functional nanoparticles of the present invention, the freezing of cells can be controlled by delivering the functional nanoparticles into the cells and affecting the water-ice behavior such as ice crystallization inside and outside the cells.

Cell-penetrating peptide is a kind of signal peptide, for example, 11 amino acid b-galactosidase (120 kDa) protein present in TAT protein, Antennapedia (Penetratin) derived from Drosophila protein, HSV-1 virus VP22, and Pep-1 derived from Simian Virus 40 large antigen T, but is not limited thereto.

In addition, peptides in which several cationic amino acids such as Arginine and Lysine are repeatedly linked may be included, such as poly Arginine and poly Lysine, but the present invention is not limited thereto.

For example, the cell-penetrating peptide may include, but is not limited to, Hph-1, Vectocell, Lactoferrin, Sim-2, LPIN3, 2IL-1a, dNP2, and the like.

The functional nanoparticles of the present invention can reduce or inhibit ice crystal growth and/or formation by being introduced into, or in contact with, edible products, thereby providing texture, taste, and effectiveness of frozen edible products including vegetables. It can improve the shelf life. And the functional nanoparticles of the present invention can inhibit and/or reduce the formation of ice crystals associated with freezing or supercooling of objects or substances including food products.

Accordingly, the present invention provides a functional food comprising functional nanoparticles in another aspect.

The functional food of the present invention can reduce, or even completely prevent, or at least minimize this ice crystal growth process by including the functional nanoparticles.

Foods include: low-fat spreads, mayonnaise, yogurt, bakery, margarine, reconstituted fruit, jam, fruit products, fruit filling, ripple, fruit sauce, fruit stew, coffee bleach, instant fruit dessert, confectionery (e.g. marshmallow) , potato based foods (e.g. chips, french fries and croquettes), processed foods (e.g. casseroles and stews) and fine foods (e.g. dressings including salad dressings; ketchup, vinaigrette dressings and soups) and the like, but is not limited thereto.

Foods also include raw, processed or pasteurized foods [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 milks, creams, Fermented dairy products, cheeses, butters, condensed milk products, cheese spreads, pasteurized liquid eggs, ice cream mixes, soy products, pasteurized liquid eggs, confectionery products, fruit products, and fat-based or water-containing stuffing It may be food, etc., but is not limited thereto.

In addition, the food may be a confectionery product such as, but not limited to, bread, cake, fine bakery, and dough.

The functional food of the present invention may be a form in which the functional nanoparticles are incorporated in the whole food and/or a form applied to the surface of the food product, but is not limited thereto.

The functional food of the present invention can be prepared in various forms such as tablets, capsules, powders, granules, liquids, pills, powders, flakes, pastes, syrups, gels, jellies, bars, etc., It can also be manufactured in a form.

The functional food of the present invention may further include components commonly added during food production within the scope of the object of the present invention, for example, it may further include protein, carbohydrate, fat, nutrients, seasonings and flavoring agents, etc. .

Examples of the carbohydrate include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose, oligosaccharides and the like; and polysaccharides, such as conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol, but is not limited thereto.

The flavoring agent may further include, but is not limited to, natural flavoring agents such as taumatin and stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents such as saccharin and aspartame. .

In addition to the above, the functional food of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol , a carbonation agent used in carbonated beverages, and the like.

In addition, the functional food of the present invention may contain flesh for the production of natural fruit juice, fruit juice beverage, and vegetable beverage. These components may be used independently or in combination. The ratio of these additives is not very important, 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.

It can be used suitably according to other conventional methods. The mixing amount of the active ingredient may be appropriately determined according to the purpose of its use (prevention, health treatment). In general, when preparing food or beverage, it may 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, based on the total weight of the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be adjusted below the above range.

The functional food of the present invention may further include a stabilizer. Stabilizers include polypeptides such as gelatin; plant extracts such as gum arabic, gum ghatti, gum karaya, gum tragacanth; 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 furceleran; pectins such as 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.

In addition, the functional food of the present invention is a sugar, for example, sucrose, fructose, dextrose, lactose, corn syrup, sugar alcohol; Or it may contain other raw materials, for example, a color and a fragrance.

In the functional nanoparticles of the present invention, a greater number of cells were found to be alive after thawing compared to the case where cells were frozen and thawed using DMSO (FIG. 8). thus, In another aspect, the present invention provides a composition for controlling freezing comprising functional nanoparticles effective for freezing storage of biological samples (eg, biological cells or extracts thereof, etc.).

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 cells, and cell extracts.

According to an embodiment of the present invention, the biological sample may be a mammalian cell, and includes a wide range of cells such as oocytes, embryos, leukocytes, red blood cells, platelets, pancreatic islets and hepatocytes; skin tissue, bone marrow tissue, corneal tissue and other broad types of tissue; And liver, kidney, heart, brain, lung, pancreas, spleen, ovary and a wide range of types of organs such as stomach; may include, but is not limited to.

According to an embodiment of the present 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 present invention, and the addition of the composition is It may be useful in situations where cell integrity upon subsequent thawing is important or desirable (eg, tissue culture deposits). That is, it is possible to minimize the loss of intrinsic properties or intrinsic morphology due to the freeze-thaw process.

In addition, according to an 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 a biological sample (eg, cells). There is (Fig. 8).

The composition of the present invention may be in various forms such as solid, semi-solid, fluid, gas, and the like. For example, it may be a fluid, but is not limited thereto.

The compositions of the present invention may further contain any of a wide range of mixtures of salts, sugars, ions and other nutrients included in electrolyte solutions known to be useful for preserving biologicals. These include tissue culture media, organ perfusate, and the like.

In addition, the present invention provides a cosmetic composition or a dermatological preparation comprising the functional nanoparticles of the present invention.

By including the functional nanoparticles of the present invention in cosmetic compositions or topical dermatological preparations, the optimal temperature of cellular enzymes is lost due to a dramatic climate- and weather-induced temperature drop, thereby causing changes in cell physiology in the cellular and extracellular space. It can prevent or improve skin structure and cell damage caused by the damaged cold.

According to one embodiment of the present invention, the skin structure and cell damage is cold, wind and/or UV light induced skin damage, skin erythema and skin pulling feeling and increased sensory sensitivity, temperature-sensitive skin, environment including, but not limited to, negative changes in the 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).

In addition, the composition, cosmetic composition, and topical dermatological formulation of the present invention may further include at least one of stabilizers, emulsifiers, surfactants and other additives known to those skilled in the art.

Other additives include, but are not limited to, anti-corrosive agents, antioxidants, anti-discoloration agents, antimicrobial agents, emulsion stabilizers, and the like.

Hereinafter, the present invention will be described in more detail by way of Examples. The following examples are for illustrative purposes only, and do not limit the scope of the present invention.

Example

Example 1 Synthesis of threonine surface-modified gold nanoparticles (20 nm)

In 1 ml of a 20 nm diameter gold nanoparticle solution (Abs = 1.0 at 520 nm), 80 uL (1.0 mg/ml) of threonine peptide (peptide with cysteine bonded to 5 threonine ends; Formula 1) was added. and shake at room temperature for 12 hours. The supernatant is removed through centrifugation (12000 rpm/15 min), and 1 ml of distilled water is added to the sediment that has settled to the bottom to redisperse the nanoparticles. The analysis results of the synthesized nanoparticles are shown in FIG. 6 .

As a result of the ice crystal growth experiment, anisotropic ice crystal growth patterns were observed. This is a result of the interference effect of the ice crystal growth by the nanoparticles (result: Fig. 7).

Example 2 Synthesis of threonine surface-modified gold nanoparticles (50 nm)

To 1 ml of 50 nm diameter gold nanoparticle solution (Abs = 1.0 at 530 nm), 80 μl (1.0 mg/ml) of threonine peptide (peptide with cysteine attached to 5 ends of threonine) was added at room temperature. Shake for 12 hours. The supernatant is removed through centrifugation (12000 rpm/15 min), and 1 ml of distilled water is added to the sediment that has settled to the bottom to redisperse the nanoparticles. The analysis results of the synthesized nanoparticles are shown in FIG. 6 . As a result of the ice crystal growth experiment, anisotropic ice crystal growth patterns were observed. This is a result of the interference effect of the ice crystal growth by the nanoparticles (result: Fig. 7).

Example 3 Synthesis of threonine-modified rod-shaped gold nanoparticles (width 10 nm, length 35 nm)

To 1 ml of a rod-shaped gold nanoparticle solution (Abs = 1.0 at 780 nm), 80 μl (1.0 mg/ml) of a threonine peptide (a peptide with cysteine bonded to 5 threonine ends) was added at room temperature for 12 hours. Shake for a while. The supernatant is removed through centrifugation (12000 rpm/15 min), and 1 ml of distilled water is added to the sediment that has settled to the bottom to redisperse the nanoparticles. The analysis results of the synthesized nanoparticles are shown in FIG. 6 .

As a result of the ice crystal growth experiment, anisotropic ice crystal growth patterns were observed. This is a result of the interference effect of the ice crystal growth by the nanoparticles (result: Fig. 7).

Example 4. Confirmation of temperature hysteresis

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 put in water were frozen at a temperature of 0° C. or less.

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, it was confirmed that the freezing point and the melting point were different because the nanoparticles combined with the ice crystals and prevented the growth of the crystals. That is, temperature hysteresis was observed in the experimental group, and it was confirmed that the functional nanoparticles of the present invention interfered with the growth of ice crystals.

Example 5. Freezing control effect of nanoparticles (cell experiment)

1. Cell Culture Method

For mESC cells, DMEM/high glucose, 200 mM _L- glutamine, 1% Penicillin/Streptomycin, 15% FBS, HEPES (pH 7.3), MEM Nonessential Amino Acids, 1000 U/ml media were used, and subcultured every 2 days. . In order to subculture mESC cells, 0.1% Gelatin was applied to a 60 cm culture dish at 37° C., 5% CO ₂ , and used after coating for 3 hours or more. TC1 cells were cultured in DMEM-High glucose, 10% FBS, 1% Penicillin/Streptomycin, and subcultured every 3 days.

2. Cell Freezing and Thawing Experiment

After washing the mESCs with 1 ml PBS, 0.4 ml of 0.25% Trypsin was added. After 1 minute, 3.6 ml of the culture medium was added to neutralize. After centrifugation at 1,200 rpm for 5 min, the media was carefully removed. 0.5 ml of freezing media (70% DMEM/high glucose, 20% FBS, 10% DMSO) was mixed with the cell pellet, and then transferred to a cryovial. Cryovials were placed in a freezing container and stored in -80°C deep freezer for 2 days. After 2 days, the cell stock vial was taken out of the deep freezer, and then 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 medium was removed. After mixing the cells with 4 ml of media, they were added in a 60 cm culture dish and then cultured.

TC1 cells were washed with 1 ml PBS, and then 2 ml of 0.25% Trypsin was added. After 1 minute, 2 ml of the culture medium was added to neutralize. After centrifugation at 1,200 rpm for 5 min, the media was carefully removed. 0.5 ml Freezing media (90% FBS, 10% DMSO) was ^{mixed with the cell pellet (2x10 5} ), and then transferred to a cryovial. Cryovials were placed in a freezing container and stored in -80°C deep freezer for 2 days. After 2 days, the cell stock vial was removed from the deep freezer, and then rapidly thawed at 37° C. for 1 minute, followed by the addition of 4.5 ml of media. After centrifugation at 1,200 rpm for 5 minutes, the culture medium was removed. After mixing the cells with 2 ml of media, they were added to a 6 well culture plate and then cultured. Cell freezing was performed with 10% 20 nm (or 10% 40 nm) Polythreonine5-AuNP instead of 10% DMSO in the above experiment ( FIG. 5 ). As shown in FIG. 5 , it can be confirmed that Poly threonine5-AuNP penetrated into the cell during freezing.

3. Defrosting process using light

Instead of rapidly thawing at 37°C for 1 minute, 520 nm laser (10 mw/cm2) was applied at 0°C to slowly thaw, and then 4.5 ml of media was added. After centrifugation at 1,200 rpm for 5 minutes, the culture medium was removed. After mixing the cells with 2 ml of media, they were added in a 6-well culture plate and then cultured (result: FIG. 8).

4. Cell Staining Experiment

After the mESC cells were cultured for 2 days, the media was removed. Cells were washed 1 or 3 times using PBS. After making 0.2% or 0.1% using Trypan blue and PBS, it was added to a 60 cm culture dish, and the cells were checked using a microscope.

TC1 cells were cultured for 2 days, and then the media was removed. Cells were washed with PBS. Cells were fixed with 4% Paraformaldehyde/sucrose for 10 minutes, and then stained with 0.5% crystal violet solution for 30 minutes. After 30 minutes, the staining solution was removed from the 6 well culture plate using running water. Cell status was confirmed using a microscope. As a result of comparing the number of live cells, it was confirmed that a greater number of cells were alive when 20 nm or 40 nm gold nanoparticles were added compared to the case where cells were frozen and thawed using DMSO (result: FIG. 8).

Claims (9)

nanoparticles; And an amino acid or peptide bound to the surface of the nanoparticles; Freezing control composition comprising functional nanoparticles comprising a.
The method according to claim 1, wherein the nanoparticles are colloidal nanoparticles, the composition for controlling freezing.
The composition for controlling freezing according to claim 1, wherein the nanoparticles are gold.
The method according to claim 1, wherein the amino acid is at least one selected from the group consisting of threonine, valine and serine, the composition for controlling freezing.
The composition for controlling freezing according to claim 1, wherein the amino acid is threonine.
The method according to claim 1, wherein the peptide comprises a compound represented by Formula 1, the composition for controlling freezing:
[Formula 1]

The method according to claim 1, wherein the functional nanoparticles further comprise a cell-permeable peptide, freezing control composition.
A functional food comprising the functional nanoparticles of any one of claims 1 to 7.
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