Composition for ice inhibition

Abstract

To provide a composition for antifreezing capable of exhibiting excellent ice recrystallization inhibiting effect, increasing a survival rate of cells during cryopreservation of the cells, and maintaining a texture of food even when used in the freezing of food.SOLUTION: The present invention relates to a composition for antifreezing including a gold nanostructure in which at least a portion thereof is concave, thereby exhibiting excellent ice recrystallization inhibiting effect, increasing a survival rate of cells during cryopreservation of the cells, and maintaining a texture of food even when used in the freezing of food.SELECTED DRAWING: Figure 30

Description

Technical Field

[0001] The present invention relates to a composition for suppressing ice formation.

Background Art

[0002] A cryoprotective agent (CPA) is a compound that can reduce or inhibit the formation of ice crystals in a solution exposed to a temperature below 0°C when present in the solution. Current CPAs include small molecules (sometimes called permeable CPAs), synthetic polymers, and antifreeze proteins.

[0003] Currently, organ transplantation is the best treatment for end-stage organ failure in terms of survival rate, quality of life, and cost-effectiveness. However, unfortunately, there is a significant gap between the supply and demand of organ transplants. This gap causes a decline in the quality of life during the long waiting period for patients with debilitating diseases and has become a major medical barrier. The significant shortage of organs is due to a considerable amount of waste generated by the absence of a reliable preservation method. In fact, more than 50% of lungs, pancreases, and hearts remain unprovided from deceased donors.

[0004] To properly preserve an organ, the organ must be washed with a preservation solution to remove blood and stabilized. Even after being stabilized in the preservation solution, the time available for organ allocation, transportation, and transplantation after removal from the donor is limited (6 to 12 hours). This short time results in most organs being provided to local patients. This is because a match with a remote patient often cannot be confirmed within the limited time. As a result of this shortage, despite laws prohibiting the sale of human organs in almost all countries, illegal organ sales and human trafficking have increased to supply the demand.

[0005] Current permeating CPAs used in organ preservation generally include, among others, ethylene glycol, 1,2-propanediol, dimethyl sulfoxide, formamide, glycerol, sucrose, lactose, and D-mannitol. To reduce or inhibit the growth of ice crystals at organ preservation temperatures, the effective concentration of permeating CPAs has to be very high (often more than 60% is required). At such high concentrations, these compounds can be toxic to the tissue to be preserved, and the substantial removal of CPAs during warming before transplantation can sometimes cause irreversible cell death.

[0006] Other CPAs used to reduce or inhibit the formation of ice crystals include synthetic polymers and antifreeze proteins. Similar to permeating CPAs, these each have drawbacks. For example, synthetic polymers cannot penetrate cell membranes. For this reason, synthetic polymer CPAs can only control the formation of extracellular ice. To effectively preserve biological samples, the formation of ice crystals has to be controlled both inside and outside of cells. Naturally occurring antifreeze proteins, such as proteins isolated from fish, plants, or insects, are very effective at preventing the formation of ice, but currently available antifreeze proteins are of low purity and very expensive. Additionally, the use of antifreeze proteins to preserve biological samples introduces a potential source of immunogenicity.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0008] An object of the present invention is to provide a composition for suppressing ice formation.

[0009] Another object of the present invention is to provide a composition for freezing cells or tissues for increasing the survival rate of cells.

[0010] Furthermore, an object of the present invention is to provide a composition for freezing foods that can maintain the texture even when the foods are frozen.

Means for Solving the Problems

[0011] 1. An anti-icing composition containing a gold nanostructure at least partially recessed.

[0012] 2. The composition according to item 1 above, wherein the structure has at least one surface with a recess, and is a tetrahedron, hexahedron, octahedron, decahedron, dodecahedron, icosahedron, tetrahexahedron, hexoctahedron, rhombic dodecahedron, cylindrical, saddle-shaped or cap-shaped.

[0013] 3. The composition according to item 1 above, wherein the structure has at least one surface with a recess, and is a tetrahedron, hexahedron, octahedron, decahedron, dodecahedron, icosahedron, tetrahexahedron, hexoctahedron or rhombic dodecahedron.

[0014] 4. The composition according to item 1 above, wherein the size of the structure is 10 nm to 1,000 nm.

[0015] 5. The composition according to item 1 above, wherein the absolute value of the surface curvature of the recess is 0.1×10 -2 nm -1 ~5×10 -2 nm -1 is.

[0016] 6. The composition according to item 1 above, wherein the surface of the structure is modified to increase hydrophilicity.

[0017] 7. A composition for freezing cells or tissues containing the composition according to any one of items 1 to 6 above.

[0018] 8. A composition for food freezing, comprising any one of the compositions of Items 1 to 6 above.

[0019] 9. A method for freezing cells or tissues, comprising the step of exposing target cells or tissues to sub-zero temperatures in the presence of the composition of Item 7 above.

Advantages of the Invention

[0020] The composition of the present invention is excellent in the effect of suppressing ice recrystallization. Thereby, the cell survival rate during cryopreservation of cells can be increased, and the texture of the food can also be maintained when used for food freezing.

Brief Description of the Drawings

[0021]

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[0022] Hereinafter, the present invention will be described in detail.

[0023] The present invention relates to a composition for inhibiting ice formation.

[0024] Ice crystals can grow by ice recrystallization, which means the process of growing from smaller ice crystals to larger ice crystals. This growth occurs by the mechanism of Ostwald ripening. Ostwald ripening can proceed by the mechanism of melting-diffusion-recrystallization or sublimation-diffusion-condensation due to the pressure caused by the difference in surface energy between normal temperature and the crystal. In other words, the growth of ice crystals does not occur by the sticking together of ice crystals, but by the melting of small ice crystals between the crystals, diffusion towards larger ice crystals, and then re-freezing as part of the larger ice crystals.

[0025] Inhibition of ice formation means the action of preventing ice from forming, slowing down the ice formation rate, preventing ice recrystallization, slowing down the ice recrystallization rate, or maintaining the small size of ice crystals.

[0026] The composition for inhibiting ice formation of the present invention contains a gold nanostructure with at least a part being concave.

[0027] As shown in FIG. 2, when at least a part of the gold nanostructure is recessed and comes into contact with the growth surface of ice crystals, a liquid water layer is formed on the surface that abuts against the recess without ice growth, and ice is extruded outside the recess, and an ice growth inhibition effect can be exhibited in that portion.

[0028] The gold nanostructure has at least one surface with a recess. Having a recess means that an ice growth inhibition effect is exhibited at least in that portion, so it suffices that at least a part of at least one surface has a recess. The recess can exist on one or more surfaces and up to the entire surface.

[0029] The gold nanostructure can have a structure such as a tetrahedron, hexahedron, octahedron, decahedron, dodecahedron, icosahedron, tetrahexahedron, hexoctahedron, rhombic dodecahedron, cylindrical shape, saddle shape, or cap shape, but is not limited thereto.

[0030] The gold nanostructure may have a size of, for example, 10 nm to 1,000 nm, specifically 20 nm to 800 nm, and more specifically 40 nm to 700 nm, but is not limited thereto. For example, when the gold nanostructure is a polyhedron, the size may be the length of one side, and when it is a cap shape, the size may be the diameter, but is not limited thereto. More specifically, when the gold nanostructure is a polyhedron, the size may be 20 nm to 200 nm, and when it is a cap shape, the size may be 10 nm to 1,000 nm, but is not limited thereto.

[0031] The gold nanostructure has, for example, an absolute value of the surface curvature of the recess of 0.1×10 -2 nm -1 ~5×10 -2 nm -1 and may specifically be 0.3×10 -2 nm -1 ~3×10 -2 nm -1 but is not limited thereto.

[0032] The gold nanostructure may have a cetyl trimethylammonium chloride group or a cetyl trimethylammonium bromide group as a functional group on its surface.

[0033] In addition, the surface of the gold nanostructure can be modified and can have hydrophilic, hydrophobic or amphoteric functional groups. In that case, the ice suppression effect is reduced or increased, and ice formation can be adjusted more finely.

[0034] Examples of the hydrophilic functional group include, but are not limited to, a hydroxy group, a carboxy group, an amino group, a carbonyl group, a phosphate group, an ammonium group, an ester group, an imide group, a thioimide group, a keto group, an ether group, an indene group, a sulfonyl group, a polyethylene glycol group, etc. Examples of the hydrophobic functional group include, but are not limited to, a C1-C30 alkyl group, a C3-C30 cycloalkyl group, a C6-C30 aryl group, a C2-C30 heteroaryl group, a halogen group, a C1-C30 ester group, a halogen-containing group, etc. Examples of the amphoteric functional group include, but are not limited to, a hydroxy C1-C10 alkyl group, a peptide containing threonine, etc.

[0035] The functional group may be formed on the surface of the gold nanostructure by modifying the surface of the gold nanostructure with, for example, the hydrophilic, hydrophobic or amphoteric functional group and a compound having a -SH functional group at the end, but is not limited thereto.

[0036] The present invention also relates to a composition for freezing cells or tissues containing the above composition.

[0037] When cells or tissues are cryopreserved, during the process of thawing the frozen cells or tissues during subsequent use, ice recrystallization progresses cell membrane damage and cell dehydration, damaging the cells and tissues. Organisms living in a low-temperature environment may be more easily damaged by ice recrystallization.

[0038] The compositions of the present invention are applicable to all cells that are normally frozen for storage and used, for example, prokaryotic cells; eukaryotic cells; microorganisms; animal cells; cancer cells, sperm; eggs; stem cells including adult stem cells, embryonic stem cells, and induced pluripotent stem cells; blood cells including cord blood, white blood cells, red blood cells, and platelets; and tissue cells including kidney cells, liver cells, and muscle cells, but are not limited thereto.

[0039] Also, tissues are applicable to all tissues that are normally frozen for storage and used, and for example, all tissues such as the cornea, kidney, heart, small intestine, pancreas, lung, and liver can be used without limitation.

[0040] The present invention also relates to a method for freezing cells or tissues, which includes the step of exposing target cells or tissues to sub-zero temperatures in the presence of the above compositions.

[0041] When freezing target cells or tissues in the presence of the above compositions, recrystallization of ice during subsequent thawing can be inhibited, preventing damage to the cells or tissues.

[0042] The present invention also relates to a composition for freezing food containing the above composition, and a method for freezing food, which includes the step of exposing food to sub-zero temperatures in the presence of the above composition.

[0043] The compositions of the present invention are applicable to all frozen foods, and by using them, a decrease in the texture of the food during thawing can be minimized.

[0044] Hereinafter, examples will be given to specifically describe the present invention in detail.

[0045] Examples 1. Simulation To investigate the difference in ice growth due to the surface shape of particles, a computer simulation was performed. As materials, a regular hexahedral gold nanoparticle with a concave surface and a spherical gold nanoparticle with a convex surface were used (Figure 1).

[0046] As a result, in the regular hexahedral gold nanoparticle with a concave surface, a liquid water layer was formed on the surface in contact with the concave part without ice growth, and ice was extruded outside the concave part, showing an ice growth inhibition effect in that part, while the spherical gold nanoparticle did not show such a phenomenon (Figures 2 and 3).

[0047] 2. Fabrication of Gold Nanostructures (1) Gold Concave Nanocube (AuCC) with a Concave Surface The materials used for fabrication are as follows. - Sodium borohydride (NaBH 4 ), ≥98.0% - Gold(III) chloride hydrate (HAuCl 4 ), ≥99.9% - Cetyltrimethylammonium chloride (CTAC) solution, 25 wt% in H 2 O - Silver nitrate (AgNO 3 ), 99.9999% - Hydrochloric acid solution (HCl), 1.0 N - L-Ascorbic acid, ≥99%

[0048] For fabrication, 0.60 ml of 10 mM NaBH 4 was dissolved in water and then cooled in a refrigerator. Then, for seed formation, 0.25 ml of 10 mM HAuCl 4 and 10.00 ml of 100 mM CTAC were mixed. Then, the prepared NaBH 4Mix them quickly, stir for 1 minute, and then let stand for 2 hours. Then, for the production of the growth solution, 0.50 ml of 10 mM HAuCl 4 0.10 ml of 10 mM AgNO 3 , 0.20 ml of 1.0 M HCl, and 0.10 ml of 100 mM ascorbic acid were mixed.

[0049] After that, the prepared seeds were diluted from ×1 / 10 to ×1 / 10 4 and then 0.1 ml of it was added to the prepared growth solution. After leaving it to stand until the reaction ended, it was washed with a centrifuge (3,000 rpm, 10 min) (Figure 4).

[0050] By diluting and growing the seed concentration, gold nanocubes of various sizes can be produced. Specifically, when the same amount of growth solution is applied to the seed solution, the gold ions in the growth solution adhere to the surface of the seed and grow into nanocubes. This depends on the number of seeds. For example, the fewer the number of seeds, the more gold ions adhere to one seed, resulting in the production of larger-sized gold nanocubes. Using the above method, nanocubes with the specifications shown in Figure 5 were produced.

[0051] It can be confirmed that the produced nanocubes have concave surfaces (Figure 6). Their size and absorption spectrum are as shown in Figures 7 to 9.

[0052] Also, AuCC40 (size 40 nm) was obtained at a concentration of 4.54×10 10 particles / ml, AuCC70 (size 70 nm) was obtained at a concentration of 1.00×10 10 particles / ml, and AuCC170 (size 170 nm) was obtained at a concentration of 13.79×10 8 particles / ml. The particle size distribution is as shown in Figure 10.

[0053] (2) Gold Nanocap (Au Cap) The materials used for production are as follows. - Tetraethyl orthosilicate (TEOS), ≥99.999% - Ammonium hydroxide solution (NH 4 OH)), ~25% NH 3 basis - Ethanol (C 2 H 5 OH)), ≥99.9% - Clean glass (2.5×2.5 cm) - Gold plate, 5 - inch radius - Hydrofluoric acid solution (HF), 35%

[0054] After mixing 6.34 g of ethanol and 1.86 g of TEOS, it was added drop - by - drop to a pre - prepared solution of 16 g of ethanol + 2.64 g of NH4OH for reaction. After 24 hours of reaction, it was washed with a centrifuge (3,000 rpm, 30 min) to prepare silica beads. Then, 0.10 ml of a 5 mg / ml silica solution was dropped onto a clean glass substrate (2.5×2.5 cm) and dried. Then, using a metal ion DC / RF sputtering device, gold was ionized and stacked on the substrate to a thickness of 20 nm. By sonication, the silica particles placed on the substrate were removed, and a 5% HF solution was mixed to dissolve the internal silica particles, leaving only the outer gold nanocap particles.

[0055] By the above method, gold nanocap particles with a diameter of 500 nm were prepared, and the amount of the TEOS solution was reduced to create even smaller - sized nanocap particles (Figure 5). Conversely, increasing the amount of the TEOS solution can create even larger - sized nanocap particles.

[0056] The fabricated nanocaps can be confirmed to have a concave surface (Figure 11). Au Cap100 (size 100 nm) was obtained at a concentration of 2.36×10 8 particles / ml, and Au Cap500 (size 500 nm) was obtained at a concentration of 2.60×10 8 particles / ml. The size distribution is as shown in Figure 12.

[0057] (3) Spherical Gold Nanoparticle (AuNS) with a convex surface (without a concave surface) The materials used for fabrication are as follows. - Gold(III) chloride hydrate (HAuCl 4 ), ≥99.9% - Sodium citrate tribasic dihydrate, ≥99.0%

[0058] 1.0 g of HAuCl 4 was dissolved in 50 ml of deionized water. Then, 0.5 g of sodium citrate was dissolved in 5 ml of deionized water. The prepared HAuCl 4 solution was heated to boiling at 120 °C. When the solution boiled, the prepared sodium citrate solution was added and reacted for 10 minutes. After the reaction, the solution was cooled to room temperature and then filtered through 0.20 μm and stored refrigerated (Figure 13). It can be confirmed that the fabricated particles are convex spherical particles (Figure 13).

[0059] (4) Surface modification The surfaces of the gold nanocubes with sizes of 40, 70, and 100 nm fabricated in (1) were modified. The gold nanocubes were fabricated using CTAC and have cetyl trimethylammonium chloride groups on their surfaces. These were reacted with the following compounds respectively to obtain surface-modified gold nanocubes.

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[0061] Referring to FIG. 14, it can be confirmed that since the sulfhydryl group of the thiol forms an Au-S bond with the gold particles in the reaction, the surface is modified during FTIR measurement and the sulfhydryl peak disappears.

[0062] In addition, it was confirmed that the surface was modified and a change occurred in the absorption spectrum due to the Surface Plasmonic Resonance (SPR) effect on the surface of the gold particles, and the larger the surface area, the greater the change width (FIGS. 15 to 17).

[0063] 3. Confirmation of Whether Intracellular Insertion is Possible It was confirmed whether AuCC40 could be inserted into HSC-3 cells. Specifically, after adding 100 μl of AuCC40 at a concentration of 4.54×10 9 particles / ml to the medium of living cells (HSC-3) and reacting for about 2 hours, the nano-sized cube particles enter the cell membrane and finally penetrate into the cell interior. The entered gold cube particles can be observed by an optical microscope due to the scattering effect. As a result, it was confirmed that gold nanocubes were inserted into the cells (FIG. 18).

[0064] 4. Evaluation of Ice Inhibition Effect The effect of ice inhibition was evaluated by observing the effect of suppressing ice recrystallization. Immediately after rapid cooling, ice has very small (a few micrometers) ice domains, and as time passes, the size of the ice domains increases due to the recrystallization phenomenon. To quantify this, after rapid cooling using the splat method and liquid nitrogen, the samples were exposed to a temperature of -6°C for 30 minutes and the area of the ice domains was measured. The splat method is a cooling method standardized for observing the suppression of ice recrystallization. Water droplets were dropped from a height of 1.5 m above a substrate rapidly cooled with liquid nitrogen to form ice in a thin film shape.

[0065] Figure 19 shows the ice crystallization inhibition effect of AuCC40 modified with 6-Mercapto-1-hexanol (C6OH) at 7.54×10 -11 M. Figure 20 shows the ice crystallization inhibition effect of AuCC40 modified with 6-Mercapto-1-hexanol (C6OH) at 7.54×10 -12 M. Figure 21 shows the ice crystallization inhibition effect of AuCC40 modified with 6-Mercapto-1-hexanol (C6OH) at 7.54×10 -13 M. Figure 22 shows the ice crystallization inhibition effect of AuCC40 modified with 6-Mercapto-1-hexanol (C6OH) at 7.54×10 -14 M. Figure 23 shows the ice crystallization inhibition effect of AuNS80 modified with 6-Mercapto-1-hexanol (C6OH) at 1.66×10 -11 M. Figure 24 shows the ice crystallization inhibition effect of AuNS80 modified with 6-Mercapto-1-hexanol (C6OH) at 1.66×10 -12 M. Figure 25 shows the ice crystallization inhibition effect of AuNS80 modified with 6-Mercapto-1-hexanol (C6OH) at 1.66×10 -13 M. Figure 26 shows the ice crystallization inhibition effect of AuNS80 modified with 6-Mercapto-1-hexanol (C6OH) at 1.66×10 -14 M.

[0066] Referring to it, it can be confirmed that the material modified with hydrophilic functional groups shows the effect of suppressing ice recrystallization, and the degree of ice recrystallization varies depending on the shape and size of the material. This result was quantified by the method shown in Figure 27.

[0067] Specifically, more than 10 large-sized ice domains were selected, and the area was calculated using the particle analysis tool of the ImageJ program. Among them, the average area of the top 10 ice with the largest area was obtained, and the area was calculated in the same way for the same sample more than 3 times and used as the final average value. The RI value is (average ice area of water containing the material) / (average ice area of pure water), and the value was derived by comparing with pure water. When the RI value approaches 0, it means that the average ice area is very small, indicating excellent ice crystallization inhibition performance.

[0068] The results are shown in Figs. 28 and 29. Referring to them, in the case of AuCC, the RI value is very small and it has excellent ice suppression performance, while in the case of AuNS, a much larger RI value was shown.

[0069] Fig. 30 shows the results of RI quantification of AuCC, Fig. 31 shows that of surface-modified AuCC, and Fig. 32 shows that of AuCap.

[0070] Referring to Fig. 30, the smaller the amount of gold seeds added during fabrication, the larger the particles formed. So, increasing the particle size makes it difficult to obtain particles at a high concentration, but it can be confirmed that particles of each size all show an ice suppression effect.

[0071] Referring to Fig. 31, it can be confirmed that in the case where no surface modification is performed (CTAC) and in the case where the surface is modified with C6OH, the ice suppression performance is the most excellent.

[0072] Referring to Fig. 32, in the case of AuCap, an RI value approximated to that of water was shown. This is judged to be because AuCap is manufactured by a sputtering method on a substrate, so its concentration is lower than that of the method of synthesizing particles in a solution, and the concentration is not sufficient to show an excellent RI value.

Claims

1. comprising a gold nanostructure with at least a part being recessed, the structure has at least one surface with a recess, and is a tetrahedron, hexahedron, octahedron, decahedron, dodecahedron, icosahedron, tetrahexahedron, hexoctahedron or rhombic dodecahedron, a composition for inhibiting ice formation.

2. The recess on the at least one surface of the structure is surface-modified with any one of 2-mercapto-1-ethanol (C2OH), 3-mercapto-1-propanol (C3OH), 3-mercapto-2-propanol (C2OH-CH3), 6-mercapto-1-hexanol (C6OH). The composition according to Claim 1.

3. The structure is formed such that its diameter is 40 to 100 nm. The composition according to Claim 2.

4. The structure has a diameter of 10 nm to 1,000 nm. The composition according to Claim 1.

5. The absolute value of the surface curvature of the concave portion is 0.1×10 －２ nm －１ to 5×10 －２ nm －１ The composition according to claim 1, wherein the composition is such.

6. The surface of the structure is modified to increase its hydrophilicity. The composition according to Claim 1.

7. A composition for freezing cells or tissues, comprising the composition according to any one of Claims 1 to 6.

8. A composition for freezing food, comprising the composition according to any one of Claims 1 to 6.

9. A method for freezing cells or tissues, comprising the step of exposing the target cells or tissues to a sub-zero temperature in the presence of the composition according to Claim 7.

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