Method for preventing spontaneous oxidation of antioxidant using aptamer, aptamer-based control of the release rate of active ingredient in the hydrogel, material and use thereof

Abstract

The present disclosure relates to a method for preventing spontaneous oxidation of an antioxidant, material thereof and uses thereof. More particularly, the present disclosure relates to a method for preventing oxidation of an antioxidant using aptamer specifically binding to its target antioxidant and aptamer-based control of the release rate of active ingredient in the hydrogel. Aptamer having such activity can have versatile applications such as cosmeceuticals and health beverages. As an example, we provide in the present disclosure the establishing method of aptamer targeting for Vitamin C and verification of its prevention of spontaneous oxidation of Vitamin C. We also provide the detailed method for trapping such aptamer-active ingredient complex in the hydrogel. It is expected that an aptamer-trapped hydrogel of the present disclosure has functions of controlling a release rate of the active ingredient through the aptamer-based sensing of specific substance released from the skin according to the skin conditions.

Claims

1. A method of preventing oxidation of an antioxidant by treating the antioxidant with an aptamer.

2. The method of claim 1, wherein the antioxidant is a material selected from the group consisting of vitamin C, vitamin A, retinol, vitamin E, astaxanthin, resveratrol, polyphenol, coenzyme Q10, a peptide, and oil.

3. The method of claim 1, wherein the aptamer includes a base sequence as set forth in SEQ ID NO: 1.

4. The method of claim 1, wherein when the antioxidant is vitamin C, the aptamer inhibits oxidations of second and third OH groups of a lactone ring of the vitamin C.

5. Aptamer preventing oxidation of an antioxidant.

6. The aptamer of claim 5, wherein the antioxidant is a material selected from the group consisting of vitamin C, vitamin A, retinol, vitamin E, astaxanthin, resveratrol, polyphenol, coenzyme Q10, a peptide, and oil.

7. The aptamer of claim 5, wherein the aptamer includes a base sequence as set forth in SEQ ID NO: 1.

8. A method for preparing an aptamer-trapped hydrogel, the method comprising the steps of: a) binding an amine group to the aptamer of claim 5; b) silanizing a hydroxyl group of a hydrogel monomer with 3-glycidoxypropyltrimethoxysilane (3-GPTMS) having an epoxy group, and then binding an amine group binding to the aptamer to the epoxy group; and c) polymerizing the hydrogel monomer.

9. The method of claim 8, wherein the method includes the step of binding a biotin to the amine group binding to the aptamer of the step a), then reacting the same with a particle having streptavidin, and then mixing the particle having the aptamer with the hydrogel monomer on hydrogel polymerization reaction.

10. The method of claim 8, wherein the hydroxy group of the hydrogel is bound to the amine group or a carboxyl group attached to the aptamer by a chemical method.

11. An aptamer-trapped hydrogel prepared by claim 8.

12. The aptamer-trapped hydrogel of claim 11, wherein a hydrogel pack has an aptamer attached to a surface of the hydrogel, and a specific ingredient is attached to an end of the aptamer.

13. The aptamer-trapped hydrogel of claim 12, wherein the specific ingredient is an ingredient having effects of skin aging prevention, wrinkle removal, whitening, or moisture.

14. A cosmetic composition comprising the aptamer-trapped hydrogel of claim 11.

15. A method for controlling release of a skin active material according to an amount of a target material released from a skin, comprising the steps of: applying the aptamer-trapped hydrogel of claim 11 to the skin to allow the aptamer binding to an ingredient from the hydrogel to penetrate into the skin; and releasing the ingredient after binding the target material with the aptamer to which the ingredient is bound.

16. The method of claim 15, wherein the target material is ATP.

17. A method for preparing an aptamer-trapped hydrogel, the method comprising the steps of: d) binding an amine group to the aptamer of claim 6; e) silanizing a hydroxyl group of a hydrogel monomer with 3-glycidoxypropyltrimethoxysilane (3-GPTMS) having an epoxy group, and then binding an amine group binding to the aptamer to the epoxy group; and f) polymerizing the hydrogel monomer.

18. A method for preparing an aptamer-trapped hydrogel, the method comprising the steps of: g) binding an amine group to the aptamer of claim 7; h) silanizing a hydroxyl group of a hydrogel monomer with 3-glycidoxypropyltrimethoxysilane (3-GPTMS) having an epoxy group, and then binding an amine group binding to the aptamer to the epoxy group; and i) polymerizing the hydrogel monomer.

19. An aptamer-trapped hydrogel prepared by claim 9.

20. An aptamer-trapped hydrogel prepared by claim 10.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] FIG. 1 shows the reduction state of vitamin C through the hydrogen bonding of the hydroxyl group of vitamin C with bases constituted in an aptamer (RNA or DNA),

[0042] FIGS. 2 to 4 show that the three aptamers of the present disclosure prevent oxidation of vitamin C by hydrogen peroxide, more details

[0043] FIGS. 5 to 10 are graphs showing dissociation constants (KD) for AA (ascorbic acid) and DHA (dehydroascorbic acid) of an aptamer of the present disclosure,

[0044] FIGS. 11 and 12 illustrate a method of producing a functional smart hydrogel using an aptamer of the present disclosure, and

[0045] FIGS. 13 to 15 illustrate a process for aptasensing of a hydrogel in which an aptamer of the present disclosure is trapped.

DETAILED DESCRIPTION

[0046] Hereinafter, the present disclosure will be described in detail with reference to non-limiting Examples. The following Examples are intended to illustrate the present disclosure, and the scope of the present disclosure is not to be construed as being limited to the following Examples.

Example 1: Construction of a Group of Aptamers Binding to Reduced Vitamin C

[0047] The systematic evolution of ligands by exponential enrichment (SELEX) was carried out under the following conditions, finding the aptamer which selectively binds to the reduced ascorbic acid from DNA aptamer library including 10.sup.13 of aptamers. To proceed with SELEX while the reduced state of ascorbic acid was maintained, glutathione was added while maintaining a pH of about 5.5.

[0048] Under these conditions, more than 99% of the vitamin C was maintained in the reduced L-ascorbic acid state, rather than the oxidized dehydroascorbic acid (DHA). Under the above reaction conditions, SELEX was carried out, and the entire selected aptamers were subjected to Next Generation Sequencing. As an analysis result, aptamers composed of 3000 or more of secondary structure group were obtained.

Example 2: Quantitative Analysis of Anti-Oxidation of Vitamin C by Aptamer

[0049] Twenty individual aptamers were selected according to the type of the secondary structure, and the experiment regarding prevention of oxidation of vitamin C was carried out. After the aptamer dissolved in the annealing buffer was heated to 95° C., the secondary structure of the aptamer was formed as the temperature was gradually lowered to room temperature. Then, the mixture was mixed and reacted with the reduced L-ascorbic acid for 30 minutes so as to allow the mixture to bind to the L-ascorbic acid. Then, hydrogen peroxide solution was added to provide the oxidation condition. Oxidation of L-ascorbic acid was measured by adding OPDA (o-phenylenediamine) as a fluorescent dye. The level of DHA production can be quantitatively analyzed by measuring the amount of fluorescence from DHA-OPDA produced by the reaction of DHA, an oxide of L-ascorbic acid, with OPDA. Under the above conditions, the amount of fluorescence of DHA-OPDA was measured every 34 seconds for 25 minutes.

[0050] All three aptamers among them prevented oxidation of vitamin C by hydrogen peroxide. The #1, #2, and #3 aptamers, respectively, prevented oxidation of about 40%, about 20%, and about 40%. Based on these experiments and other experiences, it can be concluded that the three aptamers react directly to vitamin C and thus prevent oxidation of vitamin C (See FIGS. 2 to 4).

Example 3: Determination of Steady-State Solution Dissociation Constant (KD) of Aptamer of the Present Disclosure for Ascorbic Acid (AA) and Dehydroascorbic Acid (DHA)

[0051] The dissociation constants were determined using microscale thermophoresis (MST). The present Example includes MST data and KDs computed for the three aptamers in assay buffers for both targets. All reagents, including ascorbic acid and dehydroascorbic acid, were purchased from Sigma-Aldrich (St. Louis, Mo.), and deionized water was treated with Chelex-100 resin for 1 hour to remove accidental metals prior to buffer preparation. After Chelex treatment, the water was sprayed with nitrogen gas for 10 minutes to minimize oxygen and then kept sealed. This water was used in all aqueous solutions. The final buffer included 50 mM sodium acetate, pH 5.5, 1 mM MgCl.sub.2, and 0.05% Tween-20. Both AA and DHA were analyzed in 1:1 passages dilution from e5 μM to 153 pM (final) (for aptamers #3 & #1) and from 50 μM to 1.53 nM (final) (for aptamer #2), respectively in buffer. The final concentration of each Cy5-conjugated aptamer is 20 nM. Each technical second dilution was measured twice on a Monolith NT.115 MST device from NanoTemper Technologies GmbH (Munich, Germany).

[0052] The results are shown in FIGS. 5 to 10. Each data point is shown with a mean and fitted curve (black line) in the drawings. The vertical dashed line in each graph represents KD.

[0053] The following conclusions were deduced from the above results.

[0054] 1) aptamers #3 and #1 have better selectivity for AA vs. DHA, while aptamer #2 has slightly better selectivity for DHA than AA.

[0055] 2) aptamer #3 has the best selectivity for AA vs. DHA among the three aptamers.

[0056] 3) aptamer #1 was the best for the protection of AA from oxidation but had the minimum selectivity for AA vs. DHA.

Example 4: Trapping of Aptamer on Hydrogel

[0057] The method of trapping the aptamer of the present disclosure on the hydrogel is a way to trap hydrothermal particles in the hydrogel by first attaching biotin to the amine group attached to the aptamer, then functionalizing them with the particle having streptavidin binding thereto, and then mixing them at a rate of 20% on calcination of the hydrogel. This method does not cause chemical bonding between the aptamer and the hydrogel, and it is relatively simple to implement.

[0058] Another method is to chemically attach the amine or carboxy group attached to the aptamer to the hydroxyl group of the hydrogel, which chemically binds the aptamer to the hydrogel.

[0059] In addition, various types of bonds can be brought by modifying the chemical composition of the hydrogel, or by changing the functional group to be bonded to the aptamer.

[0060] The specific trapping method includes the steps of a) binding the amine group to the aptamer, b) silanizing the hydroxyl group of the hydrogel monomer with 3-glycidoxypropyltrimethoxysilane (3-GPTMS) having an epoxy group, and then binding amine group binding to the aptamer to the epoxy group, and c) polymerizing the hydrogel monomer so that the aptamer-trapped hydrogel can be prepared.

[0061] Meanwhile, a collagen hydrogel, which is currently widely used due to moisturizing effects, can be made into an aptamer-collagen hydrogel in the same manner as described above, and it can add a function of more slowly releasing ingredients after sensing (detecting).

[0062] For example, a smart sensing (detecting) function can be added so that teprenone or caprylic acid, which is used to prevent skin aging, is gradually supplied to the skin with an aptamer-collagen hydrogel or is released according to the amount of cytokine related to skin aging, which is released from the cell.