KR101947315B1 - Polyoxalate nanoparticles containing tyrosol - Google Patents

Abstract

The present disclosure relates to nanoparticles containing an oxalate copolymer having tyrosol as a first monomer and HO-A-OH as a second monomer. The A provides nanoparticles, which are a divalent group derived from one selected from alkane, alkene, alkyne, a cycloalkane, a cycloalkane, an aryl of C_2-C_10 and combinations thereof, and thus can exhibit anti-aging activity and anti-inflammatory activity.

Description

POLYXALATE NANOPARTICLES CONTAINING TYROSOL < RTI ID = 0.0 >

The present disclosure relates to nanoparticles comprising an oxalate copolymer having thiazole as a first monomer and HO-A-OH as a second monomer, wherein A is a C ₂ -C ₁₀ alkane, alkene, Wherein the divalent group is a divalent group derived from one selected from the group consisting of alkynes, cycloalkanes, cycloalkenes, cycloalkynes, aryls, and combinations thereof. The disclosure also relates to hydrogels, cosmetic compositions, anti-aging compositions and anti-inflammatory compositions comprising the nanoparticles.

The skin protects the body from external stimuli such as ultraviolet rays, harmful substances and pathogens, and prevents moisture loss in the body. The skin is largely composed of epidermis, dermis, and subcutaneous tissue. The stratum corneum, which is the outermost layer of the epidermis, functions as a skin barrier to protect the skin from external stimuli. The normal pH range of the stratum corneum is 5.0 - 6.0. In the weakly acidic environment, β-glucoserine progesterase, which is a lipogenesis enzyme, and acid sphingomyelin degrading enzyme are activated to help form the stratum corneum. Thus, the pH of the stratum corneum plays an important role in maintaining the homeostasis of the skin barrier, and pathogens such as bacteria help prevent parasitism. However, problems arise if the pH of the skin is outside the normal range. When the pH is 6.0 or higher, the lipid of the stratum corneum is micellized, and when it is less than 4.5, the structure becomes abnormal. Therefore, the abnormal pH environment of the skin surface interferes with the balance and binding force between the homeostasis and the stratum corneum of the skin, and the weakened skin is prone to skin lesions such as inflammation. When inflammatory skin diseases such as acne, atopy and the like occur, hyperactive oxygen is produced and chronic skin lesions increase cancer incidence and accelerate skin aging.

ROS (Reactive Oxygen Species, ROS) is significantly superoxide anion (O ₂ ^-), hydroxyl radicals (· OH) radical species and hydrogen peroxide (H ₂ O _2), singlet Oxygen, such as ⁽¹ O ₂₎ And non-radical species such as. In particular, H ₂ O ₂ plays an important role in the survival, growth, maintenance and differentiation of cells, but it causes oxidative stress to the skin when it is produced excessively. In addition, it generates strong active oxygen through in vivo metal ion and pantone reaction. Excessive active oxygen also consumes antioxidants in the skin, destroying antioxidant and protective functions. In skin with a balance of antioxidants due to active oxygen, lipid peroxidation is initiated and oxidative damage of proteins and DNA is caused, causing multiple skin lesions and promoting skin aging. have. Specifically, active oxygen is considered to be the most important cause of skin aging, and attempts have been made to prevent skin aging by reducing the amount of active oxygen (Jae-Ki Hong, 7, No. 2, 2009) ). Therefore, studies for reducing oxidative stress by lowering the amount of this active oxygen continue

Harmful substances, pathogens, and active oxygen, etc., cause macrophages in the skin to initiate an inflammatory reaction, which can cause oxidative damage. Particularly, NO is a free radical generated by the reaction of NOS (Nitric Oxide Synthases) and L-arginine, which causes inflammation of macrophages. In addition, NO reacts with active oxygen radicals to generate peroxynitrite, which is a powerful oxidizing agent, to cause disease. The amount of NO is known to increase depending on the amount of iNOS expression. In addition, inflammatory responses are caused by increased expression of TNF-α and COX-2 by oxidative stress. Thus, active oxygen can cause a variety of inflammatory skin diseases, and is particularly utilized as a biomarker for the treatment of such diseases because it is involved in inflammatory diseases.

Under these circumstances, the present inventors have found a cosmetic composition exhibiting antioxidative activity and anti-inflammatory activity, which protect cells against oxidative stress by lowering the amount of active oxygen, and completed the present invention.

Preparation and Release Behavior of Atorvastatin Calcuim - Encapsulated Polyoxalate Microspheres, Polymer (Korea) Kim, Hyun Gu Lee, Jaewon Yang, Jin Young Park, Seung Chul Kim, Dong-Kwon Lim, Dongwon Lee and Gilson Khang ), 38 (5), 656, (2014)

A main object of the present disclosure is nanoparticles comprising an oxalate copolymer having tyrosol as a first monomer and HO-A-OH as a second monomer, wherein A is a C ₂ -C ₁₀ alkane Is a divalent group derived from one selected from the group consisting of alkene, alkene, cycloalkane, cycloalkene, cycloalkane, aryl, and combinations thereof.

Still another object of the present disclosure is to provide a hydrogel comprising the nanoparticles.

Another object of the present disclosure is to provide a cosmetic composition comprising the nanoparticles.

It is also an object of the present invention to provide an anti-aging composition comprising the nanoparticles and an anti-inflammatory composition.

The present invention provides, as a first aspect, a nanoparticle comprising an oxalate copolymer having a thiazole as a first monomer and HO-A-OH as a second monomer, wherein A is a C ₂ -C ₁₀ Is a divalent group derived from one selected from the group consisting of alkanes, alkenes, alkynes, cycloalkanes, cycloalkenes, cycloalkenes, aryls, and combinations thereof.

In one embodiment, the oxalate copolymer is represented by the following formula:

(2)

In the above formula, n and m are each independently an integer of 1 or more.

In one embodiment, the oxalate copolymer nanoparticles further comprise an antioxidant.

In one embodiment, the antioxidant is quercetin.

In one embodiment, the nanoparticles are used as drug delivery vehicles.

As a second embodiment of the present invention, hyaluronic acid; Hydroxyethylcellulose; And a hydrogel comprising nanoparticles according to the first aspect.

In one embodiment, the nanoparticles further comprise quercetin.

As a third aspect of the present invention, there is provided a cosmetic composition comprising nanoparticles according to the first aspect.

A fourth aspect of the present invention provides an anti-aging composition comprising nanoparticles according to the first aspect.

A fifth aspect of the present invention provides an anti-inflammatory composition comprising nanoparticles according to the first aspect.

The nanoparticles according to the present disclosure can selectively release tyrosol under oxidative conditions, thereby having anti-aging activity and anti-inflammatory activity. Therefore, the cosmetic composition containing the same may have sedative effects on skin aging and skin inflammation. In addition, the nanoparticles may additionally contain a drug other than tyrosol, which may also be released together with the tyrosol under oxidative conditions. Therefore, the nanoparticles can be usefully used as a drug delivery vehicle.

1 is a ¹ H-NMR result of the copolymer according to the present disclosure and its decomposition product. (A) is the data of the copolymer according to Production Example 1, and (B) is the data of the decomposition products obtained after treating the copolymer according to Production Example 1 with hydrogen peroxide, as confirmed in Experimental Example 1-2.
2 is an SEM image and particle size distribution diagram of the copolymer nanoparticles and degradation products thereof according to the present disclosure. (A) and (B) are SEM images and particle size distribution diagrams of the copolymer nanoparticles according to Production Example 1, (C) and (D) SEM image and particle size distribution of decomposition products obtained after treating hydrogen peroxide with coalesced nanoparticles.
FIG. 3 shows Nile Red release behavior of the nanoparticles determined in Experimental Example 2. FIG. Values are mean SD (n = 3).
Fig. 4 shows the evaluation of the cytotoxicity of the copolymer according to Preparation Example 2 as confirmed in Experimental Example 2. Fig. (A) is the cell viability measured when HaCaT cells are treated with QTPOX at various concentrations, and (B) is the cell viability measured when RAW 264.7 cells are treated with QTPOX at each concentration.
Fig. 5 is a fluorescence microscopic observation result obtained in Experimental Example 4. Fig.
FIG. 6 shows the results of fluorescence intensity measurement confirmed in Experimental Example 4. FIG. * P <0.05 and ** P <0.01 for the ROS intensity for each cell.
Fig. 7 shows the protective effect of cell damage as confirmed in Experimental Example 6. Fig. (A) shows the observed cell viability after treating each material with HaCaT cells, and (B) shows the relative fluorescence intensity of the observed ROS after treating each material with HaCaT cells. (C) shows the observed cell viability after treatment with RAW 264.7 cells, and (D) shows the relative fluorescence intensity of the observed ROS after treatment with RAW 264.7 cells. * P < 0.1 and ** P < 0.01 for each H ₂ O ₂ treated group.
Fig. 8 shows the inhibitory activity of the inflammatory inducer as confirmed in Experimental Example 7-1. (A), (B), and (C) show the expression levels of COSX-2 and RAW 264.7 cells, respectively. * P < 0.1 and ** P < 0.01 for each LPS treated group.
Fig. 9 shows the inhibitory activity of the inflammatory cytokine production confirmed in Experimental Example 7-2. (A) shows the amount of IL (interleukin) -1β produced and (B) the amount of TNF (tumor necrosis factor) -α produced after treatment with RAW 264.7 cells. * P < 0.1 and ** P < 0.01 for each LPS treated group.
10 is a photograph and SEM image of the hydrogel obtained in Experimental Example 8. Fig.
11 shows the particle release behavior of the hydrogel determined in Experimental Example 8. FIG. Values are mean SD (n = 3).

The present disclosure is, as a first aspect, a nanoparticle comprising an oxalate copolymer having thiazole as a first monomer and HO-A-OH as a second monomer, wherein A is a C ₂ -C ₁₀ Is a divalent group derived from one selected from the group consisting of alkanes, alkenes, alkynes, cycloalkanes, cycloalkenes, cycloalkenes, aryls, and combinations thereof.

In the oxalate copolymer, the hydroxy group of the first monomer, thiolsole, reacts to form polyoxalate, which is included as a part of the copolymer. Also, each hydroxy group of HO-A-OH, which is the second monomer, also forms an oxalate bond to form polyoxalate and to include it as a part of the copolymer. Specifically, such a copolymer can be represented by the following chemical formula 1:

[Chemical Formula 1]

In the above formula, n and m are each independently an integer of 1 or more. Preferably, n and m may each independently be 1 to 100. Specifically, n may be an integer of 5 to 20, 6 to 15, or 7 to 10, and m may be an integer of 10 to 90, 20 to 70, 30 to 50, and 35 to 45. Wherein A is selected from the group consisting of C ₂ -C ₁₀ , C ₆ -C ₁₀ , C ₇ -C ₉ , or C ₈ alkane, alkene, alkyne, cycloalkane, cycloalkene, cycloalkane, aryl, Lt; / RTI &gt; Specifically, A is a C ₂ -C ₁₀ , C ₆ -C ₁₀ , C ₇ -C ₉ , or C ₈ alkylene, alkenylene, alkynylene, cycloalkylene, cycloalkenylene, cycloalkynylene, aryl Or a combination thereof. These alkanes, alkenes and alkynes may be linear or branched. In one embodiment, A may be - (CH ₂ ) _x -C _y H _2y - (CH ₂ ) _z - in the combination of cycloalkane and alkane, wherein x, y, 10 &lt; / RTI &gt;

In one embodiment, the copolymer may be one in which the first monomer and the second monomer are each produced by reacting with an oxalyl halide such as oxalyl chloride. In one embodiment, the oxalate copolymer may comprise a polyoxalate obtained by reacting 1,4-cyclohexanedimethanol with a second monomer and reacting it with oxalyl chloride . The oxalate copolymer may be represented by the following chemical formula 2:

(2)

In the above formula, n and m are each independently an integer of 1 or more.

That is, the copolymer of Formula 2 is a copolymer of polyoxalate containing tyrosol as a monomer and polyoxalate containing 1,4-cyclohexanedimethanol as a monomer.

Polyoxalate (POX), which contains 1,4-cyclohexane dimethanol as a monomer, is known to be non-toxic, non-inflammatory, biocompatible and biodegradable in vivo. Also, the monomer, 1,4-cyclohexane dimethanol, is a non-toxic, biodegradable material, and oxalic acid is a small molecule that exists abundantly in the human body and is also harmless to human body. On the other hand, the bioavailability of tyrosols, which are the monomers of the copolymers of this disclosure, is also commonly reported. The molar ratio of the polyoxalate produced from the first monomer to the polyoxalate generated from the second monomer may be from 1: 1 to 1:10, from 1: 2 to 1: 8, or from 1: 3 to 5. In one embodiment, for the preparation of the copolymer, thiazole and 1,4-cyclohexane dimethanol were added in a molar ratio of 1: 4, respectively, and the ratio of each polyoxalate in the resulting copolymer was also 1: 4 Respectively.

The oxalate copolymer may be prepared in the form of nanoparticles through an emulsion process. Specifically, the size of the nanoparticles may be 100 to 1000 nm, 200 to 800 nm, or 400 to 600 nm. In one embodiment, the nanoparticles have an average size of 516 nm. The oxalate copolymer may have an average molecular weight of 1,000 to 15,000, an average molecular weight of 5,000 to 10,000, and an average molecular weight of 7,000 to 9,000.

Oxalate copolymers according to the present disclosure can decompose in an oxidizing environment to release harmless carbon dioxide and water. At this time, thiazole is released together to show a cell protection effect from the oxidative environment. That is, the amount of active oxygen in the cell can be effectively reduced to exhibit anti-aging activity. In one embodiment, it was confirmed that the oxalate copolymer was decomposed and its shape changed upon H ₂ O ₂ treatment (FIG. 2). In addition, the nanoparticles may have an anti-inflammatory activity and have an effect of soothing the skin stimulated by inflammation. Furthermore, the nanoparticles can be used as a drug delivery vehicle because they can release an active substance, e.g., a drug, selectively in an oxidative environment. In one embodiment, oxidative stress was induced in human keratinocytes and inflammatory cells, respectively, and the amount of reactive oxygen generated was observed. Oxalate copolymer nanoparticles loaded with a hair dye, Nile red, Respectively. As a result, it was confirmed that the nanoparticles were decomposed according to the generation of active oxygen due to oxidative stress and released nile red (FIGS. 5 and 6).

The oxalate copolymer nanoparticles may further comprise an antioxidant, an anti-inflammatory agent or a combination thereof. The antioxidant or anti-inflammatory agent may be selected from the group consisting of quercetin, Kaempferol, Luteolin, Apigenin, Epigallocatechin gallate (EGCG), Epigallate catechin (EGC) , Or glycosides thereof. Quercetin, a representative vegetable polyphenol compound, has strong antioxidative and radical scavenging properties comparable to other flavonoids, and has a physiological function such as anti-allergic action and anti-inflammatory action. The quartetin-bearing oxalate copolymer nanoparticles (QTPOX) containing thiols can decompose in an oxidative environment like the TPOX to release tyrosol and quercetin. In one embodiment, the QTPOX enhances the cell survival rate when treated with keratinocytes and inflammatory cells of H ₂ O ₂ -treated human cells, respectively, and it has been confirmed that the QTPOX exhibits a cytoprotective effect and reduces the amount of active oxygen. That is, it was confirmed that anti-aging activity was obtained by reducing the amount of active oxygen in the cells (FIG. 7). (POX) which does not contain tyrosol and quercetin. Therefore, the nanoparticles containing both substances exhibit such anti-aging activity by selectively decomposing in the oxidative environment Respectively. That is, the QTPOX nanoparticles exhibit anti-aging activity with H ₂ O ₂ -sensitive.

In addition, the nanoparticles may have anti-inflammatory activity. In one embodiment, when the QTPOX nanoparticles are treated, the amounts of NO produced and the amounts of proteins iNOS and COSX-2 that induce the production of NO involved in the inflammatory reaction were measured. As a result, (Fig. 8). In addition, it was confirmed that the QTPOX nanoparticles effectively reduced the amount of cytokine involved in the inflammatory reaction in one embodiment (FIG. 9).

A second aspect of the present disclosure provides a hydrogel comprising nanoparticles according to the first aspect. Specifically, hyaluronic acid; Hydroxyethylcellulose; And a hydrogel comprising the oxalate copolymer nanoparticles. The hydrogel has a three-dimensional network structure composed mainly of hydrophilic polymer chains, and is a flexible formulation having hydrodynamic characteristics similar to biological tissues. They do not dissolve in an aqueous solution but can swell to contain a large amount of water and can easily disperse the drug, which can be used as a very useful drug delivery system. The hydrogel not only facilitates skin permeation of the drug through skin hydration but also is mainly used in the form of a patch and is suitable for topical application of the drug. It is also being investigated as a means to form complexes with other systems such as liposomes, micelles, nanoparticles, and to improve stability and penetration of the drug into the skin.

Furthermore, the hydrogel can be used in cosmetics. The hydrogel capable of transferring nanoparticles having anti-aging activity and anti-inflammatory activity can be used to prevent skin aging and to calm the skin. When such a hydrogel is used as a cosmetic, it can be provided in the form of a mask sheet, an eye patch, an acne patch, a paste, or the like.

The hydrogel according to the present disclosure may be made of hyaluronic acid and hydroxyethylcellulose. Hyaluronic acid (HA) is a hydrophilic polymer composed of N-acetyl-D-glucosamine and glucuronic acid units, and is known as a major component of ECM (extracellular matrix). Hydroxyethyl cellulose (HEC), one of the cellulose derivatives, is a polymer with improved solubility over cellulose and has biocompatibility characteristics. These can be used to prepare a hydrogel for transdermal delivery of a drug. Such a hydrogel comprises oxalate copolymer nanoparticles according to the first aspect, which may be located within the three-dimensional network of the hydrogel. Such a hydrogel may act as a drug delivery vehicle that releases nanoparticles in the body.

A third aspect of the disclosure provides a cosmetic composition comprising nanoparticles according to the first aspect. As described above, the nanoparticles according to the present disclosure have anti-aging activity and anti-inflammatory activity, and thus can effectively protect the skin when used in a cosmetic composition. In particular, the cosmetic composition according to the present disclosure can effectively protect against aging by protecting cells in the skin from the oxidative environment generated in the cells from external stimuli. In addition, the cosmetic composition effectively alleviates skin irritation induced by external stimuli, and can act to calm the skin.

The cosmetic compositions of the present disclosure may be in the form of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansers, oils, powder foundations, emulsion foundations, wax foundations, And may be formulated and provided in a selected form.

The cosmetic composition of the present disclosure may further comprise a cosmetically acceptable carrier.

The type of cosmetically acceptable carrier of the present disclosure is not particularly limited so long as it does not impair the activity and properties of the cosmetic composition of the present application, and any cosmetically acceptable carrier conventionally used in the art can be used. Non-limiting examples of the cosmetically acceptable carrier include saline, sterilized water, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and the like. These may be used alone or in combination of two or more. The cosmetically acceptable carrier of the present application may comprise a non-naturally occuring carrier.

The cosmetically acceptable carriers of the disclosure vary according to the formulation of the cosmetic composition.

When the formulation of the present disclosure is a paste, a cream or a gel, it is possible to use as the carrier component an animal oil, a vegetable oil, a wax, a paraffin, a starch, a tracer, a cellulose derivative, polyethylene glycol, silicon, bentonite, silica, talc, zinc oxide And the like may be used, but the present invention is not limited thereto.

When the cosmetic composition of the present disclosure is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder and the like may be used as the carrier component, But are not limited to, propellants such as hydrofluorocarbons, propane / butane or dimethyl ether.

When the formulation of the present disclosure is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent may be used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl Benzoate, propylene glycol, 1,3-butyl glycol oil and the like can be used. Particularly, it is possible to use a cottonseed oil, peanut oil, corn oil, olive oil, castor oil and sesame oil, glycerol aliphatic ester, polyethylene glycol or sorbitan Of fatty acid esters may be used, but are not limited thereto.

When the formulation of the present disclosure is in the form of a suspension, the carrier component may be a suspending agent such as water, a liquid diluent such as ethanol or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracant, but are not limited thereto.

When the cosmetic composition of the present disclosure is a soap, the carrier component may include an alkali metal salt of a fatty acid, a fatty acid hemiester salt, a fatty acid protein hydrolizate, an isethionate, a lanolin derivative, an aliphatic alcohol, a vegetable oil, May be used, but is not limited thereto.

When the formulation of the present disclosure is a pack, a pack off pack containing polyvinyl alcohol or the like, a wash off containing a pigment such as kaolin, talc, zinc oxide, or titanium dioxide in a general emulsified cosmetic, Pack, or mask sheet pack, and is not particularly limited thereto.

The cosmetic composition of the present disclosure further comprises ingredients that are included in conventional skin external agent components such as water, a surfactant, a humectant, a lower alcohol having 1 to 6 carbon atoms, a chelating agent, a bactericide, an antioxidant, an antiseptic, .

As a fourth aspect of the present invention, there is provided an anti-aging composition comprising nanoparticles according to the first aspect. The ability to reduce the amount of active oxygen in the cells of the nanoparticles according to the present disclosure as described above can exhibit anti-aging activity.

A fifth aspect of the disclosure provides an anti-inflammatory composition comprising nanoparticles according to the first aspect. As described above, since the nanoparticles according to the present disclosure exhibit the ability to reduce or alleviate the inflammatory reaction through inhibition of proteins, cytokines, and the like related to the inflammatory reaction in cells in which an inflammatory reaction has been induced from external stimuli, Can be used. In particular, inflammatory skin diseases can be prevented and alleviated. Herein, inflammatory skin diseases are collectively referred to as skin diseases in which inflammation is the main lesion. The inflammatory skin diseases are composed of seborrhoeic dermatitis, contact dermatitis, systemic lupus erythematosus, allergic dermatitis, acne, eczema, acne, urticaria, psoriasis, irritable lupus, chronic simplex poisoning, But it is not limited thereto.

The disclosure provides, as a sixth aspect, a quasi-drug composition comprising nanoparticles according to the first aspect. In one embodiment, the quasi-drug composition protects the skin from oxidative environment to prevent skin aging. In one embodiment, the quasi-drug composition alleviates inflammation resulting from skin irritation and soothes the skin.

As used herein, the term " quasi-drug " refers to products that are less active than drugs, such as those used for diagnosis, treatment, improvement, alleviation, treatment or prevention of diseases in humans or animals. For example, Quasi-drugs are products that are used for the purpose of treating or preventing diseases or diseases of humans or animals, products which are mild or have no direct action on the human body.

The quasi-drug composition of the present disclosure may be manufactured in a form selected from the group consisting of a body cleanser, a foam, a soap, a mask, an ointment, a cream, a lotion, an essence and a spray, but is not limited thereto.

When the nanoparticles of the present disclosure are used as a quasi-drug additive, the nanoparticles of the present disclosure may be added as they are or may be used together with other quasi-quasi-drugs or quasi-drug components, and they may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be appropriately determined depending on the purpose of use.

A seventh aspect of the disclosure provides a method for preparing a mixed solution comprising: preparing a mixed solution comprising 1,4-cyclohexanedimethanol and thiazole; And adding oxalyl chloride to the mixed solution to obtain an oxalate copolymer solution. Specifically, the process utilizes a condensation reaction between oxalyl chloride and the hydroxyl groups of 1,4-cyclohexane dimethanol and thiolsole as follows.

[Reaction Scheme 1]

The method may further comprise adding quercetin to the oxalate copolymer solution.

The preparation of the mixed solution containing 1,4-cyclohexane dimethanol and thiazole is to prepare a mixed solution of the diol material, which is contained as a monomer in the copolymer. Wherein the 1,4-cyclohexanedimethanol and thiolsol may be included in a molar ratio of 1: 1 to 1:10, 1: 2 to 1: 8, or 1: 3 to 5. The solvent of these mixed solutions may be any solvent that can be used in the art as long as it can dissolve 1,4-cyclohexane dimethanol and thiazole, and non-limiting examples include tetrahydrofuran (THF) and the like Organic solvents. This step can be carried out under a nitrogen atmosphere. At this time, it may further include the step of adding an organic base catalyst. Organic base catalysts may include organic amines, specifically weak base catalysts such as triethylamine.

The step of adding oxalyl chloride to the mixed solution to obtain the oxalate copolymer solution is to react the monomer in the mixed solution with oxalyl chloride to form a copolymer of polyoxalate. This step can be carried out under a nitrogen atmosphere and can be carried out for 5 to 10 hours. As long as the solvent of the reaction solution can dissolve 1,4-cyclohexane dimethanol, thiazole, oxalyl chloride, any solvent which can be used in the art can be used, and a non-limiting example is tetrahydrofuran ( THF) and the like.

The step of adding quercetin to the oxalate copolymer solution is a step of supporting quercetin on the nanoparticles. Non-limiting examples of the solvent of the oxalate copolymer solution include an organic solvent such as dichloromethane (DCM), and examples of the solvent of the quercetin solution include hydrophilic solvents such as ethanol and water. The step may include homogenizing a mixed solution of the copolymer and quercetin by adding the solution to a poly (vinyl alcohol) solution. The homogenization can be carried out using an ultrasonic mill and a homogenizer, from which emulsion-type nanoparticles can be obtained. Followed by lyophilization.

In the following, embodiments will be described in detail with reference to the accompanying drawings. However, various modifications may be made in the embodiments, and the scope of the patent application is not limited or limited by these embodiments. It is to be understood that all changes, equivalents, and alternatives to the embodiments are included in the scope of the right.

The terms used in the examples are used for descriptive purposes only and are not to be construed as limiting. The singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, the terms "comprises" or "having" and the like refer to the presence of stated features, integers, steps, operations, elements, components, or combinations thereof, But do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, or combinations thereof.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this embodiment belongs. Terms such as those defined in commonly used dictionaries are to be interpreted as having a meaning consistent with the contextual meaning of the related art and are to be interpreted as either ideal or overly formal in the sense of the present application Do not.

Throughout this specification, "%" used to denote the concentration of a particular substance is intended to include solids / solids (wt / wt), solid / liquid (wt / The liquid / liquid is (vol / vol)%.

Substances used

The materials used are as follows:

1,4-cyclohexanedimethanol, 4- (2-hydroxyethyl) phenol (tyrosol) (purchased from Sejian City, Tokyo, Japan);

Sodium hydroxide (NaOH, assay = 98.0%) (purchased from OCI, Seoul, Korea);

Dichloromethane (purchased from Merck, Germany);

Triethylamine (purchased from Showa, Japan);

Sodium hydrogen phosphate dihydrate (NaH ₂ PO ₄ .2H ₂ O) (purchased from Junsei chemical, Tokyo, Japan);

Hyaluronic acid (HA, Mw 0.8 MDa) (purchased from Bioland, Cheonan, Korea).

Hydroxyethyl cellulose (HEC, average Mw 90,000 Da), divinyl sulfone (DVS), sodium chloride (NaCl), sodium phosphate dibasic dodecahydrate (Na ₂ HPO ₄ .12H ₂ O ), Chloroform D (CLCD ₃ ), hydrogen peroxide solution, quercetin, oxalyl chloride, poly (vinyl alcohol) (MW 13000-21000), tetrahydrofuran anhydride and hydrogen peroxide solution were all available from Sigma Aldrich (St. Louis, Mo., USA) .

Solvents such as ethanol were used for analytical grade reagents and distilled water was purified by Milli-Q.

Preparation Example 1: Preparation of thiazole-polyoxazoline copolymer (TPOX)

1,4-cyclohexanedimethanol (21.6 mmol) and thiazole (5.4 mmol) were dissolved in 20 mL of tetrahydrofuran (THF) at a molar ratio of 4: 1 in a nitrogen gas atmosphere. Then 60 mmol of triethylamine was added dropwise at 4 占 폚. On the other hand, 27.45 mmol of oxalyl chloride was dissolved in 25 mL of THF, added dropwise to the above solution, and reacted at room temperature for 6 hours. This also proceeded in the nitrogen environment. After 6 hours, the solvent was distilled off under reduced pressure, dissolved in dichloromethane (DCM) and washed with distilled water. The water was removed with sodium sulfate and the solvent was removed by vacuum distillation. This was dissolved in a small amount of DCM and precipitated using low temperature hexane to obtain a TPOX polymer.

The chemical structure was confirmed by ¹ H NMR (Fig. 1). The NMR used was 400 MHz NMR and was purchased from Varian Inc (USA). The hydrogen of the hydroxyl group of 1,4-cyclohexane dimethanol before the reaction was shown to be 3.5 ppm, but it did not appear in the NMR peak of the product due to formation of oxalate ester bond (Fig. 1 (A)). On the other hand, the hydrogen of the methylene group adjacent to the oxalate ester bond portion of the resulting polymer was 4.2 ppm (peak e). The peaks of the two aromatic hydrogens were 7.1 and 7.5 ppm (peaks a and b), and the benzylic hydrogen was 3.0 ppm (peak c). These results indicate that the condensation reaction of oxalyl chloride with the diol group of 1,4-cyclohexanedimethanol and the diol group of thiolsol respectively. That is, 1,4-cyclohexane dimethanol and thiazole were synthesized in the form of a polyoxalate containing a peroxalate ester bond. The molar ratio of 1,4-cyclohexane dimethanol and thiazole was found to be 4: 1, which was the same as that of TPOX.

The average molecular weight of the synthesized polymer was confirmed by gel permeation chromatography (GPC) (Waters 1515, 2414, 717, Waters Corporation, US) to be ~ 8835 Da and the polydispersity index was 2.56.

Production Example 2: Preparation of quercetin-containing thiazole-polyoxazoline copolymer (QTPOX) nanoparticles

A TPOX solution was prepared by dissolving 100 mg of TPOX according to Preparation Example 1 in 1 mL of DCM. Then, 5 mg quercetin was dissolved in 300 쨉 l ethanol and added to the TPOX solution. The mixture was added to 5 mL of a 10% (w / v)% poly (vinyl alcohol) solution and homogenized in the form of an emulsion using an ultrasonic sonicator and a homogenizer. The organic solvent in the emulsion was removed with a reduced pressure evaporator, and the pellet was collected by centrifugation (1000 x g) for 5 minutes. It was washed with distilled water and lyophilized at -80 ° C (yield ~ 52%).

Preparation Example 3: Preparation of QTPOX-loaded HA-HEC hydrogel

HA (0.225 g) and HEC (0.075 g) (mass ratio 3: 1) were added to 10 mL of 0.02 M NaOH solution and stirred for 3 hours at room temperature to completely dissolve. Divinyl sulfone (DVS), a crosslinking agent, was added and mixed thoroughly with stirring. The crosslinking agent was added at a molar ratio of 1: 1 per repeating unit of the polymer, and the crosslinking reaction was allowed to proceed at room temperature for 24 hours. Excessive distilled water was used to remove unreacted crosslinking agent and reactants. The prepared hydrogel was cut to an appropriate size and freeze-dried for 48 hours.

Experimental Example 1: Analysis of TPOX nanoparticles

Experimental Example 1-1. Morphology analysis of TPOX nanoparticles

The morphology of TPOX nanoparticles according to Preparation Example 1 was observed. Scanning Electron Microscopy (SEM) (FEI-Inspect F50, FEI compay, Hillsboro, USA) was used and the voltage was 15 kV. The particle size and distribution were measured using the Otsuka ELS-Z series (Otsuka Electronics, Japan), which analyzes the particle size using light scattering. He-Ne lasers were used and data were processed by cumulative analysis. The observed SEM image is shown in FIG. 2 (A), and the diameter of the nanoparticles was found to be 516 nm on average, and its particle distribution is shown in FIG . 2 (B) . The particle size was measured using dynamic light scattering.

Experimental Example 1-2. Determination of decomposition properties according to hydrogen peroxide

The effect of the TPOX nanoparticles produced according to Preparation Example 1 on hydrogen peroxide, that is, the hydrolysis reaction with hydrogen peroxide, was confirmed. The TPOX nanoparticles according to Preparation Example 1 were treated with 10 mM H ₂ O ₂ for 1 hour, cultured, lyophilized at -80 ° C., and the degradation products were measured by NMR (FIG. 1 (B)). The respective peaks of the product were confirmed to correspond to the peaks of the reactants, 1,4-cyclohexane dimethanol and thiazole, respectively.

In order to observe morphological changes due to hydrogen peroxide treatment, the TPOX nanoparticles according to Preparation Example 1 were treated with ₁₀ mM H ₂ O ₂ for 1 hour. The products decomposed in the same manner as in Experimental Example 1-1 were observed and shown in (C) and (D) of FIG. 2. The average size of the hydrogen peroxide treated nanoparticles was 1135.2 nm, which was much higher than that of the pre-treated nanoparticles. As shown in FIG. 2, many holes were formed on the outer periphery of the nanoparticles as compared with the hydrogen peroxide-untreated nanoparticles. POX It is known that oxalyl ester bonds are decomposed by H ₂ O ₂ and CO ₂ is generated. It is considered that TPOX is also released by the decomposition of oxalyl ester bond by H ₂ O ₂ , releasing CO ₂ and decomposing the thiols.

Experimental Example 2: Preparation of TPOX nanoparticles in vitro  Drug release studies

To identify drug release characteristics of TPOX nanoparticles, Nile red, a water soluble fluorescent material, was used. Nile red has lipophilic properties such as quercetin and was used to confirm the release characteristics. In Preparation Example 2, nanoparticles were prepared by carrying Nile Red in place of quercetin. Then, 1 mg of the TPOX nanoparticles (1 mg) was dispersed in 1 mL of the buffer, followed by collecting with stirring at 37 ° C. In order to evaluate the sensitivity of TPOX nanoparticles to drug release behavior at various pH and H ₂ O ₂ conditions, the buffer pH was 5 or 7 and the H ₂ O ₂ concentrations were 0, 0.1, 1, 5 , And 10 mM, respectively. 100 μl of the mixture was collected and centrifuged (1000 × g) for 5 minutes. The pellet was dissolved in the same volume of fresh buffer and added again. The supernatant collected over time was measured using a fluorescence ELISA reader. The excitation wavelength of Nile red of 543 nm and the emission wavelength of 630 nm were used, which are shown in FIG.

The release rate was slightly higher at pH 7 than at pH 5. However, the release behavior of TPOX nanoparticles was more affected by H ₂ O ₂ than by pH. In particular, when 10 mM H ₂ O ₂ was treated, it was confirmed that 90% or more of nile red was released at 60 minutes. At 100 μM and 1 mM H ₂ O ₂ , Nile Red was released steadily. The cumulative release (%) was 9% (H ₂ O ₂ untreated), 24% (100 μM) 35% (1 mM), 51% (5 mM) and 77% . The time for 50% cumulative release is about 10-20 minutes depending on the concentration of each H ₂ O ₂ treatment. On the other hand, in the absence of H ₂ O ₂ , the release rate of Nile Red was slow to 6 hours and cumulative drug release was less than 50% even at 24 hours.

The increase in the emission amount according to the H ₂ O ₂ concentration determined in FIG. 3 is attributed to the decomposition of TPOX due to H ₂ O ₂ , ie, the decomposition of H ₂ O ₂ into oxalic acid ester bonds and the release of Nile Red . This can be seen from the particle shape confirmed in FIG. 2 above.

Experimental Example 3: Evaluation of cytotoxicity

Human keratinocyte HaCaT cells and inflammatory RAW 264.7 cells were cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% fetal bovine serum (PAA, Austria), 1% penicillin-streptomycin (PAA, Austria ) At 37 ° C and 5% CO ₂ . HaCaT and RAW 264.7 cells are seeded 1 × 10 ⁴ cells / well in 96 well plates were incubated for 24 hours. Then, QTPOX according to Preparation Example 2 was treated with various concentrations (13-200 占 퐂 / mL) for 48 hours and then washed twice with PBS to obtain 3- (4,5-dimethylthiazol-2-yl) The cell viability was determined using 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT). The control shown in the formula refers to the UVA irradiated group without sample treatment, and the experiment refers to the experimental group irradiated with UVA after sample treatment.

) (3)Cell survival rate (%) = (

) (3)

The measured results are shown in Fig. Viability was significantly reduced by 200 ㎍ / mL QTPOX nanoparticles in all cell lines, but remained at 95% of the control at 100 ㎍ / mL. Therefore, concentrations below 100 μg / mL were used for all of the following experiments.

Experimental Example 4: Drug Release Study of ROS-Sensitive TPOX Nanoparticles

First, we observed the cell damage and the amount of active oxygen by oxidative stress.

RAW 264.7 and HaCaT cells were plated in black 8-well plates at 1 × 10 ⁵ cells for 24 hours. After washing once with PBS, the cells were treated with 20 μM DCFH-DA (dichlorofluorescein-diacetate) (Sigma, USA) for 30 minutes and washed three times with PBS. DCFH-DA is a fluorescent reagent mainly used for measuring the amount of active oxygen generated in cells.

Then, H ₂ O ₂ was treated with HaCaT cells, and the concentration and amount of H ₂ O ₂ were treated with 30 mL at a concentration of 4 mM, which was a predetermined cell viability of 50%. RAW 264.7 cells were treated with 2 μg / mL LPS for 4 h. The cells were treated with 50 μg / mL of TPOX nanoparticles loaded with Nile red, treated for 30 minutes, and washed three times with PBS. Nile red was observed using fluorescence microscopy (Motic, Hong Kong) at 543 nm (excitation) and 630 nm (emission) and DCF at 490 nm (excitation) and 530 nm 5 (ROS (DCF) portion). FIG. 6 graphically illustrates the ROS generation amount / Nile red emission of FIG. 5 using the ImageJ program.

In the untreated HaCaT or RAW 264.7 cells treated with Nile Red-loaded TPOX particles, less active oxygen was present in the cell, and Nile Red was also almost completely absent. On the other hand, RAW 264.7 transfected with Nile red more than HaCaT cells, suggesting that the phagocytic action of macrophages caused the TPOX particles to enter the cells and release the drug.

When oxidative stress of H ₂ O ₂ and LPS induced cells caused a large number of active oxygen (green) in the cells, it was confirmed that Nile red (red color) was introduced into the cells. Especially, the more active oxygen was generated, the more Nile red was introduced. This appears to be due to the decomposition of TPOX particles due to the hydrogen peroxide generated during the inflammatory reaction, resulting in a significant increase in drug release.

The ratio of the amount of introduced nile red / the amount of generated ROS is shown together with the respective graphs in Fig. In HaCaT cells stimulated with hydrogen peroxide, the value was 1.5, whereas in RAW 264.7 cells stimulated with LPS, the value was 1.9. That is, the amount of Nilre red transferred was higher than that of the generated ROS level. In particular, Nilre red captured in TPOX nanoparticles was released more in RAW 264.7 cells than in HaCaT cells. These results suggest that rapid drug release in RAW 264.7 cells is due to the faster degradation of TPOX nanoparticles due to the phagocytosis of inflammatory cells and the production of ROS induced by LPS. As shown in FIG. 3, the drug release behavior of TPOX nanoparticles increases in proportion to the concentration of hydrogen peroxide.

These results demonstrate that the H ₂ O ₂ -sensitive nanoparticles, TPOX particles, vary in the amount of drug released depending on the degree of damage to the cell, and thus can selectively release the drug in the damaged cell.

Experimental Example 5: Measurement of quercetin trapping efficiency in QTPOX nanoparticles

The quercetin collection efficiency of the particles was measured by taking a certain amount of the particle suspension and removing the quercetin not captured in the particles by using a 1.2 μm syringe filter (Minisart CA 26 mm). After that, a calibration curve of quercetin was prepared using a UV / Vis spectrophotometer and the quercetin concentration, which did not pass through the filter, was measured. The loading efficiency of the particles was calculated using the following equation.

(1)Loading efficiency (%) =

(One)

T: initial quercetin concentration (μg / mL),

P: Quercetin concentration (쨉 g / mL) which did not pass through a 1.2 쨉 m syringe filter,

The loading efficiency of the nanoparticles obtained using the above equation was 16%.

Experimental Example 6: Preparation of H ₂ O ₂ Effect of QTPOX nanoparticles on cell injury and reactive oxygen production

H ₂ O ₂ can easily penetrate all cell membranes and its reactivity is relatively weak compared to other ROS. When the skin is exposed to ultraviolet rays, the resulting ROS (such as · OH and H ₂ O ₂ ) damages biomolecules and cell membranes through high oxidative power. QTPOX nanoparticles were treated with HaCaT and RAW 264.7 cells in amounts of 10 μg, 50 μg and 100 μg, respectively, and then H ₂ O ₂ was treated to measure the cell viability and the amount of active oxygen. The concentration and time of H ₂ O ₂ used were 4 mM 30 min for HaCaT cells and 1 mM 6 hr for RAW 264.7 cells, and cell viability was obtained by MTT assay.

For comparison, the same amount of H ₂ O ₂ was treated alone, or after treatment with 100 μg of tyrosol, the same amount of H ₂ O ₂ , the same amount of H ₂ O ₂ after treating with 50 μg of quercetin, H ₂ O ₂ treatment, POX 50㎍ same amount of H ₂ O ₂ treatment, TPOX 50㎍ post-treatment post-treatment was observed the results of the things that the same amount of H ₂ O ₂ treatment together. The results are shown in Fig.

The results of HaCaT cells are shown in Fig. 7 (A). H ₂ O ₂ by the control (H ₂ O ₂ alone) process showed a 53% cell viability compared to the viability of the non-treatment group (Control). The QTPOX particle treated group showed a concentration-dependent cytoprotective effect except for 10 μg. Compared with the treatment with 50 ㎍ of TPOX in order to confirm the effect of quercetin released from QTPOX particles, it showed 11% higher cytoprotective effect than TPOX when treated at the same concentration. TPOX was 8% higher than POX, even at the same concentration, compared with 50 ㎍ POX treatment to confirm the antioxidative effect of thiolsol released from TPOX particles. That is, both tyrosol and quercetin exhibit protective effects on cells damaged by oxidative stress.

The results of RAW 264.7 cells are shown in Fig. 7 (C). The cell viability of RAW 264.7 cells treated with H ₂ O ₂ was 57%, and QTPOX particles showed a cell-viability and concentration-dependent cytoprotective effect with increased cell viability at all treated concentrations than the group treated with hydrogen peroxide. The nanoparticles showed cell protection effect in the order of QTPOX>TPOX> POX at the same concentration of 50 ㎍. Similarly, it can be seen that both tyrosine and quercetin exhibit protective effects on cells damaged by oxidative stress.

These results demonstrate that tyrosols bound to quaternary and TPOX moieties on QTPOX particles contribute significantly to oxidative stress induced cell damage. In particular, although the content of single substances contained in QTPOX nanoparticles was much lower than that of single treatment (25% tyrosine binding, 16% quercetin uptake), higher cytoprotective effects than single substances alone It looked. This shows that the drug delivery effect of the nanoparticles is excellent. In addition, quercetin and tyrosol-free POX particles showed a higher cytoprotective effect in RAW 264.7 cells than in HaCaT cells, although they were weak. This suggests that macrophages exert phagocytosis on foreign substances in the range of 0.5-3 ㎛, and POX easily migrates into macrophages and more effectively erases active oxygen.

In addition, the antioxidant effect of nanoparticles against oxidative stress induced by H ₂ O ₂ in HaCaT and RAW 264.7 cells, that is, the effect of inhibiting active oxygen was also observed. In the previous experiment, the same procedure was performed except that the H ₂ O ₂ concentration was changed to 4 mM or 1 mM instead of 200 μM, and the treatment time was all 30 minutes. After treatment of each substance, the cells were washed twice with PBS, and cultured in serum-free medium for 2 hours. The amount of active oxygen was measured by a DCFH-DA assay, and the inhibition rates thereof were shown in FIGS. 7B and 7D Respectively.

The antioxidant effect was similar to the cytoprotective effect confirmed earlier. These results demonstrate that the antioxidant cytoprotective effect of QTPOX particles on oxidative stress contributes to the nanoparticle-supported quercetin, oxalate structures and tyrosols released by H ₂ O ₂ .

Experimental Example 7: Evaluation of anti-inflammatory efficacy

Experimental Example 7-1. Inhibition of inflammatory response-induced protein expression

NO is known to be an inflammatory substance and is stimulated by iNOS (inducible nitric oxide synthases). In the process of inflammation, COX-2 produces PGE2, an inflammatory factor. Inhibition of the expression of inflammatory factors such as iNOS and COX-2 is very important in protecting the skin from inflammation and cancer development. Therefore, it was used to confirm the degree of inhibition of inflammation.

Expression of the inflammatory factors iNOS (nitric oxide synthase) and COX-2 (Cyclooxygenase-2) was measured by Western blot analysis. RAW 264.7 cells stimulated with LPS were dissolved in RIPA buffer and the amount of protein in the lysate was quantitated. 40 μg of the lysate was electrophoresed and transferred to a PVDF membrane. (Novus Biologicals, USA), COX-2 (R & D Systems, USA) and β-actin (Cell signaling, USA) were used as the primary antibodies and rabbit and mouse secondary antibodies Respectively.

Specifically, RAW 264.7 cells were added to 24-well plates at 1 × 10 ⁶ cells and cultured for 24 hours. Treated with 100 μg of tyrosol, 50 μg of quercetin, 50 μg of POX, 50 μg of TPOX, 10 μg of QTPOX, 50 μg of QTPOX, and 100 μg of QTPOX for 4 hours, and 2 μg / Respectively. In order to measure the antioxidant and anti-inflammatory effects, the amounts of NO production, iNOS and COX-2 expression were evaluated. NO concentration was measured by ELISA reader using colorimetric assay using Griess reaction. 100 μl of the supernatant was collected and placed in 100 μl of a grease reagent, reacted at room temperature for 10 minutes, and then measured at 570 nm. IL-1β and tumor necrosis factor-α (TNF-α), which are inflammatory cytokines, were measured by ELISA kit (R & D system, USA) using cell supernatants.

As a result, the amount of NO produced is shown in Fig. 8 (A). In RAW 264.7 cells treated with LPS alone, the amount of NO production increased to 30 μM, and when QTPOX particles were treated, they showed a significant concentration-dependent reduction in all treated animals. When 50 μg of QTPOX particles were treated, NO production was inhibited by 56% and NO production was similar to that of untreated group at 100 μg. In particular, 50 μg of QTPOX particles were twice as high as 100 μg of tyrosol and slightly more inhibitory to NO formation than 50 μg of quercetin. In order to confirm the anti-inflammatory effects of quercetin released from QTPOX particles, 50 μg of quercetin-free TPOX particles were treated, which were twice as effective as QTPOX particles of the same concentration. In order to examine the antioxidative effect of thiolsol released from TPOX particles, 50 μg of POX particles not containing thiols were treated and they showed 18% higher NO production rate than TPOX particles. Interestingly, POX particles not containing thiazole and quercetin slightly inhibited NO production, presumably due to the antioxidative effect of oxalate and H ₂ O _{2 on} POX particles. These results suggest that quercetin, TPOX bound tyrosol and oxalate moieties captured by QTPOX significantly reduce NO production in LPS - stimulated macrophages and thus have anti - inflammatory effects.

In addition to antioxidant activity, it was confirmed whether the inhibitory effect of QTPOX particles on NO production was due to the inhibition of iNOS production. LPS-stimulated macrophages were treated with QTPOX particles to evaluate the expression level of iNOS (Fig. 8 (B)). INOS was significantly increased in macrophages treated with LPS alone. QTPOX particles reduced the production of iNOS in a concentration-dependent manner without a decrease in β-actin. In particular, the amount of iNOS expression in 100 ㎍ QTPOX particles was similar to that of the untreated group. However, POX particles not containing quercetin and tyrosine did not significantly affect the expression of iNOS. These results demonstrate that the NO production inhibitory effect of QTPOX particles is due to inhibition of iNOS expression.

Additionally, QTPOX particles inhibited COX-2 expression in LPS-stimulated RAW 264.7 cells in a concentration-dependent manner (Fig. 8 (C)). In conclusion, it was confirmed that QTPOX nanoparticles not only have antioxidative effects but also inhibit the expression of proteins that induce inflammatory responses.

Experimental Example 7-2. Inhibition of inflammatory cytokine production

TNF-α and IL-1β are cytokines that induce inflammatory responses in macrophages, which are produced by macrophages after being stimulated by LPS. The effect of QTPOX particles on the production of TNF-α and IL-1β, inflammatory cytokines due to the inflammatory response of LPS-stimulated macrophages, was confirmed.

Treated with 100 μg of tyrosol, 50 μg of quercetin, 50 μg of POX, 50 μg of TPOX, 10 μg of QTPOX, 50 μg of QTPOX and 100 μg of QTPOX for 4 hours respectively, and 2 μg / Respectively. The amounts of TNF-α and IL-1β were confirmed by an ELISA kit and are shown in FIG.

TNF-α and IL-1β produced by macrophages increased significantly when LPS alone was treated. QTPOX particles attenuated the production of TNF-α and IL-1β due to LPS in cells in a concentration-dependent manner. When QTPOX particles were 50 μg, the production of TNF-α and IL-1β was weakened by about 50%, which was almost the same as that of the untreated group at 100 μg. In contrast, TPOX particles showed a slight inhibitory effect at 50 μg and POX particles showed little effect.

These results suggest that the inhibitory effect of TPOX nanoparticles on the production of inflammatory cytokines is mediated by tyrosols and TPOX nanoparticles have a high drug delivery efficiency. The TPOX nanoparticle delivery system may have contributed to a synergistic anti-inflammatory effect through H ₂ O ₂ scavenging activity by polyoxalate and antioxidant activity by released thiols after degradation of nanoparticles in LPS-stimulated RAW 264.7 cells . This transfer mechanism was shown to be synergistic anti - inflammatory effect of H ₂ O ₂ cleavage by oxalate and specific tyrosine transfer to damaged cells. 50 μg of quercetin showed a very high anti-inflammatory effect of QTPOX particles. However, considering that the content of quercetin carried on the QTPOX particles is 16%, it can be seen that the anti-inflammatory effect was very small. This suggests that QTPOX is very useful as a carrier, and the excellent anti-inflammatory effect of QTPOX particles is expected to have H ₂ O _2- specific action.

Experimental Example 8: Preparation of HA-HEC hydrogel in vitro  Drug release studies

The HA-HEC hydrogel was cross-linked by the reaction of the hydroxyl groups of HA and HEC with the divinyl group of DVS used as a cross-linking agent. Carboxyl group (-COOH) of hyaluronic acid (HA) is a pH-carboxylate (-COO ^-) is ionized to form the higher and the emission efficiency becomes higher by increasing the size of each pore are flaring due to their electrostatic repulsion . These pH-sensitive hydrogels can be rapidly treated by increasing drug release rate in skin lesions with high pH, that is, inflammatory skin. The network structure of these hydrogels was observed through SEM (TESCAN VEGA 3, Cranberry TWP, PA, USA). Figure 10 shows an image of HA-HEC hydrogel and a cross-section of HA-HEC hydrogel. The prepared hydrogel showed a porous structure in which pores of about 100 to 200 μm were connected to each other and was confirmed to be in the form of a super-porous hydrogel (SPH) having pores having a diameter of several tens to several hundreds of microns. As a result, HA-HEC hydrogel is considered to be very convenient as a reservoir and carrier capable of retaining a large amount of nano-sized QTPOX particles.

To confirm the drug release behavior of these hydrogels, QTPOX particles were loaded into the hydrogel. The dried hydrogel was loaded with QTPOX particles at room temperature for 6 hours. After removing the hydrogel from the mixed solution of the particles, the liquid on the surface was removed using a filter paper. The QTPOX particle-loaded hydrogel was immediately immersed in the release medium (pH 7, pH 5 phosphate buffer). The emission medium (100 μL) was taken at regular time intervals and a new medium (100 μL) was added to maintain a constant volume. Release studies were conducted for 12 hours. The concentration of the drug was analyzed by using a calibration curve of Q-POX particles using a UV / Vis spectrophotometer. Absorbance was measured at the maximum absorption wavelength (309 nm) of QTPOX particles. The amount of QTPOX particles captured in the hydrogel matrix was calculated by the following equation.

(2)

(2)

IE: incorporating efficiency,

V1: Volume (mL) of the initial particle suspension,

V2: Volume (mL) of the remaining particle suspension,

C1: concentration of initial particles (μg / mL),

C2: concentration of remaining particles (μg / mL),

The collection efficiency of the substances in the hydrogel is related to the swelling power. The HA-HEC hydrogel has an open-cell structure, which is rapidly absorbed by the capillary force of water and drugs, and is mostly absorbed in the form of free water. When absorbed in the form of pre-water, it has good mobility because there is no interaction between polymer chain and drug, and it is thought to contribute to absorption and dissipation of drug. According to the swelling method, the QTPOX particle solution was collected in the hydrogel, and the collection efficiency was about 32.7%.

The release behavior of HA-HEC hydrogel for QTPOX particles by pH was shown (FIG. 11). The cumulative particle emission over time was calculated in consideration of the collection efficiency of each gel. In both pH 5 and pH 7, the release of particles continued to increase, reaching 35.8% at pH 5 and 72.0% at pH 7 after 12 hours. About 2 times more particles were released at pH 7 than pH 5 because the carboxyl (-COOH) group of the HA moiety of the HA-HEC hydrogel was ionized to form carboxylate (-COO ^- ) when the pH was increased, The electrostatic repulsive force is caused to expand the pores of the hydrogel network to result in faster particle diffusion. These results suggest that the release rate of HA-HEC hydrogels carrying QTPOX nanoparticles will increase in skin lesions with weakly basic pH.

Although the embodiments have been described with reference to the drawings, various technical modifications and variations may be applied to those skilled in the art. For example, it is to be understood that the techniques described may be performed in a different order than the described methods, and / or that components of the described systems, structures, devices, circuits, Lt; / RTI &gt; or equivalents, even if it is replaced or replaced.

Claims (10)

The tee sol of a first monomer, and an HO-A-OH as a drug delivery system comprising the oxalate (oxalate) copolymer as the second monomer, and A is C ₂ -C ₁₀ alkane, alkene, alkyne, cycloalkyl A divalent group derived from one selected from the group consisting of alkanes, cycloalkenes, cycloalkynes, aryls, and combinations thereof.
The drug delivery system according to claim 1, wherein the oxalate copolymer is represented by the following formula (2):
(2)

In the above formula, n and m are each independently an integer of 1 or more.
The drug delivery system of claim 1, further comprising an antioxidant, an anti-inflammatory agent or a combination thereof.
4. The drug delivery system according to claim 3, wherein the antioxidant is quercetin.
Hyaluronic acid;
Hydroxyethylcellulose; And
A hydrogel comprising the drug delivery system according to any one of claims 1 to 4.
The cosmetic composition according to any one of claims 1 to 3, wherein the cosmetic composition comprises an oxalate copolymer having thiazole as the first monomer and HO-A-OH as the second monomer. The A is a C2-C10 alkane, alkene, alkyne, cycloalkane, Cycloalkane, aryl, and combinations thereof.
An antioxidant composition comprising an oxalate copolymer comprising thiazole as a first monomer and HO-A-OH as a second monomer, wherein A is a C2-C10 alkane, alkene, alkyne, cycloalkane, cycloalkane , Cycloalkyne, aryl, and combinations thereof.
8. The anti-aging composition of claim 7, wherein the anti-aging composition further comprises an anti-oxidant.
An anti-inflammatory composition comprising an oxalate copolymer comprising thiazole as a first monomer and HO-A-OH as a second monomer, wherein A is a C2-C10 alkane, alkene, alkyne, cycloalkane, , Cycloalkyne, aryl, and combinations thereof.
10. The anti-inflammatory composition of claim 9, wherein the anti-inflammatory composition further comprises quercetin.

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