KR102645449B1 - Nanoparticles comprising resveratrol - Google Patents

Abstract

The present invention provides polyvinylpyrrolidone-polyacrylic acid-calcium carbonate nanoparticles containing resveratrol, a method for producing the same, and a cosmetic composition containing the same.
The polyvinylpyrrolidone-polyacrylic acid-calcium carbonate nanoparticle containing resveratrol of the present invention is a calcium carbonate crystal known to not only contain a large amount of the active ingredient resveratrol, but also to have a small and uniform particle size and high drug delivery efficiency. It may contain many forms of batite and calcite. In addition, the nanoparticles have high stability and can increase the stability, cell protection and antioxidant efficacy, skin absorption and transdermal permeability of the active ingredient resveratrol, and can increase safety by lowering the cytotoxicity of resveratrol at high concentrations. It has been shown to have an anti-inflammatory effect and can be very useful as a highly functional cosmetic material.

Description

Nanoparticles containing resveratrol {NANOPARTICLES COMPRISING RESVERATROL}

The present invention relates to nanoparticles that can improve the usefulness of resveratrol as a cosmetic ingredient, a method for producing the same, and a cosmetic composition containing the same. More specifically, the nanoparticles are polyvinylpyrrolidone-polyacrylic acid containing resveratrol. -It is a calcium carbonate nanoparticle.

As people's interest in health and appearance rapidly increases, companies' efforts to find cosmetic compositions to satisfy this interest are rapidly increasing. High-functional cosmetics are also expressed as cosmeceuticals, which is a new word combining the words cosmetics and pharmaceuticals, meaning ‘skin medicine’ that combines the concept of skin treatment with existing cosmetics. Recently, ingredients that are effective in anti-aging complex functions (wrinkle improvement, whitening), hair loss prevention/hair growth promotion, and atopic dermatitis treatment using various natural active substances have been continuously developed, but the skin penetration rate is still low and the performance is not properly expressed. there is. In particular, the existing transdermal absorption promotion method does not allow natural active substances to be delivered into the skin due to the skin barrier, so there is an urgent need to develop new cosmetic materials that can overcome this problem.

High-functional nano cosmetics are cosmetics containing nano-sized nano bioparticles or nano bio structures. Nano bio particles are used to carry active substances and absorb them into the skin to improve skin regeneration and function. It refers to a special cosmetic product that maintains moisturization and improvement of the skin by applying it to block external harmful rays or substances such as ultraviolet rays, infrared rays, and fine dust. It is known that the use of nanomaterials has advantages in texture, stain resistance, aesthetics, stability, and UV protection compared to products containing the same material of different sizes.

At the beginning of the technology development of high-functional cosmetics, the development of bio-cosmetics using active substances with high functionality and biosafety was common, but recently, high-functional nano cosmetics using nanotechnology are leading the market.

Currently, most cosmeceutical products, including functional cosmetics, can display efficacy effects, but most of the active ingredients do not pass through the stratum corneum of the skin, so the perceived efficacy is low and there is a lack of safety control technology, leading to great consumer dissatisfaction. In addition, most active ingredients are poorly soluble, and although stability and skin absorption rates are being improved through technologies such as nano-sized liposomes, they are not sufficient, and synthetic stabilizing substances do not fully realize the effect.

Therefore, there is an urgent need to develop technology to improve the delivery of active ingredients in actual cosmetics, and the development of high-performance nanobiomaterial cosmetics technology with high skin absorption rate and high stability is needed more than anything else.

Meanwhile, resveratrol is a type of phenolic compound that has recently received the most attention, and is present in large quantities in grape skins. Resveratrol exhibits various physiological and pharmacological effects, including strong antibacterial, antiviral, antioxidant, antithrombotic and anti-inflammatory effects. Accordingly, it has been confirmed to be helpful in cardiovascular protection, cancer prevention, and anticancer activity, proving its potential as a cancer prevention agent, and is also in the spotlight as an ingredient in foods and cosmetics.

However, resveratrol is a poorly soluble ingredient and is used very limitedly in water-soluble processing of beverages. Due to its structural instability, the trans form changes to the cis form with low bioactivity when exposed to light, heat, oxygen, etc. Because it is metabolized quickly and has a short half-life in the body, its bioavailability is very low when ingested, and it has the problem of being toxic at high concentrations. Therefore, in order to expand and develop the resveratrol industry, there is an urgent need to develop technology to increase the bioavailability and safety of resveratrol.

The inventors of the present invention conducted research to increase the usefulness of resveratrol as a cosmetic ingredient, and through this, developed polyvinylpyrrolidone-polyacrylic acid-calcium carbonate nanoparticles containing resveratrol and completed the present invention.

It is revealed that this invention is the result of a local innovation project based on local government-university cooperation conducted in 2020 with the support of the National Research Foundation of Korea with funds from the Ministry of Education.

Registered Patent Publication No. 10-2029685 (2019.10.10) Public Patent Publication No. 10-2019-0010212 (2019.01.30)

The technical problem to be achieved by the present invention is to provide nanoparticles that can increase the usefulness of resveratrol as a cosmetic ingredient, especially transdermal permeability, stability and safety.

The technical problem to be achieved by the present invention is not limited to the technical problem mentioned above, and other technical problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

In order to achieve the above technical problem, one aspect of the present invention provides polyvinylpyrrolidone-polyacrylic acid-calcium carbonate nanoparticles containing resveratrol.

In one embodiment, the nanoparticles may have a diameter of 10 nm to 150 nm and a polydispersity index of 0.35 or less.

In one embodiment, the nanoparticles may be in the form of vaterite or calcite.

Another aspect of the present invention includes mixing resveratrol, polyvinylpyrrolidone, and polyacrylic acid; and mixing a calcium carbonate precursor into the mixture. A method for producing polyvinylpyrrolidone-polyacrylic acid-calcium carbonate nanoparticles is provided.

In one embodiment, the step of mixing resveratrol, polyvinylpyrrolidone, and polyacrylic acid includes: a first mixture of resveratrol mixed with an organic solvent; and a second mixture of polyvinylpyrrolidone and polyacrylic acid mixed in an aqueous solvent; may be prepared by preparing each and then mixing the first mixture and the second mixture. Here, resveratrol in the first mixture may be included in an amount of 0.5 to 10%. The second mixture may contain 0.1 to 10% of polyvinylpyrrolidone, and the second mixture may contain 0.05 to 5% of polyacrylic acid. The ratio (v/v) of the first mixture and the second mixture may be 1:99 to 20:80.

In one embodiment, the step of mixing the precursor of calcium carbonate with the mixture of resveratrol, polyvinylpyrrolidone, and polyacrylic acid includes sequentially adding calcium salt and carbonate to the mixture of resveratrol, polyvinylpyrrolidone, and polyacrylic acid. Or it may be mixed at the same time.

Another aspect of the present invention provides a cosmetic composition containing the polyvinylpyrrolidone-polyacrylic acid-calcium carbonate nanoparticles.

In one embodiment, the composition may contain 0.1 μM or more of resveratrol.

The polyvinylpyrrolidone-polyacrylic acid-calcium carbonate nanoparticles containing resveratrol according to one embodiment of the invention not only contain a large amount of the active ingredient resveratrol, but also have a small and uniform particle size and high drug delivery efficiency. It was confirmed that it contained a large number of vaterite and calcite, which are known calcium carbonate crystal forms. In addition, the nanoparticles have high stability and can increase the stability, cell protection and antioxidant efficacy, skin absorption and transdermal permeability of the active ingredient resveratrol, and can increase safety by lowering the cytotoxicity of resveratrol at high concentrations. It has been found to have anti-inflammatory effects.

Therefore, the nanoparticles of the present invention not only have excellent long-term stability and are useful for industrial use of resveratrol, but also increase the skin absorption rate and transdermal permeability of resveratrol in the cosmetics field, reduce cytotoxicity, and have excellent antioxidant, cytoprotective, and anti-inflammatory effects. It can be useful as an ingredient that can improve skin regeneration, skin elasticity, and whitening.

The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the description or claims of the present invention.

Figure 1 is an image of resveratrol stock solution (a) and polyvinylpyrrolidone-polyacrylic acid-calcium carbonate (PVP-PAA-CaCO ₃ ) nanoparticles (b) containing resveratrol (RSV) prepared according to Example 1. .
Figure 2 shows the results of analyzing the encapsulation rate of RSV in PVP-PAA-CaCO ₃ nanoparticles according to adjustment of the ratio between PAA-CaCl ₂ and Na ₂ CO ₃ when manufacturing nanoparticles according to Example 1.1.
Figure 3 shows PVP-PAA-CaCO ₃ nanoparticles containing resveratrol alone (RSV), polyacrylic acid (PAA), polyvinylpyrrolidone (PVP), and RSV prepared according to Example 1.1 (RSV-PAA in the figure, These are the results of FT-IR comparative analysis (a) for PVP (noted as PVP) and FT-IR analysis (b) for PVP-PAA-CaCO ₃ nanoparticles alone.
Figure 4 is a scanning electron microscope (SEM) (a) and transmission electron microscope (TEM) (b) images (some particle sizes indicated) of PVP-PAA-CaCO ₃ nanoparticles containing RSV prepared according to Example 1.1. .
Figure 5 shows the results of X-ray crystallography (XRD) analysis of PVP-PAA-CaCO ₃ nanoparticles containing RSV prepared according to Example 1.1.
Figure 6 shows the results of measuring the size of PVP-PAA-CaCO ₃ nanoparticles containing RSV prepared according to Example 1.1 using dynamic light scattering (DLS).
Figure 7 shows the results of a comparative analysis of the effects of resveratrol alone (a) and PVP-PAA-CaCO ₃ nanoparticles containing RSV (b) on cell survival of HaCaT cell lines at each concentration.
Figure 8 shows the results of a comparative analysis of the effects of resveratrol alone (a) and PVP-PAA-CaCO ₃ nanoparticles containing RSV (b) on cell survival of the NIH-3T3 cell line at each concentration.
Figure 9 shows the results of analyzing the effect of Rose Bengal by concentration on the survival rate of HaCaT and NIH-3T3 cell lines.
Figure 10 shows the results of analyzing the effect of treatment with resveratrol alone at each concentration after treatment with Rose Bengal at an IC ₅₀ concentration on the survival rate of HaCaT and NIH-3T3 cell lines.
Figure 11 shows the results of analyzing the effect of treatment with PVP-PAA-CaCO ₃ nanoparticles containing RSV by concentration after treatment with Rose Bengal at an IC ₅₀ concentration on the survival rate of HaCaT and NIH-3T3 cell lines.
Figure 12 shows the results of analyzing the effect of H ₂ O ₂ by concentration on the survival rate of HaCaT and NIH-3T3 cell lines.
Figure 13 shows the results of analyzing the effect of treatment with resveratrol alone at different concentrations after treatment with H ₂ O ₂ at an IC ₅₀ concentration on the survival rate of HaCaT and NIH-3T3 cell lines.
Figure 14 shows the results of analyzing the effect of treatment with RSV-containing PVP-PAA-CaCO ₃ nanoparticles at different concentrations after treatment with H ₂ O ₂ at an IC ₅₀ concentration on the survival rate of HaCaT and NIH-3T3 cell lines.
Figure 15 shows the results of measuring the antioxidant capacity of PVP-PAA-CaCO ₃ nanoparticles containing RSV by concentration by DPPH assay.
Figure 16 shows the results of Western blot analysis of the effect of PVP-PAA-CaCO ₃ nanoparticles containing RSV on LPS-induced COX-2 and iNOS expression.
Figure 17 shows the results of HPLC analysis of the transdermal permeability of PVP-PAA-CaCO ₃ nanoparticles containing resveratrol and RSV.

Polyvinylpyrrolidone-polyacrylic acid-calcium carbonate nanoparticles according to one aspect of the present invention contain resveratrol.

There is increasing interest in the fabrication of materials capable of stabilizing and delivering drugs or unstable biological substances such as proteins, peptides or nucleic acids for the purposes of pharmaceutical, cosmetic and chemical applications (e.g. gene therapy, aroma or drug encapsulation). In this regard, calcium carbonate (CaCO ₃ ) as a drug carrier is widely used for manufacturing and complexing due to its high biocompatibility as a template. Calcium carbonate particles can have a variety of sizes, from tens of nanometers to several micrometers. In addition, spherical or polygonal calcium carbonate structures can be formed through particleization or surface coating, and various substances such as drugs, proteins, and enzymes can be encapsulated through the pores of calcium carbonate. In other words, calcium carbonate can protect and stabilize the active ingredients of the material, so it can be applied to synthetic substances or complexes containing natural bioactive ingredients used as active ingredients in various drugs as well as functional cosmetics. In addition, calcium carbonate can very easily combine with various substances used in producing calcium carbonate particles, such as stabilizers, and by combining these substances, chemical and physical properties can be changed to have the necessary functions.

Polyacrylic acid (PAA) is a polymer and is one of the substances that can be used as a stabilizer when producing calcium carbonate nanoparticles. At this time, PAA can greatly affect the shape of the crystal. It has been reported that at low concentrations it does not affect the shape formation of calcium carbonate crystals, and at high concentrations it inhibits the fixation of carbon dioxide and crystallization of calcium carbonate. PAA is known to be good for use in nanoparticles for bioapplications due to its high stability and biocompatibility.

Polyvinylpyrrolidone (PVP) is a bulky, low-toxic nonionic polymer with C=O, C-N, and alkyl groups as functional groups, and is widely used as a stabilizer in the production of nanoparticles. PVP is amphipathic, as it contains a pyrrolidone moiety, a strong hydrophilic part, and an alkyl group, a weak lipophilic group, and is known to prevent aggregation when producing nanoparticles. When generating nanoparticles, the length of PVP is related to the stability of the nanoparticle and the protection efficiency of the material encapsulated within the nanoparticle, and it has been reported that PVP can affect the shape of the nanoparticle. In particular, PVP has properties that make it suitable for use in biocompatible nanoparticles, such as being physically and chemically inert over a wide pH range and increasing tissue permeability.

Resveratrol (or 3,5,4′-trihydroxy- trans -stilbene) is the active ingredient of the nanoparticles, and as mentioned above, has strong antioxidant, antibacterial, anti-inflammatory and whitening effects, preventing skin aging and improving elasticity. It has high functionality as a cosmetic ingredient useful for skin care, maintaining skin health and preventing blemishes. However, resveratrol has limitations in industrial use due to problems such as poor solubility and low stability.

The inventors of the present invention conducted research to solve the above-mentioned limitations of resveratrol, and as a result, polyvinylpyrrolidone-polyacrylic acid-calcium carbonate nanoparticles containing resveratrol not only increase the stability of resveratrol, but are also used in cosmetics. The present invention was completed by confirming that the material can have various additional advantages. That is, the nanoparticles not only contained a large amount of the active ingredient resveratrol, but also contained a large amount of vaterite and calcite, which are calcium carbonate crystals known to have small and uniform particle sizes and high drug delivery efficiency. The nanoparticles were found to have the anti-inflammatory effect of resveratrol and increase skin absorption and transdermal permeability. In particular, it was unexpectedly found to increase the cell protection and antioxidant effects of the active ingredient, resveratrol.

In one embodiment, the nanoparticles may have a diameter of 10 nm to 150 nm and a polydispersity index of 0.35 or less. Preferably, the diameter of the nanoparticles may be 30 to 100 nm, more preferably 35 to 70 nm.

In one embodiment, the nanoparticles may be in the form of vaterite or calcite. The ratio of vaterite and calcite among the nanoparticles may be 6:4 to 9:1, preferably 7:3 to 8:2. Calcium carbonate nanoparticles are divided into calcite, aragonite, and vaterite depending on their shape. Calcite is the most common form of calcium carbonate because it has a hexahedral structure and is the most stable. Aragonite is a needle-shaped or orthorhombic particle that is formed under high temperature conditions above 60-80 ℃ or in the presence of magnesium ions. Vatterite is a spherical particle and is more metastable than calcite. Generally, in the process of producing calcium carbonate, amorphous calcium carbonate (ACC) is created first, and a metastable form of vaterite is created through the dissolution and recrystallization process of ACC. In addition, stable calcite is created through the dissolution and recrystallization process of batite. Batterite and calcite are calcium carbonate nanoparticles that have a higher specific surface area, higher solubility, higher dispersion power, and smaller specific gravity than aragonite, and can be used for more diverse purposes than aragonite. In one embodiment of the present invention, the polyvinylpyrrolidone-polyacrylic acid-calcium carbonate nanoparticles containing resveratrol contain a large number of vaterite or calcite, which are known to be efficient in terms of delivery of active ingredients. , Among them, the proportion of battery lights with higher efficiency was found to be high.

Herein, polyvinylpyrrolidone-polyacrylic acid-calcium carbonate nanoparticles containing resveratrol, polyvinylpyrrolidone-polyacrylic acid-calcium carbonate nanoparticles or PVP-PAA-CaCO ₃ nanoparticles containing RSV, RSV- PAA, PVP and 'the nanoparticles' are all used equally to refer to the polyvinylpyrrolidone-polyacrylic acid-calcium carbonate nanoparticles containing resveratrol of the present invention.

A method for producing polyvinylpyrrolidone-polyacrylic acid-calcium carbonate nanoparticles according to another aspect of the present invention includes mixing resveratrol, polyvinylpyrrolidone, and polyacrylic acid; and mixing a precursor of calcium carbonate into the mixture. At this time, the polyvinylpyrrolidone may have a molecular weight of 1 to 500 KDa, preferably 10 to 100 KDa. The polyacrylic acid may have a molecular weight of 0.5 to 1000 KDa, preferably 1 to 100 KDa.

In one embodiment, the step of mixing resveratrol, polyvinylpyrrolidone, and polyacrylic acid includes: a first mixture of resveratrol mixed with an organic solvent; and a second mixture of polyvinylpyrrolidone and polyacrylic acid mixed in an aqueous solvent; may be prepared by preparing each and then mixing the first mixture and the second mixture. Here, resveratrol in the first mixture may be included in an amount of 0.5 to 10%. The second mixture may contain 0.1 to 10% of polyvinylpyrrolidone, and the second mixture may contain 0.05 to 5% of polyacrylic acid. The ratio (v/v) of the first mixture and the second mixture may be 1:99 to 20:80.

The step of mixing the precursor of calcium carbonate with the mixture of resveratrol, polyvinylpyrrolidone, and polyacrylic acid involves sequentially or simultaneously mixing calcium salt and carbonate with the mixture of resveratrol, polyvinylpyrrolidone, and polyacrylic acid. ; or mixing calcium salt with the mixture of resveratrol, polyvinylpyrrolidone and polyacrylic acid and then adding carbon dioxide at 100°C or higher; It may be selected from among, and preferably, calcium salt and carbonate may be mixed sequentially or simultaneously with the mixture of resveratrol, polyvinylpyrrolidone, and polyacrylic acid. The calcium salt may be water-soluble, and is preferably calcium chloride, calcium hydroxide, calcium acetate, calcium phosphate, calcium citrate, calcium lactate, calcium gluconate, calcium alginate, calcium nitrate, calcium carbonate, calcium bromide, calcium bisulfite, and sulfuric acid. It may be selected from calcium, calcium sulfide, calcium bicarbonate, and combinations thereof, and more preferably calcium chloride. The carbonate may be water-soluble, preferably selected from sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, ammonium carbonate, calcium bicarbonate, magnesium bicarbonate, and combinations thereof, and more preferably sodium carbonate. . The sequential mixing may be in the order of calcium salt and carbonate, or in the order of carbonate and calcium salt.

A cosmetic composition according to another aspect of the present invention includes polyvinylpyrrolidone-polyacrylic acid-calcium carbonate nanoparticles containing the above-described resveratrol.

At this time, the cosmetic composition can be prepared in the form of a general emulsified formulation or solubilized formulation. For example, lotion such as softening lotion or nourishing lotion; Emulsions such as facial lotion or body lotion; Creams such as nourishing cream, moisturizing cream or eye cream, etc.; essence; cosmetic ointment; spray; gel; pack; sunscreen; makeup base; Foundation, such as liquid type, solid type, or spray type; powder; Make-up removers such as cleansing creams, cleansing lotions, and cleansing oils; Cleansing agents such as cleansing foam, soap, body wash, etc.; It may have a dosage form such as:

In addition, the cosmetic composition may contain, in addition to the nanoparticles, fatty substances, organic solvents, solubilizers, thickeners, gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, Commonly used in the cosmetics field are ionic emulsifiers, non-ionic emulsifiers, fillers, sequestering agents, chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic activators, lipophilic activators or lipid vesicles. May contain adjuvants used.

For example, when the formulation of the present invention is a paste, cream or gel, the carrier ingredients include animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide. It can be used.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as the carrier component. In particular, when the formulation is a spray, chlorofluorohydrocarbon and propane may be used as carrier ingredients. /May contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent, or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, or fatty acid ester of sorbitan.

When the formulation of the present invention is a suspension, the carrier ingredients include water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, and microcrystals. Cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used.

When the formulation of the present invention is a cleansing formulation containing a surfactant, the carrier ingredients include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, and fatty acid amide. Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester can be used.

When the cosmetic composition of the present invention is a soap, a surfactant-containing cleansing formulation, or a surfactant-free cleansing formulation, it can be applied to the skin and then wiped off, removed, or washed with water. As a specific example, the soap is liquid soap, powdered soap, solid soap, and oil soap, the surfactant-containing cleansing formulation is cleansing foam, cleansing water, cleansing towel, and cleansing pack, and the surfactant-free cleansing formulation is cleansing cream. , cleansing lotion, cleansing water, and cleansing gel, but are not limited thereto.

In one embodiment, the composition may contain 0.05 μM or more of resveratrol, preferably 0.1 μM or more, more preferably 1 μM or more, and most preferably 5 μM to 10 mM. Resveratrol in the composition is preferably contained in the polyvinylpyrrolidone-polyacrylic acid-calcium carbonate nanoparticles of the present invention, and may be included by adding the nanoparticles to the composition. As described above, the content of resveratrol in the nanoparticles can be adjusted during the manufacturing process of the nanoparticles, and accordingly, considering the amount of resveratrol contained, the final concentration of resveratrol in the composition is within the above range. The amount of the nanoparticles included in the composition can be calculated and added.

When the composition containing the resveratrol-containing polyvinylpyrrolidone-polyacrylic acid-calcium carbonate nanoparticles is commercialized as a cosmetic product, the active ingredient resveratrol remains on the skin for a short period of time as a wash-off agent such as a makeup remover or cleanser. In the case of (wash-off) type cosmetics, the active ingredient may be included at a relatively high concentration. On the other hand, in the case of leave-on type cosmetics such as lotion, emulsion, cream or essence, where the active ingredients stay on the skin for a long period of time, it is okay to include the active ingredients at a lower concentration than wash-off type cosmetics. something to do.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are for illustrating the present invention, and the scope of the present invention is not limited thereto.

<Example>

1. Polyvinylpyrrolidone-polyacrylic acid-calcium carbonate containing resveratrol (RSV) (PVP-PAA-CaCO ₃ ) Nanoparticle manufacturing

Example 1.1: PVP-PAA-CaCO containing RSV ₃ Nanoparticle manufacturing

1) Dissolve Resveratrol (RSV) in Ethanol at a concentration of 5%, 2) Dissolve 1% PVP (molecular weight 55,000 Da) and 0.5% PAA (molecular weight 5,100 Da) in distilled water, then mix 1) and 2) at 5:95. After mixing (v/v), centrifugation was performed at 12000 rpm for 1 minute at 4°C, and the supernatant (polyacrylic acid particles containing RSV, PAA particles) was obtained. PAA-CaCl ₂ was prepared by mixing 50 μl of CaCl ₂ (2 M) with 10 μl of the above PAA particles. PVP-PAA-CaCO ₃ nanoparticles containing RSV were prepared by adding 50 μl of 0.052 M Na ₂ CO _{3 to} the PAA-CaCl 2 , then centrifuged at 12000 rpm for 1 minute at 4°C and supernatant (A ) was removed and a pellet (containing PVP-PAA-CaCO ₃ nanoparticles containing RSV) was obtained.

Example 1.2: PVP-PAA-CaCO containing RSV ₃ Confirmation of encapsulation rate of active ingredient RSV in nanoparticles

The active ingredient resveratrol must be encapsulated in large quantities in nanoparticles to increase its effectiveness when used as a cosmetic. In preparing PVP-PAA-CaCO ₃ nanoparticles containing RSV as in Example 1.1, the ratio of PAA-CaCl ₂ and Na ₂ CO ₃ was adjusted to increase the encapsulation rate of the active ingredient RSV that can be encapsulated in the nanoparticles. It becomes different. Encapsulation efficiency (EE) is calculated by the following equation.

EE = 100 - (Supernatant (A) RSV)/(Initial RSV *100)

As a result of measuring the encapsulation rate of resveratrol by adjusting the ratio of PAA-CaCl ₂ and Na ₂ CO ₃ , EE was found to be up to 80% or more at a ratio of 10, as shown in Figure 2. If the ratio is increased, the EE can be higher, but since convergence appears as the concentration increases, a ratio of 10 was selected, and in the following experiments, PVP-PAA-CaCO ₃ nanoparticles containing RSV prepared at a ratio of 10 were used. did.

Example 1.3: PVP-PAA-CaCO containing RSV prepared according to Example 1.1 ₃ FT-IR analysis of nanoparticles

FT-IR is an FT-IR test method that allows qualitative and quantitative analysis of organic substances and inorganic compounds by checking the light absorbance and transmittance of an infrared beam and using the sample's unique fingerprint area. PVP-PAA-CaCO ₃ nanoparticles containing RSV (RSV-PAA, PVP in Figure 3), poly(acrylic acid), PAA, polyvinylpyrrolidone (PVP), and resveratrol. FT-IR analysis was performed on (RSV). The results were as shown in Figure 3, and when the FT-IR results of the four materials were compared as shown in Figure 3a, the difference in the dotted line showed that PAA and PVP reacted to form PAA and PVP compounds. , In particular, RSV peak did not appear in RSV-PAA and PVP, confirming that RSV was encapsulated in nanoparticles (RSV-PAA and PVP).

As a result of examining the FT-IR analysis data for PVP-PAA-CaCO ₃ nanoparticles containing RSV in more detail (FIG. 3b), vaterite peaked at 1465 cm ^-1 , 874 cm ^-1 , and 740 cm ^-1 . ) type CaCO ₃ nanoparticles were confirmed to have been manufactured, and it was confirmed that calcite type was also included with a peak of 1409 cm ^-1 ,707 cm ^-1 . As a result of analysis of other peaks, RSV-PAA and PVP (2971 cm-1, 3348 cm-1) used in the CaCO ₃ nanoparticle manufacturing process were confirmed.

Example 1.4:R.S.V.PVP-PAA-CaCO containing ₃ Electron microscopic image analysis of nanoparticles

Electron microscope images were analyzed to confirm the shape and size of PVP-PAA-CaCO ₃ nanoparticles. For scanning electron microscope (SEM) image analysis, the nanoparticles were spread evenly by attaching them to carbon tape, then coating them with platinum (Pt) and using an FE-SEM device. For transmission electron microscopy (TEM) image analysis, the nanoparticles were dispersed in 100% Ethanol and fixed on a Cu grid, then dried in an oven for 24 hours so that the particles were completely fixed on the grid surface, and the dried sample A FE-TEM instrument was used. The results of analyzing the SEM and TEM images of PVP-PAA-CaCO ₃ nanoparticles showed that there were many CaCO ₃ nanoparticles in the form of vaterite with a size of 100 nm or less, as shown in Figure 4.

Example 1.5: PVP-PAA-CaCO containing RSV ₃ X-ray crystallography (XRD) analysis of nanoparticles

After settling the PVP-PAA-CaCO ₃ nanoparticles containing RSV on an XRD specimen holder made of PAAM, the results of analysis using an

Example 1.6: PVP-PAA-CaCO containing RSV ₃ Dynamic light scattering (DLS) analysis of nanoparticles

To analyze RSV-containing PVP-PAA-CaCO ₃ nanoparticles using dynamic light scattering, the nanoparticles were dispersed in distilled water and particle size and charge were measured using a zeta sizer, using a polystyrene cuvette. The particle size and charge were measured using a folded capillary zeta cell. As a result of checking the size of the PVP-PAA-CaCO ₃ nanoparticles containing RSV, it was confirmed that the nanoparticle size distribution was gathered in a narrow area in a narrow bell shape, as shown in Figure 6. These nanoparticles were found to be made at about 48 nm (average 48.64 nm), and the polydispersity index (PI) was calculated to be 0.2284, indicating that the nanoparticles were uniform in size.

Example 1.7: PVP-PAA-CaCO containing RSV ₃ Stability analysis of nanoparticles

Resveratrol has low stability, especially in UV and high pH conditions, where it decomposes and/or isomerizes rapidly in minutes, under weakly basic and sunlight conditions, within hours, and under neutral and slightly acidic conditions, within several days. It is known. In particular, the thermal stability is low, for example, at room temperature, it has been reported that more than half of the resveratrol in the sample loses stability within 5 days, and more than 90% decomposes and/or isomerizes after 10 days (Zupancic et al., Eur J. Pharm. Biopharm. (2015)).

The stability test of PVP-PAA-CaCO ₃ nanoparticles containing RSV was conducted using a constant temperature and humidity chamber at a temperature of 25°C and a humidity of 60%. For the above nanoparticles, the degree of precipitation, particle size, potential, and content of main components (encapsulation ratio) were measured on the day of manufacture and at 1 month, 2 months, and 3 months after manufacture, respectively, and the results shown in Table 1 were obtained.

When to measure On the day of manufacture 1 month after manufacturing 2 months after manufacturing 3 months after manufacturing precipitate doesn't exist doesn't exist doesn't exist doesn't exist Particle size (nm) 48 ±1.7 49 ±0.5 51±2.3 50±1.1 Polydispersity Index 0.037 0.24 0.33 0.018 Zeta potential -7.7 -7.5 -9.4 -8.1 image Enclosure rate 81.75% 82.02% 81.95% 81.88%

Looking at the results, no precipitates were observed not only on the day of manufacture but also 3 months after, the size was also within the error range at all measurement times, and the size of the nanoparticles was uniform, with the polydispersity index value remaining low. appeared to be maintained. In addition, the encapsulation rate of the active ingredient, resveratrol, was also at a similar level at all measurement time points, confirming that PVP-PAA-CaCO ₃ nanoparticles containing RSV have high stability and can stably maintain the active ingredient, resveratrol.

2. PVP-PAA-CaCO containing RSV ₃ Measuring the cytoprotective effect of nanoparticles

RSV has a cell-protecting effect, but is known to be cytotoxic at high concentrations. To determine how the RSV-containing PVP-PAA-CaCO ₃ nanoparticles of the present invention affect the above effects of RSV, HaCaT (human epidermal keratinocyte cell line) and NIH-3T3 (Mouse embryonic fibroblast cell line) The experiment was conducted using two cell lines. At this time, the cell line was attached to a 96-well plate for cell culture at ¹ It was a mixture of % FBS (fetal bovine serum) and 1% PS (penicillin-streptomycin), and culture was performed under conditions of 37°C and 5% CO ₂ . After confirming that each cell line was stabilized, resveratrol alone or PVP-PAA-CaCO ₃ nanoparticles containing RSV were treated so that the resveratrol concentration was 0.625, 1.25, 2.5, 5, 10, 20, and 50 μM, respectively. The results of measuring cell viability using the CCK-8 assay kit after 30 minutes of incubation were shown in Figures 7 and 8.

Looking at the results, when the HaCaT cell line was treated with resveratrol alone, cell viability increased at 0.625, 1.25, 2.5, and 5 μM, but this effect tended to decrease as the treatment concentration increased, and at 50 μM, cell viability actually decreased. It was confirmed that the was lowered. On the other hand, in the case of PVP-PAA-CaCO ₃ nanoparticles containing RSV of the present invention, the cell survival rate generally tends to increase as the treatment concentration increases, showing that the cell survival rate can be statistically significantly increased even when the treatment concentration is high. Confirmed. In addition, when the NIH-3T3 cell line was treated with resveratrol alone, the overall cell survival rate decreased as the treatment concentration increased, and treatment at high concentrations of 20 and 50 μM significantly lowered the cell viability, whereas PVP-PAA containing RSV significantly lowered the cell viability. In the case of -CaCO ₃ nanoparticles, this toxic effect did not appear in the entire treatment concentration range.

In summary, it can be seen that the toxicity that appears when resveratrol alone is treated at high concentrations no longer appears in the PVP-PAA-CaCO ₃ nanoparticles containing RSV of the present invention, thereby increasing the safety of resveratrol. This is because, in previous tests on the nanoparticles, it has been shown that resveratrol is well released from the nanoparticles when the nanoparticles are treated with cells (data not included in this specification), so it was encapsulated in the nanoparticles. As a result, it was confirmed that the effect was not caused by lowering the concentration of resveratrol being processed.

3. PVP-PAA-CaCO containing RSV against oxidative stress ₃ Measuring the cytoprotective effect of nanoparticles

Example 3.1. Rose Bengal Processing

To determine the effect on the antioxidant effect of resveratrol when made with PVP-PAA-CaCO ₃ nanoparticles containing RSV of the present invention, Rose Bengal and HaCaT (human keratinocyte cell lines, which form radicals by visible light) The experiment was conducted using two cell lines: epidermal keratinocyte cell line) and NIH-3T3 (mouse embryonic fibroblast cell line). At this time, the cell line was attached to a 96-well plate for cell culture at ¹ It was a mixture of % FBS (fetal bovine serum) and 1% PS (penicillin-streptomycin), and culture was performed under conditions of 37°C and 5% CO ₂ .

After confirming that each cell line was stabilized, Rose Bengal was treated at each concentration, cell viability was measured using a CCK-8 assay kit, and the IC ₅₀ of Rose Bengal was derived (FIG. 9). The IC ₅₀ of Rose Bengal against HaCaT cell line and NIH-3T3 cell line was found to be 10 μM and 16 mM, respectively. To confirm the protective effect of PVP-PAA-CaCO ₃ nanoparticles containing RSV against oxidative stress, tests were conducted using each concentration of Rose Bengal.

Resveratrol alone and PVP-PAA-CaCO ₃ nanoparticles containing the RSV of the present invention were treated at each concentration and incubated for 30 minutes, and then treated with medium containing Rose Bengal at an IC ₅₀ concentration for each cell line and irradiated with luminescent light for 30 minutes. And cell viability was confirmed using the CCK-8 assay kit.

As a result, when resveratrol was treated alone, the protective effect against oxidative stress induced by Rose Bengal on HaCaT and NIH-3T3 cell lines could not be confirmed (FIG. 10). On the other hand, when treated with PVP-PAA-CaCO ₃ nanoparticles containing RSV of the present invention, the cell survival rate can be significantly increased in almost all concentration ranges in both cell lines treated with Rose Bengal (FIG. 11), especially Rose Bengal It was found that the cell survival rate could be increased to 80-90% of the untreated cell group, confirming a greater protective effect against oxidative stress than when treated with resveratrol alone.

Example 3.2. Hydrogen peroxide (H ₂ O ₂ )process

In order to determine the effect on the antioxidant effect of resveratrol when made with PVP-PAA-CaCO ₃ nanoparticles containing RSV of the present invention, hydrogen peroxide (H ₂ O ₂ ) and HaCaT (human keratinocyte cell line, epidermal keratinocyte cell) The experiment was conducted using two cell lines: line) and NIH-3T3 (mouse embryonic fibroblast cell line). At this time, the cell line was attached to a 96-well plate for cell culture at ¹ It was a mixture of % FBS (fetal bovine serum) and 1% PS (penicillin-streptomycin), and culture was performed under conditions of 37°C and 5% CO ₂ .

After confirming that each cell line was stabilized, each cell was treated with H ₂ O ₂ at different concentrations, cell survival was measured using a CCK-8 assay kit, and the IC ₅₀ of H ₂ O ₂ was derived (FIG. 12). The IC ₅₀ of H ₂ O ₂ for HaCaT cell line and NIH-3T3 cell line was 1 μM and 6.25 μM, respectively. To confirm the protective effect of PVP-PAA-CaCO ₃ nanoparticles containing RSV against oxidative stress, tests were conducted using each concentration of H ₂ O ₂ .

Resveratrol alone and PVP-PAA-CaCO ₃ nanoparticles containing RSV of the present invention were treated at each concentration and incubated for 30 minutes, then treated with medium containing H ₂ O ₂ at an IC ₅₀ concentration for each cell line and incubated for 30 minutes. , Cell viability was confirmed using the CCK-8 assay kit. As a result, when resveratrol was treated alone, the protective effect against oxidative stress induced by H ₂ O ₂ applied to HaCaT and NIH-3T3 cell lines could not be confirmed (FIG. 13). On the other hand, when treated with PVP-PAA-CaCO ₃ nanoparticles containing RSV of the present invention, it was shown that the cell survival rate could be significantly increased in both cell lines treated with H ₂ O ₂ over the entire concentration range (FIG. 14). , a greater protective effect against oxidative stress was confirmed than when treated with resveratrol alone. However, the effect is weaker than the protective effect against Rose Bengal, which is likely because resveratrol has a lower H ₂ O ₂ scavenging activity than other antioxidants.

4. PVP-PAA-CaCO containing RSV ₃ Measurement of antioxidant capacity of nanoparticles (DPPH test method)

Free radicals have active electrons, so they are very unstable and highly reactive, and this reactivity damages skin cells and accelerates aging. Therefore, inhibiting the production of free radicals can inhibit the aging of skin cells. The antioxidant ability of PVP-PAA-CaCO ₃ nanoparticles containing RSV of the present invention was confirmed using the DPPH test method that can confirm free radical scavenging ability. At this time, the nanoparticles were mixed at a final resveratrol concentration of 1.25, 2.5, Each was treated to 5, 10, 20, 50, and 100 μM, incubated for 30 minutes, and then DPPH antioxidant assay kit (Dojindo D678) was used.

As a result, as shown in Figure 15, compared to the control group not treated with the nanoparticles, in the case of the cell group treated with the nanoparticles, the free radical scavenging ability was found to increase as the final concentration of the active ingredient, resveratrol, increased. In particular, it was confirmed that the antioxidant effect was about 22% higher when the concentration of RSV was 50 μM, and about 50% higher at 100 μM RSV.

5. PVP-PAA-CaCO containing RSV ₃ Measurement of anti-inflammatory effects of nanoparticles

COX-2 (Cyclooxygenase-2) is an enzyme induced by growth factors, inflammatory factors, oxidative stress, and cytokines, and causes disease by inducing or worsening inflammatory reactions. iNOS (inducible nitric oxide synthase) is expressed by inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and lipopolysaccharide (LPS), producing excessive amounts of nitric acid (NO). do. As a result, NO, a type of free radical, causes cell damage and induces chronic inflammation and cell death. Resveratrol is known to have an anti-inflammatory effect, and to determine whether it can have this effect even in the state of PVP-PAA-CaCO ₃ nanoparticles containing RSV, an inflammatory response was induced in cells with LPS, and the inflammatory response as described above was induced. The effect on the expression of the molecules COX-2 and iNOS was investigated.

Raw264.7 cells were treated with PVP-PAA-CaCO ₃ nanoparticles containing RSV of the present invention at various concentrations for 30 minutes, and then treated with 1 μg of LPS. After culturing for 16 hours, cell lysate was obtained and protein quantification was performed using BCA quantitation. Then, an equal amount of 30 μg of protein was loaded and separated by SDS-polyacrylamide gel electrophoresis. The proteins separated by electrophoresis were transferred to a PVDF membrane, treated with COX-2 and iNOS antibodies, and the results were confirmed by luminescence on an X-ray film (Figure 16a), and the signal intensity was quantified using the image-J program. (FIG. 16b), at this time, the expression levels of COX-2 and iNOS in the control group not treated with LPS or RSV were calculated to be 1, respectively.

As a result, in the group treated only with LPS, COX-2 was expressed approximately 3.5 times and iNOS was expressed approximately 5.5 times compared to the control group. In comparison, when PVP-PAA-CaCO ₃ nanoparticles containing LPS and the RSV of the present invention were treated at a resveratrol concentration of 50 μM, COX-2 and iNOS expressions were reduced by about 2 each compared to the control group, and LPS and RSV of the present invention were reduced by about 2 each. In the group treated with PVP-PAA-CaCO ₃ nanoparticles containing resveratrol at a concentration of 100 μM, COX-2 expression was lowered to 1.3 times and iNOS expression was lowered to 1-fold compared to the control group, resulting in PVP-containing RSV of the present invention. It was confirmed that anti-inflammatory and antioxidant activities improved as the concentration of PAA-CaCO ₃ nanoparticles increased.

6. PVP-PAA-CaCO containing RSV ₃ Confirmation of transdermal penetration rate of nanoparticles

The transdermal penetration rate of PVP-PAA-CaCO ₃ nanoparticles containing RSV was confirmed using a Franz diffusion cell. At this time, the skin tissue used in the experiment was extracted from an ICR mouse killed by cervical dislocation. After filling the receptor chamber with the receptor phase (EtOH: PBS = 50:5 w/w), the donor and receptor phases were placed with the stratum corneum facing upward. The skin was fixed in between, and the experiment was conducted while maintaining the temperature at 37°C using a constant temperature water bath. 0.2 ml of a sample containing resveratrol alone or PVP-PAA-CaCO ₃ nanoparticles containing the RSV of the present invention containing resveratrol at the same concentration was added to the skin surface through a donor, and then 0.5 ml over time. The receptor phase was collected and analyzed through the sampling port, and the same amount of receptor phase was replenished into the chamber immediately after collection. After 24 hours, HPLC analysis was performed on the stratum corneum (stratum corneum, SC), skin, and transdermal layer. The results are shown in Figure 17.

Looking at the results, when treated with resveratrol alone (control group) and PVP-PAA-CaCO ₃ nanoparticles containing RSV (experimental group), the percentage of skin penetration compared to the resveratrol in the initial sample was about 1.1% and 17%, respectively. It was found that the PVP-PAA-CaCO ₃ nanoparticles containing RSV of the present invention penetrated the skin at a much higher rate. In addition, when the skin was treated with resveratrol alone and PVP-PAA-CaCO ₃ nanoparticles containing the RSV of the present invention, about 11% and 13.6% of resveratrol was detected, respectively, and the proportion that did not pass through and remained in the stratum corneum was They were about 7.0% and 10.5%, respectively. Through the above test results, PVP-PAA-CaCO ₃ nanoparticles containing the RSV of the present invention containing resveratrol increase not only the absorption rate across different skin layers but also the skin penetration rate, and in particular, the amount penetrated through the skin is compared to the control group. It was confirmed that it was about 17 times higher. The difference in transdermal permeation between the control and experimental groups as above appears to be partly due to the low room temperature stability of resveratrol. This is because in the control group treated with resveratrol alone, the ratio of total resveratrol detected compared to the initial sample, that is, the stratum corneum, skin, and transdermal permeation This can be confirmed by the fact that the sum of the ratios of the contents itself is low. On the other hand, in the case of PVP-PAA-CaCO ₃ nanoparticles containing the RSV of the present invention containing resveratrol, the stability of the active ingredient resveratrol increased, and the HPLC results showed that the ratio of total resveratrol detected compared to the initial sample was high. there is.

In summary, the PVP-PAA-CaCO ₃ nanoparticles containing the RSV of the present invention containing resveratrol are known to not only contain a large amount of the active ingredient resveratrol, but also have a small and uniform particle size and high drug delivery efficiency. It contained a large number of batite and calcite in the form of CaCO ₃ crystals. In addition, the nanoparticles have high stability and can increase the stability, cell protection and antioxidant efficacy, skin absorption and transdermal permeability of the active ingredient resveratrol, and can increase safety by lowering the cytotoxicity of resveratrol at high concentrations. It was found to have an anti-inflammatory effect, confirming its high usefulness as a highly functional cosmetic material.

The description of the present invention described above is for illustrative purposes, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential features of the present invention. will be. Therefore, the embodiments described above should be understood in all respects as illustrative and not restrictive. For example, each component described as single may be implemented in a distributed manner, and similarly, components described as distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the claims described below, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be construed as being included in the scope of the present invention.

Claims (11)

delete delete delete mixing resveratrol, polyvinylpyrrolidone and polyacrylic acid; and
mixing a precursor of calcium carbonate into the mixture;
Including,
The step of mixing a precursor of calcium carbonate with the mixture of resveratrol, polyvinylpyrrolidone, and polyacrylic acid,
A third mixture comprising calcium salt mixed with the mixture of resveratrol, polyvinylpyrrolidone, and polyacrylic acid; and mixing carbonates,
The third mixture and carbonate are mixed in a volume ratio of 1:8 to 1:10,
A method for producing polyvinylpyrrolidone-polyacrylic acid-calcium carbonate nanoparticles, wherein the calcium salt and carbonate are mixed at a molar ratio of 3:1 to 5:1.
According to paragraph 4,
The step of mixing resveratrol, polyvinylpyrrolidone, and polyacrylic acid,
A first mixture of resveratrol mixed with an organic solvent; and
A second mixture of polyvinylpyrrolidone and polyacrylic acid mixed in an aqueous solvent; prepared by mixing the first mixture and the second mixture, respectively, of polyvinylpyrrolidone-polyacrylic acid-calcium carbonate nanoparticles. Manufacturing method.
According to clause 5,
A method for producing polyvinylpyrrolidone-polyacrylic acid-calcium carbonate nanoparticles, wherein the first mixture contains 0.5 to 10% by weight of resveratrol.
According to clause 5,
Polyvinylpyrrolidone in the second mixture is contained in an amount of 0.1 to 10% by weight,
A method for producing polyvinylpyrrolidone-polyacrylic acid-calcium carbonate nanoparticles, wherein the second mixture contains 0.05 to 5% by weight of polyacrylic acid.
According to clause 5,
A method for producing polyvinylpyrrolidone-polyacrylic acid-calcium carbonate nanoparticles, wherein the ratio (v/v) of the first mixture and the second mixture is 1:99 to 20:80.
delete A cosmetic composition comprising the polyvinylpyrrolidone-polyacrylic acid-calcium carbonate nanoparticles of claim 4.
According to clause 10,
A cosmetic composition comprising 0.1 μM or more of resveratrol.