KR102449677B1 - Method for manufacturing biotin-containing nanoliposome - Google Patents

Abstract

The method for producing biotin-containing nanoliposomes according to an embodiment of the present invention is characterized in that the encapsulation rate of biotin is improved by using a pH adjusting agent and high pressure emulsification treatment. Using the biotin-containing nanoliposome production method of the present invention, it is possible to provide a biotin-containing nanoliposome that is physically stable and has excellent encapsulation rate and transdermal absorption rate, and it can be applied to various cosmetic compositions.

Description

Method for manufacturing biotin-containing nanoliposome

The present invention relates to a method for producing biotin-containing nanoliposomes, and more particularly, to a method for producing biotin-containing nanoliposomes with a high encapsulation rate by adjusting the pH by adding arginine.

Biotin is one of the B-complex vitamins called vitamin B7. In 1927, M. A. Boas of Germany observed that when an excessive amount of raw egg white was fed to experimental animals, symptoms such as dermatitis and hair loss appeared, and a substance preventing these symptoms existed in the liver of animals.

In 1931, P. Gorgy of Germany obtained an effective substance by concentrating a factor for preventing dermatitis and hair loss, and this substance was named vitamin H after the German word for skin (Haut). After that, it was proved that vitamin H is the same substance as biotin, and the chemical structure was also revealed, and it was chemically synthesized by S. A. Harris and colleagues in 1944.

Biotin contains sulfur and is a vitamin with low solubility in water and serves as an essential adjuvant for carboxylase enzymes in several metabolic pathways. Among the protein synthesis functions of biotin, it has been reported that, in particular, the function of keratinogenesis contributes to the growth of healthy nails and hair. Biotin has become a new trend for consumers who want healthy hair and nails as a relatively economical and effective beauty product. So far, research on biotin formulation at home and abroad is in its infancy, and there has been no reported case of a technology that can improve both stability and solubility.

Therefore, in order to develop biotin as food and cosmetics and to solve the related technical problems, stabilization technology, that is, formulation technology, is required so that it does not deteriorate in the manufacturing and distribution process and can maintain its original activity.

Recently, interest in liposome formulations as a transdermal delivery system that can be stably absorbed into the skin while maintaining the activity of active ingredients in cosmetics is increasing. Liposomes are spherical vesicles composed of phospholipids, which can encapsulate water-soluble and oil-soluble components at the same time, and thus many studies are being conducted as drug delivery carriers for active ingredients such as vitamins or drugs. The double phospholipids are a major component in the biological membrane, and the phospholipid membrane constituting the liposome also has physiological functions and properties similar to those of the biological membrane. Therefore, liposomes are skin-friendly and have excellent safety, and thus are being applied as effective drug delivery systems in pharmaceuticals and cosmetics fields.

Accordingly, in order to industrialize the liposome formulation, stability, reproducibility of encapsulation rate, uniformity of particle distribution, etc. must be supplemented. As a way to supplement these problems, E. Mayhew et al. etc.) to report a method for preparing liposomes of small particle size from a high-concentration lipid suspension. There have also been reports on the production of micro- and nano-sized liposomes with a narrower particle size distribution in a reproducible manner, focusing on the use of continuous flow microfluidics.

Liposomes can be broadly divided into MLV (Multilamella vesicle) and ULV (Unilamella vesicle) according to the size and shape of the endoplasmic reticulum according to the type and characteristics of the liposome. In particular, ULV can be classified into LUV (Large unilamella vesicle), SUV (Small unilamella vesicle), and GUV (Giant unilamella vesicle) according to the size. SUVs range from 20 to 100 nm, LUVs from 100 to 1,000 nm, and GUVs from 1,000 nm or greater. MLV can be divided into OLV (Oligo lamella vesicle), MLV, MVV (Multi vesicular vesicles) according to the type of membrane (Lamella) of the endoplasmic reticulum. OLV is 100 to 500 nm, MLV is 500 to 1,000 nm, MVV is 200 to 3,500 nm in size, and is classified according to various sizes and shapes.

In addition, the size of the endoplasmic reticulum is an important parameter that determines the half-life of the liposome, and the size and number of bilayers affect the degree of drug encapsulation in the liposome. Therefore, liposomes are typically classified based on their size and number of bilayers, and recently, a new type of liposome named double liposome and MVV has been reported. Liposomes prepared by this technique are known to enhance drug protection against various enzymes.

When nano-technology is applied to the liposome formulation, the size of the nano-vesicles, which serves to deliver specific active ingredients into the skin, becomes smaller than the cells constituting the skin. Therefore, it is known that when functional cosmetics containing nano-sized particles are used, active ingredients are absorbed and delivered more deeply into the skin.

KR 10-0753437 B1 (2007.09.14. Announcement)

It is an object of the present invention to provide a method for producing biotin-containing nanoliposomes, which improves the solubility of biotin and the stability of nanoliposomes by containing biotin, a water-soluble vitamin with low solubility, in nanoliposomes, which is a kind of drug delivery system. .

In addition, an object of the present invention is to encapsulate biotin, which is a low-solubility anti-hair loss agent component used in cosmetics, in nanoliposomes to find out the effect and availability of nanoliposomes, which are known as a method of delivering active ingredients through the skin. To determine the effect on the stability and solubility of

The method for producing biotin-containing nanoliposomes of the present invention is characterized in that the encapsulation rate of biotin is improved by using a pH adjusting agent and high pressure emulsification treatment.

The manufacturing method of the biotin-containing nanoliposomes includes the steps of: (a) preparing a first mixed solution by mixing phospholipids, cholesterol, and alcohol; (b) preparing a second mixed solution by mixing a preservative, biotin, a pH adjusting agent, and purified water; (c) heating and dissolving the first mixed solution of step (a) and the second mixed solution of step (b), respectively; (d) mixing and emulsifying the first mixed solution and the second mixed solution to prepare a composition comprising liposomes; and (e) high-pressure emulsification of the liposome of step (d) to prepare a nanoliposome.

The phospholipid may be lecithin.

Step (c) may be performed at a temperature of 70 to 75 °C for 1 to 10 minutes.

Step (d) may be performed for 5 to 15 minutes at 2,500 to 3,500 rpm by adding the first mixed solution to the second mixed solution in a home mixer.

The pH adjusting agent may be at least one selected from the group consisting of arginine, sodium hydroxide (NaOH), potassium hydroxide (KOH), and triethanolamine.

The pH adjusting agent may be used in an amount of 0.001 to 0.5% by weight based on the total weight% of the composition containing the liposome.

Step (e) may be performed 1 to 3 times at a temperature of 40 to 45° C. and a pressure of 650 to 750 bar.

In addition, the present invention may provide a biotin-containing nanoliposome prepared by the above preparation method.

The nanoliposome may have a particle size of 100 to 250 nm, a polydispersity index of 0.2 to 0.5, and a zeta potential of -80 to -30 mV.

The encapsulation rate of biotin in the nanoliposome may be 25% or more.

The transdermal absorption rate of the biotin may be 30% or more.

In addition, the present invention may provide a cosmetic composition for promoting transdermal absorption comprising the nanoliposome.

Using the biotin-containing nanoliposome production method of the present invention, it is possible to provide a biotin-containing nanoliposome that is physically stable and has excellent encapsulation rate and transdermal absorption rate, and it can be applied to various cosmetic compositions.

1 schematically shows the liposome structure and the capture model of a lipophilic or hydrophilic drug.
2 schematically shows an apparatus for producing nano-sized dispersed particles using high pressure.
3 is a sensory evaluation of biotin nanoliposomes (#3) after 1 day, 1 week, 1 month and 3 months at room temperature.
4 is a sensory evaluation of biotin nanoliposomes (#4) after 1 day, 1 week, 1 month and 3 months at room temperature.
5 shows the particle size of the nanoliposome (#2) over time (measured three times and expressed as an average value, * p < 0.05, ** p < 0.01).
Figure 6 shows the result value of the polydispersity index (PDI) of the nanoliposome (#2) over time (measured three times and expressed as an average value, * p < 0.05, ** p < 0.01).
7 shows the zeta potential value of the nanoliposome (#2) according to time (measured three times and expressed as an average value, * p < 0.05).
8 shows the particle size of the biotin-containing nanoliposome (#4) according to time (measured three times and expressed as an average value, * p < 0.05, ** p < 0.01).
9 shows the zeta potential of the biotin-containing nanoliposome (#4) with time (measured three times and expressed as an average value, * p < 0.05).
10 A shows a cryo-TEM image of a biotin-containing nanoliposome (#4-3) to which arginine is not added, and FIG. 10 B is an arginine-added biotin-containing nanoliposome according to the present invention. It shows the cryo-TEM image of (#4-4).

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those of ordinary skill in the art to which the present invention pertains can easily carry out the present invention. However, the present invention may be embodied in several different forms and is not limited to the embodiments described herein. Throughout the specification, like reference numerals are assigned to similar parts.

In the present invention, liposomes are spherical vesicles composed of phospholipids, which can encapsulate water-soluble and oil-soluble components at the same time, so it means a drug delivery carrier for active ingredients such as vitamins or drugs.

It is an object of the present invention to provide a method for producing biotin-containing nanoliposomes, which improves the solubility of biotin and the stability of nanoliposomes by containing biotin, a water-soluble vitamin with low solubility, in nanoliposomes, which is a kind of drug delivery system. .

In addition, an object of the present invention is to encapsulate biotin, an anti-hair loss agent component with low solubility used in cosmetics, in nanoliposomes to find out the effect and availability of nanoliposomes, which are known as a method of delivering active ingredients through the skin. To determine the effect on the stability and solubility of

The method for producing biotin-containing nanoliposomes according to an embodiment of the present invention for achieving the above object is characterized in that the encapsulation rate of biotin is improved by using a pH adjusting agent and high pressure emulsification treatment.

Specifically, the method for preparing the biotin-containing nanoliposomes includes the steps of: (a) preparing a first mixed solution by mixing phospholipids, cholesterol, and alcohol; (b) preparing a second mixed solution by mixing a preservative, biotin, a pH adjusting agent, and purified water; (c) heating and dissolving the first mixed solution of step (a) and the second mixed solution of step (b), respectively; (d) mixing and emulsifying the first mixed solution and the second mixed solution to prepare a composition comprising liposomes; and (e) preparing a nanoliposome by high-pressure emulsification of the liposome of step (d).

In the present invention, the step of (a) preparing a first mixed solution by mixing phospholipids, cholesterol, and alcohol may be prepared by mixing the respective raw materials, and is not particularly limited.

In the present invention, the step (b) of preparing a second mixed solution by mixing a preservative, biotin, a pH adjusting agent and purified water may be prepared by mixing the respective raw materials, and is not particularly limited.

In the present invention, the step of (c) dissolving the first mixed solution of step (a) and the second mixed solution of step (b) by heating, respectively, can be performed at a temperature of 70 to 75 ° C. for 1 to 10 minutes. have.

According to the present invention, the step (d) of preparing a composition comprising liposomes by mixing and emulsifying the first mixed solution and the second mixed solution is, in a Homer mixer, the first mixed solution is added to the second mixed solution, and 2,500 ~ It can be carried out at 3,500 rpm for 5 to 15 minutes.

The pH adjusting agent may be used in an amount of 0.001 to 0.5% by weight based on the total weight% of the composition containing the liposome.

The pH adjusting agent may be at least one selected from the group consisting of arginine, sodium hydroxide (NaOH), potassium hydroxide (KOH), and triethanolamine, and preferably arginine.

Specifically, the amount of each raw material used is, with respect to the total weight% of the composition containing the liposome, 2 to 3% by weight of the phospholipid, 0.25 to 1% by weight of the cholesterol, 10 to 20% by weight of the alcohol, 0.1 to 0.5% by weight of the preservative %, 0.05 to 0.007% by weight of the pH adjuster, and the remaining amount of purified water.

The phospholipid may be lecithin, and specifically, soybean lecithin, distearoylphosphatidylcholine, hydrogenated soybean lecithin, egg lecithin, dioleoylphosphatidylcholine, hydrogenated egg lecithin, dielaidoylphosphatidylcholine, dipalmitoylphosphatidylcholine, and It may be at least one selected from the group consisting of dimyristoylphosphatidylcholine.

The alcohol may be ethyl alcohol, but is not limited thereto.

The biotin (Biotion, chemical formula: C ₁₀ H ₁₆ N ₂ O ₃ S) is a water-soluble vitamin B series, vitamin B7 (vitamin B7), vitamin B8 (vitamin B8), vitamin H (vitamin H), or coenzyme R ( coenzyme R). Biotin can also be used as a hair and skin conditioning agent in topical formulations such as shampoos, conditioners, soaps, bath oils and salts, and makeup, where biotin is a carboxylic acid.

The preservative may be one selected from the group consisting of imidazolidinyl urea, methylparaben, propylparaben and butylparaben.

The arginine (Arginine, chemical formula: C ₆ H ₁₄ N ₄ O ₂ ) is one of α-amino acids, and L-arginine is one of the amino acids constituting the protein, and is a protein protamine present in sperm of fish. In addition, as a metabolic pathway in the body, it is a component of the ornithine cycle, and serves to decompose into urea and ornithine by the action of arginase. As a basic amino acid, it exhibits strong alkalinity and acts as an acidity regulator.

If the content is out of the above-mentioned range, the encapsulation rate of biotin may be lowered. Accordingly, there is a problem that the transdermal absorption rate of biotin is lowered, so the above range is preferable.

In addition, when biotin is encapsulated by liposomes, there is a problem that stability is lowered, so the above range is preferable.

According to the present invention, the step (e) of preparing nanoliposomes by high-pressure emulsification treatment of the composition containing the liposomes of step (d) is performed 1 to 3 times at a temperature of 40 to 45° C. and a pressure of 650 to 750 bar. It can be done, preferably at a temperature of 40 to 45 ℃, it is preferable to carry out twice at a pressure of 700 bar.

If the high pressure emulsification treatment conditions are exceeded, the above range is preferable because there are problems in that turbidity increases over time, biotin is precipitated, and the size of particles increases.

Biotin-containing nanoliposomes according to another embodiment of the present invention are prepared by the above method, have a particle size of 100 to 250 nm, a polydispersity index of 0.2 to 0.5, and a zeta potential of -80 to -30 mV characterized in that

By having the zeta potential in the above range, the repulsive force between the colloidal particles increases, and the colloidal state is stabilized.

In addition, the dispersion stability of the nano-dispersion can be improved, and there is an effect of not only physical stabilization but also electrical stabilization.

In the present invention, the nanoliposome may have an encapsulation rate of biotin of 25% or more.

In the present invention, the transdermal absorption rate of the biotin may be 30% or more.

According to another embodiment of the present invention, there is provided a cosmetic composition for promoting transdermal absorption comprising biotin-containing nanoliposomes.

The cosmetic composition for promoting transdermal absorption comprising biotin-containing nanoliposomes according to an embodiment of the present invention contains 0.1 to 20% by weight of the biotin-containing nanoliposomes, preferably 1 to 15% by weight, based on the total weight of the composition. may include.

The cosmetic composition for promoting transdermal absorption comprising the biotin-containing nanoliposome is an organic solvent, a solubilizer, a thickening agent, a gelling agent, an emollient, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, a surfactant, water, It may additionally contain ionic or nonionic emulsifiers, fillers, chelating agents, preservatives, vitamins, blocking agents, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or adjuvants commonly used in cosmetics. have.

In addition, the cosmetic composition for promoting transdermal absorption comprising the biotin-containing nanoliposome is not particularly limited in its formulation. For example, softening lotion, astringent lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, pack, powder, body lotion, body cream, body oil, body Essence, makeup base, foundation, hair dye, shampoo, rinse, body wash, ointment, may be formulated as a cosmetic composition such as a patch or spray. Each of these formulations may contain a variety of bases and additives necessary and appropriate for the formulation of the formulation.

Hereinafter, the present invention will be described in more detail by way of Examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.

<Example>

1. Preparation of materials

Lecithin (Lipoid S 75-3, Lipoid, Germany), cholesterol (Cholesterol JP, Nippon Fine Chemical Co., LTD, Japan) and arginine (L-arginine, Ajinomoto, Japan) were prepared to control the pH.

Butylene glycol (1,3-Butylene glycol, Daicel, Japan) and ethanol (Ethyl alcohol, Daejung, Korea) were prepared as solvents, and purified water was purified using a distilled water maker (Pure RO 130, Human Co., Korea). prepared (< 3 μS /cm).

Biotin (Biotin, Sigma-Aldrich, MO, USA) was prepared in HPLC grade with a purity of 99% or higher, and methanol, purified water, acetonitrile and sodium hexanesulfonate used as HPLC solvents for biotin content analysis were Sigma-Aldrich (MO, USA). ) All products were prepared in HPLC grade.

To prepare liposomes, a homomixer (T.K. Auto homomixer mark Ⅱ 2.5, Tokushukika, Japan) and a high pressure emulsifier (Nanodisperser, NLM1000, Ilshin autoclave, Korea) were used, and a pH meter (Orion star A111, Thermo scientific) was used for pH measurement. , USA) were used. A Zetasizer (Zetasizer Nano ZS system, Malvern Instrument Ltd., UK) was used to measure the particle size, zeta potential and polydispersity index (PDI) of the liposome, and the biotin encapsulation rate and biotin in the nanoliposome were quantified. HPLC (Agilent 1100, Agilent Technologies, USA) was used for this.

2. Preparation of liposomes

Preparation Examples #1-1 to #4-3 described in the following tables are comparative examples, and #4-4 is an example.

1) Preliminary experiment for preparing liposomes

① Preliminary experiment for preparing nanoliposomes under solvent conditions

In order to prepare nanoliposomes, an oil phase and a water phase composed of lecithin, cholesterol, butylene glycol and ethanol are each dissolved by heating to 70 to 75 ° C. 3,000 rpm, and emulsified for 10 min to prepare liposomes.

Here, in order to confirm the formation of nanoliposomes, liposomes were prepared with the composition shown in Table 1 below, and the prepared liposomes were subjected to high pressure emulsification treatment at 40 to 45° C. at a pressure of 700 bar and the number of passes twice to prepare nanoliposomes. did.

Ingredients (wt%) #1-1 #1-2 #1-3 #1-4 paid
(Oil phase) lecithin 2.5 2.5 2.5 2.5 cholesterol 0.5 0.5 0.5 0.5 Butylene Glycol 20.0 30.0 - - ethanol - - 10.0 15.0 Awards
(Water phase) Distilled water 76.7 66.7 86.7 81.7 preservative 0.3 0.3 0.3 0.3

② Preliminary experiment for manufacturing nanoliposomes under high pressure emulsification conditions

In order to select the high-pressure emulsification conditions, high-pressure emulsified nanoliposomes were prepared with the composition and high-pressure emulsification conditions in Table 2 below.

Here, it was carried out in the same manner as in the preliminary experiment of the liposome.

Ingredients (wt%) #2-1 #2-2 #2-3 #2-4 #2-5 #2-6 paid
(Oil phase) lecithin 2.5 2.5 2.5 2.5 2.5 2.5 cholesterol 0.5 0.5 0.5 0.5 0.5 0.5 ethanol 15.0 15.0 15.0 15.0 15.0 15.0 Awards
(Water phase) Distilled water 81.7 81.7 81.7 81.7 81.7 81.7 preservative 0.3 0.3 0.3 0.3 0.3 0.3 High pressure emulsifier conditions
(pressure/frequency) 300/1 300/2 500/1 500/2 700/1 700/2

2) Preparation of nanoliposomes

① Preparation of biotin-containing nanoliposomes

To prepare biotin-containing nanoliposomes, biotin-containing nanoliposomes were prepared under the composition and high pressure emulsification conditions of Table 3 below.

At this time, the high-pressure emulsification condition was performed at a pressure of 700 bar and the number of passages twice.

Ingredients (wt%) #3-1 #3-2 #3-3 #3-4 paid
(Oil phase) lecithin 2.5 2.5 2.5 2.5 cholesterol 0.5 0.5 0.5 0.5 Butylene Glycol 20.0 30.0 - - ethanol - - 10.0 15.0 Awards
(Water phase) Distilled water 76.6 66.6 86.6 81.6 biotin 0.1 0.1 0.1 0.1 preservative 0.3 0.3 0.3 0.3

② Preparation of biotin-containing nanoliposomes with arginine added

In order to prepare biotin-containing nanoliposomes to which alginine was added, biotin-containing nanoliposomes to which arginine was added were prepared under the composition and high pressure emulsification conditions of Table 4 below.

(The preparation of liposomes and nanoliposomes was carried out in the same manner as in the preliminary experiment.)

Ingredients (wt%) #4-1 #4-2 #4-3 #4-4 paid
(Oil phase) lecithin 2.5 2.5 2.5 2.5 cholesterol 0.5 0.5 0.5 0.5 ethanol 15.0 15.0 15.0 15.0 Awards
(Water phase) Distilled water 81.7 81.64 81.6 81.54 biotin - - 0.1 0.1 Arginine - 0.06 - 0.06 preservative 0.3 0.3 0.3 0.3

<Experimental example>

1. Liposome Stability Assessment

1) Sensory evaluation of formulation stability by temperature

Each of the prepared nanoliposomes was stored at room temperature (25 ℃), cold (4 ℃), and constant temperature (40 ℃), and the change over time was measured for each day, 1 week, 1 month, and 3 months, respectively. was confirmed by the transparency, color, separation and biotin precipitation (crystallization).

2) pH measurement

2 g of each sample was diluted with 30 mL of purified water, and the pH of the nanoliposome was measured at 25 °C with a pH meter.

3) Measurement of particle size and zeta potential

The average particle size, zeta potential, and polydispersity index of nanoliposomes were measured using a zetasizer using a dynamic light scattering method.

At this time, the temperature was kept constant at 25 ℃, and the measurement was carried out with the stock solution for accuracy. In addition, in order to confirm the stability, measurements were made at intervals of 1 day, 1 week, 1 month, and 3 months.

4) Biotin content analysis

Biotin content was analyzed and quantified by HPLC system.

At this time, C18 (150 × 4.6 mm) was used for the column, the detector was a UV detector (200 nm), the flow rate was 1.0 mL/min, the oven temperature was 40° C., and acetonitrile/purified water (0.02% phosphoric acid) as the mobile phase. = 15/85 mixed solvent was used for analysis. Biotin standards and biotin-containing nanoliposomes were analyzed by dilution with methanol (0.05% phosphoric acid) solvent.

2. Measurement of liposome encapsulation rate

The biotin encapsulation rate of nanoliposomes was measured using a dialysis membrane method (DMM).

Prepare by aliquoting nanoliposomes (1.0 mL), putting them in a dialysis membrane bag, which has a molecular weight limit of 300 Da, and sealing it completely with a clip. did. After 5 hours, the outer solution that passed through the membrane bag and the inner solution that did not pass through the membrane bag were prepared by aliquoting, respectively. Each solution was filtered with a 0.45 μm Syringe filter, and then quantified under the same conditions as for biotin content analysis in an HPLC system. In order to obtain the encapsulation rate through this, first, the biotin content in the sample passing through the dialysis membrane bag and the biotin content of the biotin-containing nanoliposome were compared and calculated by Equation 1 below. This can be confirmed by the content of biotin that is not encapsulated in the liposome. Second, the biotin content in the sample that did not pass through the dialysis membrane bag and the biotin content of the biotin-containing nanoliposome were compared and calculated by Equation 2 below.

Here, A is the biotin ratio (%) of the nanoliposome outside the membrane, B is the biotin ratio (%) of the nanoliposome inside the membrane, C ₁ is the amount of biotin of the initial nanoliposome (%), and C ₂ is the biotin ratio of the nanoliposome outside the membrane (%) The amount of biotin (%), C ₃ is the amount of biotin (%) of the nanoliposome inside the membrane.

3. Measurement of in vitro transdermal absorption (Franz diffusion cell method)

The transdermal absorption rate of biotin-containing nanoliposomes was measured using the Franz diffusion cell system (FDC-6T, Logan Instrument, USA).

The transdermal absorption rate was measured using artificial skin (Strat-M membrane, Merck Millipore, USA), and the membrane was prepared by fixing the membrane between the donor and receptor phases with the stratum corneum facing up. 50% ethanol was filled in the receptor chamber, and the temperature was maintained at 37 ± 1 °C through a constant temperature water bath while the measurement was in progress. After 24 hours, the biotin content in the sample collected from the receptor chamber was measured using HPLC. In addition, in order to measure the biotin content remaining in the membrane after 24 hours, the membrane was washed 3 times with PBS, cut off the part that did not come into contact with the receptor phase, and then the remaining part was cut using scissors. The minced membrane was placed in 10 mL of 50% ethanol, extracted and treated using an ultrasonic cleaner for 1 hour to measure the biotin content remaining in the membrane.

4. Measurement of the formation of biotin-containing nanoliposomes

The shape of the nanoliposome was measured using a cryogenic transmission electron microscope (TEM, Transmission Electron Microscope, Tecnai F20 G2, FEI Company, USA).

First, the sample was prepared by pretreatment using a cryo system.

Next, using a jet freezing device (JFD 030, BALTEC, Pfaffkkon ZH, Switzerland), the sample was rapidly frozen using liquid nitrogen refrigerant at a high pressure of 2,100 bar or higher. The sample in a physically completely fixed state by the freeze-fixing method was cut out through a freeze-fracture/etching system (MED 020 GBE, BALTEC, Pfaffikon ZH, Switzerland) and dehydrated at -90 ° C. The shape was measured in TEM while maintaining 120 °C.

4. Statistical verification

The measurement was repeated 3 times, and the measurement results were expressed as mean ± standard deviation. Significance was carried out by Student's t -test, and was expressed as * p < 0.05, ** p < 0.01 according to significance.

<Results and considerations>

1. Stability analysis of formulations

1) Analysis of formulation stability and pH measurement results by temperature

Most of the nanoliposome experimental products (#3, #4) showed a reddening phenomenon over time, and it was confirmed that the turbidity also increased. The reddening phenomenon is due to lecithin used in the preparation of liposomes, and the increase in turbidity is due to precipitation of biotin and increase in particle size.

After one week of sensory evaluation, precipitation/precipitation did not occur in all nanoliposomes #1 without biotin (Data not shown), but all of nanoliposomes #3 in which biotin was added showed precipitation. Through this, it was confirmed that there is a problem in that biotin is precipitated in the nanoliposome regardless of the solvent.

However, the biotin-added nanoliposome #4-4 did not show a precipitation phenomenon, and it was confirmed that the nanoliposome #3 and nanoliposome #4-3 were more transparent ( FIGS. 3 and 4 ).

It can be confirmed that, in the case of nanoliposome #4-4 in which the pH is increased by adding arginine, biotin precipitation in the nanoliposome and discoloration in the nanoliposome are alleviated, thereby improving the stability of the nanoliposome.

In addition, from the pH measurement results, it was confirmed that the biotin-containing nanoliposomes with increased pH had higher stability than the biotin-containing nanoliposomes that did not.

Table 5 below shows the pH and sensory evaluation of nanoliposomes.

Sample pH Sensory evaluation (after 1 week at room temperature) #3-1 4.48 ± 0.12 deposition #3-2 4.77 ± 0.18 deposition #3-3 4.62 ± 0.20 deposition #3-4 4.88 ± 0.24 deposition #4-1 7.12 ± 0.36 clear #4-2 9.17 ± 0.18 clear #4-3 4.88 ± 0.21 deposition #4-4 5.62 ± 0.12 clear

2) Particle size and zeta potential analysis

① Result of preliminary experiment for preparing nanoliposomes under solvent conditions

As a result of measuring the particle size and zeta potential of the nanoliposome using a zetasizer, it was confirmed that the particle size and zeta potential were different depending on the solvent used for the preparation. It was confirmed that the dispersion stability of the liposome particles was higher because the particle size was relatively smaller than that of the formulation using the liposome, the absolute value of the zeta potential was large, and the electrostatic repulsive force between the particles was relatively large.

In addition, in observation over time, the nanoliposomes in which ethanol was selected as the solvent showed a stable tendency in particle size and zeta potential evaluation compared to the nanoliposomes in which butylene glycol was selected as the solvent. It was selected as a solvent.

② Analysis of results of preliminary experiment for manufacturing nanoliposomes under high pressure emulsification conditions

As a result of the experiment for selecting the high pressure emulsification conditions, the high pressure (bar) of the high pressure emulsifier and the more the number of treatments, the overall particle size decreased. At low pressure, when the number of treatments was increased (1→2), the particle size decreased significantly (#2-1, #2-2), but when the pressure was relatively high, even if the number of treatments was increased, the initial particle size decreased. The decrease was not large (#2-3, #2-4, #2-5, and #2-6). In addition, it was confirmed that the particle size increased with the lapse of time, and the particle size was 100 to 250 nm (FIG. 5).

Polydispersity index (PDI) showed a tendency to decrease overall as the pressure of the high pressure emulsifier was high and the number of treatments increased. Unlike the particle size, at low pressure, when the number of treatments is increased, the initial decrease in PDI is small (#2-1, #2-2), but when the pressure is relatively large, when the number of treatments is increased, the initial decrease in PDI is slightly It can be seen that they appear larger (#2-3, #2-4, #2-5, and #2-6). In addition, it can be seen that the PDI decreases with the lapse of time, and the polydispersity index (PDI) was 0.2 to 0.5 ( FIG. 6 ).

The measured zeta potential value to confirm the dispersion stability of the nano-dispersion did not show a tendency to increase or decrease according to the pressure of the high pressure emulsifier and the number of treatments. Also, it is confirmed that the absolute value decreases or increases with the passage of time, including an error. However, the zeta potential in nanoliposome #2 is about -80 to -30 mV, which is measured to be more than ±30 mV, which is a typical stable range of zeta potential, confirming that it is electrostatically stable (FIG. 7).

In order to select high-pressure emulsification conditions, sensory evaluation of formulation stability by temperature, particle size, PDI, and zeta potential of the nanoliposomes were measured. As a result, the pressure at high pressure emulsification was 700 bar, and the number of treatments was determined twice.

③ Analysis of experimental results of biotin-containing nanoliposomes

It can be confirmed that the nanoliposome in which ethanol is relatively selected as the solvent in the biotin-added nanoliposome is better than the nanoliposome in which butylene glycol is selected as the solvent in sensory evaluation of formulation stability over time, particle size and zeta potential according to temperature. .

However, in sensory evaluation after 1 week of storage at room temperature, biotin was precipitated in all 4 experimental samples (#3). In addition, it was confirmed that the pH of the nanoliposome was significantly lower than that of the previous experimental products in the range of 4.0 to 5.0. It can be confirmed that biotin is precipitated after 1 week due to an increase in the rate at which the biotin component is relatively precipitated due to the decrease in solubility in the nanoliposome. This phenomenon was also confirmed in cold and constant temperature test samples.

④ Analysis of arginine-added biotin-containing nanoliposome experiment results

As a result of measuring the pH of #4-2 and #4-4 to which arginine was added, the pH was increased. In particular, the pH was 5.62 in the biotin-containing nanoliposome #4-4, despite the addition of biotin at room temperature, after 1 week. In the sensory evaluation, it was confirmed that the precipitation phenomenon did not appear like nanoliposomes without biotin added. In addition, in the case of nanoliposomes in which the pH was increased by adding arginine, redness did not appear even after time. This means that arginine not only increases the solubility of biotin by binding with biotin, but also has an effect on the components of lecithin.

It can be confirmed that the particle size of the nanoliposomes (#4-4) to which biotin and arginine are added is also the smallest in observation over time (FIG. 8). In addition, as in the nanoliposome (#2), the zeta potential value with time decreases or rather increases, but it is judged that an error is included, but when compared with the biotin-added nanoliposome (#4-3), the arginine-added nano It was confirmed that the zeta potential of the liposome (#4-4) was larger ( FIG. 9 ).

Therefore, nanoliposomes containing 0.1% biotin and 0.06% arginine (#4-4) under the nanoliposome manufacturing conditions (pressure 700 bar, number of times of treatment twice) were the most stable in the time-dependent formulation stability sensory evaluation, particle size, and zeta potential evaluation. can be checked

⑤ Biotin content analysis

Stability was improved in terms of physical properties by adding arginine to nanoliposomes containing biotin. In order to check whether chemical stability is maintained by the addition of arginine to biotin, HPLC measurement by concentration of biotin aqueous solution and HPLC measurement by concentration of arginine-added aqueous solution were performed.

Although the HPLC analysis result of the biotin aqueous solution and the HPLC analysis result of arginine-added biotin showed similar results, it was confirmed that the biotin content decreased by about 12 to 14% when arginine was added, and the experimental results are shown in the table below. 6 is shown.

Sample in water Amount of biotin Biotin 0.04% 0.0438 ± 0.0012 Biotin 0.02% 0.0211 ± 0.0008 Biotin 0.01% 0.0105 ± 0.0014 Biotin 0.005% 0.0051 ± 0.0001 Biotin 0.001% 0.0010 ± 0.0002 Biotin 0.04% + Arginine 0.0024% 0.0361 ± 0.0021 Biotin 0.02% + Arginine 0.0012% 0.0176 ± 0.0014 Biotin 0.01% + Arginine 0.0006% 0.0087 ± 0.0007 Biotin 0.005% + Arginine 0.0003% 0.0044 ± 0.0005 Biotin 0.001% + Arginine 0.00006% 0.0008 ± 0.0001

2. Analysis of biotin-containing nanoliposome encapsulation rate measurement results

Unencapsulated and encapsulated biotin contents in the nanoliposomes without arginine (#4-3) and arginine-added nanoliposomes (#4-4) can be calculated from Equations 1 and 2.

As a result of calculating the encapsulation rate and conversion yield of biotin by substituting the biotin component content into Equation 1 and Equation 2 through HPLC analysis, in the case of nanoliposome #4-3, the encapsulation rate was 5.60% and the recovery rate was 96.29%. indicated. In addition, nanoliposome #4-4 showed an encapsulation rate of 27.64% and a recovery rate of 110.20%.

Table 7 below shows the biotin-containing nanoliposome encapsulation rate by the Dialysis membrane method.

sample Amount % Encapsulation %
(Capsulation efficiency) Nanoliposome #4-3 (initial) 0.1072 ± 0.0001 5.60 ± 0.17 Nanoliposome #4-3 (outside the membrane) 0.0974 ± 0.0187 Nanoliposome #4-3 (inside membrane) 0.0060 ± 0.0002 Nanoliposome #4-4 (initial) 0.1147 ± 0.0001 27.64 ± 0.41 Nanoliposome #4-4 (external membrane) 0.0947 ± 0.0003 Nanoliposome #4-4 (inside membrane) 0.0317 ± 0.0004

Through this, it was confirmed that the biotin encapsulation rate was increased by about 5 times in the biotin-containing nanoliposome to which arginine was added as compared to the biotin-containing nanoliposome in which the arginine was not added. In addition, it can be confirmed that the solubility increase without precipitation of biotin according to the addition of arginine can improve not only physical stability but also the encapsulation rate of biotin in preparing biotin-containing nanoliposomes.

3. Skin absorption rate results of biotin-containing nanoliposomes

The in vitro transdermal absorption rate evaluation of biotin was performed twice using baotin nanoliposomes (#4-4) with arginine added. After 24 hours using the Franz diffution cell method using artificial skin, biotin Giafid was measured by HPLC in doner, receptor, and membrane, respectively. The transdermal absorption rate (%) and recovery rate (%) of biotin were calculated using Equations 3 and 4 below for each of the measured biotin analysis results.

[Equation 3]

[Equation 4]

(Where D is the skin absorption rate (%) of biotin, E is the recovery rate (%) of biotin, F ₁ is the amount of biotin (μg) of the initial nanoliposome (#4-4), and F ₂ is the receptor (Receptor) ) is the amount of biotin in the state (㎍), F ₃ is the amount of biotin in the membrane (㎍), and F ₄ is the amount of biotin in the donor state (㎍).)

Table 8 below shows the content and diffusion rate of biotin-containing nanoliposomes (#4-4).

Sample sampling location sheep(%) Nanoliposome #4-4 initial state
(Original phaes)_F1 95.28 ± 0.47 donor status
(Donor phase)_F4 50.65 ± 3.28 Membrane_F3 - Receptor phase_F2 32.50 ± 2.58

As a result of measuring the transdermal absorption rate of biotin-containing nanoliposomes (#4-4) through the Franz diffusion cell method, 32.50 μg of biotin was detected in the receptor phase after 24 hours, and residual biotin in the membrane was not detected. Through this, the transdermal absorption rate of the biotin-containing nanoliposome was confirmed to be 34.11%, and the recovery rate was confirmed to be 87.27%.

4. Analysis of the shape of biotin-containing nanoliposomes

As a result of Cryo-TEM measurement to observe the shape of the biotin-containing nanoliposome, the particle size was similar to the particle size measured with a zetasizer, about 100 to 150 nm, and it was confirmed that it had a ULV shape (FIG. 10). ).

In addition, in the case of nanoliposomes encapsulating biotin, needle-shaped biotin was precipitated in a crystalline state both inside and outside the liposome, whereas biotin-containing nanoliposomes containing arginine hardly showed a crystalline state of biotin. .

Therefore, it can be confirmed that the arginine-added biotin-containing nanoliposomes prepared by the present invention are stable without biotin precipitation.

As a result, the biotin-containing nanoliposomes prepared according to an embodiment of the present invention have a particle size (particle size over time, 3 months) of 100 to 250 nm, polydispersity index 0.2 to 0.5, zeta potential -80 to -30 mV And, it can be confirmed that the size of the nanoliposome is about 100-150 nm by measuring the cryo-TEM image, and the nanoliposome of the ULV type is formed in a small range in the LUV of 100-1000 nm.

Claims (14)

(a) preparing a first mixed solution by mixing phospholipids, cholesterol and ethanol;
(b) preparing a second mixed solution by mixing a preservative, biotin, a pH adjusting agent, and purified water;
(c) heating and dissolving the first mixed solution of step (a) and the second mixed solution of step (b), respectively;
(d) mixing and emulsifying the first mixed solution and the second mixed solution to prepare a composition containing liposomes; and
(e) high-pressure emulsification treatment of the composition containing the liposome of step (d) to prepare a nanoliposome,
The pH adjusting agent is arginine, and is used in an amount of 0.001 to 0.5% by weight based on the total weight% of the composition,
The step (e) is performed 1 to 3 times at a temperature of 40 to 45 ° C, and a pressure of 650 to 750 bar,
Method for producing biotin-containing nanoliposomes. delete According to claim 1,
The phospholipids are
characterized in that lecithin (Lecithin),
Method for producing biotin-containing nanoliposomes. According to claim 1,
Step (c) is,
characterized in that it is carried out for 1 to 10 minutes at a temperature of 70 to 75 ℃,
Method for producing biotin-containing nanoliposomes. According to claim 1,
Step (d) is,
In a home mixer, the first mixed solution is added to the second mixed solution, characterized in that it is performed for 5 to 15 minutes at 2,500 to 3,500 rpm,
Method for producing biotin-containing nanoliposomes. delete delete According to claim 1,
Step (e) is,
Characterized in that the temperature of 40 ~ 45 ℃, performed 1 to 3 times at a pressure of 650 ~ 750 bar,
Method for producing biotin-containing nanoliposomes. Prepared by the method of any one of claims 1, 3, 4, 5 and 8,
Biotin-containing nanoliposomes. 10. The method of claim 9,
The nanoliposome has a particle size of 100 to 250 nm, a polydispersity index of 0.2 to 0.5, and a zeta potential of -80 to -30 mV,
Biotin-containing nanoliposomes. 10. The method of claim 9,
characterized in that the encapsulation rate of biotin in the nanoliposome is 25% or more,
Biotin-containing nanoliposomes. 10. The method of claim 9,
characterized in that the transdermal absorption rate of the biotin is 30% or more,
Biotin-containing nanoliposomes. Including the biotin-containing nanoliposome of claim 9,
A cosmetic composition for promoting percutaneous absorption. The cosmetic composition for promoting transdermal absorption according to claim 13, wherein the cosmetic composition comprises 0.1 to 20% by weight of nanoliposomes based on the total weight of the composition.

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