KR102737831B1 - Cosmetic composition for improving skin wrinkles containing exosomes - Google Patents

Abstract

The present invention relates to a cosmetic composition for improving skin wrinkles containing exosomes and, more specifically, to a cosmetic composition containing: 100 parts by weight of an effective ingredient; and 0.1 to 10 parts by weight of a polymersome containing exosomes, wherein the effective ingredient contains sodium hyaluronate, collagen, and hydrolyzed elastin. A cosmetic composition for improving skin wrinkles, comprising the above ingredients, can be applied to the MTS method, and exhibits an excellent skin wrinkle improvement effect due to an improved skin penetration effect and a skin soothing effect without causing skin troubles.

Description

{COSMETIC COMPOSITION FOR IMPROVING SKIN WRINKLES CONTAINING EXOSOMES}

The present invention relates to a cosmetic composition for improving skin wrinkles containing exosomes, and more specifically, to a cosmetic composition for improving skin wrinkles containing exosomes which is applicable to an MTS method and exhibits an excellent skin wrinkle improvement effect due to an improvement in skin penetration effect, and also exhibits a skin soothing effect without causing skin trouble.

Professor Dan Belsky's research team at Duke University in the United States conducted a 12-year follow-up study of 954 people and discovered that aging begins at age 26 and progresses most rapidly when the body age reaches 38. The research team conducted the study on adults living in New Zealand who were born between 1972 and 1973. In order to measure the inside of the body, they used indicators such as kidneys, liver, lungs, metabolic and immune functions, cholesterol levels, cardiopulmonary functions, and dental conditions. They also measured body age using 18 items in total, including the length of telomeres, which control cell division at the end of chromosomes and determine aging, and the condition of capillaries behind the eyes. As aging begins, skin regeneration cells also decrease at the age of 25. In addition to aging, the skin of modern people, which is exposed to various external harmful environments and stress and suffers various damages, experiences wrinkles, loss of elasticity, dryness, and other skin aging symptoms as physiological activity decreases with age. As the average life expectancy increases, the need for cosmetics to improve these aging symptoms has become greater. Based on the development of biochemistry and molecular biology, small amounts of signaling substances such as growth factors that exist in our bodies have been discovered, and the theory of aging in our bodies is being reestablished based on this. In addition, these signaling substances decrease in our bodies as we age, and this shows that the decrease in collagen, elastin, and hyaluronic acid is closely related to our aging. As these three components decrease with age, skin aging phenomena such as decreased skin elasticity, wrinkles, and pigmentation appear. Various cosmetics are being developed to improve phenomena such as collagen destruction, decreased collagen synthesis, loss of elastin, and melanin pigmentation due to decreased skin physiology, which occur in the skin epidermis and dermis.

The skin, the largest organ in the human body, has an area of 18 m2, a thickness of 2 to 4 mm, and a weight of 3 kg. It is composed of several different layers, each with a specialized function, and the main layers are the epidermis and dermis. The epidermis is the outermost layer of the skin and is divided into five layers: the stratum corneum, the stratum lucidum, the stratum granulosum, the stratum spinosum, and the stratum basale.

Between the transparent layer and the granular layer in the epidermis is a moisture barrier, which is a special chemical barrier that prevents the penetration of foreign substances from the outside and prevents moisture from escaping from the inside of the skin. Due to this moisture barrier, not only foreign substances but also water cannot penetrate inside, which makes it difficult for the effective ingredients of cosmetics to penetrate deep into the skin. The dermis layer accounts for more than 90% of the skin tissue, so it is called the dermis because it is the real skin that determines the shape of the skin. The dermis layer is divided into the papillary layer and the reticular layer. The reticular layer, located below the papillary layer, is a hard, irregular mesh-shaped connective tissue that makes up most of the dermis and is composed of collagen, elastin, hyaluronic acid, etc.

Meanwhile, the MTS (Microneedle Therapy System), a physical percutaneous absorption method, is a microneedle with established safety and effectiveness in the human body, and has been proven to be an effective method for promoting the absorption of cosmetic formulations. It is a device that physically creates a very fine planned passage of 0.25 mm to 0.5 mm to increase the absorption rate of effective ingredients by more than 80%. Even if it is a low-molecular cosmetic, it is difficult to exceed 10% in simple application, so micro-gaps are physically created to increase the absorption rate. Even if it is proven to be safe with home-use needles, wounds may occur due to micro-needles.

Based on the development of biochemistry and molecular biology, small amounts of signaling substances such as growth factors that exist in our bodies have been discovered, and the theory of aging in the body is being reestablished based on this. In addition, these signaling substances decrease in the body as we age, and it has been revealed that the decrease in collagen, hyaluronic acid, and elastin is closely related to our aging. Collagen is a type of protein composed of amino acids, and it acts as a foundation for cell activity by binding cells and tissues like glue, and exists between cells in the body to connect all cells.

Elastin is a spring-shaped light protein that exists between collagen and is entangled in a marriage tissue. Because it has a spring structure, if the elastin content decreases, the structure becomes loose, making it easy for the skin to sag and wrinkles to form. Hyaluronic acid is a type of acidic mucopolysaccharide that is widely present in the connective tissue of animals such as eye cells, chicken combs, and synovial fluid, and is a high molecular weight substance with N-acetyl-D-glucosamine and D-glucuronic acid as repeating units. It has a large molecular weight and very high water absorption capacity compared to other mucopolysaccharides. It is reported that 1 g of hyaluronic acid occupies a volume of about 1 L in water. Before skin aging, the physiological activity of skin cells is active. In the fibroblasts in the dermis, collagen synthesis and metabolism are active, and the synthesis of glycoproteins including hyaluronic acid and elastin, GAGS, is also active, giving the skin elasticity, softness, flexibility, and moisture. However, the skin of modern people is exposed to various external harmful environments and stresses, and is damaged in various ways, and as they age, the physiological activity of the skin decreases and the recovery speed of damaged skin slows down, causing various skin aging phenomena such as loss of elasticity, dryness, wrinkle formation, pigmentation, liver spots, and dullness. To solve these skin problems, a cosmetic composition containing the relevant raw materials may be required.

In addition, liposomes are widely used not only in the pharmaceutical field but also in cosmetics and food because they have the advantage of being able to stably deliver physiologically active ingredients. They are used as injections such as intravenous injections and spinal injections, and are also used as ointments applied to wounds. This is to promote skin regeneration by allowing the effective ingredients in the ointment to penetrate into the dermis rather than the epidermis, which is the outer surface of the skin. This property is also being used in the cosmetics field. The usefulness of liposomes in cosmetics is that they encapsulate water-soluble active ingredients with low absorption and affinity for hydrophobic skin into liposomes made of phospholipids, and they have the property of improving the problem of skin absorption while passing through the moisture barrier of the epidermis. Liposomes are largely divided into multilamellar vesicles (MLV) and unilamellar vesicles (ULV). ULV can be divided into large unilamellar vesicles and small unilamellar vesicles. MLV is 500–5,500 nm (nanometers) in size and the amount that can be incorporated into liposomes is 5–15%, LUV is 200–800 nm in size and the amount that can be incorporated into liposomes is 36–65%, and SUV is 20–150 nm in size and the amount that can be incorporated into liposomes is 0.5–1.0%. LUV can contain the largest amount of physiologically active substances, but it is unstable, and the structure of human skin is a multilamellar structure, so MLV has an affinity for the skin, but its size is too large to penetrate the skin. Therefore, liposome particles used in cosmetics must have stability and effectiveness for delivery into the skin, so it is thought that it is higher when it is in the SUV form. In addition, the penetration rate can be increased by splitting the molecular weight of high molecular substances larger than the skin layer, such as collagen, elastin, and hyaluronic acid, through the liposome method. The particle size should be at least hydrophilic and a phospholipid similar to the skin to deliver the active ingredients deep into the skin.

Korean Patent Registration No. 10-0564108 (2006.03.20.) Korean Patent Registration No. 10-2224032 (2021.03.02.)

The purpose of the present invention is to provide a cosmetic composition for improving wrinkles in the skin, containing exosomes, which is applicable to the MTS method and exhibits an excellent wrinkle improvement effect due to improved skin penetration effect, as well as a skin soothing effect without causing skin trouble.

The object of the present invention is achieved by providing a cosmetic composition for improving skin wrinkles containing exosomes, comprising 100 parts by weight of an effective ingredient and 0.1 to 10 parts by weight of polymersomes containing exosomes, wherein the effective ingredients contain sodium hyaluronate, collagen and hydrolyzed elastin, and the sodium hyaluronate, collagen and hydrolyzed elastin are emulsified to a size of 120 to 200 nanometers.

According to a preferred feature of the present invention, the effective ingredients are composed of glycerin, purified water, caprylic/capric triglyceride, centella asiatica extract, hydrogenated lecithin, cholesterol, sodium hyaluronate, collagen, hydrolyzed elastin, polylactic acid, and human-derived stem cell culture solution.

According to a more preferred feature of the present invention, the effective ingredient comprises 100 parts by weight of glycerin, 18 to 22 parts by weight of purified water, 7 to 9 parts by weight of caprylic/capric triglyceride, 2.5 to 3 parts by weight of centella asiatica extract, 2.5 to 3 parts by weight of hydrogenated lecithin, 0.5 to 1.0 parts by weight of cholesterol, 0.1 to 0.15 parts by weight of sodium hyaluronate, 0.1 to 0.15 parts by weight of collagen, 0.1 to 0.15 parts by weight of hydrolyzed elastin, 0.1 to 0.15 parts by weight of polylactic acid, and 0.01 to 0.02 parts by weight of a human-derived stem cell culture solution.

According to a more preferred feature of the present invention, the purslane extract is prepared by mixing 200 to 1,000 parts by weight of a lower alcohol having 1 to 4 carbon atoms with 100 parts by weight of purslane and extracting the mixture.

According to a further preferred feature of the present invention, the cosmetic composition for improving skin wrinkles containing the exosome is applied in one formulation selected from the group consisting of a toner, an eye cream, a moisturizing cream, a cleansing product, a serum, and an ampoule.

The cosmetic composition for improving wrinkles in the skin containing exosomes according to the present invention is applicable to the MTS method and not only exhibits an excellent wrinkle improvement effect due to the improvement of skin penetration effect, but also exhibits an excellent effect of providing a cosmetic composition that exhibits a skin soothing effect without causing skin trouble.

Hereinafter, preferred embodiments of the present invention and the properties of each component will be described in detail. However, this is intended to be a detailed description to enable a person having ordinary skill in the art to which the present invention pertains to easily carry out the invention, and does not mean that the technical idea and scope of the present invention are limited thereby.

The cosmetic composition for improving skin wrinkles containing exosomes according to the present invention is composed of polymersomes containing exosomes in the active ingredient, and is preferably composed of 0.1 to 10 parts by weight of polymersomes containing exosomes per 100 parts by weight of the active ingredient.

The above effective ingredients include sodium hyaluronate, collagen, and hydrolyzed elastin, and the effective ingredients containing the above ingredients exhibit excellent skin anti-aging and wrinkle improvement effects.

In addition, the sodium hyaluronate, collagen and hydrolyzed elastin above serve to supplement the sodium hyaluronate, collagen and hydrolyzed elastin components that decrease in the dermis layer, which decreases with age, thereby providing an anti-aging and wrinkle-prevention effect on the skin.

In addition, it is preferable to liposomeize the above-mentioned sodium hyaluronate, collagen, and hydrolyzed elastin through liposome method so that they can penetrate deep into the skin. At this time, it is preferable to liposomeize them by mixing polymersomes containing exosomes as described above.

In addition, it is preferable that the above sodium hyaluronate, collagen and hydrolyzed elastin are stirred in a high-pressure homogenizer while heated to a temperature of 75 to 80°C, cooled to 40°C, and emulsified using a microfluidizer (MFD) so that the average particle size is 120 to 200 nanometers.

As described above, if the microemulsion device is used for processing 7 to 10 times, the average particle size will be approximately 120 to 150 nanometers, and if the microemulsion device is used for processing 10 to 20 times, the average particle size will be 150 to 200 nanometers.

Sodium hyaluronate, collagen and hydrolyzed elastin exhibiting the above-mentioned average particle size have significantly improved skin penetration effects.

At this time, it is preferable that the effective ingredients are composed of glycerin, purified water, caprylic/capric triglyceride, centella asiatica extract, hydrogenated lecithin, cholesterol, sodium hyaluronate, collagen, hydrolyzed elastin, polylactic acid, and human-derived stem cell culture solution, and the effective ingredients are 100 parts by weight of glycerin, 18 to 22 parts by weight of purified water, 7 to 9 parts by weight of caprylic/capric triglyceride, 2.5 to 3 parts by weight of centella asiatica extract, 2.5 to 3 parts by weight of hydrogenated lecithin, 0.5 to 1.0 parts by weight of cholesterol, 0.1 to 0.15 parts by weight of sodium hyaluronate, 0.1 to 0.15 parts by weight of collagen, 0.1 to 0.15 parts by weight of hydrolyzed elastin, It is more preferable that the composition comprises 0.1 to 0.15 parts by weight of polylactic acid and 0.01 to 0.02 parts by weight of a human-derived stem cell culture medium.

If the content of sodium hyaluronate is less than 0.1 part by weight relative to 100 parts by weight of the above glycerin, the anti-aging effect is reduced, and if the content of sodium hyaluronate exceeds 0.15 parts by weight, the viscosity of the cosmetic composition for improving wrinkles manufactured through the present invention excessively increases, making it difficult to apply it to the MTS method.

In addition, the extract of the purslane is contained in an amount of 0.1 to 5 parts by weight, and has excellent wound healing and skin regeneration effects, and has effects such as improving inflammation, soothing the skin, and anti-inflammatory effects, thereby playing a role in improving skin troubles.

At this time, the above-mentioned purslane extract is prepared by mixing 200 to 1,000 parts by weight of lower alcohol having 1 to 4 carbon atoms with 100 parts by weight of purslane and extracting it. The purslane extract prepared through the above process, in addition to the above-mentioned effects, exhibits effects such as inhibition of NO activity, reduction of histamine secretion, reduction of RANTES content, reduction of collagenase activity, and reduction of MMP-1 expression level, thereby imparting anti-inflammation, anti-wrinkle, anti-allergy, etc. effects and exhibiting excellent skin elasticity improvement and skin wrinkle improvement effects.

If the content of the above-mentioned purslane extract is less than 0.1 parts by weight, the above-mentioned effect is minimal, and if the content of the above-mentioned purslane extract exceeds 5 parts by weight, the above-mentioned effect is not significantly improved, but the formulation stability of the cosmetic composition may be reduced, which is not preferable.

The above human-derived stem cell culture medium refers to stem cells obtained from adults, excluding embryonic stem cells, and includes, but is not limited to, stem cells originating from the base of blood and tissues, including fat, bone marrow, umbilical cord blood, and peripheral blood.

The use of human-derived stem cells in the cosmetics field utilizes growth factors as their metabolites. Growth factors are proteins that stimulate cell proliferation and differentiation by binding to receptors on the surface of cells, and include EGF (epidermal growth factor), TGF (transforming growth factor), HGF (hepatocyte proliferation factor), FGF (fibroblast growth factor), IGF (insulin-like growth factor), SOD (antioxidant enzyme), collagen, and fibronectin.

The polymersome containing the exosome is contained in an amount of 0.1 to 10 parts by weight, and is manufactured by adding palmitoyl tripeptide to a mixture of exosomes and polyethylene glycol in a ratio of 5:2, and exhibits an advantage in that the polymersome does not adsorb to the molecular bilayer vesiculopeptide and the thin film of the polymersome through self-association of biocompatible and biodegradable polymers similar to lipids, thereby fundamentally blocking denaturation of the effective ingredient as well as the effective substance such as the peptide inside, thereby completely maintaining the inherent activity.

The above polymersomes have a particle diameter of 300 to 1000 nanometers and are synthesized in a size that can be absorbed by the skin. The palmitoyl tripeptide is preferably contained in an amount of 0.0001 to 1 wt% based on the total weight of the polymersomes. The polymersomes composed of the above components not only enhance the skin penetration effect of the effective ingredient, but also further enhance the skin aging prevention effect because the polymersomes themselves contain the palmitoyl tripeptide component.

If the content of the above polymersome is less than 0.1 parts by weight, the above effect is minimal, and if the content of the above polymersome exceeds 10 parts by weight, the content of the effective ingredient is relatively excessively reduced, so not only is the effect of improving skin wrinkles reduced, but also the viscosity of the cosmetic composition becomes excessively high, making it difficult to apply it to the MTS method.

The cosmetic composition for improving skin wrinkles, comprising the above ingredients, is not particularly limited in its formulation, but considering the frequency of use or the area to which it is applied on the skin for the effect of improving skin wrinkles, it is preferable to apply it in one formulation selected from the group consisting of toner, eye cream, moisturizing cream, cleansing product, serum, and ampoule.

Hereinafter, a method for manufacturing a cosmetic composition for improving skin wrinkles containing exosomes according to the present invention and the properties of a cosmetic composition manufactured by the method will be described with examples.

<Manufacturing Example 1>

Sodium hyaluronate, collagen, and hydrolyzed elastin were each heated to 78°C, stirred in a high-pressure homogenizer, cooled to 40°C, and used as a microemulsifier (Microfluidizer, MFD) to have an average particle size of 120 to 200 nanometers.

<Manufacturing Example 2> Manufacturing of a bottle gourd extract

A centella asiatica extract was prepared by mixing 100 parts by weight of centella asiatica and 800 parts by weight of ethanol having a mass concentration of 50%, placing the mixture in a microwave oven, and heating it at 90 W for 5 minutes.

<Manufacturing Example 3> Manufacturing of effective ingredients

An effective ingredient was prepared by mixing 73.99 g of glycerin, 15 mL of purified water, 6 g of caprylic/capric triglyceride, 2 g of hydrogenated lecithin, 0.6 g of cholesterol, 0.1 g of sodium hyaluronate, 0.1 g of collagen, and 0.1 g of hydrolyzed elastin prepared through the above Manufacturing Example 1, 2 g of the centella asiatica extract prepared through the above Manufacturing Example 2, 0.1 g of polylactic acid, and 0.01 g of human-derived stem cell culture medium (EGF, epidermal growth factor).

<Manufacturing Example 4> Manufacturing of polymersomes containing exosomes

After dissolving exosomes and polyethylene glycol in a 5:2 weight ratio in chloroform, 1 wt% of palmitoyl tripeptide-5 was added based on the total weight of the reaction mixture, and polymersomes were synthesized using microfluidics technology. After removing the chloroform and curing, polymersomes containing exosomes were manufactured.

<Example 1>

5 parts by weight of polymersomes containing exosomes manufactured through Manufacturing Example 4 were mixed with 100 parts by weight of the effective ingredient manufactured through Manufacturing Example 3, and stirred at a speed of 150 rpm for 10 minutes to manufacture a cosmetic composition for improving skin wrinkles containing exosomes.

<Example 2>

The same procedure as in Example 1 was followed, except that 0.1 part by weight of the polymersome containing exosomes manufactured through Manufacturing Example 4 was mixed to manufacture a cosmetic composition for improving skin wrinkles containing exosomes.

<Example 3>

The same procedure as in Example 1 was followed, except that 10 parts by weight of the polymersomes containing exosomes manufactured through Manufacturing Example 4 were mixed to manufacture a cosmetic composition for improving skin wrinkles containing exosomes.

<Comparative Example 1>

The active ingredient was prepared by mixing 73.99 g of glycerin, 15 mL of purified water, 6 g of caprylic/capric triglyceride, 2 g of hydrogenated lecithin, 0.6 g of cholesterol, 0.1 g of sodium hyaluronate, 0.1 g of collagen, and 0.1 g of hydrolyzed elastin.

The properties of a cosmetic composition for improving skin wrinkles using polylactic acid (PLLA) manufactured through the above Example 1 were measured and are shown in Table 1 below.

<Table 1>

In addition, the skin toxicity of the cosmetic composition for improving skin wrinkles containing exosomes manufactured through Example 1 of the present invention was measured and shown in Table 3 below.

[However, skin toxicity was determined using the ISO 10993-5 method and graded based on the criteria in Table 2 below. A survival rate of 70% or higher is determined to be non-cytotoxic.

* Cell line: Vero Cell (African Green Monkey, KCLB No - 10081)

* Reagents used: Dubelcco’s Modified Eagle’s Medium (DMEM, Sigma), Kanamycin sulfate (SAMCHUN), Sodium bicarbonate (DUKSAN), Antibiotic-Antimyotic (GenDEPOT), Fetal bovine serum (GenDEPOT)

* Solvent: DMEM medium + 10% FBS

* Test conditions: After contacting the cells and sample lysate for 24 hours, treat with Thiazolyl Blue Tetrazolium Bromide reagent and measure absorbance at 570 nm.

* Control group: Negative control group - Phosphate Buffer Saline, Positive control group - Dimethyl sulfoxide, Solvent control group - Solvent

* How to display results: The survival rate (%) was calculated using the following formula.

Survival rate (%) = {(Sample treatment group OD570 / Sample untreated group OD570) / (Sample untreated group OD570 - Sample untreated group OD650)} × 100]

<Table 2>

<Table 3>

As shown in Table 3 above, it can be seen that the cosmetic composition for improving wrinkles containing exosomes manufactured through Example 1 of the present invention has very low skin toxicity.

In addition, the wrinkle improvement effect and skin soothing effect of the cosmetic composition for wrinkle improvement containing exosomes manufactured through Examples 1 to 3 and Comparative Example 1 were measured and shown in Table 4 below.

{However, the effect of improving skin wrinkles was measured using a method in which the cosmetic composition was manufactured and applied to 50 women in their 30s to 50s with wrinkles around the eyes at a dose of 2g per day for 4 weeks, and the degree of improvement was measured using a 5-point scale and expressed as the average value.

5 points: Much improved, 4 points: Somewhat improved, 3 points: No improvement, 2 points: Worse, 1 point: Much worse

In addition, the skin soothing effect was measured using a method in which 40 female test subjects in their 10s to 40s with skin rashes applied 2g of the manufactured cosmetic composition to the area where the rash occurred every day for 4 weeks, and the degree of improvement was measured using a 5-point scale and expressed as the average value.

5 points: much improvement, 4 points: some improvement, 3 points: no improvement, 2 points: worse, 1 point: much worse}

<Table 4>

As shown in Table 4 above, it can be seen that the cosmetic compositions manufactured through Examples 1 to 3 of the present invention exhibit superior skin wrinkle improvement and skin soothing effects compared to the cosmetic composition manufactured through Comparative Example 1.

Therefore, the cosmetic composition for improving skin wrinkles containing exosomes according to the present invention is applicable to the MTS method and not only exhibits an excellent skin wrinkle improvement effect due to the improvement of skin penetration effect, but also provides a cosmetic composition that does not cause skin troubles due to low skin toxicity and exhibits a skin soothing effect.

Claims (5)

It consists of 100 parts by weight of an effective ingredient and 0.1 to 10 parts by weight of a polymersome containing exosomes.
The above effective ingredients contain sodium hyaluronate, collagen and hydrolyzed elastin.
The above sodium hyaluronate, collagen and hydrolyzed elastin are emulsified to 120 to 200 nanometers.
The above effective ingredients are composed of 100 parts by weight of glycerin, 18 to 22 parts by weight of purified water, 7 to 9 parts by weight of caprylic/capric triglyceride, 2.5 to 3 parts by weight of centella asiatica extract, 2.5 to 3 parts by weight of hydrogenated lecithin, 0.5 to 1.0 parts by weight of cholesterol, 0.1 to 0.15 parts by weight of sodium hyaluronate, 0.1 to 0.15 parts by weight of collagen, 0.1 to 0.15 parts by weight of hydrolyzed elastin, 0.1 to 0.15 parts by weight of polylactic acid, and 0.01 to 0.02 parts by weight of a human-derived stem cell culture solution.
The above human-derived stem cell culture solution is composed of at least one selected from the group consisting of TGF (transforming growth factor), HGF (hepatocyte growth factor), FGF (fibroblast growth factor), IGF (insulin-like growth factor), SOD (antioxidant enzyme), collagen, and fibronectin.
A cosmetic composition for improving skin wrinkles containing exosomes, characterized in that the polymersome containing the exosome is prepared by adding palmitoyl tripeptide to a mixture of exosomes and polyethylene glycol in a ratio of 5:2.
delete delete In claim 1,
A cosmetic composition for improving skin wrinkles containing exosomes, characterized in that the above-mentioned purslane extract is prepared by mixing 100 parts by weight of purslane and 200 to 1,000 parts by weight of lower alcohol having 1 to 4 carbon atoms and extracting the mixture.
In claim 1 or 4,
A cosmetic composition for improving skin wrinkles containing the exosome is characterized in that the cosmetic composition for improving skin wrinkles containing the exosome is applied in one formulation selected from the group consisting of a toner, an eye cream, a moisturizing cream, a cleansing product, a serum, and an ampoule.