KR102630617B1 - Antibacterial liposome composition containing conjugate of pheophorbide a and lipid derivative of polyethylene glycol - Google Patents

Abstract

The present invention relates to an antibacterial liposome composition containing a conjugate of pheophorbid A and a lipid derivative of polyethylene glycol, and more specifically, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine- 0.0001 to 5% by weight of conjugate of N-[amino (polyethylene glycol)-2000] and pheophorbid A, 1 to 10% by weight of polyglyceryl-3-methylglucose distearate, 1 to 10% by weight of polysorbate 80 , 5 to 40% by weight of cetyl palmitate, 1 to 10% by weight of vegetable oil, 2 to 15% by weight of N-methyl-2-pyrrolidone, and the remaining amount of water.

Description

Antibacterial liposome composition containing a conjugate of pheophorbide A and a lipid derivative of polyethylene glycol {ANTIBACTERIAL LIPOSOME COMPOSITION CONTAINING CONJUGATE OF PHEOPHORBIDE A AND LIPID DERIVATIVE OF POLYETHYLENE GLYCOL}

The present invention relates to an antibacterial liposome composition containing a conjugate of pheophorbid A and a lipid derivative of polyethylene glycol, and more specifically, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine- 0.0001 to 5% by weight of conjugate of N-[amino (polyethylene glycol)-2000] and pheophorbid A, 1 to 10% by weight of polyglyceryl-3-methylglucose distearate, 1 to 10% by weight of polysorbate 80 , 5 to 40% by weight of cetyl palmitate, 1 to 10% by weight of vegetable oil, 2 to 15% by weight of N-methyl-2-pyrrolidone, and the remaining amount of water.

Efforts and research are active to utilize transdermal delivery systems in functional cosmetics in order to improve transdermal penetration efficiency while stabilizing substances that can show excellent effects but are less stable. These transdermal delivery systems are mainly applied by making vesicles containing effective substances using materials such as surfactants, lipids, and polymers. Among these, lipids are widely used as cosmetic materials due to their advantage because they are components of biological membranes.

New design methods for innovative nanoparticles that enable photo-based treatment, such as photodynamic therapy (PDT) and photothermal therapy (PTT), are being developed. In photothermal therapy using visible light, when a wavelength of an appropriate length is irradiated, an activated oxygen reaction occurs through a complex chemical reaction, and through this, the cell death process proceeds simultaneously. Photodynamic therapy (PDT) is a technology that uses a photo-sensitive material to treat incurable diseases such as cancer or treat diseases such as acne without surgery. Such photodynamic therapy has been actively researched since the early 20th century, and is currently being used to increase immunity in the diagnosis and treatment of cancer, autologous bone marrow transplantation, antibiotics, AIDS treatment, skin transplant surgery, and treatment of arthritis, and its application. The scope is gradually expanding. Photodynamic therapy involves administering a photosensitive substance, which is a substance that reacts sensitively to light, into the body. When light is irradiated from the outside, singlet oxygen or free radicals are generated through a chemical reaction between the abundant oxygen in the body and external light. It is a treatment method that uses the principle that free radicals are generated, and these singlet oxygen or free radicals destroy various lesion sites or cancer cells by inducing apoptosis.

Photosensitive substances used in photodynamic therapy include porphyrin derivatives, chlorin, bacteriochlorin, phthalocyanine, and 5-aminolevulinic acid derivatives. It is known.

Chlorophyll is most abundant in plants, and generally exists in two types, a and b, in higher plants in a ratio of 3:1 or 3:2. Chlorophyll has an excellent anti-inflammatory effect and has been used to treat wounds, burns, and ulcers, and has also been used as a health-promoting food. In addition, plant extracts containing chlorophyll are used as cosmetic raw materials to manage skin damage.

Chlorophyll exists in various derivatives, including pheophytin, pheophorbide, chlorin, and phytanic acid. When an acidic temperature is applied, the magnesium in the chlorophyll molecules is released and changed into yellow-green pheophytin. When the enzyme chlorophyllase acts on this, the phytol group is removed by hydrolysis. It becomes yellow-brown pheophorbide. On the other hand, when chlorophyllase first acts on chlorophyll, it turns into green pheophorbid, and then when heat is applied under acidic conditions, it turns into pheophorbid.

As an example of a composition using such chlorophyll a and pheophorbid a, Korean Patent Application No. 10-2014-0067083 discloses a composition for improving skin condition containing chlorophyll a or pheophorbid a. In addition, previous studies have confirmed that chlorophyll, pheophytin, pyropheophytin, and pheophorbid derivatives exhibit potential tumor suppressive effects.

Republic of Korea Patent Application No. 10-2014-0067083

The technical task of the present invention is to provide a composition that can be effectively used in the treatment of acne, etc. by efficiently delivering pheophorbid A, a photosensitivity substance, into the skin and exhibiting excellent antibacterial activity.

In order to achieve the above object, the present invention is a conjugate of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000] and pheophorbide A 0.0001 to 5% by weight, polyglyceryl-3 methylglucose distearate 1 to 10% by weight, polysorbate 80 1 to 10% by weight, cetyl palmitate 5 to 40% by weight, vegetable oil 1 to 10% by weight, N- An antibacterial liposome composition comprising 2 to 15% by weight of methyl-2-pyrrolidone and the remaining amount of water is provided.

Additionally, the present invention provides a cosmetic composition containing the antibacterial liposome composition.

Hereinafter, the present invention will be described in more detail.

In the present invention, pheophorbide A (PheoA) forms a conjugate with a lipid derivative of polyethylene glycol. In the present invention, as a lipid derivative of polyethylene glycol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000](1,2-distearoyl-sn -glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]; DSPE-PEG (2000) amine) is used. In the present invention, combining pheophorbid A with DSPE-PEG (2000) amine can help stabilize pheophorbid A and efficiently deliver it into the skin. Pheophorbid A and DSPE-PEG (2000) amine complexes can be formed, for example, in the presence of catalysts dicyclohexylcarbodiimide (DCC) and N-hydroxysulfosuccinimide (sulfo-NHS). It can be obtained by reacting pheophorbid A with DSPE-PEG (2000) amine, and is shown in Scheme 1 below.

[Scheme 1]

Hereinafter, the conjugate of Pheophorbide A and DSPE-PEG (2000) amine is hereinafter abbreviated as “DSPE-PEG_PheoA”. The DSPE-PEG_PheoA synthesized in the present invention has some shortcomings in terms of stability, solubility, and skin penetration to be used as a raw material for cosmetics, so it is used by making liposomes.

The liposome composition of the present invention contains 0.0001 to 5% by weight of DSPE-PEG_PheoA, preferably 0.001 to 4% by weight, and more preferably 0.005 to 3% by weight. In the present invention, if DSPE-PEG_PheoA is included in less than 0.0001% by weight, the antibacterial effect of DSPE-PEG_PheoA may be weak, and if it is included in more than 5% by weight, there may be difficulties in forming liposomes.

The liposome composition of the present invention contains 1 to 10% by weight, preferably 1.5 to 8% by weight, and more preferably 2 to 2% by weight of polyglyceryl-3 methylglucose distearate as a nonionic surfactant. Contains 6% by weight. In the present invention, if polyglyceryl-3 methylglucose distearate is included in less than 1% by weight or more than 10% by weight, there may be a problem in liposome formation.

The liposome composition of the present invention contains 1 to 10% by weight, preferably 1.5 to 8% by weight, and more preferably 2 to 6% by weight of polysorbate 80 (polyoxyethylene (20) sorbitan monooleate) as a nonionic surfactant. % includes. In the present invention, if polysorbate 80 is included in less than 1% by weight or more than 10% by weight, there may be a problem in liposome formation.

The liposome composition of the present invention contains 5 to 40% by weight of cetyl palmitate, preferably 8 to 35% by weight, and more preferably 10 to 30% by weight. In the present invention, cetyl palmitate can increase the affinity of liposomes with the skin and help penetrate the skin. In the present invention, if cetyl palmitate is included in less than 5% by weight, the skin affinity and skin penetration of the liposome may be poor, and if it is included in more than 40% by weight, there may be problems in liposome formation.

The liposome composition of the present invention contains 1 to 10% by weight of vegetable oil, preferably 1.5 to 8% by weight, and more preferably 2 to 7% by weight. In the present invention, vegetable oils play a role in helping liposomes penetrate the skin, such as macadamia oil, sunflower seed oil, olive oil, camellia oil, castor oil, jojoba oil, almond oil, apricot seed oil, green tea oil, and meadowform seed. (meadowfoam seed) oil, argan oil, or mixtures thereof may be used, but are not limited thereto. In the present invention, if the vegetable oil is included in less than 1% by weight, there may be a problem with liposome penetration into the skin, and if it is included in more than 10% by weight, there may be a problem in liposome formation.

The liposome composition of the present invention contains N-methyl-2-pyrrolidone and water as a solvent. The liposome composition of the present invention contains 2 to 15% by weight of N-methyl-2-pyrrolidone, preferably 4 to 13% by weight, and more preferably 5 to 10% by weight. In the present invention, water is included, for example, in an amount of 10 to 89.9999% by weight or 38 to 78.995% by weight.

The liposome composition of the present invention may additionally contain additives such as excipients, sweeteners, and flavors, depending on the need.

In the present invention, the particle diameter of the liposome is preferably 100 to 400 nm.

According to another aspect of the present invention, a cosmetic composition containing the liposome composition of the present invention is provided. In the present invention, the cosmetic composition may be formulated into, for example, skin, lotion, body lotion, cream, essence, hair mist, etc., but is not limited thereto. The cosmetic composition preferably contains 1 to 30% by weight of the liposome composition according to the present invention. In the present invention, if the cosmetic composition contains less than 1% by weight of the liposome composition, the antibacterial effect may be minimal, and even if the cosmetic composition contains more than 30% by weight, the effect of treating acne, etc. due to the antibacterial action increases in proportion to the addition. It is difficult to expect more and is not economically desirable.

The liposome composition of the present invention can effectively treat acne, etc. by showing excellent antibacterial activity by stably and efficiently delivering pheophorbid A, a photosensitivity substance, into the skin.

Figure 1 shows the results of measuring the particle diameter size of the liposome of the present invention using Photal ELS-Z.
Figure 2 shows the results of measuring the zeta potential of the liposome of the present invention using Photal ELS-Z.
Figure 3 shows the results of measuring liposome stability of the present invention.
Figure 4 is an enlarged photograph of the liposome composition of the present invention using a freezing electron microscope.
Figure 5 shows the results of measuring the skin penetration rate of the liposome composition of the present invention using confocal microscopy.
Figure 6 is a graph showing the antibacterial activity of DSPE-PEG_PheoA of the present invention compared to erythromycin.

Hereinafter, the present invention will be described in detail through examples. However, the examples are merely illustrative to aid understanding of the present invention and are not intended to limit the scope of the present invention.

Manufacturing example : DSPE -PEG_ of PheoA manufacturing

DSPE-PEG (2000) Dissolve 50 mg of amine in 10 ml of chloroform, 12.85 mg of Pheo A, and the catalyst N,N'-dicyclohexylcarbodiimide (N,N'- 6.63 mg of dicyclohexylcarbodiimide (DCC) and 3.69 mg of N-hydroxysulfosuccinimide (sulfo-NHS) were added to 10 ml of chloroform and stirred for 4 hours. Next, the Pheo A solution dissolved in chloroform was mixed with DSPE-PEG (2000) amine dissolved in chloroform and reacted at room temperature for 48 hours. Afterwards, the conjugate prepared by evaporating chloroform at 40°C for 1 hour was refrigerated and stored at 4°C until use.

Example One: DSPE -PEG_ PheoA containing liposome Preparation of composition

Polyglyceryl-3 methylglucose distearate, cetyl palmitate, macadamia oil, and DSPE-PEG_PheoA were dissolved in N-methyl-2-pyrrolidone and heated to 80°C. Polysorbate 80 and purified water were heated to 80°C or higher and then passed through a high-pressure microemulsifier at 1,000 bar five times in succession, followed by cooling and defoaming to obtain a liposome composition.

Comparative example : DSPE -PEG_ PheoA Containing general emulsion manufacturing

With the composition shown in Table 2 below, each component was introduced into a container and dissolved at a temperature of 80°C, then mixed for 5 minutes using a homomixer, cooled, and defoamed to obtain an emulsion.

Example 2: liposome containing of skin manufacturing

A skin containing the liposome of the present invention was prepared with the composition shown in Table 3 below.

Example 3: liposome Preparation of lotion containing

A lotion containing the liposome of the present invention was prepared with the composition shown in Table 4 below.

Example 4: liposome containing of body lotion manufacturing

A body lotion containing the liposome of the present invention was prepared using the composition shown in Table 5 below.

Example 5: liposome Preparation of cream containing

A cream containing liposomes of the present invention was prepared with the composition shown in Table 6 below.

Example 6: liposome Preparation of essence containing

An essence containing the liposome of the present invention was prepared with the composition shown in Table 7 below.

Example 7: liposome containing of hair mist manufacturing

A hair mist containing the liposome of the present invention was prepared with the composition shown in Table 8 below.

Experiment example One: liposomal Particle distribution measurements

The particle distribution of the liposome prepared in Example 1 was measured using Photal ELS-Z and is shown in Figure 1. As a result of the measurement, it was found that the average particle size was 211.23 nm.

Experiment example 2: liposomal Stability measurements

To measure the stability of the liposome prepared in Example 1, zeta potential was measured using Photal ELS-Z, and the results are shown in Figure 2. As a result of the measurement, it was found that the particle potential was stable at -52.66 mV.

Experiment example 3: liposomal Stability measurements

The stability of the liposome prepared in Example 1 was measured using Turbiscan, and it was found that the liposome was stable (FIG. 3).

Experiment example 4: Amidum particle photography

The liposome prepared in Example 1 was photographed. Since the particle size of the liposome was so fine that it was impossible to measure with a general optical microscope, it was photographed using freeze-fracture scanning electron microscopy (FIG. 4). From Figure 4, it was seen that the liposomes were well formed with uniform size.

Experiment example 5: transdermal Absorption promotion effect experiment

Female hairless guinea pigs (strain IAF/HA-hrBR) about 8 weeks old were used. The abdominal skin of a guinea pig was cut and placed in a Franz-type diffusion cell (Lab fine instruments, Korea) for testing. After adding 50 mM phosphate buffer (pH 7.4, 0.1M NaCl) to the receptor container (5 ml) of the Franz-type diffusion cell, the diffusion cell was mixed and dispersed at 32°C and 600 rpm, and the liposome of Example 1 And 50 ㎕ of the general emulsion of the comparative example was placed in the donor container. Absorption and diffusion were carried out according to the scheduled time, and the skin area where absorption and diffusion occurred was made to be 0.64 cm ² . After the absorption and diffusion of the active ingredient is completed, wash off the emulsion that was not absorbed and remains on the skin with dried kimwipes or 10 ml of ethanol, and use a tip-type homogenizer to absorb and spread the active ingredient. After grinding the skin, the amount of DSPE-PEG_PheoA absorbed into the skin was extracted using 4 ml of dichloromethane. Afterwards, the extract was filtered through a 0.45 ㎛ nylon membrane filtration membrane, and the content was measured by HPLC under the following conditions. The results are shown in Table 9.

As can be seen from Table 9 above, it was found that the liposome of the present invention had superior transdermal absorption compared to a typical emulsion.

Experiment example 6: Confocal Measurement of skin penetration rate using a microscope

Each sample was applied to the dorsal skin area of nude mice (n=3), where the concentration of PheoA was 1 mg/ml, and PBS buffer (pH 7.4) was used as the receptor medium. After processing the material using the Franz Cell permeation method, experimental skin tissue was obtained and skin penetration rate was evaluated using confocal microscopy. As a result, it was found that the liposome of Example 1 penetrated about 8 times better than a regular emulsion.

Experiment example 7: DSPE -PEG_ of PheoA Antibacterial activity measurement

DSPE-PEG_PheoA was dissolved in dimethyl sulfoxide (DMSO) to prepare a sample with a concentration of 0.0001%. Propionibacterium acnes , a representative acne-causing bacteria, was diluted in the prepared sample to ² After reacting for 16, 20, 24, and 28 hours, the viable count of Propionibacterium acnes for each sample was measured at an absorbance of 625 nm, and the results are shown in Figure 6.

As can be seen from Figure 6, when 0.0001% of DSPE-PEG_PheoA was added, the bacteria were almost killed in all measurement time sections. In addition, DSPE-PEG_PheoA showed almost the same effect as erythromycin, which is currently used as an anti-acne antibiotic. These results are very high when using DSPE-PEG_PheoA as a medication or topical acne treatment, considering that oral medications are usually taken once every 8 hours, and ointments or topical medications are used within 2-4 hours. This means it can be effective.

Staphylococcus sapropiticus, Staphylococcus hominis, Staphylococcus xylosus, Staphylococcus haemolyticus, Staphylococcus warneri, and Staphylococcus aureus of DSPE-PEG_PheoA using the same method as above. The minimum concentration showing antibacterial activity against U.S. epidermidis, Staphylococcus epidermidis, and Micrococcus luteus was measured, and the results are shown in Table 10 below.

Claims (7)

0.0001 to 5% by weight of a conjugate of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000] of the following formula (1) and pheophorbead A, 1 to 10% by weight of polyglyceryl-3 methylglucose distearate, 1 to 10% by weight of polysorbate 80, 5 to 40% by weight of cetyl palmitate, 1 to 10% by weight of vegetable oil, N-methyl-2-p. Antibacterial liposome composition comprising 2 to 15% by weight of rolidone and the balance of water:
[Formula 1]
. delete The method of claim 1, wherein the vegetable oil is a group consisting of macadamia oil, sunflower seed oil, olive oil, camellia oil, castor oil, jojoba oil, almond oil, apricot kernel oil, green tea oil, meadowform seed oil, argan oil, and mixtures thereof. An antibacterial liposome composition selected from the group consisting of The method of claim 1, wherein the conjugate of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000] and pheophorbid A is 0.005 to 3% by weight. , 2 to 6% by weight of polyglyceryl-3-methylglucose distearate, 2 to 6% by weight of polysorbate 80, 10 to 30% by weight of cetyl palmitate, 2 to 7% by weight of vegetable oil, N-methyl-2 -An antibacterial liposome composition comprising 5 to 10% by weight of pyrrolidone and 38 to 78.995% by weight of water. The antibacterial liposome composition according to claim 1, wherein the liposome diameter is 100 to 400 nm. A cosmetic composition comprising the antibacterial liposome composition of any one of claims 1 and 3 to 5. The cosmetic composition according to claim 6, comprising 1 to 30% by weight of an antibacterial liposome composition.