WO2019117666A1 - Composition comprising melittin nano particles for preventing, treating, and improving cancer - Google Patents

Abstract

The present invention relates to a composition which comprises melittin nano particles, is highly targeted to cancer, has a long half-life in blood and is for preventing, treating, and improving cancer.

Description

Composition for prevention, treatment and improvement of cancer comprising melitin nanoparticles

The present invention relates to a composition for prevention, treatment and improvement of cancer comprising melittin nanoparticles.

Bee venom is one of the folk remedies that activates the immune function of the human body and has been used as a treatment for various pain and inflammatory diseases. Melittin is a major constituent of the dried bee venom, 40 to 50% of which consists of 26 amino acids. Melitin has been reported to have various effects such as anticancer activity, inhibition of bacterial growth and sterilization, anti-inflammation, analgesic action and immunity enhancement (Korean Patent Laid-Open Publication No. 10-2012-0074362, Korean Patent Publication No. 10-2010-0055025, Korean Patent Publication No. 10-1992-7003080).

However, melitin acts nonspecifically on cells to degrade all of the phospholipid layers of normal cells. Since melittin acts rapidly on red blood cells and induces strong hemolytic action even at low concentrations, there is a problem in that it is industrially used .

Accordingly, the inventors of the present invention have investigated a method for increasing the selectivity of melitin to cancer cells, and found that when melittin is coated with a specific component and nanotized, the selectivity to cancer cells is high and the half-life of blood is increased. Completed.

It is an object of the present invention to provide a melitin-containing composition having high selectivity to cancer cells.

According to an aspect of the present invention,

A core comprising melitin; And

The coating layer covering the core

The present invention also provides a composition for preventing and treating cancer,

Further, according to the present invention,

A core comprising melitin; And

The coating layer covering the core

The present invention provides a food composition for preventing and / or improving cancer.

The composition of the present invention has high selectivity to cancer cells and has an effect of increasing blood half-life.

Fig. 1 shows a schematic view of particles according to the coating material.

2 is a schematic diagram showing a method for producing a nano emulsion.

3 is a schematic diagram showing a method for evaluating cytotoxicity against erythrocytes in X-Vivo.

Figure 4 shows the survival rate of HEK293 cells according to the concentration of free melittin.

FIG. 5 shows the degree of hemolysis of red blood cells according to the concentration of free melittin.

FIG. 6 shows the inhibition rate of cell proliferation of MDA-MB-231 cells according to the concentration of free melittin.

FIG. 7 shows the differences in particle size (A), PDI (B), scattering intensity (C) and zeta potential (D) of the nanoparticles according to the chitosan content. Averages with different characters are significantly different (p <0.05).

Figure 8 shows the difference in particle size (A), PDI (B), scattering intensity (C) and zeta potential (D) of the nanoparticles according to the coating composition. Averages with different characters are significantly different (p <0.05).

Figure 9 shows the difference in particle size (A), PDI (B), scattering intensity (C) and zeta potential (D) of the nanoparticles according to PLGA, PEG 2000 and DSPE PEG used in the coating. Averages with different characters are significantly different (p <0.05).

FIG. 10 shows the difference in the collection efficiency (A) and the content efficiency (B) of the nanoparticles according to PEG 2000 and DSPE PEG used in the coating.

Figure 11 shows the survival rate of HEK293 cells according to PLGA, PEG 2000 and DSPE PEG used for coating.

Figure 12 shows the degree of hemolysis of red blood cells according to PLGA, PEG 2000 and DSPE PEG used in the coating.

Figure 13 shows cell proliferation inhibition rates for MDA-MB-231 cells according to PLGA, PEG 2000 and DSPE PEG used for coating. The averages in the upper case are the same samples, and the lower case at the same concentration is quite different (p <0.05).

Fig. 14 shows the ratio of cancer cell growth inhibitory activity to the normal cytotoxicity according to PLGA, PEG 2000 and DSPE PEG used for coating.

Figure 15 is a confocal micrograph of MDA-MB-231 cells according to PLGA, PEG 2000 and DSPE PEG used for coating.

Figure 16 shows the apoptosis region of MDA-MB-231 cells according to PLGA, PEG 2000 and DSPE PEG used for coating. (A-1, B-1, C-1, D-1: 30 min culture, A-2, B-2, C-2 and D-

Figure 17 shows the difference in particle size (A), PDI (B), scattering intensity (C) and zeta potential (D) of HPH nanocapsules according to PEG 2000 and DSPE PEG used in the coating. Averages with different characters are significantly different (p <0.05).

Figure 18 shows the difference in the collection efficiency of HPH nanocapsules according to PEG 2000 and DSPE PEG used in the coating. Averages with different characters are significantly different (p <0.05).

According to the present invention,

A core comprising melitin; And

The coating layer covering the core

The present invention also relates to a composition for preventing and treating cancer.

Further, according to the present invention,

A core comprising melitin; And

The coating layer covering the core

And to a composition for preventing and / or improving cancer.

Further, according to the present invention,

To the subject

A core comprising melitin; And

The coating layer covering the core

The present invention provides a method for preventing and treating cancer, comprising the step of administering a composition for preventing and treating cancer comprising particles comprising the above-mentioned particles.

Further, according to the present invention,

To the subject

A core comprising melitin; And

The coating layer covering the core

The present invention also relates to a method for preventing and / or improving cancer, which comprises administering a food composition for preventing and / or improving cancer.

Further, according to the present invention,

A core comprising melitin; And

The coating layer covering the core

&Lt; RTI ID = 0.0 &gt; and / or &lt; / RTI &gt;

Further, according to the present invention,

A core comprising melitin; And

The coating layer covering the core

To a cancer preventing and improving particle.

Further, according to the present invention,

A core comprising melitin; And

The coating layer covering the core

For the prevention, treatment and improvement of cancer.

Hereinafter, the present invention will be described in detail.

Melitin

The melitin of the present invention may be naturally occurring melittin or synthetic melittin. Preferably, the melitin of the present invention is a naturally occurring melittin, more preferably a melittin derived from bee venom.

core

The particles of the present invention comprise a core comprising melitin. The core may further comprise other ingredients known in the art to increase the stability of the melitin outer particle, affinity with the coating layer, drug delivery, and the like.

Coating layer

The coating layer of the present invention is a layer coated with a core containing melamine. The coating layer may comprise polyethylene glycol, chitosan or polylactide-co-glycolide as the coating material, preferably polyethylene glycol, more preferably polyethylene glycol, chitosan and polylactide-co- Ride. Preferably, the polyether glycol is a non-ionic, hydrophilic polymer having a molecular weight of 1800 to 2500, preferably 2000. It is also preferred that the polylactide-co-glycolide (DSPE-PEG) is an anionic amphipathic polymer having a molecular weight of 2700 to 3000, preferably 2806. The coating layer melitin and the coating material are preferably mixed at a weight ratio of 1: 300 to 460.

particle

The present invention relates to a core comprising melitin; And a coating layer covering the core. The particles preferably have a size of 180 to 350 nm, more preferably 200 to 320 nm.

The particle has a higher degree of selectivity calculated by the following formula (4) than that of melitin having no coating layer. In the expression 4, &quot; inhibitory activity against cancer cells &quot; means &quot; cytotoxicity against cancer cells &quot;, and &quot; cytotoxicity against normal cells &quot; means &quot; cytotoxicity against normal cells &quot;. Therefore, the particles of the present invention overcome the toxicity problem of normal cells, which is a disadvantage of melitin which does not have a coating layer.

<Formula 4>

In addition, the particles of the present invention are more stable in blood than Melitin having no coating layer, and have a long half-life in vivo.

cancer

The present invention relates to a composition for preventing and treating cancer, a composition for prevention and improvement of cancer, and the like, which comprise the particles of the present invention. The cancer refers to a cancer classified into malignant tumor, cancer in the medical field. For example, the cancer may be colon cancer, stomach cancer, lung cancer, esophageal cancer, thyroid cancer, laryngeal cancer, pancreatic cancer, liver cancer, skin cancer, breast cancer and the like.

Composition for cancer prevention and treatment

The present invention relates to a composition for preventing and treating cancer comprising particles of the present invention. The composition for preventing and treating cancer is a pharmaceutical composition.

The pharmaceutical composition of the present invention may contain 0.01 to 80% by weight, preferably 0.02 to 65% by weight of the particles. However, this can be increased or decreased according to the needs of the medicinal person, and can be appropriately increased or decreased according to the situation such as the diet, nutritional status, disease progression, degree of obesity and the like.

The pharmaceutical composition of the present invention can be administered orally or parenterally and can be used in the form of a general pharmaceutical preparation. Preferred pharmaceutical preparations are those for oral administration such as tablets, hard or soft capsules, liquids, suspensions, etc., and parenteral administration preparations such as injections and intravenous injections. These pharmaceutical preparations may contain conventional pharmaceutically acceptable carriers, For example, an excipient, a binder, a disintegrant, a lubricant, a solubilizer, a suspending agent, a preservative or an extender, and the like. Preferably, the pharmaceutical preparation can be administered intravenously with a suspension for injection.

The dosage of the pharmaceutical composition comprising the particles of the present invention may be determined by a specialist depending on various factors such as the patient's condition, age, sex, and complications, but is generally from 0.1 mg to 10 g per kg of the adult, Can be administered in a dose of from 10 mg to 5 g. Also, the daily dosage of the pharmaceutical composition per unit dosage form, or a half, 1/3 or 1/4 dose thereof, may be contained, and may be administered 1 to 6 times per day. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, the amount may be less than the above range, and the active ingredient may be used in an amount of more than the above range since there is no problem in terms of safety. At this time, the particles may be separated and used in powder form or in the form of a nano emulsion.

Food composition for cancer prevention and improvement

The present invention relates to a food composition for prevention and improvement of cancer comprising particles of the present invention.

The food of the present invention may be a health supplement food, a health functional food, a functional food, and the like, but is not limited thereto, and includes the addition of particles of the present invention to natural foods, processed foods, and general food ingredients.

The food composition of the present invention can be used appropriately as it is, or it can be used in combination with other foods or food compositions. The amount of the active ingredient to be mixed can be suitably determined according to its intended use (prevention, improvement, or therapeutic treatment). In general, the particles of the present invention may be added in an amount of 0.1 to 70% by weight, preferably 2 to 50% by weight, based on 100% by weight of the raw material of food or beverage in the production of food or beverage. The effective dose of the particles may be used in accordance with the effective dose of the pharmaceutical composition, but may be less than the above range for health and hygiene purposes or long-term intake for health control purposes. It can be used in an amount exceeding the above range. At this time, the particles may be separated and used in powder form or in the form of a nano emulsion.

 There is no particular limitation on the kind of the food. The food composition may be used in the form of tablets, hard or soft capsules, liquids, suspensions, and the like, which may contain conventional excipients, such as excipients in the case of oral preparations, Binders, disintegrators, lubricants, solubilizers, suspending agents, preservatives or extenders.

Examples of the food to which the particles can be added include dairy products including meats, sausages, breads, chocolates, candies, snacks, confections, pizza, ramen noodles, gums, ice cream, soups, drinks, tea, Alcoholic beverages and vitamins complex, and other nutrients, but the present invention is not limited to these kinds of foods.

object

The present invention includes a method for preventing and treating cancer, comprising administering the composition or particles of the present invention to a subject. The invention also encompasses methods of preventing and / or improving cancer, comprising administering the composition or particles of the present invention to a subject.

The subject is a mammal, including a human, who is at high risk for developing cancer or diagnosed with cancer. Preferably, the subject is a person who is at high risk for developing cancer or who has been diagnosed with cancer, more preferably a patient diagnosed with cancer, and even more preferably a patient with breast cancer.

<Term>

In the present invention, "free melittin" means non-nanoparticulate melittin, i.e., uncoated melittin.

In the present invention, the melittin nanoparticle means a capsule having a size of nano unit coated with a coating layer of melittin.

Advantages and features of the present invention and methods of achieving them will become apparent with reference to the embodiments described in detail below. It should be understood, however, that the invention is not limited to the disclosed embodiments, but is capable of many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, To fully disclose the scope of the invention to those skilled in the art, and the invention is only defined by the scope of the claims.

&Lt; Materials and methods >

material

Melittin was purchased from Tocris Bioscience (Tocris Bioscience, Ellisville, MO, USA). PLGA (poly D, L-lactide-co-glycolide) was purchased from Sigma-Aldrich Chemical Co. Resomer® RG 752 H from St. Louis, MO, USA was used. Insoluble low molecular weight chitosan (CS, 50,000-190,000 Da) and PEG 2000 were purchased from Sigma-Aldrich Chemical Co. DSPE PEG (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethyleneglycol) -2000]) was purchased from Avanti Polar Lipids (Avanti Polar Lipids, Alabaster, AL, USA)

Statistical analysis

All experiments were repeated at least 3 times and the results were compared and analyzed by one-way ANOVA using Statistical Package for the Social Science (SPSS, Version 21.0, SPSS Inc. Chicago, IL, USA) At the p <0.05 level, Duncan's multiple range test was used to verify significance for the mean.

&Lt; Example 1 > Preparation of nanocapsules

<1-1> Production of nanocapsules by particle type

Melitin was coated with various coating materials to prepare nanocapsules as shown in Table 1 (Fig. 1).

Preparation of oil solution

First, melittin was dissolved in 20% by weight of acetonitrile to prepare melittin stock solution (5 mg / mL). 100 mg of PLGA was dissolved in 3 mL of chloroform and added to 10 mg of the melittin mother liquor to prepare an oil solution.

Preparation of aqueous solution

The PVA solution was prepared by adding polyvinyl alcohol (PVA) to distilled water to 12% (w / v) and stirring at 500 rpm (500 rpm). The chitosan solution was completely dissolved in 1 wt% glacial acetic acid. 8 mL of the PVA solution and 4 mL of the chitosan solution were mixed to prepare a PVA / chitosan solution.

On the other hand, PVA / chitosan / PEG 2000 solution was prepared by adding PEG 2000 to the PVA / chitosan solution so that the PEG 2000 was 10% (w / w) of the PVA.

Also, PVA / chitosan / DSPE PEG solution was prepared by adding DSPE PEG to the PVA / chitosan solution so that the DSPE PEG was 10% (w / w) of the PVA.

	PLGA (mg)	Chitosan (mg)	PEG 2000 (mg)	DSPE PEG (mg)
Nano Capsule 1	100	-	-	-
Nano Capsule 2	100	21.6	-	-
Nano Capsule 3	100	21.6	24	-
Nano Capsule 4	100	21.6	-	24

Nano emulsion manufacturing

3 mL of the oily solution and 12 mL of the aqueous solution were mixed and ultrasonicated (70%, 15 seconds) in a low temperature water bath (VC 505, Vibracell Sonics, Newton, USA). The mixture was stirred (500 rpm) for 24 hours to remove the organic solvent to prepare an emulsion solution. The emulsion solution was separated using Amicon Ultra-15 (50K, Millipore Co., MA, USA) to remove the PVA from the emulsion solution and to screen the nanoparticles (14,000 g force, 15 min, 25 ° C) . Then, the separated emulsion solution was washed three times with distilled water, and distilled water was added thereto to make a final volume of 12 mL, thereby preparing a nano-emulsion in the form of a dispersion in which melittin-coated nanoparticles were dispersed (FIG. 2) . In the following experiments, nanoparticles were used in the nanoemulsion state. The four types of nanoparticles 1 to 4 commonly have PLGA as an oil phase portion and PVA as a base portion. In addition, chitosan, PEG 2000, and DSPE PEG are selectively added to the water phase portion . In the case of Nano Capsule 2, PLGA was added to the oil phase, and PVA / chitosan solution containing PVA and chitosan was used as the nano emulsion in which the oil phase and water phase were emulsified.

&Lt; Example 2 > Measurement of physical properties of nanoparticles

Particle size, particle polydispersity index (PDI), derived count rate (RI), and the like of the nanocapsules were measured using a nano particle size analyzer (Zetasizer Nano ZS, Malvern Instruments, Ltd., Malvern, Worcestershire, UK) , DCR) and zeta potential were measured.

&Lt; Example 3 > Measurement of collection efficiency and content efficiency of melitin

Measurement of the absorption efficiency of melitin

To measure the amount of free nanoparticle free melittin, the solution passed through the filter was separated during centrifugation to select nanoparticles in the preparation of nanoemulsion. The solution separated by centrifugation was analyzed as bicinchoninic acid (BCA) method as non-nanocapsulated free melilin. The entrapment efficiency (EE) was calculated using Equation 1 below.

<Formula 1>

Measuring the content efficiency of melitin

To measure the content efficiency of melitin contained in the nanoparticles, melittin was extracted from the lyophilized nanoparticles. The nanoparticles were dispersed in acetonitrile, stirred for 48 hours (37 ° C, 100 rpm), and centrifuged. The content of melittin in the supernatant separated by centrifugation was analyzed using the BCA method. The loading efficiency (LE) of melitin was calculated using Equation 2 below.

<Formula 2>

Example 4 Culture of Breast Cancer Cells (MDA-MB-231) and Kidney-Derived Normal Cells (HEK293)

MDA-MB-231 cells were cultured in RPMI medium 1640 (+ L-Glutamine) supplemented with 1% penisiline-streptomycin and 10% inactivated FBS (fetal bovine serum) HEK293 cells were cultured in MEM (Minimum Essential Medium) supplemented with 1% penicillin-streptomycin and 10% inactivated FBS. Both cells were cultured in a CO 2 incubator under constant conditions (37 ° C, 5% CO2, 95% humidity). When Confluence reaches 75-80%, the medium is removed and the cells are rinsed with phosphate buffered saline (PBS). Cells were harvested using 0.25% trypsin-EDTA and centrifuged at 1,000 rpm for 3 minutes to collect the cell pellet. After removing the supernatant, the precipitated cell pellet was redispersed in 3 to 4 mL of medium and cultured in a 75 cm2 T-flask. After 48 hours, the suspension was replaced with fresh medium to remove floating cells that did not adhere to the surface of the flask. Cells are generally cultured at 6-8 days intervals. Cells collected at the time of subculture are dispersed in 3 to 5 mL medium, mixed with trypan blue staining reagent at 1: 1, The number of cells was adjusted and the number of cells was adjusted.

Example 5: Safety evaluation of melittin nanoparticles

<5-1> Evaluation of cytotoxicity in Ex vivo

The safety of nanoparticles and free melittin against cytotoxicity was analyzed by MTT (3- (4,5-dimethyl-2-tetrazolyl) -2,3-diphenyl tetrazolium bromide) assay using HEK293 cells . The MTT assay is based on the principle that a blue-purple formazan is formed from MTT by succinate-dehydrogenase, an enzyme in the cell's mitochondria.

HEK293 cells cultured in Example 4 were diluted to a concentration of 5.5 X 104 cells / mL, and 180 μL was added to each well of a 96-well plate and cultured for 24 hours. After culturing for 24 hours, the cells were injected with the nanosized dispersion according to the concentration of the final melittin, and cultured for 24 hours. Then, the pretreated cells were treated with MTT reagent and incubated for 4 hours. After centrifugation at 1,500 rpm for 5 minutes, the supernatant was removed, and dark blue formazan was dissolved in DMSO to measure the degree of color development at 540 nm. The cytotoxicity of the melittin nanodispersion to normal cells was examined by comparing the cell viability with the control group (non-treated group with melittin nano dispersion).

<5-2> Evaluation of red cell cytotoxicity in Ex vivo

Blood for ex vivo experiments related to the red cell cytotoxicity of nanoparticles was collected by injection needle into the central artery of the rabbit. In this experiment, male rabbits weighing 2 to 3 kg were used and were raised in an animal laboratory. The blood volume was calculated based on the recommended maximum volume of blood collected (10% of the circulating blood volume) once every two weeks Were collected and used for experiments. The anticoagulant citrate dextrose (ACD) was used as the anticoagulant. Blood was centrifuged at 240 xg for 10 minutes to separate red blood cells. The first separated red blood cells were centrifuged at 37 ° C in PBS, washed twice, and then washed in the same manner as described above, and diluted with PBS at 37 ° C for subsequent experiments (FIG. 3).

Red cell cytotoxicity was measured by the degree of hemoglobin color developed when red blood cells became hemolytic using red blood cell hemolysis. The red cell suspension was adjusted to 1 ~ 2 X 106 cells / mL using a hemocyte counter. The red cell suspension and nanodispersion were mixed at a weight ratio of 1: 1 and incubated at 37 ° C for 30 minutes. After centrifugation (980 x g) for 10 minutes, the supernatant was collected and measured for color development at 540 nm. The relative hemolysis was calculated by setting 1% Triton x-100 as a control, and the toxicity evaluation on the red blood cells was analyzed according to the following formula 3.

<Formula 3>

Blank (negative control): RBC + PBS

Triton (positive control): RBC + 1% Triton X-100

<5-3> Evaluation of anticancer activity in Ex vivo

Measurement of anticancer activity of nanoparticles prepared through optimal particle production conditions was performed by MTT analysis using MDA-MB-231 cells (breast cancer cells). The cultured MDA-MB-231 cells were diluted to a cell concentration of 5.5 × 10 4 cells / mL, and 180 μL of each was diluted in a 96-well plate and cultured for 24 hours. After culturing for 24 hours, the cells were injected with the nanosized dispersion according to the concentration of the final melittin, and cultured for 24 hours. Then, the pretreated cells were treated with MTT reagent and incubated for 4 hours. After centrifugation at 1,500 rpm for 5 minutes, the supernatant was removed, and the dark blue formazan was dissolved in DMSO to measure the color development at 540 nm. The inhibitory rate of cell inhibition was compared with the control group through the absorbance value, and the anticancer activity of the nanodispersion on breast cancer cells was analyzed. In addition, the selectivity indicating the ratio of the growth inhibitory activity to the target cancer cells was calculated, and the degree of targeting was analyzed as shown in Equation 4 below. &Quot; inhibitory activity against cancer cells &quot; means &quot; cytotoxicity against cancer cells &quot;, and &quot; inhibitory activity against cancer cells &quot;

<Formula 4>

<Example 6> Cell permeation observation

<6-1> Cellular permeability analysis using Confocal laser scanning microscope (CLSM)

Confocal fluorescence microscopy was used to qualitatively evaluate the nanoparticle permeability of MDA-MB-231 cells. Seeded at a density of 4 x 10 &lt; 5 &gt; cells / mL in a 6-well plate with cover glass. The seeded cells were cultured in a CO 2 incubator at 37 ° C and 5% CO 2, and the medium was replaced every 48 hours. After confluence of 50 ~ 70% was reached, the cells were washed with PBS after the formation of the appropriate cell monolayer was completed.

The standard nanoparticle dispersion labeled with the fluorescent substance coumarin-6 and the medium were treated with 0.5 mL and 2 mL each in each well and cultured for 2 hours. After the incubation was completed, the sample was removed, the cells were washed twice with PBS, and the cells were fixed with 70% ethanol (pH 7.1 in PBS) for 15 minutes.

Then, 70% ethanol was removed and washed with PBS. Cell nuclei of MDA-MB-231 cells were stained with propidium iodide (PI) and cultured for 30 minutes.

After completion of the staining, remove cover glass with MDA-MB-231 cells from the 6-well plate and place the mounting solution on the dropped slide glass. And dried for 1 to 2 hours. To measure the degree of penetration of nanoparticles into MDA-MB-231 cells, a 50% confocal laser scanning microscope (TCS SP5, Leica, Mannheim, Germany) was used. Fluorescence intensity of the nanoparticles was analyzed by Image software system (NIH, Bethesda, MD, USA).

<6-2> Measurement of apoptosis by FACS analysis

Total cell apoptotic cells / field (%) were measured by flow cytometry (FACS) to determine the total cell death process of MDA-MB-231 cells of melittin-containing nanoparticles. MDA-MB-231 cells were seeded in 25 cm &lt; 2 &gt; T-flasks at a density of 1 X 106 cells / mL.

The inoculated cells were incubated for 1 to 4 days at 37 ° C and 5% CO 2 in a CO2 incubator until confluence of 70-90% was reached. When the optimal cell concentration was reached, the medium was removed and the PBS was washed with the cells.

Subsequently, 6 mL of the nanodispersion mixed with the same volume as the medium was treated with cells, followed by incubation at intervals of 30 minutes and 60 minutes. After completion of the cultivation, the sample was removed, washed with PBS, treated with 0.5 mL of 0.25% trypsin, and the cells were desorbed, centrifuged at 1,000 rpm for 3 minutes, and then the supernatant was removed.

The cell pellet was treated with 0.5 mL of binding buffer to disperse the cells, and 5 μL annexin V-FITC conjugate and 10 μL propidium iodide solution were added and shaded for 10 min. Total cell apoptosis cells / field (%) were measured using a flow cytometer (FACSCanto I, Becton Dickinson, Heidelberg, Germany) and the FACS Diva (BD Biosciences). We analyzed 10,000 events per sample.

<Result>

Experimental Example 1: Safety of Melitin

<Experimental Example 1-1> Ex vivo cytotoxicity

Cell viability of HEK293 cells treated with melitin was measured by MTT assay for the cytotoxicity of free nanoparticle free melittin.

As a result, it was confirmed that the survival rate was 66% at 100 μg / mL and the toxicity was increased in HEK293 cells in a concentration-dependent manner (FIG. 4), as compared with the cell survival rate of the control group not treated with the sample.

<Experimental Example 1-2> Ex vivo cytotoxicity against erythrocytes in Ex vivo

When free nano - encapsulated melitin was treated at different concentrations, the degree of hemoglobin color development caused by hemolysis of red blood cells was measured at 540 nm to evaluate the toxicity to red blood cells. Hemolysis (red cell hemolysis,%) was analyzed by comparison with a red cell suspension and a control group treated with 1% Triton x-100.

As a result, hemolysis from 10 ug / mL to 10% was observed, and it was confirmed that it was cytotoxic to erythrocytes in a concentration-dependent manner (FIG. 5).

Experimental Example 2: Antitumor activity of melitin (ex vivo anticancer effect)

MDA-MB-231 cells were treated with free melittin at various concentrations, and cell inhibition rate was measured by MTT assay to evaluate the anti-cancer activity of melitin itself on breast cancer cells.

As a result, it was confirmed that the inhibitory rate of melitin itself on breast cancer cell proliferation increased from 20% to 80% in a concentration-dependent manner (FIG. 6). The low concentration range of less than 5 μg / mL, in which anti-cancer activity is expressed with relatively low cytotoxicity and red cell cytotoxicity, is measured by measuring the intrinsic antitumor activity of free melittin, which is not nanoparticle-free, To prepare nanoparticles.

<Experimental Example 3> Influence of coating material on particle characteristics of co-nanocapsule

<Experimental Example 3-1> Particle characteristics according to chitosan content

In order to determine the amount of chitosan added during the preparation of nanoparticles using chitosan (CS), PEG and DSPE PEG, particle characteristics of PEG nanoparticles were observed according to the content of chitosan. This was performed by observing the particle characteristics according to the content of chitosan using PLGA / chitosan / PEG 2000 nanoparticles containing no melitin. The nanoparticles include 100 mg of PLGA, 7.2 to 28.8 mg of chitosan and 24 mg of PEG 2000.

The particle size showed a U - shaped tendency, and the smallest particle size was observed when the chitosan content was 14.4 mg / mL. At 7.2 mg, which is the lowest content of chitosan, the binding force at the time of particle formation was also lower and the particle size was increased. Conversely, at 28.8 mg, it was determined that a relatively thick layer was formed due to high chitosan content (FIG. 7A).

PDI was stable at a low level of 0.3, and all of the experimental groups showed a stable value of less than 0.3, with no significant difference between the results (FIG. 7B).

Scattering intensity (DCR) is the amount of intensity of light scattered from the particles, which is affected by the number and size of particles produced. In the present invention, since the scattering strength was remarkably increased at 28.8 mg of chitosan, it was estimated that relatively large nanoparticles were formed in this condition under different conditions (FIG. 7C).

On the other hand, in general, the zeta potential is determined to be more stable when the absolute value is larger than 30 mV. The zeta potential tended to increase with increasing chitosan content. In particular, chitosan contents of 7.2 and 14.4 mg showed almost zero values, but 21.6 mg and 28.8 mg showed stable zeta potentials with high absolute values of 30 mV or more (Fig. 7D).

Finally, the optimum chitosan content was determined to be 21.6 mg, which is a relatively small particle size of 261 nm and exhibits stability in terms of zeta potential.

<Experimental Example 3-2> Particle characteristics according to coating material

In order to analyze the effects of PLGA, chitosan, PEG, and DSPE PEG on the particle properties of nanocapsules, blank nanoparticles without melittin were prepared and their particle characteristics were observed. Blank nanoparticles were prepared in four types in the same manner as nanocapsules 1 to 4 except that no melittin was added. In addition, coumarin-6-containing nanoparticles for measurement of cell-absorbing ability were prepared in the same manner as in Example 6, except that coumarin-6, which is a fluorescent substance, was used instead of melitin.

As a result, the particle size tended to increase with the encapsulation of chitosan, PEG, and DSPE PEG in addition to PLGA (Fig. 8A).

PDI was determined to have a stable value of 0.3 or less for all four types of nanoparticles, and it was determined that the most uniform particle distribution was formed by the PLGA-coated nanoparticles (FIG. 8B).

The scattering intensity was also found to be the highest in the test group coated with PLGA (Fig. 8C).

Zeta potential refers to the electrical double layer potential on the surface of nanoparticles and is an indicator of the stability of the colloidal system. Zeta potentials have been found to have significantly increased absolute values in nanoparticles coated with chitosan (CS), PEG, and DSPE PEG (abbreviated to "DSPE" in the present invention) as compared to PLGA nanoparticles, It was judged to be influential. (Fig. 8D).

<Experimental Example 4>

<4-1> Melitin-containing nanocapsules

In order to observe the physical properties of nanoparticles containing melitin, PLGA, PEG and DSPE PEG particles containing melitin were prepared in the same manner as in Example 1, and then the particle size and PDI, scattering strength, physical properties of zeta potential Were measured.

<4-2> Particle characteristics of melitin-containing nanocapsules

As a result of the particle size measurement, the PLGA nanoparticles were measured at 203 nm, and the particle size was slightly increased with the addition of chitosan (CS) and PEG, and the PEG and DSPE PEG nanoparticles showed 319 nm and 279 nm, respectively. When the two types of PEG were compared, the size of the DSPE PEG nanoparticles was measured to be about 40 nm smaller than that of the PEG nanoparticles (FIG. 8A).

The PDI was measured to be less than 0.3 for all particle types and exhibited a uniform particle distribution (Figure 9B).

As in the case of blank nanoparticles not containing melitin, PLGA nanoparticles exhibited the highest scattering intensity (FIG. 9C).

The zeta potential showed a PLGA nanoparticle value of -13 mV while the PEG and DSPE nanoparticles showed a stable zeta potential of + 33.3 mV and +30.2 mV, respectively, with an absolute value of 30 mV or more (FIG. 9D).

<4-3> Physical properties

Collection efficiency

The addition of chitosan and PEG to PLGA significantly increased the collection efficiency of melitin, and 95% and 94%, respectively, of PEG and DSPE PEG nanoparticles were not significantly different 10A).

Content efficiency

The content efficiency was not significantly different between PEG and DSPE PEG nanoparticles, and was measured as 17.2% and 17.8%, respectively (FIG. 10B).

<Experimental Example 5> Safety inspection of nanoparticles

<Experimental Example 5-1> Ex vivo cytotoxicity

Cytotoxicity of PLGA, PEG, and DSPE PEG nanoparticles containing melittin was observed as in Example 1. Three types of nanodispersions were used to compare HEK 293 cell viability after treatment with various concentrations of melitin.

As a result, the survival rate of HEK293 cells treated with melitin nanoparticle-free melitin (free melittin) as well as melanocytes encapsulated with PLGA, PEG, and DSPE PEG was significantly higher than that of the control group without sample treatment Of the patients. The results showed that the three kinds of nanoparticles of PLGA, PEG and DSPE PEG showed a slightly higher cytotoxicity compared to free melittin without nanoparticles, It was judged that there was no toxicity to the cells (Fig. 11).

<Experimental Example 5-2> Evaluation of cytotoxicity against erythrocytes in Ex vivo

Red blood cell hemolysis test

When the PLGA, PEG, and DSPE PEG nanoparticles diluted to the final melittin concentration range were treated with red blood cells, the amount of hemolysis of the red blood cells was analyzed and the toxicity to the red blood cells was observed.

As a result, hemolysis (%) was less than 1% in PLGA, PEG, and DSPE PEG-coated nanoparticles at the concentration range of 0 to 2 μg / mL of the final nanoparticle content. It did not exist. However, hemolysis (%) of PEG and DSPE PEG was 10% and 39%, respectively, at the concentration of melittin 3 μg / mL, indicating that the amount of erythrocyte hemolysis was higher in DSPE. This was attributed to the fact that due to the structural factors of the phospholipid-polymerized DSPE PEG, the affinity to the cells was high and the toxicity of melitin was rapidly expressed (FIG. 12).

<Experimental Example 5-3> Ex vivo anticancer activity evaluation

MDA-MB-231 cells were treated with diluted PLGA, PEG, and DSPE PEG nano-dispersions in accordance with the final concentration of melittin, and the inhibition rate of cell proliferation after 24 hours was analyzed to observe the anticancer activity against MDA-MB-231 cells Respectively.

As a result, overall antitumor activity was significantly increased with increasing concentration of both free melittin and three nanoparticles. There was no significant difference between the three types of nanoparticles at the concentration of 0.25 μg / mL of melitin. However, the concentration of each of the three types of nanoparticles was significantly higher than that of free melittin. As the concentration increased, the difference between the nanoparticles became clear. Compared with PLGA, PEG and DSPE PEG nanoparticles showed significantly higher anticancer activity than PEG, and PEG had the highest anticancer activity (Fig. 13). Therefore, melitin-containing PEG nanoparticles were most effective in terms of anticancer activity.

On the other hand, the ratio of the toxicity to HEK293 cells and the growth inhibitory activity of MDA-MB-231 cells was measured because there was a problem of decomposing phospholipid bilayer of normal normal cell membrane by acting on cells nonspecifically due to the nature of melittin.

As a result, PLGA nanoparticles showed relatively low selectivity compared to free melittin, while PEG and DSPE PEG nanoparticles showed relatively high selectivity. Particularly, the selectivity of the PEG-coated nanoparticles was the highest, and the highest selectivity ratio when treated with 1.0 ug / mL of PEG was considered to be the most effective concentration for expressing the anticancer effect . On the other hand, the selectivity of DSPE PEG was relatively lower than that of PEG, which is similar to that mentioned above. Due to the structural factors of DSPE PEG polymerized with phospholipids, affinity to normal cells as well as cancer cells is high at the same time, Was expressed in HEK293 cells (Fig. 14).

<Experimental Example 6> Cell permeation observation

<Experimental Example 6-1> Cell permeability analysis using Confocal laser scanning microscope (CLSM)

PLGA, PEG, and DSPE PEG nanoparticles capturing coumarin-6 under the same particle preparation conditions as the melittin nanoparticles were prepared and then treated with MDA-MB-231 cells. Namely, coumarin-6-trapping nanoparticles were prepared in the same manner as the nanoparticles of Experimental Example 5 except that coumarin-6 was used instead of melitin. Subsequently, the nucleus was stained with PI to visually confirm the extent of the intracellular standard uptake, and the form absorbed in the cytoplasm was observed.

As a result, it was observed that green fluorescence appeared around the nucleus of the red dyed nucleus and was absorbed into the cell. The mean pixel intensity of Free Coumarin-6 and PLGA, PEG, and DSPE PEG was calculated by Image software, respectively. As a result, it was confirmed that the intracellular absorption ability in all the nanoparticles was higher than that in Free Coumarin. On the other hand, when the nanoparticles were compared with each other, it was confirmed that when coated with PEG and DSPE PEG, the cell permeability was higher than PLGA (FIG. 15). It was determined that the effect of chitosan showed positive positivity and high cell permeability among common constituents of PEG and DPSE PEG particles.

<Experimental Example 6-2> Measurement of apoptosis through FACS analysis

Total cellular suicide cells / field (%) using flow cytometry (FACS) at intervals of 30 minutes and after 60 minutes after the treatment of free melittin and melitin-containing nanoparticles, ) Were measured.

As a result, the cell suicide area treated with PLGA exhibited similar activity to free melitin at 30 and 60 min incubation time. On the other hand, in the case of PEG and DSPE PEG, the cell suicide area was similar between the two particles, and it was 17.5 and 27.8% at 30 minutes and 30.8 and 35.9% at 60 minutes, respectively. Promoting cell apoptosis in MB-231 cells (Fig. 18). This is similar to the cell permeability measured previously by CLSM. In other words, PEG and DPSE PEG particles having higher cell permeability than PLGA were found to enhance anticancer activity.

conclusion

In the present invention, particle characteristics and cytotoxicity of three kinds of nanoparticles containing melitin, and anticancer activity and cell permeability of breast cancer cells were observed in order to analyze the effect of melitin on nano-encapsulation of breast cancer cells.

The particle size was formed from 200 to 320 nm depending on the particle type, and each particle showed a uniform particle distribution.

In the case of anticancer effect, all nanoparticles showed higher activity than free melittin without nanoparticles, and PEG and DSPE PEG nanoparticles showed the highest anticancer effect. However, in the case of toxicity, DSPE PEG showed hemolysis from 3 ug / mL and showed relatively high toxicity compared to PEG 2000.

In addition, PEG has higher selectivity ratio than DSPE PEG, indicating that it has high selectivity for breast cancer cells. Therefore, the three types of nanoparticles were less toxic, and PEG was the most effective anticancer drug against breast cancer. Optimum melittin concentration was determined to be 1 ug / mL with high selectivity ratio.

<Experimental Example 7>

<Experimental Example 7-1> Production of nanoparticles using high pressure homogenizer

In this experiment, the nanoparticles were prepared by using a high pressure homogenizer (HPH) to determine the possibility of particle size reduction by using PEG and DSPE PEG, which were the most active among the three particle types. The nanoparticles were prepared by the same method as in Example 5 except that a process of homogenization using a high pressure homogenizer (Nano DeBEE, BEE international, Inc., South Easton, Mass., USA) . The homogenization conditions were fixed at 3 cycles and the pressures were measured at 10,000 and 20,000 psi, respectively.

<Experimental Example 7-2> HPH particle particle characteristics

The results of measuring the particle characteristics of HPH particles are shown in Fig. As a result of measuring the particle size, it was possible to achieve a small particle size of 157 to 174 nm by adding the homogenization process to the HPH without significant difference according to the pressure. Which was reduced by about 100 to 150 nm in particle size prior to homogenization (Fig. 17A).

PDI was measured to be less than 0.3 in all particle types and was measured with a uniform particle distribution without significant difference and showed a more stable dispersion with a lower PDI value than the nanoparticles before homogenization (Figure 17B).

The scattering intensity was measured at 10,000 psi, similar to the scattering intensity before homogenization, and at 20,000 psi, it was measured to be lower than 10,000 psi, suggesting that more nanoparticles were formed at 10,000 psi (FIG. 17C).

Zeta potentials were generally reduced before homogenization and HPH-DSPE nanoparticles prepared at 10,000 psi exhibited high zeta potentials similar to the previous ones. Although it differs depending on the particle type, it was presumed that HPH process at high pressure and temperature affected particle formulation (Fig. 17D).

&Lt; Experimental Example 7-3 >

The collection efficiency of HPH nanoparticles was lower than that of conventional nanoparticles (94-95%). It was also found that the melitin capture efficiency was significantly higher when prepared at 10,000 psi than 20,000 psi (Fig. 18). It was concluded that the adsorption efficiency was decreased due to the loss of collected melitin as the pressure was increased during treatment with HPH.

The present invention relates to a composition for prevention, treatment and improvement of cancer comprising melitin nanoparticles having a high tolerance to cancer and a long half-life in blood.

Claims (13)

1. A core comprising melitin; And

   The coating layer covering the core

   Wherein the composition comprises a particle comprising a particle comprising a particle.
2. The method according to claim 1,

   Wherein the coating layer comprises polyethylene glycol.
3. The method according to claim 1,

   Wherein the particles have a size of 180 to 350 nm.
4. The method according to claim 1,

   Wherein the particle has a higher degree of selectivity calculated by the following formula (4) than that of melitin having no coating layer.

   <Formula 4>

   
5. The method according to claim 1,

   Wherein the particles have higher stability in blood than melitin having no coating layer.
6. The method according to claim 1,

   Wherein the particle has a longer half-life in vivo than melitin having no coating layer.
7. The method according to claim 1,

   Wherein the melitin is melittin derived from bee venom.
8. The method according to claim 1,

   Wherein the coating layer comprises polylactide-co-glycolide or chitosan.
9. The method according to claim 1,

   Wherein the composition for preventing and treating cancer is in the form of a suspension for injection.
10. A core comprising melitin; And

    The coating layer covering the core

    Wherein the composition comprises a particle comprising a particle containing at least one particle.
11. To the subject

    A core comprising melitin; And

    The coating layer covering the core

    Comprising administering to the subject a composition for preventing and treating cancer, the composition comprising a particle comprising a particle comprising the compound of formula (I).
12. To the subject

    A core comprising melitin; And

    The coating layer covering the core

    Comprising administering a food composition for preventing and / or improving cancer comprising particles comprising:
13. A core comprising melitin; And

    The coating layer covering the core

    For the prevention, treatment and improvement of cancer.

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