KR101927949B1

KR101927949B1 - multicomponet hetero-multivesicular vesicles, use thereof and preparation method thereof

Info

Publication number: KR101927949B1
Application number: KR1020170027553A
Authority: KR
Inventors: 임용범; 권수현
Original assignee: 연세대학교 산학협력단
Priority date: 2017-03-03
Filing date: 2017-03-03
Publication date: 2019-02-26
Also published as: KR20180100967A

Abstract

The present invention provides a new type of multiple vesicles composed of multiple components through hierarchical self assembly of an annular peptide and a lipid bilayer vesicle which can encapsulate a variety of physiologically active substances Therefore, it has high performance, that is, sophisticated, as well as excellent in thermal stability and has new physical properties such as electric double layer.
Therefore, the multiple vesicles of the present invention can be utilized as various organelles and artificial cells that perform complex functions.

Description

&Lt; Desc / Clms Page number 1 > Multicomponent multi-vesicles, their use and methods of making them.

The present invention relates to multiple vesicles, and more particularly to multifunctional multi-vesicles consisting of multiple components of a cyclic peptide and a phospholipid, its use and a method of preparing the same.

In general, among the candidate drugs that are expected to be potentially valuable, there are substances that are not able to enter the development phase due to their low solubility. The process of solubilizing a drug that is insoluble in water or an aqueous solution by any manipulation is called solubilization, which can increase the absorption of the drug. Conventional techniques using solubilization include ethanol or a surfactant, or in the form of a salt when an ionized group is present. In recent years, techniques using liposomes, microemulsions, cyclodextrins, and the like have been developed.

Currently, there are liposomes or vesicles in the spotlight. The liposome or vesicle is a spherical bio-nanomaterial having an inner space, and has a merit that a desired substance can be enclosed in the inner space and a drug can be delivered to a desired place. They also have the advantage that they can be widely applied not only in the field of drugs but also in the transfer of functional substances to skin cells in the cosmetics industry, encapsulation and transportation of various nutrients in the food industry.

However, there is a disadvantage that liposomes as carriers are limited in their use due to their low structural stability, and that only one substance can be trapped and moved to a desired location.

In order to overcome these disadvantages, we tried to make a liposome containing different substances into one liposome and simultaneously move and react various substances. For example, in the non-patent document 1, multiple liposomes have been disclosed, but there is no progress other than that, and the method described in the above paper is also very difficult. It is not only possible to make only experts who have made many years, There are big disadvantages.

Another example is the structure of the multivesicular body (MVBs) in the biological system. It plays an important role in protein sorting, targeting, degradation and recycling in the endocytosis and exocytosis processes. Although the functions and biogenetic principles of some formulas are not clearly explained, attention is focused on multimers having these high performance functions. This is also mostly produced using only one type of supramolecular building block and is only produced by an emulsion-based process, which is able to dynamically maintain yield in a metastable state rather than a thermodynamic equilibrium state It has disadvantages.

In order to solve the above-mentioned problems, it has been found that a complicated and hierarchical self-assembled nanostructure having a high performance function can be simply manufactured in the present invention.

Non-Patent Document 1. Boyer, C., and Zasadzinski, JA., Multiple Lipid Compartments Slow Vesicle Contents Release in Lipases and Serum, ACS Nano., 1 (3): 176-82 (2007)

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems, and it is an object of the present invention to provide a method of manufacturing a semiconductor device, which comprises (a) a first vesicle comprising a lipid bilayer structure; And

(b) a second vesicle comprising at least one annular peptide located in the first vesicle interior space,

The second vesicle (b) is a spherical vesicle of a bilayer structure formed by self-assembly of a cyclic peptide, the cyclic peptide is a hydrophilic segment, a hydrophobic self-assembling segment and a linker Lt; / RTI >

And a fatty acid is bound to the N-terminal, C-terminal or both terminal ends of the cyclic peptide.

Another object of the present invention is to provide a drug delivery system comprising the multiple vesicles.

Still another object of the present invention is to provide a process for producing multiple vesicles comprising the steps of:

a) preparing an amphipathic peptide;

b) removing the protecting group at the terminal of the amphipathic peptide, self-assembling the peptide to prepare a cyclic peptide, and then binding a fatty acid to the cyclic peptide,

c) self-assembling the peptide functionalized with the fatty acid produced in step b) to prepare a second vesicle,

d) dissolving the phospholipid in a solvent, drying with a rotary evaporator, and then adding the second vesicle prepared in step c) and

e) repeating the cycle of d) freezing and thawing the solution at least once more.

Through the step e), it is possible to form the first veycicle and induce the second veycle to be sealed in the inner space of the first veycicle.

In order to accomplish the above object, the present invention provides a method of manufacturing a semiconductor device, comprising: (a) providing a first vesicle comprising a lipid bilayer structure; And (b) a second vesicle comprised of at least one annular peptide located in the first vesicle interior space, wherein (a) the first vesicle is a spherical vesicle comprising a lipid bilayer (B) the second vesicle is a spherical vesicle of a bilayer structure formed of a cyclic peptide, wherein the cyclic peptide is composed of a hydrophilic segment, a hydrophobic self-assembling segment and a linker , And a fatty acid is bound to the N-terminal, C-terminal or both terminal ends of the cyclic peptide.

And the second vesicles each have a separate independent region.

The lipid is a phospholipid.

The phospholipid is selected from the group consisting of egg yolk lecithin (phosphatidylcholine), soybean lecithin, lysolecithin, sphingomyelin, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, diphosphatidylglycerol, cardiolipin, natural phospholipids of plasmarogens, hydrogenation products dicetylphosphate obtainable from these phospholipids by conventional methods, distearoylphosphatidylcholine, dioloylphosphatidylethanolamine, dipalmitoylphosphatidylcholine, Phosphatidylethanolamine, dipalmitoylphosphatidylserine, eleostearoylphosphatidylcholine, eleostearoylphosphatidylethanolamine, and a mixture of fatty acids that can be obtained by lipid synthesis and hydrolysis thereof. Select from It is characterized by more than one.

The cyclic peptide is characterized by being present in a concentration ranging from 1.5 to 30 [mu] M above the critical aggregation concentration (CMC).

Wherein the hydrophilic segment comprises an arginine rich motif (ARM) domain that forms an alpha -helical structure, the hydrophobic self-assembled fragment is 4 to 6 amino acid residues comprising four tryptophan, An ethylene glycol-based linker, or a linker comprising 1 to 6 amino acid residues.

Wherein the hydrophilic fragment comprises the amino acid sequence of SEQ ID NO: 1 below.

[SEQ ID NO: 1]

TRQARRNRRRRWRR

The hydrophobic self-assembled fragments include the following SEQ ID NO: 2 or SEQ ID NO: 3.

[SEQ ID NO: 2]

GWWWW

[SEQ ID NO: 3]

WWGWW

The cyclic peptide is characterized by being represented by the following formula (1) or (2).

The fatty acid may be a saturated fatty acid or an unsaturated fatty acid, and the saturated fatty acid may be selected from the group consisting of butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, eicosanoic acid and docosano And the unsaturated fatty acid may be any one or more selected from the group consisting of oleic acid, linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid, docosahexanoic acid and erucic acid.

The first veycicle has an average diameter in the range of 1 to 10 mu m and the second veycicle has an average diameter in the range of 10 to 500 nm.

The multiple vesicles are characterized by having an electric double layer.

Another object of the present invention is to provide a method for producing multiple vesicles comprising the following steps.

a) preparing an amphipathic peptide,

The step c) is characterized in that the peptide functionalized with the fatty acid is present in a concentration ranging from 1.5 μM to 30 μM with a critical aggregation concentration (CAC) or higher.

The cycle of freezing and thawing is characterized in that a freezing process at-200 to 150 ° C for 1 to 10 minutes and a thawing process at 40 to 60 ° C for 1 to 10 minutes are repeatedly performed.

The present invention provides a new type of multiple vesicles composed of multiple components through hierarchical self assembly of an annular peptide and a lipid bilayer vesicle which can encapsulate a variety of physiologically active substances Therefore, it has high performance, that is, sophisticated, as well as excellent in thermal stability and has new physical properties such as electric double layer.

The multiple vesicles can be easily and conveniently prepared through self-assembly according to the differential thermal stability between the cyclic peptide of the present invention and the lipid bilayer.

Therefore, the multiple vesicles of the present invention can be utilized as various organelles and artificial cells that perform complex functions.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A is a schematic view schematically showing the overall structure of multiple vesicles according to the present invention. FIG.
Figure 1b is a schematic diagram showing the pathway of multiple vesicles according to the present invention in intracelluar. In this case, CPP is a cell-penetrating peptide, NLS is a nucleus localization signal, HFIP is hexafluoroisopropanol, GFPr is a green fluorescent probe (carboxyfluorescein), RFPr (Red fluorescent probe (rhodamine B, RDB)), AEX is anion exchange chromatography, and CEX is cation exchange chromatography.
2A is a diagram illustrating a process of synthesizing a cyclic peptide according to the present invention.
FIG. 2b is a diagram illustrating a mechanism for producing a cyclic peptide functionalized with a fatty acid according to the present invention.
FIG. 3A is a graph showing HPLC results of a cyclic peptide building block (cpBB) prepared from Preparation Example 2 and functioning as a purified fatty acid.
3B is a graph showing MALDI-TOF MS spectral results of a cyclic peptide (cpBB) prepared from Preparation Example 2 and functionalized with purified fatty acid. The average molecular weight (MW) of cpBB at this time is calc'd 3607.36 and obs'd 3607.28.
4 is a view illustrating a process of manufacturing a second veycicle through self-assembly according to the present invention.
FIG. 5 is a view illustrating a process of preparing multiple vesicles according to the present invention.
FIG. 6 shows the critical aggregation concentration (CAC) of the cyclic peptide (cpBB) functionalized with fatty acid prepared in Preparation Example 2. FIG. 6A is a graph showing trp (tryptophan) fluorescence intensities measured in water according to the concentration (μM) of the cyclic peptide functionalized with fatty acid prepared in Production Example 2. FIG. 6B is a graph showing the trp This is a log scale showing fluorescence intensity in the 360 ㎚ category in order to confirm whether the cyclic peptide exhibits a concentration dependent tendency. An extrapolated linear regression line of point intersection was used to calculate the CAC.
7A, 7B and 7C are negative negative stain TEM photographs of the second vesicle of Preparation Example 3 prepared by self-assembly from the cyclic peptide (cpBB) functionalized with fatty acid of Preparation Example 2 .
Figure 8 is an AFM image for the second vesicle of Preparation Example 3, on mica. At this time, the left side is a height image and the right side is a phase image. Here, the concentration of the cyclic peptide functionalized with fatty acid of Preparation Example 2 used for preparing the second vesicle of Preparation Example 3 was 20 μM.
FIG. 9A is a graph showing the fluorescence intensity of calcein measured after a period of time after treatment with Triton X-100 (1%, v / v) to the first vesicle (LV) FIG.
FIG. 9B shows the fluorescence intensity of calcein measured after a period of time after treatment with Triton X-100 (1%, v / v) to the second vesicle (cPV) of preparation example 3 in which calcein was captured, FIG.
FIG. 9c shows the results of the second vesicle (cPV) of Preparation Example 3 in which calcein was captured and the first vesicle (LV) and Triton X-100 (1%, v / v) And a calcein leakage assay (%) as a function of time.
FIG. 9D is a graph showing the calcein leakage assay measured before and after performing 5 freeze-thaw cycles (FTCs) on the second vesicle (cPV) of Preparation Example 3 in which calcein was captured. The freeze-thaw cycle (FTCs) was performed at -196 ° C for freezing and at 55 ° C for thawing.
Figure 10 is the CD spectrum for the second vesicle of Preparation Example 3 under various temperature conditions. The cyclic peptide functionalized with the fatty acid of Preparation Example 2 used for preparing the second vesicle of Preparation Example 3 was 20 [mu] M.
11A is an AFM image of the first veycicle prepared from Production Example 4. Fig.
11B is an AFM image of the dual-MVVs (cpBB 20 uM + EYPC (egg yolk L-a-phosphatidylcholine) 3 mg / ml) prepared in Preparation Example 5.
12 is an AFM image for a dual-MVV manufactured from Production Example 5. Fig. The left side of the drawing is the height image, the right side is the phase image, and the graph on the upper side of the drawing shows the height in the red line area displayed on the drawing.
13 is a cryo-TEM image of a multi-vesicle (dual-MVV) prepared from Preparation Example 5. Fig. The cyan line represents the circumference of the first vecicle and the yellow line represents the circumference of the second vecicle.
Fig. 14 is a confocal laser scanning microscope (CLSM) image for a multi-vesicle (quad-MVV) encapsulating the fluorescent substance of Production Example 6. Fig. In this case, Bg is an image of the background, Gr is an image of green fluorescence, and Rd is an image of red fluorescence. However, since the background image did not show actual fluorescence and there was a long exposure time before the green fluorescence image was taken, the background fluorescence in the green fluorescence image was observed outside of the multiple vesicles will be. Bar = 10 μm
Figure 15 shows the size distributions (a, c, e) for the first vecicle, the second vecicle and the multiple vecicles and the zeta potentials (b, d, (g, h) of each vesicle derived from the zeta potential results. (D) dynamic light scattering (DLS) data of the second veycles prepared from the preparation example 3, c) dynamic light scattering (DLS) data of the first veycles prepared from the production example 4, e) (DLS) data of the multiple vesicles. B) the zeta potential data of the second vesicle prepared in Production Example 3, d) the zeta potential data of the first vesicle prepared in Production Example 4, f) the zeta potential data of the multiple vesicles prepared in Production Example 5 Potential data. Mean + sd (standard deviation) (n = 6). All of the above data were measured on distilled water at 25 ° C.
Figure 16 is intended to observe how multiple vesicles according to the present invention are differentially absorbed into cells.
Specifically, FIG. 16A shows the results of treatment of cells with a second vesicle (cPV) capturing FAM (carboxyfluorescein) prepared from 1) of Production Example 6, followed by shooting a bright field image and a fluorescence image with a confocal microscope, It is an image superimposed on this.
FIG. 16B is an image obtained by treating cells with a first vesicle prepared in Production Example 4 capturing RDB (rhodamine B) and then photographing bright field and fluorescence images using a confocal microscope, and then superimposing the images.
FIG. 16C is an image obtained by treating cells with multiple vesicles encapsulating a fluorescent substance prepared in Production Example 6 and then photographing bright field and fluorescence images using a confocal microscope, and then superimposing the images.
17 is a schematic diagram showing the absorption path of a second vesicle, a first vesicle, and a multiple vesicle according to the present invention in intracelluar, analogous to the results of Fig. 16. Fig. In this case, CPP is a cell-penetrating peptide, NLS is a nucleus localization signal, HFIP is hexafluoroisopropanol, GFPr is a green fluorescent probe (carboxyfluorescein), RFPr (Red fluorescent probe (rhodamine B, RDB)), AEX is anion exchange chromatography, and CEX is cation exchange chromatography.

Hereinafter, various aspects and various embodiments of the present invention will be described in more detail.

As used herein, the term "peptide" means a linear molecule in which amino acid residues are joined together by peptide bonds.

Representative amino acids and their respective abbreviations include alanine (Ala, A), isoleucine (Ile, I), leucine (Leu, L), methionine (Met, M), phenylalanine (Phe, F) P), tryptophan (Trp, W), valine (Val, V), asparagine (Asn, N), cysteine (Cys, C), glutamine (Gln, Q), glycine ), Threonine (Thr, T), tyrosine (Tyr, Y), aspartic acid (Asp, D), glutamic acid (Glu, E), arginine (Arg, R), histidine ).

The peptides of the present invention can be prepared according to chemical synthesis methods known in the art, particularly solid-phase synthesis techniques (Merrifield, J. Amer. Chem. Soc. 85: 2149-54 (1963) ; Stewart, et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co .: Rockford, 111 (1984)).

An aspect of the present invention provides a method of preparing a pharmaceutical composition comprising: (a) providing a first vesicle comprising a lipid bilayer structure; And (b) a second vesicle comprised of at least one annular peptide located in the first vesicle interior space, wherein (a) the first vesicle is a spherical vesicle comprising a lipid bilayer , (B) the second vesicle is a double-membrane spherical vesicle formed of a plurality of annular peptides, the annular peptide is a hydrophilic segment, a hydrophobic self-assembling segment, and a linker And a fatty acid is bound to the N-terminal, C-terminal or both terminal ends of the cyclic peptide. Its structure is shown in more detail in Fig.

In the present invention, "multivesicular vesicles (MVV)" refers to a multicyclic vesicle in which at least one second vesicle smaller than the first vesicle is captured in a first vesicle of one lipid bilayer do. Its structure is shown in Fig. Unlike conventional multilamellar, the present invention is a form in which a plurality of vesicles are dispersed in a vesicle inner space.

More specifically, the first and second vesicles were prepared with different components. Therefore, since the first vesicle and the second vesicle each have separate independent regions, it is preferable that the first vesicle and the second vesicle are separated from each other by one or more The carrier material can be sealed.

The lipid constituting the first vesicle may be a phospholipid. This is characterized by being in the form of a vesicle as shown in Fig. The phospholipid is selected from the group consisting of egg yolk lecithin (phosphatidylcholine), soybean lecithin, lysolecithin, sphingomyelin, phosphatidic acid, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, diphosphatidylglycerol, cardiolipin, natural phospholipids of plasmarogens, hydrogenation products dicetylphosphate obtainable from these phospholipids by conventional methods, distearoylphosphatidylcholine, dioloylphosphatidylethanolamine, dipalmitoylphosphatidylcholine, Phosphatidylethanolamine, dipalmitoylphosphatidylserine, eleostearoylphosphatidylcholine, eleostearoylphosphatidylethanolamine, and a mixture of fatty acids that can be obtained by lipid synthesis and hydrolysis thereof. Select from It may be more than one. The phospholipid is preferably an egg yolk lecithin (phosphatidylcholine). It is excellent in structural stability, can bind various ligands and receptors to the surface, has a thermal stability difference when mixed with the following cyclic peptide, It is possible to manufacture multiple vesicles controlled to have a desired structure through self-assembly.

Refers to a "sandwich-like" structure composed of amphiphilic lipid molecules (phospholipids) arranged as two molecular layers with a hydrophobic part of the lipid and a polar part outside the surface inside the surface.

Characterized in that it has an inner space isolated from the medium by a spherical first vesicle made of the lipid bilayer and can stably capture or encapsulate at least one second vesicle in the inner space.

Wherein the second vesicle is a spherical vesicle of a bilayer structure formed of a plurality of annular peptides, wherein the annular peptide comprises a hydrophilic segment, a hydrophobic self-assembling segment and a linker , A fatty acid may be bonded to the N-terminal, C-terminal or both terminal ends of the cyclic peptide.

Specifically, the second vesicle is an inner membrane formed by the cyclic peptide; And an outer membrane formed on the outer circumferential surface of the inner membrane formed by connecting the plurality of annular peptides, wherein the annular peptide is bonded by hydrophobic interaction and pi-pi interactions (pi-pi stacking).

The inner membrane has hydrophobic self-assembled fragments arranged in the direction of the outer membrane, the outer membrane has hydrophobic self-assembled fragments arranged in the direction of the inner membrane, and the inner membrane and the outer membrane have hydrophobic interactions between the hydrophobic self- The structure may be maintained. This type of molecular alignment and molecular alignment maximizes molecular alignment density and charge density, and allows stable stabi- lization of the alpha-helix secondary structure.

Therefore, as described above, the second vesicle made of the cyclic peptide is characterized by being a double vesicular spherical vesicle.

The hydrophilic fragment includes not only an alpha -helical structure that plays an important role in recognizing a specific molecule, but also an amino acid sequence containing more than 50%, preferably 60% or more of positively charged arginine residues. It does not. Since the hydrophilic fragment is completely constricted in the cyclic peptide, the alpha -helical structure is not solved and stably maintained, and the base for allowing multiple vesicles to have an electric double layer can be provided.

More specifically, the hydrophilic fragment includes an arginine rich motif (ARM) domain forming an alpha -helical structure, more preferably a human immunodeficiency virus type 1 having an alpha helical structure (hereinafter referred to as HIV- 1, which contains an amino acid sequence derived from the Rev protein, which is involved in the release of the intron-containing virus mRNA outside the nucleus, which encodes a protein essential for HIV-1 replication, It is already known that they have specific binding characteristics to each other and do not coagulate with each other.

In addition, when the ARM domain of the Rev protein is used, it can achieve a role of inhibiting the actual Rev protein.

More preferably, the hydrophilic alpha-helical domain may comprise the amino acid sequence of [SEQ ID NO: 1].

[SEQ ID NO: 1]

TRQARRNRRRRWRR

The hydrophobic self-assembled fragments are included in the cyclic peptide to form a double-wall structure by hydrophobic interaction with the hydrophobic self-assembled fragments of other cyclic peptides. In the present invention, four hydrophobic self-assembled fragments Or consisting of 4 to 6 amino acid residues comprising it.

The hydrophobic self-assembled fragment may comprise SEQ ID NO: 2 or SEQ ID NO: 3 below.

[SEQ ID NO: 2]

GWWWW

[SEQ ID NO: 3]

WWGWW

The cyclic peptide having the structure as described above may be bound without a linker, but preferably includes a linker having a length not to interfere with the molecular recognition specificity of the cyclic peptide in view of flexible characteristics. Wherein the linker comprises an amino acid (G) n consisting of glycine, an amino acid consisting of glycine-serine (e.g., (GS) n, (GSGS) n and (GGGS) n wherein n is at least one integer) An amino acid consisting of alanine, an amino acid consisting of alanine-serine, and another flexible linker such as a tether for a shaker potassium channel. However, glycine is most preferred because glycine can effectively access the phi-psi space and is less restrictive than other amino acid residues with long chains than alanine. Since serine is hydrophilic, a globular glycine chain can be dissolved. Therefore, a flexible linker composed of an amino acid composed of glycine-serine is most preferable.

That is, the cyclic peptide can be represented by the following formula (1) or (2).

[Chemical Formula 1]

(2)

The fatty acid may be a saturated fatty acid or an unsaturated fatty acid, and the saturated fatty acid may be selected from the group consisting of butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, eicosanoic acid and docosano And the unsaturated fatty acid may be any one or more selected from the group consisting of oleic acid, linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid, docosahexanoic acid and erucic acid. Due to the flexible alkyl chains of these fatty acids, it is possible to enhance the structural rigidity of the cyclic peptide and to satisfy structural deficiencies to provide more closely bound cyclic peptides. Also, when multiple vesicles are formed, they act as a nanocontainer to prevent the substances contained in the first vesicle from being released to the outside. Saturated fatty acids are most preferably used for this effect, and stearic acid is most preferably used.

At this time, the fatty acid may be modified to react with an amine of the cyclic peptide. Preferably, the terminal of the fatty acid is succinimidyl succinate, succinimidyl propionate, succinimidyl butano Octadecanoate, N-hydroxysuccinimide, benzotriazole carbonate, maleimide, and OPSS.

The multiple vesicles according to the present invention are multi-component and multidimensional self-assembled nanostructures manufactured through self-assembly using different physical and charge characteristics among the components. Multiple vesicles of multicompartment can be prepared according to the charge characteristics and the differential physical properties according to the kinds of the components.

Since the formation of multiple vesicles is based on the principle of thermodynamic self-assembly, not only the stability to freezing and thawing cycles but also the structural stability of the similar nanostructures produced by the emulsion-based kinetic method is remarkably excellent, Since it has independent multiple compartments by the second vesicle, it is possible to encapsulate various transport materials and has an advantage of having high functionality.

Firstly, the present invention relates to a method wherein the cyclic peptide forms a small second vesicle in the lumen and the lipid forms the outermost large vesicle, so that the first and second vesicles of the different components form one , Different carrier materials may be enclosed within the multiple vesicles.

Since the multiple vesicles of the present invention are produced through self-assembly, they are carried out in a pure solution. Therefore, unlike the oil-based emulsion method, a large amount of organic solvent having toxicity is not left, It does not exhibit toxicity.

The first vesicles may have an average diameter in the range of 1 to 10 mu m and the second vesicles may have an average diameter in the range of 10 to 500 nm.

The multiple vesicles may have an electrical double layer, and more particularly the multiple vesicles have a zeta potential in the range of + 5-15 [deg.]. Generally, the first vesicles of the multiple vesicles should have near-neutral zeta potential because they are composed of charge neutral phospholipids. However, at least one second vesicle charged in the present invention is trapped in the first vesicle interior space And each second vesicle has its own stern layer, so that it is expected that uneven distribution of ions will occur in the lumen of the phospholipid. In sum, the nonuniformly charged lumen generates a potential across the outermost first veycicle and attracts negatively charged counter ions to the outer surface of the first veycicle.

Therefore, multiple vesicles behave as positively charged particles, even though they have a chemically natural surface, because they have a slipping plane on the outer surface as described above. This makes it possible to perform an uncommon multifunctional function (see FIG. 15 h).

Another aspect of the present invention relates to a drug delivery system comprising the multiple vesicles.

The present invention relates to a drug delivery vehicle comprising a rigid multi-vesicle form, wherein the multiple vesicles having the structure described above are characterized in that the multiple vesicles have an internal space between the first and second vesicles, , Which encapsulates any one or more of the same or different transport materials in an independent region of a multi-vesicle, and which is applied to the human body, It can be used as a drug delivery system.

In addition, liposomes composed of lipid components or structures such as vesicles and polymer particles have poor stability and are not easy to store, have a complicated manufacturing process, take too long a time for the manufacturing process, Or bio-toxicity due to the organic solvents used during the manufacturing process. However, since the multiple vesicles of the present invention are made of multiple constituents formed by a first vesicle composed of lipid and a second vesicle composed of a cyclic peptide, excellent structural stability in freezing and thawing cycles It is easy to store, easy to manufacture and easy to use, and does not use organic solvents, so it is not biocompatible or harmful to human body.

In the drug delivery system, surface charge plays an important role in determining the efficiency of delivering the carrier substance. Generally, in the case of liposomes, the charge carriers are not negatively charged because of the negative charge. Because the cell membrane is negatively charged, repulsion occurs.

Therefore, the multi-vesicle of the present invention has a positive zeta potential, unlike the liposome, despite the fact that the first vesicle present at the outermost periphery is composed of lipids, and thus has a general advantage of liposome, Can be obtained. In addition, the multiple vesicles of the present invention can provide a more promising approach because they achieve the effects described above without treating the first vesicle with any hydrophilic ligand.

The multiple vesicles of the present invention have a completely new absorption path that can not be obtained when the first vesicle and the second vesicle are present alone. For example, when a peptide having cell permeability is used (a conventional cell permeable peptide Particularly a peptide having an arginine-rich motif) has an absorption path similar to that of the present invention, and when it is prepared as a multiple vesicle, it is not absorbed into the nucleus but is concentrated around the nuclear membrane, Can be designed to release the vesicles and materials into the cytoplasm and secondly to have a differential absorption pathway in which the second vesicle is dense in the nuclear membrane of the cell and can concentrate the second vesicle material near the nuclear membrane . This series of processes is shown in more detail in Fig.

The carrier material may be any one or more carriers selected from the group consisting of proteins, peptides, nucleic acid molecules, saccharides, lipids, nanoparticles, compounds, and fluorescent materials.

The drug delivery system of the present invention is characterized in that multiple vesicles are formulated using a pharmaceutically acceptable carrier. Since the multiple vesicles of the present invention described above are used, In order to avoid the excessive complexity of the specification according to the present invention.

The drug delivery system of the present invention can be administered orally or parenterally. In the case of parenteral administration, the drug delivery system can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, and transdermal administration.

A suitable dose of the drug delivery vehicle of the present invention may vary depending on various factors such as formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and reactivity, , But it may be an oral dose of preferably 0.001 to 100 mg / kg (body weight) per day.

The drug delivery vehicle can be prepared in unit dosage form by intramuscular injection into a multi-dose container by formulating it with a pharmaceutically acceptable carrier and excipient according to a conventional method. At this time, the formulations may be any one selected from the group consisting of solutions, suspensions, powders, granules, tablets and capsules.

Another aspect of the present invention relates to a process for preparing multiple vesicles comprising the steps of:

a) preparing an amphipathic peptide,

First, a linear amphipathic peptide is prepared prior to the production of the cyclic peptide. FIG. 2 is a view illustrating a process of synthesizing a cyclic peptide according to the present invention and a cyclic peptide functionalized with a fatty acid.

Reference can be made to chemical synthesis methods, particularly solid-phase synthesis techniques, which are known in the art for preparing linear amphipathic peptides, first of all (Merrifield, J. Amer. Chem Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem. Co .: Rockford, 111 (1984)).

The amphipathic peptide comprises a hydrophilic segment, a hydrophobic self-assembling segment and a linker, wherein the hydrophilic segment comprises an alpha -helical structure that plays an important role in recognizing a specific molecule But is not limited to, an amino acid sequence containing 50% or more, preferably 60% or more of positively charged arginine residues. When the amphipathic peptide is cyclized to form a cyclic peptide, the hydrophilic fragment is completely constricted in the cyclic peptide, so that the α-helical structure is not stably maintained, and the multiple vesicles form an electric double layer And the like.

[SEQ ID NO: 1]

TRQARRNRRRRWRR

The hydrophobic self-assembled fragment is contained in an amphipathic peptide. This is a part that makes it possible to form a bilayer structure by hydrophobic interaction with hydrophobic self-assembled fragments of other cyclic peptides when the amphipathic peptide is produced as a cyclic peptide through cyclization. In the present invention, 4 Or a tryptophan amino acid residue thereof.

[SEQ ID NO: 2]

GWWWW

[SEQ ID NO: 3]

WWGWW

The amphipathic peptide having the structure as described above may be bound without a linker, but preferably includes a linker having a length that does not interfere with the molecular recognition specificity of the amphipathic peptide in terms of flexible characteristics. Wherein the linker comprises an amino acid (G) n consisting of glycine, an amino acid consisting of glycine-serine (e.g., (GS) n, (GSGS) n and (GGGS) n wherein n is at least one integer) An amino acid consisting of alanine, an amino acid consisting of alanine-serine, and another flexible linker such as a tether for a shaker potassium channel. However, glycine is most preferred because glycine can effectively access the phi-psi space and is less restrictive than other amino acid residues with long chains than alanine. Since serine is hydrophilic, a globular glycine chain can be dissolved. Therefore, a flexible linker composed of an amino acid composed of glycine-serine is most preferable.

Next, the end-protecting group of the amphipathic peptide synthesized in the step a) is removed and self-assembled to prepare a cyclic peptide, and then the fatty acid is bound to the cyclic peptide.

The fatty acid is preferably in a reaction-active form so as to be able to react with the amine of the cyclic peptide. To this end, the fatty acid is reacted with N-hydroxysuccinimide (NHS) esters or sulfo N-hydroxysuccinimide sulfo-N-hydroxysuccinimide (Sulfo-NHS) esters and the like. In some cases, maleimide (MAL) or orthopyridyl disulfide (OPSS) may be used so as to react with a thiol group (see FIG. 2B).

The organic solvent is not particularly limited as long as it is not an organic solvent adversely affecting the lipid and the cyclic peptide. Examples of the organic solvent include ethyl ether, acetic acid, acetonitrile, methyl ethyl ketone, chloroform, (DMF), formaldehyde, dimethylsulfoxide, isopropanol, methanol, tetrahydrofuran, tetrahydrofuran (THF), dimethylsulfoxide , At least one organic solvent selected from the group consisting of ethanol, propanol, 1,3-butylene glycol, propylene glycol, glycerin, 1,2-pentanediol, di-panthenol, dipropylene glycol, acetone and DIPEA may be used .

The step b) may be carried out at 20 to 100 ° C for 12 to 50 hours.

Next, c) self-assembling the peptide functionalized with the fatty acid produced in step b) to prepare a second vesicle.

Wherein the peptide functionalized with fatty acids has a concentration ranging from 1.5 μM to 30 μM which is equal to or higher than a critical aggregation concentration (CAC). There is a disadvantage in that the self-assembly process is not properly performed when the concentration is less than 1.5 μM or the concentration is more than 30 μM.

The peptide functionalized with the fatty acid was dispersed in an aqueous solution and the ultrasonic waves were treated for 10 to 30 minutes to self-assemble a second vesicle having a controlled structure.

Thereafter, d) the phospholipid is dissolved in an organic solvent, and the solvent is evaporated and decompressed to form a lipid film. At this time, the temperature can be carried out at 20 to 40 占 폚.

Finally, e) a second vesicle prepared in step c) is added to the lipid film, and the cycle of freezing and thawing is repeated at least once to prepare multiple vesicles.

In the case of the carrier material to be enclosed in the second vesicle, the carrier material is dissolved in water, dissolved in an aqueous solution, dissolved in an organic solvent and administered together in step c).

In the case of a carrier material to be sealed in the inner space between the first veycicle and the second veycicle, the carrier material may be dissolved in water, dissolved in an aqueous solution, dissolved in an organic solvent, and administered in step e) .

The cycle of freezing and thawing is characterized in that a freezing process at-200 to 150 ° C for 1 to 10 minutes and a thawing process at 40 to 60 ° C for 1 to 10 minutes are repeatedly performed, , Multiple vesicles having excellent structural stability against freezing and high temperature can be obtained.

Hereinafter, the present invention will be described in more detail with reference to Examples and the like, but the scope and content of the present invention can not be construed to be limited or limited by the following Examples. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit and scope of the present invention as set forth in the following claims. It is natural that it belongs to the claims.

In addition, the experimental results presented below only show representative experimental results of the embodiments and the comparative examples, and the respective effects of various embodiments of the present invention which are not explicitly described below will be specifically described in the corresponding part.

material

Fmoc-amino acids, Dde-Lys (Fmoc) -OH and coupling reagents were purchased from Novabiochem (Germany) or Anaspec (USA). Common chemical samples were purchased from Sigma-Aldrich (USA) and Merck (Germany). (Fmoc-8-amino-3,6-dioxaoctyl) succinamic acid (Fmoc-PEG-Suc-OH or Fmoc-Ebes-OH) which is a linker based on the above oligoethylene glycol was purchased from Anaspec Respectively.

HPLC solvents were purchased from Fisher Scientific (USA). Chloroform was purchased from Duksan (Korea).

Manufacturing example 1. Ring type Of peptide synthesis

One) Peptides synthesis

The peptides were synthesized using the standard Fmoc protocol using a 0.1 mmol scale Tribute peptide synthesizer (Protein Technologies, USA). The standard amino acid protecting group was synthesized except for Dde-Lys (Fmoc) -OH. Initially, it was preloaded with 2-chlorotrityl resin (Novabiochem, Germany) using Fmoc-Ebes-OH (Anaspec, USA). In the present invention, the peptides shown in Table 1 below were synthesized and used.

division Fused Peptide Nomenclature Sequence (N-> C) Production Example 1 SEQ ID NO: 1 H-WWKWW-Ebes-TRQARRNRRRRWRR-COOH

2) Cyclization

The synthesized peptide was deprotected at the N-terminal Fmoc-functional group for head-to-tail cyclization. Protected peptide fragments (20 μmol) were separated from the resin using cleavage cocktail [acetic acid / 2,2,2-trifluoroethanol (TFE) / methylene chloride (MC) (2: 2: 6)]. After an interval of ~ 1 to 2 hours, the resin was removed by filtration and the filtrate was recovered (4 mL x 2).

Finally, the resin was washed three times with a cleavage cocktail for complete recovery. Hexane was added to the filtrate to remove the acetic acid using hexane as the azeotrope. The evaporation process was repeatedly treated to obtain a protected peptide fragment prepared through the above synthesis step as a white powder. The evaporation process is carried out by dissolving the peptide fragment in methylene chloride (MC), adding hexane, and then evaporating at 35 ° C to 20 ° C using a rotary evaporator (EYELA, Tokyo Rikakikai Co., Ltd., Japan) Respectively. The peptide was cyclized by head-to-tail cyclization between the N-terminal amine and the C-terminal carboxylic acid functional group.

During the cyclization process, 20 pmol (1 equiv.) Of the protected peptide fragment and 4 equivs of N, N-diisopropylethylamine (DIPEA) were dissolved in N, N-dimethylformamide ) (20 mL) and transferred to a ciliner. Next, 1 equiv of 1- [bis (dimethylamino) methylene] -1H-1,2,3-triazolo [4,5-b] pyridinium 3-oxid hexafluorophosphate (HATU) was dissolved in N, N-dimethylformamide 20 ml and then transferred to another ciline. In order to achieve pseudo-high dilution conditions, 1-hydroxybenzotriazole (NMP) was added to N, N-dimethylformamide (DMF, 20 ml) at a rate of 0.06 ml / min using a syringe pump 1-hydroxybenzotriazole, HOBt) and 0.1 equiv of HATU were mixed together to prepare a reaction mixture. When the reaction mixture is thoroughly mixed, it is immediately mixed overnight. N, N-Dimethylformamide (DMF) was then evaporated using a rotary evaporator at 60 ° C and the residue was dissolved in dichloromethane and then quenched to a powder of quartzed protected peptide fragments (four times) -Butyl methyl ether / hexane was added.

(V / v) hydrazine / N, N-dimethylformamide (hydrazine / DMF) was added to the cyclized protected peptide fragment obtained in the above procedure four times to remove the Dde functional group bound to the lysine 2 min per cycle). Subsequently, Dde removed from 2% (v / v) hydrazine / N, N-dimethylformamide (hydrazine / DMF) remaining in the solution was removed using rotary steam. Followed by pulverization using methylene chloride (MC) and tert-butyl methyl ether / hexane to obtain the cyclic peptide fragment (Chemical Formula 1) shown in FIG.

Manufacturing example 2. Stearic acid Functionalized cyclic peptides ( cpBB )

1) Preparation of formula 3

(3)

The compound represented by Formula 3 was prepared through N-hydroxysuccinimide ester of stearic acid. Specifically, the esterification reaction is carried out using an equimolar amount (800 μmol) of stearic acid and N-hydroxysuccinimide.

Specifically, stearic acid and N-hydroxysuccinimide are reacted in the presence of N, N'-diisopropylcarbodiimide (1 equiv.) In tetrahydrofuran, THF) (100 ml) were homogeneously mixed at room temperature and equilibrated. The precipitated product was removed through filtration using a filter paper, and the filtered filtrate was dried using a rotary evaporator. The succinimide ester was dissolved in 5 ml of 60% ethanol at 60 ° C and recrystallized by cooling at room temperature to remove other impurities. The crystals obtained were obtained by filtration using filter paper and dried in air.

The yield was 203.4 mg (533.1 [mu] mol, 66.6%).

^{1 H NMR (250 MHz, CDCl} 3) d 0.88 (t, 3H, CH 3 CH 2), 1.25 (s, 28H, CH 3 (CH 2) 14 CH 2), 1.68-1.81 (m, 2H, CH 2 CH ₂ CO), 2.60 (t, 2H, CH ₂ CO), 2.84 (s, 4H, CO ( CH ₂ ) ₂ CO).

2) Stearic acid N- Hydroxysuccinimide ester( stearic acid N-hydroxysuccinimide ester) ( Manufacturing example 2-1) and annular Peptides ( Manufacturing example 1).

20 μmol (1 equiv.) Of the cyclic peptide synthesized from Preparation Example 1 was mixed with 5 equiv and 3 DIPEA 10 equiv of the formula 3 prepared from Preparation Example 2-1) in dimethylformamide (DMF, 2 ml) To prepare a reaction mixture. The reaction mixture was stirred overnight, and dimethylformamide (DMF) was evaporated using a rotary evaporator. The obtained product was dissolved in methylene chloride (MC), and tert-butyl methyl ether / hexane). The powder obtained through the above process was treated with a cleavage cocktail (TFA / TIS / water; 95: 2.5: 2.5) for 3 hours and pulverized with tert -butyl methyl ether, To obtain a cyclic peptide functionalized with one stearic acid (hereinafter also referred to as cpBB). The resulting cpBB was purified using reverse-phase (RP-HPLC) (water / acetonitrile with 0.1% TFA) and the molecular weight of the peptide was determined by matrix-assisted laser desorption / ionization time-of-flight MALDI-TOF) mass spectrometry using a Microflex LRF20 instrument (Bruker, Germany). These results are shown in Figs. 3 (a) and 3 (b).

The concentration of the peptide was determined using a spectrophotometer. Water / acetonitrile (1: 1) was used as the solvent. The molar extinction coefficient of tryptophan (5502 M ^-1 cm ^-1 ) at 280 nm Respectively.

The term cpBB in the present invention refers to designed cyclic peptide building blocks, which in the examples and preparations represent peptides functionalized with fatty acids.

Manufacturing example 3. Second Of cyclic peptide vesicles (cPV) Produce

A second vesicle having a controlled structure through self-assembly is manufactured according to the following protocol. During the preparation of the second vesicles, the concentration of the cyclic peptide functionalized with fatty acids was generally 20 [mu] M.

First, the peptide functionalized with fatty acids (cpBB) was dissolved in 30% 1,1,1,3,3-hexafluoro-2-propanol, HFIP) (HFIP / water, vol%, 100 [mu] l). This was lyophilized and the peptide functionalized with fatty acids was rehydrated with distilled water (100 μl). Next, the solution was sonicated at room temperature for 20 minutes using a cup-horn sonicator (Qsonica, USA) to produce a second vesicle having a controlled structure through self-assembly. This process is shown in FIG. In FIG. 4, only the first self-assembly was performed without the step of adding GFPr (fluorescent chromophore).

Manufacturing example 4. First Vesicular Produce.

The first solution was prepared by dissolving egg yolk L-α-phosphatidylcholine (EYPC) in a 2: 1 chloroform / methanol (100 μl) solvent. The first solution was evaporated using an erection during rotation at 35 DEG C to prepare 300 mu g of a lipid film and hydrated with distilled water. The hydrated lipid film was subjected to five freeze-thaw cycles (FTC) to prepare a spherical vesicle (first vesicle) consisting of a lipid bilayer.

Manufacturing example 5. Multiple Vesicle (dual- MVV ) Produce.

The first solution was prepared by dissolving egg yolk L-α-phosphatidylcholine (EYPC) in a 2: 1 chloroform / methanol (100 μl) solvent. The first solution was evaporated using an erection during rotation at 35 DEG C to prepare 300 mu g of a lipid film. The lipid film was hydrated with a second vesicle (cPV) (100 μl) solution prepared from Preparation Example 3 and subjected to 5 freeze-thaw cycles (FTC) , And at least one second vesicle was enclosed in a spherical vesicle (first vesicle) composed of a lipid bilayer. At this time, the freezing is repeated with liquid nitrogen at -195 ° C for 5 minutes, and thawing is carried out in a water bath at 55 ° C for 5 minutes. At this time, the multiple vesicles were produced through the above-described process, unless otherwise specified.

If the concentration of cpBB used to prepare the second vesicle and the concentration of EYPC used to make the first vesicle are different, or if you want to clarify, the concentration of cpBB and EYPC used next to the multiple vesicle .

Manufacturing example 6. Multiplexing with fluorescent material Of the quad-MVV Produce.

1) The second capturing FAM Vesicle Produce

A second vesicle prepared from Preparation Example 3 was rehydrated with distilled water containing 200 uM FAM (carboxyfluorescein) to prepare a second vesicle capturing FAM (carboxyfluorescein).

Anion exchange column filled with 0.5 mg / ml (50)) of anion exchange resin (Dowex ㄾ 1X4 chloroform, 100-200 mesh, Sigma-Aldrich) was used to remove unencapsulated FAM Respectively.

2) Quadrature Vesicular Produce

The second vesicle (cPVs-FAM) capturing the purified FAM was mixed with EYPC (3 mg / ml) and rhodamine B (ROD; 200 mM) and subjected to 5 freeze-thaw cycles (FTC) (LV-RDB) in which the quad-MVV (i.e., the second vesicle (cPVs-FAM) capturing FAM and the RDB 'were captured).

The second vesicle (cPV-FAM) and rhodamine B (RDB), which captured positively charged FAM, were removed using a cation exchange column. The cationic acylation column (Dowex 50WX2 hydrogen form 100-200 mesh, Sigma-Aldrich) was packed with 5 ml of silage and 0.5 mg / ml of cation exchange resin.

At this time, in case of taking only Vesicle alone using a confocal microscope, and in the experiment in which the first vesicles captured by RDB were treated with the cells, they were prepared under PBS environment, but the remaining experiments were made in almost distilled water environment.

Experimental Example 1. Cyclic form functionalized with fatty acids Of peptide Critical Aggregation Concentration ( CAC ) minute three

1) critical aggregation concentration, CAC ) Analysis method

A cyclic peptide functionalized with fatty acid prepared in Preparation Example 2 on the basis of 500 ml of distilled water was prepared by dissolving it in various concentrations. The steady-state fluorescence spectrum for tryptophan fluorescence was determined using a PerkinElmer LS-55 fluorescence spectrophotometer (PerkinElmer LS-55 fluorescence spectrophotometer). At this time, the sample was placed in a quartz cuvette having a length of 1 cm, and excitation was measured at 280 nm. The excitation and emission slit with a nominal bandpass of 10 nm was used, and the critical aggregation concentration (CAC) of the second vesicle in the cyclic peptide functionalized with fatty acids was calculated as by plotting the logarithm of the concentration of the cyclic peptide functionalized with fatty acids and calculating the concentration at which the slope changes.

2) CAC Results analysis

In order to confirm whether molecular aggregates can be formed in an appropriate concentration range, the critical aggregation concentration (CAC) in distilled water was measured and analyzed. The results are shown in FIGS. 6A and 6B. The critical aggregation concentration (CAC) was determined by confirming concentration-dependent changes in tryptophan fluorescence.

The calculated critical aggregation concentration (CAC) of the fatty acid functionalized peptide (cpBB) (Preparation Example 2) was 1.5 μM, and the aggregation tendency was relatively strong. Based on this, all subsequent studies were performed at concentrations above the critical aggregation concentration (CAC).

Experimental Example 2. The second Vesiche About TEM analysis

One) TEM (transmission electron microscope) measurement method

2 [mu] l of the second vesicle (20 [mu] M) solution prepared from Preparation Example 3 is placed on the EM-grid. The EM-grid was covered with a newly glow-discharged continuous carbon film. The EM-grid with the second vesicle applied was then air-dried completely. Then, 3 μl of a 1% (w / v) uranyl acetate solution was added, and 1 minute later, the excess solution was removed using a filter paper to prepare a test piece. After thorough air drying, the dried test specimens were measured using a JEOL-JEM 2010 instrument operating at 120 kV (JEOL Ltd., Tokyo, Japan). Images were recorded using a OneView CMOS camera (Gatan Inc., Pleasanton, Calif., USA) and the images were analyzed using Digital Micrograph software.

2) TEM analysis

The second vesicles prepared from Preparation Example 3 were measured by TEM and are shown in Figs. 7A, 7B and 7C. 7A, 7B and 7C are negative negative stain TEM photographs of the second vesicle of Preparation Example 3 prepared by self-assembly from the cyclic peptide (cpBB) functionalized with fatty acid of Preparation Example 2 . At this time, the cyclic peptide functionalized with the fatty acid of Preparation Example 2 used to prepare the second vesicle of Preparation Example 3 was 20 [mu] M, and the bar in the figure indicates 100 nm.

As shown in Figs. 7A, 7B, and 7C, the second vesicle of Preparation Example 3 showed that the cyclic peptide functionalized with the fatty acid of Production Example 2 was self-assembled to produce a typical nanosecond structure with a size of several tens of nanometers Respectively.

Although the critical aggregation concentration differs depending on the cyclic peptide to be synthesized, it contains not only the alpha -helical structure used in the present invention, but also the positively charged arginine residue at 50% or more, preferably 60% or more And a hydrophobic self-assembled fragment comprising 4 to 6 amino acid residues comprising 4 hydrophilic segments containing 4 tryptophan amino acid residues, and a peptide having a structure comprising a linker moiety and a fatty acid, have similar critical aggregation concentrations, Under the self-assembling conditions of 3, a sufficiently spherical second vesicle can be prepared.

7a and 7b, it was confirmed that the second vesicles of Preparation Example 3 were spherical nanoparticles having a typical size of 50 to 150 nm and 70 to 110 nm on average.

Considering the molecular length of the peptide functionalized with the fatty acid of Production Example 2 in the fully expanded state (~ 4-6 nm), the spherical nanostructures observed in Figures 7a, b, and c show a bilayer structure It is a vesicle in the form of a follicle.

Experimental Example 3. Second Vesiche About Atomic power microscope( AFM ) analysis

One) Atomic power Atomic force microscopy, AFM ) How to measure

5 μl of the sample was applied on each mica and then completely dried to measure the AFM. The images were measured in noncontact mode using an NX10 instrument (Park Systems, Korea), AFM scans were performed at a scan rate of 1.0 Hz, and data were acquired using XEN software.

2) Atomic power Microscope analysis

Figure 8 is an AFM image for the second vesicle of Preparation Example 3, on mica. At this time, the left side is a height image and the right side is a phase image. Here, the concentration of the cyclic peptide functionalized with fatty acid of Preparation Example 2 used for preparing the second vesicle of Preparation Example 3 was 20 μM.

As shown in FIG. 8, it was confirmed that the second vesicle of Preparation Example 3 was formed into a spherical nanostructure through self-assembly of the cyclic peptide functionalized with fatty acid of Preparation Example 2.

Experimental Example 4. First Vesicel and Second Vesicular Stability evaluation

One) calcein Capturing the second Vesicular Produce

First, a mixture of 20 μM of cyclic peptide functionalized with fatty acid of Preparation Example 2 and 200 μM calcein from 30% HFIP (500 μl) was lyophilized, hydrated with distilled water and incubated for 20 minutes at room temperature with an ultrasonic injector -horn sonicator) to prepare a solution of a second vesicle (20 [mu] M, 500 [mu] l) containing calcein. And dialyzed overnight using a dialysis membrane (3500 Da MWCO, spectrum laboratories, USA) to remove unincorporated calcein in the second vesicle. At this time, distilled water was used as a dialysis buffer and dialysis buffer was changed three times during the dialysis. The second vesicle capturing the calcein prepared above was mixed with 1% (vol.) Triton X-100 at room temperature.

2) calcein The first to capture Vesicular Produce

Except that a 2: 1 chloroform / methanol solution containing 200 μM calcein was used instead of the 2: 1 chloroform / methanol solution in the first vesicle production process of Production Example 4, To prepare a first vesicle in which calcein was captured. Unincorporated calcein was removed using a NAP ^TM -5 column (GE Healthcare, UK) with distilled water as elution buffer.

3) Leakage assay

The fluorescence spectrum of the stable state was measured using a PerkinElmer LS-55 fluorescence spectrophotometer by placing the sample in a 1 cm path length quartz cuvette. The sample was excited at 495 nm to measure calcein fluorescence. To analyze the time-lapse emission data, the percentage of fluorescence intensity emitted at other points through the equation below was calculated.

[Formula 1]

% release = 100 ( I _t - I ₀ ) / ( I _∞ - I ₀ )

Here, I is the fluorescence intensity at a specific time point, I _o is the fluorescence intensity before the surfactant treatment, and I _∞ is the fluorescence intensity when the structure of the vesicle is completely destroyed.

4) Leakage analysis results

The structural and thermal stability of the second vesicles prepared from Preparation Example 3 and the first vesicles prepared from Preparation Example 4 were compared. For this purpose, a fluorescence probe (calcein) was entrapped in the inner space of the second vesicle of Production Example 3 and the vesicle of Production Example 4. Free dye molecules not captured in the vesicles were separated using size exclusion chromatography (SEC).

A surfactant Triton X-100 was added to a second vesicle (preparation example 3) and a first vesicle (preparation example 4) in which a fluorescent probe calcein was captured, and the vesicle membrane was disrupted, and a leakage fluorescent probe calcein ) Were analyzed for stability and time.

FIG. 9A is a graph showing the fluorescence intensity of calcein measured after a period of time after treatment with Triton X-100 (1%, v / v) to the first vesicle (LV) FIG.

FIG. 9B shows the fluorescence intensity of calcein measured after a period of time after treatment with Triton X-100 (1%, v / v) to the second vesicle (cPV) of preparation example 3 in which calcein was captured, FIG.

FIG. 9c shows the results of the second vesicle (cPV) of Preparation Example 3 in which calcein was captured and the first vesicle (LV) and Triton X-100 (1%, v / v) And a calcein leakage assay (%) as a function of time.

FIG. 9D is a graph showing the calcein leakage assay measured before and after performing 5 freeze-thaw cycles (FTCs) on the second vesicle (cPV) of Preparation Example 3 in which calcein was captured. The freeze-thaw cycle (FTCs) was performed at -196 ° C for freezing and at 55 ° C for thawing.

As shown in Figs. 9A to 9C, the first vesicle of Preparation Example 4 confirmed that calcein was completely released within 2 minutes, whereas the second vesicle of Preparation Example 3 required 30 minutes to completely release calcein Respectively. As a result, it was confirmed that the second vesicle of Preparation Example 3 was significantly superior to the first vesicle of Preparation Example 4 in structural stability due to membrane breakage.

Further, as shown in Fig. 9D, the second vesicle of Production Example 3 is also remarkably excellent in thermal stability. In general, freeze-thaw is used to destroy and reuse liposomes as part of liposome preparation methods. However, the second vesicle according to the present invention confirmed that there was almost no leakage of calcein trapped in the inner space despite the repeated freeze-thaw cycle. That is, it can be seen that the second vesicle of Production Example 3 has excellent stability even after heat change, and permeability to small molecules is maintained as it is.

Experimental Example 5. Second Vesicular CD spectrum

One) Circular polarization Dichroism Spectroscopy dichroism (CD) spectroscopy).

The second vesicle (20 μM, 250 μl) of Preparation Example 3 was measured for CD spectra using a Chirascan ^™ Circular Dichroism spectrometer equipped with a Peltier temperature controller (Applied Photophysics, UK). The CD spectra of the samples were recorded at wavelengths from 190 nm to 260 nm using a 1 mm path length cuvette at various temperatures (4 to 94 ° C). At this time, the scan was performed under conditions of temperature change from 4 ° C. to 94 ° C. (FIG. 10A) and from 94 ° C. to 4 ° C. (FIG. 10B), and the experiment was repeated three times in total. The results were averaged, (molar ellipticity) is calculated for each amino acid residue.

2) Results

Figure 10 is the CD spectrum for the second vesicle of Preparation Example 3 under various temperature conditions. The concentration of the cyclic peptide functionalized with fatty acid of Preparation Example 2 used for preparing the second vesicle of Preparation Example 3 was 20 μM.

As shown in FIG. 10, the second vesicle (Preparation Example 3) prepared from the cyclic peptide functionalized with fatty acid according to the present invention (Preparation Example 2) It can be seen that it has high structural stability against thermal change. That is, it can be confirmed that the structure is almost maintained at 1 to 100 ° C. In addition, it can be confirmed that the second vesicle structure according to the present invention is reversibly changed according to the thermal change.

Experimental Example 6. First Vesicle And multiplex Vesicular Thermal Stability Analysis

(20 μM + 3 mg / ml) prepared in Preparation Example 5 and the first vesicle (3 mg / ml) prepared in Preparation Example 4 were used as samples. Was measured by the atomic force microscope measurement method of Experimental Example 3-1 and is shown in FIG.

Fig. 11A is an AFM image of the first vesicle prepared in Production Example 4, Fig. 11B is an AFM image of the dual-MVVs (cpBB 20 uM + EYPC (egg yolk L-a-phosphatidylcholine) 3 mg / m).

As shown in Fig. 11, it was confirmed that the first vesicle of Production Example 4 retained the structural stability even after the freeze-thaw cycle. As a result, it can be seen that a multi-vesicle made of a more elaborate binary component can be produced.

Specifically, freeze-thawing process in a defrost cycle, if the first lipid component chopping a glass transition temperature higher than the temperature (T _g) of the constituting the cyclohexane is not particularly limited, in the present invention hayeoteumeuro use EYPC -15 ~ -7 ℃ Gt; 55 C. < / RTI >

11b, the presence of a first vesicle having a diameter of several hundred nanometers and a second vesicle having a diameter of several tens of nanometers was confirmed. In addition, a second vesicle was observed in the first vesicle interior space. Therefore, it can be seen that the second vesicles are embedded in the inner space of the first vesicles despite being composed of different constituents.

Experimental Example 7. Multi Vesiche Structural Analysis

1) Purification of multiple vesicles of Preparation Example 5

In order to clarify the structural analysis of the final finished multi-vesicle, which was not subjected to purification and separation processes, in the previous experiment, a multi-vesicle without impurities was prepared by removing the unreacted second vesicle impurity. Cation exchange chromatography (CEX) was used to separate the second vesicle impurities unreacted with the multiple vesicles.

Specifically, to separate the unreacted second vesicle not captured in the lipid vesicle (first vesicle) from the multi-vesicle (dual-MVV) prepared in Preparation Example 5, it was immersed in a cation exchange resin (Dowex 50WX2 chloroform (chloride form 100-200 mesh, Sigma-Aldrich) was used. The cation exchange mini-column was prepared by placing some glass wool fibers on the bottom of a 5 ml syringe and placing 0.5 g / ml of the washed and swollen cation exchange resin in distilled water (500 μl) in the syringe .

The multiple vesicles of the finally isolated Preparation Example 5 were measured by the atomic force microscope measurement method of Experimental Example 3-1), and the results are shown in Fig.

2) Results of AFM analysis

12 is an AFM image for a dual-MVV manufactured from Production Example 5. Fig. The left side of the drawing is the height image, the right side is the phase image, and the graph on the upper side of the drawing shows the height in the red line area displayed on the drawing.

As shown in Fig. 12, it was confirmed that only the pure vesicles of Preparation Example 5 were separated after the purification process through the cation exchange resin. It was also confirmed that a first vesicle having a diameter of several hundred nanometers and a second vesicle having a diameter of several tens of nanometers were present in the inner space thereof.

3) Results of cryo-TEM analysis

The multi-vesicle structure of the present invention was analyzed through cryo-TEM (cryo-TEM; 300 kV, -196 ° C) measurement. At this time, the sample was frozen using liquefied ethane in a container cooled with -196 占 폚 liquid nitrogen for cryogenic TEM.

13 is a cryo-TEM image of a multi-vesicle (dual-MVV) prepared from Preparation Example 5. Fig. The cyan line represents the circumference of the first vecicle and the yellow line represents the circumference of the second vecicle.

As shown in Figure 13, a second vesicle of 10 to 500 nm in size within a first vesicle having a diameter of from a few hundred nanometers to an average of 1 [mu] m to 10 [mu] m, as in the AFM image results Can be confirmed. Thus, the structure of the multiple vesicles of the present invention can be reconfirmed.

Experimental Example 8. Multiplexing with fluorescent material Vesiche Structural Analysis

One) Confocal Laser scanning microscope measurement method

To measure confocal laser scanning microscopy (CLSM), 15 μl of the sample was mounted between the slide and cover glass and sealed with a nail polish. Images were measured using an LSM700 confocal laser scanning microscope (Carl Zeiss, Germany) and the data were analyzed using ZEN software. The results are shown in Fig.

2) Analysis results

Fig. 14 is a confocal laser scanning microscope (CLSM) image for a multi-vesicle (quad-MVV) encapsulating the fluorescent substance of Production Example 6. Fig. In this case, Bg is an image of the background, Gr is an image of green fluorescence, and Rd is an image of red fluorescence. However, since the background image did not show actual fluorescence and there was a long exposure time before the green fluorescence image was taken, the background fluorescence in the green fluorescence image was observed outside of the multiple vesicles will be. Bar = 10 μm

As shown in Fig. 14, a quad-MVV encapsulated with a fluorescent substance prepared in Production Example 6 contained fluorescence (fluorescence) differently colored in separate compartments (or inner spaces) of the first veycicle and the second veycicle, It was confirmed that the probe was collected.

First, a negatively charged green fluorescent probe (carboxyfluorescein (FAM)) was captured in the second vesicle, and unreacted residual FAM was removed via anion exchange chromatography. Secondly, a second vesicle encapsulating a green fluorescent probe and a positively charged red fluorescent probe (rhodamine B, hodamine B; RDB) were simultaneously added to the phospholipid film. Next, a freeze-thaw cycle was performed. Through this process, a first vesicle encapsulating a red fluorescent probe; At least one second vesicle captured in the first vesicle interior space; And a green fluorescent probe (FAM) sealed in the inner space of the second vesicle.

That is, the second vesicles are captured in the first vesicle inner space, so that the first vesicles and the second vesicles have independent compartments (or inner spaces). It can be seen that the second vesicle has a section (or inner space) independent of the first vesicle and the first vesicle has an independent remaining section (or inner space) except the second vesicle.

In other words, since the second vesicle inner space is separated from the first vesicle, the substance existing in the inner space of the second vesicle does not leak into the inner space of the first vesicle, and the substance existing in the inner space of the first vesicle Are separated from each other and are not absorbed into the interior space of the second vecicle.

As shown in FIG. 14, it was confirmed that both green and red fluorescence were locally observed in the multiple vesicle structure prepared in Production Example 6, indicating that the second vesicle in which the green fluorescent probe was encapsulated in the multiple vesicles and the red fluorescence probe Indicating that both exist.

Furthermore, it was confirmed that green and red fluorescence were observed spatially separated in the multiple vesicles of Preparation Example 6, and that the first vesicle and the second vesicle had independent internal spaces, It can be seen that the material can be sealed. From these results, it can be seen that the multiple vesicles according to the present invention can have multiple vesicles of the four components as shown in FIG.

EXPERIMENTAL EXAMPLE 9. Analysis of size distribution and surface charge state of multiple vesicles

1) Dynamic Light scattering dynamic light scattering (DLS) and zeta Potential How to measure

1 ml of the second vesicle (cPV) (20 [mu] M) solution prepared in Preparation Example 3 and 1 ml of the first vesicle prepared in Preparation Example 4 were prepared by performing freeze-thaw cycles (3 mg / ml) . In addition, multiple vesicles (dual-MVV) (20 μM cyclic peptide functionalized with fatty acid of Preparation Example 2, 3 mg / ml phospholipid) prepared from Preparation Example 5 were prepared. Each of these samples was dissolved in distilled water. DLS and z-potential were performed at 25 ° C using a Nano ZS particle analyzer (Malvern Instruments Inc., UK) equipped with an HE-Ne laser operating at 633 nm.

2) Analysis of results

Figure 15 shows the size distributions (a, c, e) for the first vecicle, the second vecicle and the multiple vecicles and the zeta potentials (b, d, (g, h) of each vesicle derived from the zeta potential results. (D) dynamic light scattering (DLS) data of the second veycles prepared from the preparation example 3, c) dynamic light scattering (DLS) data of the first veycles prepared from the production example 4, e) (DLS) data of the multiple vesicles. B) the zeta potential data of the second vesicle prepared in Production Example 3, d) the zeta potential data of the first vesicle prepared in Production Example 4, f) the zeta potential data of the multiple vesicles prepared in Production Example 5 Potential data. Mean + s.d (standard deviation) (n = 6). All of the above data were measured on distilled water at 25 ° C.

As shown in Fig. 15, a dual vesicle made from Preparation Example 5 has a second vesicle having a diameter of several tens to several hundreds of nm in the inner space, and the second vesicle has a value of + 37.1 ± 1.3 (Zeta potential of positive).

These zeta-potential values show a reasonable correlation when the second vesicle is composed of cyclic peptides functionalized with fatty acids containing a large number of arginine residues.

In the present invention, it is preferred that the second vesicle has a zeta potential of + 30 ㎷ or more, because it has good colloidal stability and forms a stable dispersion in the solution. If the zeta potential of the second vesicle is less than +30 ㎷, the stability of the colloid is lowered and the dispersibility of the second vesicle may be lowered.

A multimodal size distribution was observed for the first vesicles prepared from Preparation Example 4. [ Specifically, it was confirmed that the average diameter of the first veycles was 1 to 10 탆, which was much larger than that of the second veycles.

The first vesicle was also measured with a zeta potential (-6.2 ± 1.0 ㎷) consistent with electrically neutral lipids.

Multiple vesicles were found to have an average diameter of about 1 to 10 μm, similar to diameters found in the first vesicle major population.

As shown in FIG. 15, the multiple vesicles were expected to have a nearly neutral zeta potential because the outer surface was composed of charge neutral phospholipids, but it was observed to have a totally different zeta potential (+ 10.6 ± 1.2 ㎷) . This can be seen as the formation of a region called the electric double layer by the uneven distribution of the counter-ions and co-ions near the surface of the charged second vesicle and the first vesicle.

15 (g)), the first layer (the fixed layer or the stern layer) formed on the surface of the second vesicle has a negatively charged negative Ions. On the other hand, in the second layer (diffusion layer) with a low charge density, the surface will be pulled to the point where the anion is referred to as a slipping plane. Finally, it can be seen that the zeta potential measured at the second vecicle is the zeta potential at the slipping plane of the second vecicle in the bulk fluid.

Based on this, the zeta potential of multiple vesicles can be seen to be more complex than the ion distribution around the second vesicles.

It can be expected that each second vesicle present in the inner space of the multiple vesicles has its own first layer (stern layer), resulting in uneven distribution of ions in the phospholipid lumen. Together, the unevenly charged lumen generates potential across the outermost phospholipid bilayer and attracts negatively charged counter ions to the outer surface of the double vesicles.

Thus, multiple vesicles, as described above, have a slipping plane on the outer surface, so that the double vesicles act as positively charged particles, even though they have a chemically natural surface.

Experimental Example 10. First Vesicle , The second Vesicle And multiplex Vesicular Intracellular absorption pathways (or pathways)

To compare the characteristics of the first vesicles, the second vesicles and the multiple vesicles according to the present invention for cellular internalization.

1) Cell culture

HeLa human uterine cancer cells were purchased from ATCC Cell Bank (ATCC, Manassas, VA, USA). HeLa cells were grown in RPMI 1640 (United Search Partners, Inc.) supplemented with inactivated 10% (v / v) bovine serum (FBS, United Search Partners, Austin, TX, USA) and 1% (v / v) antibiotic- Austin, TX, USA). The culture conditions were maintained in a humidified atmosphere of 5% CO ₂ and 95% air at 37 ° C. To observe fluorescence emission by confocal laser scanning microscopy (CLSM), a T75 flask (Nalge Nunc International, Naperville, IN, USA).

2) Confocal Microscopic measurement method

1 x 10 ⁴ cells were cultured in a Lab-Tek glass chamber slide (Nalge Nunc International, Naperville, IN, USA) in a 35 mm cell culture dish. After 24 hours, the glass chamber slides were taken out of the culture dish and loaded onto the chamber. The chamber was then attached to a microscope. The chamber maintained the temperature at 37 占 폚. 270 μl of OptiMEM medium was injected into the chamber, treated with 30 μl of the sample, and incubated for 4 hours. Fluorescence images were taken, digitized, and stored in a computer for later analysis.

At this time, the sample contained a second vesicle capturing FAM (carboxyfluorescein) prepared from 1) of Production Example 6, a first vesicle of Production Example 4 capturing RDB (rhodamine B), and a fluorescent substance of Production Example 6 Is enclosed in a multi-vesicle.

3) Analysis results

FIG. 16 is a view for observing how multiple vesicles according to the present invention are differentially absorbed into cells. Specifically, FIG. 16A is a graph for capturing FAM (carboxyfluorescein) prepared from 1) of Production Example 6 (CPV) was treated with cells, and bright field images and fluorescence images were photographed with a confocal microscope and then superimposed on the images. FIG. 16B shows the results obtained from Preparation Example 4 capturing RDB (rhodamine B) FIG. 16C shows an image obtained by photographing a bright vein image by confocal microscope after treating the prepared first vecicle with cells, and FIG. 16C is a view showing an image obtained by overlapping multiple vesicles , Cells were subjected to confocal microscopy, and bright field images and fluorescence images were taken, and then superimposed on the images.

16D is an image obtained by photographing green and red fluorescence signals of a cell treated with multiple vesicles enclosed with a fluorescent substance manufactured in Production Example 6 using a confocal microscope and then superimposing the image. The image interpolated in Fig. 16D is an enlarged image of Fig. 16D.

As shown in FIG. 16A, in HeLa cells treated with a second vecicle (cPV) capturing FAM (carboxyfluorescein) prepared from Preparation Example 6-1, green fluorescence was observed inside the nucleus of the cells Respectively.

The second vesicle (cPV) capturing the FAM (carboxyfluorescein) prepared from the preparation example 6-1 thereby has a corona form and is composed of an ARM peptide derived from Rev protein (SEQ ID NO: 1) , Cell permeability (CPP) and nuclear locus signal characteristic (NLS), indicating that a large amount of green fluorescence is detected in the nucleus of HeLa cells.

On the other hand, as shown in FIG. 16B, in the cells treated with the first vecicle prepared in Production Example 4 capturing RDB (rhodamine B), it was confirmed that red fluorescence was specifically observed in the cytoplasm of the cells Respectively.

Taken together, the above results indicate that the multiple vesicles composed of the second vesicle and the first vesicle release the substance encapsulated in the first vesicle in the cell, the substance encapsulated in the second vesicle in the nucleus, It can be expected that it will have an absorption path to be released.

16C and 16D, however, it can be seen that the multiple vesicles of the present invention have absorption paths completely different from the above-mentioned predictions.

Specifically, observation of the cells treated with the dual-MVV enveloped with the fluorescent substance prepared in Preparation Example 6 revealed that a majority of yellow fluorescent signals were observed in the cytoplasm.

At this time, the yellow signal is a signal that appears as green (FAM of the second vecicle) and red (RDB of the first vecicle) fluorescence exists in colocalization.

The majority of the quad-MVV encapsulating the fluorescent material remains intact in the cell, and the payload from the quad-MVV is destroyed and released to be observed as green and red fluorescence .

That is, considering the results of FIG. 16A, since the second vesicle has nuclear locus signal (NLS) activity and cell permeability due to the ARM peptide (SEQ ID NO: 1) derived from Rev protein and the corona form, The red fluorescence in the second vesicle (cPV) emitted from the multi-vesicle (quad-hMV) encapsulating the fluorescent material should be present inside the nucleus.

However, it can be seen that the multiple vesicles encapsulated with the fluorescent substance in the present invention are only densely packed in the perinuclear region without the second vesicles that have been emitted across the inside of the cell nucleus .

In summary, the multi-vesicles (dual-MVV) encapsulating the fluorescent substance according to the present invention have absorption paths completely different from the intracellular absorption pathways of the first and second vecils, respectively, . In other words, the ARM peptide (SEQ ID NO: 1) derived from the coronary form of the second vecicle and the Rev protein constituting it has been found to be dependent on a specific intracellular location (cytoplasm, nucleus etc.) It means that the absorption path of the active site is changed and becomes inactive and has a completely different absorption pathway than when it exists alone.

When a peptide having intracellular permeability is produced using this property as a second vesicle, the multiple vesicles of the present invention firstly release the second vesicles and substances into the cytoplasm by the first vesicle, Can be designed to have a differential absorption pathway in which the second vesicle is dense in the nuclear membrane of the cell to allow intense release of the second vesicle material near the nuclear membrane. This series of processes is shown in more detail in Fig.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> multicomponet hetero-multivesicular vesicles, use thereof and preparation method thereof <130> HPC7067 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> arginine rich motif <400> 1 Thr Arg Gln Ala Arg Arg Asn Arg Arg Arg Arg Trp Arg Arg 1 5 10 <210> 2 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> hydrophobic domain <400> 2 Gly Trp Trp Trp Trp 1 5 <210> 3 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> hydrophobic domain <400> 3 Trp Trp Gly Trp Trp 1 5

Claims

(a) a first vesicle comprising egg yolk lecithin; And
(b) a second vesicle comprising at least one annular peptide located in the first vesicle interior space,
(B) the second vesicle is a double-walled spherical vesicle formed of a plurality of annular peptides,
The cyclic peptide comprises a hydrophilic segment represented by SEQ ID NO: 1, a hydrophobic self-assembled segment represented by SEQ ID NO: 2 or 3, and a linker,
Wherein the steric acid is bound to the N-terminal, C-terminal or both terminal ends of the cyclic peptide.

delete

The method according to claim 1,
Wherein said cyclic peptide is present in a concentration ranging from 1.5 to 30 [mu] M above the critical aggregation concentration (CMC).

delete

The method according to claim 1,
Wherein the cyclic peptide is represented by the following formula (1) or (2).
[Chemical Formula 1]

(2)

delete

The method according to claim 1,
Wherein the first vesicles have an average diameter in the range of 1 to 10 mu m and the second vesicles have an average diameter in the range of 10 to 500 nm.

The method according to claim 1,
Wherein the multiple vesicles have an electrical double layer.

The method according to claim 1,
Wherein the multiple vesicles have a zeta potential in the range of + 5-15 < RTI ID = 0.0 > p. &Lt; / RTI >

A drug delivery system comprising multiple vesicles according to claim 1.

15. The method of claim 14,
Wherein the multiple vesicles are filled with at least one carrier material which is the same or different from each other in the inner space between the first vesicle and the second vesicle and in the independent region of the second vesicle.

a) preparing an amphipathic peptide consisting of a hydrophilic segment represented by SEQ ID NO: 1, a hydrophobic self-assembled segment represented by SEQ ID NO: 2 or 3, and a linker;
b) removing the protecting group at the terminal of the amphipathic peptide, self-assembling the peptide to prepare a cyclic peptide, and then binding stearic acid to the cyclic peptide;
c) self-assembling the peptide functionalized with the stearic acid produced in step b) to prepare a second vesicle;
d) dissolving egg yolk lecithin in an organic solvent, evaporating the solvent and decompressing the solvent to form a lipid film; And
e) adding the second vesicle prepared in step c) to the lipid film, and then performing a cycle of freezing and thawing the vesicle at least once or more to prepare multiple vesicles. A method for producing multiple vesicles.

17. The method of claim 16,
Wherein the step c) is carried out at a concentration ranging from 1.5 μM to 30 μM, wherein the peptides functionalized with stearic acid have a critical aggregation concentration (CAC) or higher.

17. The method of claim 16,
Wherein the freezing and thawing cycle is repeatedly performed at a temperature of-200 to 150 ° C for 1 to 10 minutes and a process of defrosting at 40 to 60 ° C for 1 to 10 minutes.