KR101811267B1 - Cell-like Liposome - Google Patents

Abstract

The present invention provides a cell-like liposome and a drug delivery system using the same. According to the present invention, the cell-like liposome can secure stability and affinity similar to actual cells, can efficiently deliver to specific organs in vivo, and plays a key role in a drug delivery system using liposomes.

Description

Cell-like liposome < RTI ID = 0.0 >

The present invention relates to cell-like liposomes.

In biology, the extracellular matrix is primarily an organization responsible for the structural support of animals. The extracellular matrix belongs to the connective tissue of the animal. The extracellular matrix is composed of interstitial matrix and basement membrane (Kumar, Abbas, Fausto; Robbins and Cotran: Pathologic Basis of Disease; Elsevier; 7th ed). The intercellular matrix is a substrate that fills the space between the various cells. (2004). <Tissues> Tissues are a mixture of polysaccharide gels and protein fibrils that are packed between cells, helping to buffer the extracellular matrix (Alberts B, Bray D, Hopin K, Johnson A, Lewis J, Raff M, Roberts K, and Cancer>). The basement membrane is made up of thin paper, with epithelial tissue on it.

The components of the extracellular matrix are produced by the cells and secreted to the extracellular matrix through exocytosis. The newly generated extracellular matrix is secreted and integrated into the existing extracellular matrix. The extracellular matrix is composed of an interlocking mesh of fibrous proteins and glycosaminoglycans. The extracellular matrix is composed of proteoglycans such as heparan sulfate, chondroitin sulfate and keratan sulfate, non-proteoglycan polysaccharides such as hyaluronic acid, fibers such as collagen and elastin, fibronectin and laminin.

The cytoskeleton is an intracellular substrate that supports cell morphology and function. In eukaryotes, the substrate exhibits a dynamic structure that can rapidly assemble or degrade according to the requirements of cells composed of three major proteins (McKinley et al. (2015).) Human Anatomy (4th Ed. New York: McGraw Hill Education, p. The main protein is a microtubule composed of actin and a microtubule composed of tubulin and an intermediate filament (Wickstead B and Gull K (Aug 2011). The evolution of the cytoskeleton. The Journal of Cell Biology 194 (4): 51325 and Gunning PW et al. (Jun 2015). "The evolution of compositionally and functionally distinct actin filaments ". Journal of Cell Science 128 (11): 20092019).

Liposomes are spherical vesicles with at least one lipid bilayer. Liposomes have been used to deliver nutrients and medicines. Liposomes are biocompatible because they have a structure similar to biological membranes, and they are widely used as medicament transporters for more effectively delivering hydrophilic drugs because they have the advantage of enclosing hydrophilic drugs inside with a closed double layer structure (Xiong, XB et Pharm Res., 2005, 22, 933. and Gabizon, AA et al., Pharm Res., 1993, 10, 703.). However, liposomes are not only easily absorbed by the reticuloendothelial system in the liver or spleen after administration, but also cause structural instability due to adsorption of proteins and aggregation of liposomes in the blood, resulting in leakage of the encapsulated drug, . In order to stabilize the liposome structure, various liposome surface modifications have been actively studied (Seo, DH et al., Polymer (Korea), 2005, 29, 277. Park, YJ et al. , 28, 502.)

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have tried to prepare cell-like liposomes having the strength and flexibility of cell membranes similar to actual cells. As a result, the present invention has been completed by preparing a phospholipid membrane containing an anionic lipid, an extracellular matrix bound to the anionic lipid, and a cytoskeleton located within the phospholipid membrane, and confirming its structural stability.

Accordingly, an object of the present invention is to provide a cell-like liposome.

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

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a pharmaceutical composition comprising: (a) a phospholipid membrane comprising an anionic lipid and a neutral lipid; (b) an extracellular matrix ionically bound to the anionic lipid and located on the surface; And (c) a cell-like liposome comprising a cytoskeleton within the phospholipid membrane.

The present inventors have tried to prepare cell-like liposomes having the strength and flexibility of cell membranes similar to actual cells. As a result, a liposome composed of a phospholipid membrane containing an anionic lipid, an extracellular matrix bound to the anionic lipid, and a cytoskeleton located inside the phospholipid membrane was prepared and its structural stability was confirmed.

One of the main features of the present invention is that the phospholipid membrane constituting the cell-like liposome of the present invention comprises an anionic lipid.

As used herein, the term &quot; anionic lipid &quot; means any amphipathic lipid having at least one negative charge within the pH range of 4.0 to pH 8.0. The anionic lipids include any anionic lipids known to those of ordinary skill in the art.

According to one embodiment of the present invention, the anionic lipid may be selected from the group consisting of Dioleoyl phosphatidylserine (DPS), dimyristoyl phosphatidyl glycerol (DMPG), dipalmitoyl phosphatidyl glycerol (DPPG), diethylenetriamine pentaacetic acid (DTPA) dipalmitoyl-tartarate-2,3-diglutaric acid), DSTSA (1,4-disteroyl-tartarate-2,3-disuccinic acid), CHHDA (2-carboxyheptadecanoyl heptadecylamide), DMPS (Dimyristoylphosphatidylserine), DPPS (Dipalmitoylphosphatidylserine) Oleoylphosphatidylserine), DOPG (Dioleoylphosphatidylglycerol), POPG (Palmitoyl-oleoylphosphatidylglycerol), DMPA (Dimyristoylphosphatidic acid), DPPA (Dipalmitoylphosphatidic acid), DOPA (Dioleoylphosphatidic acid), POPA (Palmitoyl-oleoylphosphatidic acid), CetylP (cholesterol hemisuccinate). &lt; / RTI &gt;

According to another embodiment of the present invention, the anionic lipid is at least one anionic lipid selected from the group consisting of DOPS, DMPG, DPPG, DTPA, DPTGA, DSTSA and CHHDA.

According to a particular embodiment of the invention, the anionic lipid is DOPS.

The anionic lipid constituting the phospholipid membrane of the extracellular matrix delivery liposome of the present invention includes any anionic lipids known to those of ordinary skill in the art.

The lipid constituting the phospholipid membrane of the cell-like liposome of the present invention includes neutral lipids in addition to anionic lipids.

As used herein, the term &quot; neutral lipid &quot; means a lipid that has an uncharged, neutral zwitterion form within the pH range of 4.0 to pH 8.0. Such neutral lipids include any neutral lipids known to those of ordinary skill in the art.

According to one embodiment of the present invention, the neutral lipid is selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-palmitoyl-2-oleoyl-sn- glycero-3-phosphoethanolamine 2-oleoyl-sn-glycero-3-phosphocholine, DPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl- glycero-3-phosphocholine, DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), SM (N-palmitoyl-D-erythro-sphingosylphosphorylcholine), DLPE phosphatethanolamine, DiPPE (2-diphytanoyl-sn-glycero-3-phosphoethanolamine), phosphatidyl choline, phosphatidyl ethanolamine, tetraether lipid, ceramide, At least one neutral lipid selected from the group consisting of sphigolipid, diacryl glycerol and glyceride.

According to another embodiment of the present invention, the neutral lipid is at least one neutral lipid selected from the group consisting of DOPC, POPE, cholesterol, DSPC, DPPC, POPC and DOPE.

According to a particular embodiment of the invention, the neutral lipid is DOPC, POPE and cholesterol.

Neutral lipids constituting the phospholipid membranes of the cell-like liposomes of the present invention include any neutral lipids known to those of ordinary skill in the art.

The cell-like liposome of the present invention has a phospholipid membrane composed of an anionic lipid and a neutral lipid.

According to one embodiment of the present invention, the phospholipid membrane comprises 1-30 mole% anionic lipid.

According to another embodiment of the present invention, the phospholipid membrane comprises 1-25 mol%, 1-20 mol%, 5-25 mol% or 5-20 mol% anionic lipids.

According to certain embodiments of the present invention, the phospholipid membrane comprises 10 to 20 mole% anionic lipids.

Another of the main features of the present invention is that the extracellular matrix is bound to the anionic lipid constituting the phospholipid membrane of the cell-like liposome through ionic bonding and the polymerization reaction of the extracellular matrix induces self-assembly, Surface. The monomer is induced by the anionic lipid and the derived monomers are introduced into the oligomer through self-assembly, that is, polymerization reaction. For example, collagen is induced as a monomer to form a multimer by polymerization reaction. In the case of fibronectin, monomers are converted into a monomer, followed by structural change of the monomer, unfolding of the folding structure, and unfolding of the fibronectin Constitute a multimer.

According to one embodiment of the present invention, the extracellular matrix is selected from the group consisting of fibronectin, collagen, laminin, elastin, integrin and glycosaminoglycan Lt; RTI ID = 0.0 &gt; extracellular &lt; / RTI &gt;

According to another embodiment of the present invention, the extracellular matrix is at least one extracellular matrix selected from the group consisting of fibronectin, collagen, laminin and elastin.

According to a particular embodiment of the invention, the extracellular matrix is one or more extracellular matrix selected from the group consisting of fibronectin and collagen.

The extracellular matrix delivery liposome of the present invention comprises 1-30 mole% anionic lipids, 70-99 mole% neutral lipids, and extracellular matrix.

According to one embodiment of the present invention, the phospholipid membrane of the extracellular matrix-transferring liposome is composed of DOPC, POPE, DOPS and cholesterol.

According to another embodiment of the present invention, the phospholipid membrane comprises 1-30 mol% of DOPS.

According to a particular embodiment of the present invention, the phospholipid membrane comprises 30-70 mol%, 1-30 mol%, 1-30 mol% and 10-40 mol% of DOPC, POPE, DOPS and cholesterol.

Another aspect of the present invention includes a cytoskeleton inside a phospholipid membrane composed of an anionic lipid and a neutral lipid.

According to an embodiment of the present invention, the cytoskeleton is selected from the group consisting of actin, vimentin, glial fibrillary acdic protein, neurofilament, keratin, a nuclear lamina, and tubulin.

In order to induce the polymerization reaction of the cytoskeleton of the cell-like liposome of the present invention, an ion-permeable carrier may be used.

As used herein, the term &quot; ion-permeable carrier &quot; refers to a species in which an ion reversibly binds. Many ion-permeable carriers are lipophilic independent bodies that transport ions through the cell membrane.

According to one embodiment of the present invention, the ion-permeable carrier is selected from the group consisting of Beauvericin, Calcimycine or A23187, Carbonyl cyanide m-chlorophenyl hydrazone, Gramicidin A, Ionomycin, Lasalocid, Monensin, Nigericin, Nonactin, Nystatin, Salinomycin, and Valinomycin. At least one ion-permeable support selected from the group consisting of

The ion-permeable carrier includes any ion-permeable carrier known to those skilled in the art.

According to one embodiment of the present invention, g-actin, which is a cytoskeleton of a monomeric form induced in a cell-like liposome, is transported through an ion-permeable carrier to polymerize with f-actin.

The cell-like liposome of the present invention is a nano-sized liposome.

According to one embodiment of the present invention, the cell-like liposome is 10-500 nm in size.

According to another embodiment of the present invention, the cell-like liposome is 10-400 nm, 10-300 nm, 10-200 nm or 50-150 nm in size.

When another functional substance is additionally introduced into the cell-like liposome, various functions of imaging or chemically / biologically (for example, cell tracking and cancer treatment) can be performed. (Such as an antibody, a protein, an antigen, a peptide, a nucleic acid, an enzyme, or a cell) or a chemically active substance (e.g., a single molecule, a polymer, an inorganic support, a drug, etc.) is covalently bonded to the cell- Bond, a hydrophilic bond, a van der Waals bond, an electrostatic bond, or a hydrophobic bond. Such binding can be indirectly done to cell-like liposomes and multifunctional ligands coated directly on the surface or on cell-like liposomes.

Further, a contrast agent and an active substance may be used together with the carrier. Examples of additional biomolecules include antibodies, proteins, antigens, peptides, nucleic acids, enzymes, cells and the like, preferably proteins, peptides, DNA, RNA, antigens, hapten, avidin, Streptavidin, neutravidin, protein A, protein G, lectin, selectin, hormone, interleukin, interferon, growth factor, tumor necrosis factor, endotoxin, lymphotoxin, urokinase, streptokinase But are not limited to, tissue plasminogen activator, hydrolytic enzyme, oxidoreductase, degrading enzyme, bioactive enzyme such as isomerizing enzyme and synthetic enzyme, enzyme coauthor, and enzyme inhibitor.

The chemically active material includes various functional monomers, polymers, inorganic substances, drugs, and the like. Examples of such monomolecules include, but are not limited to, anticancer drugs, antibiotics, vitamins, drugs including folic acid, fatty acids, steroids, hormones, purines, pyrimidines, monosaccharides and disaccharides. Examples of the chemically active chemical polymer include polymers and copolymers such as dextran, carbodextran, polysaccharide, cyclodextran, pullulan, cellulose, starch, glycogen, carbohydrate, monosaccharide, disaccharide, and oligosaccharide, polyphosphazene, Polylactide, polylactide-co-glycolide, polycaprolactone, polyanhydride, polymalic acid, and derivatives of polymalic acid, polyalkylcyanoacrylates, polyhydroxybutyrates, polycarbonates But are not limited to, polyacrylic acid esters, polyorthoesters, polyethylene glycols, poly-L-lysine, polyglycolide, polymethylmethacrylate, polymethylether methacrylate, and polyvinylpyrrolidone.

Examples of the chemically active inorganic material include metal chalcogen compounds, inorganic ceramic materials, carbon materials, II / VI semiconductors, III / V semiconductors, and IV semiconductors, metals, or composites thereof. ZnS, ZnTe, Si, GaAs, ZnSe, ZnSe, ZnSe, ZnSe, ZnSe, ZnSe, ZnSe, ZnSe, Al, Au, Pt, Ag, Cu, and the like.

According to another aspect of the present invention, there is provided a drug delivery system comprising a drug encapsulated in the cell-like liposome.

The drug carrier comprising the liposome of the present invention and the drug encapsulated in the liposome may be administered together with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are those conventionally used in the field of the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, poly But are not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The drug carrier comprising the liposome of the present invention and the drug encapsulated in the liposome is preferably parenterally administered. In the case of parenteral administration, it can be administered by intravenous infusion, intramuscular injection, intralesional injection, or intracranial injection. Suitable doses may be variously prescribed by factors such as the formulation method, the mode of administration, the age, weight, sex, pathological condition, food, time of administration, route of administration, excretion rate, and responsiveness of the patient. The drug delivery vehicle comprising the liposome of the present invention and the drug encapsulated in the liposome comprises a therapeutically effective amount of the particles of the present invention. The term &quot; therapeutically effective amount &quot; means an amount sufficient to treat a disease of therapeutic interest and is generally 0.0001-100 mg / kg.

The drug carrier comprising the liposome of the present invention and the drug encapsulated in the liposome may be a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable carrier according to a method that can be easily carried out by those skilled in the art. May be prepared in unit dosage form by formulation with excipients, or may be prepared by penetration into a multi-dose container. The formulations may be in the form of solutions, suspensions, or emulsions in oils or aqueous media, or in the form of extracts, powders, granules, tablets, or capsules, and may additionally contain dispersing or stabilizing agents.

According to another aspect of the present invention, the present invention provides a method of producing a cell-like liposome comprising the steps of:

(a) preparing a phospholipid membrane composed of an anionic lipid, a neutral lipid, and an ionophore;

(b) introducing a cytoskeleton into the phospholipid membrane; And

(c) introducing an extracellular matrix to the surface of the phospholipid membrane.

According to one embodiment of the present invention, the introduction of the cytoskeleton of the step (b) is carried out by electric application.

According to the following embodiment, the cytoskeleton introduced in the step (b) is a single molecule. Cations are introduced through the ion-permeable carrier of the phospholipid membrane to induce the formation of cytoskeletal bundles so as to have cytoskeletons similar to actual cells.

According to an embodiment of the present invention, the extracellular matrix of step (c) is introduced in combination with an anionic lipid constituting the phospholipid membrane.

Since the method for producing the cell-like liposome of the present invention has the same phospholipid membrane structure as the cell-like liposome, the description common to both of them is omitted in order to avoid the excessive complexity of the present specification.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides a cell-like liposome and a drug delivery system using the same.

(b) Cell-like liposomes of the present invention exhibit improved strength and flexibility.

(c) The cell-like liposome of the present invention assures the stability and affinity similar to actual cells, can efficiently transfer to specific organs in vivo, and plays a key role in a drug delivery system using liposomes.

Figure 1 shows the size of the large liposome.
Figure 2 shows a schematic and fluorescent image showing the process of inducing extracellular matrix in macrophage.
Fig. 3 shows fluorescence images of macrophage-induced cytoskeleton and cytoskeleton-induced macrophage-liposomes.
Fig. 4 shows fluorescence images of cell shrinkage with increasing sucrose concentration of Hela cells.
Figure 5 shows the results of increasing the stiffness and elasticity of collagen-induced liposomes and fibronectin-induced liposomes.
6A and 6B show the results of increasing the strength of the cytoskeleton-containing liposome.
Figures 7a and 7b show fluorescence images in which the extracellular matrix (ECM) and the cytoskeleton are introduced into the membrane and inside of the liposome.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example 1: Fabrication of liposome containing extracellular matrix

Induction of extracellular matrix using negative charge

Lipids constituting the phospholipid membrane of liposomes were purchased from Avanti lipid. Collagen, an extracellular matrix, was purchased from sigma-aldrich and fibronectin from cytoskeleton, inc.

First, in order to assemble the phospholipid film forming the negative charge to the outside through self-assembly, the following lipid composition including negatively charged DOPS was selected. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE): 1,2-dioleoyl-sn-glycero-3-phospho- L-serine (DOPS): cholesterol (chol) = 4: 1: 1: 2 (mol). The reaction was carried out in chloroform (CHCl ₃ ) at the same ratio as 1 mg lipid / ㎖, coated on ITO glass, and then the organic solvent was removed with nitrogen, and the organic solvent was completely removed for about 1 hour. After incubation with a solution of 0.2 M sucrose, 2 mM MES, pH 4.2 (for collagen induction) or 0.2 M sucrose, 2 mM Tris-HCl, pH 7.4 (for induction of fibronectin) ) Or 24 hours (at the time of induction of fibronectin), followed by application of 1.5 V, 10 Hz alternating current to both electrodes for about 4 hours to prepare giant liposome of 10-100 탆 (FIG. 1).

The left side of Fig. 2 is a liposome synthesized by the above-mentioned method. The synthesized liposome contains DOPS and contains negative charge, which is used to induce collagen or fibronectin. Specifically, a 1 mg / ml collagen solution was denaturated at 80 ° C to form monomolecules. The resulting solution was added to a liposome solution, and reacted at 37 ° C for 30 minutes. Then, the solution was adjusted to pH To 7.4. Fibronectin was dissolved in PBS buffer at 1 mg / ml and then reacted at 37 캜 for 24 hours. During the reaction, 99% humidity was maintained to avoid osmotic pressure outside and inside of the liposome. The right part of FIG. 2 is a liposome in which collagen or fibronectin is induced by the above method. Glycero-3-phosphoethanolamine-N- (lissamine rhodamine B sulfonyl) was used for fluorescence tagging of liposomes, HiLyte 488 was used for FITC fluorescence tagging for collagen, and HiLyte 488 for fibronectin.

Example 2: Production of liposome containing cytoskeleton (F-Actin) in liposome

Cytoskeleton formation inside liposome through ion-permeable ionophore

The lipid constituting the phospholipid membrane of liposomes was purchased from Avanti lipid, and the cytoskeleton actin was purchased from cytoskeleton, inc. Actin, the cytoskeleton, is present in the form of filaments, which are monomolecular and F-actin, and the F-actin structure is the cytoskeleton within the cell. First, liposomes containing G-actin were synthesized in liposomes, and divalent cations were permeated through the ion-permeable carrier into the phospholipid membrane to induce self-assembly by F-actin.

The following lipid compositions were selected to produce liposomes that permeate divalent cations to the phospholipid membranes. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE): 1,2-dioleoyl-sn-glycero-3-phospho- 2% calcium ionophore was contained in L-serine (DOPS): cholesterol (chol) = 4: 1: 1: 2 (mol). As in Example 1, chloroform (CHCl ₃ ) was reacted with 1 mg lipid / ml, coated on ITO glass, and then the organic solvent was removed with nitrogen, and the remaining organic solvent was completely removed. After that, the cells were reacted with 0.2 M sucrose, 2 mM Tris-HCl, 0.2 mM NaATP, 10 μM G-actin and 2 μM rhodamine phalloidin pH 7.4 solution. Then, 1.5 V and 10 Hz alternating current Electricity was applied for about 4 hours to complete a large liposome of 10-100 탆. Thereafter the reacted in 0.2 M glucose, 2 mM Tris-HCl, 10 mM CaCl 2 pH 7.4 to be divalent cations (Ca ^{+ 2)} is transported into the liposome was converted to G- actin as F- actin. The right part of FIG. 3 is a liposome in which the cytoskeleton is induced in the above manner. As the fluorescent tagging of liposomes, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine-N- (7-nitro-2-1,3-benzoxadiazol- The cytoskeleton (inc) was used.

Example 3: Observation of liposome structural modification by osmotic pressure

Observation of liposome structure change by sucrose concentration gradient

Liposomes composed exclusively of lipid are sensitive to extrinsic pressure and microenvironmental changes, and liposomes are destroyed immediately. In order to confirm the structural support of the extracellular matrix or the cytoskeletal liposome produced in the present invention, the structure change was observed after osmotic pressure was generated by increasing the concentration of sucrose on the outside. In the experiment of FIG. 4, Hela cells were fluorescently tagged and then 1 M sucrose solution was added to the outside to increase the external concentration. The cells were shrunk by increasing the concentration. In the same manner, changes in the extracellular matrix, that is, collagen or fibronectin-induced liposomes, were observed. As a result, it was confirmed that collagen-induced liposomes increased the stiffness and the fibronectin-induced liposome elasticity was increased (FIG. 5). The cytoskeleton-containing liposome was observed to maintain its structure even when the liposome was damaged according to the external environment (FIGS. 6A and 6B). In order to test the flexibility of the extracellular matrix-induced liposomes and the liposomes composed only of the previously-proposed phospholipids, the intensities were determined by varying the external osmotic pressure of the liposomes. The phospholipid structure of the control liposome was composed of the same (DOPC): (DOPS): (DOPS): (chol) = 4: 1: 1: 2 (mol) as the extracellular matrix- The condition is also the same as the liposome containing the same negative charge. 1M sucrose solution was added to the synthesized bare liposomes, and the osmotic pressure difference between the outside and inside of the liposome was 0.05 O Osm / kg, and the change was observed. For easy observation, fluorescence images were obtained by adding alexa-488 dye to 1M sucrose solution. As shown in FIGS. 6A and 6B, when the osmotic pressure was externally applied, the control liposome immediately disappeared, but the extracellular matrix-induced liposome maintained its shape.

As a result, various changes in the characteristics of liposomes can increase the structural support in vivo environment and influence upon drug delivery through liposomes, thus providing a biocompatible and stable drug delivery system, medical treatment, Will play a pivotal role in the study of the relationship between skeleton and cell membrane.

Example 4 Production of Liposome Containing Extracellular Substrate and Intracellular Skeleton

The lipid constituting the phospholipid membrane of liposomes was purchased from Avanti lipid, and the cytoskeleton actin was purchased from cytoskeleton, inc. Actin, a cytoskeleton, exists in the form of a monomolecular state of G-actin and a filament of F-actin, and the F-actin structure is a cytoskeleton in the cell. First, liposomes containing G-actin were synthesized in liposomes, and divalent cations were permeated through the ion-permeable carrier into the phospholipid membrane to induce self-assembly by F-actin.

The following lipid compositions were selected to produce liposomes that permeate divalent cations to the phospholipid membranes. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE): 1,2-dioleoyl-sn-glycero-3-phospho- (Calcium ionophore) and magnesium ionophore (magnesium ionophore) in L-serine (DHPS): cholesterol (chol) = 2: 1: 1: 1 mol. As in Example 1, chloroform (CHCl ₃ ) was reacted with 1 mg lipid / ml, coated on ITO glass, and then the organic solvent was removed with nitrogen, and the remaining organic solvent was completely removed. After that, the cells were reacted with 0.2 M sucrose, 2 mM Tris-HCl, 0.2 mM NaATP, 10 μM G-actin and 2 μM Acti-stain 670 phalloidin pH 7.4 solution. For about an hour to complete a giant liposome of 10-100 [mu] m containing G-actin (41 kDa). G-actin is introduced into the liposome as the phospholipid membrane is swelled by electrical application. Actin was then reacted with 0.2 M glucose, 2 mM Tris-HCl, 10 mM CaCl ₂ 10 mM MgCl ₂ pH 7.4 to allow divalent cations (Mg ^{2 +} , Ca ^{2 +} ) to be transported into the liposomes, . &Lt; / RTI &gt; The right part of FIG. 7 is a liposome in which the cytoskeleton is induced by the above method. Liposomes were labeled with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- (lysamine rhodamine B sulfonyl) (ammonium salt) and Actin-stain 670 phalloidin (cytoskeleton, inc) Respectively.

 Laminin (cytoskeleton, inc), an extracellular matrix, was induced through the negative charge after the formation of the cytoskeleton inside the liposome through an ionophore. The synthesized cytoskeletal liposomes contained negative charges including DOPS and induced laminin. Laminin was dissolved in PBS buffer at 1 mg / ml and reacted at 37 ° C for 24 hours. During the reaction, 99% humidity was maintained to avoid osmotic pressure outside and inside of the liposome. Laminin was fluorescently tagged with HiLyte 488.

As shown in FIG. 7, it was confirmed that the phospholipid membrane of the liposome was labeled in red, and the laminin bound to the phospholipid membrane surface was labeled in green. The cytoskeleton formed a single actin molecule with actin bundles using divalent cations and is shown in white (Fig. 7A). Using a confocal microscope, Z-stack images of spherical liposomes were obtained and the overall structure was confirmed using a 3D-rendering technique (FIG. 7B). The extracellular matrix was coated on the outside of the liposome, and some of the extracellular matrix was found to be partially present through diffusion, and the cytoskeleton was found to be entirely present in the liposome.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (9)

(a) a phospholipid membrane composed of an anionic lipid, a neutral lipid, and an ionophore; (b) an extracellular matrix ionically bound to the anionic lipid and located on the surface; And (c) a cell-like liposome containing a cytoskeleton inside the phospholipid membrane,
Wherein the extracellular matrix is at least one selected from the group consisting of fibronectin, collagen and laminin, and the cytoskeleton is actin.
The method according to claim 1, wherein the anionic lipid is selected from the group consisting of dioleoyl phosphatidylserine (DPS), dimyristoyl-phosphatidyl glycerol (DMPG), dipalmitoyl-phosphatidyl glycerol (DPPG), diethylenetriamine pentaacetic acid (DTPA), 1,4-dipalmitoyl- 2,3-diglutaric acid), DSTSA (1,4-disteroyl-tartarate-2,3-disuccinic acid), CHHDA (2-carboxyheptadecanoyl heptadecylamide), DMPS (Dimyristoylphosphatidylserine), DPPS (Dipalmitoylphosphatidylserine), POPS (Palmitoyl-oleoylphosphatidylserine DOPG (Dioleoylphosphatidylglycerol), POPG (Palmitoyl-oleoylphosphatidylglycerol), DMPA (Dimyristoylphosphatidic acid), DPPA (Dipalmitoylphosphatidic acid), DOPA (Dioleoylphosphatidic acid), POPA (Palmitoyl-oleoylphosphatidic acid), CetylP (Cetyl phosphate) and CHEMS Wherein said lipid is at least one anionic lipid selected from the group consisting of:
The method of claim 1, wherein the neutral lipid is selected from the group consisting of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), POPE (1-palmitoyl-2-oleoyl-sn- glycero-3-phosphoethanolamine), cholesterol , DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3 glycero-3-phosphoethanolamine, SM (N-palmitoyl-D-erythro-sphingosylphosphorylcholine), DLPE (1,2-dilauroyl- , 2-diphytanoyl-sn-glycero-3-phosphoethanolamine, phosphatidyl choline, phosphatidyl ethanolamine, tetraether lipid, ceramide, sphigolipid Wherein the lipid is at least one neutral lipid selected from the group consisting of diacrylic acid, glycerol, diacryl glycerol, and glyceride.
3. The composition of claim 1, wherein the ion-permeable carrier is selected from the group consisting of Beauvericin, Calcimycin or A23187, Carbonyl cyanide m-chlorophenyl hydrazone, Gramicidin A, Ionomycin ), Lasalocid, monensin, nigericin, nonactin, nystatin, salinomycin, and valinomycin. Lt; RTI ID = 0.0 &gt; 1, &lt; / RTI &gt;
The cell-like liposome according to claim 1, wherein the extracellular matrix further comprises at least one selected from the group consisting of elastin, integrin and glycosaminoglycan.
The method of claim 1, wherein the cytoskeleton is selected from the group consisting of vimentin, glial fibrillary acdic protein, neurofilament, keratin, nuclear lamina, tubulin). &lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt;
The cell-like liposome according to claim 1, wherein the cell-like liposome is 10-500 nm in size.
7. A drug delivery system comprising a drug encapsulated in the cell-like liposome of any one of claims 1 to 7.
(a) preparing a phospholipid membrane composed of an anionic lipid, a neutral lipid, and an ionophore;
(b) introducing a cytoskeleton into the phospholipid membrane; And
(c) an extracellular matrix is bound to the anionic lipid via ionic bonding, and the polymerization reaction of the extracellular matrix induces self-assembly to induce additional extracellular matrix to the surface, Introducing an extracellular matrix into the surface of the phospholipid membrane, the method comprising:
Wherein the extracellular matrix is at least one selected from the group consisting of fibronectin, collagen and laminin, and the cytoskeleton is actin.

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