WO2015060504A1 - Method for inclusion of drug in membrane vesicle through cell modification using membrane fusogenic liposome and drug delivery method using same - Google Patents

Abstract

The present invention relates to a membrane vesicle in which a prepared drug is included using a membrane fusogenic liposome (MFL), a membrane vesicle preparation method, and a drug delivery method using the membrane vesicle. Specifically, the membrane vesicle of the present invention allows hydrophilic and hydrophobic drugs to be simultaneously included therein, is isolated from a cell to facilitate more efficient and selective drug delivery to neighboring cells, and is safe as a nanomaterial derived from a living body, and a step for directly entrapping the drug in the membrane vesicle is unnecessary. Therefore, the membrane vesicle can be effectively used for preventing and treating a variety of cancers and degenerative diseases in which the membrane vesicle is known to play an important role.

Description

 【Specification】

 [Name of invention]

 Method of incorporating drug into cell membrane blebs through cell transformation by cell membrane-bound liposomes and method of delivery of drug using same

Technical Field

 The present invention utilizes the principle that the drug delivered to the cell using a membrane fumeogenic liposome (MFL) is naturally mounted on the cell membrane blebs secreted from the cell, and the method for producing a cell membrane vesicle in which the drug is entrapped. It relates to a drug delivery method using cell membrane blebs.

Background Art

Membrane blebs are small vesicles of cell membrane structure that are secreted from various types of cells. The diameter of cell membrane blebs is reported to be approximately 30-500 nm. Axosome, one of the cell membrane vesicles, has been observed in electron microscopic studies to originate in specific compartments in cells called multivesicular bodies (MVBs) and to be released or secreted out of cells, rather than falling directly off the plasma membrane. In other words. When polycystic and plasma membrane fusion occurs. Such vesicles are released into the extracellular environment, called exosomes. In addition to these exosomes, microvesicles (niicrovesicle) generated by directly deriving cell membranes are one of the cell membrane blebs. Membranes It is not clear what molecular mechanism these cell membrane vesicles are made of, but it is not only red blood cells, but also B-lymphocyte T-lymphocytes, dendritic cells. Various types of immune cells, including platelets, macrophages, and tumor cells, are also known to produce and secrete cell membrane blebs in the living state. Membrane blebs are known to be released separately from many other cell types under both normal and pathological conditions. In addition, cell membrane blebs are known to contain major histocompatibility (MHC) and heat shock protein (HSP), which are important immunologically. Membrane vesicles play an important role in cancer development, and the transport of biological molecules of cell membrane vesicles contributes to immune evasion, angiogenesis and cancer metastasis. Cellular blebs-mediated si RNA therapy has been reported to be effective in Alzheimer's disease, with 60% transcription inhibition and 62% translational inhibition (Alvarez-Ervi ti et al., Nat Biot echnol, 2011). . By mixing the cell membrane vesicles isolated from the cells with curcuniin, the curcumin encapsulated in the cell membrane vesicles showed a longer circulatory period than if not present in the cell membrane blebs. Treatment with septic shock mouse model by lipopolysaccharide (l ipopolysacchar id LPS) (Sun D et al. Molecul ar therapy, 2010).

Therefore, efforts have been made to enclose a drug in a cell membrane vesicle composed of a double lipid membrane structure in vivo. Until now, a method of separating a cell membrane vesicle from a cell and exposing the drug in vitro to the separated cell membrane bleeding is performed. Although this has been reported, the drug is entrapped on the surface of the cell membranes, and thus, aggregat ions still occur and precipitation occurs during ultracentrifugation, making it difficult to separate the cell membranes, resulting in structural damage to the protein or cell membranes. There have been several limitations in the simultaneous inclusion of hydrophilic and hydrophobic drugs. Therefore, the present inventors are not enveloping the drug in the separated cell membrane blebs, but by separating and collecting the cell membrane vesicles in which the drug is naturally contained within the cell, thereby preventing cell membrane blistering phenomenon and maintaining the intrinsic properties of the cell membrane blebs. In the meantime, we have devised a way to detect drugs. Accordingly, the present inventors have attempted to develop a new method for incorporating drugs into cell membrane blebs secreted from cells (all vesicles composed of cell membranes secreted from cells including exosomes), and have been described as membrane-linked liposomes (membrane fusogeni cl iposomes; MFLs). If the hydrophilic drug is delivered cytoplasmic hydrophobic drug to the cell membrane, the two drugs delivered to the cell membrane will be naturally contained in the intracellular membrane and the membrane. As a result, hydrophilic Alternatively, the cell membrane-bound liposomes containing the hydrophobic drug were treated with the cells to confirm that the cell membrane vesicles containing the hydrophilic or hydrophobic drug were secreted from the treated cells. The cell membrane blebs prepared in this way originate in vivo and ensure safety. In addition to being able to deliver hydrophobic agents and hydrophilic drugs at the same time, 5 separate from conventional membrane membrane blisters followed by inclusion of drugs. There is little risk of damaging external proteins of cell membrane blebs, and biosafety liposomes are used, making them easier to apply to in vivo experiments and clinical trials. The cell membrane blebs enveloping the drug of the present invention can be effectively used for various diseases in which cell membrane blebs are known to play an important role by effectively treating primary and metastatic lesions.

DETAILED DESCRIPTION OF THE INVENTION [Technical Problems] An object of the present invention is to provide a cell with liposomes incorporating a desired drug in or outside a living body.

15 is treated to provide a cell membrane blister containing the drug from the cell.

 Still another object of the present invention is to provide a method for producing the cell membrane blebs.

 Still another object of the present invention is to provide a liposome capable of releasing cell membrane blebs enveloping a drug from a cell.

Another object of the present invention is to provide a drug delivery method using the liposomes or cell membrane blebs.

 Still another object of the present invention is to provide a pharmaceutical composition for water delivery, including the liposomes or cell membrane blebs.

It is another object of the invention to provide a pharmaceutical composition for ^cancer, 25 for prevention or treatment containing the liposomes or membrane property blisters.

 Still another object of the present invention is to provide an antibiotic comprising the liposome or cell membrane blebs.

Still another object of the present invention is to provide a light containing the liposomes or cell membrane blebs To provide a sensitizer.

 Still another object of the present invention is to provide the use of the liposomes or cell membrane vesicles for use as a drug delivery composition, a pharmaceutical composition for preventing or treating cancer, an antibiotic, or a photosensitizer.

 Still another object of the present invention is to provide a drug delivery method comprising administering to the individual an effective amount of the liposomes or the cell membrane blebs.

 Still another object of the present invention is to provide a method for treating cancer, antibacterial or antifungal method, or method for enhancing photosensitivity, comprising administering a pharmaceutically effective amount of the liposomes or the cell membrane blebs to a subject.

[Technical Solution] To solve the above problem. The present invention

 1) Consists of double lipid membrane. Hydrophobic drug in the double lipid membrane. Preparing a liposome that encapsulates a hydrophilic drug inside the double lipid membrane and fuses the cell membrane to secrete the hydrophobic drug or a cell membrane vesicle containing the hydrophilic drug;

 2) treating the cells with liposomes of step 1) to release the cell membrane blebs from the cells; And

 3) It provides a method for producing a cell membrane vesicle containing a drug comprising the step of collecting the cell membrane vesicles released in step 2).

 In addition, the present invention consists of a double lipid membrane. It provides a liposome comprising a hydrophobic drug in the bilipid membrane, a hydrophilic drug in the bilipid membrane, and fusion to the cell membrane to secrete the cell membrane vesicles containing the hydrophobic drug or the hydrophilic drug.

 In addition, the present invention, the bilipid membrane is composed of a lipid combined with PEG and D0TAP. Provides membrane-bound liposomes (MFL) enclosed with hydrophilic or hydrophobic drugs.

In addition, the present invention comprises the liposomes or the cell membrane vesicles Provided is a composition for drug delivery.

 Also. The present invention provides a pharmaceutical composition for preventing or treating cancer comprising the liposomes or the cell membrane blebs.

 The present invention also provides an antibiotic comprising the liposome or the cell membrane blebs.

 The present invention also provides a photosensitizer comprising the liposomes or the cell membrane blebs.

 The present invention also provides the use of the liposomes or the cell membrane vesicles for use as a composition for drug delivery.

 The present invention also provides the use of the liposomes or the cell membrane blebs for use as pharmaceutical compositions for the prevention or treatment of cancer.

 The present invention also provides the use of said liposomes or said cell membrane blebs for use as antibiotics.

 The present invention also provides the use of said liposomes or said cell membrane blebs for use as photosensitizers.

 Also. The present invention provides a drug delivery method comprising administering an effective amount of the liposomes or the cell membrane blebs to a subject.

 Also. The present invention provides a method for treating cancer, comprising administering to a subject a pharmaceutically effective amount of the liposomes or the cell membrane blebs.

 The present invention also provides an antimicrobial or antifungal method comprising administering to a subject a pharmaceutically effective amount of said liposomes or said cell membrane blebs.

 In addition, the present invention provides a method for enhancing photosensitivity comprising administering a pharmaceutically effective amount of the liposomes or the cell membrane blebs to a subject.

[Effective Effects] The cell membrane blebs of the present invention can simultaneously enclose a hydrophilic drug and a hydrophobic drug, and since the cell membrane vesicles (exosomes) are separated from the cells, more efficient and selective drug delivery to surrounding cells is possible. In addition, as a nanomaterial originated from living bodies By eliminating the step of encapsulating the drug directly into the cell membrane, which is safe. The cell membrane blebs may be usefully used to treat various cancers and degenerative diseases in which cell membrane blebs are known to play an important role. [Brief Description of Drawings]

 1 is B16F10. CT26, Hela cell line treated with Dil (red) cell membrane-bound liposomes (MFL), non-binding liposomes (NFL) and PLGA nanoparticles (NP), respectively, for 30 minutes and observed under confocal microscopy (blue: hoechst Nuclei stained with).

 . FIG. 2 is a diagram showing the results of a fusion assay performed after HeLa cells treated with membrane-bound liposomes (MFL) and non-binding liposomes (NFL):

 FD: fluorescence increase of R18 dye (dye increases with binding to cell membrane).

 Figure 3 shows that cell membrane-bound liposomes (MFL) contain a hydrophobic material (Dil or ZnPc) in the bilipid membrane for delivery to the cell membrane, and a hydrophilic material (calcein) inside the liposome for delivery into the cytoplasm. It is a figure explaining what to do.

 4 is a diagram showing the results of observing Dil and calcein in Hela, B16F10, and CT26 cancer cell lines in which MFL or NFL containing Dil and calcein were added together and incubated for 30 minutes.

 Figure 5 is Hela regardless of the type of liposomes treated. B16F10. The amount of cell membrane blebs in CT26 cancer cell line is similar.

 Figure 6 is a diagram confirming the efficiency delivered to the cell membrane vesicles Dil clad in the double lipid membrane of liposomes.

 7 is a diagram confirming the efficiency of delivery of the calcein trapped inside the liposomes to the cell membrane blebs.

 FIG. 8 is a diagram illustrating cell membrane blebs in cancer cell lines of Hela, B16F10, and CT26 treated with membrane-bound liposomes (MFL) by transmission electron microscopy (TEM).

9 is Hela, B16F10 treated with cell membrane-bound liposomes (MFL). Dynamic light scattering was used to determine the size of the membranes in each cancer cell line. Measurement result. It is confirmed that the size of the cell membrane blebs is an average of 80 to 100 nm. 10 is Hela, B16F10. The trend of distribution of cell membrane bleeding proteins according to the presence or absence of cell membrane-bound liposome (MFL) treatment in each cancer cell line of CT26 was confirmed by Coomassie Brilliant Blue staining. 11 is Hela treated with cell membrane-bound liposomes (MFL). The figure showing the result of confirming the expression level of the exosome-specific protein CD63 by Western blot in the cell membrane blebs isolated from B16F10 and CT26 cancer cells.

 FIG. 12 shows Hela, B16F10 and CT26 cells treated with cell membrane-bound liposomes (MFL), non-binding liposomes (NFL) and PLGA nanoparticles (containing only NP, Dil), followed by separation of cell membrane blebs (400 yg). Fig. 3 shows the results of measuring Dil fluorescence intensity.

 FIG. 13 shows that cell membrane-bound liposomes (MFL) or non-binding liposomes (NFL) incorporating Dil and calcein phosphorus were treated with cells in the upper filter for 30 minutes, and all cells and unreacted liposomes were removed. Cell membrane blebs released from the cells treated with liposomes over time are delivered to the cells through 400 nm pores, and when the fluorescent material is delivered to the cells by membrane-bound liposomes, the cells release the cells. Shows that the fluorescent material is transferred to another cell by.

 FIG. 14 is a diagram comparing the fluorescence intensities of substances (Dil and Calcein) delivered by liposomes (MFL and NFL) in the media of the upper filter and the cells of the lower transwell.

 FIG. 15 is a diagram showing that Calcean or Dil fluorescence was not observed when Hela cells were treated with a medium in which cell membrane blebs were removed.

 16 is a diagram showing the results observed by confocal microscopy of Hela, B16F10 and CT26 cells treated with cell membrane-bound liposomes (MFL) and non-binding liposomes (NFL) containing a photosensitive agent (ZnPc).

 17 is a graph showing the results of measuring the fluorescence intensity of the photosensitive agent (ZnPc) in the separated membranes (400 yg).

FIG. 18 shows ZnPc-bound membrane-bound liposomes (MFL) or non-binding liposomes (NFL) treated with cells in the upper filter for 30 minutes and not reacted with cells. ₀

 After removing all liposomes, the cell membrane vesicles released from the upper cells treated with liposomes were transferred to the lower cells through 400 nm pores for 48 hours, and when ZnPc was delivered to the cells by the membrane-bound liposomes, ZnPc is transferred to another cell by the cell membrane blebs that the cells release, followed by a 660 nm laser (l aser source) treatment for 5 minutes for the entire cell in the lower transwell where ZnPc is delivered by the cell membrane blebs. Shows a method of confirming the viability of the cells.

 19 is a result of the same method as in FIG. Cell viability with and without laser treatment in the cells of the upper filter and the lower transwell is compared.

 20 is a schematic diagram of a method for naturally enclosing a drug in a cell membrane bleeding secreting a drug through cell transformation by a cell-binding liposome.

[Best form for implementation of the invention]

 Hereinafter, the present invention will be described in detail. The present invention provides a membrane vesicle (membrane ves i c le), specifically exosomes containing the drug.

 Since the cell membrane blebs of the present invention are secreted from the cell membrane, they are composed of the double lipid layer of the cell membrane.

 The liposome of the present invention (l iposome) is composed of a double lipid membrane of the cell, the hydrophobic drug inside the double lipid membrane, and the hydrophilic drug inside the double lipid membrane. The cell membrane may be fused to secrete the cell membrane vesicles containing the hydrophobic drug or the hydrophilic drug.

 The liposome's bilipid membrane is composed of components similar to the cell's bilipid membrane, and specifically, it is preferably composed of lipids bound to base lipid (base l ipi d) and PEG (po ethylene glycol; polyethylene glycol), but not limited thereto. Do not.

Thus the cell membrane blebs of the present invention may enclose a hydrophilic drug or a hydrophobic drug. As a double lipid membrane structure existing in a living body, there is an advantage in that a coagulation phenomenon occurring in a synthetic liposome does not occur. Since the cell membrane blebs are a substance in vivo, aggregation does not occur in vivo as compared to the synthesized liposomes, and thus, drug delivery into the cells can be facilitated because they easily enter the cells by endocytosis of the cells.

 It is also possible to move toward specific purposes and targets rather than by irregular diffusion or circulation of cell membrane blebs (Peinado et al., Nature medicine, 2012; Zhu et al., Small, 2012: Mittelbrunn et al., Nature communi cat ions, 2011).

 The size of the cell membrane blebs is preferably 80 to 100 mm 3 (see FIG. 9), but is not limited thereto.

 The cell membrane blebs may enclose hydrophilic drugs or hydrophobic drugs, respectively or simultaneously.

 The cell membrane blebs can be transferred to cell membrane blebs special target tissues or cells by known methods.

 The hydrophilic drug or hydrophobic drug may be a cell membrane dye photosensitiser, an anticancer agent. It is preferred that it is an antibiotic (ant ibiot ics), but is not limited thereto.

 The anticancer agent is calcein (Calcein), gemcitabine (genicitabine), busul fan (Busul fan), chlorambucil (Ch 1 or ambuc il), cyclophosphamide (Cyc 1 ophospham ide). Melphalan, Cisplat in, Hosfamide, Cytarabine, 5-Plutouracil (5-FU), Methotrexate;

MTX). Daunorubicin, Adriamycin, Vinblastine, Vincr ist ine. Bindesine, Procarbazine, Tamoxifen, Megestes are selected from the group consisting of acetate ester, flutamide and goreline acetate (Zoladex). It is preferably one, but is not limited thereto.

The cancer is lung cancer, testicular cancer, bladder cancer, prostate cancer. Breast cancer. Ovarian cancer cervical cancer. It is preferably selected from the group consisting of pancreatic cancer, skin cancer gastric cancer and liver cancer, but is not limited thereto. The antibiotics penicillin (penicillin) antibiotics, cephalosporin (cephalosporine) antibiotics, macrolide (niacrol ide) antibiotics, tetracycline (tetracycline) antibiotics, ^"Quebec nolron (quinolone) antibiotics. Antihistamines, antibacterial agents, clindamycin. Metronidazole ᅳ chloramphenicol, Actinomycin D (Act inomycin-D), Bleomycin (Bly lycin) and mitomycin-C (Mitomycin-C) is preferably any one selected from the group Or not limited thereto. In addition, the present invention provides a method for producing a cell membrane bleeding containing a drug consisting of the following steps:

 1) preparing a liposome comprising a bilipid membrane, a hydrophobic drug in the bilipid membrane, a hydrophilic drug in the bilipid membrane, and fusion to the cell membrane to secrete the hydrophobic drug or a cell membrane blister containing the hydrophilic drug;

 2) treating the cells with liposomes of step 1) to release the cell membrane blebs from the cells; And

 3) collecting the cell membrane blebs released in step 2).

 Liposomes enter the cell by intracellular corrosion. Deliver the entrapped drug.

 Liposomes of the invention do not enter the cell but bind to the cell membrane.

 The bilipid membrane is preferably composed of a lipid bound to a basic lipid and PEG, but is not limited thereto.

 The basic lipid of the present invention is composed of the same components as the cell membrane, and is preferably composed of one or more phospholipids from the group consisting of all phospholipids of the phosphatidicolin and phosphoethanolamine series.

The PEG of the present invention may be commercially available, the terminal is composed of a methoxy group (methoxy group), the length of the chain (chain) is preferably 2,000, but is not limited thereto. Usually when PEGylating liposomes. It has the effect of preventing the destruction of RES by phagocytic cells, Has the advantage of increasing.

 Lipids in which the basic lipid and PEG are bound are preferably 50 to 80 parts by weight and 1 to 20 parts by weight. It is not limited to this.

 The size of the liposome is preferably 115 to 125 mm 3, but is not limited thereto.

 Liposomes are usually degraded by the cellular corrosion of immune cells. The inclusion of PEG as a constituent of liposomes inhibits cellular corrosion by cells and increases the circulation (Bailon P et al. Biocon jugate Chem .. 2001 ).

 The circulator of the liposome is preferably 330 minutes to 350 minutes, but is not limited thereto.

 The double lipid membrane may further include a positively charged lipid.

 Basic lipid of the present invention: PEG-bound lipid: Positively charged lipid is 60 to 90 parts by weight: 1 to 10 parts by weight: 5 to 30 parts by weight. Preferably it is 70-85 weight part: 1-5 weight part: 10-25 weight part. More preferably, it is 70-80 weight part: 1-5 weight part: 15-25 weight part.

 The positively charged lipid of the present invention,

 18 ： 1 TAP (1, 2-d i 01 eoy 1 -3- 1 r i me t hy 1 amnion i um-pr opane),

 14: 0 TAP (l, 2-dimyristoyl-3-trimethyl amnion i um-pr opane),

 16 ： 0 TAP (1, 2-d i pa 1 m i t oy 1 -3- 1 r i me t hy 1 ammon i um-propane),

 18: 0 TAP (1,2-stearoyl-3-tr imethyl ammon i um-propane),

 18: 0 DDAB (D i me t hy 1 d i oc t adecy 1 animon i urn),

 12: 0 EPC (l, 2-di 1 aur oy 1-s / rg 1 ycer ο-3-e t hy 1 hosphocho 1 ine),

 14: 0 EPC (1, 2-d i myr i s t oy 1 -5 / rg 1 ycero-3-e t hy 1 phosphocho 1 ine),

 14 ： 1 EPC (1, 2-d i myr i s t o 1 eoy 1 -sn-g 1 ycer ο-3-e t hy 1 hosphocho 1 ine),

 16: 0 EPC (1,2-dipalmi toyl-s / 7-glycero-3-ethylphosphochol ine),

 18: 0 EPC (1.2-distearoyl-5 /?-Glycero-3-ethylphosphochol ine),

 18: 1 EPC (1, 2-d i o 1 eoy 1-sn-g 1 ycer ο-3-e t hy 1 hosphocho 1 ine).

16: 0-18: 1 EPC (1-pa 1 mit oy 1 -2-o 1 eoy 1-sn-g 1 ycer ο-3-et hy 1 phosphocho 1 ine) It is preferably one or more selected from the group consisting of D0TMA (1, 2-di-0-oct adeceny 1-3-tri methyl ammoni um propane), but is not limited thereto. The surface potential of the liposomes is (+) 50 mV, but the surface potential of the cell membrane-bound liposomes of the present invention is 10 to 20 niV. If the surface potential of the liposome is 10 mV or less, the cell potential does not bind to the cell membrane, and if the surface potential of the liposome is 20 mV or more, it enters the cell by intracellular corrosion. Preferably, the surface potential of the liposome is 12 to 20 mV, more preferably 16 to 20. mV.

 The liposome is more preferably a membrane-bound liposome (membrane fusogeni Cl iposome; MFL), but is not limited thereto.

 The bilipid membrane of the cell membrane-bound liposome is PEG and D0TAP (N— [1- (2, 3-D i 01 eoy 1 oxy)] -N, N. N-1 ri me t hy 1 ammon i uni propane methyl su l hate, consisting of a lipid bound to N- [l- (2, 3-dioleoyloxy)]-Ν, Ν, Ν-trimethylammonium propane methyl sulfite). Hydrophilic or hydrophobic drugs can be entrapped.

 Lipid to which the PEG and D0TAP are bound is preferably 3 to 8 parts by weight and 15 to 48 parts by weight, but is not limited thereto.

 The drug entrapped in the liposome is preferably at least one of a hydrophilic drug and a hydrophobic drug. Cell membrane dyes, photosensitive agents, anticancer agents. It is preferably one of antibiotics, but is not limited thereto.

 The anticancer agent is calcein, gemcitabine, busulfan, chlorambucil. Cyclophosphamide, melfaran, cisplatin, ifosfamide, cytarabine, 5-fluorouracil, methotrexate, actinomycin-D, bleomycin, daunorubicin, adriamycin, mitomycin-C, vinblastine, Vincristine. It is preferably, but is not limited to, any one selected from the group consisting of bindecine, procarbazine, tamoxifen, megestrol acetate, pollutamide, and gocethelin acetate.

 The cancer is lung cancer, testicular cancer, bladder cancer, prostate cancer, breast cancer ovarian cancer, cervical cancer, pancreatic cancer, skin cancer. It is preferably selected from the group consisting of gastric cancer and liver cancer, but is not limited thereto.

The antibiotic is a penicillin-based antibiotic. Cephalosporin antibiotics, macrolides Antibiotics, tetracycline antibiotics, quenolone antibiotics, antihistamines, antibacterial agents, clindamycin, metronidazole, chloramphenicol, actinomycin-D, bleomycin and mitomycin—are preferably selected from the group consisting of C, but It is not limited.

 The cell membrane-bound liposomes of the present invention have excellent binding ability to cell membranes, and hydrophobic drugs or hydrophilic drugs contained when treated in cells are delivered to the cell membrane and cytoplasm, respectively, and the drug is released at high yield in the cell membrane blebs secreted from the cell membrane. To be enclosed. In addition, the secreted cell membrane blebs easily enter the surrounding cells, thereby delivering drugs entrapped in the cell membrane fusion liposomes into the cells.

 As described above, by administering cell membrane-bound liposomes in vivo, the cell membrane-bound liposomes are excellent drug delivery agents.

 In addition, in the present invention, by administering the cell membrane-bound liposomes in which the drug is contained in the cells in vitro, the drug-embedded cell membranes may be secreted and collected and administered to the human body to deliver the drug to a desired site. The present invention also provides a composition for drug delivery comprising the liposomes or the cell membrane blebs.

 Also. The present invention provides a pharmaceutical composition for preventing or treating cancer comprising the liposomes or the cell membrane blebs.

 The present invention also provides a pharmaceutical composition for antibiotics comprising the liposomes or the cell membrane blebs.

 Also. The present invention provides a pharmaceutical composition for photosensitizers comprising the liposomes or the cell membrane blebs. The present invention also provides the use of the liposomes or the cell membrane blebs for use as compositions for drug delivery.

 Also. The present invention provides the use of the liposomes or the cell membrane blebs for use as pharmaceutical compositions for preventing or treating cancer.

In addition, the present invention is the liposome or the cell membrane for use as an antibiotic Provides the use of blisters.

 The present invention also provides the use of said liposomes or said cell membrane blebs for use as photosensitizers. The present invention also provides a drug delivery method comprising administering to the individual an effective amount of the liposomes or the cell membrane blebs.

 Also. The present invention provides a method for treating cancer, comprising administering to a subject a pharmaceutically effective amount of the liposomes or the cell membrane blebs.

 The present invention also provides an antimicrobial or antifungal method comprising administering to a subject a pharmaceutically effective amount of said liposomes or said cell membrane blebs.

In addition, the present invention provides a method for enhancing photosensitivity comprising administering a pharmaceutically effective amount of the liposomes or the cell membrane blebs to a subject. In a specific embodiment of the present invention. The cell membrane-binding liposomes of the present invention are significantly non-binding liposomes (NFLs) that do not bind to the cells or compared to the biodegradable PLGA nanoparticles, which are frequently used as carriers of hydrophobic substances, have a significantly higher binding capacity with the cell membrane. Excellent (see FIG. 1). In addition, compared with the non-binding liposomes, it was confirmed that the size and surface potential of the cell membrane-bound liposomes decreased and the half-life increased about 6 times (see Table 1). In addition, as a result of testing the binding ability by treating the cancer cells with MFL and NFL, it was confirmed that MFL binds to the cell membrane at a significant level (see Fig. 2). In addition, MFL or NFL containing Di l and calcein were added to cancer cells and cultured. Measurement of the fluorescence intensity of Di l and calcein in the washed cells. In the case of MFL, Di l was found to be specific in the cell membrane and calcein in the cytoplasm. The fluorescence remained even though the liposomes were washed and removed. The cell membrane-bound liposomes encountered were confirmed to carry Di and calcein in the cells (see FIG. 4). In addition, it was confirmed that the cell membrane blebs isolated from various cancer cells treated with MFL and NFL had similar amounts of cell membrane blebs regardless of the type of liposomes treated (see FIG. 5). Di l inside the cell membranes according to the amount treated with NFL The fluorescence measurement results confirmed that the cell membrane binding ability of the MFL is more effective than absorbed into the cell (see FIG. 12). As a result of measuring the fluorescence of Di l and calcein in the cell membrane blebs, significantly higher fluorescence intensity was observed in the case of treatment with the non-binding liposomes and the treatment with the cell membrane-bound liposomes compared to the non-treatment group (Fig. 6 and FIG. 7). In addition, as a result of observing the appearance of the cell membrane vesicles in the cells treated with cell membrane-bound liposomes by transmission electron microscopy, it was confirmed that similar to the appearance of cell membrane vesicles in conventional cells reported previously (see FIG. 8), On the cell membrane bleeding size, it was confirmed that there exists a similar distribution trend in the range of 80 to 100 nm on average (see FIG. 9). Dean: white matter contained in isolated cell membrane blebs was observed by size separation in SDS-PAGE gel, and no significant protein distribution difference was detected according to the presence or absence of FL treatment (see FIG. 10). It was confirmed that the protein CD63 showed a significant expression in common (see FIG. 11).

 In a specific embodiment of the present invention, the result of comparing the fluorescence intensity with respect to the substance contained in the liposome using a medium. In the case of using MFL, it was confirmed that the entrapped material was efficiently transported in the lower medium by a significant level (see FIGS. 14 and 15). In addition, in order to confirm whether MFL can deliver anticancer drugs to cell membrane blebs, an MFL containing a photosensitive agent (ZnPc) was prepared and treated to cells to confirm that the MFL binds well with the cell membrane (FIG. 16 and FIG. 17). Reference) . Comparing the cell viability of the upper filter medium and the lower transwell medium with or without photosensitive agents. By the transfer of cell membrane vesicles containing photosensitizers secreted from cells treated with cell membrane-bound liposomes entrapped with photosensitizers to significant levels in the lower transwell. The laser causes significant photodynamic destruction of the cells in both the upper filter and the lower transwell. It was confirmed that the photosensitive agent can be used for photodynamic cancer treatment by treating the cell membrane-bound liposomes entrapped with cancer cells (see FIG. 19).

therefore. The cell membrane-bound liposomes of the present invention have excellent cell membrane-binding ability and, in the case of being treated by cells, simultaneously transport the hydrophobic and hydrophilic agents contained in the surrounding plasma membrane and cytoplasm to enable efficient and selective drug delivery. Rather, it is a nano material derived from living organisms Since it does not occur and circulation is good, it can be usefully used for the treatment of various cancers and degenerative diseases in which cell membrane blebs are known to play an important role. Pharmaceutically acceptable carriers included in the composition for drug delivery of the invention are, for example, one or more water, saline, phosphate buffered saline. Dextrin, glycerol. Ethane as well as combinations thereof. Such compositions may be formulated to provide fast release, or sustained or delayed release of the active ingredient after administration. Pharmaceutically acceptable carriers can be prepared according to a variety of factors well known to those skilled in the art. For example, the specific bioactive material used, its concentration, stability and intended bioavailability; Diseases and Conditions or Conditions to be Treated with the Cellular Binding Liposomes of the Invention: Subject, age to be treated. Size and general condition; Factors such as, but not limited to, the route used to administer the composition, such as nasal, oral, ocular topical, transdermal and muscular, should be considered. Pharmaceutically available carriers which are generally used for the administration of physiologically active substances other than the oral route of administration include aqueous solutions comprising D5W, textose and physiological salts within 5% of the volume. Pharmaceutically available carriers may also include additional ingredients that can enhance the stability of the active ingredients such as preservatives and antioxidants.

 Also. The dosage of the composition for drug delivery of the present invention is administered at a dose effective for the intended treatment. Therapeutically effective amounts required to treat or inhibit the progress of a particular medical disease can be readily determined by one skilled in the art using preclinical and clinical studies known in the medical arts. In the present invention, the therapeutically effective amount is directed to a clinician or researcher, specific tissue, system. And the amount of active ingredient which causes a biological or medical reaction in an animal or human. As the route of administration of the composition for drug delivery of the present invention, any suitable route of administration may be used. The cell membrane-bound liposomes according to the present invention may be fused to cell membranes or tissue membranes and introduced into cells by cell corrosion, but are not limited thereto. Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are only for illustrating the present invention. The contents of the present invention It is not limited by the Example.

Example 1 Cell Culture

Cancer cells (HeLa, B16F10, CT26), fibroblasts (L929) and macrophages (Raw 264.7) have a common humidity of 100%. The cells were cultured in an incubator (Thermo scientific) at 5% C0 ₂ and temperature 37 ° C. Tumor cells were also cultured in a lower chamber of membrane filters and transwells. Specifically, HeLa cells, B16F10 skin cancer cells and Raw 264.7 macrophages were treated with 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin / streptomycin (penici 11 in / streptomycin; Life Technologies Inc. Cultured in DMEM medium (Dulbecco's Modified Eagle's Medium; Wei gene, Daegu, Korea), including Carlsbad, CA, USA. CT26 colorectal adenocarcinoma cells and L929 fibroblasts contained RPMI 1640 medium containing 10% (v / v) FBS and W (v / v) penicillin / straptomycin (Roswell Park Memorial Institute; Wei gene, Daegu, Korea). Incubated at.

<Example 2> Characterization of liposomes according to the ratio of liposome composition

 Instead of the conventional method of separating exosomes and binding fluorescent markers, the exosomes originate in the cell membrane, and the cell membranes are stained with hydrophobic dyes, and then the cell membrane blebs are released in vivo by separating the cell membranes. Even using drugs and fluorescent markers that are present. In order to contain drugs or fluorescent markers in cell membrane blebs secreted in vivo, liposomes having the property of binding to cell membranes were selected.

Specifically. First, all the lipids dissolved in the organic solvent (Avanti polar lipids, Alabaster, Alabama USA) were mixed and the organic solvent was completely removed. A lipid cake was then made, and then liposomes were formed through a hydrat process. Finally, an extrusion method known in the art was performed using a 100 ηηι membrane filter (membrane filtter) to make the liposomes about 100 nm in size. In addition, it was prepared using an emulsion formation method well known in the art to produce PLGA nanoparticles. Also, The amount of PEG (polyethylene glycol) is fixed and binds to the cell membrane most effectively by varying the amount of D0TAP (N- [l- (2,3-Dioleoyloxy)]-N, NN- trime thy 1 ammonium propane methyl sulphate) Liposomes having quantitatively were screened using a fusion assay and confocal microscopy (confocal microscopy; TCS SP8 X. Leica. Wetzlar, Germany) to confirm cell membrane binding. Specifically, X 63 oi 1 -immersion optical lens (objective lense) was measured using the liposomes were treated with the cells for 30 minutes, cells and non-reflective liposomes were removed, and the cells were imaged in a live state. Incorporate R18 (self-quenching lipid dye octadecyl rhodamine B, Invitrogen, Grand Island, NY, USA) into liposomes, and then process the cells into real-time using a spectrofluorometer (Molecular Device, Sunnyvale, CA) in real time. The binding capacity was quantitatively analyzed by measuring the amount of increase in fluorescence. In addition, the size and surface potential of liposomes were measured by dynamic light scattering with Zetasizer Nano ZS90 (Malvern Instruments, Worcestershire, UK).

 As a result, as shown in Figure 1 and Table 1, by adjusting the size and surface potential by varying the composition ratio of liposomes, compared to liposome L4 in which the ratio of D0TAP in liposomes is 0, by increasing the ratio of D0TAP surface It was confirmed that the potential value could be adjusted from 15 to 17 nW (Table 1). In addition, the results measured in cells treated with liposomes. The fluorescence intensity value of Dil was also measured for L4 consisting of DMPC and PEG-PE. Compared to this, by increasing the ratio of D0TAP, the fluorescence intensity of Dil also increased significantly as the surface potential of liposomes increased. (FIG. 1).

Table 1 § ^■ Comparison of Lipocytic Toxicity of Various Liposomes

Example 3 Confirmation of Cell Membrane Binding Ability of Membrane Fusogenic Liposome (FL)

 Liposomes with the highest cell membrane binding ability among the liposomes compared to <Example 2> were named cell membrane binding liposomes, and a fusion test was performed to confirm cell membrane binding ability.

Specifically, the <Example 1> and the inclusion membrane binding a Hela, CT26 and B16F10 a Dil dyed in red in the cancer cell lines cultured in the same conditions, sex liposomes (MFL) 0.14 10- ⁶ mol (mole), the key synthetic liposomes After treatment with (NFL) 0.14 × 10 ⁶ moles and PLGA nanoparticles (NP) 35 / g for 30 minutes each, the cells and uncoated liposomes were removed. After 48 hours, cell membrane blebs were separated from the supernatant of cells using an ultracentrifuge. Each B16F10 treated with MFL, NFL and PLGA nanoparticles. CT26, To determine the extent of liposome binding to cell membrane in Hela cancer cells. Samples were observed under confocal microscopy. In addition, the HeLa cells were subjected to a fusion test in the same manner as in <Example 2> of the samples treated with MFL and NFL for 30 minutes. Also. Cell membrane-bound liposome bilipid membrane was prepared to contain a hydrophobic material (Dil or ZnPc), and to include a hydrophilic material (Calcein: Calcein) inside the liposome (FIG. 3). Then, Hela, B16F10. Dil in CT26 cancer cell line And after putting together MFL or NFL containing calcein and incubated for 30 minutes, Dil and calcein were observed in the washed cells. In addition, the size and surface potential of liposomes were measured in the same manner as in <Example 2>. The circulator was injected with liposomes and PLGA into the tail vein of the mouse, and blood was collected from the mouse for a predetermined time, and the fluorescence intensity of Dil present in the blood was measured by a spectrofluorometer (Molecular Device, Sunnyvale, CA). The circulator was calculated by calculating the half-life based on the measured values.

As a result. As shown in FIG. 2, Table 2 and FIG. 4, cell membrane-bound liposomes do not bind to cells and are incorporated into nonfusogenic liposomes (NFL) or biodegradable PLGA nanoparticles, which are often used as carriers of hydrophobic substances. It was confirmed that the binding to the cell membrane significantly better (Fig. 2A). In addition, compared with the non-binding liposomes, the cell membrane-bound liposomes had an average size of 4.5 nm, a surface potential of about 301, and a half-life of about 6 times (Table 2). HeLa cells were treated with MFL and NFL, and then tested for binding ability, and it was confirmed that MFL had a significant level of cell membrane binding capacity (FIG. 2B). In addition, MFL or NFL containing Dil and calcein were put together in three different cancer cell lines and incubated, and then observed Dil and calcein fluorescence intensity in the washed cells. In Dil red fluorescence cell membrane in the case of the MFL, the green fluorescence of the knife-old it was confirmed that the specific light emission in the cytoplasm, removed by washing the liposomes though the fluorescence is left as _a, membrane facing fabric to Dil and knife-old Binding liposomes were found to efficiently transport Dil and calcein to the cells (FIG. 4).

[Table 2] Comparison of Physical Properties of Membrane-bound and Non-binding Liposomes

Example 4 Identification of Physical Characteristics of Cellular Blasts Isolated from Cells Treated with Membrane Binding Liposomes

 In the cell delivery of Dil and calcein through treatment of the cell membrane-bound liposomes identified in <Example 3>, to confirm whether Dil and calcein are delivered to cells and included in the cell membrane blebs, Dil and Cal Separation of cell membrane blebs from cells treated with cell membrane-bound liposomes or non-binding liposomes enclosed with sane. Volume, shape of cell membranes. The size and extent of fluorescence of Dil and calcein in cell membrane blebs were compared.

Specifically, the cell membrane-bound liposomes or non-binding liposomes containing Dil and calcein in the same manner as in <Example 3> were treated with Hela. CT26 and B16F10 cancer cell lines were incubated for 48 hours. First, fractional centrifugation was performed to separate cell membrane blebs. Specifically, the cell culture cultured for 48 hours was harvested, and then cells and debris were removed by centrifugation for 20 minutes at 10,000 g for 30 minutes at 200 g for 5 minutes at 300 g at 4 ^° C. Then, using a ultracentrifuge (ultracentrifuge, Ultra 5.0, Hani 1 science. Incheon, South Korea) to separate the cell membrane bleeding in the same manner as in <Example 3> under the conditions of 4 ^° C. 120 minutes, 110.000 g. Cell membrane blebs were isolated. The isolated cell membrane blebs were resuspended using PBS. Then, the amount of protein of the isolated membrane vesicles was measured by the cancer cells (Hela, CT26 and B16F10) for the membrane-bound liposome (MFL) treated group, non-binding liposome (NFL) treated group or untreated control, respectively. Cell membrane properties using BCA protein assay kit (Thermo Scientific, Walt ham, MA. USA) The amount of cell membrane blebs was indirectly measured by measuring the amount of protein present in the blebs. In addition, in order to check whether the isolated cell membrane blebs are stable in vitro, the separated cell membrane blebs are placed in PBS and a culture medium, and then transmission electron microscopy (TE) (JEM-3011, [JEOL] Ltd, Tokyo, Japan) and the shape was observed, and the size of cell membrane blebs was measured by dynamic light scattering. Specifically, the cell membrane blebs were separated, and the diameter of the cell membrane blebs contained in the PBS state was measured using a DLS apparatus. The intensity of fluorescence of dil and calcein contained in the amount of solution corresponding to 400yg of cell membrane blebs isolated from cells treated with MFL, NFL or PLGA nanoparticles (containing only NP and Dil) was measured using a spectrophotometer. Measured. In the case of Dil, the excitation was measured at 530 nm and the emission at 570 nm, and in the case of calcein at 490 nm and the emission at 520 nm. At this time, in the MFL or the NFL throughput in the cell, the NFL's composition because PEG is not so much compared to the MFL amount is absorbed by the cells treated with the same amount and MFL (0.14 X 10 ^-6 mole), or all (Fig. 12 NFL), or lower the amount of liposomes to be absorbed by the cells to treat (NFL (small amount) in Figure 12; 0.07 X 10 ^"6 mol). The extent of Dil delivery to cell membrane blebs was compared.

As a result, as shown in FIG. Hela, B16F10 and CT26 cancer cell lines were treated with MFL or NFL, and then membrane membrane blisters were isolated. Regardless of the type of liposomes treated, the amount of cell membrane blebs was confirmed to be similar (FIG. 5). In addition, as shown in FIG. 12, when NFL was treated with an amount similar to MFL (large amount), a significantly high Dil fluorescence was measured inside the cell membrane, but the amount of NFL was decreased so that the same amount was absorbed into the cells. As a result, it was confirmed that the membrane binding ability of MFL was more effective than the intracellular uptake in fluorescence marker (Dil) delivery to the cell membrane (FIG. 12). 6 and 7, as shown in FIG. In the degree of fluorescence of both dil and calcein, it was confirmed that the fluorescence intensity was significantly higher when treated with non-binding liposomes and when treated with cell membrane-bound liposomes compared to the untreated group (FIG. 6 and 7). In addition, as shown in Figures 8 and 9, the appearance of the cell membrane vesicles in cells treated with cell membrane-bound liposomes As a result of the transmission electron microscopy, it was confirmed that the cell membrane blebs in the conventional cells reported previously similar to (Fig. 8), the average size of the cell membrane vesicles showed a slight difference depending on the cell line, but on average It was confirmed that there was a similar distribution trend in the range of 80 to 100 nm (FIG. 9).

Example 5 Analysis of Immunohistochemical Characteristics of Isolated Cellular Blasts

 In order to confirm immunobiochemical specificity of the cell membrane blebs obtained by separating physical properties from Example 4, SDS-PAGE and western blot were performed on the separated cell membrane blebs to obtain protein profiles. Confirmed. Specifically, after separating the membranes from the cells cultured by treatment with MFL or NFL as in <Example 4>, the distribution pattern of the isolated membranes blister protein separated by size with SDS—PAGE gel and then Kumar Visualization was performed by Cooniassie Brilliant Blue staining. Western blot was performed to confirm the expression level of the exosome-specific protein CD63 in the isolated cell membrane blebs. Specifically. Western blots were performed using the isolated cell membrane blebs samples as follows: Cell lysates were centrifuged at low temperature. A sample (Cleared lysate) except for debris was obtained. The protein loaded on the SDS PAGE gel was then transferred to the membrane. The localization reaction was performed on the membrane using the following primary antibody (anti—CD63 antibody; System Biosceinces, Mountain View. CA, USA) as a probe. Secondary antibodies conjugated with HRP (secondary antibody Goat ant i-Rabbit HRP; System Biosceinces, Mountain-View, CA, USA) were further reacted and the bands of the proteins were visualized.

 As a result, as shown in FIG. 10 and FIG. 11, the isolated cell membrane blebs were separated by size on an SDS-PAGE gel, followed by Coomasibrilliblib staining. As a result, the distribution of cell membrane vesicle proteins with or without MFL treatment was observed. No significant difference was found (FIG. 10), and the exosome-specific protein CD63 showed a large amount of exosomes in the cell membrane vesicles isolated by showing a significant expression in common.

Example 6 Confirmation of Migration of Cell Membrane Binding Liposomes to Adjacent Cells „ _T

 24 For use as photodynaniic therapy (PDT), to determine if cell membrane-bound liposome treatment moves drug entrapped in liposomes to adjacent cells more effectively than non-binding liposome (NFL) treatment through cell membrane blebs Experiments were performed in which cancer cells were cultured in a lower chamber of membrane filters and transwells.

 Specifically, as shown in Figure 13, the cell membrane-bound liposomes (MFL) or non-binding liposomes (NFL) in which Dil and calcein phosphors were enclosed were treated with cells in the upper filter for 30 minutes and did not react with the cells. After removing all of the liposomes, the cell membrane vesicles released from the upper cells treated with liposomes were transferred to the lower cells through the 400 nm pores for 48 hours. Experiments were performed showing that the fluorescent material was delivered to another cell by the blowing membranes.

 As a result, as shown in FIG. 14 and FIG. 15, the fluorescence intensity of the material (Dil and calcein) encapsulated in liposomes in the medium of the upper filter treated with MFL or NFL and the medium of the lower transwell was compared. In the lower medium, when the MFL was used than the NFL, the entrapped material exhibited significantly higher fluorescence intensity, indicating that the MFL efficiently transported the entrapped material to neighboring cells (FIG. 14). . Also. Hela cells were treated with medium in which cell membrane blebs were removed. It was confirmed that no calcein or Dil fluorescence appeared (FIG. 15).

Example 7 Delivery Confirmation of Photosensitive Agent

In photodynamic therapy of cancer. The cytotoxic R0S produced by photosensitize! "And visible light combined with oxygen kills malignant cells through the process of apoptosis or necrosis and suppresses the tumor microvasculature. It is known to stimulate the host immune system (Castano, et al. Nat Rev Cancer. 2006), as shown in Example 6 above that cell membrane-bound liposomes can effectively transport drugs entrapped in liposomes. Likewise, the drug entrapped in the liposome was changed to a photosensitive agent to confirm whether the photosensitive agent was delivered. Specifically, cell membrane-bound liposomes (MFL) containing a photosensitive agent (ZnPc; Sigma aldhirich, St. Louis, MO, USA) 6.75 in Hela, B16F10 and CT26 cells cultured in the same manner as in <Example 1> and Unbound liposomes (NFL) were treated and then observed by confocal microscopy (TCS SP8 X; Leica. Wetzlar, Germany). Then, the fluorescence intensity of the photosensitive agent (ZnPc) was measured in the isolated cell membrane blebs 400.

 As shown in Fig. 16 and 17, MFL containing a photosensitive drug for treating cancer by phototherapy (ZnPc) was prepared and treated in the cells, it was confirmed that MFL binds well to the cell membrane and is isolated from these cells One cell membrane blebs (exosomes) were confirmed to contain the photosensitive agent was delivered (Figs. 16 and 17).

<Example 8> Confirmation of cell viability of cells treated with cell membrane-bound liposomes with or without laser treatment

 To find out the potential for use in combination with laser therapy. Cell viability test was performed with or without laser treatment.

 Specifically, as shown in Figure 18, ZnPc-enclosed cell membrane-bound liposomes (MFL) or non-binding liposomes (NFL) to the cells in the upper filter for 30 minutes after removing all the cells and liposomes did not react. The cell membrane vesicles released from the upper cells treated with liposomes for 48 hours are transferred to the lower cells through 400 nm pores. When ZnPc is transferred to the cells by the membrane-bound liposomes, they are released by the membranes. Is a diagram showing that ZnPc is delivered to another cell. In addition, an experiment was performed to confirm the viability of the cells by performing a 660 m laser (laser source) treatment for 5 minutes on all the cells in the lower transwell to which ZnPc was delivered by the cell membrane blebs. MTT (Sigma aldhirich, St. Louis, MO, USA) solutions were treated and MTT toxicity assays were performed according to methods well known in the art.

As a result, as shown in FIG. 19, the cell survival rate according to the presence or absence of laser treatment in the medium of the upper filter and the lower transwell treated with MFL or NFL was compared. As a result of <Example 7> Cell Membrane Binding with Photosensitive Agent Cell membrane blebs containing photosensitive agents isolated from cells treated with liposomes are transferred to the lower transwells to a significant level. It was confirmed that the laser caused significant photodynamic destruction of the cells in both the upper filter and the low transwell (FIG.

19). Through this, it was confirmed that the photosensitive agent can be used for treating cancer by phototherapy by treating the cell membrane-bound liposomes encapsulated with phototherapy.

Claims

【Scope of Claim】 【Claim 1】 1) It is composed of a double lipid membrane, and contains a hydrophobic drug in the double lipid membrane and a hydrophilic drug inside the double lipid membrane, and produces a liposome (l iposome) that fuses with the cell membrane and secretes a membrane vesicle that encapsulates the hydrophobic drug or hydrophilic drug. steps; 2) Treating cells with the liposome of step 1) to release membrane vesicles from the cells; and 3) A method for producing a membranous blister containing a drug, comprising the step of collecting the membranous blister released in step 2). 【Claim 2】 The method of claim 1, wherein the double lipid membrane is composed of a lipid to which base lipid (base l ipid) and PEG (polyethylene glycol) are bonded. 【Claim 3】 In paragraph 2. A method for producing a drug-containing membranous blister, wherein the basic lipid and the PEG-bound lipid are used in an amount of 50 to 80 parts by weight and 1 to 20 parts by weight. 【Claim 4】 In paragraph 2. A method for producing a drug-containing membranous bleb, wherein the bilipid membrane further contains positively charged lipids. [Claim 5] The drug-containing membranous bleb according to claim 4, wherein the basic lipid: PEG-bound lipid: positive charge-loaded lipid is 60 to 90 parts by weight: 1 to 10 parts by weight: 5 to 30 parts by weight. Manufacturing method. [Claim 6] The drug-containing membranous bleb according to claim 5, wherein the basic lipid: PEG-conjugated lipid: the positively charged lipid is 70 to 85 parts by weight: 1 to 5 parts by weight: 10 to 25 parts by weight. Manufacturing method. 【Claim 7】 According to paragraph 1. A method of producing a drug-containing cell membrane bleb, wherein the liposome has a surface potential of 12 to 20 mV. 【Claim 8】 A membranous bleb containing a hydrophobic drug or a hydrophilic drug prepared by the method of claim 1. 【Claim 9】 It is composed of a double lipid membrane. Hydrophobic drugs in lipid membranes. A liposome characterized by encapsulating a hydrophilic drug inside the double lipid membrane and fusing with the cell membrane to secrete a membranous vesicle encapsulating the hydrophobic drug or hydrophilic drug. 【Claim 10】 The drug-containing liposome according to claim 9, wherein the liposome has a surface potential of 12 to 20 mV. 【Claim 11】 The drug-containing liposome according to claim 9, wherein the double lipid membrane is composed of a basic lipid and a lipid bound to PEG. 【Claim 12】 In clause 11. The lipid to which the basic lipid and PEG are bound is 50 to 80. Liposomes containing a drug, characterized in that the amount is 1 to 20 parts by weight. [Claim 13] The drug-containing liposome according to claim 11, wherein the double lipid membrane further includes lipid loaded with positive charge. [Claim 14] The method of claim 13, wherein the basic lipid: PEG-conjugated lipid: positively charged lipid is 60 to 90 parts by weight: 1 to 10 parts by weight: Liposome containing drug, characterized in that 5 to 30 parts by weight. [Claim 15] The drug-containing liposome according to claim 14, wherein the basic lipid: PEG-conjugated lipid: positively charged lipid is 70 to 85 parts by weight: 1 to 5 parts by weight: 10 to 25 parts by weight. 【Claim 16】 The double lipid membrane is PEG and D0TAP (N-[l-(2,3-Dioleoyloxy)]-N,N,N- t rime thy 1 ammonium propane methyl sulphate; !^-[1-(2,3-dioleoyloxy) Si)]ᅳ!^.! 1^- A cell membrane-bound liposome (membrane fusogenic liposome; MFL) composed of lipids bound to trimethylammonium propane methyl sulfite) and encapsulated with hydrophilic or hydrophobic drugs. 【Claim 17】 The cell membrane-bound liposome according to claim 16, wherein the lipid to which PEG and D0TAP are bound is 3 to 8 parts by weight and 15 to 48 parts by weight. [Claim 18] The method of claim 16, wherein the drug is a cell membrane dye or a photosensitizer. A cell membrane-bound liposome characterized as an anticancer agent and antibiotic. 【Claim 19】 The method of claim 18, wherein the anticancer agent is Calcein, gemcitabine, Busulfan, Chlorambuci 1, Cyclophosphamide, Melphalan, and cisplatin. (Cisplat in), Ifosfamide, Cytarabine, 5-fluorouracil (5-FU), Methotrexate (MTX), Daunorubicin, Adriamycin, Vin Vinblastine, Vincristine, Vindesine, Procarbazine, Tamoxifen, Megesterol acetate, Fhitamide and goserelin acetate A cell membrane-bound liposome, characterized in that it is any one selected from the group consisting of (Goserel ine acetate, Zoladex). [Claim 20] The method of claim 18, wherein the antibiotic is a penicillin-based antibiotic, a cephalosporin-based antibiotic, a macrolide-based antibiotic, a tetracycline-based antibiotic, or a quinolone-based antibiotic. , antihistamines, antibacterial agents. Any one selected from the group consisting of clindamycin, metronidazole, chloramphenicol, Act inomycin-D, Bleomycin and Mitoniycin-C A cell membrane-bound liposome characterized by: 【Claim 21】 A composition for drug delivery comprising the liposome of claim 9 or 16 or the cell membrane vesicle of claim 8. [Claim 22] A pharmaceutical composition for preventing or treating cancer comprising the liposome of claim 9 or 16 or the cell membrane vesicle of claim 8. [Claim 23] In Article 18, the cancers include lung cancer, testicular cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, and cervical cancer. A cell membrane-bound liposome selected from the group consisting of pancreatic cancer, skin cancer, stomach cancer, and liver cancer. [Claim 24] Antibiotics containing the liposome of claim 9 or 16 or the membranous vesicle of claim 8. 【Claim 25】 A photosensitizer comprising the liposome of claim 9 or 16 or the cell membrane vesicle of claim 8. [Claim 26] The liposome or agent of claim 9 or 16 for use as a drug delivery composition Use of paramembranous blisters in paragraph 8. 【Claim 27】 Use of the liposome of claim 9 or 16 or the cell membrane vesicle of claim 8 for use as a pharmaceutical composition for preventing or treating cancer. [Claim 28] Use of the liposome of claim 9 or 16 or the membranous vesicle of claim 8 for use as an antibiotic. 【Claim 29】 Use of the liposome of claim 9 or 16 or the cell membranous vesicle of claim 8 for use as a photosensitizer. [Claim 30] A drug delivery method comprising administering an effective amount of the liposome of claim 9 or 16 or the cell membrane vesicle of claim 8 to a subject. 【Claim 31】 A cancer treatment method comprising administering a pharmaceutically effective amount of the liposome of claim 9 or 16 or the cell membrane vesicle of claim 8 to a subject. [Claim 32] An antibacterial or antifungal method comprising administering a pharmaceutically effective amount of the liposome of claim 9 or 16 or the cell membrane vesicle of claim 8 to a subject. 【Claim 331 A method for enhancing photosensitivity comprising the step of administering a pharmaceutically effective amount of the liposome of claim 9 or 16 or the cell membrane vesicle of claim 8 to a subject.