KR101586036B1 - Method of drug loading in cell-derived membrane vesicles by engineering parent cells with membrane fusogenic liposomes and drug delivery method using thereof - Google Patents

Abstract

The present invention relates to a cell membrane vesicle encapsulated with a drug prepared using membrane fusogenic liposome (MFL), a method for producing the cell membrane vesicle and a drug delivery method using the cell membrane vesicle, , The cell membrane vesicle of the present invention can simultaneously encapsulate a hydrophilic drug and a hydrophobic drug, and it is a cell membrane vesicle isolated from a cell, so as to enable more efficient and selective drug delivery to peripheral cells, Because of the safety of the material and the step of enclosing the drug directly in the cell membrane vesicle, there is no need for the cell membrane vesicle to be useful for the prevention and treatment of various cancer and degenerative diseases known to play an important role in cell membrane vesicles.

Description

FIELD OF THE INVENTION The present invention relates to a method of delivering a drug to a cell membrane vesicle through cell deformation by cell membrane-bound liposomes and a method of delivering a drug using the cell membrane-bound vesicles thereof}

The present invention is based on the principle that a drug delivered to a cell using a membrane fusogenic liposome (MFL) is naturally mounted on a cell membrane vesicle secreted from the cell, and a method for preparing a cell membrane vesicle encapsulating a drug and To a method of drug delivery using cell membrane vesicles.

Cellular vesicle is a small vesicle of cell membrane structure that is secreted from various kinds of cells. The diameter of the cell membrane vesicles is reported to be approximately 30-500 nm. One of the cell membrane vesicles, exosomes, was observed not to be removed directly from the plasma membrane in electron microscopic studies but to be released or secreted out of the cell, originating from specific compartments, called multivesicular bodies (MVBs). That is, when fusions between the polycapellum and the plasma membrane occur, such vesicles are released into the extracellular environment, which is called exosomes. In addition to these exosomes, microvesicles, which are formed by direct extraction of cell membranes, are also one of the vesicular membranes. Cell membrane It has not been clear what kind of molecular mechanism this cell membrane vesicle is made. However, it is not clear whether the cell membrane is involved in various kinds of immune cells including B-lymphocytes, T-lymphocytes, dendritic cells, platelets, macrophages, Cells are also known to produce and secrete vesicular vesicles in a live state. Cellular vesicles are known to be released from many different cell types under both normal and pathological conditions, both of which are known. Cellular vesicles are also known to contain major histocompatibility (MHC) proteins and heat shock proteins (HSP), which are immunologically important proteins.

Cellular vesicles play an important role in cancer development, and transport of biological molecules of vesicular vesicles contributes to immune avoidance, angiogenesis and cancer metastasis, and studies related to cell membrane vesicles in the cancer field are actively under way. Cellular vesicle-mediated siRNA therapy has been applied to Alzheimer's disease and has been reported to exhibit 60% transcriptional inhibition and 62% translation inhibition (Alvarez-Erviti et al., Nat Biotechnol, 2011). By mixing the cell membrane vesicles isolated from the cells with curcumin, the curcumin encapsulated within the cell membrane vesicles exhibited a longer cycling period than when they were not present in the cell membrane vesicles. (Sun D et al., Molecular therapy, 2010). Lipopolysaccharide LPS has been shown to be effective in the septic shock mouse model.

Thus, efforts have been made to encapsulate the drug in cell membrane vesicles composed of a biomolecular dual lipid membrane structure. Up to now, cell membrane vesicles have been isolated from the cells, and in vivo ex vivo drug adsorption However, since the drug is encapsulated on the surface of the cell membrane vesicles, agglutination is still caused and the cell membrane vesicle is difficult to separate due to sedimentation during ultracentrifugation, the structure of the protein or cell membrane is damaged, And the impossibility of simultaneous inclusion of hydrophobic drugs. Therefore, the present inventors not only encapsulate the drug in the separated cell membrane vesicles but also collect the cell membrane vesicles naturally contained in the cells inside the cell to collect the cell membrane vesicles and prevent the cell membrane vesicle aggregation phenomenon, I have come up with a way to probe the drug.

Accordingly, the present inventors have developed membrane fusogenic liposomes (MFLs) in order to develop a new method for containing a drug in cell membrane vesicles (all vesicles composed of cell membranes secreted from cells including exosome) It is expected that the two drugs delivered in this way will naturally encapsulate the inside of the cell membrane and the membrane, respectively, if the hydrophilic drug is transferred to the cytoplasm and the hydrophobic drug is transferred to the cell membrane. As a result, it was confirmed that cell membrane vesicles containing hydrophilic or hydrophobic drugs were secreted from the treated cells by treating the cells with cell membrane-binding liposomes containing hydrophilic or hydrophobic drugs. The cell membrane vesicles produced in this manner are originated from the living body to ensure safety, can simultaneously deliver the hydrophobic preparation and the hydrophilic drug, and unlike the conventional method in which the cell membrane vesicles are separated and the drug is encapsulated, And the use of biosafe liposomes makes it easier to apply in vivo experiments and clinical applications. The cell membrane vesicles encapsulating the drug of the present invention can effectively treat primary lesions and metastatic lesions and thus can be effectively used for various diseases known to play an important role in cell wall vesicles.

An object of the present invention is to provide a cell membrane vesicle in which a drug is encapsulated from a cell by treating the cell with a liposome encapsulating a desired drug in and / or out of the living body.

It is a further object of the present invention to provide a method for preparing said cell membrane vesicles.

Yet another object of the present invention is to provide a liposome capable of releasing a vesicular vesicle enveloped with a drug from a cell.

It is another object of the present invention to provide a drug delivery method using the liposome or cell membrane vesicle.

It is another object of the present invention to provide a pharmaceutical composition for drug delivery comprising the liposome or cell membrane vesicle.

It is another object of the present invention to provide a pharmaceutical composition for preventing or treating cancer comprising the liposome or cell membrane vesicle.

It is another object of the present invention to provide antibiotics comprising the liposomes or cell membrane vesicles.

It is another object of the present invention to provide a photosensitizing agent comprising the liposome or a cell membrane vesicle.

In order to solve the above object,

1) preparing a liposome which is composed of a double lipid membrane and which encapsulates a hydrophobic drug in the lipid membrane and a hydrophilic drug in the lipid membrane and fuses with the cell membrane to secrete the hydrophobic drug or a cell membrane vesicle encapsulating the hydrophilic drug ;

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

And 3) collecting the cell membrane vesicles released in step 2). ≪ Desc / Clms Page number 2 >

The present invention also provides a liposome composition comprising a lipid membrane characterized by comprising a hydrophilic drug and a hydrophilic drug fused to a lipid membrane and a hydrophilic drug inside the lipid membrane to secrete the hydrophobic drug or a hydrophobic drug, .

In addition, the present invention provides a membrane lipid membrane (MFL) comprising lipid bound to PEG and DOTAP and encapsulating a hydrophilic or hydrophobic drug.

In addition, the present invention provides a drug delivery composition comprising the liposome or the cell membrane vesicle.

The present invention also provides a pharmaceutical composition for preventing or treating cancer comprising the liposome or the cell membrane vesicle.

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

In addition, the present invention provides a photosensitizer comprising the liposome or the cell membrane vesicle.

Since the cell membrane vesicle of the present invention can simultaneously encapsulate a hydrophilic drug and a hydrophobic drug and is a cell membrane vesicle (exosome) isolated from a cell, not only it enables more efficient and selective drug delivery to peripheral cells, The cell membrane vesicles are useful for the treatment of various cancers and degenerative diseases which are known to play an important role in cell membrane vesicles, because it is safe with the originating nanomaterial and the step of enclosing the drug directly in the cell membrane vesicle is not necessary.

Figure 1 shows that B16F10, CT26, cell membrane-binding liposomes (MFL), noncontiguous liposomes (NFL) and PLGA nanoparticles (NP) containing DiI (red) in the Hela cell line were each treated for 30 minutes, Observation (blue: hoechst stained nucleus) is the limit.
FIG. 2 is a graph showing the results of fusion assay of HeLa cells treated with cell membrane-bound liposomes (MFL) and non-synthetic liposomes (NFL)
FD: Increase in fluorescence of R18 dye (fluorescence increases with binding to cell membrane).
Figure 3 shows that a membrane-bound liposome (MFL) contains a hydrophobic substance (Dil or ZnPc) in the lipid bilayer for transferring to the cell membrane, and a hydrophilic substance (Calcein) is contained in the liposome for delivery into the cytoplasm Fig.
FIG. 4 is a graph showing the results of observing dil and calcein in washed cells after culturing for 30 minutes with MFL or NFL containing dil and calcein in Hela, B16F10, CT26 cancer cell lines.
Figure 5 shows that the amount of cell membrane vesicles in Hela, B16F10, CT26 cancer cell lines is similar regardless of the type of liposomes treated.
FIG. 6 is a view for confirming the efficiency of the transfer of the Dil encapsulated in the lipid bilayer lipid membrane into cell membrane vesicles.
FIG. 7 shows the efficiency of the delivery of calcein encapsulated in liposomes into cell membrane vesicles.
FIG. 8 shows TEM images of cell membrane vesicles in cancer cell lines of Hela, B16F10, and CT26 treated with cell membrane-binding liposomes (MFL).
FIG. 9 shows the results of dynamic light scattering measurement of the cell membrane vesicle size in cancer cell lines of Hela, B16F10, and CT26 treated with cell membrane-bound liposomes (MFL) 100 nm, respectively.
FIG. 10 shows the distribution patterns of cell membrane vesicle proteins according to presence or absence of cell membrane-binding liposome (MFL) treatment in tumor cells of Hela, B16F10 and CT26, respectively, by Coomassie Brilliant Blue staining.
11 shows the results of western blot analysis of the expression level of exo-specific protein CD63 on cell membrane vesicles isolated from Hela, B16F10, CT26 cancer cells treated with cell membrane-bound liposomes (MFL) .
Figure 12 shows that Hela, B16F10 and CT26 cells were treated with cell membrane-bound liposomes (MFL), non-lytic liposomes (NFL) and PLGA nanoparticles (NP, containing only Dil) ≪ tb > DiI < tb > fluorescence intensity < SEP >
FIG. 13 shows that the cells in the upper filter were treated with the cell membrane-bound liposomes (MFL) or non-synthetic liposomes (NFL) encapsulating DiI and calcein fluorescent material for 30 minutes, and all of the cells and unreacted liposomes were removed, The results show that the cell membrane vesicles released from the liposome-treated upper cell are transported to the lower cell through the 400 nm hole. When the cell membrane-bound liposome transfers the fluorescent substance to the cell, And the fluorescent substance is transferred to another cell.
FIG. 14 is a graph comparing fluorescence intensities to substances (Dil and Calcein) delivered by the liposomes (MFL and NFL) in the culture medium of the upper filter and the cells of the lower transwell as a result of the same method as shown in FIG.
Fig. 15 shows that Hela cells were treated with media from which cell membrane vesicles had been removed, indicating that neither calcine nor Dil fluorescence was observed.
FIG. 16 is a graph showing the results of treatment with cell membrane-binding liposomes (MFL) and non-synthetic liposomes (NFL) containing photosensitizer (ZnPc) in Hela, B16F10 and CT26 cells and observation with a confocal microscope.
17 is a graph showing the result of measuring the fluorescence intensity of a photosensitizer (ZnPc) in the separated cell membrane vesicles (400 μg).
FIG. 18 shows the results of treatment of cells in the upper filter for 30 minutes with membrane-bound liposomes (MFL) or non-synthetic liposomes (NFL) encapsulated with ZnPc and removing the cells and unreacted liposomes, followed by treatment with liposomes And the cell membrane vesicles released from the upper cell are transferred to the lower cell through the 400 nm hole. When the ZnPc is transferred to the cell by the cell membrane-binding liposome, And then the whole cells in the lower transwell where ZnPc was delivered by the cell membrane vesicles were treated with a laser source of 660 nm for 5 minutes to confirm the viability of the cells.
FIG. 19 is a graph comparing cell survival rates with and without laser treatment in the cells of the upper filter and the lower transwell as a result of the same method as shown in FIG. 18.
20 is a schematic diagram of a method of naturally encapsulating a cell membrane vesicle secreted from a cell through cell deformation by a three membrane-bound liposome.

Hereinafter, the present invention will be described in detail.

The present invention provides a membrane vesicle encapsulating a drug, specifically an exosome.

Since the cell membrane vesicle of the present invention is secreted from the cell membrane, it is composed of a double lipid layer of the cell membrane.

The liposome of the present invention is composed of a double lipid membrane of a cell. The liposome includes a hydrophobic drug inside the lipid membrane and a hydrophilic drug inside the lipid membrane. The liposome fuses with the cell membrane to form a cell membrane vesicle covering the hydrophobic drug or the hydrophilic drug. Can secrete.

The lipid bilayer lipid membrane is composed of components similar to the lipid bilayer of cells, and is preferably composed of a lipid having a base lipid and a polyethylene glycol (PEG) bonded thereto, but is not limited thereto.

Therefore, the cell membrane vesicle of the present invention is capable of encapsulating a hydrophilic drug or a hydrophobic drug and has a dual lipid membrane structure existing in vivo, so that there is no coagulation phenomenon occurring in a synthetic liposome.

Since the cell membrane vesicle is a substance in vivo, aggregation does not occur in vivo as compared with the synthesized liposome, and it easily enters the cell by intracellular endocytosis of the cell, thereby facilitating drug delivery into the cell.

(Peinado et al., Nature medicine, 2012; Zhu et al., Small, 2012; Mittelbrunn et al., 2002), but also to move towards specific targets and targets rather than moving by irregular diffusion or circulation of cell- Nature communications, 2011).

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

The cell membrane vesicles may encapsulate a hydrophilic drug or a hydrophobic drug, respectively, or simultaneously.

The cell membrane vesicles can be transferred to cell membrane vesicular target tissues or cells by known methods.

The hydrophilic drug or the hydrophobic drug is preferably a cell membrane dye, a photosensitiser, an anti-cancer agent, or antibiotics, but is not limited thereto.

The anticancer agent may be selected from the group consisting of Calcein, gemcitabine, Busulfan, Chlorambucil, Cyclophosphamide, Melphalan, Cisplatin, (6-fluorouracil (5-FU), methotrexate (MTX), daunorubicin, adriamycin, vinblastine, vincristine, From the group consisting of Vincristine, Vindesine, Procarbazine, Tamoxifen, Megesterol acetate, Flutamide and Gosereline acetate (Zoladex) But it is not limited thereto.

The cancer is preferably selected from the group consisting of lung cancer, testicular cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, skin cancer, gastric cancer and liver cancer.

The antibiotic may be selected from the group consisting of penicillin antibiotics, cephalosporine antibiotics, macrolide antibiotics, tetracycline antibiotics, quinolone antibiotics, antihistamines, which is any one selected from the group consisting of clindamycin, metronidazole, chloramphenicol, Actinomycin-D, Bleomycin and mitomycin-C But is not limited thereto.

The present invention also provides a method for preparing a drug-containing cell membrane vesicle comprising the steps of:

1) preparing a liposome which is composed of a double lipid membrane and which encapsulates a hydrophobic drug in the lipid membrane and a hydrophilic drug in the lipid bilayer and fuses with the cell membrane to secrete the hydrophobic drug or a cell membrane vesicle covering the hydrophilic drug;

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

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

The liposomes enter the cell by intracellular phagocytosis, thereby delivering the encapsulated drug.

The liposomes of the present invention can not enter into cells and bind to cell membranes.

Preferably, the dual lipid membrane is composed of a lipid to which a basic lipid and PEG are bonded, 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 at least one phospholipid from the group consisting of phosphatidicolin and phosphoethanolamine phospholipids, but is not limited thereto.

The PEG of the present invention can be a commercially available one, and it is preferable that the terminal is composed of a methoxy group and the chain has a length of 2,000, but the present invention is not limited thereto. Generally, when the liposomes are PEGylated, they have the effect of preventing the RES from being destroyed by the phagocytic cells and increasing the residence time in the blood.

The base lipid and the lipid to which the PEG is bound are preferably 50 to 80 parts by weight and 1 to 20 parts by weight, but are not limited thereto.

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

Liposomes are usually degraded by intracellular phagocytosis of immune cells. When PEG is incorporated as a constituent of liposomes, intracellular phagocytosis by cells is inhibited and the circulation is increased (Bailon P et al., Bioconjugate Chem., 2001) .

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

The dual lipid membrane may further include positively charged lipids.

The base lipid of the present invention: lipid bound with PEG: the lipid loaded with positive charge is 60 to 90 parts by weight: 1 to 10 parts by weight: 5 to 30 parts by weight. Preferably 70 to 85 parts by weight: 1 to 5 parts by weight: 10 to 25 parts by weight, more preferably 70 to 80 parts by weight: 1 to 5 parts by weight: 15 to 25 parts by weight.

The lipid loaded with the positive charge of the present invention,

18: 1 TAP (1,2-dioleoyl-3-trimethylammonium-propane),

14: 0 TAP (1,2-dimyristoyl-3-trimethylammonium-propane),

16: 0 TAP (1,2-dipalmitoyl-3-trimethylammonium-propane),

18: 0 TAP (1,2-stearoyl-3-trimethylammonium-propane),

18: 0 DDAB (Dimethyldioctadecylammonium),

12: 0 EPC (1,2-dilauroyl-sn-glycero-3-ethylphosphocholine)

14: 0 EPC (1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine)

14: 1 EPC (1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine)

16: 0 EPC (1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine)

18: 0 EPC (1,2-distearoyl-sn-glycero-3-ethylphosphocholine)

18: 1 EPC (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine)

16: 0-18: 1 EPC (1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine) and

DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), but the present invention is not limited thereto.

The surface potential of the liposome is (+) 50 mV, while the surface potential of the cell membrane-binding liposome of the present invention is 10 to 20 mV. If the surface potential of the liposome is 10 mV or less, it does not bind to the cell membrane. If it is 20 mV or more, it enters the cell by intracellular phagocytosis. Preferably, the surface potential of the liposome is 12 to 20 mV, more preferably 16 to 20 mV.

The liposome is more preferably membrane fusogenic liposome (MFL), but is not limited thereto.

The lipid bilayer membrane of the cell membrane-binding liposomes is composed of PEG and DOTAP (N- [1- (2,3-Dioleoyloxy)] - N, N, N-trimethylammonium propane methyl sulphate; Yloxy)] - N, N, N-trimethylammonium propane methyl sulfite), and a hydrophilic or hydrophobic drug may be encapsulated.

The lipid to which PEG and DOTAP are bonded is preferably 3 to 8 parts by weight and 15 to 48 parts by weight, but not limited thereto.

The drug encapsulated in the liposome is preferably at least one of a hydrophilic drug and a hydrophobic drug, and is preferably any one of a cell membrane dye, a photosensitizer, an anticancer drug, and an antibiotic, but is not limited thereto.

The anticancer agent may be selected from the group consisting of calcine, gemcitabine, bosulfate, chlorambucil, cyclophosphamide, melphalan, cisplatin, ipospamide, cytarabine, 5-fluorouracil, methotrexate, actinomycin-D, bleomycin, It is preferably any one selected from the group consisting of dunorubicin, adriamycin, mitomycin-C, vinblastine, vincristine, vindesine, procarbazine, tamoxifen, megestrol acetate, flutamide and goserelin acetate But is not limited thereto.

The cancer is preferably selected from the group consisting of lung cancer, testicular cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, skin cancer, gastric cancer and liver cancer.

The antibiotic may be selected from the group consisting of a penicillin antibiotic, a cephalosporin antibiotic, a macrolide antibiotic, a tetracycline antibiotic, a quinolone antibiotic, an antihistamine, an antibacterial agent, a clindamycin, a metronidazole, a chloramphenicol, an actinomycin-D, a bleomycin and a mitomycin -C, and the like. However, the present invention is not limited thereto.

The cell membrane-binding liposome of the present invention has an ability to bind to a superior cell membrane. When the cell membrane-treated liposome is treated with a cell, the hydrophobic drug or the hydrophilic drug is delivered to the cell membrane and the cytoplasm, To be sealed. In addition, the secreted vesicular vesicles easily enter the surrounding cells, thereby transferring the drug encapsulated in the vesicle-mediated liposome into the cells.

As described above, since the cell membrane-binding liposome is administered in vivo, the cell membrane-binding liposome is an excellent drug delivery agent as a next step.

In addition, in the present invention, a cell membrane-binding liposome encapsulating a drug in a cell in vitro can be administered to secrete a cell membrane vesicle encapsulating the drug, collect the cell membrane vesicle, and administer it to the body to deliver the drug to a desired site.

In addition, the present invention provides a drug delivery composition comprising the liposome or the cell membrane vesicle.

The present invention also provides a pharmaceutical composition for preventing or treating cancer comprising the liposome or the cell membrane vesicle.

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

In addition, the present invention provides a pharmaceutical composition for a photosensitizer comprising the liposome or the cell membrane vesicle.

In a specific embodiment of the present invention, the cell membrane-binding liposome of the present invention is compared with biodegradable PLGA nanoparticles, which are frequently used as a nonfusogenic liposome (NFL) or a carrier of a hydrophobic substance, (Fig. 1). ≪ tb > < TABLE > In addition, it was confirmed that the size and surface potential of the cell membrane-binding liposome were decreased and the half-life was increased about 6-fold (see Table 1) as compared with the non-synthetic liposome. In addition, MFL and NFL treatment of the cancer cells revealed that MFL binds to the cell membrane at a significant level (see FIG. 2). In addition, when MFL or NFL containing dil and calcein were cultured in cancer cells, the fluorescence intensity of dil and calcein in the washed cells was measured. As a result, in the case of MFL, Dil was found in the cell membrane, Specific luminescence was confirmed, and the fluorescence remained even though the liposome was washed away. As a result, the membrane-binding liposome encapsulating calcein as a hydrophobic drug as a hydrophobic drug carried dil and calcein in the cells (See FIG. 4).

In addition, it was confirmed that the amount of cell membrane vesicles was similar regardless of the type of liposome treated in the cell membrane vesicles isolated from various cancer cells treated with MFL and NFL (see FIG. 5). DiI type light intensity measurement inside the cell membrane vesicle according to the amount treated with NFL showed that the cell membrane binding ability of MFL was more effective than that of the inside of the cell (see FIG. 12). As a result of measuring the fluorescence of the membranes of the vesicle and calcein, the fluorescence intensity was significantly higher when the cell membrane-binding liposome was treated compared to the non-crystallized liposome treated group and the untreated group (FIG. 6 And Fig. 7). In addition, the appearance of the cell membrane vesicles in the cells treated with the cell membrane-binding liposomes was confirmed by a transmission electron microscope to be similar to that of the cell membranes in the conventional cells (see FIG. 8) It was confirmed that the cell membrane vesicle size was present in a similar distribution tendency within the range of 80 to 100 nm on average (see FIG. 9). Proteins contained in the separated cell membrane vesicles were separated by size in SDS-PAGE gel, and no significant difference in protein distribution was observed depending on the presence or absence of MFL treatment (see FIG. 10). The exosome-specific protein CD63 showed a common expression level (see Fig. 11).

In a specific example of the present invention, the fluorescence intensity of the material encapsulated in the liposome was compared using the medium. As a result, when the MFL was used, it was confirmed that the inclusion material was significantly higher in the lower medium, (Fig. 14 and Fig. 15). In order to confirm whether MFL can also deliver anticancer drugs to cell membrane vesicles, it was confirmed that an MFL containing a photosensitizer (ZnPc) was prepared and treated with cells to bind to the cell membrane (FIGS. 16 and 17 Reference). As a result of comparing the cell survival rate with the presence or absence of the photosensitizing treatment in the medium of the upper filter and the culture medium of the lower transwell, the cell membrane vesicle containing the photosensitizing agent secreted from the cells treated with the cell membrane-binding liposome encapsulating the photosensitizer By moving to the lower transwells at significant levels, the laser significantly destroys the cells in both the upper filter and the lower transwell, and by treating cell membrane-bound liposomes encapsulating the photosensitizer with cancer cells, (See Fig. 19).

Therefore, the cell membrane-binding liposome of the present invention has an ability to bind to an excellent cell membrane, and when the cell is treated, the hydrophobic agent and the hydrophilic agent contained therein are simultaneously transported to the surrounding plasma membrane and cytoplasm, thereby enabling efficient and selective drug delivery In addition, it can be used for the treatment of various cancer and degenerative diseases which are known to play an important role because cell wall vesicles play important role because they are safe and coagulation phenomenon does not occur in the living body and circulation is good.

Pharmaceutically acceptable carriers in the drug delivery compositions of the present invention include, for example, one or more of water, saline, phosphate buffered saline, dextrin, glycerol, ethanol as well as combinations thereof. Such compositions may be formulated to provide a rapid release, 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, including, for example, the particular physiologically active material employed, its concentration, stability and intended bioavailability; Diseases and conditions or conditions to be treated with the cell membrane-binding liposomes of the present invention; The subject, age, size and general condition to be treated; But are not limited to, the route used to administer the composition, for example, nasal, oral, ocular, topical, transdermal, and muscular factors. In general, pharmaceutically acceptable carriers for administration of physiologically active substances other than the oral administration route include aqueous solutions containing D5W, dextrose and physiological salts within 5% of the volume. Pharmaceutically acceptable carriers may also contain additional ingredients which may enhance the stability of the active ingredients such as preservatives and antioxidants.

In addition, the dose of the drug delivery composition of the present invention is administered in a dose effective for the intended treatment. The therapeutically effective amount required to treat or inhibit the progression of a particular medical disorder can be readily determined by those skilled in the art using preliminary clinical and clinical studies known in the medical arts. In the present invention, the therapeutically effective amount refers to the amount of the active ingredient that causes the biological or medical reaction of a specific tissue, system, and animal or human, to be of clinical or research interest. The administration route of the drug delivery composition of the present invention may be any suitable administration route. The cell membrane-binding liposome according to the present invention may be fused to a cell membrane or a tissue membrane and may be introduced into the cell by intracellular phagocytosis, but is not limited thereto.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the examples.

< Example  1> Cell culture

Cancer cells (HeLa, B16F10, CT26), fibroblasts (L929) and large progenitor cells (Raw 264.7) were commonly cultured in an incubator (Thermo scientific) under the conditions of 100% humidity, 5% CO 2 and 37 ° C. In addition, tumor cells were cultured in a lower chamber of a membrane filter and a transwell. Specifically, HeLa cells, B16F10 skin cancer cells and Raw 264.7 macrophage cells were treated with 10% (v / v) FBS (fetal bovine serum) and 1% (v / v) penicillin / streptomycin (Life Technologies Inc.). (Dulbecco's Modified Eagle's Medium; Welgene, Daegu, Korea) containing 10% FBS, Carlsbad, CA, USA. CT26 colon adenocarcinoma cells and L929 fibroblasts were cultured in RPMI 1640 medium (Roswell Park Memorial Institute; Welgene, Daegu, Korea) containing 10% (v / v) FBS and 1% (v / v) penicillin / streptomycin .

< Example  2> Liposome  Depending on the composition ratio Liposomal  Identify characteristics

In view of the fact that the exosome originated from the cell membrane instead of the existing method of binding the fluorescent marker after separating the exosome, the cell membrane was dyed with a hydrophobic dye, and then the cell membrane vesicle was isolated and the cell membrane vesicle The liposomes which have the property of binding to the cell membrane without entering into the cell are selected to contain drugs or fluorescent markers in the vesicular secreted in vivo by using the property that a drug or a fluorescent marker exists.

Specifically, lipids (Avanti polar lipids, Alabaster, Alabama USA) dissolved in an organic solvent were all mixed and then the organic solvent was completely removed. Then a lipid cake was made and then the liposomes were formed through a hydration process. Finally, extrusion methods known in the art were performed using a 100 nm membrane filter to make the liposomes about 100 nm in size. Further, in order to produce PLGA nanoparticles, an emulsion forming method well known in the art was used. In addition, the amount of polyethylene glycol (PEG) is fixed and the most effective binding to the cell membrane is achieved by varying the amount of DOTAP (N- [1- (2,3-Dioleoyloxy)] - N, N, N-trimethylammonium propane methyl sulphate (TCS SP8X, Leica, Wetzlar, Germany) were used for quantitative determination of liposomes by fusion assay and confocal microscopy (confocal microscopy; TCS SP8X, Leica, Wetzlar, Germany). Specifically, it was measured using an X 63 oil-immersion optical lens, and the liposomes were treated with the cells for 30 minutes, and the liposomes which had not reacted with the cells were removed. Then, the cells were imaged in a live state. R18 (self-quenching lipid dye octadecyl rhodamine B, Invitrogen, Grand Island, NY, USA) was added to the liposomes and the cells were treated with real time spectrophotometer (Molecular Device, Sunnyvale, CA) The binding capacity was quantitatively analyzed by measuring the increase of fluorescence. In addition, the size and surface potential of liposomes were measured by dynamic light scattering with a Zetasizer Nano ZS90 (Malvern Instruments, Worcestershire, UK).

As a result, as shown in Fig. 1 and Table 1, by controlling the size and surface potential by varying the composition ratio of the liposomes, the ratio of DOTAP was increased as compared with the liposome L4 in which the ratio of DOTAP in the liposome was 0, It was confirmed that the potential value could be adjusted to 15 to 17 mV (Table 1). In addition, the fluorescence intensities of DiI were measured in L4 composed of DMPC and PEG-PE alone, and the ratio of DOTAP was increased. As the surface potential of liposome increased, DiI (Fig. 1). The fluorescence intensities of the cells were also significantly increased.

< Example  3 > Membrane-binding liposomes ( membrane fusogenic liposome ; MFL ) Cell membrane binding ability

The liposome having the highest cell membrane-binding ability among the liposomes as described in Example 2 was designated as a cell membrane-binding liposome, and a fusion test was performed to confirm cell membrane-binding ability.

Specifically, 0.14 x 10 < -6 > mol (mole) of cell membrane-binding liposomes (MFL) encapsulated with DiI stained in red in Hela, CT26 and B16F10 cancer cells cultured under the same conditions as in Example 1, 0.14 x 10-6 moles of liposome (NFL) and 35 mu g of PLGA nanoparticles (NP) were treated for 30 minutes each, and then the liposomes which had not reacted with the cells were removed. After 48 hours, the cell membrane vesicles were separated from the supernatant of cells using an ultracentrifuge. To confirm the degree of binding of liposomes to cell membranes in each of the B16F10, CT26 and Hela cancer cells treated with MFL, NFL and PLGA nanoparticles, the samples were observed with a confocal microscope. In addition, a fusion test was performed on HeLa cells treated with MFL and NFL for 30 minutes in the same manner as in Example 2 above. In addition, a hydrophobic substance (Dil or ZnPc) was contained in the cell membrane-binding liposome dual lipid membrane and a hydrophilic substance (Calcein) was contained in the liposome (FIG. 3). Then, MFL or NFL containing Dil and calcein were added together in Hela, B16F10, CT26 cancer cell lines, and cultured for 30 minutes. Then, dil and calcein were observed in the washed cells. The size and surface potential of the liposome were measured in the same manner as in <Example 2>. In the circulation period, liposome and PLGA were injected into the tail vein of a mouse, and blood was collected from a mouse for a predetermined time, and the fluorescence intensity of DiI present in the blood was measured with a spectrofluorometer (Molecular Device, Sunnyvale, CA). The half-life period was calculated based on the measured values and the circulation period was calculated.

As a result, as shown in FIG. 2, Table 2 and FIG. 4, the cell membrane-binding liposome was found to be a nonfusogenic liposome (NFL) which enters into the cell without binding to cells or biodegradable PLGA nano Lt; RTI ID = 0.0 &gt; (FIG. 2A). &Lt; / RTI &gt; In addition, it was confirmed that the size of the cell membrane-binding liposome was 4.5 nm on average, the surface potential was about 30%, and the half-life was about 6 times as compared to the non-synthetic liposome (Table 2). HeLa cells were treated with MFL and NFL and then tested for binding potency, indicating that MFL had a significant level of cell membrane-binding ability (FIG. 2B). In addition, when MFL or NFL containing Dil and Calcene were cultured in three different cancer cell lines, the fluorescence intensity of Dil and Calcane in the washed cells was observed. As a result, in the case of MFL, , It was confirmed that the green fluorescence of the calcein emits light specifically in the cytoplasm. Even though the liposome was washed and removed, the fluorescence remained, so that the cell membrane-binding liposome encapsulating the dil and calcein was added to the cells, (Fig. 4).

Example 4: Physical properties of cell membrane vesicles isolated from cells treated with cell membrane-bound liposomes

In order to confirm whether the dil and calcein were delivered to the cells and contained in the cell membrane vesicles in the cell transfer of the dil and calcein through the treatment of the cell membrane-binding liposome identified in Example 3 above, Cellular vesicles were isolated from cells treated with cell membrane - bound liposomes or non - cellulosic liposomes encapsulating sine, and the amount, shape and size of cell membrane vesicles, and the degree of fluorescence emission of dil and calcein in cell membrane vesicles were compared.

Specifically, cell membrane-binding liposomes or non-synthetic liposomes encapsulating Dil and calcein were treated in the same manner as in Example 3, and Hela, CT26 and B16F10 cancer cells were cultured for 48 hours. First, fractional centrifugation was carried out to separate the vesicular membranes. Specifically, the cell culture incubated for 48 hours was harvested, and cells and debris were removed by centrifugation at 4 ° C for 30 minutes at 300 g for 5 minutes to 200 g for 20 minutes to 10,000 g at 300 ° C. The cell membrane vesicles were then separated using an ultracentrifuge (Ultra 5.0, Hanil science, Incheon, Korea) under the conditions of 4 ° C, 120 min and 110,000 g in the same manner as in Example 3, The vesicles were separated. The isolated cell membrane vesicles were resuspended using PBS. Then, the amount of the separated plasma membrane vesicles was measured for cancer cell lines (Hela, CT26 and B16F10) for cell membrane-bound liposomal (MFL) -treated, noncontiguous liposomal (NFL) treated or untreated control. The amount of plasma membrane vesicles was indirectly measured by measuring the amount of protein present in the vesicular vesicles using the BCA protein assay kit (Thermo Scientific, Waltham, Mass., USA). In order to confirm whether the separated cell membrane vesicles were stable in vitro, the separated cell membrane vesicles were placed in PBS and culture medium, and then analyzed by transmission electron microscopy (TEM) (JEM-3011, JEOL Ltd., Tokyo, Japan), and the size of the vesicular vesicles was measured by dynamic light scattering. Specifically, the cell membrane vesicles isolated from the cell membrane vesicles and stored in the PBS state were measured for their diameters using a DLS instrument. Fluorescence intensities of DiI and calcein contained in the amount of solution corresponding to 400 μg of cell membrane vesicles isolated from cells treated with MFL, NFL or PLGA nanoparticles (NP, containing only Dil) were analyzed using spectrophotometric instrument Respectively. For DiI, excitation was measured at 530 nm, emission at 570 nm, for calcein, stimulation at 490 nm and emission at 520 nm. At this time, since the amount of MFL or NFL treated in the cells is much higher than that of MFL because NFL has no PEG in its composition, it is treated with the same amount (0.14 x 10 ^-6 mol) as MFL (NFL (small amount) in FIG. 12; 0.07 x 10 ^-6 mol) for the same amount of liposome to be absorbed into the cells. The degree of DiI delivery to cell membrane vesicles was compared.

As a result, as shown in Fig. 5, after the treatment of MFL or NFL with Hela, B16F10 and CT26 cancer cell lines, the cell membrane vesicles were separated, and the amount of cell membrane vesicles was similar regardless of the types of liposomes treated (Fig. 5). In addition, as shown in Fig. 12, when the NFL was treated in an amount similar to the MFL (large amount), the DiI fluorescence amount was significantly high in the cell membrane vesicle, but the amount of NFL was small As a result of the treatment, it was confirmed that the cell membrane-binding ability of MFL was more effective than the uptake of fluorescence marker (DiI) into cell membrane vesicles into the cells (Fig. 12). 6 and 7, the degree of fluorescence emission of both dil and calcein was significantly increased in the case of treating the cell membrane-binding liposome as compared with the case of treatment with the non-synthetic liposome and the case of the non-treated liposome (Fig. 6 and Fig. 7). In addition, as shown in FIGS. 8 and 9, the appearance of the cell membrane vesicles in the cells treated with the cell membrane-binding liposomes was confirmed by transmission electron microscope to be similar to that of the previously reported cell membranes in normal cells (Fig. 8). It was confirmed that the average cell size was slightly different depending on the cell line in the cell membrane vesicle size, but the cells were present in a similar distribution pattern in the range of 80 to 100 nm on average (Fig. 9).

<Example 5> Immuno-biochemical characterization of isolated cell membrane vesicles

SDS-PAGE and western blot were performed on the separated cell membrane vesicles to confirm the immunochemical characteristics of the cell membrane vesicles isolated in the above Example 4 and confirming the physical characteristics, Respectively.

Specifically, cell membrane vesicles were isolated from cells cultured by treatment with MFL or NFL as described in Example 4, and then the distribution pattern of the separated vesicular vesicle proteins was separated by SDS-PAGE gel, And visualized by Coomassie Brilliant Blue staining. Western blot was performed to confirm the expression amount of exo-specific protein CD63 on the separated cell membrane vesicles. Specifically, Western blotting was performed using the separated cell membrane vesicle samples as follows: The cell lysate was centrifuged at a low temperature, and a sample (cleared lysate) except for the debris was obtained. SDS PAGE The protein loaded on the gel was transferred to the membrane, and hybridization was performed using the following primary antibody (anti-CD63 antibody: System Biosciences, Mountain View, CA, USA) as a probe on the membrane. HRP-conjugated secondary antibody (secondary antibody Goat anti-Rabbit HRP; System Biosceinces, Mountain View, Calif., USA) was further reacted and the band of the protein was visualized.

As a result, as shown in FIG. 10 and FIG. 11, the separated cell membrane vesicles were separated by SDS-PAGE gels, and then stained with Coomassie Brilliant Blue. As a result, the distribution of cell membrane vesicle proteins (FIG. 10), and it was confirmed that exoose-specific protein CD63 had a common expression amount, and that the exosome was contained in the separated cell membrane vesicles.

< Example  6> cell membrane binding Liposomal In the forage  Confirmation of migration to adjacent cells

To use PDT as a photodynamic therapy (PDT), we investigated whether cell membrane-bound liposome treatment more effectively transports liposomally entrapped drugs to neighboring cells than noncontiguous liposome (NFL) treatment through cell membrane vesicles, Experiments were performed in which the cancer cells were cultured in a low chamber of a membrane filter and a transwell.

Specifically, as shown in Fig. 13, cell membrane-bound liposomes (MFL) or non-synthetic liposomes (NFL) encapsulated with DiI and calcein fluorescent materials were treated for 30 minutes in cells in the upper filter, After removal of all of the liposomes, the cell membrane vesicles released from the upper cells treated with the liposome for 48 hours are transferred to the lower cells through the 400 nm hole. When the fluorescent substance is transferred to the cells by the cell membrane-binding liposome, Experiments were carried out to show that the fluorescent substance was transferred to another cell by the vesicle blast.

As a result, as shown in Figs. 14 and 15, fluorescence intensities of the substances (Dil and Calcein) encapsulated in the liposome were compared in the medium of the upper filter treated with MFL or NFL and the medium of the transwell below thereof , It was confirmed that when the MFL was used in the lower medium in the case of using the MFL, the fluorescence intensity of the inclusion material was significantly higher than that of the NFL in the lower culture medium, so that the MFL efficiently transported the inclusion material to the adjacent cells ). In addition, it was confirmed that Hela cells were treated with a medium from which cell membrane vesicles had been removed, so that neither calcine nor Dil fluorescence appeared (FIG. 15).

< Example  7> Optic  Confirm delivery

In the photodynamic therapy of cancer, cytotoxic ROS produced by the combination of photosensitizer and visible light with oxygen kills malignant cells through apoptosis or necrosis, (Castano et al., Nat Rev Cancer, 2006). It is known to stimulate the host immune system. Thus, in Example 6, it was confirmed that the cell membrane-binding liposome was able to effectively transfer the drug encapsulated in the liposome, and the drug encapsulated in the liposome was replaced with a photosensitizer to confirm the delivery of the photosensitizer.

Specifically, membrane-bound liposomes (MFL) containing 6.75 μg of photosensitizer (ZnPc; Sigma aldrich, St. Louis, Mo., USA) were added to Hela, B16F10 and CT26 cells cultured in the same manner as in Example 1, And noncrystalline liposomes (NFL) were treated and then observed with a confocal microscope (TCS SP8X; Leica, Wetzlar, Germany). The fluorescence intensity of the photosensitizer (ZnPc) was measured at 400 μg of the separated cell membrane vesicles.

As shown in FIG. 16 and FIG. 17, MFL containing photopherin (ZnPc) for cancer treatment by phototherapy was prepared and treated with cells to confirm that MFL binds well to the cell membrane. It was confirmed that the above-mentioned photosensitizer was also transferred to a cell membrane vesicle (exosome) (FIGS. 16 and 17).

< Example  8> Cell membrane binding with and without laser treatment Liposomes  Cells of treated cells Survival  Confirm

To investigate the possibility of using laser treatment, we performed cell viability test with and without laser treatment.

Specifically, as shown in Fig. 18, cell membrane-bound liposomes (MFL) or non-synthetic liposomes (NFL) encapsulated with ZnPc were treated for 30 minutes in cells in the upper filter, and the cells and unreacted liposomes were removed, And the cell membrane vesicles released from the liposome-treated upper cell are transferred to the lower cell through a 400 nm hole. When ZnPc is delivered to the cell by the cell membrane-binding liposome, Is transferred to another cell. In addition, an experiment was performed to examine the viability of the cells in the lower transwell where the ZnPc was delivered by the cell membrane vesicle by performing a laser source treatment with 660 nm for 5 minutes. MTT (Sigma aldrich, St. Louis, Mo., USA) solution was treated and MTT toxicity test was performed according to methods well known in the art.

As a result, as shown in Fig. 19, the cell survival rates according to the presence or absence of the laser treatment were compared in the medium of the upper filter treated with MFL or NFL and the culture medium of the lower transwell. As a result, The cell membrane vesicles containing photosensitizers isolated from cells treated with cell membrane-bound liposomes encapsulated with photosensitizers migrate to the lower transwells at significant levels, so that the lasers are significantly reduced in both the upper filter and the lower transwell (Fig. 19). It was confirmed that the cell membrane - binding liposome encapsulated with photosensitizer could be applied to cancer therapy by phototherapy by treating cancer cells.

Claims (25)

1) preparing a liposome which is composed of a double lipid membrane and which encapsulates a hydrophobic drug in the lipid membrane and a hydrophilic drug in the lipid membrane and fuses with the cell membrane to secrete the hydrophobic drug or a cell membrane vesicle encapsulating the hydrophilic drug ;
2) treating the cells with the liposomes of step 1) to release the cell membrane vesicles from the cells; And
3) collecting the cell membrane vesicles released in step 2). &Lt; Desc / Clms Page number 20 &gt;
The method of claim 1, wherein the bilayer lipid membrane is composed of a lipid having a base lipid and a polyethylene glycol (PEG) bonded thereto.
[Claim 3] The method according to claim 2, wherein the lipid to which the basic lipid and the PEG are bound is 50 to 80 parts by weight and 1 to 20 parts by weight, respectively, based on the weight of the total lipid lipid.
3. The method of claim 2, wherein the bilayer lipid membrane further comprises positively charged lipid.
5. The pharmaceutical composition according to claim 4, wherein the lipid to which the basic lipid: PEG is bound: lipid loaded with positive charge is 60 to 90 parts by weight: 1 to 10 parts by weight: 5 to 30 parts by weight Gt;
Claim 6 has been abandoned due to the setting registration fee. 6. The pharmaceutical composition according to claim 5, wherein the lipid to which the basic lipid: PEG is bound: lipid loaded with positive charge is 70 to 85 parts by weight: 1 to 5 parts by weight: 10 to 25 parts by weight Gt;
[3] The method of claim 2, wherein the basic lipid is at least one phospholipid selected from the group consisting of phosphatidicolin and phosphoethanolamine phospholipids.
4. The method of claim 4, wherein the lipid loaded with the positive charge is selected from the group consisting of 18: 1 TAP (1,2-dioleoyl-3-trimethylammonium-propane), 14: 0 TAP (1,2-dimyristoyl-3-trimethylammonium- : 0 TAP (1,2-dipalmitoyl-3-trimethylammonium-propane), 18: 0 TAP (1,2-stearoyl-3-trimethylammonium-propane), 18: 0 DDAB (Dimethyldioctadecylammonium) Glycero-3-ethylphosphocholine), 14: 0 EPC (1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine), 14: 1 EPC (1,2-dimyristoleoyl- ethylphosphocholine), 18: 0 EPC (1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine), 18: 0 EPC (1,2- distearoyl- 2-dioleoyl-sn-glycero-3-ethylphosphocholine), 16: 0-18: 1 EPC (1-palmitoyl-2-oleoyl-sn- glycero-3-ethylphosphocholine), DOTMA -3-trimethylammonium propane) and DOTAP (N- [1- (2,3-Dioleoyloxy)] -N, N, N-trimethylammonium propane methyl sulphate). My Way.
The method of claim 1, wherein the liposome has a surface potential of 12 to 20 mV.
A cell membrane vesicle in which a hydrophobic drug and a hydrophilic drug prepared by the method of claim 1 are simultaneously enclosed.
A cell membrane vesicle encapsulating a hydrophobic drug prepared by the method of claim 2.
A cell membrane vesicle in which the hydrophilic drug prepared by the method of claim 2 is enclosed simultaneously.
Lipid bound with PEG: basic lipid with respect to the total lipid lipid membrane part: lipid loaded with positive charge: 70 to 85 parts by weight: 1 to 5 parts by weight: 10 to 25 parts by weight,
A cell membrane-binding liposome characterized in that a hydrophobic drug is contained in the lipid bilayer and a hydrophilic drug is contained in the lipid bilayer, and the cell membrane vesicle secreted by the hydrophobic drug or the hydrophilic drug is fused to the cell membrane.
14. The cell membrane-bound liposome according to claim 13, wherein the liposome has a surface potential of 12 to 20 mV.
14. The cell membrane-binding liposome according to claim 13, wherein the basic lipid is at least one phospholipid selected from the group consisting of phosphatidicolin and phosphoethanolamine phospholipids.
14. The method of claim 13, wherein the lipid loaded with the positive charge is selected from the group consisting of 18: 1 TAP (1,2-dioleoyl-3-trimethylammonium-propane), 14: 0 TAP (1,2-dimyristoyl-3-trimethylammonium- : 0 TAP (1,2-dipalmitoyl-3-trimethylammonium-propane), 18: 0 TAP (1,2-stearoyl-3-trimethylammonium-propane), 18: 0 DDAB (Dimethyldioctadecylammonium) Glycero-3-ethylphosphocholine), 14: 0 EPC (1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine), 14: 1 EPC (1,2-dimyristoleoyl- ethylphosphocholine), 18: 0 EPC (1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine), 18: 0 EPC (1,2- distearoyl- 2-dioleoyl-sn-glycero-3-ethylphosphocholine), 16: 0-18: 1 EPC (1-palmitoyl-2-oleoyl-sn- glycero-3-ethylphosphocholine), DOTMA -3-trimethylammonium propane) and DOTAP (N- [1- (2,3-Dioleoyloxy)] -N, N, N-trimethylammonium propane methyl sulphate). Little.
14. The method of claim 13, wherein the lipid loaded with the positive charge is selected from the group consisting of DOTAP (N- [1- (2,3-Dioleoyloxy)] - N, N, N-trimethylammonium propane methyl sulphate; Diolureoyloxy)] - N, N, N-trimethylammonium propane methyl sulfite).
14. The cell membrane-binding liposome according to claim 13, wherein the drug is a cell membrane dye, a photosensitizer, an anti-cancer agent, or an antibiotic.
Claim 19 is abandoned in setting registration fee. 19. The method of claim 18, wherein the anticancer agent is selected from the group consisting of Calcein, gemcitabine, Busulfan, Chlorambucil, Cyclophosphamide, Melphalan, cisplatin Cisplatin, ifosfamide, cytarabine, 5-fluorouracil (5-FU), methotrexate (MTX), daunorubicin, adriamycin, Vincristine, Vindesine, Procarbazine, Tamoxifen, Megesterol acetate, Flutamide, and Gosereline acetate. Zoladex). &Lt; / RTI &gt;
Claim 20 has been abandoned due to the setting registration fee. 19. The method of claim 18, wherein the antibiotic is selected from the group consisting of a penicillin antibiotic, a cephalosporine antibiotic, a macrolide antibiotic, a tetracycline antibiotic, a quinolone antibiotic, From the group consisting of antihistamines, antimicrobial agents, clindamycin, metronidazole, chloramphenicol, Actinomycin-D, Bleomycin and Mitomycin-C Wherein the liposome is one selected from the group consisting of liposomes and liposomes.
13. A drug delivery composition comprising the liposome of claim 13 or the cell membrane vesicle of claim 10.
A pharmaceutical composition for preventing or treating cancer comprising the liposome of claim 13 or the cell membrane vesicle of claim 10.
The pharmaceutical composition according to claim 22, wherein the cancer is selected from the group consisting of lung cancer, testicular cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, pancreatic cancer, skin cancer, stomach cancer and liver cancer Composition.
13. A antibiotic comprising the liposome of claim 13 or a vesicular vesicle of claim 10.
13. A photosensitiser comprising the liposome of claim 13 or a cell membrane vesicle of claim 10.

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