Preparation of liposome fusion-induced tumor-targeting vesicle and use thereof

Abstract

A cell exosome, which contains, on the membrane surface thereof, one or more phospholipids of a high-fluidity liposome and a surface protein of a cancer cell.

Description

Preparation and application of liposome fusion-induced tumor-targeting vesicles Technical Field

[0001] This disclosure relates to an exosome, particularly an exosome with tumor targeting and whose membrane surface comprises phospholipids derived from highly fluid liposomes and proteins on the surface of cancer cells. Background Technology

[0002] CN1665488A and US20040213766A both disclose an artificial liposome or lipid vesicle that can undergo absorption, endocytosis, fusion, and lipid exchange with the cell membrane; they also disclose that the liposome composition can be selected from a variety of components including DOPC and DOPE. Previous studies have also disclosed that liposomes can induce membrane exchange with the cell membrane through temperature and membrane ion charge control (Zhu T, Jiang Z, Ma Y. Lipid exchange between membranes: effects of membrane surface charge, composition, and curvature. Colloids Surf B Biointerfaces 2012, 97, 155-161). However, the aforementioned studies do not disclose the composition and ratio of DOPC and DOPE in the liposomes in this disclosure, nor do they indicate that it can increase the production of outer membrane vesicles.

[0003] Meanwhile, other studies have mentioned that liposome co-incubation with cancer cells can increase the generation of extracellular vesicles (Emam, SEAndo, ​​El., Lila, ASA et al. Liposome co-incubation with cancer cells secreted exosomes (extracellular vesicles) with different protein expressions and different uptake pathways, Sci. Rep. 2018, 8, 14493), but the liposomes and culture methods used are different from those disclosed in this paper, and the mechanism is not mentioned in that literature. Bacteria have been used as a model in the past, but this invention uses mammals as a model.

[0004] Exosome research and its potential applications are receiving increasing attention; however, the yield of exosomes produced by the biogenic method is extremely low, completely failing to meet the needs of scientific research and clinical applications. This invention, as a method for producing artificial exosomes, can significantly improve the bottlenecks faced by the development of related fields. Furthermore, the exosomes produced by this invention have been proven to have specific tumor targeting, are simple to prepare, and do not involve the use of toxic chemicals, demonstrating enormous potential for future commercialization. Summary of the Invention

[0005] This disclosure can increase the yield of tumor-targeting cell exosomes, which is an artificial production method. Compared with traditional biological production methods, the increased yield makes its related scientific research and clinical applications more feasible.

[0006] The results of this disclosure also support the excellent tumor targeting properties of the exosomes disclosed herein, and these exosomes can encapsulate various nanomedicines or serve as drug carriers for targeted tumor therapy. Furthermore, the exosomes produced by the method of this disclosure also have extended applications in immune activation and non-invasive detection.

[0007] The exosomes disclosed herein possess cell-specific targeting characteristics. That is, when placed in a cellular environment with the same cell type as the source cells, the exosomes are further internalized by the source cells. Therefore, the exosomes disclosed herein can be used in the preparation of drug carriers and membrane-coated nanomaterials. Drugs can be administered to patients via intravenous injection and further accumulate in the tumor region for targeted tumor therapy. The encapsulation of the drug and nanomaterials can be achieved using ultrasound-assisted methods.

[0008] This disclosure provides a cell exosome, wherein the membrane surface of the cell exosome comprises: phospholipids of one or more highly fluid liposomes; and surface proteins of cancer cells.

[0009] The "highly fluid liposomes" described in this disclosure refer to liposomes whose phospholipid fatty acid chains are C12 to C20 and whose fatty acid chains are unsaturated. High fluidity makes the membrane of the liposomes unstable, easily deformed and remodeled, and therefore easier to fuse with cells.

[0010] In this disclosure, "the fatty acid chain is unsaturated" means that the fatty acid chain contains one or more carbon-carbon double bonds.

[0011] The term "cancer cell" as used in this disclosure refers to cells, stem cells, or derivatives thereof derived from cancer or tumors.

[0012] In one embodiment, the phospholipids of the highly fluid liposomes are selected from the group consisting of: 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dilauroyl-sn-glycero-3-phosphocholine. glycero-3-phosphocholine (DLPC), 1,2-tetradecanoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine ( 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), phosphatidylethanolamine (PE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (1,2-dioleoyl-sn-glycero-3-phosphoserine) Leoyl-sn-glycero-3-phosphoserine (DOPS), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), and 1',3'-bis[1,2-dioleoyl-sn-glycero-3-phospho]-sn-glycerol (TOCL).

[0013] In a preferred embodiment, the phospholipids of the highly fluid liposomes are derived from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0014] The high-fluidity liposomes DOPC and DOPE disclosed herein have a composition ratio of 50:50 to 100:0. In a preferred embodiment, the composition ratio of DOPC and DOPE is 60:40 to 85:15. In a more preferred embodiment, the composition ratio of DOPC and DOPE is 70:30.

[0015] In one embodiment, the cancer cells include breast cancer cells, lung cancer cells, liver cancer cells, or cervical cancer cells. In a preferred embodiment, the cancer cells are breast cancer cells, liver cancer cells, or cervical cancer cells.

[0016] This disclosure also provides a pharmaceutical composition comprising the aforementioned exosomes, wherein the exosomes contain an anticancer drug or nanomaterials.

[0017] In one embodiment, the anticancer drug or the nanomaterial is encapsulated on the surface of the anticancer drug or the nanomaterial by ultrasound-assisted encapsulation of the cell exosomes. The ultrasound-assisted method involves mixing the aforementioned cell exosomes with the nanomaterial to form a mixed solution, then oscillating the mixed solution in an ultrasonic oscillation tank for 10 minutes, followed by placing it in an ice bath for 5 minutes, and repeating the cycle three times.

[0018] In one embodiment, the anticancer drug carrier or the exosomes of the encapsulated nanomaterial are administered to the patient via intravenous injection.

[0019] The anticancer drugs used in this disclosure are lipophilic or hydrophilic anticancer drugs. In a preferred embodiment, the anticancer drug is a hydrophilic anticancer drug. In a more preferred embodiment, the anticancer drug is doxorubicin (DOX). The lipophilic drug can be carried by being modified with a hydrophilic agent and embedded in a vesicle shell.

[0020] In one embodiment, the pharmaceutical composition is administered via intravenous injection.

[0021] This disclosure further provides the use of a composition in the preparation of a medicament for treating cancer, wherein the composition comprises the aforementioned exosomes, which further contain an anticancer drug.

[0022] In one embodiment for pharmaceutical preparation, the cancer includes breast cancer, lung cancer, liver cancer, or cervical cancer. In a preferred embodiment, the cancer cells are breast cancer cells, liver cancer cells, or cervical cancer cells.

[0023] This disclosure further provides a method for preparing the aforementioned cell exosomes, comprising: (a) mixing highly fluid liposomes with cancer cells and co-culturing them in a low-nutrient culture medium; and (b) collecting the cell exosomes secreted by the cancer cells in step (a), wherein the membrane surface of the cell exosomes has phospholipids of one or more of the highly fluid liposomes and surface proteins of the cancer cells.

[0024] In one embodiment, the cancer cells include breast cancer cells, lung cancer cells, liver cancer cells, or cervical cancer cells. In a preferred embodiment, the cancer cells are breast cancer cells, liver cancer cells, or cervical cancer cells.

[0025] In one embodiment, the low-nutrient culture medium is a protein- and glucose-free culture medium used for cell culture.

[0026] The anticancer drug used in this disclosure is a lipophilic or hydrophilic anticancer drug. In a preferred embodiment, the anticancer drug is a hydrophilic anticancer drug. In a more preferred embodiment, the anticancer drug is doxorubicin. Attached Figure Description

[0027] Figure 1 shows a comparison of the yield of exosomes extracted from 4T-1 breast cancer cell cultures using traditional biological methods and the liposome fusion-induced membrane exchange (LIME) method. The control group was the traditional biological method, which did not involve the addition of liposomes.

[0028] Figure 2 is a schematic diagram of the LIME mechanism.

[0029] Figure 3 shows an electron microscope image of the gold rod@silica shell nanomaterial.

[0030] Figure 4 shows the protein staining and elemental analysis. Protein analysis was performed on 4T-1 breast cancer cell lysate, liposomes, gold rod@silica nanoshells (Pure Au@SiO2), exosomes containing 4T-1 breast cancer cell surface proteins (Pure 4T-1membrane-integrated liposome), and exosomes encapsulating gold rod@silica nanoshells and doxorubicin and containing 4T-1 breast cancer cell surface proteins (Au@SiO2-doxorubicin@membrane-integrated liposome, Au@SiO2-DOX@MIL) to examine whether the surface of each liposome carries specific surface proteins.

[0031] Figure 5 shows the elemental analysis diagrams. Elemental analysis was performed on the nanomaterials coated with 4T-1 breast cancer cell surface proteins using electron microscopy. The analysis revealed the presence of nitrogen (from proteins) and phosphorus (from phospholipids) on the surface of the 4T-1 breast cancer cell lysate, pure liposomes, pure Au@SiO2 gold rod@silica nanoshells, pure 4T-1 membrane-integrated liposomes, and Au@SiO2-doxorubicin@membrane-integrated liposomes and Au@SiO2-DOX@MIL, all encapsulated with gold rod@silica nanoshells and doxorubicin.

[0032] Figure 6 shows the uptake ratio analysis of different nanomaterials by 4T-1 breast cancer cells. Using 4T-1 breast cancer cells as a model, the gold concentration of the uptake nanomaterials was identified by atomic absorption spectrometry. Uptake ratio analysis was performed on gold rods@silica nanoshells + doxorubicin (Au@SiO2-doxorubicin, Au@SiO2-DOX), liposomes encapsulating gold rods@silica nanoshells and doxorubicin (Au@SiO2-doxorubicin@Liposome, Au@SiO2-DOX@Liposome), and exosomes encapsulating gold rods@silica nanoshells and doxorubicin and containing 4T-1 breast cancer cell surface proteins (Au@SiO2-doxorubicin@4T-1membrane-integrated liposome, Au@SiO2-DOX@4T-1MIL).

[0033] Figure 7 shows the uptake ratios of different cells of exosomes encapsulating gold rods@silica nanoshells and doxorubicin, and containing surface proteins of 4T-1 breast cancer cells. These represent NIH / 3T3 embryonic fibroblasts, HeLa cervical cancer cells, and 4T-1 breast cancer cells, respectively.

[0034] Figure 8 shows the feeding of HepG2 liver cancer cells and Huh-7 liver cancer cells with unencapsulated HepG2 liver cancer cell exosomes and nanomaterials with encapsulated HepG2 liver cancer cell exosomes. Detailed Implementation

[0035] The following embodiments are non-limiting and represent only various aspects and features of this disclosure.

[0036] Example 1: Preparation and Mechanism of Tumor-Targeting Exosomes

[0037] In this embodiment, highly fluid liposomes co-constructed with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) were used to provide phospholipids. Under low-nutrient culture conditions without fetal bovine serum (FBS) and glucose, the target breast cancer cells 4T-1 were mixed with the highly fluid liposomes. After the culture dishes were placed on a shaker in an incubator and shaken for 24 hours, exosomes were collected using conventional methods, including extracting the culture solution, centrifuging at low speed to remove suspended cells, removing excessively large impurities by simple filtration, and further purifying the exosomes (MIL) by ultracentrifugation and ultrafiltration, as shown in Figure 1. A visible increase in product was produced after one day of culture.

[0038] The low-nutrient medium is RPMI-1640 medium containing 1% 100x penicillin-streptomycin solution and lacking protein and glucose.

[0039] The culture medium used for 4T-1 breast cancer cells was RPMI-1640 containing 1% 100x penicillin-streptomycin solution and 10% fetal bovine serum (FBS).

[0040] The high-fluidity liposomes DOPC and DOPE disclosed herein have a composition ratio of 70:30.

[0041] The process of mixing target breast cancer cells 4T-1 with highly fluid liposomes is called liposome fusion-induced membrane exchange (LIME), as shown in Figure 2. Its characteristic is that when cells are under starvation (nutrient deficiency), they will further absorb more phospholipids from the environment to replace the original membrane-generating mechanism, and the highly fluid liposomes can further enhance their fusion with the cells. Furthermore, the excessive incorporation of highly fluid phospholipids into the cell membrane will further cause cell membrane instability, thereby promoting the secretion of exosomes. During this process, surface proteins of the original cells may also be carried away. It is noteworthy that the artificial exosomes obtained through this method possess the surface characteristics of the source cells, thus exhibiting an inherent ability to integrate with the source cells. Therefore, the artificial exosomes disclosed in this paper demonstrate the application potential of tumor-targeted drugs and membrane-coated nanoparticles.

[0042] Example 2: Coating with nanomaterials

[0043] In this embodiment, gold rod@silica nanoshell (Au@SiO2-DOX) is used as a representative nanomaterial. As shown in Figure 3, exosomes are mixed with nanomaterials to form a mixed solution. The mixed solution is then agitated in an ultrasonic oscillation tank for 10 minutes, followed by standing in an ice bath for 5 minutes. This cycle is repeated three times to encapsulate the exosomes disclosed in this invention on the surface of the nanomaterial, forming exosomes encapsulated with gold rod@silica nanoshell (Au@SiO2-DOX@MIL).

[0044] Example 3: Protein Identification and Elemental Analysis

[0045] This embodiment confirms through protein identification and elemental analysis that the cell surface proteins of the disclosed exosomes are successfully coated onto the surface of nanomaterials. As shown in Figures 4 and 5, protein analysis was performed on 4T-1 breast cancer cell lysate, liposomes (Pure Liposome), gold rod@silica nanoshells (Pure Au@SiO2), exosomes with 4T-1 breast cancer cell surface proteins (Pure 4T-1 membrane-integrated liposome), and exosomes encapsulating gold rod@silica nanoshells and doxorubicin and possessing 4T-1 breast cancer cell surface proteins (Au@SiO2-doxorubicin@membrane-integrated liposome, Au@SiO2-DOX@MIL) (Figure 4). The results show that the surface of each type of liposome is successfully coated with specific surface proteins. Elemental analysis (Figure 5) was performed using electron microscopy to analyze the elemental composition of the nanomaterials encapsulated by the liposomes.

[0046] Example 4: Cell Experiment

[0047] As shown in Figures 6 and 7, in this embodiment, Au@SiO2-DOX and Au@SiO2-DOX@MIL were fed to 4T-1 breast cancer cells and HeLa cervical cancer cells. It is worth noting that the proportion of 4T-1 breast cancer cells internalized by the nanomaterial (Au@SiO2-DOX@MIL) encapsulated with exosomes was significantly increased. At the same time, both 4T-1 breast cancer cells and HeLa cervical cancer cells successfully took up Au@SiO2-DOX@MIL.

[0048] In this embodiment, HepG2 liver cancer cells and Huh-7 liver cancer cells were also fed HepG2 liver cancer cell exosomes encapsulated with nanomaterials. As shown in Figure 8, HepG2 liver cancer cells and Huh-7 liver cancer cells were fed unencapsulated HepG2 liver cancer cell exosomes and nanomaterials encapsulated with HepG2 liver cancer cell exosomes, respectively. The results showed that the proportion of nanomaterials encapsulated with cell exosomes was significantly increased by the HepG2 liver cancer cells.

[0049] This embodiment demonstrates that the exosomes disclosed herein possess excellent tumor targeting capabilities, providing a novel possibility and enormous commercial potential for related developments.

[0050] The exosomes disclosed herein have target specificity for the source cells. That is, when the exosomes are placed in a cellular environment with the type of source cells, the exosomes will be further internalized by the source cells. Therefore, the exosomes disclosed herein can be used in the preparation of drug carriers and membrane-coated nanomaterials. The drugs can be administered to patients via intravenous injection and will further accumulate in the tumor area for tumor-targeted therapy.

[0051] Those skilled in the art will readily understand that this disclosure is highly suitable for achieving the foregoing objects and obtaining the foregoing objects and advantages, as well as those inherent therein. The processes and methods described above for producing them represent preferred embodiments, are exemplary, and do not limit the scope of this disclosure. Modifications and other uses will be apparent to those skilled in the art. These modifications are included within the spirit of this disclosure and are defined by the scope of the claims.

Claims

1. An extracellular vesicle, wherein the membrane surface of the extracellular vesicle comprises: one or more phospholipids of a high fluidity liposome; and a surface protein of a cancer cell.

2. The extracellular vesicle of claim 1, wherein the high fluidity liposome refers to the fatty acid chain of the phospholipid on the liposome is C 12 to C 20 , and the fatty acid chain has a high degree of unsaturation.

3. The extracellular vesicle of any one of claims 1, wherein the phospholipid of the high fluidity liposome is selected from the group consisting of 1,2-dioleoyl-sn-glycero-3- phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3- phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1- stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), Phosphatidylethanolamine (PE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dioleoyl-sn- glycero-3-phosphoserine (DOPS), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), and 1',3'-bis[1,2-dioleoyl-sn-glycero-3-phospho]-sn-glycerol (TOCL).

4. The extracellular vesicle of any one of claims 1, wherein the phospholipid of the high fluidity liposome is 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

5. The extracellular vesicle of any one of claims 1, wherein the phospholipid of the high fluidity liposome is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

6. The extracellular vesicle of any one of claims 1, wherein the phospholipid of the high fluidity liposome is 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC).

7. The extracellular vesicle of any one of claims 1, wherein the phospholipid of the high fluidity liposome is 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC).

8. The extracellular vesicle of any one of claims 1, wherein the phospholipid of the high fluidity liposome is 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC).

9. The extracellular vesicle of any one of claims 1, wherein the phospholipid of the high fluidity liposome is 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC).

10. The extracellular vesicle of any one of claims 1, wherein the phospholipid of the high fluidity liposome is Phosphatidylethanolamine (PE).

11. The extracellular vesicle of any one of claims 1, wherein the phospholipid of the high fluidity liposome is 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE).

12. The extracellular vesicle of any one of claims 1, wherein the phospholipid of the high fluidity liposome is 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS).

13. The extracellular vesicle of any one of claims 1, wherein the phospholipid of the high fluidity liposome is 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG).

14. The extracellular vesicle of any one of claims 1, wherein the phospholipid of the high fluidity liposome is 1',3'-bis[1,2-dioleoyl-sn-glycero-3-phospho]-sn-glycerol (TOCL).

4. The extracellular vesicle of claim 3, wherein the phospholipid of the high fluidity liposome is from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

5. The extracellular vesicle of any one of claims 1, wherein the cancer cell comprises a breast cancer cell, a lung cancer cell, a liver cancer cell, or a cervical cancer cell.

6. A pharmaceutical composition comprising the extracellular vesicle of claim 1, wherein the extracellular vesicle comprises an anticancer drug or a nanomaterial.

7. Use of a composition in the manufacture of a medicament for treating cancer, wherein the composition comprises the extracellular vesicle of claim 1, and the extracellular vesicle further comprises an anticancer drug.

8. The use of claim 7, wherein the cancer comprises a breast cancer, a lung cancer, a liver cancer, or a cervical cancer.

9. A method of preparing the extracellular vesicle of any one of claims 1 to 5, comprising: (a) mixing a high fluidity liposome with a cancer cell, and co-culturing in a low nutrient medium; and (b) collecting the extracellular vesicle secreted by the cancer cell in step (a), wherein the membrane surface of the extracellular vesicle has one or more phospholipids of the high fluidity liposome and surface proteins of the cancer cell.

10. The method of claim 9, wherein the low nutrient medium is a medium without protein and glucose. ​ ​

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